Nusantara Bioscience vol. 6, no. 1, April 2014 by Nusantara Bioscience

Tilia platyphyllos photo by Andy Hamilton

| Nus Biosci | vol. 6 | no. 1 | pp. 1-106 | May 2014 | | ISSN 2087-3948 | E-ISSN 2087-3956 |

| Nus Biosci | vol. 6 | no. 1 | pp. 1-106 | May 2014 | | ISSN 2087-3948 | E-ISSN 2087-3956 | I S E A

J o u r n a l

o f

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N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060101

Vol. 6, No. 1, pp. 1-6 May 2014

Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia) KAVEH RAHMANIFARAH1,â&#x2122;Ľ, BAHAREH SHABANPOUR1, AMIR REZA SHAVIKLO2, MEHRAN AALAMI3 1

Department of Fishery, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran. Tel/fax. +98 171 2227867, â&#x2122;Ľ email: k.rahmanifarah@gmail.com 2 Department of seafood processing, Iranian Fisheries Research Organization, Tehran, Iran 3 Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran. Manuscript received: 31 January 2014. Revision accepted: 10 April 2014.

Abstract. Rahmanifarah K, Shabanpour B, Shaviklo AR, Aalami M. 2014. Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia). Nusantara Bioscience 6: 1-6. The effect of protein extraction procedures (leached mince and heated suspension) on selected properties of fish protein powder (proximate composition, pH, color, density, viscosity, fat adsorption, emulsifying capacity, emulsifying stability, foaming capacity, foaming stability, WBC, protein solubility in water, hygroscopicity, Trichloroacetic acid (TCA)-soluble peptides and free sulfhydryl groups) was investigated. Results showed that Fish protein powder (FPP) produced by leaching mince (LM) have higher protein, moisture, ash, pH, L*, viscosity, emulsion capacity, emulsion stability, foam capacity, foam stability, water binding capacity (WBC), protein solubility, hygroscopicity, TCA soluble peptides and free sulfhydryl group content than heated suspension (HS) (P<0.05). However, HS had higher fat and density in comparison with LM (P<0.05). No significant differences between a*, b* and fat adsorption were observed (P>0.05). Overall, it was observed that high temperature during heating of suspension in HS method makes possible protein denaturation and aggregation. Consequently, based on functional, chemical and physical properties, extraction of fish protein by leaching process was found to be suitable for the production of fish protein powder. Key words: Fish protein powder, functional properties, kilka, leaching, suspension

INTRODUCTION

is regarded as an excellent source of high quality protein, particularly the essential amino acids lysine and methionine (Sathivel and Bechtel 2006). Many protein-rich seafood byproducts have a range of dynamic properties (Phillips et al. 1994) and can potentially be used in foods as binders, emulsifiers, and gelling agents (Sathivel et al. 2004). The FPP, kept above 0oC has many advantages in food trade such as ease of handling, low distribution costs, convenient storage and ease in mixing with other ingredients (Shaviklo

Common kilka (Clupeonella cultiventris caspia) is one of the most important economic fishes in the Caspian Sea (Figure 1). It belongs to the Clupeidae family (Nelson 1998). The processing of small pelagic fish is associated with difficulties. Kilka have a dark and sensitive muscle, small size and high value of fat that cause difficulty in processing. Therefore, because of these unsuitable properties more than 95% of the Kilka resource in Iran has no direct human applications. Methods for processing small pelagic fish such as Kilka that counteract these difficulties and facilitate utilization of their valuable proteins have been missing. Typically, most Kilka are dried at high temperature and powdered for livestock and poultry feed. The functional properties of proteins are a major interest as they affect the usability of the proteins in different food applications. Sathivel et al. (2004) reported that protein powders from herring and arrow tooth were good sources of high quality fish protein with many Figure 1. Common kilka (Clupeonella cultiventris caspia) (photo: Yuriy Kvach) desirable functional properties. Fish

N U SAN TA RA B IOSC IE NC E 6 (1): 1-6, May 2014

et al. 2010). The minced fish can be dried via different drying procedures. Cordova-Murueta et al. (2007), Huda et al. (2001) and Shaviklo et al. (2010) applied hot air drying, vacuum drying and spray drying, respectively to dry the minced fish. However, these methods have some advantage of fish drying, but freeze drying is the best method for drying fish mince (Cordova-Murueta et al. 2007; Huda 2001). Extraction of fish proteins to produce FPP can be turned into a solvent extraction method (Liston and Pigott 1971), pH shifting method (Hultin and Kelleher 1999), Enzyme/acid hydrolysis (Hoyle and Merritt 1994) and Mince leaching (Shimizu 1965) (surimi) by adding four times the weight of water and has excellent functional properties such as the ability to form kamaboko gels (Huda et al. 2001; Niki et al. 1983; Shaviklo 2013). Moreover, Sathivel et al. (2003) introduces the new method to extract fish protein that heated the fish suspension and dried after separation. In this study, we compare the leaching process with heated suspension to investigate some functional and chemical properties of dried leached mince and heated suspension. MATERIALS AND METHODS Materials Kilka fish was caught from Caspian Sea and transported to Amir Abad beach in the Caspian Sea Water (CSW) system. Afterwards, the fish was packed in polystyrene boxes with ice and brought to the laboratory. FPP production Samples of Kilka were washed, gutted, headed and deboned with bone separator (Bone Separator Farayazan Andishan, Iran). After mincing, fish mince was split into two parts. One part was used to prepare leached mince. The ratio of mince to water was 1: 5 and the temperature was kept under 10 °C during processing. The mixture was stirred well with a stainless steel spatula for 15 min. The slurry was passed through cheesecloth (Shimizu 1965). At this stage leached mince was prepared. Another protein extraction method was done according to the method of Sathivel et al. (2003). A 500 g portion of each ground fish part was mixed with an equal volume of distilled water and homogenized in an Ultraturrax homogenizer (IKA, T25, digital Germany) for 2 min. The mixture was continuously stirred for 60 min at 85 °C. The heated suspension was centrifuged at 2560 g for 15 min, resulting in three separate phases: the semi solid phase at the bottom containing insoluble protein, bone, and skin; the heavy liquid phase in the middle containing soluble proteins, and the light liquid phase at the top, containing crude lipids. The heavy liquid middle layer was separated and collected. Leached mince (LM) and heated suspension (HS) were mixed with 2% (w/w) sucrose and 0.2 % (w/w) sodium tripolyphosphate as lyoprotectants using a silent cutter (Saya, Pars Khazar, Iran) for 5 min. After mixing, protein

extracts were freeze-dried for 72 h. The resulting FPP samples Milled and placed in zip lock plastic and stored at -80 °C until analyzed. Proximate composition Crude protein, ash, moisture and lipid content of samples were analyzed by the method of AOAC (1990). The 5 g of samples was dried in an oven at 105oC until constant weights were achieved and moisture content was calculated. Samples were then extracted using a Soxhlet extraction (416 SE, Gerhardt, Germany) with petroleum ether to determine oil content. Protein content was determined using the Kjeldahl method (Gerhardt, Vap 40, Germany). Ash content was determined by holding samples overnight at 550oC. pH pH of FPP was determined by blending 5 g of samples with 20 mL of distilled water for 30 second using an Ultra Turrax tissue homogenizer (T25 IKA-Ultra-Turrax, Germany). The pH of suspension was recorded by using a combined glass electrode with a digital pH meter (728 pH Lat Stirrer, Metrohm). Color FPP sample color was evaluated using the colorimeter (Lovibond CAM-system, England 500). CIE (Commission Internationale de l’Eclairage) L* (lightness), a* (red to green), and b* (yellow to blue) were measured. All samples were kept at room temperature in a plastic bag for more than 2 h to eliminate the effects of various temperatures at measurement. Density Density was determined in triplicate for each sample by placing the sample in a pre-weighed 10 mL graduated cylinder up to the 10 mL mark with gentle tapping. The graduated cylinder weighed again and the density was calculated as g powder per mL volume (Venugopal et al. 1996). Viscosity The viscosity of FPP was measured using a Brookfield synchro-lectric viscometer model LVT (Brookfield Ltd, Cooksville, ON, Canada). A sample solution containing 10 % protein was prepared from each FPP and homogenized with Ultra Turrax homogenizer (T25 IKA-Ultra-Turrax, Germany). The solution was kept refrigerated overnight. It was homogenized the day after, before running viscosity measurements. The viscosity was measured routinely at 60 rpm using spindle No. 3. The values were recorded after 30 s of rotation of the spindle in the dispersion. Fat adsorption The fat adsorption capacities were determined by the methods of Shahidi et al. (1995). This test was performed in triplicate and fat adsorption was expressed as the volume (mL) of fat adsorbed by 1 g of protein.

RAHMANIFARAH et al. – Production of protein powder from Clupeonella cultiventris caspia

Emulsification properties Emulsifying capacity was measured using the procedure described by Yatsumatsu et al. (1972). The powder (1 g) was added to 25 mL of distilled water and 25 mL of sunflower oil. The mixture was then mixed with Ultra Turrax homogenizer (T25 IKA-Ultra-Turrax, Germany) for 1 min and transferred to the 50-mL calibrated centrifuge tube. The tube containing the sample was then centrifuged at 7500 g for 5 min. The emulsifying capacity was calculated by dividing the emulsion volume after centrifugation by the original emulsion volume and then multiplied by 100. Emulsifying stability was determined by the same procedure, except that, before centrifugation the emulsion was heated at 90 ◦C for 30 min followed by cooling in tap water for 10 min (Yatsumatsu 1972). Foaming properties Determination of foaming capacity was done following the method of Miller and Groninger (1976) with slight modification. Forty milliliter of 1 % protein aqueous dispersion was mixed thoroughly using an Ultra Turrax (T25 Ika-Ultra-Turrax, Germany) at 10,000 rpm for 2 min. The total volume of the protein dispersion was measured immediately after 30 sec. The difference in volume was expressed as the volume of the foam. Foam stability was determined by measuring the fall in volume of the foam after 1 h. Water binding capacity WBC was measured using the method described by the American Association of Cereal Chemists (AACC 1981) with slight modifications. 1 g FPP was weighed into centrifuge tubes. Subsequently, 40 mL deionized water was added and mixture was left to stand for 30 min at room temperature and then the sample was centrifuged at 5000 g for 15 min. The weight of supernatant after centrifuge was recorded. WBC was expressed as the ratio of weight gained per unit weight of fish protein powder (AACC 1981). Protein solubility in water To determine protein solubility, 1 g FPP was dispersed in 20 mL of deionized water. The mixture was stirred at room temperature for 30 min with intermittent stirring and then the sample was centrifuged at 5000 g for 15 min. Protein content in the supernatant and in the sample was determined. Protein solubility was calculated as follows: Protein content in supernatant Solubility = -------------------------------------------x 100 Total protein content in sample

Hygroscopicity For the hygroscopicity, about 1 g of FPP was placed in a desiccator containing a saturated solution of Na2SO4 to establish a relative humidity of 81 % (Jaya and Das 2004). After keeping the sample at 25oC for a week, the hygroscopic moisture (%) was calculated using the equation:

Where a (g) was the amount of the sample, Wi was the moisture content in the powder before the measurement and b (g) was the powder weight increase. All the measurements were made in three replicates. TCA-soluble peptides TCA-soluble peptides were determined according to the method of Visessanguan et al. (2004). FPP samples (1 g) were homogenized with 29 mL of cold 5% (w/v) TCA with an Ultra Turrax (T25 Ika-Ultra-Turrax, Germany) and kept at 4oC for 1 h, followed by centrifugation at 12,000 g for 15 min at 4oC. TCA-soluble peptides in the supernatant were measured by the method of Biuret using bovine serum albumin (BSA) as a standard. The results are the average of three determinations and expressed as micromole tyrosine/ gram sample. Free sulfhydryl groups The concentration of free sulfhydryl groups (SH) of the FPP samples was determined using Ellman’s reagent (50, 5-dithiobis (2-nitrobenzoic acid), DTNB) (Sigma-Aldrich. Milan, Italy). Changes in free sulfhydryl groups were measured in triplicate as reported by Beveridge et al. (1974). Briefly, FPP 1.5 g was diluted to 10 mL with 1% (p/v) NaCl in tris-glycine buffer (10.4 g tris, 6.9 g glycine, 1.2 g EDTA per liter, pH 8.0). A volume of 2.9 mL of 0.5% SDS in tris-glycine buffer was added to 0.1 mL of diluted egg white and 0.02 mL of Ellman’s reagent (4 mg/mL DTNB in tris-glycine buffer) to develop a color. After 15 min, absorbance was measured at 412 nm using a UV-VIS spectrophotometer. The concentration of free sulfhydryl groups (l mg-1) was calculated from the following equation:

Where A412 is the absorbance at 412 nm; C is FPP concentration (mg/mL); D is the dilution factor; and 73.53 is derived from 106/(1.36×104); 1.36×104 is the molar absorptive and 106 is the conversion factor from molar basis to µM/mL and mg solid to g solid respectively (Ellman 1959). Data analysis All data presented are means ± standard deviations. Assays were conducted in triplicate and the statistical significance of differences between means (P<0.05) was determined by Student’s t-tests. The analysis was carried out using SPSS 16.0 for Windows software package. RESULTS AND DISCUSSION Table 1 depicts proximate composition and pH value of trial group. Results show that LM has higher protein content than HS (P<0.05) but the fat content in the LM was

N U SAN TA RA B IOSC IE NC E 6 (1): 1-6, May 2014

lower than HS (P<0.05). Better protein extraction in LM protein for herring protein powders (Sathivel et al. 2004), than HS could be due to higher fat depletion during 3.7 to 7.3 mL of oil/g protein for hydrolyzed herring byleaching process. Hence fat reduction leads to an overall product proteins (Sathivel et al. 2003) and 2.86 to 7.07 mL increase in protein content in LM. Fat content of HS in this of oil/g protein for Atlantic salmon protein hydrolysate work is similar to the fat content of insoluble fraction in the (Kristinsson and Rasco 2000). The mechanism of fat study of Sathivel et al. (2008). Sathivel et al. (2006) binding capacity is thought to be mainly because of the observed protein extraction with solubilizing leads to physical entrapment of the oil (Sathivel and Bechtel 2008). The ability of proteins to form stable emulsions is increase lipid scattering in suspension and therefore fat reduction in extracted protein. Kahn et al. (1974) reported a important for interaction between proteins and lipids in number of variables influencing solubility and protein many food systems. It has been reported that proteins with extraction efficiency from fish tissues, including both hydrophilic and hydrophobic residues act as concentration and particle size of suspended tissues, emulsifiers and when the protein has a balance between extraction time, temperature, pH, and the type and these residues the emulsion capability is optimal concentration of salts used for extraction. Moisture and ash (Damodaran 2008). Emulsion capacity and emulsion content in the LM was higher than HS (P<0.05). Higher stability are shown in Figure 2. LM had higher emulsion moisture might be partly due to higher cohesion of leached capacity than HS (P<0.05). It could be explained by more mince during freeze drying in comparison with heated protein denaturation during protein extraction in HS suspension. Heating process in HS extracting method leads procedure while protein exposed to 85˚C for 60 min. to protein denaturation and aggregation. Lower ash value in Emulsion capacity and stability of Kilka protein powder in HS may be as results of better separation process during this work are lower than Arrow tooth Flounder and Herring protein extraction. However, in LM some bone section that reported by Sathivel et al. (2004). Gauthier et al. probably causes an increase in ash content. Additionally, (1993) stated that factors such as protein solubility and hydrophobicity also play major roles in emulsifying pH in the LM was higher than HS (P<0.05). As can be seen in Table 2, Lightness of LM was higher properties. Both FPP had lower foam capacity in comparison with than HS (P<0.05). Millard reaction during heating and lower moisture content throughout drying could be the other report (Shaviklo et al. 2010). The molecular reason for lower lightness of HS group. Redness and yellowness in the HS group were higher than in the LM Table 1. Proximate composition and pH of leached mince (LM) and heated suspension group, but these differences were not (HS) significant (P>0.05). Density of HS was higher than LM (P<0.05) Protein Fat Moisture Ash Sample pH because of lower pores. The higher % % % % a b a a the processing temperature, the LM 74.85±1.6 8.17±1.7 3.53±0.3 7.08±0.3 7.93±0.1a b a b b HS 68.35±0.1 24.66±0.6 2.31±0.1 3.61±0.3 7.51±0.1b higher the shrinkage of the material Different letters (a-b) represent significant differences between protein extraction method leading to lower levels of pores (P<0.05, n = 3). Values are means±SD. (Rahman et al. 2002). Density of freeze-dried Kilka protein powder in this study was higher than the freezeTable 2. Color, density, viscosity and fat adsorption of leached mince (LM) and heated dried lizard fish, thread fin bream suspension (HS) and purple spotted big-eye prepared by Huda et al. (2001). In the current Fat Density Viscosity study LM had a higher viscosity than adsorption Sample L* a* b* g/ml Pa HS (P<0.05). Higher viscosity in LM oil/g a a a b attributes better protein quality in LM 68.20±0.1 4.30±0.0 2.46±0.4 0.39±0.1 1.93±0.1a 3.67±0.3a HS 62.20±0.9b 4.83±0.5a 2.70±0.1a 0.43±0.1a 1.20±0.1b 3.2±0.4a comparison with HS. The viscosity Different letters (a-b) represent significant differences between protein extraction method of all FPP samples was lower than (P<0.05, n = 3). Values are means±SD. that of Saithe reported by Shaviklo et al. (2010). This may be as results of lower quality in functional properties Table 3. WBC, protein solubility, hygroscopicity, TCA soluble peptides and free of Kilka protein than Saithe. Fat sulfhydryl group content of leached mince (LM) and heated suspension (HS) adsorption is an important functional characteristic of ingredients used in TCA-soluble Free Protein Hygrothe meat and confectionery WBC peptides sulfhydryl solubility scopicity Sample industries. Results show no (%) (micromole group (%) (%) tyrosine/g) (micromole/g) significant differences among LM 637.1±38.3a 30.21±6.0a 3.05±0.1a 5.49±0.3a 0.86±0.3a different extraction method (P>0.05) b b b b HS 226.3±13.7 25.46±2.7 2.22±0.1 4.73±0.2 0.29±0.1b (Table 2). Fat adsorption capacity Different letters (a-b) represent significant differences between protein extraction method values have been reported that (P<0.05, n = 3). Values are means±SD. WBC: water binding capacity ranged from 3.9 to 11.5 mL of oil/g

RAHMANIFARAH et al. – Production of protein powder from Clupeonella cultiventris caspia

properties relevant to foaming are similar to those required for emulsification (Panyam and Kilara 1996). Foam ability is an important functional property of proteins by which proteins form a flexible cohesive film to entrap air bubbles. In this study LM shows higher foam capacity than HS (P<0.05) (Figure 2). Proteins that rapidly unfold and adsorb at the freshly formed air/liquid interface during bubbling exhibit improved foam ability (Damodaran 1997). Foam expansion is mainly related to the solubility of proteins (Kinsella 1979). The more the proteins are soluble, the more the protein available to form the flexible cohesive film to entrap the air and hence the higher foam expansion. In this study extraction method in the HS group had lower protein solubility; therefore higher foam capacity of LM could be explained. Foam stability of FPP reported by Shaviklo et al. (2010) was higher than observed in this work (Figure 3). Formed foam was dropped after about few min and slight foam was absorbed at 1 h.

Figure 2. Emulsion capacity and emulsion stability of leached mince (LM) and heated suspension (HS). Different letters (a-b) represent significant differences between same factor (P<0.05, n = 3). Values are means±SD.

Figure 3. Foam capacity and foam stability of leached mince (LM) and heated suspension (HS). Different letters (a-b) represent significant differences between same factor (P<0.05, n = 3). Values are means±SD.

Results of current study depicted higher amount of WBC and protein solubility in the LM group than HS (P<0.05) (Table 3). At higher drying temperatures, WBC and protein solubility could be decreased as a result of protein denaturation (Huda et al. 2000). High temperature

during heating of suspension in HS method makes possible protein denaturation and aggregation. From other studies, it is clear that WBC is closely related to fish species (Huda et al. 2001), amount of Lyoprotectants (Matsuda 1971), different techniques and processes used for drying, and the interaction between these factors (Roos 2002). Mean WBC of LM in this study was 637 % while in other study reported by Shaviklo et al. (2010) on freeze dried and spray dried fish protein powder on Saithe ranged between 300350 % (Shaviklo et al. 2010). Hygroscopicity of fish protein powder in LM group was higher than HS group (P<0.05). The hygroscopicity can be defined as the ability of a food to absorb the moisture from a high relative humidity environment and has been related either to the porosity of the powder (Nadeau et al. 1995) or the amorphous glassy state of the sugars present in the food (Roos 2002). With regard to the density of FPP, it obviously appeared that LM has more porous than HS therefore porosity could be the reason for higher hygroscopicity of LM in this work. LM had higher TCA-soluble peptides than HS (P<0.05). TCA-soluble peptides for LM and HS were 5.49 and 4.73 respectively. High TCA-soluble peptide content indicated a greater hydrolysis and degradation of muscle proteins. Leached mince extraction might produce small peptides, resulting in an increase in TCA-soluble peptide content. Myofibrillar protein degradation, especially myosin, resulted in reduction in molecular weight and the loss of structural domains, which are essential for molecular interaction and binding (Visessanguan and An 2002). However, lower TCA-soluble peptide content in HS than LM, could be explained by higher aggregation of HS protein affected by heating denaturation. Sulfhydryl groups are considered to be the most reactive functional group in proteins. Free sulfhydryl groups of trial treatments (Micromole/g) are depicted in Table 3. The free sulfhydryl group method has been widely used in order to evaluate protein oxidation and more precisely cysteine oxidation. Cysteine oxidation can induce protein cross links by the formation of intermolecular disulfide bridges, thus, the higher the free sulfhydryl groups the lower cysteine oxidation (Lara et al. 2011). Free sulfhydryl groups in current work shows significant differences between trial groups (P<0.05). The free sulfhydryl groups content of the LM and HS were 0.86 and 0.26 respectively. Protein oxidation is also associated with a decrease in sulphydryl groups, which are converted into disulphides (Batifoulier et al. 2002). Cysteine residues in proteins occur as the free sulphydryl form or oxidized system. Oxidation induced by a massive free radical production had the edge on the reduction process leading to the decrease of the free sulfhydryls (Soyer et al. 2010). Heat treatment could induce the SH/S-S exchange reaction and lead to an increase S-S group level during suspension heating. Visschers and De Jongh (2005) had reported that cysteine residues and disulphide bonds had important contributions to the aggregation of proteins. Increase in the S-S group level during processing could induce changes in myofibrillar proteins structure and lead to protein aggregation.

N U SAN TA RA B IOSC IE NC E 6 (1): 1-6, May 2014 CONCLUSION

The overall results of this study have shown that protein extraction without heating process gives better quality in fish protein powder. Extraction of protein with the leaching process shows better fat suspension and therefore higher protein extraction. Functional and physico-chemical properties of FPP extracted with leached process were higher than for the heated suspension procedure. Consequently, based on functional, chemical and physical experiments, extraction of fish protein by leaching process was found to be suitable for the production of fish protein powder. REFERENCES AOAC. 1990. Official Methods of Analysis, Association of Official Analytical Chemists, Washington DC, USA. American Association of Cereal Chemists [AACC]. 1981. Technical committee. Method for water hydration capacity of plant protein material. 1981. Cereal Foods World 26: 291-293. Batifoulier F, Mercier Y, Gatellier P, Renerre M. 2002. Influence of vitamin E on lipid and protein oxidation induced by H2O2-activated MetMb in microsomal membranes from turkey muscle. Meat Sci 61: 389-395. Beveridge T, Toma SJ, Nakai S. 1974. Determination of free SH-and SS groups in some food proteins using Ellman’s reagent. J Food Sci 39: 49-51. Cordova-Murueta JH, Navarrete-del TMA, Garcia-Carreno FG. 2007. Concentrates of fish protein from by catch species produced by various drying processes. Food Chem. 100: 705-711. Damodaran S. 1997. Food proteins: an overview. In: Damodaran S, Paraf A (eds). Food proteins and their applications. Marcel Dekker, New York. Damodaran S. 2008. Amino acids, peptides and proteins. In: Damodaran S. (ed). Fennema’s Food Chemistry. 4th ed. CRC Press/Taylor & Francis, New York. Ellman GD. 1959. Tissue sulfhydryl groups. Arch Biochem and Biophys 82: 70-77. Gauthier SF, Paquin P, Pouliot Y, Turgeon S. 1993. Surface activity and related functional properties of peptides obtained from whey proteins. J Dairy Sci 76: 321-328. Hoyle NT, Merritt JH. 1994. Quality of fish protein hydrolysates from herring (Clupea harengus). J Food Sci 59: 76-79. Huda N, Aminah A, Babji AS. 2000. Nutritional quality of surimi powder from Threadfin bream. J Muscle Foods 11: 99-109. Huda N, Aminah A, Babji AS. 2001. Functional properties of surimi powder from three Malaysian marine fish. Int J Food Sci Technol 36: 401-406. Hultin HO, Kelleher SD. 1999. Process for isolating a protein composition from a muscle source and protein composition. U.S. Patent no. 6,005,073. Jaya S, Das H. 2004. Effect of malt dextrin, glycerol monostearate and tricalcium phosphate on vacuum dried powder properties. J Food Eng 63: 125-134. Kahn LN, Berk Z, Pariser ER, Goldblith SA, Flink JM. 1974. Squid protein isolate: effect of processing conditions on recovery of yields. J Food Sci 39: 592-595. Kinsella JE. 1979. Functional properties of soy proteins. J Am Oil Chem Soc 56: 242-57. Kristinsson HG, Rasco BA. 2000. Biochemical and functional properties of Atlantic salmon (Salmo salar) muscle proteins hydrolyzed with various alkaline proteases. J Agric Food Chem 48: 657-666. Lara MS, Gutierrez JI, Timón M, Andrés AI. 2011. Evaluation of two natural extracts (Rosmarinus officinalis L. and Melissa officinalis L.)

as antioxidants in cooked pork patties packed in MAP. Meat Sci 88: 481-488. Liston J, Pigott GM. 1971. Fish protein concentrate. In: 3rd international congress; food science and technology. Proceedings SOS/ 70, Institute of Food Technologists, Chicago, IL. Matsuda Y. 1971. Influence of platen temperature of freeze-drying on the kamaboko-forming ability of lyophilized kaen-surimi. Bull Jpn Soc Sci Fish 37: 135-145. Miller R, Groninger HS. 1976. Functional properties of enzyme modified fish protein deviates. J Food Sci 41: 268-272. Nadeau JP, Puiggali JR. 1995. Sechage: des processus physiques aux procedes industriels. 1st Ed.; Technique et Documentation-Lavoisier, Paris Cedex. Nelson G, Paxton JR, Eschmeyer WN. 1998. Encyclopedia of Fishes. Academic Press, San Diego. Niki H, Kato T, Deya E, Igarashi S. 1983. Water holding capacity, emulsifying capacity and storage ability of active fish protein powder. Bull Jpn Soc Sci Fish 49: 91-96 Panyam D, Kilara A. 1996. Enhancing the functionality of food protein. Trends Food Sci Tech 7: 120-125. Phillips LG, Whitehead DM, Kinsella J. 1994. Structure Function Properties of Food Proteins. Academic Press: San Diego, CA. Rahman MS, Al-Amri OS, Al-Bulushi IM. 2002. Pores and physicochemical characteristics of dried tuna produced by different methods of drying. J Food Eng. 53: 301-313. Roos YH. 2002. Importance of glass transition and water activity to spray drying and stability of dairy powders. Lait. 82: 475-484. Sathivel S, Bechtel PJ, Babbitt J, Prinyawiwatkul W, Negulescu II, Reppond KD. 2004. Properties of Protein Powders from Arrow tooth Flounder (Atheresthes stomias) and Herring (Clupea harengus) Byproducts. J Agr Food Chem 52: 5040-5046. Sathivel S, Bechtel PJ, Babbitt J, Smiley S, Crapo C, Reppond KD, Prinyawiwatkul W. 2003. Biochemical and functional properties of herring (Clupea harengus) byproduct hydrolysates. J Food Sci. 68: 2196-2200. Sathivel S, Bechtel PJ. 2006. Properties of soluble protein powders from pollock. Inter J Food Sci Tech 41: 520-529. Sathivel S, Bechtel PJ. 2008. A comparison of physical and rheological properties of arrow tooth flounder protein made using three different Extracting Processes. J Food Biochem 32: 557-575. Shahidi F, Han XQ, Synowiecki J. 1995. Production and characteristics of protein hydrolysates from capelin (Mallotus villosus). Food Chem 53: 285-293. Shaviklo AM. 2013. Development of fish protein powder as an ingredient for food applications: a review. J Food Sci Technol. DOI. 10.1007/s13197-013-1042-7. Shaviklo GR, Thorkelsson G, Kristinsson HG, Arason S, Sveinsdottir K. 2010. The influence of additives and drying methods on quality attributes of fish protein powder made from saithe (Pollachius virens). J Sci Food Agric 90: 2133-2143. Shimizu, Y. 1965. Manufacturing method of leached meat. Japanese Patent, Showa 40, 21224. Soyer A, Ozalp B, Dalmıs U, Bilgin V. 2010. Effects of freezing temperature and duration of frozen storage on lipid and protein oxidation in chicken meat. Food Chem 120: 1025-1030. Venugopal V, Chawla SP, Nair PM. 1996. Spray-dried protein powder from threadfin bream: preparation, properties and comparison with FPC type-B. J Muscle Foods 7: 55-71. Visessanguan W, An H. 2002. Effects of proteolysis and mechanism of gel weakening in heat-induced gelation of fish myosin. J Agr Food Chem 48: 1024-1032. Visessanguan W, Benjakul S, Riebroy S, Thepkasikul P. 2004. Changes in composition and functional properties of proteins and their contributions to Nham characteristics. Meat Sci 66 (3): 579-588. Visschers RW, De Jongh HJ. 2005. Disulphide bond formation in food protein aggregation and gelation. Biotech Advan 23: 75-80. Yatsumatsu K, Sawada K, Moritaka S, Misaka M, Toda J, Woda T. 1972. Whipping and emulsifying properties of soybean products. Agric Biol Chem 36: 719-727.

N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060102

Vol. 6, No. 1, pp. 7-12 May 2014

Optimization of in vitro sterilization protocol for obtaining contamination-free cultures of Tilia platyphyllos 1

VAHIDEH PAYAMNOUR1, KAMAL GHASEMI BEZDI2,♥, MOSTAFA MEHRDAD1, AKRAM AHMADI1,♥♥ Department of Forest Ecology, Faculty of Forest Sciences, Gorgan University of Agricultural Sciences & Natural Resources, Gorgan, Golestan, Iran. Tel. /Fax. +981714427050, ♥♥email: ahmadi.1870@gmail.com 2 Cotton Research Institute of Iran (CRII), Beheshti St, P.O. Box 49175-483, Gorgan, Golestan, Iran. Tel: +98171-2254960. Fax: +98171-2227781, ♥ email: kghasemibezdi@yahoo.com Manuscript received: 28 November 2013. Revision accepted: 4 April 2014.

Abstract. Payamnour V, Ghasemi Bezdi K, Mehrdad M, Ahmadi A. 2014. Optimization of in vitro sterilization protocol for obtaining contamination free cultures of Tilia platyphyllos. Nusantara Bioscience 6: 7-12. Tilia platyphyllos is one of threatened species of Caspian forests. Tissue culture techniques are applied for culture, regeneration and genetic resources preservation. Utilizing an accurate sterilization procedure is important to reduce the cost, time and energy. The aim of this present study was to provide optimization of in vitro sterilization protocol to obtain contamination-free cultures of T. platyphyllos. Explants were collected randomly from the best individuals of T. platyphyllos, which were located in Tooskestan forest of Gorgan, Iran. Results revealed that the optimum protocol for sterilization was when explants were exposed in pre-sterilizing solution of 600 mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 5% commercial sodium hypochlorite (NaOCl) solution (5% Cl activated) for 20 minutes and then explants were exposed in sterilizing solution containing 600 mg L-1 ascorbic acid and 10% sodium hypochlorite. Key words: Explants, pre-sterilization, sterilization, Tilia platyphyllos

INTRODUCTION The Linden trees, from Tilia genus belong to the Salicaceae family and Malvales order (Sabeti 1965; Sadati 2002). Generally, Tilia genus includes woody plants (about 30-40 species) and 10 species of Linden are distributed in moderate areas of northern parts of the earth, 4 of which 4are found in Europe (Magherini and Nim 1993). Tilia platyphyllos is the only species of Tilia genus in Iran, which is one of the important species and has wide distribution (Mozafarian 1998). Natural site of T. platyphyllos is located in the northern forests of Iran (Sabeti 1965). Tilia platyphyllos is hydro-phobe and hard on the land that prefers cold or moderate climate and also, distributes its root system (Kunneman and Albers 1991; Haller 1995). Its longevity is average (Moghadasi 2001) that in some species, reaches to 500-1000 years (Kunneman and Albers 1991; Haller 1995). Generally, the fruits are grayish, nutshaped and oval-shaped capsule that matures in autumn to winter, but may also remain on the tree. Each includes a pod that contains a seed (Brinkman 1974; Pigott and Huntley 1981). External coating contains a layer with low fiber. Linden can be included as a medical plant because its flower and leaves have medical usage (Moghadasi 2001). The honey produced by bees extracting its nectar is very famous in the world (Haller 1995). Its wood has the highest efficiency in the sculpture due to its beauty and softness. Usually, these trees are planted in many parks and roadsides in Europe, because they grow so fast and make

the landscapes beautiful (Edlin 1976). Tilia platyphyllos can be planted with beech or hornbeam for rehabilitation of destroyed northern forests of Iran (Sadati et al. 2007). Due to the many problems that exist in germination of T. platyphyllos seeds, techniques such as tissue culture are important for restoration and conservation of this genetic resource (Sadati 2002; Shahrzad 1997; Hartman et al. 1990; Bewley and Black 1985). The first study of tree tissue culture was conducted in the mid-1920s (Ghasemi Bezdi and Ahmadi 2009). Due to the presence of many microorganisms in the explants, contamination reveals in cultures despite the use of sterilization methods (Skirvin et al. 1999; Martinez and Wang 2009). Tilia platyphyllos is valuable broad-leaved forest tree that much research should be done in tissue culture because the seed germination is negligible due to the hard seed coat. The first reports of the T. platyphyllos tissue culture was presented by Barker (1969). He proceeded through callus culture of Tilia americana. Chalupa (1984) was one of those who were very active in T. platyphyllos tissue culture and has done much research in this field. At first, he worked on Tilia cordata, and used the lateral buds to grow in vitro culture, in 1984. Due to many difficulties in plant tissue culture, especially in forest trees, optimizing the sterilization procedure of plant material seems necessary (Osterc 2004). Analogous investigations were carried out about sterilizing forest trees explants and achieving standard method for sterilization (Osterc 2004; Meier-Dinkel 1986; Preil 1997; Langens-Gerrits 1998). In Linden, due to the difficulties in propagating even through laboratory procedures, some

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researches have been done (Shahrzad 1997; Mehrdad et al. 2007, 2011). Shahrzad (1997) harvested the twigs of T. platyphyllos from three provinces of Tehran (botanic garden), Mazandaran (Waz forests) and Gilan (Shafaroud forest) in different seasons. She found that the best method for sterilization of this species was washing explants with 70% ethanol, with 1 and 2% sodium hypochlorite (NaOCl) and 1% HgCl2 and the best time to harvest buds was from the fall to early winter. Also, she stated that suitable buds for culturing were apical buds or near the terminal end buds. Also, Mehrdad et al. (2011) studied the micropropagation of Tilia begonifolia, in vitro. Therefore, this experiment was undertaken with an objective of finding out the optimized in vitro sterilization protocol to obtain contamination-free cultures of T. platyphyllos.

the medium was adjusted to 5.7 before autoclaving, and ascorbic acid added to the medium after autoclaving. The buds were used as explants for accessing best protocol to obtain contamination free-cultures of T. platyphyllos. Different treatments of time and material were used for pre-sterilizing and sterilizing of explants. Then, viability (in percent) of explants was investigated after sterilizing and transferring to a MS basal medium using a completely randomized design with three replications. Data obtained from this research were analyzed statistically using one-way analysis of variance (ANOVA) and the significant differences among means were assessed using Duncan’s multiple range test with MSTATC software. RESULTS AND DISCUSSION

MATERIALS AND METHODS In the present research, twigs of Tilia platyphyllos were used as a source plant. Twigs were harvested from Tooskestan forest of Gorgan, Iran, transferred to the laboratory and were cleaned. At first, water and soap water were used to eliminate surface pollution. In order to reduce pollution, pre-sterilizing was carried out, in which explants were set at a definite volume of sterilized water (dependent on explants volume) with 300 and 600 mg L-1 ascorbic acid, 2 and 4 g L-1 captan (1, 2, 3, 6-tetrahydro-Ntrichloromethyl thiophthalmide) fungicide and 5% and 10% commercial sodium hypochlorite (NaOCl) and then were shacked in 10, 20 and 30 minutes (Table 1). Then, explants were exposed to running water for washing. Table 1. Type and amount of used compound at pre-sterilizing solutions Code of presterilizing solution 1 2 3 4 5 6 7 8

Ascorbic acid (mg L-1) 300 300 300 300 600 600 600 600

Captan fungicide (g L-1) 2 2 4 4 2 2 4 4

Commercial sodium hypochlorite (%) 5 10 5 10 5 10 5 10

Then, to achieve suitable sterilizing of explants and successful culture, the effects of different concentrations of commercial sodium hypochlorite (0, 10, 20, 30, 40 and 50%) and time of sterilizing (5, 10 and 20 minutes) were investigated. Likewise, different concentrations (0, 300 and 600 mg L-1) of ascorbic acid in the media were studied to eliminate the harmful effects of phenol compound that exist in explants at micro-propagation culture. The MS medium (Murashige and Skoog 1962) containing 30 g L-1 sucrose and 6.7 g L-1 agar was used for all experiments. The pH of

Variance analysis results of different factors in order to pre-sterilizing of explants, ascorbic acid amounts at sterilizing solutions, necessary time for sterilizing treatments, sodium hypochlorite concentrations of sterilizing solutions and interaction effects of these factors on viability (in percent) of explants are shown in Table 2. According to the results, viability (percent) of explants under pre-sterilizing factors, ascorbic acid amount, sterilizing time, sodium hypochlorite concentration and their interaction effects was significant (p≤0. 01). Table 2. Variance analysis of pre-sterilizing effect, ascorbic acid, time of pre-sterilizing, sodium hypochlorite concentration and their interaction effects on viability percent of T. platyphyllos explants Source of variation Degree of Mean of (S.O.V) freedom (df) Squares (MS) Pre-sterilizing (P) 7 0.220** Ascorbic acid (S) 2 0.192** P×S 14 0.030** 0.788** Time of sterilizing (T) 2 P×T 14 0.038** S×T 4 0.059** P×S×T 28 0.008** Sodium hypochlorite 5 2.111** concentration (N) P×N 35 0.035** S×N 10 0.024** P×S×N 70 0.005** T×N 10 0.057** P×T×N 70 0.006** S×T×N 20 0.008** P×S×T×N 140 0.003** Error 864 0.003 Total 1295 Note: **): significantly different at the 1% level.

The presented data in Table 3 showed the results obtained from means comparisons of pre-sterilizing factor effect as ascorbic acid amount and their interaction with T. platyphyllos explants viability by Duncan’s multiple range

PAYAMNOUR et al. – In vitro sterilization protocol for Tilia platyphyllos

test at the 5% level. As presented, factors were investigated at five different groups. No. 7 pre-sterilizing treatment was the best (first group) and showed 17.6% viability, which consisted of 600 mg L-1 Ascorbic acid, 4 g L-1 captan fungicide and 5% commercial sodium hypochlorite. No. 8 treatment showed 16.1% viability and placed in second group Duncan’s multiple range test which consisted of 600 mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 10% commercial sodium hypochlorite. Table 3 clearly indicated that these two groups revealed higher viability than other groups. In contrast, No. 1 pre-sterilizing treatment showed the minimum percentage of explants viability (6.7%) which contained at least concentration of ascorbic acid, captan fungicide and sodium hypochlorite. The results of mean comparison of ascorbic acid amount effect on T. platyphyllos explants (Table 3) showed that under study factors set at two different groups with the highest percentage of explants viability (13.8%) were observed when higher amount of ascorbic acid (600 mg L1 ) were used. The results clearly indicated that, 0 and 300 mg L-1 ascorbic acid were non-significant and placed in the second group with 10.1 and 10.2% viability, respectively. From the mentioned results, it can be concluded that with the increase of ascorbic acid until 300 mg L-1, viability increased a little, but the viability showed significant increase when ascorbic acid changed from 300 to 600 mg L-1. The interaction between pre-sterilizing factor and ascorbic acid in sterilizing solution on T. platyphyllos explants viability (Table 3) revealed that data were classified into 18 groups according to viability. The highest viability percentage (24.1) at No. 7 pre-sterilizing treatment was observed that contained 600 mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 5% commercial sodium hypochlorite and sterilizing solution with 600 mg L-1 ascorbic acid showed significant increase viability than other treatments. On the other hand, No. 1 pre-sterilizing treatment containing 300 mg L-1 ascorbic acid, 2 g L-1 captan fungicide and 5% commercial sodium hypochlorite and sterilizing solution without ascorbic acid showed the minimum amount of viability. Table 3. The means comparison of explants viability (%) of T. platyphyllos in relation to pre-sterilizing factor effect, ascorbic acid amount and their interactions PreMean of preAscorbic acid (mg L-1) sterilizing sterilizing 0 300 600 code factor 1 4.4n 6.4mn 9.3i-l 6.7 e 2 7.4lm 10.1k-h 11.0g-k 9.5 d h-k lm 3 9.9 7.5 12.9d-g 10.1 d klm f-i 4 8.3 11.5 14.8cd 11.5 c lm klm efg 5 7.3 8.4 12.5 9.4 d h-l jkl fgh 6 9.4 9.0 11.8 10.1 d c c-f a 7 15.4 13.5 24.1 17.6 a 8 18.9b 15.3c 14.3cde 16.1 b b b 10.2 13.8 a 10.1 Mean of ascorbic acid Note: Means separated by Duncan’s multiple range test at alpha = 0.05. Means with the common letters were not significantly different (at the 5 % level) and they were in the same group.

The results obtained from a mean comparison of sterilizing time effect on T. platyphyllos explants viability are presented in Table 4. Time of 5 minutes for sterilizing with 15.4% viability placed in the first group, 10 minutes with 11.9% in the second group and 20 minutes with 6.9% viability in the third group. Therefore, it could be concluded that the viability percentage of explants decreased with the increasing time of sterilizing, and existing material at a sterilizing solution had a negative effect on explants viability. Thereupon, it is better to reduce the treating time with sterilizing solutions, if it is possible. The data of the interaction between pre-sterilizing factors and time of exposing at sterilizing solution on T. platyphyllos explants viability are shown in Table 4, that indicated the highest value of free-contamination explants (24.8% and 24.3%, respectively) were obtained when explants were at a sterilizing solution for 5 minutes (No. 7 and No. 8 pre-sterilizing codes). It was evident that the presterilizing solution had less effect than sterilizing time. On the other hand, with the increase of captan fungicide and ascorbic acid in pre-sterilizing solution, viability increased, but increasing the time more than optimum time, negative effects of solution increased and viability decreased extremely. As, with increasing the time from 5 to 10 minutes, viability decreased and with increasing the time to 20 minutes, viability decreased even more. Table 4. The means comparison of explants viability (%) of T. platyphyllos in relation to pre-sterilizing factor effect, time of sterilizing and their interactions PreMean of preTime of sterilizing (minute) sterilizing sterilizing 5 10 20 code factor 1 7.3ij 6.9ij 6.1jk 6.7 e 2 10.6fgh 11.4efg 6.5j 9.5 d def fgh j 3 12.8 12.5 6.7 10.1 d c def j 4 15.5 12.6 6.6 11.5 c cd gh k 5 14.6 9.4 4.2 9.4 d cde gh j 6 13.5 10.2 6.5 10.1 d a b gh 7 24.8 18.5 9.6 17.6 a 8 24.3a 15.2c 9.0hi 16.1 b a b c Mean 15.9 11.89 6.88 Note: Means separated by Duncan’s multiple range test at alpha = 0.05. Means with the common letters were not significantly different (at 5% level) and they were in the same group.

The data in Table 5 showed the mean comparison of the sodium hypochlorite effect on viability of T. platyphyllos explants. The viability of explants at 10% and 20% sodium hypochlorite was 25.6% and 19.5% at sterilizing solution, respectively. Whereas, no explants were alive in sterilizing solution without sodium hypochlorite and 100% of them were contaminated. It is quite clear that increasing commercial sodium hypochlorite, explants viability decreased and too much of commercial sodium hypochlorite might cause the viability till zero. Thus, it can be concluded that sodium

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hypochlorite is necessary, but should be used in minimum amount. Concerning the interaction between the pre-sterilizing factors and the sodium hypochlorite amount on explants viability (Table 5), the data indicated that the highest viability (40.9%) was related to pre-sterilizing No. 7 and 10% sodium hypochlorite. Viability difference between the first and second groups showed the effect of commercial sodium hypochlorite in pre-sterilizing solution that demonstrated the minimum necessity of using commercial sodium hypochlorite at pre-sterilizing solution. To study the negative effects of commercial sodium hypochlorite we increased up to 50% at sterilizing solution and it caused a remarkable reduction of explants viability. In all presterilizing method, without using sodium hypochlorite in sterilizing solution, there was not any viability observed. Table 5. The means comparison of explant viability (%) of T. platyphyllos in relation to pre-sterilizing factor effect, sodium hypochlorite and their interactions PreSodium hypochlorite (%) sterilizing code 0 10 20 30 t

jkl

o-r

Mean of presterilizing 50 factor 2st 6.7 e 4.14rst 9.5 d 3.7grs 10.1 d 2.2rst 11.5 c 2.8rst 9.4 d 1.7st 10.1 d 2.6rst 17.6 a 2.4rst 16.1 b 2.6e -

1 0 15.9 12.41 5.5 4.2p-s 2 0t 21.8ef 14.4hij 9.63lmn 7.96mno 3 0t 2fg 17.2gh 11.1klm 8.3mno 4 0t 26.7d 20.2fg 3.3ijk 6.8n-q 5 0t 2fg 15.7hi 10.9klm 7nop t e 6 0 23.5 17.6gh 11klm 6.7n-q 7 0t 41a 30.4c 2fg 12jkl t b cd fg 8 0 36 28 2 11klm Mean of 0f 25.6a 19.5b 12.7c 8d sodium hypochlorite Note: Means separated by Duncan’s multiple range test at alpha = 0.05. Means with the common letters were not significantly different (at the 5% level) and they were in the same group.

sterilizing solution without using sodium hypochlorite did not show any viability. Table 6. The means comparison of explant viability (%) of T. platyphyllos in relation to ascorbic acid, time of sterilizing and their interactions Mean of presterilizing factor 10.1 b 10.2 b 13.8 a

Time of sterilizing (minute)

5 10 20 0 11.8c 11.2cd 7.4e Ascorbic acid (mg 300 14.7b 10.1d 5.7f -1 a b L ) 14.3 7.5e 600 19.6 a b Mean 19.8 11.9 6.9c Note: Means separated by Duncan’s multiple range test at alpha = 0.05. Means with the common letters were not significantly different (at 5% level) and they were in the same group.

Table 7. The means comparison of explant viability (%) of T. platyphyllos in relation to ascorbic acid, sodium hypochlorite and their interactions Sodium hypochlorite (%) 0

10 h

20 b

30 c

40 e

50 f

O 22.9 17.3 11.5 6.9 2.2g Ascorbic 0 acid (mg 300 0h 22.8b 17.6c 11.1e 7.2f 2.4g L-1) 600 0h 31.1a 23.5b 15.4d 7.8e 3g Mean 0f 25.6a 19.5b 12.7c 8d 2.6e Note: Means separated by Duncan’s multiple range test at alpha = 0.05. Means with the common letters were not significantly different (at the 5% level) and they were in the same group.

Table 8. The means comparison of explant viability (%) of T. platyphyllos in relation to time of sterilizing (minute), sodium hypochlorite and their interactions Sodium hypochlorite (%)

As for the interaction effect between ascorbic acid treatment and time of exposing explants to sterilizing solution on explants viability (Table 6), the data indicated that an increase of ascorbic acid in sterilizing solution had a direct effect (increasing viability) and time of exposing explants to sterilizing solution had the reverse effect (reducing viability) on explants viability. The highest viability (19.6%) was observed in 5 minutes-exposure to the sterilizing solution containing 600 mg L-1 ascorbic acid. Also, the minimum viability (5.7%) was observed in 20 minutes-exposure to the sterilizing solution containing 300 mg L-1 ascorbic acid. The data obtained from the interaction between ascorbic acid amount and sodium hypochlorite at sterilizing solution on explant viability (Table 7) pointed out that the highest viability (31.1%) was related to 600 mg L-1 ascorbic acid and 10% sodium hypochlorite. It can be concluded that the minimum usage of commercial sodium hypochlorite and the maximum amount of ascorbic acid (600 mg L-1) was necessary to increase explant viability. The explants in

Mean of presterilizing factor 10.1 b 10.2 b 13.8 a

Mean of presterilizing factor 15.8 a 11.9 b 6.9 c

0h 31.6a 24.5b 17.8d 14e 4.4g Time of 5 sterilizing 10 0h 25.8b 21.8c 13.96e 7.2f 2.6g (minute) 20 0h 19.4d 12.1e 6.25f 2.7g 0.8h Mean 0f 25.6a 19.5b 12.7c 8d 2.6e Note: Means separated by Duncan’s multiple range test at alpha = 0.05. Means with the common letters were not significantly different (at the 5% level) and they were in the same group.

The results showed in Table 8 revealed that the highest percentage of viability (31.6%) was related to treatment with 10% sodium hypochlorite for 5 minutes. Other treatments pointed out that the time of sterilizing, increased the negative effects of sodium hypochlorite in sterilizing solution and with the increasing time and sodium hypochlorite, viability decreased (severely). Also, the results of the mean comparison of interaction effects between pre-sterilizing factors, ascorbic acid amounts, time of sterilizing and sodium hypochlorite amounts in sterilizing solution (the data were not shown)

PAYAMNOUR et al. â&#x20AC;&#x201C; In vitro sterilization protocol for Tilia platyphyllos

implied that No. 7 pre-sterilizing with 600 mg L-1 ascorbic acid in sterilizing solution containing 10% sodium hypochlorite for 10 minutes and 5 minutes showed 68.3% and 65% viability, respectively and they were the optimum treatments for sterilization of T. platyphyllos explants. Meanwhile, Viability was not observed in treatments without sodium hypochlorite and also with high concentration of sodium hypochlorite (over 50%). Discussion Tilia platyphyllos is one of the valuable and high longevity (sometimes until 1000 years) plant species that exists individually under ecological and economical value of beech and oak trees (Sabeti 1965). These typical trees are planted in parks and streets in Europe and America. The leaves of this tree which are processed in tea and the honey produced by bees which extract nectar from their flowers are popular in the world, as well (Sadati et al. 2007). This species have been distributed from 100 to 1600 meters and even, sometimes, to 2400 meters above sea level (Sheikh Al-Eslami and Namiranian 2002). Because of the wide range of altitude distribution and genetic variability preservation of this species and efforts in preserving the natural structure of Iran forests, the importance of this species is clear for everyone. Many studies demonstrated that producing T. platyphyllos seedlings have encountered serious problems such as weak germination in nurseries until now. It was an interesting fact that although mature seeds are healthy, their germination is low (Tabari et al. 2007). Although there are problems, using biotechnology tools such as tissue culture might be a proper solution. Tissue culture and regeneration of T. platyphyllos are difficult and the success of regeneration is very low. Hence, regeneration may also be an effective technique to multiply stock plants of this species. One of important problems in case of T. platyphyllos or every plant, which their explants collected from in vivo special forest areas, is the control of contamination at in vitro condition. It is necessary to note, that pre-sterilizing is preliminary stage that can reduce contamination of explants in vitro. Although, this stage was conducted in laminar airflow, which is not sterile, but this contamination could not be included in the data in the independent stage for calculations. According to this point and achieved experiments in this study, it could not eliminate presterilizing effect from calculations and considered it as one of dependent on sterilizing stage. Therefore, with used material at pre-sterilizing, eight treatments of presterilizing were adopted and included in this investigation. The present study demonstrated that the best sterilization method was using 4 g L-1 captan and 600 mg L-1 ascorbic acid and 5% v/v commercial sodium hypochlorite. In general, increasing chlorox concentration increased percentage of contamination-free explants that presented results were coordinated with those of Abou Dahab et al. (2005). Sharifkhani et al. (2011) demonstrated that the most suitable concentration of sodium hypochlorite for sterilization is 5%, which agrees with these results.

A wide range of contamination has been indentified in plant tissue culture that causes considerable economical losses (George 1993; Altan et al. 2010). Fungi are from contaminations in plant tissue culture that may arrive with explants, airborne or enter a culture (Babao et al. 2001; Altan et al. 2010). In the present study, a fungicide was used to reduce fungal contaminations observed during the research. Observed fungi were identified and were reported in the other research that consisted of Penicillium sp., Fusarium sp. and Alternaria sp. (Mehrdad et al. 2010). Shahrzad (1997) reported that using fungicide reduced remarkably fungal contaminations, which agrees with these results. The effectiveness of sterilization may depend on the type, concentration and time of treatment with the sterilant (Roxas et al. 1996; Tomaszeska-Sowa and Figas 2014). The best sterilization method was found for a solution containing 600 mg L-1 ascorbic acid and subsequently, 10% v/v commercial sodium hypochlorite while the optimal time was 5 minutes. The explant viability decreased with the increment of sterilization time and materials in sterilizer solution that have a negative effect on explant viability. Researchers working on sodium hypochlorite concentration and percentage of the surviving explants (Sharifkhani et al. 2011) agree with mentioned results. Also, Researchers working with NaOCl reported that using sodium hypochlorite for 5-10 minutes was considered a suitable time (Miller and Lipschutz 1984; Naik and Chandra 1993; Gopal et al. 1998; Villafranca 1998; Badoni and Chauhan 2010). Due to poisonous effects resulted from high concentration of sodium hypochlorite, all explants except complete seeds that contained embryo preservative membrane turned yellow and dried after several days. Preservative membrane causes material transferring, slowly. Sodium hypochlorite is necessary, but it should be used at least amount. Increment of sodium hypochlorite concentration will decrease the viability of T. platyphyllos explants. Similar results were recorded by Shahrzad (1997) using sodium hypochlorite and sterilizing time, but in that project HgCl2 was used. However, Shahrzad (1997) reported that the optimum concentration of sodium hypochlorite 1% and 2%, but in this study, the best concentration of this sterilizer solution was 0.5%. CONCLUSION In sterilization of T. platyphyllos, it was concluded that superabundant of phenolic compound was reduced by using ascorbic acid. The optimum protocol was using presterilizing solution of 600 mg L-1 ascorbic acid, 4 g L-1 captan fungicide and 5% commercial sodium hypochlorite (NaOCl) solution for 20 minutes. The results of present research will be applicable for propagation in large scale, conservation, breeding and further genetic improvement programs.

N U SAN TA RA B IOSC IE NC E 6 (1): 7-12, May 2014 REFERENCES

Abou Dahab AM, Afaf Habib MA, Hoseini YA, Gabr AMM. 2005. Effect of some sterilization treatments and growth regulators on Ruscus hypoglossum L. Arab J Biotech 8 (1): 127-140. Altan F, Burun B, Sahin N. 2010. Fungal contaminants observed during micropropagation of Lilium candidum L. and the effect of chemotherapeutic substances applied after sterilization. Afr J Biotechnol 9 (7):991-995. Babao LM, Yorgancılar M, Akbudak MA. 2001. Doku kültürü: Temel laboratuar teknikleri (Plant tissue culture: Basic laboratory techniques). In: Babaolu M, Gürel E, Özcan S (eds), Bitki. Badoni A, Chauhan JS. 2010. In Vitro Sterilization Protocol for Micropropagation of Solanum. Academia Arena 2(4): 24-27. Bewley DJ, Black M. 1985. Seeds physiology of development and Germination. Plenum press, New York. Brinkman KA. 1974. Tilia, basswood, linden. In: Schopmeyer CS, tech. coord. Seeds of woody plants in the United States. Agric. Handbk 450. Washington, DC: USDA Forest Service: 810B812. Chalupa V. 1984. In vitro propagation of oak (Quercus robur L.) and linden (Tilia cordata Mill.). The Journal of the Biologia Plantarum 26 (5): 374-377. Chalupa V. 1984. Vegetativní množení dubu Quercus robur L., lípy Tilia cordata Mill. a jeřábu Sorbus aucuparia L. in vitro. Lesnictví 30: 1019-1028. Edlin HL 1976. Trees, Woods and Man. Collins, London. George EF. 1993. Plant Propagation by Tissue Culture, Part 1, Techonology, England: Exegetics Ltd. Ghasemi Bezdi K, Ahmadi A. 2009. Cells and Tissue Biotechnology. Makhtoumgholi Fraghy publications, Iran. Gopal J, Minocha JL, Dhaliwal HS. 1998. Microtuberization in potato (Solanum tuberosum L.). Plt Cell Rep 17: 794-798. Haller JM. 1995. Tilia americana, linden: a neglected jewel. Arbor Age 15(7): 32-33. Hartman HT, Kester L, Dale E. 1990. Plant propagation, Principals and practices. Printice Hall New Gersey. Kunneman BPAM, Albers MRJ. 1991. Linden trees (Tilia spp.). In: Bajaj YPS, ed. Biotechnology in agriculture and forestry. Trees 3(16):152163. Langens-Gerrits M, Albers M, de Klerk GJ. 1998. Hot-water treatment before tissue culture reduces initial contamination in Lilium and Acer. Plant Cell Tiss Org 52: 75-77. Magherini R, Nin S. 1993. Experiments on seed germination of some Tilia spp. ISHS Acta Horticulturae 331. Martinez JC, Wang K. 2009. A sterilization protocol for field-harvested maize mature seed used for in vitro culture and genetic transformation. Maize Genetics Cooperation Newsletter 83. Mehrdad M, Payamnoor V, Ghasemi Bezdi K. 2007. Investigation of regeneration maount of some Tilia begonifolia explants for sexual propagation by tissue culture method. First national symposium of forest and wood sciences and paper 716-722. Mehrdad M, Payamnoor V, Ghasemi Bezdi K. 2011. The Study of Tilia begonifolia micropropagation in vitro.70 pp. A Thesis for M.Sc. in Silviculture and Forest Ecology [Persian]. Mehrdad M, Payamnour V, Ghasemi Bezdy K, Kavousi MR and Zhand S. 2010. Isolation and identification of fungi accompanied with seed using in Linden (Tilia begonifoila) micro-propagation. Second International symposium of climate change and dendrochronology in Caspian ecosystems.

Meier-Dinkel A. 1986. In vitro Vermehrung ausgewählter Genotypen der Vogelkirsche (Prunus avium L.). Allgemeine Forst- und Jagdzeitung 157: 139-144. Miller SA, Lipschutz L. 1984. Potato in: Ammirato PV, Evans D, Sharp WR, Yamada Yasuguki (eds.), Handbook of plant tissue culture. McMillan, New York. Moghadasi D. 2001. Investigation the most important ecological characteristics of linden (Tilia begonifolia). [M.Sc.-Thesis], University of Gorgan. Mozafarian V. 1998. Dictionary of Iranian plants. Publication of Moaser Farhang, Iran. Murashige T, Skoog F. 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497. Naik PS, Chandra R. 1993. Use of tissue culture technique in crop improvement with special reference to potato. CPRI, Shimla Osterc G, Lutharz Z, Stampar F. 2004. The importance of the sterilization procedure for producing vigorous cherry plants (Prunus sp.) in vitro. Acta Agriculturae Slovenica 83(1): 45-51. Preil W. 1997. In-vitro-Vermehrung von Gehölzen: In: Krüssmann, G. (eds.): Die Baumschule. Parey, Berlin, Germany. Roxas NJL, Tashiro Y, Miyazaki S, Isshiki S, Takeshita A. 1996. In vitro Propagation of Higo Chrysanthemum (Dendranthema x grandiflorum (Ramat.) Kitam.); Bull. Fac Agr Saga Univ 80:57-67. Sabeti H. 1965. Trees and Shrubs of Iran. University of Tehran publication. Sadati A, Emadyan SFA, Jalilvand H, Mokhtari J, Tabari M. 2007. The effect of topography on the distribution of linden (Tilia platyphyllos) from the Waz forest of Mazandaran. J Agr Sci Nat Res 41:443-451. Sadati A. 2002. Investigation of site demand and quantification and qualification characters of linden specie (Tilia platyphyllos) in Waz forests, Mazandaran. [M.Sc. Thesis]. Mazandaran University, Iran [Persian]. Shahrzad Sh. 1997. Linden (Tilia platyphyllos) propagation through tissue culture. Final report on research projects. Research Institute of Forests and Pastures. Sharifkhani A, Halimi MS, Maheran AA. 2011. An alternative safer sterilization method for explants of Aloe vera barbadensis Mill. 2nd International Conference on Chemical Engineering and Applications IPCBEE vol. 23. Sheikh Al-Eslami A, Namiranian M. 2002. Review of qualitative and quantitative characteristics in Linden at Western forests of Mazandaran. J Univ Agric Sci 8 (3):73-78. Skirvin RM, Motoike S, Norton MA, Ozgur M, Al-Juboory K, McMeans OM. 1999. Workshop in micropropagation establishment of contaminant-free perennial plant in vitro. In Vitro Cell Dev Biol Pl 34: 278-280. Tabari M, Tabandeh A. 2007. Stratified seed germination response of Linden (Tilia platyphyllos) to irrigation and seed planting depth. J Sci Res For Poplar Iran 15(2):144-151. Tomaszewska-Sowa M, Anna Figas A. 2014. Environmental and ecological aspects of cultivation of selected energy and herbal plants propagated in vitro culture. Infrastruct Ecol Rural Areas 4 (3): 14571465. Villafranca MJ, Vermendi J, Sota V, Mingo-Castel AM. 1998. Effect of physiological age of mother tuber and number of subcultures on in vitro tuberization of potato (Solanum tuberosum L.). Pl Cell Rep 17:787-790.

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ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060103

Vol. 6, No. 1, pp. 13-18 May 2014

Hydrophysical, chemical and microbial properties of imported green waste composts 1

SAIFELDIN A.F. EL-NAGERABI1,♥, ABDULKADIR E. ELSHAFIE2, HUDA ALBURASHDI2 Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, P.O. Box 33, Postal Code 616,Birkat Al Mouz, Nizwa, Oman, Tel. +968 96365051, Fax. +968 25443050,♥e-mail: nagerabi@unizwa.edu.om 2 Department of Biology, College of Science, Sultan Qaboos University, P.O. Box 36, AlKhoudh, Postal Code 123,Oman Manuscript received: 12 February 2014. Revision accepted: 20 March 2014

Abstract. El-Nagerabi SAF, Elshafie AE, Alburashdi H. 2014. Hydrophysical, chemical and microbial properties of imported green waste composts. Nusantara Bioscience 6: 13-18. To study the hydrophysical, chemical and microbial properties of the imported green waste composts (GWCs) and their suitability as an alternative to agrochemicals, four types of GWCs (Florabella, Mikskaar, Potgrond, and Shamrock) were selected. All composts showed normal physical properties, except weed seeds in Shamrock. The germination indexes comparable to the standard (90%) were 100% for Mikskaar followed by Florabella (97%), Potgrond (95%), and Shamrock (92%). Variations in physico-chemical properties were shown as acidic pH 5.1-6.5 (standard 5-8), electrical conductivity (EC) 0.8-1.8 mScm-1 (standard 0.0-4.0 mScm-1), moisture content (MC) 54-70.5% (standard 35-60%) and water holding capacity (WHC%) 400800%. The chemical properties were expressed as ammonia concentrations 2871-6565 mg kg-1 (standard <500 mg kg-1), organic matter 53.3-66.2% (standard 35%). The concentrations of heavy metals (Zn, Ni, Pb, Hg, As, Cd, and Cr) were lower than the recommended levels. The bacterial colony forming unit per gram compost ranged between 330-2870 cfu/g, the most probable number (MPN) for coliform bacteria was 23-460 cfu/g, whereas the fungal cfu were 30-1800 cfu/g. Aspergillus niger was the predominant fungus recovered from all compost samples (100%), followed by A. fumigatus (75%), whereas A. sparsus, A. versicolor and yeasts (50%), and the remaining species of the genus Acremonium sp., Aspergillus flavus, A. restrictus, Cladosporium spp., and Penicillium spp. recovered from 25% of the samples. Generally, these composts revealed normal hydrophysical properties with obvious variation in moisture contents and elevated chemicals and microbial contamination. Therefore, there is an urgent need for quality control measurements and restrict abide to legislations and quarantine regulations. Keywords: Aspergillus niger, coliform bacteria, green waste composts, heavy metals, most probable number, Oman

INTRODUCTION Composts are produced by microbes and other organisms decaying of organic matter in warm and moist conditions (Salvator and Sabee 1995; Adeniran et al. 2003; Adegunloye et al. 2007; Briancesco et al. 2008). Green waste compost (GWC) is a biodegradable waste originates from almost pure plant materials such as garden trimming or garbage from vegetable and fruits (Wilson 1983; Anon 1997; Christensen et al. 2002). It improves the biological, and physic-chemical properties of the soil (Christensen et al. 2002; Dalal et al. 2009; Hartley et al. 2009), enhanced plant growth (Straatsma et al. 1994; Keeling et al. 2003; Ali et al. 2007), remediates contaminated soil (Van Herwijnen et al. 2007, 2008; Alvarenga et al. 2009; Hartley et al. 2009), and inhibits some soil borne disease (Van der Gaag 2007; Lozano et al. 2009). The modern mechanized agriculture depends on the application of large amounts of an expensive and nonecofriendly chemical fertilizers, pesticides, and herbicides which increase the crop production. Nonetheless, they destroy the diversity of biota and hazardous to human health (Khan et al. 2007). These are logistic and justifiable reasons to compost needs and importation. Therefore, attention has been directed to the safe compost industry for both environment and public health. Thus, quality control

of the composts is significantly important to promote the recycling of the organic wastes (Harada et al. 1993; Brinton 2000). It must comply with national and international compost standards (Anon 1997, 2002a, 2002b). These include moisture content, odor, carbon and nitrogen contents, phytotoxic substances, harmful elements, weeds, nutrient contents, plant pathogens and effectiveness to plant growth and soil amendment (Harada et al. 1993). They can be maintained by maturation of the compost which reflects the degree of transformation of the organic materials (Mondini et al. 2003). No single method can be adapted universally to all compost types due to the wide range of feedstock, the composting process (Barberis and Nappi 1996; Chen et al. 1996; Itävaara et al. 2002), and different chemicals in organic wastes (He et al. 1995; Benito et al. 2003). On the other hand, pathogens are generally present in sewage and household wastes which are commonly composted (Pahren 1987). This composting is an efficient method for pathogen destruction to a safe level for humans, animals and plants (Christensen et al. 2002; Dumontet et al. 1999; Van der Gaag 2007). In Oman, land is the major non-renewable resource facing the threat of soil degradation. Sustainable agriculture must be environmentally safe and produces adequate amount of quality foods with minimum fertilizers and rely mainly on the renewable resources of the farm itself (Khan

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et al. 2007; Chitravadivu et al. 2009).This is especially important for 90% of the farms in the third world, where agricultural inputs are often not available or affordable (Chitravadivu et al. 2009). Thus, compost is one of the inputs for meeting nutrient requirements of crops (Zameer et al. 2010). In Oman, green waste composts are imported from other countries under weak quarantine regulations with high cost and economic burden to the local farmers. There is an urgent need for development of strong legislations, analytical methods and well equipped entry points for controlling the import and export processes of the compost. Therefore, the present study was conducted to evaluate the hydrophysical, chemical and microbial properties of imported green waste composts in order to determine their suitability for different uses and ability to meet the standard of agrochemical properties. MATERIALS AND METHODS Compost samples collection Four imported green waste composts namely Florabella, Mikskaar, Potgrond, and Shamrock were selected and drawn according to the Gulf standard number GS0901/1997 (Anon 1997). Five samples of 1 kg each were collected from the compost bags, mixed to form composite samples and were then divided into 4 working samples and stored at 5◦C for further study. Hydrophysical characterization The samples were visually inspected for free flowing, hard lumps, objectionable odor, and color. The particle size of the composts was determined according to the Gulf standard number GS01167/2002 using three replicates of 100 g oven dried samples (Harada et al. 1993). The samples were placed on 12 mm sieve and shaken for 5 min at 100 shakes per min (Abd El-Hady and El-Dirdiry 2006). The percentage of the particles greater than 12 mm was calculated as the percent mass of the remaining materials on the top of the sieve to the mass of the original sample (Orozco et al. 1997). For phytotoxicity and the presence of the viable seeds (El-Nagerabi et al. 2012), 6 plastic pots (10 × 15 cm) were filled with the compost samples. Three pots were seeded with 100 seeds of Phaseolus mungo (mung bean) and the remaining three pots were kept without seeds. As a control, another 100 seeds of mung bean were inoculated into plastic trays with moisten cotton and incubated in the greenhouse of the Biology Department, Sultan Qaboos University. The inoculated seeds were moistened and examined for the presence of the weed seeds and germination of the mung bean seeds. The hydrogen ion concentration (pH), moisture contents (%), electrical conductivity (EC), and water holding capacity (WHC%) of the composts were detected using basic standard procedures and techniques (Wilson 1983; El-Nagerabi et al. 2012). The pH was determined in triplicate with the pH meter. The moisture content was determined by the oven method (El-Nagerabi and Elshafie 2000). Replicates of 10 g were placed in glass Petri dishes;

soft lumps were crushed with spatula and dried at 105◦C in an electric oven for 16 hours. The moisture content was determined as percentage of the initial weight. For electrical conductivity (EC), replicates of 2 g of each compost sample was added to 5 ml of distilled water and the mixture was filtered through Whatman filter paper No. 42 (Whatman International Ltd, Maidstone, UK). The EC for each filtrate was measured by electrical conductivity meter. For of the water holding capacity (WHC%), 500 g from each sample were added to pre-weighed dry sieve and pressed evenly, saturated with water, kept covered for overnight, and then the dripped water was wiped off the sieve with fine tissues. The sieve with the moistened sample was weighed, placed in desiccators, allowed to dry, and re-weighed to calculate the amount of water held by the samples. The WHC was calculated as the percentage mass of the absorbed water to the mass of the dried sample according to the Gulf standard No. GS01/2002 (Anon 2002a,b). Chemical analysis The compost samples were stored in polyethylene bags and the chemical analyses started within 48 hours after the sample collection. The organic matter (OM) was determined by measuring the loss of mass through ignition at 550◦C according to the modified combustion method (Inbar et al. 1990; Christensen et al. 2002; Petrus et al. 2009), which adopted by the Gulf standard NO. GSO1167/2002 (Anon 2002a,b). From each sample, 10 g were used instead of 5 g in order to increase the degree of the method accuracy. The samples were dried to constant mass in an oven at 105°C and cooled in desiccators to avoid moisture absorption from the atmosphere. Ten grams of each sample were kept into an oven-dried porcelain dish, placed in the furnace, then the temperature was increased to 550◦C, and samples were converted into ash. The percentage of the organic matter was calculated in triplicates as a percentage loss of mass to the mass of the original test sample. The ammonia-nitrogen contents were determined in triplicate using the Kjeldahl method (Kjeltec Foss, Tecator AB, Hogana, Sweden, N-Analyzer). For this, 0.5 g from each sample and one keltab catalyst (SeK2SO4) were added to digestion tube, mixed with 10 ml of sulfuric acid, digested for 3 hours, allowed to cool and the concentration of the ammonia was measured. For the heavy metals concentrations, 5 g from each sample were mixed with 25 ml of distilled water, filtered with Millipore filter papers, and 10 ml of the filtrate were analyzed with Inductive Couple Plasma (ICP-MS, OPTIMA, 3100RL Spectrometer, Perkin Elmer, and Norwalk, USA) (Khan et al. 2007). Enumeration of fungi and bacteria by agar plate technique Both fungi and bacteria were isolated from the compost using the agar plate method. One gram from each sample was added to 9 ml sterile distilled water, vortexed, and serial dilutions were prepared. One ml was aseptically

EL-NAGERABIet al. – Properties of green waste composts

inoculated on Potato Dextrose Agar (PDA) for fungal growth at 28◦C, for 7 days, and similarly Nutrient Agar (NA) was inoculated for bacterial growth at 37◦C, for 48 hours. After incubation, the number of colony forming units (cfu) per gram was calculated. The isolated fungi were identified using different taxonomic books and monographs (e.g. Raper and Fennell 1965; Pitt 1979; Ellis 1971, 1976; Sutton 1980; Samson et al. 1995; Barnett and Hunter 1998, 2003). The presence of coliform bacteria in the compost samples was screened using the standard table of the most probable number (MPN). Statistical analysis Duncan’s multiple range and one way ANOVA were used to compare between the four composts with p<0.05. The analysis was carried out using statistical package software SPSS (version 11.0). RESULTS AND DISCUSSION Physical properties of the composts Table 1 shows the physical characteristics of the four composts. Visually all the samples were physically uniform, free flowing, no hard lumps, dark brown to black in color, free from objectionable odor, and with particle size less than 12mm, except one viable weed seed in shamrock. It indicates the good quality of the composts, the completion of the degradation process and compost maturity as suggested by many authors (Iglesias-Jiménez and Pérez-Garcia 1992; Benito et al. 2003; Lozano et al. 2009). Nonetheless, the presence of viable weed seeds may be associated with compost immaturity and there is a high possibility for disseminating weeds (Iglesias-Jiménez and Pérez-Garcia 1992). Seed germination indexes in compost are biological method to evaluate the degree of the compost maturity of the composted materials. It shows the degree of the decomposition of phototoxic substances such as ammonia and acids produced during the early active composting stages (He et al. 1995; Wu et al. 2000). In the present study (Table 1), the germination percentages of the mung bean seeds in the four compost types ranged between 92-100%. The germination level lower than the acceptable index (>90%) can be attributed to the phytotoxic effects of the organic acid and ammonia toxicity produced during the active composting process (Wong 1985; Wu et al. 2000). In the present study, the ammonia concentration in the four composts ranged between 2871-656.5 mg kg-1 (Table 3). Similarly, the electrical conductivity (EC) which indicates the salt contents of the compost, is injurious to plant roots and prevents their growth (Cai et al. 2010). Therefore, higher level of bean seed germination may be due to acceptable electrical conductivity for Mikskaar (0.4 mScm1 ), Shamrock (1.8 mScm-1), Potgrond (0.8 mScm-1), Florabella (1.2 mScm-1), which ranged within the standard limit (0-4 mScm-1) and not harmful to the plant growth. Nonetheless, the high salt contents in the compost are associated with the high electrical conductivity and will be added to the receiving soil as beneficial to the plant growth

and soil remediation. In similar studies, it was found that the electrical conductivity varies considerably and ranged between 0.12-17.08 mScm-1 (Tang 2003). This wide range of electrical conductivity expressed the diversity of the chemical and microbial properties of the various compost products. This suggests mixing compost with vermiculite or adding less compost to the soil of low salt contents to adjust the optimum growth conditions of the amended soil. Hydrophysical properties of the compost The hydrogen ion concentration (pH) varies at the beginning of composting and ramped from 7.3 to 7.7 as the composting proceeded up to 8.8-9.6 (Adegunloye et al. 2007). The pH of the screened compost (5.1-6.4) was found to be within the recommended limit (Anon 1997, 200a, 200b; Alvarenga et al. 2009). This acidity of the composts may be due to the production of phytotoxic organic acids during immature composting, which causes immediate growth injuries (Wong 1985). Thus, the addition of this compost to soil may modify the pH of the final mix and buffer the soil pH (Adegunloye et al 2007). The moisture contents of the composts ranged between 3.1-82.7% and varied considerably with the variation in the composted materials (Tang 2003). The moisture content of the compost was considerably higher in the first 3 weeks of composting and increases significantly in the later weeks (Adegunloye et al 2007). Therefore, the addition of compost provides excellent drought resistance and great efficient water retention. In the present study (Table 2), the moisture contents of these composts (64-74%), which were higher than the acceptable limits (35-60%). The moisture content of between 50-60% was considered as the optimal level for further composting (Tiquia et al. 1996). Nonetheless, the compost with higher moisture content will inhibit aerobic degradation (shouldn’t' be decomposed any more) and enhanced the unpleasant odor from the growth of anaerobic sulfate reducing bacteria. Yet, the ideal moisture content depends on the potential uses of the compost. Water retention capacity of the substrate is generally considered as the quality determining factor (Abd El-Hady and El-Dirdiry 2006). The highest saturation of the compost is 75% and the good compost must have high water holding capacity and low filtration rate for supporting the plant growth. In the present study (Table 2), the water holding capacity (WHC%) of the tested composts was found to be more than four to eight times of their actual weight. The water holding capacity ranged between 400-800%. Therefore, these composts can be used separately or mixed with sandy soil with low water holding capacity if they satisfied the other quality control parameters and the essential plant growth requirements. Chemical properties of the compost The chemical properties expressed the diversity of various compost products and the raw materials used (Tang 2003). The total carbon content (TC) was in the range of between 16.9-51.0%. A approximately 11-27% of the total carbon was lost during the 7 days of active composting, and 62-66% during the whole composting time (Vuorienin

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optimum 40-60%) (Anon 1997).The high organic matter contents of the compost indicate the presence of uncomposted organic materials which can be degraded slowly by microorganisms and eventually used by higher plants (Bary et al. 2002). Table 1. Physical properties of the imported green waste composts Heavy metals, as harmful Composts types elements are one of the determining Properties Florabella Mikskaar Potgrond Shamrock factors for compost quality (Harada Free flowing + + + + et al. 1993). They may come from Hard lumps sewage water, organic waste, chicken Objectionable odor manure, animal dung, and the soil Normal color + + + + added. They negatively affect the Particle size (<12 mm) + + + + plant during the slow degradation Foreign viable weed seeds +* process. On the contrary, compost Germination of bean seed% 98 100 95 92 reduces the mobility of some toxic Note: * Presence of only one germinated weed seed. metals to the plants through formation of some complexes. In this Table 2. Hydrophysical properties of the imported green waste composts study (Table 3), although, there were Compost types significant variations in the heavy Properties Standards Florabella Mikskaar Potgrond Shamrock metals concentrations (Zn, Ni, Pb, Hydrogen ion concentration (pH) 5.2C* 6.4A 5.6B 5.1C 5-8 Hg, As, Cd, Cr) the levels of these -1 Electrical conductivity (mScm ) 1.2B 0.4D 0.8C 1.8A 0-4 metals were lower than the limits Moisture content (%) 65C 74A 70.5B 64C 35-60 suggested by the Gulf countries Water holding capacity (%) 400D 646C 800A 757B 100 (Anon 1997), Germany (Benito et al. Note: *Within rows, number with different upper case letters differ significantly (P<0.05). 2003), and Canada Food Inspection Agency (1997). Nonetheless, the high contents of heavy metals may be Table 3. Chemical properties and heavy metals concentration (ppm) of the imported due to the addition of these metals to composts animal feeds (Harada et al. 1993) or Compost types contamination during plant growth or Properties Standards Florabella Mikskaar Potgrond Shamrock plants themselves (Van Roosmallen Ammonia (mg/kg) 4452.3c* 6179b 2871d 6565a <500 et al. 1987).

and Saharinen 1997). Table 3 shows the total organic matter for tested composts (53.5-66.2%) is significantly higher than the standard set by the Gulf countries (35%,

Organic matter (%) 53.3b 64a 66.2a 65a 35 Copper (Cu) 0.05c 0.08b 0.12a 0.06bc 150-250 Nickel (Ni) 0.02a 0.03a 0.03a 0.03a 50-70 Lead (Pb) 0.01a 0.03a 0.03a 0.02a 120-150 Cadmium (Cd) 0.08a 0.03b 0.03b 0.02b 3-5 Arsenic (As) 0.04a 0.03a 0.05a 0.04a 15-25 Chromium (Cr) 0.45c 0.50b 0.19d 0.57a 100-150 Zinc (Zn) 30.0c 79.4b 120.3a 120.7a 350-500 Mercury (Hg) 0.00591a 0.00591a 0.00591a 0.00592a 1.5-3 Note: *Within rows, number with different lower case letters differ significantly (P <0.05). Table 4. Microbial properties of the imported green waste composts Compost types Florabella Mikskaar Potgrond Shamrock Bacteria (cfu/g) 2870A* 2580B 1720C 330D Fungi (cfu/g) 200C 270B 1800A 30D MPN (cfu/g) 460A 43C 240B 23D Acremonium sp. + Aspergillus flavus + A. fumigatus + + + A. niger + + + + A. sparsus + + A. restrictus + A. versicolor + + Cladosporium spp. + Penicillium spp. + Yeasts + + Note: *Within rows, number with different upper case letters differ significantly (P<0.05). Properties

Microbial estimates of the compost Bacteria and fungi are present in large number during composting and they are essential for slow degradation of partially decomposed organic materials (Adegunloye et al. 2007). The pathogenic fungi and bacteria were normally detected in composted household wastes, and sewage sludge (Pahren 1987). However, composting is an efficient method for destruction of pathogenic microorganisms to an acceptable level, which is safe for human, animals and plants (Dumontet et al. 1999; Christensen et al. 2002; Van der Gaag et al. 2007). But reasonable amount of microorganisms is still present in the compost at maturity (Adegunloye et al. 2007). In the present study, the average of the bacterial colony forming unit per gram of the compost were 330-2870 cfu/g, whereas the colony forming unit of fungi in the compost were 301800 cfu/g (Table 4). It is evident

EL-NAGERABIet al. â&#x20AC;&#x201C; Properties of green waste composts

that Florabella, and Mikskaar have the highest bacterial colonies, whereas Potgrond contains low numbers of fungal colonies. These large numbers of bacterial and fungal colonies (standard <1000 cfu/g) may be responsible for the slow degradation of the organic matter (Christensen et al. 2002; Benito et al. 2003; Souza Dias et al. 2009). Most of the thermophilic genus and moisture tolerant Aspergillus species were responsible for the slow degradation of the composts. In the present study (Table 4), A. niger was the predominant species recovered from all compost samples (100%) at the later stage as reported by many authors (Millner et al. 1977; Adegunloye et al. 2007; Souza Dias et al. 2009). This fungus was followed by A. fumigatus (75%), where A. sparsus, A. versicolor and yeasts (50%), and the remaining species of the genus Acremonium sp., A. flavus, A. restrictus, Cladosporium spp., and Penicillium spp. were recovered from only 25% of the samples. In similar studies, different species of Aspergillus and Penicillium were isolated from different composts (Straatsma et al. 1994; Adegunloye et al. 2007; Souza Dias et al. 2009). Pathogenic bacteria were isolated from various composts and composted materials (Christensen et al. 2002; Adegunloye et al. 2007). Fecal coliforms found in the raw materials composed of Escherichia coli, wherein the finished compost the majority of the coliforms were probably of non-fecal origin (Christensen et al. 2002). In the present results, the most probable number (MPN) was used to determine the fecal contamination of the composts. Our findings (Table 4) showed that Florabella was contaminated with coliforms (480 cfu/g), followed by Potgrond (240 cfu/g), Mikskaar (7 cfu/g) and Shamrock (4 cfu/g). The presence of coliforms in Florabella and Potgrond may indicate the possible contamination of these composts with sewage water or other animal products during the composting process. Therefore, there is a high possibility of transmission of serious diseases during handling and usage of compost in addition of expected infestation of the cultivated plants with serious devastating pathogenic bacteria and fungi. Whereas compost which has undergone well controlled composting process is safe and canâ&#x20AC;&#x2122;t transmit serious diseases. CONCLUSION It is apparent that the investigated composts were visually free from physical constraints, except one viable weed seed in Shamrock which indicates compost immaturity. The composts contain high concentrations of ammonia, elevated moisture content, large amount of total organic matters, and low level of heavy metals. The composts were contaminated with variable levels of saprophytic fungi and coliform bacteria. It is evident that these composts revealed some variations in their general properties; however, it is feasible to be used for plant growth, soil biofertilizer and soil amendment as environmentally friendly products with proper adjustment of their physical-chemical and microbial properties. There is an urgent need for setting detailed legislations, regulation policies, proper testing methods, quality control measurements,

and restricted quarantine regulations for export-import of the green waste composts. Attention should be given to the local production of high quality composts which serve the environment, waste management, and recycling industry and satisfaction of the local markets. ACKNOWLEDGEMENTS We are grateful to the authority of the Department of Biology, College of Science, Sultan Qaboos University for providing space and faculties to carry this research. Dr. Peter Cowan of the Department of Biological Sciences and Chemistry, University of Nizwa, improved the scientific content of the manuscript. Dr. Tom Hughes of University of Nizwa Writing Center proof reads the English of this article. REFERENCES Abd El-Hady M, El-Dirdiry EI. 2006. Improving hydrophysical properties of compost. J Appl Sci Res 2: 1137-1141. Adegunloye DV, Adetuyi FC, Akinyosoye FA, Doyeni MO. 2007. Microbial analysis of compost using cowdung as booster. Pakistan J Nutr 6: 506-610. Adeniran JA, Taiwo LB, Sobulo RA. 2003. Effects of organic wastes and method of composting on compost maturity, nutrient composition of compost and yield of two vegetable crops. J Sustain Agr 22: 95-101. Ali M, Griffith AJ, Williams KP, Jones DL. 2007. Evaluating the growth characteristics oflettuce in vermicompost and green waste compost. Eur J Soil Biol 43: 316-319. Alvarenga P, Goncalves AP, Fernandes RM, de Varennes A, Duarte E, Cunha-Queda AC, Vallin G. 2009. Reclamation of a mine contaminated soil using biologically reactive organic matrices. Waste Manage Res27: 101-111. Anon. 1997. Municipal solid waste compost. Standardization Organization for G. C. C. (GSO) 901/1997, Anon. 2002a. Method for testing municipal solid waste compost. Standardization Organization for G. C. C. (GSO) 1167/2002. Anon. 2002b. Test method of potting mixes (potting soil). Standardization Organization for G. C. C. (GSO) 1169/2002, Barberis R, Nappi P. 1996. Evaluation of compost stability.In: de Bertoldi M. et al, (ed). The Science of composting: Blackie Academic and Professional, Glasgow, Scotland. Barnett HL, Hunter BB. 1998. Illustrated genera of imperfect fungi. 4th. edn. Prentice-Hall, Inc., New York, USA. Barnett HL, Hunter BB. 2003. Illustrated genera of imperfect fungi. 4th. edn. Burgess Publishing Company Minneapolis, Minn. USA. Bary A, Cogger C, Sullivan D. 2002.What does compost analysis tells you about your compost? Poster at Biologically intensive and organic Farming Research Conference, 2002. Benito M, Masaguer A, Moliner A, Arrigo N,Palma RM. 2003.Chemical and microbial parameters for the characterization of the stability and maturity of pruning waste compost. Biol Fert Soils 37: 184-189. Briancesco R, Coccia A.M, Chairetti G, Della LS, Semproni M, Bonadonna L. 2008. Assessment of microbiological and parasitological quality of composted wastes: health implications. Waste Manag Res 26: 196-202. Brinton WF. 2000. Compost quality standards Guidelines. Wood and Research Laboratory 2000: 1-41. Cai H, Chen T, Liu H, Gao D, Zheng G, Zhang J. 2010. The effect of salinity and porosity of sewage sludge compost on the growth of vegetable seedlings. Sci Hort 124: 381-386. Canadian Food Inspection Agency. 1997. Standards for metals in fertilizers and supplement, Trade Memorandum T-4-93. Chen Y, Inbar Y, Chefetz B,Hadar Y. 1996. Compost and recycling of organic wastes. In: Rosen D et al, (ed). Modern agriculture and environment. Kulwer, Dordrecht, the Netherlands.

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Chitravadivu C, Balakrishnan V, Manikandan J, Elavazhagan T, Jayakumar S. 2009. Application of food waste compost on soil microbial population in groundnut cultivated soil, India. Middle-east J Sci Res 4: 90-93. Christensen KK, Carisbaek M, Kron E. 2002.Strategies for evaluating the sanitary quality of composting. J Appl Microbiol 92: 1143-1158. Dalal RC, Gibson I, Allen DE, Menzies NW. 2009. Green waste compost reduces nitrous oxide emissions from feedlot manure applied to soil. Agric Ecosyst Environ 10: 1-9. Dumontet S, Dinel H, Baloda SB. 1999.Pathogen reduction in sewage sludge by composting and other biological treatments. A review. Biol Agr Hort 16: 409-430. Ellis MB. 1971. Dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew. Ellis MB. 1976. More dematiaceous hyphomycetes. Commonwealth Mycological Institute, Kew. El-Nagerabi SAF, Elshafie AE. 2000. Incidence of see-borne fungi and aflatoxins in Sudanese lentil seeds. Mycopathologia 149: 151-156. El-Nagerabi SAF, Elshafie AE, AlRawahi HS. 2012. Physicochemical and microbial characteristics of locally processed green waste composts. Compost Sci Util 20: 120-127. Harada Y, Haga K, Osada T,Koshino M. 1993. Quality of compost produced from animal wastes. JARQ 26: 238-246. Hartley W, Dickison NM, Riby P, Lepp NW. 2009. Arsenic mobility in Brownfield soils amended with green waste compost or biochar and planted with Miscanthus. Environ Pollut 157: 2654-2662. He XT, Logan TJ, Traine SJ. 1995. Physical and chemical characteristics of selected U.S. municipal waste composts. J Environ Qual 24: 543552. Iglesias-Jiménez E, Pérez-Garcia V. 1992. Determination of maturity indices for city refuses composts. Agr, Ecosys Environ 38: 331-343. Inbar Y, Chen Y, Hadar Y. 1990. Humic substances formed during the composting of organic matter. Soil Sci Soc Am J 54: 1316-1323. Itävaara M, Venelampi O, Vikman M, Kapanen K. 2002. Compost maturity-problem associated with testing. In: Insam H et al, (ed) Microbiology of composting: Springer Verlag, Heidelberg, Germany. Keeling AA, McCallum KR,Beckwith CP. 2003. Mature green waste compost enhances growth and nitrogen uptake in wheat (Triticum aestivum L.) and oil seed rape (Brassica napus L.) through the action of water-extractable factors. Bioresour Technol 90: 127-132. Khan MAI, Ueno K, Harimoto S, Komai F, Tanak K, Ono Y. 2007. Evaluation of the physio-chemical and microbial properties of green tea waste-rice bran compost and the effect of the compost on spinach production. Plant Prod Sci 10: 391-399. Lozano J, Blok WJ. 2009. Termorshuizen AJ, Effect of compost particle size on suppression of plant diseases. Environ Eng Sci 26: 601-607. Millner PD, Marsh PB, Snowden RB, Parr JF. 1977. Occurrence of Aspergillus fumigatus during composting sewage sludge. Appl Environ Microbiol 34: 765-772. Mondini C, Dell'Abate MT, Leita L,Benedetti A. 2003. An integrated chemical, thermal, and microbiological approach to compost stability evaluation. J Environ Qual 32: 2379-2386. Orozco R, Gschwander S, Marfa O. 1997. Substrate classification from particle size analysis.Acta Hort 450: 397-403. Pahren H. 1987. Microorganisms in municipal solid waste and public health applications. CRC Crit Rev Environ Cont 17: 187-228. Petrus AC, Ahmed OH, Muhamed AN, Nassir HM, Jiwan M,Banta MG. 2009. Chemical characteristic of compost and humic acid from sago waste (Metroxylon sagu).Am J Appl Sci 6: 1880-1884.

Pitt JI. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press, New York. Raper KB, Fennell DI. 1965. The genus Aspergillus. Williams and Wilkins, Baltimore. Salvator K,Sabee WE. 1995. Evaluation of fertilizer value and nutrient release from corn and soybean residue under laboratory and greenhouse conditions. Commu Soil/Sci., Plant Anal 26: 469-484. Samson RA, Hoekstra ES, Frisval JC, Filtenborg O. 1995. Introduction to Food-Borne Fungi. Baarn, Holland Centraalbureau voor Schimmelcultures. Sarwar G, Schmeisky H, Hussain N, Muhammad S, Ibrahim M,Safdar E. 2008. Improvement of soil physical and chemical properties with compost application in rice-wheat cropping system. Pakistan J Bot 40: 275-282. Souza Dias E, Guimaraes SE, de Siqueira FG, da Silva R,Batista LR. 2009. Allergenic and toxigenic fungi in the compost of cultivation of Agaricus brasiliensis. Scientia Agrarian 10: 507-511. Straatsma G, Samson RA, Olijnsma TW, Den Camp HJMOP, Gerrits JPG, van Griensven LJLD. 1994. Ecology of thermophilic fungi in mushroom compost, with emphasis on Scytalidium thermophilum and growth stimulation of Agaricus biosporus mycelium. Appl Environ Microbiol 60: 454-458. Sutton BC. 1980. The Coelomycetes. Commonwealth Mycological Institute, London. Tang JC. 2003. Chemical and microbial properties of various compost products. Soil Sci Plant Nutr 49: 273-280. Tiquia SM, Tam NFY, Hodgkiss IJ. 1996. Microbial activity during composting of spent pig-manure sawdust litter at different moisture contents. Bioresour Technol 55: 201-206. Van der Gaag DJ, van Noort FR, Stapel-Cuijpers LHM, de Kreij C, Termorshuizen AJ, van Rijn E, Zmora-Nahum S, Chen Y. 2007.The use of green waste compost in peat-based potting mixtures: Fertilization and suppressiveness against soil borne diseases. Scientia Hort 114: 289-297. Van Herwijnen R, Hutchings TR, Al-Tabbaa A, Moffat AJ, Johns ML, Ouki SK. 2007. Remediation of metal contaminated soil with mineral-amended composts. Environ Pollut 150: 347-354. Van Herwijnen R, Ouki S.K, Al-Tabbaa A, Moffat AJ, Johns ML, Hutchings TR. 2008. The effect of two different composts on the performance and metal uptake of poplar growing on heavy metal contaminated soil. SEESOIL 17: 39-48. Van Roosmallen GREM, Lustenhouwer JWA, Oosthoek J, Senden MMG. 1987. Heavy metal sources and contamination mechanisms in compost production. Resour Conserv 14: 321-334. Vuorienin AH, Saharinen MH. 1997. Evaluation of microbial and chemical parameters during manure and straw composting in a drum composting system. Agric Ecosys Environ 66: 19-29. Wilson GCS. 1983. The physio-chemical and physical properties of horticultural substrates. Acta Hort 150: 19-32. Wong MH. 1985. Phytotoxicity of refuse compost during the process of maturation. EA, Ecol Biol37: 159-174. Wu L, Ma LQ,Martinez GA. 2000. Comparison of methods for evaluating stability and maturity of biosolids compost. J Environ Qual 29: 424429. Zameer F, Meghashri S, Copal S,Rao BR. 2010.Chemical and microbial dynamics during composting of herbal pharmaceutical industrial waste. E-Journal Chem 7: 143-148.

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ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060104

Vol. 6, No. 1, pp. 19-25 May 2014

The mycobiota associated with paper archives and their potential control 1

SAIFELDIN A. F. EL-NAGERABI1,♥, ABDULKADIR E. ELSHAFIE2, UMAIMA A. AL-HINAI2 Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, P.O. Box 33, Postal Code 616, Birkat Al Mouz, Nizwa, Oman, Tel. +968 96365051, Fax. +968, 25443050, ♥e-mail: nagerabi@unizwa.edu.om 2 Department of Biology, College of Science, Sultan Qaboos University, P.O. Box 36, AlKhoudh, Postal Code 123, Al Khoud, Muscat, Oman Manuscript received: 16 March 2014. Revision accepted: 22 March 2014.

Abstract. El-Nagerabi SAF, Elshafie AE, Al-Hinai UA. 2014. The mycobiota associated with paper archives and their potential control. Nusantara Bioscience 6: 19-25. Historical collections kept in archives and libraries represent a cultural and artistic heritage of innumerable value. Recently in Oman, more than seventy thousand documents were collected from different countries and displayed as archives showed evident sign of mold contamination. The objectives of the present study were to screen these archives for mold invasion and a test for the effective control measure. For this, 102 samples were collected from documents of different sources and incubated on potato dextrose agar (PDA) at ambient temperature (25○C±2). The isolated fungi were identified microscopically and confirmed with DNA extraction, PCR and DNA sequencing. Twenty-two fungal species belonging to 11 genera were recovered. The genus Penicillium (46.8%) was the most prevalent, followed by Aspergillus (30.7%), Cladosporium (7%), Rhizopus (4%), and Chaetomium (3.5%) whereas the remaining 6 genera represent only 8%. Eleven species were previously reported from similar substrates, whereas 11 species and one genus are new records for the mycoflora of archives. Sodium hypochlorite at 0.3-5.2% completely inhibited the fungal growth of the 10 tested fungal isolates with minimum inhibition concentration at 0.7%. Fumigation of books with 0.7-5.2% sodium hypochlorite completely inhibited all fungi without evident damage of the documents or ink discoloration. Therefore, sodium hypochlorite can be recommended as effective and eco-friendly disinfectant for archives comparable to other hazardous chemicals. Key words: Archives, biological degradation, books, ethylene oxide, sodium hypochlorite

INTRODUCTION Archives and libraries are documentation of human thought and cultural heritage on paper, parchment and other photographic and electronic supports (Maggi et al. 2000). A modern library has in its collections of rare books and archives, periodicals, newspapers, maps, video tapes, digital discs, hard discs, CD,s and DV,s (Byers 1983; Adams 2011). Paper is a wood based material extracted from the tree and made of different organic, inorganic constituents with chemical additives to improve the paper quality (Guggenheim and Martin 1995; Mabee and Roy 2003; Doncea et al. 2010; Adams 2011; Chen et al. 2011; Area and Cheradame 2011; Henniges et al. 2012). Liquid ink contains different chemicals, dyes and pigments are used to produce text, design, map or an image (AghaAligol et al. 2007). All of these materials (paper and ink) are excellent substrates for the fungal growth under favorable conditions (Byers 1983; Nittérus 2000). They are prone to physical, chemical and biological degradation and damage depending on their chemical nature and storage conditions (Area and Cheradame 2011). It is subject to degradation by different enzymes of fungi and bacteria (Benoit et al. 2012). Therefore, biodeterioration of library materials and arts work is a worldwide problem. The literature on the fungal contamination of the library materials during 1919-1977 was reviewed by Zyska (1997). Since then no review was carried out in this field and few studies were conducted. Biological degradation of paper by

molds and insects are the most frequent cause of biological problems (Eduardo 2001; Borrego et al. 2010). The fungi produce different types of enzymes such as cellulosedegrading enzymes of Trichoderma reesei (Martinez et al. 2008; Area and Cheradame 2011; Kubicek et al. 2011; Van den Brink and de Vries 2011), and pectin degradation enzymes by Aspergillus and Rhizopus species (Benoit et al. 2012). The apparent growth of these fungi depends on the environmental conditions such as humidity, pH, temperature and nutrients, which are prevailed in archives and libraries (Fassatiová et al. 1987; Fassatiová 1995; Borrego et al. 2010). Fungal contamination occurs in books, manuscript, archives, libraries and museums around the world. Zyska (1997) reported 84 fungal genera and 234 species from library materials worldwide. In Prague, 88 fungal species were isolated from the atmosphere, archive documents, walls and shelves (Fassatiová et al. 1987). More than fifty fungal species were isolated and identified from air and dust samples of 10 French archives which associated with occupational symptoms (Roussel et al. 2012). Many species of fungi were isolated from libraries in Warsaw, Poland (Gutarowska et al. 2012), about 77 species and 30 fungal genera on the documents from eight stores in Russia (Mokeeva and Budarina 1991), bio-aerosol fungi from the Doctorate Library of the University of Perugia, Italy (Ruga et al. 2008), and 21 genera and 33 species of fungi from 163 damaged archives in Assiut City, Egypt (Abdel-Mallek 1994). From the dust sample collected from mortgage registers in the court of the Polish,

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the genus Penicillium, as well as Cladosporium herbarum, Geotrichum candidum, Cephalosporium glutineum, Mucor racemosus, Trichoderma viride, and A. niger were recovered (Krysinska-Traczyk 1994). A. ustus, A. nidulans, A. versicolor, seven Penicillium chrysogenum strains, A. alternata, C. cladosporioides, Mucor racemosus, Phoma glomerata, and Trichoderma longibrachiatum were recovered from cinematographic films of Spanish archives in Madrid, Barcelona, (Abrusci et al. 2005). The most frequent species are Cladosporium indicum, Alternaria alternata, Fusarium sp., Penicillium sp., Chaetomium sp., Aspergillus sp., (Abdel-Mallek 1994; Rakotonirainy et al. 1999; Nittérus 2000; Rakotonirainy and Lavedrine 2005). Some of these fungi were associated with many health risks and human diseases such asAspergillosis of Aspergillus fumigatus (Abdel-Mallek 1994; Krysinska-Traczyk 1994; Srikanth et al. 2008; Gutarowska et al. 2012). Different methods were continuously assessed for controlling the fungal growth on books and archives such as UV, gamma radiations and some chemical disinfectants (Hanus 1985; Nittérus 2000). The UV radiation has been restricted because it does not penetrate the paper surface and may cause paper ageing (Hanus 1985). Gamma rays penetrate efficiently, but may cause paper aging and cancer (Pavon 1975; Justa 1992). Although, large number of toxic chemicals has been utilized to sanitize papers, some of these chemicals result in pigment discoloration and damage the books and artworks. Carbo gasoline, formaldehyde, thaiabendazole, ethylene oxide, and essential oils were commonly used against mold growth on books and archives (Beebe 1911; Rakotonirainy et al. 1999; Rakotonirainy and Lavédrine 2005). However, most of these chemicals have adverse effects on paper and mild fungal control. Nonetheless, thaiabendazole at 10% was very effective on fungi and does not damage the paper and artwork (Rakotonirainy et al. 1999). Formaldehyde (formalin, methyaldehyde) at 1.5% has been used in the treatment of 8.1 million books in Russia, but it was restricted due to its toxicity and irritation effect (Nittérus 2000). Ethylene oxide was a powerful sterilant in museum fumigation (Brokerhof 1989). For almost 60 years, thymol as a fungicidal has been used in paper conservative practice (Nittérus 2000). On the other hand, since 1787, sodium hypochlorite solution (NaOCl, household bleach), as an alternative to SO2 in winemaking, it is used for controlling the fungal and bacterial contamination (Yoo et al. 2011). At high concentrations, sodium hypochlorite cause skin burns and eye damage, nonetheless, at less than 4.0% it was classified as a moderate oxidizing hazard by the National Fire Protection Association (NFPA). The 0.4% is the minimum inhibitory concentration of sodium hypochlorite against Penicillium expansum (Cerioni et al. 2013). Therefore, sodium hypochlorite has been suggested as an effective alternative chemical agent against the fungal growth with negligible damage on paper and artwork (Ebling 2007; Yoo et al. 2011). In Oman, recently around 70,000 documents were collected from India, UK, Tanzania and Pakistan by the National Records and Archives Authority. They represent a big challenge of evident fungal contamination and their

subsequent control measure. Therefore, the present study was designed to identify the fungal flora invading these books and archives and searching for simple, cheap, and eco-friendly control measures. This will participate effectively in the development of national and international strategy for the collection and storage of rare books and archives which are useful to mankind. MATERIALS AND METHODS Sample collection and isolation of fungi For this study, sterile cotton swabs were used to collect 102 samples from the books and archives of the National Records and Archives Authority, Muscat, Oman. The swabs were streaked onto Potato Dextrose Agar (PDA) and incubated at room temperature (25±2○C) for 10 days. The developed fungal isolates were isolated in pure cultures for further identification to the species level. Morphological identification of the isolated fungi The fungal isolates developed in the growth media were identified macroscopically and microscopically based on their characteristics on the growth media and the morphology of sexual and asexual structures. The identification of the isolated fungi was confirmed using many taxonomic books, monographs and taxonomic papers (eg. Raper and Fennell 1965; Ellis 1971, 1976; Pitt 1979; Sutton 1980; Samson et al. 1995; Barnett and Hunter 1998, 2003). For non-sporulating fungi, mycelial fragments were inoculated on Malt Extract Agar (MEA) and incubated at 28ºC±2ºC to stimulate sporulation and were then identified to species level following the same identification method. Molecular identification of the isolated fungi The morphological identification of the isolated fungi was confirmed with the help of molecular techniques through DNA extraction, purification, polymerase chain reaction (PCR), and DNA sequencing. The internal transcribed spacer region (ITS) was used for the identification of the isolated fungi as described by many authors (Eberhardt 2010). Two primers namely ITS1 and ITS4 were consumed. Isolation and purification of the genomic DNA of fungi The DNA of the fungal isolates was extracted using Soil DNA Extraction Kits prepared by MO BIO Laboratory, Inc., (Carlsbad, California, USA) as per the manufacturer protocol with some modifications. The mycelial mat of 6 day-old fungal culture was harvested from the surface of the growth media with a sterile disposable loop, aseptically transferred into 2 ml Bead Solution tube and gently vortex. Solution S1 was heated to 60ºC and 60 µL were added, vortex, 200 µL of Solution IRS (inhibitor Removal Solution) were added, vortex at maximum speed for 15 min. The supernatant was transferred to a clean microcentrifuge tube, 250 µL of Solution S2 were added, vortex for 5 min., centrifuged for 1 min. at 10000 xg and the entire supernatant was transferred to a clean microcentrifuge tube. To the tube

EL-NAGERABIet al. – The mycobiota on paper archives

contents, 1 ml of Solution S3 was added, vortex for 5 sec., 700 µL were loaded onto a spin filter and centrifuged at 10000 xg for 1 min., and this step was repeated until all the supernatant has passed through the spin filter. From solution S4, 300 µL were added, centrifuged for 30 sec. at 10000 xg, the flow through was discarded and centrifuged again for 1 min. Spin filter was placed in a new clean tube, 50 µL of Solution S4 were added to the center of the white filter membrane, centrifuged for 30 sec., the spin filter was discarded and the isolated DNA in the tube is then ready and frozen at -20ºC for further uses. Polymerase chain reaction Polymerase chain reaction (PCR) was adopted to amplify the extracted DNA samples. For the identification of the ITS region of the isolated fungi, a set of forward primer (ITS1) and reverse primer (ITS4) were consumed (Martin and Rygiewicz 2005). Each PCR reaction mixture contains 10 µL of Promega PCR master mix, 0.4 µL ITS1 primer, 0.4 µL ITS4 primer, 2 µL of DNA sample and nuclease free water to make the final volume of 25 µL. The amplification was carried out by automated thermal cycle with thermocycling of 95○C for 10 minutes (heating and denaturing); 35 cycles at 95○C for 30 seconds; 55○C, 30 seconds; and 72○C, 60 seconds, 72○C, 10 minutes (extension). Detection and purification of PCR product For testing the quality of PCR product, a volume of 20 µL of PCR was checked by gel electrophoresis in 1.5% agarose gel in 1x Tris-borate-EDTA buffer (TBE) at 100 v for 40 min. The gel was stained with ethidium bromide and viewed under ultraviolet light to detect the presence and size of the amplified DNA product. The PCR products were purified using the EXO-SAP (Exonuclease and Shrimp Alkaline phosphatase) stored at -20○C. The EXO removes any single DNA strand or primer from the product, while the SAP removes the unconsumed dNTPs that may interfere with the sequencing. Both EXO and SAP utilize hydrolytic enzymes to remove unwanted area from the DNA fragments. Each purification reaction contained 10 μL of Exo-SAP mix (0.025 μL ExoI; 0.25 μL SAP and 9.75 μL water) and 5μL of PCR products. The reaction requires two incubation holds in the thermal cycle at 37°C for 60 minutes and 95°C for 5 minutes to deactivate the enzymes. DNA sequencing reaction After the purification of the PCR products, the sequencing was carried out with Applied Biosystems v3.1 Big Dye Cycle sequencing kit where both forward and reverse reactions were conducted separately. Each sequencing reaction contained 2μL of purified PCR product, 2 μL of primer, 1.5 μL of free-nuclease water, 2 μL of Q solution, 0.5 μL of Big Dye® terminator v3.1 5X sequencing buffer, 2 μL of dye terminator 5X cycle sequencing. The sequencing reactions were run in the BioRad Thermal Cycler with thermal conditions of 96°C for 1 min, followed by 25 cycles at 96°C for 10 seconds, 54°C for 5 seconds, and 60°C for 4 minutes. The sequences were

purified using DyeEx® 2.0 spin kit (250) for dye terminator removal (QIAGEN). After purification the product was sequence using the 3130x/Genetic Analyzer (Applied Biosystem). With the help of Bioedit program, the resulted ITS sequences were compared with the sequences of fungal isolate at the National Center for Biotechnology Information (NCBI) using BLAST search. Effect of sodium hypochlorite on fungal spores For testing the effect of sodium hypochlorite (NaOCl) on the fungal spore germination, 10 fungal species were selected, namely Aspergillus arborescens, A. clavatus, A. flavus, A. niger, Cladosporium cladosporioides, Drechslera australiensis, Erysiphe pisi, Fusarium oxysporum, Penicillium marneffi and Mycosphearella graminicola. From 7-day old fungal cultures grown on PDA, 5 loops full inocula were added to 9 ml sterile distilled water, thoroughly mixed and serial dilutions of up to 10-3 were prepared. Different concentrations of sodium hypochlorite (0.3, 0.7, 1.3., 2.6, 5.2%) were prepared. From the spore suspension of each fungus, 1 ml was added to sterile petri dish and mixed with 1 ml of different concentrations of sodium hypochlorite and 10 ml of molten cool Malt Extract Agar (MEA). As a control, similar set was prepared using sterile distilled water instead of sodium hypochlorite. Three replicates were incubated at room temperature for 10 days. Effect of sodium hypochlorite fumigation on fungal contamination of books To investigate the effect of sodium hypochlorite vapor on naturally contaminated books, 6 pages from contaminated books were placed inside a clean fish tank (29.5 × 22 × 44 cm) lined with filter papers. The tank was sprayed with 20 ml of sodium hypochlorite (5.2%), covered and sealed with Vaseline. As a control similar tank was sprayed with 20 ml of water. The tanks were incubated for 48 hours. The presence of the fungi was tested by the swab method and the observations were reported. RESULTS AND DISCUSSION The morphological and molecular identification of fungi from archives and books showed the high contamination of these documents with different fungal species (Table 1, Figure 1). The fungal contamination of the archives and books was investigated using morphological and molecular identification methods. For the molecular identification, Internal Transcribed Spacer region (ITS) which of high degree of variation than other genic regions of rDNA was used. The size of the fragment within this region is between 600-750 bp; and the resulted fragments in our results fall in this range (Figure 1). In the present study, 102 swab samples collected from different books and manuscripts show evident fungal contamination (100%). Twenty-two species which belong to 11 genera of fungi were identified (Table 1). Of these fungi the genus Penicillium (46.8%) is the most prevalent, followed by Aspergillus (30.7%), Cladosporium (7%), Rhizopus (4%), and Chaetomium (3.5%) whereas the remaining 6 genera

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represent only 8% of the isolated fungi. In similar study, species dependent where each species produces different some of these fungi were isolated from archives and enzyme such as highly efficient cellulose degrading documents (Fassatiová et al. 1987; Maggi et al. 2000; enzymes of Trichoderma reesei (Martinez et al. 2008; Nittérus 2000; Gutarowska et al. 2012; Roussel et al. Kubicek et al. 2011; Van den Brink and de Vries 2011), 2012). Filamentous fungi of 84 genera, represented by 234 and pectin degradation enzymes by Aspergillus and species, were isolated in the period 1911-1977 from books, Rhizopus species (Benoit et al. 2012). The genera of paper and other materials (Zyska 1997). In Prague, of the Aspergillus, Alternaria, Cladosporium, Curvularia, fungi isolated from atmosphere, archive documents, walls Fusarium, Mucor, Neurospora, and Penicillium were and shelves fungi, Aspergillus niger, A. versicolor, capable of degrading cellulose and excreting pigments and Cladosporium herbarum, C. cladosporioides, Alternaria acids (Borrego and Perdomo 2012). In the present study, alternata, Rhizopus arrhizus, and Penicillium chrysogenum some of these fungi were recovered from books and were the most frequent isolates followed by A. flavus, A. archives such as the species of Aspergillus niger, A. flavus, fumigatus, A. nidulans, Aureobasidium pullulans, Alternaria alternata, Penicillium sp., Rhizopus sp., and Chaetomium sp., Fusarium sp., Rhizopus stolonifer, Chaetomium globosum, Cladosporium spp., Erysiphe pisi, Ulocladium and many Penicillium species (Fassatiová et al. Phycomyces sp., and Puccinia striiformis which have a 1987). More than fifty fungal species were isolated and strong cellulolytic activity and high capability to secrete identified from air and dust samples of 10 French archives different enzyme as biodegradors. Of the isolated fungi, the which associated with occupational symptoms (Roussel et fungus A. flavus is one of the serious pathogenic and al. 2012). These fungi include Cladosporium aflatoxin producers in food and feed products (El-Nagerabi sphaerospermum, C. herbarum, C. cladosporioides, A. et al. 2012, 2013a,b,c). Most of the isolated fungi produce fumigatus, A. ochraceus, A. sydowii, A. niger, A. large numbers of spores which may be hazardous to the versicolor, Penicillium chrysogenum, P. crustosum, P. workers and the visitors of the libraries. Many of these citrinum, Rhizopus sp., Alternaria alternata, Trichoderma, fungi are associated with various human diseases such as Ulocladium and Verticillium species. In Warsaw, Poland aspergillosis of Aspergillus fumigatus (Abdel-Mallek 1994; many species of fungi were isolated from three libraries Gutarowska et al. 2012). Other studies showed that the such as Alternaria alternata, Aspergillus candidus, A. direct contact with contaminated documents with fungi was fumigatus, A. niger, Aspergillus ochraceus, Chaetomium linked to headache, fatigue, eye irritation, throat irritation indicum, Penicillium chrysogenum and Cladosporium and coughing (Roussel et al. 2012). From cultural herbarum (Gutarowska et al. 2012). The most frequent institutions at Havana University, the genera of Aspergillus species are Cladosporium indicum, Alternaria alternata, Fusarium sp., Penicillium sp., Chaetomium sp., Table 1.Incidence of fungi isolated from Omani books and archives collected from Aspergillus sp. (Abdel-Mallek 1994; different countries. Rakotonirainy et al. 1999; Nittérus 2000; Rakotonirainy and Lavédrine 2005). CFU/plate Fugal isolates Document source ×103 Inside six repositories of the National USA, India 5.5 Alternaria alternate* Archives of the Republic of Cuba, Kenya 8 Alternaria arborescens ** Aspergillus, Cladosporium, Curvularia, Kenya, Oman, Tanzania 6.6 Aspergillus clavatus ** Mucor, Neurospora, and Penicillium Oman, UK 3.5 Aspergillus flavus* genera were isolated (Borrego and Oman, Kenya 4.3 Aspergillus nidulans* Perdomom 2012). Of the 28 genera and East Africa, Kenya, Oman, Tanzania 12.6 Aspergillus niger* 31 species identified in a range of public USA 15 Aspergillus ochraceus* and private buildings including libraries, East Africa 4 Aspergillus versicolor* A. flavus, A. niger, P. citrinum, C. Tanzania 8.5 Chaetomium globosum* cladosporioides, C. sphaerospermum East Africa, Kenya 10 Cladosporium caryigenum** were the most prevalent species (Rojas Oman, USA 3.5 Cladosporium cladosporioides* and Aira 2012). In the present study, Kenya 5 Cladosporium sphaerospermum* eleven species of the isolated fungi were Oman 12 Erysiphe pisi** previously encountered from archives Oman 3 Mycosphearella graminicola** and library documents, whereas eleven Oman 5 Paecillomyces lilacinus** species and one genus were considered Oman, Tanzania, UK, USA 9.6 Penicillium chrysogenum* new to the mycoflora of archives and Kenya, Oman, Tanzania, UK, USA 17.3 Penicillium digitatum** books (Table 1). India, Oman 4 Penicillium marneffi** In order for any fungus to grow on India, Oman, Tanzania 8.6 Penicillium paxilli** the paper surface, they must produce Oman 8 Penicillium stipitatus** some types of enzymes such as Oman 3.5 Phycomyces sp.** cellulases which attack the fibers and UK 3 Puccinia striiformis** lead to the cleavage of carbohydrates Oman, Tanzania, UK 5 Rhizopus arrhizus* (Abdel-Mallek 1994; Area and Note: *: Previously isolated from library documents. **: New records Cheradame 2011). These enzymes are

EL-NAGERABIet al. – The mycobiota on paper archives

2000 1550 1400

1000 750 600 400 300 200 100 50

Figure 1. Electrophoresis of the PCR products of the fungal isolates in 1.5% agarose gel with ladder of 200bp with fragments fall between 500-750bp.

(A. flavus, A. niger, A. terreus, A. fumigatus), Penicillium, Cladosporium, Fusarium, and Monilia were identified as airborne mycoflora (Rojas et al. 2002). More than fifty fungal species were isolated and identified from air and dust samples of 10 French archives which associated with occupational symptoms (Roussel et al. 2012). These fungi include Cladosporium sphaerospermum, C. herbarum, C. cladosporioides, A. fumigatus, A. ochraceus, A. sydowii, A. niger, A. versicolor, Penicillium chrysogenum, P. crustosum, P. citrinum, Rhizopus sp., Alternaria alternata, Trichoderma, Ulocladium and Verticillium species. In Warsaw, Poland from three libraries many species of fungi were isolated such as Alternaria alternata, Aspergillus candidus, A. fumigatus, A. niger, A. ochraceus, Chaetomium indicum, P. chrysogenum and Cladosporium herbarum (Gutarowska et al. 2012). The concentration of filamentous fungi in archives is associated with health risk for workers (Krysinska-Traczyk 1994). In the present study, some of these fungi were isolated and may cause similar health hazards to the workers and the visitors. This could be avoided by controlling the fungal infestation of archives and books in libraries. Different chemicals were commonly used to compact mold growth and contamination of different substrates. However, there are numerous difficulties that prevent the disinfection of books such as gaseous penetration inability, and steam injuries of the books (Beebe 1911). Thermal fogging with alkyl dimethylbenzylammonium chloride solution has been employed for cleaning of the libraries atmosphere contaminating by fungi (Rakotonirainy et al. 1999). Several researchers have pointed out the possibility of using gamma radiation for paper disinfection (ex. Barkai et al. 1969; Pavon 1975). Thiabendazole (Thiazol-4) 2benzimidazole at 10% was an effective sanitation of atmosphere (Rakotonirainy et al. 1999). Ethylene oxide has been widely used as powerful sterilant in museum fumigation (Nittérus 2002). However, the high toxicity and carcinogenic properties prevent its application in paper conservation practice. The vapor of nine essential oils and their components showed the potential use of linalool as an alternative to chemical fungicides to disinfect is difficult to

assess, but may be useful in preventing fungal contamination in the storage area of cultural properties (Rakotonirainy and Lavédrine 2005). The vapors of thymol have been extensively used in fumigation cabinets for books and archives, but is no longer used because of its health hazard and deleterious effects on the object (Byers 1983; Isbell 1997; Rakotonirainy and Lavédrine 2005).On the other hand, sodium hypochlorite (NaOCl, household bleach) is known to be one of the most effective antimicrobial chemicals which is useful against fungal and bacterial contamination (Okungbowa and Usifo 2010; Reynolds et al. 2012). It is evidently safe and effective chemical compared to other chemical disinfectants (Ebling 2007; Yoo et al. 2011). It is widely used in food industries, despite the increasing availability of other disinfectants (Fukuzaki 2006). At 1-5.7%, NaOCl caused a 100% reduction in spore’s viability of P. brevicompactum (Ebling 2007). The minimum inhibitory concentration of NaOCl against P. expansum (apple blue mold), was 50 mg/liter (Cerioni et al. 2013). It is effective against bacterial contamination of kitchenware and labware items (Feliciano et al. 2012). Sitara and Akhter (2007) reported that 10% NaOCl was effective against seed borne mycoflora of maize including A. flavus, A. wentii, Chaetomium, Drechslera, Rhizopus, and Fusarium species. All fungal spores on grains were completely inhibited by 1-5% NaOCl (Sauer and Burroughs 1986). The use of low concentration of NaOCl (2.4%) was effective and recommended for controlling of indoor mold (Reynolds et al. 2012). In the present study, it is evident that the use of different concentrations of sodium hypochlorite (0.3-5.2%) significantly inhibited the fungal growth of the tested fungal species namely Aspergillus arborescens, A. flavus, A. niger, Cladosporium cladosporioides, Penicillium marneffi (Figure 2), and A. clavatus, Drechslera australiensis, Erysiphe pisi, Fusarium oxysporum, Mycosphearella graminicola (Figure 3). The minimum inhibitory concentration was recorded at 0.7% for all fungi. In previous studies, the minimum inhibitory concentration against the growth of spores was reported at 0.4% sodium hypochlorite (Cerioni et al. 2013). On the other hand,

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fumigation of pages from contaminated books with 5.2% sodium hypochlorite completely inhibited the fungal growth without evident effect on the paper quality comparable to spray with water. Therefore, it can be recommended as a safe control measure against fungal contamination of books and archives without adverse effect on the quality of paper text discoloration as suggested by many researchers.

inhibition concentration (MIC) of 0.7%. Therefore, NaOCl can be used as eco-friendly fumigant against mold growth on archives, books and other library materials without apparent damage to paper. ACKNOWLEDGEMENTS We thank the authority of National Records and Archives, Muscat, Oman, the Department of Biology, Sultan Qaboos University, and the Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa for facilities. We thank the University of Nizwa Writing Center for proofreading the English of this manuscript. REFERENCES

Figure 2. The inhibitory effect of different concentrations sodium hypochlorite using three replicates on P. marneffi, A. niger, Alternaria arborescens, A. flavus and C. cladosporioides.

Figure 3. The effect of different concentrations of sodium hypochlorite using three replicates on A. clavatus, F. oxysporum, Erysiphe pisi, M. graminicola and D. australiensis.

CONCLUSION It is evident that the collected archives and books by the National Records and Archives Authority of Oman from different sources are subject to high invasion with numerous molds. Twenty two species which belong to 11 genera of fungi were recovered from these documents. Of these recovered fungi, 11 species were previously isolated from similar archives and library materials, whereas 11 species and one genus are new records. Some of these fungi are of cellulolytic activity which degrades papers and associated with many health hazards. Sodium hypochlorite was found effective against mold growth with minimum

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ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060105

Vol. 6, No. 1, pp. 26-32 May 2014

Microbial water quality of coastal recreational water in the Gaza Strip, Palestine

MAZEN T. ABUALTAYEF1,♥, ABDEL FATTAH N. ABD RABOU2, ♥, AHMED A. ABU FOUL1, SAID M. GHABAYEN1, HASSAN M. ELSINWAR1

Department of Environmental Engineering, Islamic University of Gaza, P.O. Box 108, Gaza, Palestine, ♥email: mabualtayef@iugaza.edu.ps Departments of Marine Sciences and Biology, Islamic University of Gaza, P.O. Box 108, Gaza, Palestine, ♥♥email: arabou@iugaza.edu.ps

Manuscript received: 2 April 2014. Revision accepted: 24 April 2014.

Abstract. Abualtayef MT, Abd Rabou AN, Abu Foul AA, Ghabayen SM, Elsinwar HM. 2014. Microbial water quality of coastal recreational water in the Gaza Strip, Palestine. Nusantara Bioscience 6: 26-32. Wastewater disposal into the Mediterranean coast of the Gaza Strip has many negative effects, whether on the environment or on human health, thus microbiological analysis of seawater samples was carried out. The microbial analysis was confined on two types of fecal indicators (fecal coliform and fecal streptococci), in addition to a single type of bacteria (pseudomonas). This study was conducted between the beginning of July 2012 to the mid of October 2012 over an area extended from the proposed Khan Younis fishing port to Gaza fishing port, with a length of about 23 km. The study area was divided into five zones. The samples were collected in two rounds: the first round included 75 samples that collected along the study area during the summer season. The second round included 19 samples that collected in the autumn season to compare it with their counterparts that have been collected in the summer season. Laboratory analysis showed the presence of contamination in many of these samples. The results also showed that the pollution was concentrated in and surrounding the mouths of wastewater outfalls. Depending on the microbial analyses, which have been collected in the first round, the fecal coliform appeared in 61% of the samples, while fecal streptococci appeared in all samples and pseudomonas appeared in 33% of the samples. The pollutants were widespread along the study area, which are the result mainly from wastewater discharge into the sea. A risk analysis was done for season variations using the second moment method; in general, it was found that risk in both seasons was high especially in summer. Key words: Gaza Strip, microbial analysis, recreational seawater

INTRODUCTION Recreational use of water and beaches is an important feature of leisure and tourism worldwide. Recreational seawaters generally contain a mixture of pathogenic and nonpathogenic microbes derived from wastewater effluent; industrial process; farming activities and wildlife in addition to any truly indigenous microorganisms. This mixture can present a hazard to the bathers where an infective dose of the pathogen colonizes a suitable growth site in the body and leads to a disease (WHO 1998). Only about 40% of the sewage generated in the Gaza Strip is properly treated. The percentage of population served by sewerage systems is 78.9% (WASH 2011), leaving nearly half a million people unconnected to the network and dependent on alternative means for excreta disposal. Most of the wastewater treatment plants (WWTPs) in Gaza are overloaded and are working beyond their designed capacities (Abd Rabou 2011). According to an ongoing study for the de-pollution of the Mediterranean Sea findings, about 110,000 m3 per day of untreated or partially treated wastewater, mostly coming from these WWTPs and Wadi Gaza, is discharged into the Mediterranean Sea. The untreated wastewater contains many pathogens such as bacteria, viruses, and harmful parasites. These objects may find suitable environment

when they reach water bodies to multiply and spread (Castro and Huber 2007). The present disposal practices in the Gaza Strip are likely to have an adverse effect on the quality of seawater, marine biota and public health (MoEA 2001; Abd Rabou et al. 2007; Abd Rabou 2013). Microbiologically contaminated seawater, beach sand and fishes were found along the Gaza Strip coast (Afifi et al. 2000; Abed Rabou 2003; Elmanaema 2004; Elmanaema et al. 2004; Aljubb 2012; Al-Safadi 2013), and there is an evidence of sanitation-related infections in the Gaza Strip (Abu Mourad 2004; Astal 2004; El-Kichaoi et al. 2004). According to Hilles (2012) and Hilles et al. (2013), it was found that 48.1% of the seawater samples were parasitically contaminated with six species of human gastrointestinal parasites that have been discovered and determined in seawater samples in most of the study area. The species were; Entamoeba histolytica, Ascaris lumbricoides, Giardia lamblia, Strongyloides stercoralis, Hymenolepis nana and Cryptosporidium parvum. These parasitic species and many others are common in the Gaza Strip environment which is deteriorating day by day (Abd Rabou 2011). The population of the Gaza Strip continues to grow rapidly, thus increasing the amounts of poorly treated or untreated sewage being discharged into the coastal water. With a Palestinian population growth rate of around 3.5

ABUALTAYEF et al. â&#x20AC;&#x201C; Microbial water quality of coastal Gaza

percent per annum that would result in a doubling of the population in 15 years. Effective management and sustainable development of Gaza resources will be a huge challenge to the Palestinian Authority (UNEP 2003). The aim of this study is to estimate the microbial pollution (fecal coliform and fecal streptococci and pseudomonas) of recreational seawater of the Mediterranean coast of Gaza, to determine the coastal area that suitable for recreation and to raise awareness of local people towards contaminated coasts.

MATERIALS AND METHODS The Gaza Strip is a narrow piece of land lying on the eastern coast of the Mediterranean Sea. Its position on the cross road from Africa to Asia made it a target for occupied and conquerors over the centuries. The Gaza Strip is very crowed place with an area of 360 km2. The coastal area along the eastern Mediterranean Sea is about 42 km long, and about 6 to 12 km wide, bordered by Egypt from the south.

Figure 1. A. The study area location map. B. The zones of the study area along Gaza coast, C. Sample locations during summer season and D. Sample locations during autumn season.

N U SAN TA RA B IOSC IE NC E 6 (1): 26-32, May 2014

The study area expanded from the Khan Younis fishing area to Gaza fishing port (Figure 1.A), which is about 23 km. The study area covers most busy beaches along the Gaza Strip and serves 1,120,000 inhabitants. Samples of seawater were collected from different locations, starting from February 2012 to December 2012. The study area was divided into five main zones as follows: (i) Zone A extends from the proposed Khan Younis fishing port to Asdaa sewage outfall. (ii) Zone B extends from Asdaa sewage outfall to Deir Al Balah sewage outfall. (ii) Zone C extends from Deir Al Balah sewage outfall to Wadi Gaza. (iii) Zone D extends from Wadi Gaza to El Shiekh Ejleen sewage outfall. (iv) Zone E extends from El Shiekh Ejleen sewage outfall to Gaza fishing port (Figure 1.B). A total of 94 seawater samples was collected within several sampling trips from the different locations. The samples were collected on two rounds; the first round included 75 samples collected every 300 m in summer season along the study area, and the second round included 19 samples collected in autumn and cover the study area as illustrated in Figure 1.C and 1.D. Seawater samples were collected in sterile (600 mL) glass bottles according to the American Public Health Association (APHA) standard methods (APHA 1995) at approximately 15 cm below the sea surface at a point where the depth of the water is approximately 0.5 meters (based on McBride et al. 1998). The seawater sample bottles were labeled directly after the collection process, and kept at less than 10oC using an ice box, and were transferred in the laboratory and processed within 24 hours of collection. Concentrations of microbial pollutant analyzed in seawater samples by Membrane Filter Technique. All the concentrations reported are in CFU. The procedure in microbiological analysis was recommended by the APHA Standard Methods (APHA 1995). The membrane technique was used in the determination of bacteria in seawater samples. Samples passed through Gellman Millipore filter under negative pressure (vacuum). Volume of samples ranges from 100 mL to 1000 mL according to the organisms needed for isolation. These membranes were transferred by a forceps in the media and placed on the surface of the media and passed carefully to avoid any air bubbles. The plates used for isolation of fecal coliform, fecal streptococci and pseudomonas were incubated at 37oC for 24 to 48 hours, while the plates used for isolation of fecal coliform were incubated at 44oC for 24 to 48 hours. The colonies appeared on the surface of the membrane were counted and identified by the Gram stain, Biochemical tests and specific antisera.

countries, identifying waters that are not fecally-impaired has become a challenge (McLaughlin et al. 2005). Once bacteria, viruses and protozoa enter the ocean, invertebrates can efficiently concentrate these potential pathogens through filter-feeding activity (Miller et al. 2008). Sewage disposal in natural waters is a common practice among many nations (Rajagopalan 2005). Large inputs of organic matter, pathogens and nutrients from raw sewage to a weak hydrodynamic environment poses environmental and health problems of deterioration of water quality (Al Dahmi 2009). Inadequate or faulty sewerage and/or sewage treatment system are major causes of pollution in natural waters (Cimino et al. 2002). The exponential growth in urbanization through migration of people from rural and semi-urban areas to cities in search of livelihood, has contributed to the deploring sewerage situations in most major cities of the world notably in developing countries (Longe and Ogundipe 2010). Microbial contamination along the Mediterranean Coast of Gaza Based on an ongoing study for the de-pollution of the Mediterranean Sea findings, 110,000 m3/day is discharged along the coastal line of the Gaza Strip from multiple points. The main outfalls along the Gaza coast are Rafah outfall which disposes about 11,300 m3/day, Asdaa outfall in Khanyounis city which disposes raw sewage directly to the sea of about 12,000 m3/day, Deir Al-Balah outfall and Wadi Gaza, which both dispose raw sewage to the sea with a rate of 11,700 m3/day, El-Shiekh Ejleen outfall which disposes partially treated wastewater to the sea with a rate of 75,000 m3/day and Al-Shalihat outfall. Eutrophication is a process whereby water bodies, such as lakes, estuaries, or slow-moving streams receive excess nutrients that stimulate excessive plant growth (algae and nuisance plants weeds). This enhanced plant growth, often called an algal bloom, reduces dissolved oxygen in the water when dead plant material decomposes and can cause other organisms to die (Abd Rabou 2013). Nutrients can come from many sources, such as fertilizers applied to agricultural fields, erosion of soil containing nutrients, and WWTP discharges. During sample collection, it was observed the presence of the eutrophication in various areas of the beach, which can be traced to the large quantities of wastewater which discharged directly into the sea. This phenomenon is known to be harmful to the marine environment and its biota in the Gaza Strip (Abd Rabou et al. 2007). It is noted that the highest concentrations were found in the summer season in the north of the sewage outfalls higher than the concentrations in the south of the outfalls, this is due to the direction of the currents from the south to the north direction.

RESULTS AND DISCUSSION Fecal contamination of coastal marine habitat is a global problem manifesting, as increased beach closures, water contact-associated illness and shellfish harvest restrictions (Knap et al. 2002; Kumar et al. 1984). In some

Fecal coliform (FC) The results showed that the bacterial count of fecal coliform during summer season ranged from 0 to 30,000 CFU per 100 mL with a mean value of 4,795 CFU per 100 mL. The maximum value of 30,000 CFU per 100 mL was found at two locations (sample locations 48 and 57), which

ABUALTAYEF et al. â&#x20AC;&#x201C; Microbial water quality of coastal Gaza

locates in the north of Wadi Gaza and El Shiekh Ejleen sewage outfall as shown in Table 1. The results did not indicate the presence of FC at locations 6, 8 and 13 which located to the north of Asdaa sewage outfalls, it was noticed that the discharge of wastewater was stopped during the sampling collection. The concentration of FC northern the outfalls was higher than those to the south. While the bacterial count of fecal coliform during autumn season ranged from 0 to 40,000 CFU per 100 mL with a mean value of 2,995 CFU per 100 mL. The maximum value 40,000 CFU per 100 mL was found at El Shiekh Ejleen sewage outfall. Contrary to the results in summer, the results indicated the presence of FC at locations 6, 9 and 14 that located to the north of Asdaa sewage outfall and it was in operation during sampling collection in autumn. The concentration of FC southern the outfalls was higher than those to the north. Fecal streptococci (FS) The bacterial count of fecal streptococci during summer season ranged from 16 to 1,000 CFU per 100 mL with a mean value of 180 CFU per 100 and maximum value of 1,000 CFU per 100 mL was found at location 71 which is near Al Shalihat sewage outfall as shown in Table 1. Contrary to the results of FC at the locations 6, 9, and 14 which located to the north of Asdaa outfall, FS pollution appeared at these locations, since FS is more resistance to the environmental conditions. The bacterial count of fecal streptococci during autumn season ranged from 30 to 1,200 CFU per 100 mL with a mean value of 772 CFU per 100 mL. The maximum value of 1200 CFU per 100 mL was found at El Shiekh Ejleen outfall.

Pseudomonas aeruginosa The bacterial count of pseudomonas during summer season ranged from 0 to 60 CFU per 250 mL with a mean value of 4.8 CFU per 250 mL. The maximum value of 60 per 250 mL was found at the Al Shalihat sewage outfall as shown in Table 1. Generally, pseudomonas appears at the locations near the outfalls. The bacterial count of pseudomonas during autumn season ranged from 0 to 25 per 250 mL with a mean value of 2.2 CFU per 100 mL. The maximum value of 25 CFU per 250 mL was found to the north of Wadi Gaza. Contrary to the results of summer, pseudomonas did not appear at locations 71 and 73 that located near at the Al Shalihat outfall, since it was stopped discharge into the sea at that time. Water quality variation along the Mediterranean Coast of Gaza Changes in the concentrations of each of FC, FS and pseudomonas were tracked along the beach in the study area. Based on the microbiological analysis of the samples; the results were classified into five zones (Table 2): Zone A expanded from Khan Younis port to Asdaa outfall. The results show that there is no FC pollution in this zone. The maximum value of FS was 120 CFU per 100 mL. The maximum value of pseudomonas was 2 CFU per 250 mL. The average value of FS was 102 CFU per 100 mL. It should be noted that the discharge of wastewater from Asdaa outfall was stopped during sampling collection. Zone B expanded from Asdaa outfall to Deir Al Balah outfall. The FC contamination can be neglected at this zone. The maximum value of FS was 400 CFU per 100 mL and the average value was 129 CFU per 100 mL.

Table 1. Comparison between fecal coliform, fecal streptococci, and Pseudomonas aeruginosa results in summer and autumn seasons Site no. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Coordinates N E 31.38435 34.28879 31.38707 34.29211 31.38907 34.29428 31.39105 34.29644 31.39310 34.29853 31.39494 34.30083 31.39669 34.30323 31.39892 34.30666 31.40073 34.30902 31.40273 34.31118 31.40472 34.31332 31.40655 34.31564 31.40849 34.31788 31.41041 34.32019 31.41228 34.32251 31.41409 34.32483 31.41611 34.32695 31.41800 34.32917 31.42003 34.33152 31.42188 34.33383 31.42377 34.33609 31.42591 34.33808 31.42790 34.34031 31.43001 34.34221

Fecal coliform Summer Autumn 0 40 0 70 0 90 0 900 0 1000 60 1000 750 700 100 10000 10000 20 30000 20 400 40 90 3000 30000 40000 8000 0 90 10 900 0 20 10 10000 10 700 0 0 40 0 70 0 90 0 900 0 1000

Fecal streptococci Summer Autumn 100 80 400 60 240 30 350 120 110 200 150 220 50 160 100 1000 240 120 200 90 100 40 30 300 60 1200 100 40 60 40 50 30 16 40 1000 50 60 40 100 80 400 60 240 30 350 120 110 200

Pseudomonas aeruginosa Summer Autumn 1 0 0 2 0 4 0 2 2 2 0 3 1 1 1 25 5 0 0 1 6 0 0 1 1 0 0 1 1 0 0 0 0 0 60 0 13 0 1 0 0 2 0 4 0 2 2 2

N U SAN TA RA B IOSC IE NC E 6 (1): 26-32, May 2014

Zone C expanded from Deir Al Balah outfall to Wadi Gaza. The maximum values of FC and FS was 750 and 150 CFU per 100 mL, respectively. The largest values of FC occurred to the north of small outfall located at the Al Zawaida beach. The maximum value of FS occurred to the north of Deir Al Balah outfall. The average values of FC and FS was 153 and 64 CFU per 100 mL, respectively. As compared with the previous zones, there is a significant increase in the fecal contamination in this zone; because it's located between the two major wastewater outfalls. It is noted that there is a significant increase in the values closer to wastewater outfalls, whether near Deir Al Balah outfall to the south or the Wadi Gaza to the north. Zone D expanded from Wadi Gaza to El Shiekh Ejleen outfall. The maximum values of FC and FS was 40,000 and 700 CFU per 100 mL, respectively. These values occurred to the north of Wadi Gaza. This zone is the most polluted by fecal contamination due to the large amounts of wastewater discharged by Wadi Gaza and El Shiekh Ejleen outfall. It should be noted that the currents direction was from south to the north. The average values of FC and FS was 11,086 and 154 CFU per 100 mL, respectively. It is noted that the fecal contamination decreased gradually as going far from Wadi Gaza until reaching the minimum values of100 CFU per 100 mL for FC and 18 CFU per 100 mL for FS, then a significant increase occurred as be coming closer to El Shiekh Ejleen outfall. Zone E expanded from El Shiekh Ejleen outfall to Gaza fishing port. The maximum value of FC was 30,000 CFU per 100 mL which occurred to the north of El Shiekh Ejleen outfall. The maximum value of FS was 1,000 CFU per 100 mL which occurred at the El Shalihat outfall. It is noted that the pollution in the north of El Shiekh Ejleen outfall was greater than the pollution to the south. The average values of FC and FS was 4,215 and 137 CFU per 100 mL, respectively. It is noted that the fecal contamination decreased gradually as going far from El Shiekh Ejleen outfall until reaching the minimum values at 66 and 67 locations, then a significant increase occurred as be coming closer to El Shalihat outfall. Table 2. Results of microbial analysis at zone A, B, C, D, and E FC

Pseudomonas

1 2 3 4 5

Coordinates N E Zone A 31.37037 34.27432 31.37266 34.27697 31.37620 34.28045 31.37914 34.28315 31.38127 34.28516

110 120 80 100 100

6 7 8 9 10 11 12 13 14

Zone B 31.38435 31.38707 31.38907 31.39105 31.39310 31.39494 31.39669 31.39892 31.40073

100 200 400 200 250 20 10 240 40

1 -

No.

34.28879 34.29211 34.29428 34.29644 34.29853 34.30083 34.30323 34.30666 34.30902

15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

31.40273 31.40472 31.40655 31.40849 31.41041 31.41228 31.41409 31.41611 31.41800 31.42003 31.42188 31.42377 31.42591 31.42790 31.43001

34.31118 34.31332 34.31564 34.31788 34.32019 34.32251 34.32483 34.32695 34.32917 34.33152 34.33383 34.33609 34.33808 34.34031 34.34221

20 10 10 -

100 40 40 100 60 20 50 300 350 300 30 40 20 80 110

2 2

30 31 32 33 34 35 36 37 38 39 40 41 42 43

Zone C 31.43195 31.43405 31.43613 31.43807 31.44026 31.44238 31.44446 31.44567 31.44864 31.45071 31.45279 31.45487 31.45786 31.46084

34.34435 34.34634 34.34834 34.35041 34.35246 34.35442 34.35643 34.35838 34.36040 34.36243 34.36445 34.36644 34.36951 34.37257

60 90 80 60 60 700 750 20 50 50 60 60 100

150 100 100 20 25 40 40 50 50 50 60 50 60 100

1 1 1 1 1

44 45 46 47 48 49 50 51 52 53 54 55 56

Zone D 31.46292 31.46498 31.46498 31.46707 31.46915 31.47123 31.47327 31.47541 31.47748 31.47956 31.48170 31.48375 31.48599

34.37456 34.37661 34.37661 34.37864 34.38062 34.38263 34.38468 34.38657 34.38860 34.39056 34.39254 34.39459 34.39710

120 10,000 40,000 20,000 30,000 40,000 900 2,000 400 100 103 90 400

80 240 700 300 200 80 20 18 100 120 100 30 20

1 5 3 5 6 4 8 2

Zone E 31.48818 34.39903 31.49024 34.40106 31.49228 34.40311 31.49439 34.40511 31.49640 34.40725 31.49853 34.40923 31.50063 34.41120 31.50273 34.41316 31.50484 34.41512 31.50694 34.41709 31.50915 34.41895 31.51132 34.42080 31.51350 34.42268 31.51572 34.42446 31.51800 34.42619 31.52023 34.42800 31.52252 34.42968 31.52381 34.43247 31.52508 34.43374 Note: â&#x20AC;&#x153;-â&#x20AC;&#x153; = negative

30,000 9,000 8,000 6,300 90 80 800 900 700 20 120 20 3000 10,000 10,000 700 360

60 100 100 100 60 100 20 50 30 4 30 6 16 400 1,000 400 60 30 30

1 1 4 5 25 60 18 13 5 -

57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

ABUALTAYEF et al. â&#x20AC;&#x201C; Microbial water quality of coastal Gaza

Figure 2. A. Fecal coliform distribution along the study area. B. Fecal streptococci distribution along the study area. C. Pseudomonas distribution along the study area.

From the results, it is clear that there is a significant pollution in Gaza seawaters mainly from the discharge of wastewater directly into the sea. Figure 2 shows the distribution of pollutants along the study area beach based on summer season measurements. The study area beach had been classified according to suitability for recreation depending on the concentration of fecal contamination (Figure 3). This classification was created on the consideration that the contaminated areas are the areas that reached a concentration of 100 CFU per 100 mL for any of fecal coliform or fecal streptococcus. Based on Figure 8, the contamination mainly appeared at the wastewater outfalls and the surrounding areas. The Gaza city beach is the most contaminated, the reason can be traced to the huge amount of wastewater that is discharged from El-Shiekh Ejleen outfall. The risk of fecal coliform and fecal streptococci was significantly higher during summer than autumn, which were 69% , 64% and 62.17%, 62.17%, respectively. That was different for pseudomonas which was 63.31% and 65.17% during the summer and autumn, respectively. However, the risk was higher in both cases.

to suitability for recreation depending on the concentration of fecal contamination. This classification was created on the consideration that the contaminated areas are the areas that reached a concentration of 100 CFU per 100 mL for any of fecal coliform or fecal streptococcus. Based on Figure 8, the contamination mainly appeared at the wastewater outfalls and the surrounding areas. The Gaza city beach is the most contaminated, the reason can be traced to the huge amount of wastewater that is discharged from El Shiekh Ejleen outfall.

CONCLUSION Generally, the pollution was significant at the zone C, and was very severe at the zones D and E. These zones contain the major wastewater outfalls which dispose huge amounts of wastewater to the sea. Water contaminated by human excreta may contain a range of pathogens (diseasecausing) microorganisms, such as viruses, bacteria and protozoa. These organisms may pose a health hazard when the water is used for recreational activities such as swimming and other high-contact water sports. In this section, the study area beach had been classified according

Figure 3. Classification of the study area beach

N U SAN TA RA B IOSC IE NC E 6 (1): 26-32, May 2014 ACKNOWLEDGMENTS

Special thanks to Osama Eldahoudi and Yousef Hammad for sampling collection, Sami Lubbad and Yasir Albaiomi from the Public Health Laboratory, Ministry of Health, Adel Atallah and Iyad Atallah from Ministry of Agriculture and Bahaa Alagha and Atia Albursh from the Environmental Quality Affairs for their precious help and support during the study. REFERENCES Abd Rabou AN, Yassin MM, Saqr TM, Madi AS, El-Mabhouh FA, Abu Nada FM, Al-Masri MK, Doulah MH and Al-Haj Ahmad MM. 2007. Threats facing the marine environment and fishing in the Gaza Strip: Field and literature study. Theme XII: Environmental Design Trends and Pollution Control. The 2nd International Engineering Conference on Construction and Development (IECCD-II), Islamic University of Gaza, Gaza Strip, Palestine, September 3-4, 2007, 11-31. Abd Rabou AN. 2011. Environmental impacts associated with the Beit Lahia wastewater treatment plant, North Gaza Strip, Palestine. Middle-East J Sci Res 7(5): 746-757. Abd Rabou AN. 2013. Priorities of scientific research in the fields of marine environment and fishery resources in the Gaza Strip, Palestine. Priorities of Scientific Research in Palestine: Towards a National Directory of Scientific Research, Scientific Research Affairs, Islamic University of Gaza, Gaza Strip, Palestine, March 2526, 2013, 481-522. Abed Rabou IM. 2003. The impact of seawater microbial pollution on fish, beach sand and health in Gaza Strip. [M.Sc.-Thesis]. School of Public Health, Al-Quds University, Palestine. Abu Mourad T. 2004. Palestinian refugee conditions associated with intestinal parasites and diarrhea: Nuseirat refugee camp as a case study. Public Health 118 (2): 131-142. Afifi S, Elmanaema A, Shubair M. 2000. Microbiological assessment of beach quality in Gaza Strip. Egypt J Med Lab Sci 9 (1): 51-63. Al Dahmi H. 2009. Raw sewage and marine pollution information source. National Scientific and Technical Information Center, Kuwait Institute for Scientific Research, Kuwait. Aljubb AR. 2012. Impacts evaluation of effluent discharge of Khan Yunes wastewater treatment plant on beach quality for recreational purposes. [M.Sc.-Thesis]. Environmental Sciences Master Program (Environmental Monitoring and Management), Islamic University of Gaza, Gaza Strip. Al-Safadi MY. 2013. Physicochemical and microbiological characteristics of seawater in northern part of the Gaza Strip, Palestine. [M.Sc.Thesis]. Institute of Water and Environment, Al-Azhar University, Gaza Strip. APHA [American Public Health Association]. 1995. Standard methods for the examination of water and wastewater. 19th ed. APHA, AWWA, WPCF, Washington, DC.

Astal Z. 2004. Epidemiological survey of the prevalence of parasites among children in Khan Younis Governorate. Palestine. Parasitol Res J 94 (6): 449-451. Castro P, Huber ME. 2007. Marine biology. 7th ed. McGrawHill, New York. Cimino G, Puleio MC, Toscano G. 2002. Quality assessment of freshwater and coastal seawater in the Ionian area of N.E. Sicily, Italy. Environ Monit Assess 77 (1): 61-80. El-Kichaoi AY, Abd Rabou AN, Sharif FA and Al-Amassi HM. 2004. Changing trends in frequency of intestinal parasites in Gaza Strip, 1995-2000. J Islamic Univ Gaza (Nat Sci Ser) 12 (2): 121-129. Elmanaema A, Fahd M, Afifi S, Abdallah S, Bahr S. 2004. Microbiological beach sand quality in Gaza Strip in comparison to seawater. Environ Res 99 (1): 1-10. Elmanaema AA. 2004. The use of sand and seawater in the assessment of microbial quality of Gaza beach and public health. [Ph.D.Dissertation]. Womanâ&#x20AC;&#x2122;s College, Ain Shams University, Egypt & AlAqsa University, Palestine (The Joint Program for Higher Studies). Hilles AH, Al-Hindi AI, Abu Safieh YA. 2013. Is Gaza sandy shoreline region contaminated with human gastrointestinal parasites? Jordan J Biol Sci 6 (3): 205-210. Hilles AH. 2012. Assessment of parasitic pollution due to wastewater discharge in the shoreline region of the Gaza City, Palestine. [M.Sc.Thesis]. Institute of Water and Environment, Al-Azhar University, Gaza Strip. Knap A, Dewailly E, Furgal C, Galvin J, Baden D, Bowen RE, Depledge M, Duguay L, Fleming LE, Ford T, Moser F, Owen R, Suk WA, Unluata U. 2002. Indicators of ocean health and human health: developing a research and monitoring network. Environ Health Perspect 110 (9): 839-845. Kumar S, Shorenstein ML, Niven N, Stroe M. 1984. Non-01 Vibrio cholerae gastroenteritis in northern California. West J Med 140 (5): 783-784. Longe EO, Ogundipe AO. 2010. Assessment of wastewater discharge impact from a sewage treatment plant on lagoon water, Lagos, Nigeria. Res J Appl Sci Eng Technol 2 (3): 274-282. McBride GB, Salmond CE, Bandaranayake DR, Turner SJ, Lewis GW, Till DG. 1998. Health effects of marine bathing in New Zealand. Intl J Environ Health Res 8: 173-189. McLaughlin JB, DePaola A, Bopp CA, Martinek KA, Napolilli NP, Allison CG, Murray SL, Thompson EC, Bird MM, Middaugh JP. 2005. Outbreak of Vibrio parahaemolyticus gastroenteritis associated with Alaskan oysters. New England J Med 353:1463-1470. Miller MA, Miller WA, Conrad PA, James ER, Melli AC, Leutenegger CM. 2008. Type X Toxoplasma gondii in a wild mussel and terrestrial carnivores from coastal California: new linkages between terrestrial mammals, runoff and toxoplasmosis of sea otters. Intl J Parasitol 38(11): 1319-1328. MoEA [Ministry of Environmental Affairs]. 2001. Gaza coastal and marine environmental action plan. Gaza, Palestine. Rajagopalan V. 2005. Sewage pollution. Central Pollution Control Board, Ministry of Environment and Forests in India, New Delhi. UNEP. 2003. Desk study on the environment in the occupied Palestinian Territories. United Nations Environment Program, Palestine. WASH. 2011. Water for Life. Monitoring Program, Palestinian Hydrology Group, technical support from UNICEF, Palestine. WHO. 1998. Guidelines for safe recreational water environment, Vol. 1: Coastal and fresh water. World Health Organization, Geneva.

N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060106

Vol. 6, No. 1, pp. 33-38 May 2014

Six unrecorded species of Russula (Russulales) from Nagaland, India and their nutrient composition RAJESH KUMAR1,♥, ASHWANI TAPWAL2, SHAILESH PANDEY 1, RAJA RISHI1, GAURAV MISHRA1, KRISHNA GIRI1 1

Rain Forest Research Institute, P.O. 136, Jorhat 785001, Assam, India. Tel.: +91-0376-2305106, ♥e-mail: rajeshicfre@gmail.com 2 Forest Research Institute, Dehradun 248006, Uttrakhand, India Manuscript received: 10 February 2014. Revision accepted: 6 March 2014.

Abstract. Kumar R, Tapwal A, Pandey S, Raja-Rishi R, Mishra G, Giri K. 2014. Six unrecorded species of Russula (Russulales) from Nagaland, India and their nutrient composition. Nusantara Bioscience 6: 33-38. The young and matured carpophores of mushrooms were collected from the forests of Puliebzie, Zakhama, Pherma, Mankoi, Chungtia and Tigit located in different districts of Nagaland, one of the north-eastern states of India. All the species were found associated with Pinus khasya, P. caribaea, P. patula, Cryptomeria japonica, Canarium resiniferum, Dipterocarpus macrocarpus and Shorea assamica. The collected mushrooms were identified as Russula species on the basis of their macro and microscopic characteristics. The identified Russula species were also screened for their nutrient content. The protein and carbohydrate content was found to vary between 28.12 to 42.86, and 49.33 to 55 %, respectively. These species [Russula aeruginea Lindblad; -Fr., R. alnetorum Romagnesi, R. brevipes Peck, R. fragrantissima Romagnesi, R. nobilis Velen. and R. ochroleuca (Pers.) Fr Gray.] are new record from Nagaland state of India. Key words: Russula, mushroom, Nagaland, nutrient content

INTRODUCTION Nagaland is situated in the north-eastern part of India within the longitude of 93°15´ E to 25°6´ E and Latitude 25°10´ N to 27°4´ N. It comprises of eleven districts with an area of 16,579 sq.km. The altitude ranges between 1943826 m and the forest cover is about 80.33% of state's geographic area (ISFR 2011). The prominent tribes of Nagaland are Chakhesang, Angami, Zeliang, Aoo, Sangtam, Yimchunger, Chang, Sema, Lotha, Khemungan, Rengma, Konyak, Pachury and Phom. The average annual rainfall ranges between 2000 and 2500 mm and the temperature during the summer ranges between 15 and 30ºC, while in winter it falls below 4ºC. Cogent climatic conditions and diverse vegetation favor the growth of a variety of mushrooms in the forests. Most of these mushrooms (species of Russula, Agaricus, Cantharellus, Boletus etc.) form mutual symbiotic association with forest trees in the form of ectomycorrhiza, which is most important for their growth, nutrient absorption and protection of roots from pathogens (Marx 1997). The genus Russula is cosmopolitan and an ectomycorrhizal genus associated with a wide range of Gymnosperms and Angiosperms (Richardson 1970; Alexander 1981; Molina and Trappe 1982; Pillukat and Agerer 1992; Kraigher et al. 1995; Agerer 2002). Ectomycorrhiza create distinct features in roots of forest trees. These characters are preferentially dependent, influenced and fashioned by the fungal hyphae of these essentially important structures of the root system (Agerer 2002). Many species of fungi are normally involved in ectomycorrhizal association with a single tree or a single

species may involve in this association with more than one tree (Marx 1997). Wild mushrooms are richer sources of protein and have a lower amount of fat than commercial mushrooms (Barros et al. 2007). A large number of Russula species are better known for their antimicrobial and antioxidant activities and thus having medicinal significance (Mercan et al. 2006; Liu 2007; Turkoglu et al. 2007; Jain and Pande 2013). Twentythree species of the genus Russula have been reported which are associated with different forest trees of Pakistan (Ahmad et al. 1997). The diversity of Russula species has been explored in different parts of the west district of Sikkim, India (Das 2010; Das et al. 2010; Das and Verbeken 2011, 2012). Recently, three new species, i.e. R. sharmae, R. sikkimensis and R. dubdiana were reported from Sikkim, India (Das et al. 2013) We collected, identified and analyzed the nutrient content of fifteen mushroom species from different forest of Nagaland recently (Kumar et al. 2013). The diversity of Russula species has not been explored from Nagaland state of India yet. The present paper proposes six new records, i.e. R. aeruginea Lindblad; -Fr., R. alnetorum Romagnesi, R. brevipes Peck, R. fragrantissima Romagnesi, R. nobilis Velen and R. ochroleuca (Pers.) Fr. Gray from Nagaland state of India. MATERIALS AND METHODS Sample collection and diversity analysis The periodic surveys and collections were done in the forests of Lahorijan, Puliebzie, Zakhama, Pherma, Mankoi,

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Chungtia, Nongkham, Namcha and Tigit (Figure 1) during the rainy (June to September) and winter (October to December) seasons during 2010-2011. The collected samples were wrapped in wax paper and brought to the laboratory for identification and proximate analysis. Morphological and anatomical characterization was carried out under Stereo microscope (Olympus BX 50) and Compound microscopes. The taxonomic identification was carried out consulting the available literatures (Zoberi 1973; Purakasthya 1985; Agerer 1991; Alexopolous et al. 1996; Adhikari 2000, 2004). The specimens were preserved in 2% formaldehyde and kept in the museum of Forest Protection Division, Rain Forest Research Institute, Jorhat, Assam by assigning identification numbers. The frequency and density of different species have been determined by the following formulas: Number of site in which the sp. is present Freq. of fungal species (%) = ------------------------------------x 100 Total number of sites Total number of individuals of a particular species Density = ---------------------------------------------------------x 100 Total number of species

Nutrient content analysis For proximate analysis, fruiting bodies were oven dried and powdered in a Moulinex blender. The fine powder was stored in the desiccators and utilized for proximate mineral and nutrient analysis following Anthrone method (Fasidi and Kadiri 1993). Moisture content: The fresh and oven dried weight (80° C for 48 h) of each mushroom species was recorded and moisture content was determined (Raghuramulu et al. 2003) .

Figure 1. Study sites in the Nagaland state of India

Fresh weight-dry weight Moisture content (%) = ----------------------------------x 100 Fresh weight

Dry matter content: Weight obtained after gradual oven drying from 35-60°C. Crude fiber: The crude fiber content was calculated using the following equation: Crude fiber (g/100 g sample) = [100-(moisture + fat)] × (We-Wa)/Wt of sample (Raghuramulu et al. 2003). Protein content: 0.5 g of the powdered mushroom sample was extracted with 50 cm of 2% NaCl in a waterbath at 60°C for 1 h. The extract was filtered out and 50 cm of 3% copper acetate monohydrate were added to the filtrate to precipitate the protein content. The precipitated protein was then centrifuged and dissolves in 50 cm of 0.1 m NaOH. The quantity of protein in the alkaline solution was then determined using the folin-phenol method (Kadiri and Fasidi 1990). Total carbohydrate estimation: The content of the available carbohydrate was determined by the following equation: Carbohydrate (g/100 g sample) = 100-[(moisture + fat + protein + ash+ crude fiber) g/100 g] (Raghuramulu et al. 2003). Ash content: The powdered mushroom sample (3.0 g) was ashed in a Gallenkamp furnance in previously ignited and cooled crucible of known weight at 550°C for 6 h. Fairly cooled crucibles were put in desiccators and weighed (Raghuramulu et al. 2003). The ash content (g/100g) was calculated using the following equation: Weight of ash Ash content = -------------------------------x 100 Weight of sample taken

KUMAR et al. – Russula from Nagaland

Statistical Analysis: Experimental values are given as means ± standard deviation (SD). Statistical significance was determined by one-way variance analysis (ANOVA). Differences at P < 0.05 were considered to be significant. RESULTS AND DISCUSSION Six Russula species were identified from different forest of Nagaland state, India. The macroscopic and microscopic characters like shape, size, color, texture, attachment of stipe, smell, spore print and spore size were recorded (Figures 1 and 2). The description of the collected specimens is as follows: Russula ochroleuca (Pers.) Fr Gray. (ID No/RFRI/NL000362) (Figure 2A, 3A) It was found in Lahorijan, Puliebzie, Zakhama forest range. The cap was 4-10 cm in diameter, convex with a depression. The color of the cap was yellow with eventually furrowed margins. The stem was 39-70x14-24 mm long and white. The color of the flesh was white. The gills were adnexed, creamy. The spore print was whitish to pale cream. The spores measure 8-10x7-8 µm, broadly ovoid with warts up to 1.2m high, joined by numerous fine lines forming a fairly well-developed network, cystidia were absent. Russula nobilis Velen (ID No/RFRI/NL-000298) (Figure 2B, 3B) It was found in Pherma and Mankoi forest range. The cap was 3 to 9 cm in diameter, smooth, non-striate and bright red. The cap of this species generally remains convex with at most only a shallow central depression. The flesh was red or pink immediately beneath the cuticle; elsewhere the flesh was white. The gills were white and adnexed. The crowded gills of this species are very brittle indeed and easily crumble if they are handled. The spores measure 7-8x6-6.5 µm, ovoid with warts up to 0.5 µm tall and joined by narrow connectives in a nearly complete reticulum. The spore print was white. Russula alnetorum Romagnesi (ID No/RFRI/NL000351) (Figure 2C, 3C) It was found in leaf litter in the forest range of Mankoi and Chungtia. The cap was 3-5 cm, convex deviant flat centrally depressed; the surface was light violet in color. The gills were white, adnexed and narrow in front. The stem was 3-8 cm long, 2.5-4 cm thick, sturdy and solid, more or less equal, dry, smooth and whitish. The spore’s measure 7-11x6.5-l0 µm, broadly ellipsoid to subglobose, ornamented with warts 0.7-1.7 µm high. Russula aeruginea Lindblad;-Fr. (ID No/RFRI/ NL000352) (Figure 2D, 3D) It was found in Puliebzie, Zakhama and Pherma forest range. The cap was 4-9 cm across, convex then flattening or depressed, grass-green, sometimes with yellowish or brownish tinges, without any violaceous tints, with rusty spots, center darker, smooth or radially veined, peeling halfway; margin often furrowed. The stem was 40-80x7-20 mm, white to yellowish, fairly firm. Flesh white. The gills

were free. The spore print was cream. Spores elliptic, with rounded warts up to 0.6μ high, some joined by fine lines to form a very incomplete network with 0-2 meshes, 6-10x5-7 μm; cystidia cylindrical to spindle-shaped, without septa. Russula fragrantissima Romagnesi (ID No/RFRI/ NL000285) (Figure 2E, 3E) It was found in Nongkham, Namcha and Tigit forest range. The cap was 7-20 cm across, sub globose, slowly expanding with incurved margin, thick, fleshy; yellowishbrown and tuberculate-striate at the margin. The gills were adnate, close, pale yellow and narrow in front. The stem was 70-150x15-60 mm long, firm, hollow, colored as cap. The spore print was pale orange-yellow. The spores measure 6-9x5.5-7.7 µm; broadly elliptic, warts up to 1 μm with partial to complete reticulum. Russula brevipes Peck (ID No/RFRI/ NL-000282) (Figure 2F, 3F) Russula brevipes Peck, is widely distributed throughout Nagaland. It was found very common in Puliebzie, Zakhama, Pherma, Mankoi, Chungtia and Namcha forest range. It can easily be identified by its large size and white coloration which does not stain when handled. It is also known as short-stem Russula. The fungus color was whitish to dull-yellow. The cap ranged from 7 to 30 cm (312 in) in diameter, whitish to dull-yellow in color and funnel-shaped with a central depression. The gills were narrow and thin, recurrent in an attachment, nearly white when young but becoming pale yellow to buff in age, and sometimes forked near the stipe. The stem was 3-8 cm long, 2.5-4 cm thick, sturdy and solid, more or less equal, dry, smooth, whitish. The spore print was white. The spore’s measure 8-11x6.5-l0 µm, broadly ellipsoid to subglobose, ornamented with warts 0.7-1.7 μm high. Frequency of species and proximate analysis The maximum frequency occurrence was exhibited by Russula fragrantissima (66.6%) followed by Russula aeruginea (58.3%) and Russula ochroleuca (41.6%). The minimum frequency occurrence was observed with Russula alnetorum (25%). The frequency and density of different Russula species are shown in Table 1. The nutrient composition of the selected mushroom species is shown in Table 2. Fresh mushrooms contained about 90% moisture and 10% dry matter and dry mushrooms contained about 90% dry matter and 10% moisture (Chang and Buswell 1996). The moisture content of the collected mushroom samples ranges from 87.1193.90%. R. ochroleuca, R. aeruginea and R. fragrantissima have higher moisture content in comparison to other species. The dry matter content ranged from 4.10-4.62%. Crude fibres were recorded in the range of 9.81-12.13% and recorded minimum for R. fragrantissima and maximum for R. brevipes. The protein contents were ranged from 28.93% to 39.1% of dry weight (Ragunathan et al. 2003; Sanmee et al. 2003). The highest (43%) and lowest protein content (28%) was recorded in R. ochroleuca and Russula alnetorum, respectively. The carbohydrate content of edible mushrooms usually ranges from 40.6% to 53.3% of dry weight (Khanna et al. 1992;

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Figure 2.A. Russula ochroleuca, B. Russula nobilis, C. Russula alnetorum, D. Russula aeruginea, E. Russula fragrantissima, F. Russula brevipes

Figure 3.A. Russula ochroleuca, B. Russula nobilis, C. Russula alnetorum, D. Russula aeruginea, E. Russula fragrantissima, F. Russula brevipes. Bar = 10 um.

KUMAR et al. – Russula from Nagaland

Table 1. Frequency of occurrence and density of Russula species Name of the species

Family

Edible Unknown

Freq. Density (%) 58.3 108.3 25.0 66.6

Edible/ medicinal Inedible Inedible Edible/ medicinal

25.0 66.6 33.3 41.6

Association with

Use

Russula aeruginea Lindblad et Fr. Russula alnetorum Romagnesi

Russulaceae Pinus patula Russulaceae Shorea assamica/ Dipterocarpus macrocarpus/ Alnus incana Russula brevipes Peck Russulaceae Pinus khasya/ Shorea assamica Russula fragrantissima Romagnesi Russulaceae Cryptomeria japonica/ Canarium resiniferum Russula nobilis Velen. Russulaceae Pinus khasya/ Pinus caribaea Russula ochroleuca (Pers.) Fr Gray. Russulaceae Shorea assamica / Dipterocarpus macrocarpus

25.0 133.3 33.3 58.3

Table 2. Proximate composition (g/100g) of Wild Mushrooms of Russula species (mean ±SE) Mushrooms Russula aeruginea Lindblad et Fr. Russula alnetorum Romagnesi Russula brevipes Peck Russula fragrantissima Romagnesi Russula nobilis Velen. Russula ochroleuca (Pers.) Fr Gray. CD (p < 0.05) SE (m)

Moisture

Dry matter

Crude fibre

Protein

Carbohydrates

Ash

93.33±0.44 84.30 ±1.44 87.13±1.13 95.17±0.88 87.83±1.15 93.93±1.31 2.624 0.822

4.33±0.21 4.10±0.07 4.21±0.12 4.62±0.02 4.21±0.05 4.42±0.02 0.300 0.094

11.20±0.13 11.98±0.30 12.13±0.09 9.81±0.07 11.28±0.14 10.14±0.05 0.302 0.095

37.55±0.25 28.12±0.13 32.76±0.21 36.30±0.30 35.14±0.12 42.86±0.49 0.799 0.250

55.00±0.26 49.33±0.25 52.07±0.16 53.23±2.05 54.83±0.19 52.91±0.19 2.59 0.81

7.05±0.02 8.09±0.05 7.97±0.10 7.07±0.04 7.0±0.06 7.28±0.01 0.143 0.045

Ragunathan et al. 1996). In the present study, Russula species have carbohydrate content between 55-49.33%. The ash content has exhibited quite variation from 8.09-7% in different species. Several dozen species of wild fungi are sold in the market of north-eastern India and most are ectomycorrhizal (Tanti et al. 2011). Kumar et al. (2013) collected fifteen edible/ medicinal mushrooms of Nagaland and worked out them for protein, crude fiber, carbohydrate and ash content. Tapwal et al. (2013) made a collection of 30 macrofungal species belonging to 26 genera from wet evergreen forests of Assam, northeastern India and investigated their ecological relationship with higher trees and documented their utilization as per available literature. Several authors have described the taxonomy of mushrooms from various regions of the world, but an analysis reveals that 60% of the newly described fungi are from the tropics, including mushrooms and up to 55% of the mushroom species have proved to be undescribed (Hawksworth 2001). Except a dozen of species cultivated on a large scale, all the macrofungal species grow in the natural habitat and their harvest is being undertaken for the benefit in different countries including India. Nowadays the anthropogenic activity has made countries all over the world to show serious concern about the dwindling biodiversity being lost at the rate never known before. Therefore, exploration, systematics and conservation of wild mushrooms have received more attention in the present day world. The proteins of wild edible mushroom contain considerable amounts of non-essential amino acids like alanine, arginine, glycine, glutamic acid, aspartic acid, proline and serine (Manzi and Pizzoferrato 2000). The add-value arising from mushrooms are bioactive materials which lead to an increase in its consumption and therefore, stimulating

the commercialization of edible species. Having some sound knowledge of macrofungal diversity at the community and species level in the natural forests of North-eastern India is an essential component for developing and understanding their overall ecology as well as assessing the crucial niche relationships of the various elements that make up the forest biota, including the macrofungi themselves. CONCLUSION The current environmental issues of global warming and climate change would adversely affect the regeneration and growth pattern of the delicate fungi which requires a specific micro-climate. Consequently, higher nutritional quality and unique flavor of these mushrooms are likely to be lost if these wild edibles are not properly documented. A huge gap exists with respect to our knowledge of macrofungal diversity existing in the forests of northeastern India is currently a serious constraint of being able to develop any type of understanding. We hope that it may serve as a starting point for future studies. Our previous and present study is a step to decipher the macrofungal diversity, specifically in the biodiversity rich forests of Nagaland for proper planning management and conservation of biodiversity. We hope that it can serve as a starting point for future studies. ACKNOWLEDGEMENTS The authors gratefully acknowledge to Indian Council of Forestry Research and Education (ICFRE) for funding the research project: No-RFRI-39/2010-11/FP.

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N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060107

Vol. 6, No. 1, pp. 39-44 May 2014

Uniconazole effect on endogenous hormones, proteins and proline contents of barley plants (Hordeum vulgare) under salinity stress (NaCl) MOHAMED A. BAKHETA1,â&#x2122;Ľ, MOHAMED M. HUSSEIN2 1

Department of Botany, National Research Centre, El Buhouth St. Dokki 12311, Giza, Cairo, Egypt. Tel.: +20-122340355 â&#x2122;Ľ email: mbekheta@yahoo.com Department of Water Relations and Irrigation, National Research Centre, El Buhouth St. Dokki 12311, Giza, Cairo, Egypt. Manuscript received: 3 September 2013. Revision accepted: 3 February 2014.

Abstract. Bakheta MA, Hussein MM. 2014. Uniconazole effect on endogenous hormones, proteins and proline contents of barley plants (Hordeum vulgare) under salinity stress (NaCl). Nusantara Bioscience 6: 39-44. Pot experiments were carried out during two growth seasons 2010/2011 under greenhouse conditions of the National Research Centre, Dokki, Cairo, Egypt to investigate the response of barley plants (Hordeum vulgare L) grown under salinity stress (2500 or 5000 ppm) to spraying with solutions of uniconazole at 150 or 200 ppm. The obtained results showed that irrigation with saline solutions caused increases in the amounts of abscisic acid (ABA), crude protein, total soluble-protein and proline contents. The results showed that spraying barley plants grown under saline solutions with uniconazole increased endogenous hormone contents of ABA, cytokinins, crude protein, total soluble protein and proline but caused decreases in the amounts of endogenous indole acetic acid (IAA) and gibberellic acid (GA3). High protection of abscisic acid in treating plants with uniconazole and under salt stress (interaction effect) increases proline, proteins and soluble protein which has been proposed to act as compatible solutes that adjust the osmotic potential in the cytoplasm. Thus, these biochemical characters can be used as a metabolic marker in relation to salinity stress. Key words: Barley, salinity, uniconazole, abscisic acid, indole acetic acid, gibberellic acid, proline

INTRODUCTION Plants, growth and production are affected by natural stresses in the form of biotic and abiotic stresses, inversely. The abiotic stress causes loss of hundred million dollars annually, because of reduction and loss of products (Mahajan and Tuteja 2005). Salinity is the most important limiting factor for crop production and it is becoming an increasingly severe problem in many regions of the world. Plantâ&#x20AC;&#x2122;s behavioral response to salinity is complex and different mechanisms are adopted by plants when they encounter salinity. The soil and water engineering methods increase farm production in the damaged soil by salinity, but achievement of higher purposes by these methods seems to be very difficult (Yokoi et al. 2002). The high salinity of the soil affected the soil penetration, decreased the soil water potential and finally caused physiological drought (Yusuf et al. 2008). The plants under salinity condition change their metabolism to overcome the changed environmental condition. One mechanism utilized by the plants for overcoming the salt stress effects might be via accumulation of compatible osmolytes, such as proline and endogenous hormones. Production and accumulation of free amino acids, especially proline by plant tissue during drought, salt and water stress are an adaptive response. Proline has been proposed to act as a compatible solute that adjusts the osmotic potential in the cytoplasm. Thus, proline can be used as a metabolic marker in relation to stress. Since this soil salinity consider one among the several environmental stresses causing drastic changes in the growth,

physiology and metabolism of plants and threatening physiology and metabolism of plants the cultivation of plants around the globe. Salt accumulation in irrigated soils is one of the main factors that diminish crop productivity, since most of the plants are not halophytic (Jamal et al. 2011). Salt stress induces various biochemical and physiological responses in plants and affects almost all plant processes; salinity also induces water deficit biosynthesis, even in well-watered soils by decreasing the osmotic potential 1 the inhibition of gibberellic acid (Turan et al. 2009). Uniconazole [(E)-1-(4-chlorophenyl)-4, 4-dimethyl-2(1, 2, 4-triazol-l-yl)-1-penten-3-ol] is a new plant growth retardant in the triazole family. It inhibits gibberellin biosynthesis within the plant (Zhou and Leul 1999), reduces the concentration of endogenous indole-3-acetic acid, and increases the concentration of zeatin, ABA and ethylene (Izumi et al. 1988, Zhou and Leul 1999). Foliar application of uniconazole has been shown to retard leaf elongation, improve tiller number and root growth. Uniconazole applied as a foliar spray at the three-leaf stage improved plant growth, including plant height, leaf size and number, leaf area per plant and increased seed and oil yields of winter rape compared to untreated plants (Leul and Zhou 1998). It was also shown to enhance the plant photosynthetic rate, soluble protein, and total sugar concentrations (Yang et al. 2005a, b). According to Gandee et al. (1997), Zhang et al. (2001), certain interactions existed between uniconazole and N fertilizer, which affected plant growth and yield formation. Our previous

N U SAN TA RA B IOSC IE NC E 6 (1): 39-44, May 2014

study also showed some interactions between uniconazole and different cultivars, sowing dates, planting densities, and N application levels, which affected the yield formation and enhancement (Yang et al. 2004). However, the effect of uniconazole on grain proteins is uncertain and available studies have not been reported. Exogenous application uniconazole produced some benefit in alleviating the adverse effects of salt stress and they also improve germination, growth, fruit setting, fresh vegetable and seed yields and yield quality (Bekheta 2009). The use of plant growth regulators is directed in general, to improve yield quality and /or quantity of many crops through regulating and adjusting the balance of endogenous hormone level in favors of normal physiological process and in turn yield. The present investigation was conducted to study the role of uniconazole in ameliorating the adverse effects of salinity through its effects on the endogenous hormones and proline contents. MATERIALS AND METHODS Pot experiments were carried out in the greenhouse of the National Research Centre, Dokki, Cairo, Egypt during two successive growing seasons (2009/2010 and 2010/2011). Naked barley grains (Hordeum vulgare L) were obtained from the Agricultural Research Center, Ministry of Agriculture. The grains were selected for uniformity by choosing those of equal size and same color. The selected grains were washed with distilled water, sterilized with 1% sodium hypochlorite solution for about 2 min. and washed again with distilled water. Factorial experiment laid out in a randomized block design with nine replicates. Ten days after sowing, the seedlings were thinned to two seedlings per pot. In order to reduce compaction and improve drainage, the soil was mixed with yellow sand in a 1:1 proportion. Granular ammonium sulfate 20.5% N at a rate of 40 kg N ha-1 and single super-phosphate (15% P2O5) at a rate of 54 kg P2O5 ha-1 were added to each pot. The N and P fertilizers were mixed thoroughly into the soil of each pot immediately before sowing. The growth regulator used was uniconazole, a plant growth retardant manufactured by Sumitomo Chemical Company, Ltd, Japan. Fresh solutions of uniconazole (uni.) at the rate of 150 and 200 ppm were applied twice to barley plants, the 1st one spray after 30 days from sowing while the 2nd spray after two weeks later, in addition to the control group (plants sprayed with distilled water). Irrigation water consisted of three concentrations of salt (tap water as a control, 2500 and 5000 ppm) in the form of sodium chloride (NaCl) solution applied 10 days after sowing (DAS). Samples were taken after two weeks for the second application of uniconazole (60 DAS) include number of fresh plants and selected recently leaves to be used for estimation of endogenous hormones and proline contents. Determination of endogenous hormones by using GLC and HPLC Extraction Extraction and separation were essentially similar to that reported by Wasfy and Orrin (1975). The plant

material was immersed in cold 85% ethanol in glass Stoppard brown jars which was kept in deep freezer till extraction. The frozen material was homogenized in cold ethanol (85%) by an electric auto mix, then extracted by an electric stirrer with 85% ethanol at about 0°C. The solvent was changed 3 times during the extraction period of 6 hours. The 3 extracts after filtration, were combined together and concentrated under vacuum at 30-35°C to few mL which were kept in a deep freezer till required. The aqueous phase was adjusted to pH 8.8 by using NaOH (1%). The alkaline aqueous residue was shaken three times with equal quantities of ethyl acetate using separating funnel. The combined ethyl acetate fraction was evaporated to dryness and held for further purification. The aqueous fraction was acidified to pH 2.8 with HCl (1%) and shaken three times with equal volumes of ethyl acetate. The remaining aqueous phase was discarded. The combined acidic ethyl acetate phase was reduced in volume (fraction I) to be used for determination of the acidic hormones (gibberellins "GA3", auxins "IAA" and abscisic "ABA") by using GLC. The aqueous phase was adjusted to pH 5.5 with 1% NaOH and extracted three times with water-saturated n-butanol. All n-butanol phases were combined (fraction II) and reduced to 5 mL volume, then stored at-20°C for cytokinins analysis using HPLC. Methylation of endogenous hormones Diazomethane was prepared from methylamine hydrochloride according to the method described by Vogel (1975). Identification of endogenous hormone peaks: Identification of peaks was performed by comparing the relative retention time (RT) of each peak with those of IAA, GA3 and ABA standards. The relative properties of the different individual components were therefore obtained at various retention times on samples. The retention time (RT) of the peaks of authentic samples was used in the identification and characterization of the peaks of samples under investigation (Shindy and Smith 1975). Identification and determination of auxins, gibberellins, and abscisic acid The retention time (RT) of the peaks of authentic samples was used in the identification and characterization of the peaks of samples under investigation (Shindy and Smith 1975). Peak identification was performed by comparing the relative retention time of each peak with those of IAA, GAs and ABA standards. The relative properties of the different individual components were therefore obtained at various retention times on samples. HPLC of cytokinin substances The retention time (RT) of the peaks of authentic samples was used in the identification and characterization of the peaks of samples under investigation (Shindy and Smith 1975). The peak was performed by comparing the relative retention time of each peak with those of IAA, GAs and ABA standards. The relative properties of the different individual components were therefore obtained at various retention times on samples. Endogenous cytokinins fraction as zeatin was determined by HPLC isocratic UV analyzer ODS Hyparsil C18 column, 20 min gradient from

BAKHETA & HUSSEIN â&#x20AC;&#x201C; Uniconazole effect on Hordium vulgare

0.1N acetic acid. pH 2.8 to 0.1 N acetic acid in 95% aqueous ethanol, pH 4. The flow rate: 1 mL /min, detection: UV 254 nm, standards of zeatin, was used (Muller and Hilgenberg 1986). Determination of proline Amino acid free proline was determined in fresh young leaves of barley plants according to the calorimetric method described by Bates et al. (1973). Determination of crude protein and total soluble protein Determination of crude protein and total soluble protein were determined according to the modified method described by Reuveni et al. (1992) with some modifications Statistical analysis All the collected data were subjected to the proper statistical analysis as described by Gomez and Gomez (1984). RESULTS AND DISCUSSION Endogenous hormones Indole acetic acid (IAA) and Gibberellins (GA3) Data presented in Table 1 indicated that irrigated barley plants with solutions of NaCl (2500 or 5000 ppm) caused marked decreases in the endogenous amounts of IAA and GA3 in comparison with that obtained from the plants that irrigated with tap water. Regarding the influence of uniconazole alone on the endogenous levels of IAA and GA3 of barley plants, the results show that application of uniconazole at 150 or 200 ppm has the same trend of saline solutions. Concerning the interaction effect between uniconazole and salinity, the data revealed that application of uniconazole on barley plants irrigated with saline solutions (2500 or 5000 ppm) caused a marked decrease in the endogenous amounts of IAA and GA3 in comparison with that obtained from their corresponding control (plants sprayed with distilled water and irrigated with tap water). The decrements were directly proportional to the concentration used of uniconazole and salinity i.e. the highest value of decrements were obtained from the application of uniconazole and salinity at 200 and 5000 ppm respectively. These results are in harmony with that recorded by Egamberdieva (2009). Abscisic acid content (ABA) It is clear from the data presented in Table 1 that irrigated barley plants with solutions of NaCl up to 5000 ppm caused an increase in the endogenous amounts of ABA in comparison with the amounts obtained from the plants irrigated with tap water. Regarding the influence of uniconazole, the results show that application of uniconazole at 150 or 200 ppm has the same trend of saline solutions. Concerning the interaction effect of uniconazole and salinity, the data indicated that application of uniconazole on barley plants grown under salinity stress caused marked increases in the endogenous content of

ABA in comparison with that obtained from their corresponding control. Table 1. The effect of uniconazole on the contents of endogenous hormones of barley plants grown under salt stress (NaCl). Indole Abscisic Cytokinins Uniconazole Gibberellic acetic acid (CK) Âľg/g (UN) acid (GA3) mg/g fresh acid(IAA) (ABA) fresh ppm weight weight mg/g fresh mg/g weight fresh weight 130.10 48.7 0.93 339 Tap water 0 100 117.33 41.00 1.19 347 150 100.90 34.30 1.35 350 2500 ppm 0 120.05 40.10 1.38 314 100 109.11 36.21 1.58 327 150 98.71 28.90 1.79 346 5000 ppm 0 108.09 32.50 1.51 300 100 99.22 30.11 1.72 318 150 81.11 21.76 1.88 329 116.11 41.33 1.15 345 Mean values of 109.29 35.07 1.58 329 salinity 96.14 28.12 1.73 316 111.09 40.43 1.27 345 Mean values of 102.22 35.77 110 329 uniconazole 93.57 28.22 1.67 330 Salinity (NaCl) ppm

The obtained results are in harmony with Rademacher (2000) who concluded that enzymes, similar to the ones involved in GAs biosynthesis, are also importance in the formation of abscisic acid, ethylene, sterols, flavonoids, and other plant constituents. Changes in the levels of these compounds found after treatment with growth retardants can mostly be explained by side activities of such enzymes. The promotion in the growth of hormone-treated plants grown under stress conditions could be attributed to its effect on hormonal balance between the values of growth promoters and inhibitors. Application of ABA on plants grown under drought stress induced biosynthesis of gene manipulation called AtNCED3 (9-cis-epoxycarotenoid diogenase), this gene is thought to be the key enzyme in ABA biosynthesis, which in turn led to accumulation of endogenous ABA, improved drought tolerance (Iuchi et al. 2001). Under stress conditions, plants tend to increase endogenous ABA, which may contribute to the maintenance of water relations between the second and the third day of water stress treatments Yang et al. (2004). Plant adaptive responses to drought are coordinated by adjusting growth and developmental processes as well as molecular and cellular activities. The root system is the primary site that perceives drought stress signals, and its development is profoundly affected by soil water content. Various growth hormones, particularly abscisic acid (ABA) and auxin, play a critical role in root growth under drought through complex signaling networks. Here, we report that a R2R3-type MYB transcription factor, MYB96, regulates the drought stress response by integrating ABA and auxin signals. The MYB96-mediated ABA signals are integrated

N U SAN TA RA B IOSC IE NC E 6 (1): 39-44, May 2014 shoots and therefore a non-significant increase in proline content, even at the highest salinity level was observed. Recently, Heidari and Sarani (2012) recorded that salinity stress significantly increased proline and soluble carbohydrate in the leaves of chamomile Matricaria chamomilla L.

into an auxin signaling pathway that involves a subset of GH3 genes encoding auxin-conjugating enzymes. A MYB96-overexpressing Arabidopsis (Arabidopsis thaliana) mutant exhibited enhanced drought resistance with reduced lateral roots. In the mutant, while lateral root primordia were normally developed, meristem activation and lateral root elongation were suppressed. In contrast, a T-DNA insertional knockout mutant was more susceptible to drought. Auxin also induces MYB96 primarily in the roots, which in turn induces the GH3 genes and modulates endogenous auxin levels during lateral root development. We propose that MYB96 is a molecular link that mediates ABA-auxin cross talk in drought stress response and lateral root growth, providing an adaptive strategy under drought stress conditions Seo et al. (2009).

Table 2. The influence of uniconazole on amino acid (proline) of barley plants grown salt stress (calculated as Âľ mol/g F. wt.)

Cytokinins Data presented in Table 1 indicated that irrigated barley plants with solutions of NaCl caused a decrease in the endogenous levels of cytokinins in comparison with that obtained from the plants that irrigated with tap water. On the other hand, application of uniconazole caused a marked increase in the endogenous levels of cytokinins as compared with the amount obtained from the untreated plants. Concerning the interaction of uniconazole and salinity on the endogenous contents of cytokinins extracted from the plants treated with uniconazole and grown under salt stress, the obtained data indicated that application of uniconazole on barley plants grown under salinity stress caused marked increases in the endogenous contents of cytokinins in comparison with that obtained from their corresponding control. Similar results were obtained by Bekheta (2000); Zaky (2000) and Upreti and Murti (2004) and Bekheta and Ramadan (2005) on wheat, Vicia faba, beans and cotton plants respectively. In addition, SA reduced the damaging action of salinity on plant growth and accelerates reparation of the growth processes mediated by maintaining a high level of IAA, CKs and ABA, which in turn induce a wide spectrum of anti-stress reactions in plants. The high concentrations of cytokinins were observed in the leaves of cotton plants grown under drought stress could be related to delay in leaf senescence. Meanwhile, the high levels of ABA observed in plants contribute to the acceleration of leaf senescence (Efetova et al. 2007).

It is clear from the same Table 2 that application of uniconazole at two used treatments (150 or 200 ppm) caused considerable increases in proline of barley plants in comparison with that obtained from the untreated plants. The results show that application of uniconazole under salt stress led to a significant increase in the amounts of proline as compared with that obtained from their respective control. The maximum value of increments was obtained from the plants treated with uniconazole at 200 ppm and irrigated with saline water at 5000 ppm. One of the most important mechanisms of higher plants under salt-stress is the accumulation of compatible solutes such as proline. Proline accumulation in salt stressed plants is a primary defense response to maintain the osmotic pressure in a cell. Several reports show a significant role of proline in osmotic adjustment, protecting cell structure and its function in plants in salt-tolerant and salt-sensitive cultivars of many crops (Desingh and Kanagaraj 2007; Turan et al. 2007). In addition, a positive correlation was determined between proline and tissue-Na concentrations under salt stress (Bajji et al. 2001). The present study shows that uniconazole treatments induced an increase in proline concentrations in barley plants under salt stress. A similar result has been reported by Cha-Um and Kirdmanee (2009) and Turan et al. (2009).

Influence on proline content Table 2 shows that irrigation of barley plants with salinity of 2500 or 5000 ppm caused significantly increases in the endogenous content of amino acid "proline" in comparison with that obtained from their corresponding control (plants irrigated with tap water). Many investigators (Bekheta (2009); Khosravinejad et al. (2009) and Heidari and Sarani 2012) work in different plants indicated that salinity stress significantly increased proline content. Sanaullah (2000) pointed out that proline contents were significantly increased (p<0.001) in the resistant lines of wheat viz. cv. Pak-81, Lyllpur-73 and Capelle and barley viz. Jau-87 and Haider-93 with increasing concentrations of NaCl. Accumulation of proline was failed in spikes and

Uniconazole treat. 0.0 ppm Salinity ppm (NaCl) Control (tap water) 2500 ppm 5000 ppm LSD at 1%

11.03 13.90 16.23 2.56

150 ppm

200 ppm

13.56 15.66 17.78

15.76 18.09 20.00

Total crude protein and soluble protein Data recorded in Table 3 show that irrigated barley plants with saline solutions at 2500 or 5000 ppm caused significant increases in both total-N and total solublenitrogen of barley plants in comparison with that obtained from the plants irrigated with tap water. Demiral and Turkan (2005) detected that total soluble protein content of salt tolerant Oryza sativa cv Pokkali plants increased with salinity while sensitive (Oryza sativa cv. IR-28) rice cultivars showed a decrease under salt stress. Similar results were reported in salt tolerant cultivars of barley, sunflower, finger millet and rice plants, These different results of salt stress showed that the responses to salt stress depends on plant species, even in varieties of same plant species plant developmental stage, duration and severity of the salt application (Parvaiz and Satyavati 2008).

BAKHETA & HUSSEIN â&#x20AC;&#x201C; Uniconazole effect on Hordium vulgare Table 3. The effect of uniconazole on the total - N and total soluble-N of the leaves of barley plants grown under salt stress (NaCl) Salinity (S) ppm Tap water

2500

5000

LSD at 1 %

Uniconazole (UN) ppm 0 150 200 0 150 200 0 150 200

Total-protein as mg/100 g F. wt. 52.50 56.88 59.67 54.09 58.12 61.99 60.00 64.07 68.55 3.35

Total soluble protein as mg/100 g F. wt. 13.00 14.22 15.33 13.99 14.74 16.17 15.00 15.67 16.98 0.81

The obtained results indicate that application of uniconazole of all the used treatments (150 or 200 ppm) caused considerable increases in the total protein and total soluble protein of barley plants in comparison with that obtained from the untreated plants. These results are in harmony with Al-Rumaih and Al-Rumaih (2007) reported that using uniconazole on Datura significantly increased protein levels and stimulated nitrate reductase activity, particularly at lower NaCl concentrations. Application of uniconazole under salinity stress led to increase in the amounts of total proteins and total soluble protein as compared to their respective control. Proteins that accumulate in plants under saline conditions may provide a storage form of nitrogen that is re-utilized later (Singh et al. 1987) and may play a role in osmotic adjustment. They may be synthesized de novo in response to salt stress or may be present constitutively at low (Pareek-Singla and Grover 1997). It has been concluded that a number of proteins induced by salinity are cytoplasmic which can cause alterations in cytoplasmic viscosity of the cells (Hasegawa et al. 2000). A higher content of soluble proteins has been observed in salt tolerant cultivars of barley, sunflower, finger millet, and rice (Ashraf and Harris 2004). In higher plants, osmotic stress induces several proteins in vegetative tissues, which are related to late-embryogenesis-abundant (LEA) proteins. The correlation between LEA protein accumulation in vegetative tissues and stress tolerance indicates its protective role under dehydration stress (Ingram and Bartels 1996). Engineered rice plants over expressing a barley LEA gene, HVA1, under the control of rice actin 1 promoter showed better stress tolerance than did the wild type (Xu et al. 1996). CONCLUSION It is clear from this study that the ability of plants to tolerate salt stress is determined by multiple biochemical pathways that facilitate retention and /or acquisition of water, protect chloroplast functions and maintain ion homeostasis. In our study application of uniconazole at (150 or 200 ppm) on barley plants grown under salinity

stress at 2500 and 5000 ppm led to increases in the synthesis of osmotically active metabolites, total proteins, total soluble nitrogen, specific proteins, amino acid proline as well as the endogenous hormones GA3, IAA, cytokinins" and especially ABA. Proteins that accumulate in plants due to application of uniconazole on the barley plant under salinity stress may provide a storage form of nitrogen that is re-utilized later and may play a role in osmotic adjustment. Such all these compounds might be used to protect the plants against stress conditions. REFERENCES Al-Rumaih MM, Al-Rumaih MM. 2007. Physiological response of two species of Datura to uniconazole and salt stress. J Food Agric Environ 5 (3&4): 450-453 . Ashraf M, Harris PJC. 2004. Potential biochemical indicators of salinity tolerance in plants. Plant Sci 166: 3-16. Bajji M, Lutts S, Kient JM. 2001. Water deficit effects on solute contribution to osmotic adjustment as a function of leaf aging in three durum wheat (Triticum durum Defs.) cultivars performing differently in arid conditions. Plant Sci 160: 669-681. Bates LS, Waldren RP, Teare ID. 1973. Rapid determination of free proline for water stress studies. Plant Soil 39: 205-207. Bekheta MA, Abdelhamid M, El-Morsi A. 2009. Physiological response of Vicia faba L. to prohexadione calcium under saline conditions. Plant Daninha Vicosa-MG 27 (4): 769-779. Bekheta MA, Ramadan A. 2005. Influences of uniconazole and coumarin on some morphological, physiological aspects and fiber quality of cotton plants. J Agric Sci Mansoura Univ 30 (7): 3683-369. Bekheta MA. 2000. Physiological studies on the effect of uniconazole on growth and productivity of Vicia faba plants grown under different levels of salinity. [Ph.D. Dissertation]. Botany Department, Faculty of Science, Cairo University, Cairo. Cha-um S, Kirdmanee C. 2009. Effect of salt stress on proline accumulation, photosynthetic ability and growth characters in two maize cultivars. Pak J Bot 41: 87-98. Demiral TT. 2005. Comparative lipid per-oxidation, antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance. Environ Exp Bot 53: 247-257. Desingh R, Kanagaraj G. 2007. Influence of salinity stress on photosynthesis and antioxidative systems in two cotton varieties. Gen Appl Plant Physiol 33: 221-234. Efetova M, Zeier J, Riederer M, Lee C-W, Stingl N, Mueller M, Hedrich WR, Deeken R. 2007. A central role of Abscisic Acid in drought stress protection of Agrobacterium-induced tumors on Arabidopsis. Plant Physiol 145: 853-862. Egamberdieva D. 2009. Alleviation of salt stress by plant growth regulators and IAA producing bacteria in wheat. Acta Physiol Plant 31 (4): 861-864. Gandee CM, Davies WP, Gooding MJ. 1997. Interaction between nitrogen and growth regulator on bread wheat (Triticum aestivum) and spelt (T. spelta). Aspect Appl Biol 50: 219-224. Gomez KA, Gomez AA. 1984. Statistical procedures for agricultural research. 2nd ed. John Wiley & Sons, New York. Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ. 2000. Plant cellular and molecular responses to high salinity. Ann Rev Plant Physiol Plant Mol Biol 51: 463-499. Heidari M, Sarani S. 2012. Growth, biochemical components and ion content of Chamomile (Matricaria chamomilla L.) under salinity stress and iron deficiency. J Saudi Soc Agric Sci 11 (1): 37-42. Ingram J, Bartels D. 1996. The molecular basis of dehydration tolerance in plants. Ann Rev Plant Physiol Mol Biol 47: 377-403. Iuchi S, Kobayashi M, Taji T, Naramoto M, Seki M, Kato T, Tabata S, Kakubari Y, Shinozaki Y, Shinozaki K. 2001. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J 27 (4): 325-333. Izumi K, Nakagawa S, Kobayashietal M. 1988. Levels of IAA, cytokinins, ABA and ethyhene in rice plants as affected by a gibberellin biosynthesis inhibitor uniconazole. Pl Cell Physiol 29: 99-104.

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N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060108

Vol. 6, No. 1, pp. 45-48 May 2014

Effect of fungicides and bioagents on number of microorganisms in soil and yield of soybean (Glycine max) GAURAV MISHRA1,♥, NARENDRA KUMAR2, KRISHNA GIRI1, SHAILESH PANDEY1, RAJESH KUMAR1 2

1 Rain Forest Research Institute, Jorhat, Assam, India. Tel. 091 8471938089, ♥email: gaurav.mishra215@gmail.com Department of Soil Science, G.B. Pant University of Agriculture and Technology, Pantnagar-263145, Udham Singh Nagar, Uttarakhand, India.

Manuscript received: 27 April 2014. Revision accepted: 30 April 2014.

Abstract. Mishra G, Kumar N, Giri K, Pandey S, Kumar R. 2014. Effect of fungicides and bioagents on number of microorganisms in soil and yield of soybean (Glycine max). Nusantara Bioscience 6: 45-48. In field experiments, the effect of selected fungicides and bioagents on number of soil microorganisms and yield of soybean (Glycine max L. Merill) was investigated. The results showed that some of the crop protections preparations applied in the experiment (as seed dressing) increased the populations of the examined microorganisms after the harvest of crops. Maximum counts of bacteria were recorded with Thiomethaxam at 3 g kg-1 while Pseudomonas at 3 g kg-1 showed the highest population of fungi, Actinomycetes, Bradyrhizobium japonicum, PSB and Pseudomonas. The highest straw and grain yields of 3241.6 and 1439.4 kg ha-1, respectively, were recorded with Pseudomonas at 3 g kg-1. Key words: Carbendazim, carboxin, Pseudomonas, thiram, thiomethaxam, Trichoderma viride, vitavax.

INTRODUCTION Many scientists have conducted research to explore the effect of fungicide seed dressing on number of soil microorganisms and yield of various crops. Here, an attempt has been made to analyze the effect of some fungicide as well as some bioagents on soil microflora and yield with special reference to soybean (Glycine max L. Merill). Soybean is an important oil seed crop of the 21st century with high protein (40-45%) and oil content (2022%). It has an important place in the agriculture and oil economy of India by currently occupying 9.67 million ha with an estimated production and productivity of 10.22 million tons and 1006 kg ha-1 respectively. The soybean has a wide range of geographical adaptation, unique chemical composition and good nutritional value, functional health benefits and versatile end uses (food, feed and non-edible). It has a good adaptability towards a wide range of soil and climate and fetches good returns to farmers even with low level of agricultural inputs. It has also played a significant contribution to the yellow revolution in India and as a food plant it forms an important part of routine diet of people in India. Today, soybean belongs to one of most important economic crops in the subcontinent. Soybean occupied a unique position in agriculture by virtue of the ability to fix atmospheric nitrogen (300 kg N ha-1) with the help of root nodule bacteria Bradyrhizobium japonicum (Bezdicek et al. 1978). Soybean is a legume and generally does not need to be fertilized with nitrogen in case where effective homologous strains of bradyrhizobia are present in soil. This is because of a symbiotic relationship with soil bacteria called bradyrhizobia which attach themselves to the soybean root and form nodules. The efficiency of biological nitrogen fixation is markedly dependent on the mutual compatibility

of both partners, and is influenced by a number of environmental factors (Sprent and Minchin 1983; Vincent 1980). Biological nitrogen fixation with the soybean crop can be improved by seed inoculation with superior Bradyrhizobium strains, but factors that reduce the population of inoculated bradyrhizobia on the seed will directly affect the efficiency of the process. Seed treatment with fungicides has been broadly practiced as cheap insurance against seed and soil-borne pathogens, but the toxicity of most fungicides to bradyrhizobia has often been underestimated. Fungicidal seed dressing used to improve the early plant emergence are often damaging to Rhizobium applied as inoculants to legume seed. Some reports claim little damage, which may reflect the considerable variation within and in between different groups of Rhizobium in their sensitivity to fungicides (Curley 1975). Nodulation, nitrogen fixation and growth of various legumes can be inhibited by fungicides. Therefore, keeping the above points in view, the present investigation was carried to study the influence of applied fungicides and bioagents on soil microflora and yield of soybean. MATERIALS AND METHODS A field experiment was conducted during 2009-2010 at Norman. E. Borlaug Crop Research Center, Pantnagar, India to study the influence of selected fungicides and bioagents on soil microflora and yield of soybean variety JS 335. The soil of the experimental site was silty clay loam of pH 7.64 having 0.76% organic carbon, 254 kg ha-1 available nitrogen, 23.26 kg ha-1 available phosphorus and 145 kg ha-1 available potassium (Table 1). The experiment was conducted in randomized block design with three

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replications in 4 x 5 m2 plots. Soybean seed (JS 335 variety) was sown with a spacing of 45 cm between rows, at 5cm depth and 14 to 15 seeds per meter. Various field operations from preparatory tillage to the harvesting of the crop viz. ploughing and leveling, manure and fertilizer applications, irrigations, sowing and weeding were undertaken as per requirements. Table 1. Characterization of some soil physico-chemical properties Soil level (0-30 cm)

Texture Bulk pH SOC Avail. N Avail. P Avail. K density (%) (kg ha-1) (kg ha-1) (kg ha-1) Silty clay 1.37 7.6 0.76 254 23.26 145 loam

The crop was uniformly fertilized with a basal dose of nitrogen (urea), phosphorus (SSP) and potassium (MOP) at 20, 60, 40 kg ha-1, respectively at the time of sowing. Plant population was maintained to 40 plants per square meter area. Soybean seed was treated with fungicide concentrations, ranging from 2 g kg-1 to 3 g kg-1 of seed. Treatments were Thiram (T1), Thiram + Carbendazim (T2), Carboxin (T3), Vitavax (T4), Trichoderma viride (T5), Pseudomonas (T6), Thiomethaxam (T7) and control (T8). Pour plate serial dilution method was used for estimating the population of total bacteria, fungi and Actinomycetes in soil. The soil was serially diluted and aliquots of suitable dilutions were plated with the appropriate culture medium in triplicate. The culture media used were nutrient agar for bacteria, “Martin’s Rose Bengal Streptomycin Agar” media for fungi, “Ken Knight Agar” medium for Actinomycetes, yeast extract mannitol (YEM) agar for B. japonicum, Pikovskaya’s media for phosphate solubilizing bacteria (PSB) and “King’s B” media for Pseudomonas (Cleyet-Marel 1993). The population of these microorganisms in soil was computed by multiplying the mean colonies with the dilution factor used for computing population. Viable counts (g-1 soil) = (number of colony x dilution factor) Weight of soil

RESULTS AND DISCUSSION Population of soil microbes The effect of applied fungicide and bioagents on total bacterial population in the soil was not-significant in comparison to control (Table 2). Bacterial count in soil ranged from 7.5 to 8.8 x 106 CFU g-1 of soil at crop harvest. Thiomethaxam at 3 g kg-1 showed the maximum population of bacteria in soil. Seed inoculation with Thiram + Carbendazim 2:1 at 3 g kg-1, Vitavax at 3 g kg-1, T. viride at 5 g kg-1 and Thiomethaxam at 3 g kg-1 increased bacterial population in the soil of 2.7, 1.6, 2.6 and 11.1% over control treatment. Seed inoculation with Pseudomonas at 3 g kg-1 showed the maximum number of fungi (12.27 x 104 CFU g-1 of soil), 13.6% more over control. All the treatments except Pseudomonas at 3 g kg-1 showed numerically decrease in the fungal population in

comparison to control treatment. At harvest, the effect of applied fungicide and bioagents was also non-significant in comparison to control treatment. Seed inoculation with Pseudomonas at 3 g kg-1 showed the maximum number of Actinomycetes counts (11.4 x 105 CFU g-1 of soil) and showed 13.9% more Actinomycetes population over control. All the treatments showed numerically increase in Actinomycetes population in comparison to control. At harvest, the effect of applied fungicide and bioagents was not-significant in comparison to control treatment. Table 2. Effect of fungicides and bioagents on bacteria (×106), fungi (×104) and actinomycetes (×105) population at harvest of soybean Treatments Thiram at 3g kg-1 Thiram+Carbendazim 2 1 at 3g kg-1 Carboxin at 2g kg-1 Vitavax at 2g kg-1 Trichoderma viride at 5g kg-1 Pseudomonas at 3g kg-1 Thiomethaxam at 3g kg-1 Control CD 5%

ActinoBacteria Fungi mycetes 6 4 (×10 ) (×10 ) (×105) 7.88 10.52 10.74 8.14 10.61 10.06 7.55 9.65 10.64 8.05 6.95 10.06 8.13 8.88 10.52 7.88 12.27 11.42 8.80 9.65 10.57 7.92 10.80 10.02 NS 2.27 NS

At the time of harvest B. japonicum count in soil ranged from 27.7 to 35.2 x 104 CFU g-1 of soil at crop at harvest (Table 3). Pseudomonas at 3 g kg-1 showed the maximum population of B. japonicum in soil. Seed inoculation with Thiram at 3 g kg-1, Vitavax at 3 g kg-1, T. viride at 5 g kg-1 and Pseudomonas at 3 g kg-1 increased B. japonicum population in soil by 10.8, 0.6, 4.0 and 19.5% over control treatment. The effect of different fungicides and bioagents was significant in comparison to control treatment at harvesting. PSB population in soil ranged from 11.6 to 23.8 x 104 CFU g-1 of soil. Treatments having Pseudomonas at 3 g kg-1 and Thiomethaxam at 3 g kg-1 showed a significant increase of 73.1 and 54.7%, while and T. viride at 5g kg-1 showed a decrease of 17.7 in the PSB population in comparison to control treatment. The effect of applied fungicide and bioagents was also non-significant in comparison to control treatment. Table 3. Effect of fungicides and bioagents on B. japonicum (×104), PSB (×104) and Pseudomonas (×104) counts at harvest of soybean Treatments Thiram at 3g kg-1 Thiram+Carbendazim 2 1 at 3g kg-1 Carboxin at 2g kg-1 Vitavax at 2g kg-1 Trichoderma viride at 5g kg-1 Pseudomonas at 3g kg-1 Thiomethaxam at 3g kg-1 Control CD 5%

B. japonicum (×104) 32.66 28.56 27.73 29.64 30.63 35.23 29.13 29.46 NS

PseudoPSB monas 4 (×10 ) (×104) 13.8 6.9 16.4 7.9 16.1 7.5 16.3 6.5 11.6 7.4 23.8 8.0 21.3 6.7 13.7 7.3 3.2 NS

MISHRA & KUMAR â&#x20AC;&#x201C; The isozymic diversity of cardamoms

Pseudomonas population in soil ranged from 6.5 to 8.02 x 104 CFU g-1 of soil. Seed-inoculation with Pseudomonas at 3 g kg-1 gave the maximum number of Pseudomonas population and showed the numerical increase of 8.5% in comparison to control treatment. The treatments Thiram + Carbendazim 2:1 at 3 g kg-1, Carboxin at 2 g kg-1, T. viride at 5 g kg-1 showed the numerically more number of Pseudomonas count in comparison to control treatment. Grain and straw yield The given treatments significantly affected the grain yield as compared with control. The highest grain yield (1446.4 kg ha-1) was recorded in the treatment with Carboxin at 2 g kg-1 while lowest 1244.92 kg ha-1 in control. Treatments having Carboxin at 2 g kg-1 and Pseudomonas at 3 g kg-1 seed inoculation showed significant increases of 16.2 and 15.6%, respectively over control. All the treatments except Thiomethaxam at 3 g kg-1 gave a significantly higher grain yield than control. The applied treatments showed significant effect on the straw yield in comparison to control (Table 4). The highest straw yield of 3241.6 kg ha-1 was obtained with inoculation of Pseudomonas at 3 g kg-1 which was 16.5% higher than the control. All the treatments showed numerical increases in the straw yield over the control. Treatments with Carboxin at 2 g kg-1 and Pseudomonas at 3 g kg-1 were significantly better than control. Table 4. Effect of fungicides and bioagents on Grain and Straw yield of soybean Treatments Thiram at 3g kg-1 Thiram + Carbendazim 2 1 at 3g kg-1 Carboxin at 2g kg-1 Vitavax at 2g kg-1 Trichoderma viride at 5g kg-1 Pseudomonas at 3g kg-1 Thiomethaxam at 3g kg-1 Control CD 5%

Grain yield Straw yield (kg ha-1) (kg ha-1) 1428.08 3010.56 1435.54 3062.16 1446.45 3230.50 1385.60 2946.56 1373.19 2877.56 1439.46 3241.66 1316.63 2790.20 1244.92 2782.30 108.77 295.54

The fungicide and bioagents influenced the population of bacteria in soil after harvest of crops. The highest bacterial population was shown by Thiomethaxam at 3 g kg-1. Wainwright and Pugh (1974) concluded that field application of fungicides at twice the normal rate resulted in increases in bacterial population. Fungal population in soil was significantly affected by the use of fungicide and bioagents over control treatment. All fungicide treatments showed lower fungal population in comparison to control. Several studies showed that the fungal population was being reduced with the application of fungicides (Ingham 1985). Treatment with Pseudomonas at 3 g kg-1 showed the highest population of fungi in soil. This might be because inoculation of Pseudomonas enhanced phosphate supply in the soil and created conducive environment in soil for fungi. The reduction in fungal population with T. viride was due to its parasitic action on other fungi in soil. The population of Actinomycetes was not affected by the use of

fungicides and bioagents. The effect of fungicides and bioagents on B. japonicum population was found notsignificant. Gianasi et al. (2000) also found that fungicide seed treatment did not affect nitrogen fixation by B. japonicum in soybean. Highest population B. japonicum was found with Pseudomonas at 3 g kg-1. Treatments with Thiram + Carbendazim 2:1 at 3 g kg-1, Carboxin at 2 g kg-1 and Thiomethaxam at 3 g kg-1 showed lower counts of B. japonicum in comparison to control treatment. Kaur et al. (2007) reported that Carbendazim is toxic to the nodule bacterium. Thiram concentration beyond 500 Âľg mL-1 was observed to be highly toxic with respect to plant growth factors and rhizobial infection to the G. max (Bikrol et al. 2005). The results also showed that fungicides and bioagents inoculation significantly influenced PSB population in soil over control treatment. Pseudomonas at 3 g kg-1 gave the highest PSB population. All the fungicide treatment showed a lower population of PSB in comparison to Pseudomonas treatment. Decrease in PSB population with fungicides was also reported by Gaind et al. (2007) who concluded that the PSB showed decline in their viable population on prolonged contact with fungicides. Seed treatment with fungicides and bioagents have increased grain yield of soybean. Soares et al. (2004) also reported that fungicide treatments showed higher yield than non-treated plants, varying from 14.5 to 27.3%. Carboxin at 2 g kg-1 numerically increased grain yield over control treatment. Revellin et al. (1993) found that Vitavax 200FF (Carboxin and Thiram), had a small effect or no effect on the survival of B. japonicum and on the nodulation and yield of soybean. It was possibly due to the reduced infection by soil pathogens (Fusarium spp., Pythium spp. and Rhizoctonia spp.) which was significantly controlled in fungicide-treated seeds compared with untreated control and might be due to improvement in soil health. The numerical increase in straw yield was found with fungicides and bioagents over control treatment. Ekundayo (2003) also observed that fungicides did not prevent seed germination. Greater seedling emergence was obtained with fungicide-treated and inoculated seeds compared with fungicide-untreated but inoculated control. Maximum straw yield was recorded with Pseudomonas at 3 g kg-1. Zaidi and Singh (2001) observed that inoculation with B. japonicum strain SB-12 and different isolates of fluorescent Pseudomonas as well as their possible combinations significantly increased yield of soybean over control. This might be due to the reduction in infection by soil pathogens and greater seedling emergence. CONCLUSION The study able to clarify the apparent confusion regarding the response of the seed dressing with plant protection measures for the soybean crop. Agriculturally, the expectation is the increase in yield which should be judged by the success of the dressing of the selected fungicides and bioagents. The value of this study has been to highlight the importance of identifying the crop protection measure through seed dressing and it has been

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found that seed dressing with Pseudomonas is very helpful in maintaining the soil microbial population and achieving the maximum yield of this valuable oilseed crop. The finding suggests soybean seed treatment with Pseudomonas should be followed as a routine. REFERENCES Bezdicek DF, Evans DW, Adebe B, Witters RE. 1978. Evaluation of peat and granular inoculums for soybean yield and nitrogen fixation under irrigation. Agron J 70: 865-868. Bikrol A, Saxena N, Singh K. 2005. Response of Glycine max in relation to nitrogen fixation as influenced by fungicide seed treatment. African J Biotech 4 (7): 667-671. Cleyet-Marel JC. 1993. Preparation of a cultural medium for Rhizobium. In Technical Handbook on Symbiotic Nitrogen Fixation. Legume/ Rhizobium. FAO, Rome, Italy. Curley RL, Burton JC. 1975. Compatibility of Rhizobium japonicum with chemical seed protectants. Agron J 67: 807-808. Ekundayo EO. 2003. Effect of Common Pesticides Used in the Niger Delta Basin of Southern Nigeria on Soil Microbial Populations. Env Monit Assess 89 (1): 35-41. Gaind S1, Rathi MS, Kaushik BD, Nain L, Verma OP. 2007. Survival of bio-inoculants on fungicides-treated seeds of wheat, pea and chickpea and subsequent effect on chickpea yield. J Environ Sci Health B. 42 (6): 663-668.

Gianasi L, Fernandes N, Lourenco SA. 2000. Fungicides applied on soybean seed treatment and its effects on nodulation, nitrogen fixation, dry matter production and yield of soybean. Phytopathologica 26 (3): 352-355. Ingham ER. 1985. Review of the effects of 12 selected biocides on target and non-target soil organisms. Crop Protect 4 (1): 3-32. Kaur C, Maini P, Shukla NP. 2007. Effect of Captan and Carbendazim fungicides on nodulation and biological nitrogen fixation in soybean. Asian J Exp Sci 21 (2): 385-388. Revellin C, Leterme P, Catroux G. 1993. Effect of some fungicide seed treatments on the survival of Bradyrhizobium japonicum and on the nodulation and yield of soybean (Glycine max L. Merr). Biol Fertil Soils 16: 211-214. Soares RM, Rubin S de AL, Wielewicki AP, Ozelame JG. 2004. Fungicides on the control of soybean rust (Phakopsora pachyrhizi) and soybean yield. Ciencia Rural 34 (4): 1245-1247. Sprent JI, Minchin FR. 1983. Environmental factors on the physiology of nodulation and nitrogen fixation. In: Jones DG, Davies DR (eds). Temperate Legumes: Physiology, Genetics and Nodulation. Pitman Advance Publishing Programme, Boston. Vincent JM. 1980. Factors controlling the legume Rhizobium symbiosis. In: Newton WE, Orme-Johnson WH (eds). Nitrogen Fixation. Vol. 2. University Park Press, Baltimore, M.D. Wainwright M, Pugh GJF. 1974. Effect of fungicides on the numbers of microorganisms and frequency of cellulolytic fungi in soils. Pl Soil 43 (1-3): 561-572. Zaidi SFA, Singh HP. 2001. Effect of dual inoculation of fluorescent Pseudomonas and Bradyrhizobium japonicum on nutrient uptake plant growth, nodulation and yield of soybean (Glycine max (L) Merr.). Appl Biol Res 3 (1/2): 1-8.

N U S AN T AR A BIO S C IEN C E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060109

Vol. 6, No. 1, pp. 49-56 May 2014

Effect of nitrogen fertilizers on productivity of Urtica pilulifera plant HEND E. WAHBA1,â&#x2122;Ľ, HEMAIA M. MOTAWE2, ABEER Y. IBRAHIM1 1

Department of Medicinal and Aromatic Plants, Division of Pharmaceutical and Drug Industries, National Research Centre, El Buhouth St. Dokki 12622, Giza, Cairo, Egypt. Tel.: +20-33371718, Fax.: +20-33370931, ď&#x201A;Šemail: hend_wahba@yahoo.com 2 Department of Pharmacognosy, Division of Pharmaceutical and Drug Industries, National Research Centre, El Buhouth St. Dokki 12622, Giza, Cairo, Egypt. Manuscript received: 2 April 2014. Revision accepted: 25 April 2014.

Abstract. Wahba HE, Motawe HM, Ibrahim AY. 2014. Effect of nitrogen fertilizers on productivity of Urtica pilulifera plant. Nusantara Bioscience 6: 49-56. Urtica pilulifera L. have been known for a long time as a medicinal plant for treatments of many diseases, but its agricultural studies and chemical composition did not have enough researches. Two field experiments were carried out at the experimental farm of Faculty of Agriculture, Cairo University during two successive seasons 2009 and 2010, to study the effect of nitrogen dressing application (Urea, ammonium nitrate and ammonium sulfate) at different doses (0, 20, 40 and 60 N units/fed.) on growth, yield parameters and chemical composition of Urtica pilulifera plants. The results showed that the application of all nitrogen forms significantly increased the determined parameters as compared to untreated plants and the best treatment was ammonium sulfate at 60 N unit to increase quantitative and qualitative plant production. Key words: caffeic acid, lipids, nitrogen, Urtica

INTRODUCTION Urtica pilulifera L. (Roman Nettle) (Figure 1) belongs to family Urticaceae and grown as an annual or perennial plant. Urtica pilulifera have been known for a long time as a medicinal plant for treatments of many diseases. Urtica sp. contain various constituents as histamine, acetylcholine, coumaric acid, gallic acid, tannis, 5 hydroxy tryptamine, vitamins A and C and mineral salts including calcium, potassium, silicon, iron, manganese and sulfur Stuart (1982). Urtica sp. was reported as one of the most effective medicinal plants to treat benign prostate hyperplasia (Hirano et al. 1994; Vahlensiek 2002) also it is widely used folk remedy to treat hyperglycemia, hypertension, inflammation of some organs such as the uvula and uterus and fresh branches applied externally in rheumatism, uterus bleeding, anemia, wound healing and as toner tea. Urtica herb extract is useful for bladder disorder; it reduced postoperative blood loss, bucteriuria and prevented hemorrhagic and purulent inflammation following adenomectomy. The powdered leaf used as snuff stops nose bleeds and as lowering blood pressure agent as well as promoter of hair growth (Davidove et al. 1995). Due to the increasing importance of such plant, there are several categories to enhance the growth and increase the active ingredients. One of these categories is the effect of fertilization. Nitrogen is considered a master element in plant nutrition. Nitrogen uptake as ammonium compounds form serves as starting material for amino acid biosynthesis and additional N-containing compound such as pyrimidine, purine bases, chlorophyll, proteins, nucleic acid, vitamins and other organic compounds, therefore, the higher plants require larger amount of nitrogen than is any of the mineral nutrients and the absence of an external supply of nitrogen

reduced plant growth, root and stem growth also directly reduce photosynthesis, protein synthesis and respiration (Strafford 1973). Nitrogen has an important role in plant metabolism that affects quantitative and qualitative plant production by stimulating the growth and activating the vital processes in the plant to increase the active substances were studied by many investigators. Application of nitrogen at 150-200 kg/ha increased the yield, chlorophyll and Mg content of Urtica dioica (Biesiada 2003). The same trend was recorded by many scientists, Aziz (2004) on Achillea millefolium plants, Golcz et al. (2006) and Zheljazkov et al (2008) on Ocimum basilicum, Ierna et al. (2012) and Leskovar et al. (2012) on globe artichoke plants. This research aimed to evaluate the effect of sources and doses of nitrogen fertilizers on productivity and chemical constituents of Urtica pilulifera plant.

MATERIALS AND METHODS This experiment was carried out at the experimental farm of Faculty of Agriculture, Cairo University, Giza, Egypt in two successive seasons (2009 and 2010) to investigate the response of Urtica pilulifera to dressing application of (urea, ammonium nitrate and ammonium sulfate) at different doses (0, 20, 40 and 60 N unit /fed). Seeds of Urtica pilulifera were obtained from Borg ElArab location. The seeds were propagated in Faculty of Agriculture Experimental Farm, then the harvested seed was used as plant material in this experiment. The seeds were sown in different dates as well as it were sown in the nursery and directly on row 60 cm apart and 40 cm in between. The observation indicated that the best date and method of agriculture were direct sowing at the end of

N U S AN T AR A BIO S C IEN C E 6 (1): 49-56, May 2014

Figure 1. Urtica pilulifera L. (Roman Nettle)

Table 1. Physical and chemical analysis of the experimental soil Soil physical analysis

Soil chemical analysis

Total Total Cation Meq/L Anion N P Total K Na+ K+ Ca++ Mg++ HCO- SO4 ClSandy loam 55% 29.75% 14.93% 8.23 2.8 mmohs 480 ppm 37.80 ppm 35.10 ppm 9.50 0.70 14.00 8.20 4.40 25.00 13.00 Texture

Sand

Silt

Clay

E.C

September; therefore, Urtica pilulifera were sown at the end of September in both seasons. Three forms of nitrogen were applied at the rate of 0, 20, 40 and 60 units/fed and the nitrogen sources, urea (46%), ammonium nitrate (33%) and ammonium sulfate (21%) were applied as two separate side dressing. The first addition was after two months from planting and the second was after one month from the first addition. Before planting the physical and chemical properties of the soil were determined using the method of Chapman and Pratt (1978) (Table 1). The plants were fertilized with 100 kg phosphorus/fed as calcium superphosphate (15.5%) and 100 kg/fed potassium as potassium sulfate (48%). Calcium superphosphate was added during the preparation of soil, while potassium sulfate was added to the experimental plots into two equal amounts with an interval of one month starting after two months from planting date. All other horticultural practice was made up when needed. Plant height (cm), number of branches/ plant as well as fresh and dry weight of herb g/plant, fresh and dry weight of leaves g/plant were recorded for each replicate at the vegetative stage, while the total fresh and dry weight of flowers g/plant were recorded at the flowering stage. The yield of seeds was calculated as g/plant and kg/fed by collecting the seeds for each treatment at the end of growth.

Total carbohydrate percentage in herb, flowers and seeds were determined colorimetrically (Dubois et al. 1956). Total caffeic acid derivative content in the plant parts of Urtica pilulifera was determined as chicoric acid (Bauer and Wagner 1988). Total lipid content in the dried herb, roots, flowers and seeds was determined according to AOAC (1995). Urtica pilulifera were sown at the end of September in both seasons in rows 60 cm apart at 40cm in between plants in plots 2 x3 m. Each plot contained eighteen plants. The experiment included 10 treatments with three replicates. The experiment was designed in a split-split design. All obtained data were subjected to statistical analysis according to Snedecor and Cochran (1980).

RESULTS AND DISCUSSION Effect of nitrogen application on growth parameters Vegetative parameters Application of all nitrogen sources and doses increased all growth parameters (plant height, number of branches, fresh and dry weight of the root, herb and leaves) in both seasons as compared to untreated plant (Table 2 and 3).

WAHBA et al. â&#x20AC;&#x201C; Effect of nitrogen on Urtica pilulifera

Plant height. The maximum mean values of plant yield, while ammonium nitrate and ammonium sulfate gave height were resulted from ammonium sulfate followed by about the same values. However, in the second season, urea urea treatment. Regarding the effect of doses on plant treatments showed a remarkable influence on the fresh height, data in the same Table 2 show that the best weight of root as compared to ammonium nitrate and application in this concern was 60 units of nitrogen. The ammonium sulfate treatments (Table 2). effect of interaction between the sources and doses of nitrogen was Table 2. Effect of different nitrogen sources and doses on vegetative growth parameters of significant for ammonium sulfate Urtica pilulifera during 2009 and 2010 seasons. at 60 units of nitrogen /fed. These results agreed with Abbaszadeh et Number Root weight g/plant Plant height al. (2009) showed that the highest of branches / Treatments cm/plant Fresh Dry biological yield and plant height plant S S S S S S S S2 of Melissa officinalis were 1 2 1 2 1 2 1 Control 128.67 134.69 15.33 17.67 181.6 191.70 21.78 23.00 produced by applying 90 kg N ha1 Urea (N unit) 20.00 134.67 147.67 16.33 18.00 203.30 251.70 22.40 30.50 as urea. Ezz El-Din et al (2010) 40.00 146.67 150.33 19.33 21.67 241.70 298.30 30.00 38.00 reported that the maximum values 60.00 138.70 144.23 17.67 20.67 211.70 291.70 24.60 35.00 of plant height of Carum carvi Mean 140.01 14741 17.78 20.11 218.90 280.60 25.70 37.80 were obtained from the addition Ammonium 20.00 131.33 135.67 19.00 22.33 210.00 220.00 25.20 26.40 of 200kg N/fed as ammonium nitrate 40.00 133.33 144.67 22.67 23.33 228.30 246.70 27.90 30.10 nitrate. (N unit) 60.00 138.70 150.67 24.00 25.00 255.03 268.70 30.98 32.20 Number of branches/ plant. Mean 134.45 143.67 21.89 23.56 231.20 245.10 27.90 29.60 Ammonium 20.00 137.67 139.00 19.00 21.67 206.70 221.70 24.00 26.80 The fertilized plants with sulphate 40.00 141.00 150.33 19.33 22.33 226.7 236.70 27.60 28.90 ammonium nitrate were superior (N unit) 60.00 149.33 153.67 20.33 23.33 258.3 267.00 30.90 32.70 to those of plants fertilized with Mean 142.67 149.52 19.55 22.44 230.50 241.8 27.5 29.5 ammonium sulfate or urea in both Means of dose Control 128.67 134.67 15.33 17.67 191.70 181.70 23.00 21.80 seasons. On the other hand, the (N unit) 20.00 137.56 140.78 16.33 20.67 206.67 231.11 24.60 27.89 differences between ammonium 40.00 140.33 148.44 19.33 22.44 232.22 260.56 28.46 31.98 nitrate and urea were significant 60.00 142.22 149.52 17.67 23.00 241.78 275.78 27.96 33.61 while the differences between LSD (0.05) of N.S. 7.40 8.70 2.40 2.50 10.40 14.60 2.80 3.10 ammonium nitrate and LSD (0.05) of N.D. 6.88 8.54 2.20 2.34 3.60 12.30 2.00 2.40 LSD (0.05) of N.S.xN.D 6.60 6.40 2.10 2.30 8.40 1.80 1.90 1.40 ammonium sulfate were Note: S1: First season, S2: Second season, N.S.: Nitrogen source, N: Nitrogen N.D.: Nitrogen insignificant; this trend was true Doses in both seasons. As for doses of nitrogen application, the dose of nitrogen at 40 units/fed in the first Table 3. Effect of different nitrogen sources and doses on vegetative growth parameters of season and 60 units/fed in the Urtica pilulifera during 2009 and 2010 seasons second season were more effective in promoting numbers Herb weight g/plant Leaves weight g/plant of branches. Ammonium nitrate Treatments Fresh Dry Fresh Dry at 60 units/fed showed promising S1 S2 S1 S2 S1 S2 S1 S2 effects on growth parameters. The Control 1514.70 1695.00 246.30 273.48 655.33 742.33 72.09 81.66 Urea N (unit) 20.00 1903.33 1915.00 300.00 309.73 943.67 859.00 104.75 95.35 same results obtained with 40.00 239.00 2726.67 372.82 427.77 1203.00 1340.00 133.65 148.87 Ozguven and Sekeroglu (2007) 60.00 1971.67 2331.67 309.00 370.33 982.00 1103.33 108.02 121.37 showed that N 60 kg /fed nitrogen Mean 2088.00 2324.44 327.27 369.28 1042.89 1100.78 115.47 121.86 fertilization gave the highest Ammonium 20.00 2258.33 2450.00 327.50 366.04 1369.67 1358.33 150.66 149.42 value of black cumin number of nitrate N (unit) 40.00 2490.00 2771.67 370.14 416.53 1518.00 1635.00 170.02 183.12 branches. Also, Bala and 60.00 2749.33 2891.17 422.51 446.23 1583.33 1645.68 174.17 181.02 Fagbayide (2009) reported that Mean 2499.22 2704.28 373.38 409.60 1490.33 1546.33 164.95 171.19 application of N significantly Ammonium 20.00 2221.67 2496.67 363.92 41.63 1025.67 1094.00 112.82 120.34 increased plant height and sulphate N (unit) 40.00 2548.33 2785.00 404.06 450.22 1229.00 1291.00 141.01 144.59 60.00 2881.33 3145.00 473.16 515.31 1340.00 1474.67 151.42 166.64 number of branches of rosell Mean 2550.44 2808.89 413.71 460.05 1198.22 1286.56 135.08 143.86 plant. Mean of dose Control 1514.70 1695.00 246.60 273.48 655.33 742.33 72.09 81.66 Fresh and dry weight of N (unit) 20.00 2127.78 2287.22 330.47 363.46 1113.00 1103.78 122.74 121.70 root. The response of fresh and 40.00 2475.78 2761.11 382.34 431.51 1316.67 1422.00 148.23 158.86 dry weight of roots for Urtica 60.00 2534.11 2789.28 401.56 443.96 1301.78 1407.89 144.54 156.37 pilulifera to the nitrogen source LSD (0.05) of N.S. 59.00 66.00 24.30 19.70 44.00 51.00 8.60 7.50 application was varied during the LSD (0.05) of N.D. 55.00 64.21 23.13 20.18 41.76 48.06 7.90 7.27 two seasons. During the first LSD (0.05) of N.S.xN.D 58.70 64.30 12.98 11.92 34.20 30.91 7.62 7.24 season, urea treatment produced Note: S1: First season, S2: Second season, N.S.: Nitrogen source, N: Nitrogen N.D.: Nitrogen Doses the least mean values of root

N U S AN T AR A BIO S C IEN C E 6 (1): 49-56, May 2014

The response of root yield to doses of nitrogen application was with 60 unit nitrogen /fed which produced the maximum values in this concern followed by 40 units/fed. The effect of interaction between nitrogen sources and doses of the application was different in both seasons. In the first season, treating the plants with ammonium sulfate at 60 units/fed produced the maximum fresh weight of the root, while in the second season the medium level of urea (40 units/fed) was the best one in this concern. The dry weight of root gave similar trend of results as fresh weight. The treatments which encouraged the fresh weight of roots were as the same of which produced the high values of the dry weight of roots. Fresh and dry weights of herb. Ammonium sulfate significantly increased fresh weight of herb comparing to ammonium nitrate and urea in both seasons (Table 3). The heaviest mean values of herb fresh weights 2550.44 and 2808.89 g/plant (were obtained with ammonium sulfate, whereas urea treatment gave the least mean values 2088.00 and 2324.44g/plant) in the first and second seasons, respectively. Concerning the effect of nitrogen application, it is clear that the total fresh weight of herb per plant was significantly and gradually increased with increasing the dose of nitrogen up to 60 units/fed. Also the differences between doses of nitrogen were significant. In other words, application of nitrogen at 60 units/fed produced the maximum values in this concern 2534.11 and 2789.28 g/plant (followed by 40units of nitrogen/fed 2475.78 and 2761.11 g/plant (then 20 units of nitrogen/fed which produced the least value 2127.78 and 2287.22 g/plant (in the first and second season, respectively. Concerning the interaction effect between sources and doses of nitrogen significant responses were observed in both seasons. The heaviest herb fresh weights 2881.33 and 3145.00g/plant (were produced with ammonium sulfate at 60 units/fed in both seasons, respectively. The dry weight of total herb (g/plant) had a similar trend of results of fresh weights, treatments which encouraged the fresh weight were the same produced the high values of herb dry weight. The obtained results agreed with Shaheen et al (2007) showed that, treating Cynara scolymus with 100 – 120 kg N/fed as ammonium sulfate gained the best values of fresh and dry weight yield. El-Sayed et al (2012) found that the highest level of nitrogen (300kg/fed.) on Echinacea paradoxa L. significantly improved plant height, fresh and dry weight of herb, fresh and dry weight of whole plant. Fresh and dry weights of leaves. Data presented in Table 3 indicate that the most effective source of nitrogen was ammonium nitrate, which gave the highest mean values at high dose 1583.00 and 1645.67 g/plant, while the least mean values 943.67 and 859.0 g/plant were obtained from urea treatment. Differences between the three forms of nitrogen were significant. Concerning the effect of nitrogen doses on leaves fresh weight, the medium dose 40 units/fed) produced the highest values followed with 60 units/fed then 20 units/fed. These differences between low dose 20 units/fed of nitrogen and other two doses 40 and 60 units/fed were significant, while the differences between medium and high dose of nitrogen were insignificant (Table 3).

Generally, the fresh weight of leaves increased gradually with increasing the doses of ammonium nitrate and ammonium sulfate, while the medium level of urea produced the highest values as compared to high and low doses of urea. The heaviest fresh weight of leaves per plant was observed with ammonium nitrate at 60 units/fed. The same trend was observed during the two seasons with the dry weight (Table 3. The treatments which produced the heaviest fresh weights of leaves were those which produced the greatest dry weights. The differences between treatments in this concern were significant in the two seasons. These results on the effect of nitrogen on the vegetative growth of some medicinal plants are in harmony with Mousa (2000) on Ocimum basilicum, Sabra (2002) on Ocimum americanum, Biesiada et al. (2008) on Lavandula angustifolia, Abbaszadeh et al. (2009) on Melissa officinalis and El-Habbasha and Abd-Salam (2010) on Brassica napus. They found that fresh and dry weight of leaves was stimulated by increasing nitrogen rate. Flowering parameters The flowers yield. Data in Table 4. show that nitrogen sources and doses significantly affected flower yield of Urtica plants in both seasons as compared to untreated plant. The most effective treatment which gave the heaviest flower yield 272.89 and 294.44 g/plant in the first and second seasons, respectively, was ammonium sulfate. On the other hand, the differences between the source of nitrogen urea and ammonium nitrate were insignificant in both seasons. Concerning the effect of nitrogen doses on flower yield, data in Table 4) show that the low doses of nitrogen 20 units/fed gave the least mean values 248.66 g and 265.11 g/plant for first and second seasons, respectively (Table 3). Increasing the dose of nitrogen from 20 to 40 units/fed increased the flower fresh weight. On the other hand, the treatment of nitrogen at 60 units/fed produced about the same value of 40 units/fed and the differences between them were insignificant, this trend was observed in the two seasons. In regard to the interaction between nitrogen sources and doses in both seasons, the maximum values of flowers fresh weight were resulted from plants fertilized with both ammonium nitrate and ammonium sulfate at high dose (60 units/fed. Regarding the effect of the used nitrogen sources and doses on flower dry weight, the treatments which increased the fresh weight of flowers were parallel to those increased the dry weight of flowers. The same results were reported by Biesiada et al (2008) on Lavandula angustifolia found that supplying 100kg N /ha as ammonium nitrate was suitable for fresh and dry weight inflorescence yield. The seed yield The seed yield as g/plant, g/ plot and kg/fed were significantly increased with different sources and doses of nitrogen fertilizer (Table 4). Nitrogen as ammonium sulfate was superior to the other two nitrogen sources in seed production; it produced the highest mean values of seed yield followed by ammonium nitrate then urea. The differences in the seed yield kg/fed due to three forms of nitrogen were significant in both seasons. The total yield of

WAHBA et al. – Effect of nitrogen on Urtica pilulifera

seed (kg/fed) of ammonium sulfate treatment was higher seasons, addition of urea at high dose 60 units/fed than the ammonium nitrate by 14.33% and 9.76%, while produced the maximum value of total carbohydrate ammonium sulfate produced an increment in seed yield by percentage. 17.61% and 19.57% higher than urea treatment for the first Herb. The results in Table 5 show that treating the and second seasons, respectively. Regarding nitrogen plants with urea as a source of nitrogen was enhanced the doses, dose of 60 units/fed gave the maximum values in accumulation carbohydrate as compared to ammonium both two seasons. The effect of interaction between nitrate and ammonium sulfate in two seasons. As for the nitrogen sources and doses showed that both nitrogen doses of nitrogen, all doses of nitrogen significantly sources as ammonium nitrate and ammonium sulfate gradually Table 4. Effect of different nitrogen sources and doses on flowers weight and yield of seed of increased seed yield by Urtica pilulifera during 2009 and 2010 seasons increasing the applied dose. Flower weight g/plant Yield of seed Generally, the maximum values Treatments Fresh Dry g/plant g/ plot (6m) Kg/fed. of the seed yield in both seasons S1 S2 S1 S2 S1 S2 S1 S2 S1 S2 were resulted from ammonium Control 236.50 242.00 52.03 53.24 13.50 15.20 243.00 273.60 162.00 182.40 sulfate with 60 units/fed. A 20.00 258.00 264.33 57.79 59.21 17.20 18.30 309.60 329.40 206.40 219.60 Urea similar trend of results was 40.00 282.00 303.33 62.89 67.64 22.60 23.12 406.80 416.16 271.20 277.44 N (unit) reported by Refaat et al. 2000) 60.00 248.33 261.67 54.63 57.57 147.40 19.50 313.20 351.00 208.80 234.00 on Borago officinalis found that Mean 262.78 276.44 58.44 61.47 19.07 20.31 343.20 365.52 228.80 243.68 foliar nutrient with urea at 2% Ammonium 20.00 236.33 250.00 52.85 52.50 15.60 16.20 280.80 291.60 187.20 194.40 nitrate 40.00 250.00 260.00 57.20 61.02 21.30 22.80 383.40 410.40 255.60 273.60 produced the highest seed yield N (unit) 60.00 290.00 300.66 63.51 65.85 24.60 27.12 442.80 488.16 295.20 325.44 as compared to 0.5% or 1%. Mean 258.78 276.06 57.85 58.52 20.50 22.04 396.00 369.5 246.00 264.48 Kewalanand et al. (2001) treated Ammonium 20.00 251.66 281.00 57.02 62.10 19.30 20.33 347.40 365.94 231.60 243.96 dill plants with 0, 35, 70, and sulphate N 40.00 475.30 297.00 61.40 66.23 24.15 24.00 432.00 434.70 288.00 289.80 105kg N/ha. They recorded that (unite) 60.00 291.67 305.33 64.46 67.48 28.16 29.16 506.88 524.88 337.92 349.92 umbels per plant and seeds yield Mean 272.89 294.44 60.96 65.27 23.87 24.50 428.76 441.84 285.84 294.56 increased with the increase in the Control 236.50 242.00 52.03 53.24 13.50 15.20 243.00 273.60 162.00 182.40 Mean of nitrogen rate. El-Leithy et al 20.00 248.66 265.11 55.89 57.94 17.37 18.28 312.60 328.98 208.40 219.32 dose (2011) found that nitrogen 40.00 269.11 286.78 60.50 64.97 23.18 22.81 417.30 410.52 278.20 273.68 N (unit) 60.00 276.67 289.89 60.87 63.63 214.56 24.09 44.08 433.56 294.72 289.04 fertilization at 300 kg /fed LSD (0.05) of N.S. 14.60 12.0 2.60 3.10 1.80 2.00 22.00 24.65 14.60 18.40 significantly increased seed yield LSD (0.05) of N.D. 11.20 10.34 1.97 2.65 1.48 1.62 19.80 21.34 15.50 16.04 /plant of Ricinus communis L. Effect of nitrogen application on chemical composition The carbohydrate percentage All nitrogen sources and doses significantly increased the percentage of total carbohydrate content in the root, herb, flowers, and seeds in both seasons as compared to untreated plants (Table 5). Roots. From the data in Table 5 show that the urea treatment produced the maximum values in both seasons. In regard to the effect of different nitrogen applied doses, the Table 5 show that all Urea treatment produced the maximum carbohydrate percentage values in both seasons. All used doses increased the percentage of total carbohydrates in the roots. The best values were recorded with nitrogen at 40 units/fed. Regarding the interaction between nitrogen sources and doses of application in both

LSD (0.05) of N.S. 9.79 9.52 3.20 3.14 2.30 2.40 13.45 14.62 7.98 8.50 x N.D Note: S1: First season, S2: Second season, N.S.: Nitrogen source, N: Nitrogen N.D.: Nitrogen Doses

Table 5. Effect of different nitrogen sources and doses on the total carbohydrates percentage of Urtica pilulifera during 2009 and 2010 seasons. Total carbohydrate % Root % Herb % Flowers % Seed % S1 S2 S1 S2 S1 S2 S1 S2 Control 12.40 13.20 11.70 12.80 12.70 11.50 11.71 12.74 20.00 15.00 14.73 15.00 14.80 13.31 15.00 93.96 13.58 Urea N (unit) 40.00 17.00 15.20 15.40 19.10 15.82 16.10 15.76 15.97 60.00 20.00 17.20 18.80 20.00 14.69 15.10 15.25 13.48 Mean 17.33 15.71 16.40 17.97 14.61 15.40 14.99 14.34 20.00 13.40 13.40 13.10 13.90 13.50 13.00 13.56 13.872 Ammonium 40.00 158.30 15.60 15.70 16.50 14.60 14.30 13.65 16.92 nitrate N (unit) 60.00 16.51 16.20 18.40 18.50 15.64 15.40 18.28 19.20 Mean 15.07 15.07 15.73 16.30 14.58 14.23 15.17 16.65 Ammonium 20.00 13.70 13.03 14.10 14.90 14.40 13.91 14.00 14.50 sulphate N 40.00 16.00 15.90 16.30 16.90 14.80 15.60 16.26 16.49 (unit) 60.00 16.90 17.70 17.60 18.00 16.34 17.90 19.03 19.54 Mean 15.53 15.54 16.00 16.60 15.18 16.43 16.84 16.84 LSD at (0.05)- N.S. 1.16 1.06 1.33 1.36 0.94 1.22 1.16 1.17 LSD (0.05) of N.D. 0.94 1.10 1.11 1.24 0.88 1.15 1.10 1.06 LSD (0.05) of N.S. x 0.80 1.01 1.30 1.28 0.84 1.16 0.95 1.11 N.D Note: S1: First season, S2: Second season, N.S.: Nitrogen source, N: Nitrogen N.D.: Nitrogen Doses Treatments

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increased the carbohydrate content. The increments were carbohydrate content in seed of Nigella sativa. gradually with increasing the dose of nitrogen. The most promising effect on the accumulation of total carbohydrate Total caffeic acid derivatives TCAD was urea at a high level 60 units/fed. Flowers are the richest part in the total caffeic acid Flowers. Urtica pilulifera flowers highly respond to derivatives followed by the seed, then the herb while the ammonium sulfate in accumulation of total carbohydrate in roots contained the lowest values (Table 6). This was the flowers. As for nitrogen doses, the dose of nitrogen at 40 Table 6. Effect of different nitrogen sources and doses on the total caffeic acid derivatives units/fed produced the maximum content of Urtica pilulifera during 2009 and 2010 seasons. value of the total carbohydrate percent as 15.21% and 16.10% Total caffeic acid derivatives % for first and second seasons, Treatments Herb Roots Flower Seeds respectively, followed by the dose S1 S2 S1 S2 S1 S2 S1 S2 of 60 units/fed which produced Control 0.52 0.7 0.39 0.46 1.28 1.50 1.08 1.30 15.14% and 15.53%, then the low 20.00 1.39 1.28 0.99 0.89 2.04 2.64 1.22 1.96 Urea N (unit) 40.00 1.17 1.03 0.77 0.85 0.80 2.43 1.58 2.17 level 20 units/fed which 60.00 0.99 0.76 0.45 0.61 1.77 1.97 1.39 1.40 represented 14.71% and 14.57% Mean 1.18 1.02 0.14 0.78 2.20 2.35 1.40 1.85 for the first and second seasons, Ammonium 20.00 1.09 1.64 0.64 0.56 2.03 2.37 1.63 2.20 respectively. In regard to the nitrate 40.00 1.16 2.10 0.95 0.84 2.33 3.01 1.78 2.10 interaction between sources and N (unit) 60.00 1.62 2.41 0.99 1.30 2.73 3.25 1.37 1.93 doses, ammonium sulfate gave Mean 1.29 2.05 0.86 0.90 2.36 2.87 1.59 2.08 the highest response to the Ammonium 20.00 1.10 1.08 0.86 0.79 1.89 2.79 2.21 1.92 accumulation of total carbohydrate. sulphate N 40.00 1.45 1.69 0.94 0.93 2.19 2.91 2.40 2.42 Seeds. The maximum values (unit) 60.00 1.06 1.37 0.61 0.58 1.81 2.53 0.98 1.54 Mean 1.20 1.38 0.80 0.77 1.96 2.14 1.53 1.96 of the total carbohydrate in the Mean of Control 0.52 0.77 0.39 0.46 1.28 1.50 1.08 1.30 seed were recorded with concentrations 20.00 1.19 1.33 0.83 0.75 1.99 2.60 1.35 21.03 ammonium sulfate treatment, (N unit) 40.00 1.26 1.60 0.89 0.87 2.44 2.78 1.92 2.23 while urea treatment produced the 60.00 1.22 1.51 0.68 0.83 2.10 2.58 1.25 1.62 least mean values (Table 5). The LSD at (0.05) - N.S. 2.16 0.19 0.11 0.11 0.13 0.14 0.12 0.11 differences between urea and the LSD (0.05) of N.D. 0.17 0.17 0.14 0.08 0.12 0.12 0.11 0.09 other two nitrogen sources were LSD (0.05) of N.S.xN.D 0.14 0.14 0.12 0.10 0.10 0.11 0.09 0.07 significant while the difference Note: S1: First season, S2: Second season, N.S.: Nitrogen source, N: Nitrogen N.D.: Nitrogen between ammonium sulfate and Doses ammonium nitrate was insignificant. As for different Table 7. Effect of different nitrogen sources and doses on total lipid content of Urtica doses, increasing the dose of pilulifera plants during 2009 and 2010 seasons. nitrogen from 20 to 40 units/fed produced significant increments. Total lipid% The highest total carbohydrate Treatments Rotts Herb Flower Seeds was obtained as a result of 60 S1 S2 S1 S2 S1 S2 S1 S2 units/fed application. The effect Control 0.32 0.38 0.89 0.92 2.87 3.35 14.28 16.26 of interaction between sources 20.00 0.77 0.57 1.40 1.48 4.61 4.42 20.48 20.31 and doses of nitrogen was Urea N (unit) 40.00 1.03 1.04 1.78 1.80 6.54 6.51 20.03 19.86 60.00 0.98 0.95 1.60 1.57 5.83 6.07 19.64 19.84 significant in both seasons Table Mean 0.93 0.85 1.59 1.62 5.66 5.67 20.33 20.29 5. The application of ammonium 20.00 0.64 0.57 1.11 1.11 5.00 5.42 20.84 20.92 sulfate and ammonium nitrate at Ammonium nitrate 40.00 1.21 1.24 1.94 2.13 6.58 6.92 20.88 20.72 N (unit) 60 units/fed increased the total 60.00 0.921 1.10 1.84 1.73 6.47 6.63 21.00 21.17 carbohydrate in both seasons. Mean 0.93 0.97 1.63 1.66 6.02 6.32 20.62 20.65 Results are in accordance with 20.00 0.40 0.48 1.00 1.06 5.40 5.19 19.09 18.90 Ammonium sulphate Hammam (1996) fertilized anise 40.00 0.54 0.63 1.55 1.34 6.16 6.14 19.98 19.56 N (unit) 60.00 0.51 0.60 1.42 1.19 5.99 5.34 20.63 20.80 plants with nitrogen at the rates of Mean 0.48 0.57 1.32 1.20 5.85 5.56 19.90 19.75 20, 40 and 80 kg/fed He found Mean of concentrations Control 0.32 0.38 0.89 0.92 2.87 3.35 14.28 16.26 that, the contents of total (N unit) 20.00 0.60 0.54 1.17 1.22 5.00 5.01 20.17 20.01 carbohydrate, nitrogen, 40.00 093 0.97 1.76 1.76 6.43 6.52 20.30 20.05 phosphorus and potassium in herb 60.00 0.80 0.88 1.62 1.49 6.10 6.01 20.42 20.60 were increased steadily by raising LSD at (0.05)- N.S. 0.08 0.10 0.07 0.14 0.85 0.89 0.68 0.55 the rate of nitrogen fertilization. LSD (0.05) of N.D. 0.07 0.11 0.05 0.12 0.75 0.74 0.62 0.42 Khalil et al. 2001 who showed LSD (0.05) of N.S.xN.D. 0.06 0.08 0.04 0.11 0.77 0.72 0.65 0.46 that application of 40 units of Note: S1: First season, S2: Second season, N.S.: Nitrogen source, N: Nitrogen N.D.: Nitrogen Doses nitrogen increased total

WAHBA et al. – Effect of nitrogen on Urtica pilulifera

case in both seasons, regardless of the applied treatments. The total caffeic acid derivatives showed a statistically significant increment due to different sources and doses of nitrogen in both seasons as compared to untreated plants. As for nitrogen sources, ammonium nitrate has more promising effects on increasing the total caffeic acid derivatives in all parts of Urtica plants. On the contrary, urea treatment showed the least effect on increasing total caffeic acid derivatives in all plant parts. Concerning the nitrogen doses, data in Table 6 showed that the maximum values of the total caffeic acid derivatives in all parts were recorded with medium dose 40 N units/fed in both seasons while the interaction effect of different sources and doses of nitrogen on the TCAD indicated that ammonium nitrate at high dose 60 N units/fed produced the maximum values in roots and herb although ammonium sulfate at the medium dose 40 N units/fed magnified TCAD in seed than the other treatments. These results are in accordance with Mao et al 2001 who noticed that nitrogen levels significantly affected the concentrations of caffeic acid on sweet potato weevil (Coleoptera: Curculionidae). El-Sayed et al. (2012) recorded results showed that the highest level of Nitrogen (300kg/fed) was improving the content of polysaccharide, caffeic acid and alkamides of Echinacea paradoxa L. plants. Total lipid content It is evident that the lipid content is the highest in the seed 14.28 and 16.26% followed by flowers 2.87 and 3.35% while the roots contained the lowest lipid content as compared to flowers and seed (Table 7). All the used sources and doses of nitrogen significantly increased the lipid content in different plant parts in both seasons. Application of ammonium nitrate produced the highest effect on the lipid accumulation in roots, herb, flowers and seed as compared to other sources. Although all the used doses of nitrogen resulted in considerable increases in the lipid content in the plant parts, the medium dose 40 units/fed was the most effective in increases the lipid in roots, herb and flowers, while the high dose (60 units/fed was the most effective in increase the total lipid in seed, these trends have been followed in both two seasons. The maximum values of total lipid content in root, herb and flowers were resulted from ammonium nitrate at 40 units/fed while ammonium nitrate at high dose 60 N units/fed produced the highest values in seed. Our results are in harmony with Khalil et al 2001 who reported that application of nitrogen fertilizer on Nigella sativa increased total lipid content of the seed. It is clear from the mentioned results that the best treatment to enhance total carbohydrate content in all plant parts is ammonium sulfate at 60 units/fed except herb which induced by urea at 60 units/fed while the most effective treatment to augment total caffeic acid derivatives in all parts is ammonium nitrate at 60 units/fed On the other hand, the best treatment to increase the total lipid content in all plant parts was ammonium nitrate at 40 units/fed.

CONCLUSION Urtica pilulifera plants positively responded to nitrogen application. All nitrogen treatments produced significant increments in growth and determined chemical constituents as compared to untreated plants and the best source was ammonium sulfate for all parameters at the high level (60 N units/fed).

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Khalil MY, Hussein MS El-Sherbeny SE. 2001. A comparative study on the effect of some foliar fertilizers on the growth and yield of Sinapis alba and Nigella sativa plants. Cairo University, Cairo. Leskovar DI, Shinohara T, Agehara S, Patil B. 2012. Integrated approaches for annual artichoke production in South west Texas. Acta Hort 942: 235-28. Mao L, Sory RN, Hammond AM, Pater S, Labonte DR. 2001. Effect of nitrogen on resistance of sweet potato to sweet potato weevil (Coleoptera: Curculionidae) and on storage root chemistry. J Ecol Entomol 94 (5): 1285-1291. Mousa F. 2000. Studies on production of Nigella sativa L. and Ocimum Canum Sims and effect of their oils as ajuvant on foot and mouth disease vaccine [Ph.D. Dissertation]. Faculty of Agriculture, Cairo University, Cairo. Ozguven M, Sekeroglu N 2007. Agricultural practices for high yield and quality of black cumin (Nigella sativa L.) cultivated in Turkey. Acta Hort, (756): 329-337. Refaat AM, Wahba HE, El-Gengaihi S. 2000. The effect of foliar nutrient with urea and methods of planting on Borage (Borago officinalis) productivity. Egypt J Hort 27 (4): 425-438. Sabra AM. 2002. Response of Ocimum americanum, L. plants to nitrogen fertilization. [M.Sc. Thesis], Faculty of Agriculture,, Cairo University, Cairo.

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N U S AN T AR A BIO S C IEN C E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060110

Vol. 6, No. 1, pp. 57-62 May 2014

Using and comparing two nonparametric methods (CART and RF) and SPOT-HRG satellite data to predictive tree diversity distribution SIAVASH KALBI♥, ASGHAR FALLAH, SEYED MOHAMMAD HOJJATI Department of Forestry, Faculty of Natural Resources, Sari Agricultural Sciences and Natural Resources University, P.O.Box: 578, Sari, Mazandaran, Iran. Tel./Fax. +98 151 3822715, ♥email: s.kalbi63@gmail.com Manuscript received: 13 September 2013. Revision accepted: 19 December 2013.

Abstract. Kalbi S, Fallah A, Hojjati SM. 2014. Using and comparing two nonparametric methods (CART and RF) and SPOT-HRG satellite data to predictive tree diversity distribution. Nusantara Bioscience 6: 57-62. The prediction of spatial distributions of tree species by means of survey data has recently been used for conservation planning. Numerous methods have been developed for building species habitat suitability models. The present study was carried out to find the possible proper relationships between tree species diversity indices and SPOT-HRG reflectance values in Hyrcanian forests, North of Iran. Two different modeling techniques, Classification and Regression Trees (CART) and Random Forest (RF), were fitted to the data in order to find the most successfully model. Simpson, Shannon diversity and the reciprocal of Simpson indices were used for estimating tree diversity. After collecting terrestrial information on trees in the 100 samples, the tree diversity indices were calculated in each plot. RF with determinate coefficient and RMSE from 56.3 to 63.9 and RMSE from 0.15 to 0.84 has better results than CART algorithms with determinate coefficient 42.3 to 63.3 and RMSE from 0.188 to 0.88. Overall the results showed that the SPOT-HRG satellite data and nonparametric regression could be useful for estimating tree diversity in Hyrcanian forests, North of Iran. Key words: Tree diversity, random forest, classification, regression tree

INTRODUCTION Forest management and farming, along with natural disturbances like wildfire, storms, and floods have caused widespread land use changes and landscape fragmentation (Ramezani and Holm 2010). These processes may be resulted in biodiversity losses, environmental functions and ecological processes which generate and maintain soil, convert solar energy into plant tissue, regulate climatic parameters and provide multiple forest products (Isik et al. 1997). Hyrcanian forests are the individual natural ecosystem that enjoys the highest plants and animals diversity comparing with other ecosystems in Iran. They are being destroyed by degradation and conversion to other land uses. Under pressure to make informed management decisions rapidly, conservation practitioners must increasingly rely on predictive models to provide them with information on species distributions (Loiselle et al. 2003; Saatchi et al. 2000). The most accurate ways to collect biographical data on species distributions are intensive ground surveys or inventories of species in the field. However, remote sensing offers a cost-efficient means for deriving complete spatial coverage of environmental information for large areas in a consistent manner. Recent studies have indicated that remote sensing may be able to provide useful information on biodiversity (HernandezStefanoni and Dupuy 2007; Mohammadi and Shataee 2010). Dogan and Dogan (2006) tested the predictability of several biodiversity indices such as Shannon’s diversity, Simpson and richness using spatial predictor variables.

These variables are topography, geology, soil, climate, normalized difference vegetation index (NDVI), and land cover. They offered three models for Shannon’s diversity, Simpson, and richness indices. Mohammadi and Shataee (2010) investigated the possibility of estimation of tree diversity using Landsat ETM+ data in the Hyrcanian forests, North of Iran. The models for tree species richness and the reciprocal of the Simpson index were obtained with reasonable accuracy. Bawa et al. (2002) reported that there is a statistically significant relation between the species diversity and NDVI of IRS 1C imagery and NDVI may be used to characterize areas of high and low tree species richness in tropical forests where biodiversity losses is significant. The regression analysis approach has broadly been applied in ecological surveys (Lehmann et al. 2002). Linear regression is a commonly used statistical technique for modeling biodiversity because of its easy use and direct interpretability (Curt et al. 2001; Seynave et al. 2005). The development of advanced nonparametric and machine learning techniques are opening up plenty of opportunities for modeling biodiversity with greater accuracy and may be better fitted to address the mentioned problems compared with linear regression (Aertsen et al. 2010). Generalized linear models (McCullagh and Nelder 1989) and generalized additive models (Hastie and Tibshirani 1990) using presence-absence survey data have been taken much more attention recently. Moisen and Frescino (2002) investigated the performance of nonparametric techniques as CART, generalized additive models (GAM) and artificial neural networks (ANN)

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compared to parametric techniques for the prediction of several species independent forest characteristics in the interior Western United States. MARS and ANN worked best to simulated data, but less suitable for real data, in which case a LM approach often provided comparable results. Shataee et al. (2012) compared three nonparametric models include k-nearest neighbor (k-NN), support vector machine regression (SVR) and tree regression based on random forest (RF), for estimation forest structure characteristic using ASTER satellite data. Overall, they showed RF produced has better results than SVR and k-NN. The aim of this study was to compare and evaluate two statistical non-parametric (CART, RF) for modeling tree species diversity. It is also intended to investigate the relationship between the properties of satellite image spectral bands and tree species diversity; in order to predict the distribution of plant species diversity using new nonparametric methods over the study area.

MATERIALS AND METHODS Study area The study area is located in the Hyrcanian forests, the district 1 of Darabkola’s forests, Sari, North of Iran (Figure 1). The boundary of this area is located at 36° 28´-36° 33´ N and 53° 16´-53° 20´ 30´´ W. The Darabkola’s forestry plan, with about 2600 ha area, consists of natural temperate and uneven aged stands. The main tree species are Quercus Castaneifolia (chestnut-leaved oak), Carpinus betulus (hornbeam), Acer velutinum (velvet maple), Alnus subcordata (Caucasian alder), Tilia begonifolia (linden tree), Parrotia persica (Persian ironwood), Ulmus glabra (elm), Acer platanoides (Norway maple), Diospyros lotus (date pulm), Zelkova carpinifolia (Siberian elm), Fagus orientalis (Oriental beech) and Acer cappadocicum (coliseum maple). Field data Species richness and diversity indices are dependent on the size of the sample plot. Phytosociological data were collected based on a systematic sampling method during 5th June to 15th July 2010. The size and the number of quadrates were determined using the species area curve (Misra 1968). Choosing the sample size, the number of sampling units to select and measure, is a key part of planning a survey. 100 sample plots (quadrate shape) were placed using a stratified random sampling design 450 × 500 m. The sample plot size was 60 × 60 m and characteristics of trees with DBH more than 7.5 cm were measured. The geographical center of each plot was registered using a GPS Oregon 550. Diversity indices A large number of diversity indices can be used to characterize tree size diversity within a stand (Smith et al. 1992; Varga et al. 2005; Ozdemir et al. 2008). Two common approaches for measuring alpha diversity are species richness and evenness/ heterogeneity (Ojo and OlaAdams 1996). Species richness simply refers to the number

of species in the community while evenness/ heterogeneity refer to the distribution of individuals among the species. In this study, species richness wasn't considered. For the measurement of evenness/ heterogeneity, Simpson, Shannon diversity indices and the reciprocal of the Simpson index were computed for each of the sites. The more uncertainty one has about the species of an individual, the higher the diversity of the community. The proportion of a species has been based on a variety of variables to represent frequency, including the number of individuals (Niese and Strong 1992; Condit et al. 1996), basal area (Harrington and Edwards 1995; LeMay et al. 1997), stems per ha (McMinn 1992; Harrington and Edwards 1995); and biomass (Swindel et al. 1984). In this study, the proportion of basal area species is used in this index. Satellite data The SPOT-HRG data were orthorectified using 23 GCPs and DEM. The total root mean square errors (RMSE) were obtained about 0.67 for visible and near infra bands and 0.5 for the middle infra band. Pixel size of middle band was resized to 10m using nearest-neighbor resampling method. The geometric precision of the images was also verified using road vector layer and unused collected GPS control points and proved the accuracy of geometric rectification. In order to atmospheric correction, the COST general method was used for decreasing of effect of attenuation and scattering in the visible and near-infrared bands. The DNs of images were converted to radiance and then to reflectance values. The reflectance of the haze number was determined through the histogram evaluation. Image processing techniques After geometric rectification and atmospheric corrections, the most used vegetation indices were generated for probabilistic capabilities of these indices in regression modeling (Table 1). Also used of mean and variance each four bands and principal component analyses for all bands and three bands. Spectral signature extraction of the plot The pixel sizes of all used images were aggregated to 60 meters according to size of field plots (60×60) and their spectral values were averaged. Then the averaged values of main and processed images of SPOT-HRG were extracted in place of each plot. Statistical models Classification and regression tree Classification and regression tree, a statistical procedure introduced by Breiman et al. (1984), is primarily used as a classification tool, where the objective is to classify an object into two or more populations (Lee et al. 2006). Regression trees, while effective at incorporating disparate data types, non-normal distributions and non-linear relationships, do not allow for tree optimization, and accuracy may suffer in the presence of outliers and nonbalanced datasets (Lawrence et al. 2004; Barrett et al. 2010). Regression trees are hierarchical structures, where the internal nodes contain tests on the input attributes. Each

KALBI et al. – Predictive tree diversity distribution based on satellite data

Figure 1. Location of the study area in the Mazandaran Province (a) and allocation of sample plots (b) in the study area.

Table 1. Most importance Spectral vegetation indices examined in this study. Index

Equation

Reference

Normalized Ratio (NR) Simple Ratio (SR)

Red-NIR

Mohammadi et al. (2010 Birth and McVey (1968)

Difference Vegetation Index (DVI) Modified Soil Adjusted Vegetation Index (MSAVI2)

NIR-Red

Tucker (1979) Qi et al. (1994)

Normalized difference vegetation index(NDVI) Short wave infrared to visible ratio (SVR) Moisture stress index (MSI) Reduced Simple Ratio (RSR) Renormalized Difference Vegetation Index (RDVI) Normalized difference water index (NDWI)

NIR + 0.5 (NIR-RED) /(NIR+RED) SWIR/[(RED+GRN)/2] SWIR/NIR

Rouse et al. (1973) Wolter et al. (2008) Rock et al. (1986) Brown et al. (2000) Roujean and Breon (1995)

NIR-SWIR/NIR+SWIR

Global environmental monitoring index (GEMI)

Gao (1996) Pinty and Verstraete (1992)

η(1-0.25η) η= Note: SWIRmin and SWIRmax are the minimum and maximum reflectance values observed in the corresponding pixels in field plots.

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branch of an internal test corresponds to an outcome of the test, and the prediction for the value of the target attribute is stored in a leaf. Each leaf of a regression tree contains a constant value as a prediction for the target variable (Kocev et al. 2009). The resulting prediction of the tree is taken from the leaf at the end of the path. Random forest Random forest is a novel ensemble classifier; it uses a similar but improved method of bootstrap as bagging (Zhang et al. 2009). It uses the strategy of a random selection of a subset of predictors to grow each tree, where each tree is grown on a bootstrap sample of the training set. This number, m, is used to split the nodes and is much smaller than the total number of variables available for analysis (Breiman 2001). In training, the random forest algorithm creates multiple CART-like trees (Breiman et al. 1984), each trained on a bootstrapped sample of the original training data, and searches only across a randomly selected subset of the input variables to determine a split (for each node). Random forests for regression are formed by growing trees depending on a random vector such that the tree predictor takes on numerical values. However, when constructing a tree, random forest searches for only a random subset of the input features (bands) at each splitting node and the tree is allowed to grow fully without pruning (Chan and Paelinckx 2008). The random forests predictor is formed by taking the average over a number of the trees specified by the user (Lariviere and van den Poel 2005). The number of predictors used to find the best split at each node is a randomly chosen subset of the total number of predictors (Prasad et al. 2006). One of the main parameters which should be determined in RF, is a k predictor (independent variables) in each node for predicting dependent values (response). The response of each tree depends on a set of predictor values, which is independently chosen with replacement and with the same distribution of all trees in the forest, which is a subset of the predictor values of the original data set. The simplest choosing way k is calculation of root square of total independent variables (k ≤ √m, m is the number of input variables). Model evaluation and performance assessment Data were randomly split into two data sets, 70% of the data for modeling and 30% for testing. For each model that was tested, four statistics are reported; these are the squared coefficient of determination (R2) (Pearson, 1896) and adjusted coefficient of determination (adjusted R2). The validity of performances was examined using regression diagnostics metrics, i.e. root mean square error (RMSE), relative RMSE, bias and relative bias, and using the independent and unused 30 samples. In addition to, some common graphical diagnostic tools (McRoberts 2009) were used to illustrate the quality of performances.

 est  obs  m

RMSe 

i1

  est

 obsi 

i1

RMSe% 

  obs  / m m

i1

Bias  

m i1

m *100

 est  obs  i

 est  obs  m

i1

Bias% 

m *100

 obs  / m m

i1

Where est is estimated values from implementation of algorithms in m validation samples, obs is observation values and m is the number of validation samples.

RESULTS AND DISCUSSION Descriptive statistics of indices Simpson, Shannon’s diversity indices and the reciprocal of Simpson index descriptive statistics for the proportion of basal area species is provided in Table 2. The value of Simpson, Shannon’s indices, and the reciprocal of the Simpson index ranged from 0.105 to 0.86, 0.12 to 2.89 and 1.105 to 4.02, respectively. It indicates a wide range of tree species diversity in the study area (Table 2). All models were critically investigated for confounding factors and checked for all basic assumptions (Table 3). The number of predictor variables entering the models is ranging from three to five, while the predictor variables selected by each technique are not identical. The measures of performance are summarized for each model in Table 4. The best model performance was realized with highest R2, adjusted R2 and lowest RMSE, RMSEr Bias and Biasr values. In the total cases, the best goodness-of-fit, i.e. lowest values for RMSE and Bias and the highest adjusted R2, was obtained from the RF models. Discussion Hyrcanian forests comprise a diverse vegetation cover in the north of Iran and are increasingly degraded and converted to other land uses (Mohammadi et al. 2008). In this study, assessing utility SPOT5-HRG satellite images data and two different regression techniques for modeling tree diversity in Hyrcanian forest. These results are similar to those obtained in other studies (Foody and Cutler 2007; Hernandez-Stefanoni and Dupuy 2007; Mohammadi and Shataee 2010) where researchers demonstrated that satellite data can identify broad patterns of tree species diversity. In this study, the infrared index was determined to be very important to estimate the species diversity of trees and this wavelength was used due to the high reflection in the

KALBI et al. – Predictive tree diversity distribution based on satellite data

infrared spectrum (Bawa et al. 2002). Correlation coefficients between Table 2. Descriptive statistics of model and validation samples for indices species diversity and range of values in different bands corresponding Training samples Evaluation samples positive and reflects the increasing Index N Mean Min Max S.D N Mean Min Max S.D range of different wavelengths, tree Simpson 70 0.474 0.105 0.86 0.19 30 0.514 0.122 0.752 0.148 and shrub diversity also increased. Shannon’s 70 1.22 0.12 2.66 0.547 30 1.38 0.154 2.89 0.519 The dense masses, in which there is diversity more species diversity, reflect a large Reciprocal 70 2.07 1.10 4.02 0.708 30 2.27 1.22 4.02 0.735 amount of the infrared spectral range, but in sparse masses, where the Table 3. Overview of the predictor variables selected by the tree biodiversity models species diversity is low, reflected developed with two techniques. infrared is decreased because the red wavelength enters into the forest and Modelling spreads, which influences its Index Variable(s) selected by the model technique absorption and ultimately reduces its Simpson CART SVR, MSI, NR, Mean NIR reflection. RF Mean SWIR, Variance NIR, Mean Red, NR Increasing the diversity and Shannon’s CART Mean SWIR, MSI, Mean NIR, Variance Green density of the canopy tree increases diversity RF RDVI, Mean SWIR, pci3, Mean NIR, Variance NIR Reciprocal CART PCI4, MSI, Mean NIR, SVR, Variance swir the rate of reflection in this range. RF SVR, Mean swir, Mean NIR With adjusted R2 values for the best models ranging from 42.8 to 64.5, the results look satisfactory Table 4. Performance indices of all SI-models for the three tree species and two compared to other studies modelling techniques. Best model performance for every evaluation measure, is (Mohammadi and Shataee 2010; highlighted in bold. Gillespie et al. 2009; Dogan and Dogan 2006). In recent years the RF Modelling Index R2 R2adj RMSE RMSEr Bias Biasr algorithm has gained popularity as an technique effective regression method in the Simpson CART 43.6 42.8 0.188 36.9 0.038 7.4 remote sensing domain (Shataee et RF 56.8 56.2 0.15 30.1 0.038 7.4 Shannon-Wiener CART 53.7 53 0.68 53.7 -0.23 -18.6 al. 2012). The results of the present RF 61.2 60.6 0.56 43.9 -0.19 -14.9 study confirm that the RF algorithm Reciprocal CART 63.8 63.3 0.88 41.6 -0.15 -7.4 is a robust and accurate method for RF 64.5 63.9 0.84 40.06 -0.22 -10.5 the modeling satellite data. The robustness of the RF algorithm can be explained by the ability of the modeling and classification algorithm to exploit the noise in the dataset to create a more diverse classifier (Breiman 2001). In all REFERENCES cases, CART model has shown a poor result for modeling biodiversity. The results of this study have been consistent W, Kint V, Van Orshoven J, Ozkan K, Muys B. 2010. with some of previous studies (Moisen and Frescino 2002; Aertsen Comparison and ranking of different modeling techniques for Aertsen et al. 2010) that reported that CART models prediction of site index in Mediterranean mountain forests. Ecol performed worst than nonparametric regressions. This may Model 221: 1119-1130. be owed to the fact that CART models produce a stepwise Barrett K, Kasischke ES, McGuire AD, Turetsky MR, Kane ES. 2010. Modeling fire severity in black spruce stands in the Alaskan boreal response function. In case of a rather smooth relationship forest using spectral and non-spectral geospatial data. Remote Sens between predictors and response, this can lead to low Environ 114: 1494-1503. performance. Bawa K, Rose J, Ganeshaiah KN, Barve N, Kiran MC, Umashaanker R.

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N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060111

Vol. 6, No. 1, pp. 63-68 May 2014

Review: Potential production of carotenoids from Neurospora SRI PRIATNI Research Center for Chemistry, Indonesian Institute of Sciences. Jl. Cisitu, Bandung 40135, West Java, Indonesia. Tel./Fax. +62-22-2503051/222503240 ♥email: sripriatni@yahoo.com Manuscript received: 6 February 2014. Revision accepted: 22 April 2014.

Abstract. Priatni S. 2014. Review: Potential production of carotenoids from Neurospora. Nusantara Bioscience 6: 63-68. Carotenoids are abundant and widely distributed in plants, animals and microorganisms. Commercial use of carotenoids competes between microorganisms and synthetic manufacture. Carotenoids production can be increased by improving the efficiency of carotenoid synthesis in microbes. Some of the cultural and environmental stimulants are positively affecting the carotenoid content of carotenogenic strains such as Neurospora. Neurospora is a fungus that exhibits the formation of spores and conidia, the part of the cell for carotenoids biosynthesis. The Indonesian traditional fermented food, red peanut cake or oncom, especially in West Java, is produced from legume residues of Neurospora sp. This fungus has been isolated and identified as Neurospora intermedia. In order to apply this pigment for food and cosmetic colorants, encapsulation techniques of carotenoids have been developed to improve its solubility and stability. Key words: Biosynthesis, encapsulation, Neurospora, pigments

INTRODUCTION Carotenoids can not only be found as plant pigments in nature, but also in animal and microorganisms. Plants are able to synthesize carotenoids, by the presence of a little amount of biosynthetic precursors, together with derivatives of the main components in plants (Rodriguez and Kimura 2004). The roles of carotenoids in plants is to protect the photosynthetic system from excessive light, so it seems can to balance the absorption of sufficient light in this process (Cazzonelli 2011). The color of carotenoids plants are yellow, orange and red, which can be found in fruit, flowers, roots and seeds. However, carotenoids are not a sole producer of those colors, anthocyanins and quinine are also involved in it. Carotenoids content in animals are not from biosynthesize process, but usually from the accumulation of what they were eating. The color of egg yolk is due to carotenoids from the poultry feed that used intensively (Britton and Khachik 2009). Carotenogenic species of fungi are categorized in some classes and their carotenoid synthesis can be easily found in nature. Carotenoids biosynthesis in several species depend on light. This indicates that carotenoids function in these organisms is for light protecting. Generally, carotenoids in fungi are the β-carotene, γ-carotene, torulene and their hydroxy and keto derivatives (Sandmann et al. 2008). Production of carotenoids from microorganisms commercially competes with synthetic manufactured by a chemical process. The efficiency of carotenoids biosynthesis could improve by microbial stimulation. Carotenoids biosynthesis are influenced by culture conditions, the level and activity of carotenoid biosynthetic enzymes and total carbon flux through synthesizing system (Bhosale 2004). In order to achieve simple and low cost of

carotenoid production, Neurospora sp. is a potential microbial sources due to its carotenoids content and easy to cultivate on waste solid substrates. This review discussed the structural properties and biosynthesis of carotenoids. The information is used to evaluate the potential of carotenoids production from Neurospora sp. as foods and cosmetics colorant.

CAROTENOIDS STRUCTURE AND ITS PROPERTIES Carotenoids are a member of isoprenoid compounds which was synthesized by tail-to-tail linkage of two C20 geranyl-geranyl diphosphate molecules (Britton 1995). Carotenoids are generally C40 tetraterpenoids that governed from eight C5 isoprenoid units. The conjugated doublebond system in carotenoids structure is the chromophore for light-absorbing that gives these compounds an attractive color. In the metabolic system, the precursor phytoene converts into cyclic carotenes by a series of desaturation and cyclization reaction. During the initiation process of conjugated double bonds, the chromophore will lengthen (Armstrong and Hearst 1996). The carotenoid structures can be modified by several processes such as of cyclization, hydrogenation, dehydrogenation, double bond migration, chain shortening or extension, rearrangement, isomerization, introduction of oxygen function, and combination of these processes (Rodriguez and Kimura 2004). The long system of alternating double bonds and single bonds in carotenoid structure can form the central part of the molecule. The π-electrons which constitutes in a conjugated system are effectively delocalized over the

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entire length of polyene chain. Basically, the double bond in carotenoids can exist in two configurations, that is trans and cis, it depends on the position of substituent groups which linkage in the central part (Britton 1995). The unsaturated carotenoid tends to isomerization and oxidation process. Isomerization of trans carotenoids to cis configuration is promoted by heat, acids, and exposure to light (Rodriguez and Kimura 2004). The important property of carotenoids that will influence to HPLC analysis is the long conjugated polyene system that makes the all-trans isomers rigid, linear molecules. Practically, this property gives carotenoids the ability to absorb the light in visible region at 400-550 nm. According to this character, HPLC which is combined with photodiode array detector (PDAD) is suitable for identification of carotenoids. λmax of each carotenoid component and the shape of spectrum can be monitored simultaneously in HPLC chromatograms (Khachik 2009). These properties are the characteristic of the carotenoids chromophore. Generally, carotenoids absorb at three wavelengths, resulting in three-peak spectra. The number of carotenoids structure is correlated with λmax values. The greater number conjugated double bonds, the higher λmax values (Rodriguez and Kimura 2004). Carotenoids are sensitive to light, oxygen, heat, and acid degradation, due to the presence of many conjugated double bonds in the structure. Carotenoids are hydrophobic molecules with little or no solubility in water which was normally found in hydrophobic areas of cell, such as the inner core of membranes. They can access an aqueous environment when they are associated with protein or with other polar compounds. The polarity of carotenoids can be altered by interaction with a polar functional group and other molecules (Britton 1995). Carotenoids have been attributed to an antioxidant property due to their ability to quench single oxygen and interact with free radicals (Rodriguez and Kimura 2004). Many studies of carotenoids concerning the relative antioxidant activity, they concluded that there are three types of carotenoids reaction with radical species, i.e. radical addition, electron transfer to the radical and allylic hydrogen abstraction (Yeum et al. 2009). Some studies suggest that radical forming can be influenced by the structure and level of carotenoids, and also the level of oxygen and the polarity of solvent. Carotenoids radical cation mediates the isomerization and modifications of carotenoids structure. A study showed that the oxidation of canthaxanthin and 8′-apo-betacaroten-8′-al resulting the formation of radical cation followed by formation of cisisomers (Boon et al. 2010).

CAROTENOIDS PRODUCTION BY MICROORGANISMS Commercial production of carotenoids by nonphotosynthetic microorganisms is becoming an attractive prospect in the future. Production of ß-carotene from the fungus Blakeslea trispora was developed in Europe through fermentation process. At present, this product has

been used in food industry for several purposes. Carotenoids was produced by fermentation process is competing with carotenoids from synthetic chemicals process or with extraction of some natural sources (Dufossé 2009). Under certain growth conditions such as limitation of nitrogen or high salt concentration and exposed to high light intensity, the green algae Dunaliella salina can accumulate high amounts of carotenoids (up to 13% w/w). This alga accumulates β-carotene, astaxanthin, zeaxanthin, lutein and cryptoxanthin (El-Baky et al. 2007). Microalgae such as of Arthrospira, Chlorella, Dunaliella, Spirulina and Aphanizomenon have been used as functional foods due to carotenoids content. Commercially, the application of these microalgae has been increased that can be found in the form of pills, tablets and capsules. Carotenoids primarily used for dietary supplements, fortified foods, food color, animal feed and pharmaceuticals and cosmetics (Vílchez et al. 2011). By using carotenogenic microbe stimulants at certain external conditions of the cultures, hyper-production of carotenoids can be achieved with an effective cost. Several stimulators have been studied to increase of β-carotene production in Blakeslea trispora and Phycomyces blakesleeanus, which was grown under normal fermentation conditions. Irradiation of algae, fungi, and bacteria by white-light positively affect the accumulation of carotenoids. However, the intensity and protocol of illumination may depend on the microorganism properties (Bhosale 2004). Stimulation by light, temperature, chemical compound, metal ion and salt, and solvent to carotenogenesis process of Rhodotorula strains has been studied. Moreover, optimization of medium component and improvement of Rhodotorula strains are efforts to increase the carotenoids production (Heriyanto and Limantara 2009). El-Banna et al. (2012) reported that the isolation and identification of carotenoid-producing strains of Rhodotorula glutinis isolated from pin cushion flower. This strain produced 7 g/L dry biomass, 266 μg/g cellular carotenoids, 1.6 μg/L volumetric carotenoids, and 12.4% lipids, after fermented in yeast malt broth with shaking (100 rpm) at 30°C for 4 days. Carotenoid production by microorganisms can be increased efficiently by two strategies, i.e. by enhancement of biomass production and biosynthesis carotenoids. Biosynthesis of carotenoid in the cell is depending on the activity of enzymes involved in the process. This activity can be altered by using the recombinant DNA technique. Hyper-production of carotenoid in the cell can be reached by the application of recombinant DNA technique (Rodney 1997). For example, gene crtS from Xanthophyllomyces dendrorhous has been cloned simultaneously; which is responsible in the conversion of β-carotene to astaxanthin. Astaxanthin production used two kinds of enzymes i.e. αcarotene hydroxylase and α-ketolase that work on each intermediates (Martín et al. 2008). Loto et al. (2012) reported that carotenoid production was increased by disrupting the C22-sterol desaturase gene (CYP61) in X. dendrorhous. CYP61 gene encoded cytochrom P450 enzyme which is involved in mevalonate pathway. The study suggested that in X. dendrorhous, ergosterol

PRIATNI – Carotenoids from Neurospora

regulates the carotenoid biosynthesis by a negative feedback mechanism. Carotenoids production in Escherichia coli or other bacteria cannot be synthesize naturally. Introducing of carotenogenic genes to E. coli has been studied to achieve the carotenoid biosynthesis de novo. In these bacteria, other isoprenoid compounds such as dolichols and quinines are also synthesized. The important carbon flux for biosynthesis of isoprenoid compounds have been directed by the introduction of the carotenogenic genes. Plasmids containing crt genes have been constructed and expressed in E. coli in purpose to synthesize the lycopene, ß-carotene and zeaxanthin (Dufossé 2009). General terpenoid pathway of carotenoids biosynthesis in E. coli involves the geranylgeranyl diphosphate (GGDP) synthase (crtB) and phytoene synthase (crtE) gene, resulting C40 carotenoid phytoene. Desaturation and modification of phytoene desaturase (crtI) catalyzed by e.g. cyclases, hydroxylases, and ketolases, resulting many kinds of carotenoids. Acyclic and cyclic carotenoids synthesis determined by Phytoene desaturase (crtI) and lycopene cyclase (crtY) which is located at important branchpoints of the carotenoid biosynthetic pathway (Dannert et al. 2000).

CAROTENOID BIOSYNTHESIS BY Neurospora sp. Neurospora, the ascomycetes fungi is grown in tropical or subtropical countries. Neurospora species are grown and sporulates quickly on the surface of fire scorched vegetation. Five species of Neurospora have been identified in Europe are N. crassa, N. discreta, N. intermedia, N. sitophila and N. tetrasperma (Jacobson et al. 2006). Neurospora crassa has been used as a model organism to study the responses of light in eukaryotic cells. Several processes such as induction of carotenoid production, protoperithecial formation, phototropism of perithecial beaks, perithecial polarity, and circadian rhythm are controlled by blue light (Belozerskaya et al. 2012). The characteristics of these strains have been identified to have four heterothallic species with eight-spore asci and pseudo homothallic species with four spores asci. Distribution of each species has a specific pattern; however they have similarity with each other. They are similar in morphology, its conidia color are orange or yellow-orange (Perkins and Turner 1988). Neurospora produced huge number of macro conidia and are easy to recognize in nature. This fungus is grown in various substrates such as bread, burned woods, corn cobs and waste of sugarcane industries (Pandit and Maheshwari 1996). Neurospora population has been studied by collecting the strains from many regions in the world. Population of the genus was found everywhere, but the species from each region are different. Some species have similarity on the basis of vegetative morphology. The similarity of N. crassa and N. intermedia was identified when crossing with each other. The perithecia of these species have normal appearance, but N. intermedia has larger conidia than N. crassa. N. intermedia can grow on non-burned substrates (Turner et al. 2001). Accumulation of carotenoids in

Neurospora sp. show has a correlation with latitude of sampling regions. Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains which is isolated from higher latitudes. This is happened because low latitude regions receive more UV radiation than high latitude regions (Luque et al. 2012). Neurospora species has been studied by biological species recognition (BSR) and phylogenetic species recognition (PSR) methods. By BSR method, Neurospora has been well characterized and used as model organism because its sexual cycle can manipulated easily (Dettman et al. 2003). Genetic and molecular analyses were carried out to N. intermedia strain from Maddur in Southern India (Souza 2005). Molecular identification of Neurospora N-1 which isolated from Indonesian red fermented cake or oncom, was carried out based on the genetic analysis partially on ribosomal DNA included in the sub-unit of 28S rDNA (D1/D2 region) and internal transcribed spacer (ITS). The isolate was 100% homologeneity by amplification on D1/D2 region and 99% by amplification on ITS region to N. intermedia. Based on phylogenetic analysis using D1/D2 region and ITS methods, Neurospora intermedia N-1 has a close relation to N. crassa (Priatni et al. 2010). A biological phenomenon of N. crassa has been investigated to study the biosynthesis of carotenoids pathway and its regulation. Identification of the albino strains al-1 to al-3 and characterization of the responsible genes was carried out to investigate the carotenoid biosynthetic pathway in N. crassa. Condensation of two GGPP molecules produced colorless carotene-phytoene which is catalyzed by al-2 enzyme. Introduction of up to five conjugated double bonds into phytoene mediated by al-1, resulting 3,4-didehydrolycopene via phytofluene, ζcarotene, neurosporene and lycopene. These reactions were identified by using a PDA detector that monitored the changes of λmax. The data showed that the absorption shifts toward longer wavelengths, and visually the color of desaturation products changes from yellow to red colors (Sandmann et al. 2008; Estrada et al. 2008). Al-2 is bifunctional gene which contain cyclase and phytoene synthase, as found for homologous genes from X. dendrorhous, Phycomyces blakesleeanus and Mucor circinelloides (Arrach et al. 2002). The activation of two carotenogenic genes (al-1 and al-2) by light did not correlate with accumulation of carotenoids in Neurospora sp. (Olmedo et al. 2013). Accumulation of neurosporaxanthin and some carotenoid precursors give characteristic orange of conidia and mycelia in N. crassa. Ca2+ signaling pathway indicates could be involved in regulation of carotenoid production in this fungus (Deka and Tamuli 2013). Carotenoids biosynthesis pathway in Neurospora was presented in Figure 1. Identification of carotenoids in N. intermedia N-1 isolated from oncom has been carried out by using the HPLC equipped with a photodiode array (PDA) detector. Analysis of pigment extract shown that at least five carotenoid compounds were identified in spores of N. intermedia N-1 i.e. lycopene, neurosporen, γ-carotene, βcarotene and phytoene. This study suggested that

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carried also to produce the alternative poultry feed with high content of βcarotene (Nuraini et al. 2009). In order to achieve optimum physical and chemical stability, and bioavailability of carotenoids, innovative processes for their production with modern methods of encapsulation technology have been developed and investigated (Ribeiro et al. 2010). Encapsulation of carotenoids has been studied by some researcher to improve its stability and solubility. Nanoparticle formation and encapsulation of β-carotene using copolymer casein-g-dextran was studied by hydrophobic interaction. The particle core was forming by interaction between βcarotene and hydrophobic segments of casein and the hydrophilic dextran shell makes the nanoparticle stable Figure 1. Biosynthesis of carotenoids in Neurospora sp. (Arrach N et al. 2002). and dispersible in pH range 2-12 (Pan et al. 2007). The common techniques such as spray drying and inclusion complexation have been biosynthesis pathway in N. intermedia is similar with the studied for carotenoids encapsulation. Lycopene powder biosynthesis in N. crassa (Priatni et al. 2010). was obtained by encapsulation through spray-drying technique with β-cyclodextrin (β-CD). The efficiency of encapsulation was between 94 to 96% and the average EY CAROTENOIDS PRODUCTION FOR FOOD AND (encapsulation yields) was 51 ±1%. These complexes COSMETICS COLORANTS (lycopene-β-CD) were formed at a molar ratio of 1:4 (Nunes and Mercadante 2007). Carotenoids show several beneficial functions to human life such as anti-carcinogen, antioxidant, anti-inflammatory and chemo preventive agent for some cancer diseases. This pigment has been used also for food, beverages and cosmetic colorants. Nowadays, food and cosmetic industries were more interested to substitute synthetic colorants by natural pigments (Britton 1995; El-Baky et al. 2007). Most of foods and beverages which are marketed in Indonesia are colored with synthetic colorants which is not food grade and of course dangerous to health. Natural colorants will be a good alternative for synthetic colorants as long as its quality and safety are guaranteed. The potential carotenoid source in Indonesia has been explored to the palm oil which contains high amount of β-carotene (400-700 ppm). The technology separation of β-carotene in palm oil including extraction, adsorption and trans- Figure 2. Fermented product of N. intermedia N-1 on solid waste esterification process, should be applicable and low tofu (Priatni et al. 2008). processing cost (Darnoko 2008). Neurospora intermedia N-1 isolated from oncom has been studied for carotenoids The encapsulation process of lycopene in oil has been production as an alternative for food and beverages carried out using coacervation technique. Complex colorant. This study recommended that the solid waste coacervation is a spontaneous separation between two or from tofu production is the best substrate fermentation due more polymers forming an insoluble complex through the to high yield of spores production and concentration of the electrostatic interactions. On this study, the retention of total carotenoids (Priatni et al. 2008). The fermented lycopene was higher in the microcapsules compared to the product of N. intermedia N-1 on solid waste tofu free material. The potential of microencapsulation indicates production was shown on Figure 2. The fermentation of N. has greater protection to carotenoid degradation (Rochacrassa on tapioca by product and waste tofu has been Selmi et al. 2013). The other method for encapsulating this

PRIATNI – Carotenoids from Neurospora

pigment is by using supercritical technology. Some methods of supercritical technology have been developed. One interesting method is supercritical anti-solvent (SAS) method. The principle of this method is based on a rapid decrease of a solvent solubility (first solvent) by adding the second solvent as anti-solvent. The supercritical fluid or anti-solvent will saturate the first solvent through mixing and extraction process (Diego et al. 2010). Microencapsulation of carotene extracts from Neurospora sp. Spores has been carried out by using protein base as shell material. Encapsulation carotenoid extract with sodium caseinate as shell material gives the highest microencapsulation efficiency, total carotenes, and carotenes retention values, compared to soy protein isolate and milk protein isolate (Pahlevi 2008). Another encapsulation process of carotenoids is from N. intermedia N-1 by using copolymer of gelatin and maltodextrin. In this study, the average EY of carotenoids powder obtained by using spray dryer was 48%. The stability of carotenoids powder can be maintained at low humidity and dark storage. Encapsulated carotenoids from N. intermedia N-1 were stable when they are stored in brown glass at RH between 20-30% (Gusdinar et al. 2011).

CONCLUSIONS Production of carotenoids from microorganisms is an alternative for chemical process. Production of carotenoids by microorganisms will become an attractive prospect in the future. Biosynthesis of this pigment is influenced by culture condition, level and activity of carotenoid biosynthetic enzymes. Stimulation at certain external conditions to the growth of carotenogenic microbes can enhance carotenoids production. Some stimulants such as light, temperature, chemical compound, metal ion and salt, and solvent, have been used for hyper-production of carotenoids in microbes. Moreover, optimization of medium component and improvement of strains can be away to commercialize carotenoids production. Neurospora, the ascomycetes fungi are easy to grow in tropical country such as Indonesia. The carotenoids biosynthesis and its regulation of this fungus have been investigated. Neurospora intermedia which are isolated from red oncom have been studied for carotenoids production. Fermentation of N. intermedia on waste solid tofu production can produce high yield of carotenoids extract. In N. intermedia spores, at least five carotenoid compounds were identified i.e. Lycopene, neurosporen, γcarotene, β-carotene and phytoene. However, these compounds are hydrophobic molecules with little or no soluble in water, they can access an aqueous environment when they are associated with protein or with other polar compounds. Encapsulation of carotenoids from Neurospora by using suitable copolymer can increase its solubility and stability. According to some studies, the application of carotenoids from Neurospora sp. for food and cosmetic colorants is very potential in the future.

ACKNOWLEDGEMENTS The author thanks to Prof. Dr. Tutus Gusdinar, Prof. Dr. LBS Kardono, Dr. Marlia Singgih, and Dr. Tiny Agustini Koesmawati for their kind suggestions regarding the manuscript.

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Khachik F. 2009. Analysis of carotenoids in nutritional studies. In: Britton G, Liaaen-Jensen S, Pfander H, Carotenoids, Volume 5: Nutrition and Health. Springer, New York. Loto I, Gutiérrez MS, Barahona S, Sepúlveda D, Martínez-Moya P, Baeza M, Cifuentes V, Alcaíno J. 2012. Enhancement of carotenoid production by disrupting the C22-sterol desaturase gene (CYP61) in Xanthophyllomyces dendrorhous.BMC Microbiol 12: 235 Luque EM, Gutierrez G, Navarro-Sampedroa L, Olmedob M, Rodrıguez RJ, Ruger-Herreros C, Tagua VG, Corrochano LM. 2012. A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain. PLoS ONE 7 (3): e33658| www.plosone.org Martín JF, Gudiña E, Barredo JL. 2008. Conversion of β-carotene into astaxanthin: Two separate enzymes or a bifunctional hydroxylaseketolase protein? Microbial Cell Factories 7: 3 Nunes IL, Mercadante AZ. 2007. Encapsulation of lycopene using spraydrying and molecular inclusion processes. Intl J Braz Arch Biol Technol 50 (5): 893-900 Nuraini, Sabrina, Latif SA. 2009. Improving the quality of tapioca by product through fermentation by Neurospora crassa to produce βcarotene rich feed. Pakistan J Nutr 8 (4): 487-490. Olmedo M, Ruger-Herrerosb C, Luqueb EM, Corrochanob LM. 2013. Regulation of transcription by light in Neurospora crassa. A model for fungal photobiology? Fungal Biol Rev 27 Pahlevi YW, Estiasih T, Saparianti E. 2008. Microencapsulation of carotene extracts from Neurospora sp. spores with protein based encapsulation using spray drying method. J Teknologi Pertanian 9 (1): 31-39 Pan X, Yao P, Jiang M. 2007. Simultaneous nanoparticle formation and encapsulation driven by hydrophobic interaction of casein-graftdextran and β-carotene. J Colloid Interface Sci 315: 456-463 Pandit A, Maheshwari R. 1996. Life-history of Neurospora intermedia in a sugar cane field. J Biosci 21 (1): 57-79 Perkins DD, Turner BC. 1988. Neurospora from natural populations: Toward the population biology of a haploid eukaryote. Exp Mycol 12: 91-131. Priatni S, Limantara L, Heriyanto, Singgih M, Gusdinar T. 2010. Identification of carotenoids in Neurospora intermedia N-1 isolated

from Indonesian fermented peanut cake (oncom merah). Proceedings of NP-SEA. Malang. 20-21 March 2010. Priatni S, Singgih M, Kardono LBS, Gusdinar T. 2008. Carotenoids production by Neurospora sp. by utilization of the solid waste production. Proceedings of Sciences and Technology of Natural Pigment. Salatiga, 5 September 2008. Priatni S, Singgih M, Kardono LBS, Gusdinar T. 2010. Molecular identification of Neurospora sp N-1 isolated from Indonesian red fermented cake. J pure Appl Microbiol 4 (2): 527-531 Ribeiro HS, Schuchmann HP, Engel R, Walz E, Briviba K. 2010. Encapsulation of carotenoids. In: Zuidam NJ, Nedović VA. (eds.). Encapsulation technologies for active food ingredients and food processing. Springer, New York. Rocha-Selmi GA, Favaro-Trindade CS, Grosso CRF. 2013. Morphology, stability, and application of lycopene microcapsules produced by complex coacervation. J Chem, Article ID 982603. Hindawi Publishing Corporation Rodney LA. 1997. Commercial opportunities for carotenoid production by biotechnology. Pure App Chem 69 (10): 2169-2173. Rodriguez A, Kimura M. 2004. Handbook for carotenoid analysis. Harvestplus, Washington, DC Sandmann G, Takaichi S, Fraser PD. 2008. C35-apocarotenoids in the yellow mutant Neurospora crassa YLO35. Phytochemistry 69: 28862890 Souza ADD, Sultana S, Maheshwari R. 2005. Characterization and prevalence of a circular mitochondrial plasmid in senescence-prone isolates of Neurospora intermedia. Curr Genet 47: 182193 Turner BC, Perkins DD, Field AF. 2001. Neurospora from natural populations: a global study. Fungal Genet Biol 32: 67-92 Vílchez C, Forján E, Cuaresma M, Bédmar F, Garbayo I and. Vega JM. 2011. Marine carotenoids: biological functions and commercial applications. Mar Drugs 9: 319-333. Yeum KJ, Aldini G, Russell RM .and Krinsky NI. 2009. Antioxidant/prooxidant actions of carotenoids. In: Britton G, Liaaen-Jensen S, Pfander H (eds). Carotenoids, Volume 5: Nutrition and Health. Springer, New York.

N U S AN T AR A BIO S C IEN C E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060112

Vol. 6, No. 1, pp. 69-81 May 2014

Review: Mangrove hybrid of Rhizophora and its parental species in Indo-Malayan region AHMAD DWI SETYAWAN1,2,â&#x2122;Ľ, YAYA IHYA ULUMUDDIN3, PANDISAMY RAGAVAN4 1

Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University. Jl. Ir. Sutami 36A Surakarta 57 126, Central Java, Indonesia. Tel./Fax. +62-271-663375, ď&#x201A;Šemail: volatileoils@gmail.com. Program of Conservation Biology, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Indonesia, Depok 16424, West Java, Indonesia 3 Research Center for Oceanography, Indonesian Institute of Sciences (PPO-LIPI), East Ancol, North Jakarta 14430, Indonesia. 4 Institute of Forest Genetics and Tree Breeding, R.S.Puram, P.B. No 1061, Coimbatore 641002, Tamil Nadu, India.

Manuscript received: 14 November 2013. Revision accepted: 30 April 2014.

Abstract. Setyawan AD, Ulumuddin YI, Ragavan P. 2014. Mangrove hybrid of Rhizophora and its parental species in Indo-Malayan region. Nusantara Bioscience 6: 69-81. There was two putative hybrid species of mangrove in the Indo-Malayan region, namely Rhizophora x annamalayana Kathir. and R. x lamarckii Montrouz. Rhizophora x annamalayana is most recently known as a natural hybrid between R. apiculata and R. mucronata. At first, this plant is considered as R. x lamarckii, a well-known mangrove hybrid between R. apiculata and R. stylosa. Since R. stylosa is not distributed in India and Sri Lanka, the location where this species was firstly discovered, the name of the new hybrid species between R. apiculata and R. mucronata was corrected. Meanwhile, R. x lamarckii has long been known and is always found in habitat where its parents grew. Besides, the cross breeding of R. mucronata and R. stylosa has never been reported. Both are sibling species that have identical morphological characteristics, thus the hybrid cross-bred species was not observed visually and can only be distinguished through genetic studies. Key words: annamalayana, apiculata, description, Indo-Malaya, Rhizophora

INTRODUCTION There are approximately 70 species of mangrove plants distributed worldwide, divided into 20 genera (Spalding et al. 2010). Almost all mangrove species have been identified, named and classified, that the new species is commonly a hybrid of previously known species. The progress of molecular biology helps to solidify this hybrid species. Hybrids of mangrove species have been found in Rhizophora, Sonneratia, Bruguiera, Lumnitzera and Heritiera (Tomlison 1986; Zhou et al. 2005, 2008; Duke and Ge 2011; Guo et al. 2011; Ng and Chan 2012). Intermarriage also occurs among different mangrove species, but its frequency is relatively limited and generally produces sterile progeny (Chan 1996). Rhizophora L. (Rhizophoraceae R.Br.) is the most successful-growth mangrove, with the widest extent of spread and the easiest to be found (highest abundance), grows in tropical and subtropical coastal areas, characterized by the stilt roots (Duke 2006a; Giesen et al. 2006). Rhizophora growing success is mainly supported by the ability to reproduce with the help of the wind, although it can also breed with the help of insects because the flowers have an odor, color and nectar that can attract insects. Rhizophora apiculata Bl. has specialized wind pollination mechanisms; otherwise R. stylosa Griff has less specialized and need insects for pollination (Tomlinson et al. 1979). Rhizophora is still actively evolving, where sterility barriers between species are easily traversed, thus it can give birth to some putative hybrid species, namely: R. x lamarckii Montrouz in the Malay Archipelago

(Tomlinson and Womersley 1976), R. x annamalayana Kathir. in India and Sri Lanka (Kathiresan 1995, 1999), and R. x selala in Pacific islands (Tomlinson 1978; Duke 2010). Wind-pollination based breeding increases the frequency of putative hybrids (Tomlinson et al. 1979), especially Rhizophora pollen of different species have much in common morphology (Muller and Caratina 1977). Rhizophora x annamalayana is the latest known mangrove species, as a natural hybrid between R. apiculata and R. mucronata Lam (Kathiresan 1995, 1999). Previously, it has been known R. x lamarckii which is a hybrid between R. apiculata and R. stylosa (Tomlison 1986). Meanwhile, hybrids between R. mucronata and R. stylosa were never reported. Both are sibling species with very similar morphological characteristics and genetic profile similar to 96.5% (Parani et al. 1997b). Formerly, R. stylosa is a variant with the name of R. mucronata var. stylosa (Griff.) Salvoza; thus to distinguish the two hybrids are difficult and can only be identified by DNA sequences (Ng et al. 2013). Natural hybridization between species often occurs to maintain the genetic diversity and evolution that the plants can last a long time, although it can also occur as an introgression where progenies become weaker and sterile. Rhizophora x lamarckii and R. x annamalayana have so far been thought to be sterile and it always requires the presence of both parents (Duke and Blunt 1979; Chan 1996; Parani et al. 1997; Lo 2010; Ng and Chan 2012). In the hybrid species R. x annamalayana pollen viability (3%) is much lower than both the parent (100% in R. mucronata, R. apiculata in 53%) (Kavitha and Kathiresan 2011, 2012). It denies the possibility that the parent species of R. stylosa

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has been presented in India and gave birth of hybrid species before changed its distribution and was no longer found in the region (Schwarzbach and Ricklefs 2001). The accuracy of identification was instrumental in the successful restoration of mangrove forests (Ng and Szmidt 2013), where Rhizophora is widely used for this purpose (Setyawan et al. 2004). In Indo-Malaya, Rhizophora consists of three species, i.e. R. mucronata, R. stylosa (two being closely allied or sibling species) and R. apiculata, and two putative hybrids, R. x lamarckii (R. apiculata x R. stylosa) and R. x annamalayana (R. apiculata x R. mucronata) (Duke 2006b). Although it has been widely published, both the name of the hybrid species was categorized as 'unresolved' names that it is not yet possible to assign a status of either 'accepted' or 'synonym' (The Plant List 2010). The five species can be found in Indonesia. Rhizophora apiculata and R. stylosa are the most common species. Rhizophora mucronata is less common, although its global distribution is much wider than the other two species (Hou 1992, Duke 2006b; Duke et al. 2010a, b, c). Rhizophora x lamarckii is only found in locations where both parents present. Rhizophora x annamalayana (Kathiresan 1995; 1999) is recorded only once in Lombok Island, West Nusa Tenggara, and is originally named as R. x lombokensis Baba & Hayashi (Baba 1994) (Figure 1). In the coastal region of western Africa and America, R. mangle L. (syn. R. samoensis (Hochr.) Salvoza), R. racemosa G. Mey., and R. harrisonii Leechm. can be found and one hybrid is known as R. x selala (Salvoza) Toml. Rhizophora harrisonii has characteristics of both R. mangle and R. racemosa that it was initially thought to be the hybridization of the two, but recent molecular study makes it clear that it wasnâ&#x20AC;&#x2122;t (Ceron-Souza et al. 2010). While in the Pacific islands, R. mangle var. samoensis and hybrid R. x selala (R. mangle var. samoensis x R. stylosa) there can also be found. The islands are the overlapping meeting point of Rhizophora of Indo-Malayan and Rhizophora of American (Tomlison 1986; Duke 2006b). Initially, R.

samoensis is known as a separated species, but today the species is regarded as a variant of R. mangle (The Plant List 2010) (Figure 1). The name of R. x annamalayana for a hybrid between R. apiculata and R. mucronata was firstly given by Kathiresan (1995) based on specimens grown in Pichavaram, Tamil Nadu, India. Kathiresan (1999) rewrite the publication, since the first paper was not accompanied by a complete description. The existence of Rhizophora hybrid species in this place have been previously indicated by Lakshmanan and Rajeswari (1983), Muniyandi and Natarajan (1985), Singh et al. (1987) and Subramonian (1993), but they called it R. x lamarckii, which is actually the name for a hybrid of R. apiculata and R. stylosa. The molecular studies support that R. x annamalayana is derived from hybrid of R. apiculata and R. mucronata, and not of R. apiculata and R. stylosa (Parani et al. 1997; Lakshmi et al. 2002). Based on the mitochondrial genome, R. x annamalayana is related closer to R. apiculata (Parani et al. 1997), on the other hand, with microsatellites; it is related closer to R. mucronata (Kumar et al. 2011). Baba (1994) had previously named species from hybrid of R. mucronata and R. stylosa with the name of R. x lombokensis, based on specimens obtained from the Lombok Island, West Nusa Tenggara, Indonesia. However, the name is less developed and is not used in subsequent publications, despite the provisions of the International Code of Plant Nomenclature, a first-made name is prioritized (McNeill et al. 2005). At this time, the name of R. x annamalayana is widely used, but some authors write it as R. x annamalai (Lakshmi et al. 2002: Duke 2006b), because it is more proper to Latin grammar than the initial name. This hybrid species is also found in Merbok, the Malay Peninsula (Ong 2003). The main distribution of R. x annamalayana is the east coast of India (Pichavaram, Tamil Nadu), Sri Lanka and the Andaman and Nicobar Islands (Ragavan et al. 2011, 2014; Dahdouh-Guebas 2012). Meanwhile, the distribution of R. x lamarckii is much broader and its abundance is

Figure 1. Species distribution of Rhizophora in Indo-Malayan region. Note: R. stylosa, R. apiculata, R. mucronata, R. x lamarckii, R. x annamalayana (Duke et al. 2002; Duke 2006b; Giesen et al. 2006; Setyawan and Ulumuddin 2012; Lo et al. 2014).

SETYAWAN et al. â&#x20AC;&#x201C; Rhizophora hybrids and its parentage

much higher than R. x annamalayana. The last species is only found abundantly in locations where R. stylosa is not present, while in locations where the three parents of Rhizophora are present, R. x annamalayana is far below R. x lamarckii. Since, the western distribution of R. stylosa does not reach these regions, except for a little stand in Orissa and Andaman and Nicobar state (Ellison et al. 2012), the hybridization is only occurred between R. apiculata and R. mucronata, but both hybrids are thought to be present in Andaman and Nicobar Islands (ANI), due to the present of their three parents. Rhizophora hybrids are present in the mixed stands of R. apiculata and R. mucronata; in other places, the hybrids are present along with R. apiculata, R. mucronata and R. stylosa. Thus, it may be concluded that both R. x lamarckii and R. x annamalayana might be present in Andaman and Nicobar Islands. These hybrids are easily identified by their height, a large number of flowers with smooth bract and rare occurrence of propagules (Ragavan et al. 2014), and the presence of stamens in two distinct whorls i.e. outer longer stamens and inner smaller stamens (Ragavan et al. 2011). Meanwhile, in the Malay Archipelago, there are plenty of R. stylosa. In many cases, tree density of R. stylosa is higher than R. mucronata, thus more frequent hybridization between R. apiculata and R. stylosa generate R. x lamarckii. Also, the nature of the style of R. stylosa is much longer than the style of R. mucronata that is thought to increase of the success of hybridization between R. apiculata and R. stylosa rather than the hybridization between R. mucronata and R. apiculata (Setyawan and Ulumuddin 2012).

MORPHOLOGY OF RHIZOPHORA HYBRIDS Lo (2003, 2010) suggested that hybridization in Rhizophora is bidirectional and all the known Rhizophora in Indo-Malaya (R. apiculata, R. mucronata and R. stylosa) would play a maternal parent. Thus, it is essential to find the taxonomic distinction between R. stylosa and R. mucronata for better understanding of Rhizophora hybrids. But, the occurrence undefined intermediate individuals cause the uncertainty to distinguish R. stylosa and R. mucronata. The question is whether the intermediates are mixed genotypes between variants of one species or between genetically distinct, sibling species. On the current observation of ANI specimens (PR, 2014, pers. obs.), we have observed the intermediates between R. stylosa and R. mucronata only in place where both coexist. The intermediates are difficult to assign to either taxa but they can be distinguished from R. stylosa and R. mucronata by following ways, i.e. plenty of flowers less fruiting, more than 8 flowers per inflorescences, distinct leafy growth at base of bud and trichotomous inflorescence braches (Figure 2). Rest of the characters, i.e. style length, leaf shape and color resembles either R. mucronata or R. stylosa. Thus, it may conclude that intermediates are variants of one species. At the same time, we have observed some distinct specimens from sites where R. stylosa and R. mucronata coexist (Table 1). Some Rhizophora have highly leathery

leaves with rounded apex, long style and lack of advanced stage of fruits. This specimen resembles to R. mucronata by its broad leaves, and resembles to R. stylosa by its long style (5 mm) and differs from R. stylosa and R. mucronata by its ovate leaves with rounded leaf apex, leathery texture, mostly 2-6 flowered inflorescences, large dimension of mature buds (1.7 cm L, 0.7 cm W)(Figure 3). Based on the above characters this specimen may consider as hybrids between R. stylosa and R. mucronata, but we have observed only one individual each at two sites with consistent morphological characters. Similarly, we have observed Rhizophora which has 2-4 flowered inflorescences with multiple numbers of joints in inflorescences this kind of inflorescences quit different among Rhizophora Other distinguishing characters are elliptic leaves folded laterally, intermediate (2-2.5 mm), large dimension of mature bud and occurrence few mature propagules (Figure 4). In addition, some individuals resemble to R. stylosa with plenty of flowers and lack of fruiting and R. mucronata like with narrow acute leaves, longitudinally fissured bark with plenty fruiting. These observations insist that R. stylosa and R. mucronata are distinct and they can hybridize. Thus, the intermediates may be hybrids between R. stylosa and R. mucronata and hybridization is not restricted to F1 stage. The observation of hybrid between R. stylosa and R. mucronata by Ng et al. (2013) based on molecular analysis also insist the same. Moreover, Lo (2010) also mentioned that R. mucronata and R. stylosa are likely to have recently diverged and ISSR data well discriminate the two taxa as separate clusters. It is necessary to find the good specimen of R. mucronata and R. stylosa for better understanding of their role in hybridization (Figure 6). Similarly, identification of good specimen of R. x lamarckii and R. x annamalayana is needed for their taxonomic distinction (Table 2; Figure 7). In places were only R. apiculata and R. mucronata present the hybrid individual exhibit consistent morphological characters, i.e. broadly elliptic leaves, L/W ratio is <1.8, ovate bud, 4 sided in cross section, occurrence of stamens in two distinct whorls, light bark with finely fissured in both horizontal and vertical and small style. Similarly, hybrids from areas where only R. apiculata and R. stylosa present are consistent in following characters, i.e. narrowly elliptic leaves, L/W ratio is 2, ellipsoidal bud, slightly four sided, occurrence of stamens in two distinct whorls, rough dark grey bark fissured in horizontal, long style. Thus, R. x annamalayana and R. x lamarckii are distinguished by leaf shape, L/W ratio, bud shape and dimension, peduncle length, bark texture and style length. But, where all the parental species present the hybrids individuals are not exhibit consistent morphology characters (e.g. Ragavan et al. 2011) and this variation confirms the bidirectional hybridization and potential of all known Rhizophora as a maternal parent. The observation of Rhizophora with four stamens (Figure 5) and mature propagules from areas where Rhizophora hybrids present in mixed strands of R. apiculata and R. mucronata shows that hybridization is not restricted to F1 stage.

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Table 1. Diagnostic characters between R. x annamalayana and R. x lamarckii of Andaman and Nicobar Islands specimens

Table 2. Diagnostic characters between R. x annamalayana and R. x lamarckii of Andaman and Nicobar Islands specimens

Characters

R. stylosa

R. mucronata

Characters

Leaf shape

elliptic, broader towards the apex acute cuneate spike 2.02 Dark grey to black smooth finely fissured 3.35 0.23 3

ovate, broader at base broadly acute rounded spike 1.6 Brown to bark grey rough friable horizontally fissured 2.61 0.31 3

2 to 8

smooth 1 0.43 2.39 rounded ellipsoidal 3.9 0.2 0.8 0.3 folded hairy 8 4 2.5 2 1.25 30 1.5 1

smooth 1.47 0.8 1.81 rounded ellipsoidal 3.15 0.3 0.8 0.3 folded hairy 8 0.1 4.5 3 1.5 51 1.7 1.5

Leaf L 12.39 13.08 Leaf W 7.4 6.45 Leaf L/W 1.67 2 Leaf shape broadly elliptic elliptic Leaf apex acute acute Leaf base cuneate attenuate Leaf mucro spike spike Leaf mucro L 0.34 0.45 Petiole L 2.17 2.39 Petiole W 0.35 0.3 Inflorescence branch no. 1 1 No. of flowers per 2 to 4 2 to 4 inflorescences Bract condition smooth smooth Bud L 1.44 1.65 Bud W 0.86 0.8 Bud L/W 1.68 2.06 Mature bud X section four sided slightly four sided Bud shape ovate ellipsoidal Peduncle L 1.3 1.85 Peduncle W 0.5 0.4 Petal L 1.08 1 Petal W 0.36 0.3 Petal X section curved curved Petal margin slightly hairy slightly hairy Stamen No 8 to 16 8 to 16 Style L 0.12 0.3 Fruit L 4 [none] Fruit W 2.3 [none] Fruit L/W 1.73 [none] Hypocotyl L 29 [none] Hypocotyl W 1.5 [none] Collar L 1 [none] Note: Measurement in cm, L = length, W = width.

Leaf apex Leaf base Leaf mucro Leaf L/W Bark

Petiole L Petiole W Inflorescence branch no. No. of flowers per inflorescences Bract condition Bud L Bud W Bud L/W Mature bud X section Bud shape Peduncle L Peduncle W Petal L Petal W Petal X section Petal margin Stamen no. Style L Fruit L Fruit W Fruit L/W Hypocotyl L Hypocotyl W Collar L

R. x annamalayana R. x lamarckii

Figure 2. The intermediates between R. mucronata and R. stylosa. (A) Inflorescence with trichotomous branch, (B) Buds with distinct leafy growth at the base and branching points, (C) Inflorescences with more than 8 flowers.

SETYAWAN et al. â&#x20AC;&#x201C; Rhizophora hybrids and its parentage

Figure 3. Rhizophora with ovate leathery leaves and long style. A. Branchlet, B. Rounded apex, C. Inflorescences, D. Mature bud, E. Stamens and highly hairy folded petals, F. Long style, G. Stamens, H. Two leafy outgrowth at branching point, I. Single leafy outgrowth at first joint.

Figure 4. Rhizophora having inflorescences with multiple joints (A, B, C); D. Style, E. Petals, F. Stamens, G. Long mucronate, H. Branchlet with laterally folded leaves.

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Figure 5. Rhizophora with four stamens. A. Branchlet with dark green elliptic leaves, B. Inflorescence with short peduncle, C. Inflorescences with 2-6 flowers, D. Flowers with 4 stamens, E. Four sided ovary with small style (1 mm), F. Hairy thick leathery folded petal.

A.a

A.b

D.a

F.a

B.a

D.b

F.b

B.b

E.a

G.a

C.a

C.b

E.b

G.b

Figure 6. Morphological distinction between R. mucronata (left/a) and R. stylosa (right/b) of Andaman and Nicobar Islands. A. Leaves, B. Inflorescent, C. Hypocotyl, D. Flower buds, E. Dichotomic branching, F. Styles, G. Flowers

SETYAWAN et al. â&#x20AC;&#x201C; Rhizophora hybrids and its parentage

A.a

A.b

C.a

E.a

C.b

E.b

B.a

B.b

D.a

D.b

F.a

F.b

Figure 7. Morphological difference between R. x annamalayana (left/a) and R. x lamarckii (right/b) of Andaman and Nicobar Islands. A. Bark, B. Leaves, C. Mature bud, D. Stamens in two rows, E. Style length, F. Bud cross section.

KEY OF IDENTIFICATION Rhizophora Linnaeus, Sp. Pl. 1: 443. 1753. Tree or shrub with aerial roots (pneumatophore), which is usually with branches and several meters high from the trunk. Stipules are reddish, sessile, leaf like, interpetiolar, lanceolate. Stem is medium to high; up to 30-40 m, generally 5-8 m. Stem diameter above the aerial root is 1535 cm and above the substrate is 0.5 to 7 m. Stem internodes are hollow. The bark is gray to dark-gray and longitudinally fissured, sometimes it is red-brown and smooth. Mature aerial roots stick up 1-2 m, long and slender (R. stylosa) or big and strong (R. mucronata). Leaves are opposite or distichous, cauline, leathery, petiolate, simple, light or dark green, obovate, margins revolute, 6-19 cm long, 3-10 cm wide. Petiole is 1-4 mm. Leaf tip is mucronate, 1-7 mm long. Leaf blades are glabrous, elongated middle veins, hard to drop, leaf edge is blunt or serrate near top; elliptic, or obovate (usually elliptic to obovate); pinnately veined (midrib extended into a caducous point); margin is entire or serrulate near apex. Upper surface of the leaves is smooth, shiny. On undersurface, there are spots cork, evenly dispersed, not obtrusive, but are absent in R. apiculata and R. x lamarckii of the southern Papuasia and northern Australia.

Inflorescences axillary, dense cymes (simple or di- or trichasial); pedunculate, with little to many nodes, 1-2 branching and 1-many flower buds. Flowers are ebracteate; bracteoles forming a cup just below flower; usually 4 or 5 merous. In R. mucronata, R. stylosa, R. x annamalayana, R. x lamarckii, and R. apiculata consecutive interest lies in node of 1-3, 1-5, 3-5, 3-6, and 6-11 of apical buds. Perianth is with distinct calyx and corolla; 6-32; 2-whorled; isomerous. Calyx tube is ovate (to narrowly ovate), adnate to ovary, persistent; lobes 5-8. Petals tube adnate to the ovary, ovate; sessile, alternating with the calyx, lanceolate; blunt-lobed; valvate; tubular; regular; commonly fleshy (or leathery); persistent. Petals are pale yellow, lobes 4, lanceolate. Flower buds obovate, green to yellowish green, 1-2 cm long, ~1 cm wide. Corolla lobes are 4, lanceolate stripe, pink to white, furred, ~10 mm long, ~2 mm wide. Stamens are 7-12, yellow; filaments are much shorter than anthers or absent; free of one another; anthers are connivent; dorsifixed; dehiscing by longitudinal valves, introrse, locules many, dehiscing by an adaxial valve; filaments are light green, rounded, 0.5-6 mm above receptacle, two forked tip. The bud of R. x annamalayana is longer than R. x lamarckii (<1.7 mm). R. mucronata bud is longer (> 2 mm) than R. stylosa (<2 mm). Ovary is inferior, 2-lobed, apically partly

N U S AN T AR A BIO S C IEN C E 6 (1): 69-81, May 2014

surrounded by a disk, free part elongating after anthesis; style 1, sometimes very short; stigmas are 4. Bracts form a bowl just below the flower. Mature bracts of buds are green, slender (R. mucronata, R. stylosa), green, swollen (R. x annamalayana, R. x lamarckii) or bright green, swollen (R. apiculata). Peduncles 1-7 cm long, ~3 mm wide. Bracteoles in R. mucronata and R. stylosa are slender (length far exceeds its width) at the base of mature shoots, while the other is as wide as its length or wider than its length. Bracteoles of R. apiculata are swollen (wider than its length), corky brown, fused inflorescence, nodes of mature buds and flower are in axillary panicles, at 6-11 nodes below the apical bud, far below the leaves on leafy shoots. Fruit is brown, ovoid, ovoid-conic, or pyriform. Fertile seed is one in each fruit; germination is viviparous; hypocotyl is protruding to 78 cm before propagule falls. Mature fruit is pear-shaped, elongated, narrowed at the middle, smooth brown surface, elongated petal lobes (when hypocotyl will emerge). In R. apiculata, R. mucronata, R. stylosa, R. x lamarckii, and R. x annamalayana, mature fruit is respectively located in the leaf axil at 8, 3-5, 4-7, 7 (rare), and 5 (rare), below the apical bud. Hypocotyl is slender, cylindrical, elongated, green, and smooth with spots of small brown irregular lenticels, half of the distal width is quite broad, the distal tip is generally thorny, but is commonly rounded to blunt at R. apiculata. Mature hypocotyl on R. apiculata, R. mucronata, R. stylosa, R. x lamarckii, and R. x annamalayana, are

respectively located in the leaf axil at 9-13, 4-10, 4-9, none, and 8-9 (rarely) below the apical bud. Hypocotyl size is varied and inconsistent in each species, 14-80 cm long, 1-2 cm at its widest point, and 0.5-1.5 cm wide at the collar; hypocotyl elongated to ~75 cm before propagule fall (Ding Hou 1958; Ko 1983; Tomlinson 1986; Duke, 2006; Gathe and Watson 2008). Summary of morphological comparisons are shown in Table 3 and Figure 8. Nine species, tropics and subtropics; five species in Indo-Malaya, include Indonesia. 1a. Peduncle is shorter than petiole, thick, on leafless stems; flowers are 2 per inflorescence; bracteoles united, cupshaped; petals are glabrous; stamen is 9-15. 2a. Bracts are corky brown, flower are hairless ..R. apiculata 2b. Bracts are smooth green 3a. Flowers sterile; hypocotyl not formed.............. .................. R. x lamarckii (R. apiculata x R. stylosa) 3b. Flowers generally sterile; stamens in two distinct whorls; hypocotyl rarely formed ........................... ...... R. x annamalayana (R. apiculata x R. mucronata) 1b. Peduncle is usually as long as (or greater than) petiole, slender, in leaf axel; flowers are more than 2 per inflorescence; bracteoles are united at base; petals are pubescent; stamen is 6-8. 4a. Style is less than 2.5 mm (0.5-1.5 mm); anthers are sessile .............................................. R. mucronata 4b. Style isomer than 2.5 mm (4-6 mm); anthers are on a short but distinct filament .................... R. stylosa

Table 1. Morphological comparison of Rhizophora in Indo-Malaya region. Part of plant

R. apiculata

R. mucronata

R. stylosa

R. x lamarckii

R. x annamalayana

Stem

Dark gray with shallow grooves Petiole 1.5-3 cm, midrib on undersurface with a reddish tinge, lamina elliptic oblong to sublanceolate, tip with shoot elongation, the undersurface with clearly black spots

Dark gray with deep grooves Petiole 2.5-4 cm, midrib on undersurface pale green, lamina broadly elliptical to round elongated, tip with prominent mucronate spurs, undersurface with prominent black spots Branched 2-4 times, 4-8 flower buds appear on long peduncles Petals hairy, style short, 0.5-1.5 mm. Flower on long branching stalks with short style. Dark brown when ripe, ovate, 2.5-3.5 cm. Fruit large compared to sepals. Hypocotyl cylindrical, warty with pointed tip, up to 30-60 cm, yellow. Very long hypocotyl.

Reddish brown without grooves Petiole 2.5-3.5 cm, midrib on undersurface pale green, lamina ellipticwide, tip with prominent mucronate spurs, undersurface with conspicuous black spots

Gray with grooves

Brown with grooves

Petiole 2-3 cm, midrib on undersurface light green, lamina obovate-elliptical, tip with prominent mucronate spurs, undersurface with black spots that tight on mature leaves Branched 1-2 times, 2-4 flower buds appear on short peduncles Petals slightly hairy, style medium, 2-3 mm

Petiole 2-3.5 cm, midrib on undersurface light green, lamina obovate-elliptical, tip with a clear mucronate spurs, undersurface with clearly black spots

Leaves

Inflorescence

Always with a pair of buds, arise from the strong peduncles

Flowers

Petals glabrous, style 1 mm. Flower on very short stalks.

Fruits

Brown when ripe, 22.5 cm. Fruit on very short stalk almost stuck to the branch. Hypocotyl cylindrical, rounded elongated with blunt ends, up to 30 cm, slightly red color

Propagule

Branched 2-4 times, 4-8 buds appear on the elongated peduncles Petals hairy, style elongated, 4-5 mm. Flowers on long branching stalks with long style. Brown when ripe, Sterile ovate, 2 cm. Fruit not thus large compared to sepals. Hypocotyl Sterile cylindrical, warty with pointed tip, up to 30 cm, yellow

Branched 1-2 times, 2-4 flower buds appear on short peduncles Petals slightly hairy, style short, 1 mm

Generally sterile

Rarely formed

SETYAWAN et al. â&#x20AC;&#x201C; Rhizophora hybrids and its parentage

R. apiculata Stem

Stilt roots

Leaves

Inflorescence

Flower

Style

R. mucronata

R. stylosa

R. x lamarckii

R. x annamalayana

N U S AN T AR A BIO S C IEN C E 6 (1): 69-81, May 2014

Fruits

[none]

[rare]

Hypocotyl

[none]

[rare]

Figure 8. Morphological comparison of Rhizophora in Indo-Malayan region. (Photo source from P. Awale, N.C. Duke, J.L.T. Kwong, P. Ragavan, R. Tan, R. Yeo, P.B. Pelser & J.F. Barcelona, etc.).

DESCRIPTION Rhizophora apiculata Blume, Enum. Pl. Javae 1: 91. 1827. Syn. Mangium candelarium Rumph., Rhizophora candelaria DC, Rhizophora conjugata (non Linné) Arn., Rhizophora lamarckii, Rhizophora mangle (non Linné). Local names. Bakau, bakau minyak, bangka minyak, donggo akit, jangkar, abat, bangkita, bangkita baruang, kalumagus, kailau, parai (Ind.); bakau minyak, bakau tandok, bakau akik, bakau puteh, akik (Mal.); bakauan lalaki, bakauan, bakau, uakatan bakad, bakhau, bakhaw, uakatan, bakauan lalake (Philip.), duoc (Viet.), kongkang, kongkaang bai leu (Thai.), kongkang-slektoch (Camb.) Description. Trees or shrubs are erect, 3-6 (-30) m tall, and 50 cm d.b.h. Stilt-roots are up to 5 m up the stem. Bark is dark grey and chequered, usually with vertical fissures. Stipules are 4-(6)-8 cm, dropped early. Leaves are crowded at twig tips, opposite, simple, penniveined but venation barely visible, glabrous, narrowly elliptic, and leathery; are dark green with a distinct light green zone along the midrib, tinged reddish underneath. Petiole is 1.5-3.5 cm, usually tinged reddish; and is flanked by leaflets at its base, 4-8 cm. Leaf blade is elliptic-oblong to sublanceolate, 7-19 × 3-8 cm, abaxial midvein reddish, base broadly cuneate, apex acute to apiculate. Undersurface leaves are with reddish-brown spots (cork warts), except for population from southern Papuasia and northern Australia. Inflorescences are 2-flowered cymes; peduncle is 0.7-10 mm. Flowers are ca. 2 cm diam., sessile, yellow-red, placed in axillary bundles, stalk 1.4 cm long. Calyx lobes are

ovate, concave, 1-1.4 cm, apex acute. Sepals are 4, yellowred, persistent, in a recurved form on the end of the fruit. Petals are lanceolate, flat, 6-8 mm, membranaceous, glabrous, white. Petals are 4, yellow-white, membranous, flat, hairless, 9-11 mm long. Stamens are mostly 12, 4 adnate to base of petals, 8 adnate to sepals, 6-7.5 mm; anthers are nearly sessile, apex apiculate. Ovary is largely enclosed by disk, free part 1.5-2.5 mm; style is ca. 0.8-1 mm. Fruit ca. is 2.5 × 1.5 cm, apical half narrower, contain one fertile seed. Hypocotyl is cylindric-clavate, green with purple, club shaped, ca. 1.8-3.8 × 1-2 cm, blunt before falling. Flowers and fruits throughout the year (Qin and Boufford 2007; Setyawan and Ulumuddin 2012). Ecology. Grows on the deep muddy soil, gently, which is flooded by normal high tide. Avoids harder substrate mixed with sand. Also prefers tidal waterways with permanently strong freshwater input. Its stilt root branching is likely to be abnormal, caused by beetle damage on root tip. Crabs can inhibit the growth of seedlings due to gobble or peeling hypocotyl skin. Grows slowly, but is blooming throughout the year. Grows up to form a dominant 90% of the vegetation in an area. Grows abundantly in Southeast Asia and are unevenly distributed in Australia (Giesen et al. 2006). Distribution. South China (Guangxi, Hainan), Cambodia, India, Indonesia, Malaysia, Myanmar, Philippines, Sri Lanka, Thailand, Vietnam; East Africa, North Australia, New Guinea, Micronesia, West Pacific Islands. Conservation status. Least Concern ver 3.1.

SETYAWAN et al. – Rhizophora hybrids and its parentage

Uses. It is heavy to very heavy wood, and very hard that it requires careful treatment, thus it would not break, but it can be worked out with good results. It is used for pile, beams, and outrigger boats, home interior, furniture, firewood and charcoal. The bark contains up to 30% tannins. Stilt root branching is used to make the anchor, after weighted with stones. It is sometimes planted along the pond to protect levees and dikes. It is used for mangrove rehabilitation and plantation forests (Giesen et al. 2006). Notes. The leaves of Indo Malayan Rhizophora species typically have small reddish-brown spots (cork warts) on their undersurfaces. Spots are present on R. apiculata from India to Southeast Asia and northern Papuasia. However, in southern southern Papuasia and northern Australia, the spots are absent in R. apiculata and the hybrid R. x lamarckii (Duke et al. 2002). Rhizophora mucronata Lamarck ex Poiret, Encycl. 6: 189. 1804. Syn. Mangium candelarium Rumph., Rhizophora candelaria Wight & Arn., Rhizophora latifolia Miq., Rhizophora longissima Blanco, Rhizophora macrorrhiza Griff., Rhizophora mangle (non Linné) Roxb., Rhizophora mucronata var. typica Schimp. Local names. Bangka itam, dongoh korap, bakau hitam, bakau korap, bakau merah, jangkar, lenggayong, belukap, lolaro (Ind.); bakau kurap, bakau belukap, bakau gelukap, bakau jangkar, bakau hitam (Mal.); bakau, bakauan-babae, bakhau, bakhaw, bangkau, bakauan babe (Philip.), koriki, pabo, togo, tortor, totoa (PNG), dong (Viet.), kongkaang bai yai, kongkang (Thai.) Description. Trees or shrubs are erect, and up to 27(30) m, d.b.h. above highest stilt root is up to 70 cm in diam.; bark is dark to almost black, horizontally fissured. It has both stilt roots and aerial roots growing from lower branches. Stipules are 5.5-8.5 cm. Petiole is 2.5-4 cm. Leaf blade is broadly elliptic to oblong, 8.5-23 ×5-13 cm, leathery, base cuneate, apex blunt to acute. Petiole is green, 2.5-5.5 cm long, leaflets are at the base of petiole 5.5-8.5 cm. Inflorescences are forked 2-3 times, 2-5(-12)-flowered cymes; peduncle is 2.5-5 cm. Flowers are sessile. Calyx lobes are ovate, 9-14 × 5-7 mm, deeply lobed, pale yellow. Petals are lanceolate, 7-9 mm, fleshy, partly embracing stamens, margins pilose (densely hairy margins). Stamens are 8, 4 borne on base of petals, 4 borne on sepals, 6-8 mm; anthers sessile. Ovary emerges far beyond disk, free part elongate-conic, 2-3 mm; style is 0.5-1.5 mm. Fruit is dull, brownish green, elongate-ovoid, 5-7 × 2.5-3.5 cm, basally often tuberculate, apically slightly contracted. Hypocotyl is cylindrical, 30-65 cm long, up to 2 cm wide (Qin and Boufford. 2007; Setyawan and Ulumuddin 2012). Ecology. Grows up in an environment similar to R. apiculata, but is more tolerant to sandy and harder substrate. Generally grows in clusters near or on the banks of creeks and tidal estuaries, rarely far from the tides. Optimal growth occurs in deep submerged areas, on the hard ground and rich in humus. This species is one of the most important and widespread mangrove. Flowering occurs throughout the year. Seedlings are often eaten by

crab, preventing new growth. Seeds dried in the shade for a few days before planting are less favorable for crab. This process is likely to cause the accumulation of protective tannins. The presence is very abundant (Giesen et al. 2006). Distribution. Taiwan, Cambodia, India, Indonesia, Japan (Ryukyu Islands), Malaysia, Myanmar, Pakistan, Philippines, Sri Lanka, Thailand, Vietnam; East Africa, Madagascar, Southwest Asia, North Australia, New Guinea, Micronesia, West Pacific Islands. Introduced to Hawaii. Widely distributed in the Southeast Asia. Conservation status. Least Concern ver 3.1. Uses. It is heavy to very heavy wood, and very hard and strong; shrinks a lot and somewhat is difficult to work on because of its hardness. It is used for firewood and charcoal. The tannin of the bark is used for tanning and dyeing, especially to strengthen the fishing lines and rigging. Sometimes it is used to treat hematuria. It can be grown in fish ponds to protect the levees and dikes, and can be used to make fish traps (Giesen et al. 2006). Rhizophora stylosa Griffith, Not. Pl. Asiat. 4: 665, 1854. Syn. Rhizophora lamarckii, Rhizophora mucronata Lamarck var. stylosa (Griffith) Schimper. Local names. Typically the same as the name of R. mucronata, namely: bakau (Ind. and Mal.); bakau pasir (Sing.); bakauan bato, bakhaw, bangkau (Philip.). Description. Small trees or shrubs are erect and has multi- or single-trunked, often less than 8 m up to 10 m tall. Bark is reddish or pale gray, smooth to rough, fissured; it is 10-15 cm at d.b.h.; stilt-roots are up to 3 m long, and aerial roots emerge from the lower branches. Leaves are broadly elliptic; leaf blade is obovate, 6.5-12.5 × 3-4(-5.5-7.5) cm, base broadly cuneate, apex mucronate, leathery, with a regularly-spotted under surface and appointed tip. Petiole is 1-3.5 cm, with 4-6 cm long leaflets at its base. Inflorescences are axillary, flowers are forked 3-5 times, with 2- to many (up to 32) flowered; peduncle is 1-5 cm. Pedicel 5-10 mm, terete; bracteoles are brown, connate. Calyx lobes are pale-yellow, still present on the fruit, but are then recurved, lanceolate to oblong- lanceolate, 9-12 × 3-5 mm. Petals are white-yellow, 0.8-1.2 cm, involute, margin densely villous/woolly. Stamens are usually 8; filaments are short but distinct; anthers are 5-6 mm. Ovary is emerging beyond disk, free part and shallowly conic and less than 1.5 mm; style is 4-6 mm; stigma lobes are 2. Fruit is green-brown, conic, pear-shaped, 2.5-4 × ca. 2 cm. Hypocotyl is cylindrical (often mistaken for the ‘fruit’), 2040 cm, apex acute. Flowers and fruits are produced throughout the year (Qin and Boufford 2007; Setyawan and Ulumuddin 2012). Ecology. It can grow on different tidal habitats, such as mud, sand, coarse gravel and pebbles, but prefers a tidal river bank, and is also a pioneer species in coastal areas or outer part of the mangrove ecosystem. One distinctive niche is its ability to grow as the mangrove edges on small coral islands, growing on the coral substrate. This species produces flowers and fruit throughout the year. Abundance slightly common to common.

N U S AN T AR A BIO S C IEN C E 6 (1): 69-81, May 2014

Distribution. China (Guangdong, Guangxi, Hainan), Taiwan, Cambodia, Indonesia, Japan (Ryukyu Islands), Malaysia, Philippines, Singapore, Vietnam; North Australia, New Guinea, Pacific Islands. Conservation status. Least Concern ver 3.1. Uses. It can be used as wood buildings and utensils, firewood and charcoal. Australian Aborigines use it to make boomerangs, spears and ceremonial objects. Palm wine with low alcohol content and herbs to treat hematuria can be made from its fruit. Rhizophora x lamarckii Montrouz. Mém. Acad. Roy. Sci. Lyon, Sect. Sci. sér. 2, 10:201. 1860 Rhizophora x lamarckii is a hybrid of R. apiculata and R. stylosa. This species has much in common with both parents. Description. Trees or shrubs are erect and up to ca. 25 m, often with several trunks; bark is light brown, smooth to dark grey, rough, and often horizontally fissured. Stilt roots extend to 2(-6) m above the ground, extend downwards from branches. Stem base is diminished below the stilt roots. Leaves are simple, opposite, obovate-elliptic to elliptic, bright yellowish-green, waxy above and dull below, 7-15 cm long, 3-8 cm wide, yellow-green, with a pointed apex and mucronate spike to 6 mm long, and are not evenly spotted below (but old leaves are often liberally wound-spotted); petiole is 1-4 cm long. Inflorescence is axillary, 2-4-flowered (occasionally 1), borne within the leafy shoot; yellow-green or cream flowers which are held within leaf clusters; peduncle is 1-3 cm long, green, smooth; bracteoles are partly united, smooth, yellow-green except a brown crenulate rim. Flowers have 4 calyx lobes and 4 slightly hairy white petals. Petals are ~10 mm long; margins slightly incurved, sparsely hairy. Stamens are variable, usually 9-11; anthers sessile. Upper part of ovary is shallowly conical; style are ~2.5 mm long; stigma minutely 2-lobed. Fruit is rarely found beyond the immature fruit stage. Fruit is shiny, brown-olive, inverted pear-shape, pyriform, 2.5-3 cm long. Hypocotyl is rarely developed, smooth, green, 14-28 cm long, narrowly clubshaped, rounded at apex (Chan 1996; Duke 2006; Setyawan and Ulumuddin 2012). Ecology. It lives in habitat that same with those R. apiculata and R. stylosa live. Distribution. Indonesia, India, Malaysia, North Australia. Conservation status. This hybrid species has not yet been assessed for the IUCN Red List. Uses. The wood is used as fire-wood, for construction and charcoal making. Rhizophora x annamalayana Kathiresan, Environ. Ecol. 17 (2): 500, 1999 Rhizophora x annamalayana is a hybrid of R. apiculata and R. mucronata. This species has much in common with both parents. Syn. Rhizophora x annamalai K.Kathiresan (= R. apiculata x R. mucronata) Description. Trees are erect, about 25 m tall; bark brown, with vertical fissured. Leaves are simple, opposite,

with short stalks, ovate to elliptical widened, tip apiculate, base cuneate, entire, coriaceous, 10-15 x 5-9 cm. Inflorescence is axillary cyme. Flowers are 2-4, white or cream, hermaphrodite. Petals are 4 lobes, hairy. Stamens are 12 -15, sometimes are very small, staminodium or filamentous. Fruits brown, obpyriform. Hypocotyl is 30-50 cm, smooth, cylindrical. Ecology. It is commonly found at the top of the tidal area and rarely at the mid-tide. Distribution. Sri Lanka, India (Pichavaram of Tamil Nadu and Andaman and Nicobar Islands), Malaysia (Merbok, Kedah), Indonesia (Lombok, West Nusa Tenggara). Conservation status. This hybrid species has not yet been assessed for the IUCN Red List. Uses. Bark is used as a source of tannin and petroleum coke substitute for calcium carbide, and is used as firewood and medicine; tannins from the bark are used as an insect repellent. The leaves are used as animal feed. Stem with complicated stilt roots is effectively used as a tidal wave breaking to prevent abrasion and forms ideal niches for various species of fauna.

CONCLUSION In the Indo-Malayan region, there are three species of Rhizophora namely: R. apiculata, R. mucronata and R. stylosa and two putative hybrid species, namely R. x lamarckii (R. apiculata and R. stylosa) and R. x annamalayana (R. apiculata and R. mucronata). Genetically, there is also a hybrid of R. mucronata and R. stylosa, but because both are sibling species and have very similar morphologic characteristics, the progeny is morphologically indistinguishable from the parent.

ACKNOWLEDGEMENTS The work was partly supported by the Research Project of "Natuna Sea Expedition-2010" in collaboration with the Directorate General of Higher Education, Ministry of National Education, Republic of Indonesia and the Research Center for Oceanography, Indonesian Institute of Science, Jakarta.

REFERENCES Baba S. 1994. New natural hybrid in Rhizophora. Mangroves, newsletter of the International Society of Mangrove Ecosystems 13: 1. Ceron-Souza I, Rivera-Ocasio E, Medina E, Jiménez JA, McMillan WO, Bermingham E. 2010. Hybridization and introgression in New World red mangroves, Rhizophora (Rhizophoraceae). Am J Bot 97 (6): 945957. Chan EWC, Wong SK. 2009. Chemical constituents of leaves of Rhizophora x lamarckii, R. apiculata and R. stylosa. ISME/GLOMIS Elec J 7 (1): 1-2. Chan HT. 1996. A note on the discovery of Rhizophora x lamarckii in Peninsular Malaysia. J Trop For Sci9: 128-130. Ding Hou. 1958. Rhizophoraceae. Flora Malesiana Seri 1, 5: 429-493. Duke N, Kathiresan K, Salmo III SG, Fernando ES, Peras JR, Sukardjo S, Miyagi T. 2010a. Rhizophora apiculata. In: IUCN 2012. IUCN Red

SETYAWAN et al. – Rhizophora hybrids and its parentage List of Threatened Species. Version 2012.2. www.iucnredlist.org. On 15 November 2012. Duke N, Kathiresan K, Salmo III SG, Fernando ES, Peras JR, Sukardjo S, Miyagi T. 2010b. Rhizophora mucronata. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2. www.iucnredlist.org. On 15 November 2012. Duke N, Kathiresan K, Salmo III SG, Fernando ES, Peras JR, Sukardjo S, Miyagi T. 2010c. Rhizophora stylosa. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.2. www.iucnredlist.org. On 15 November 2012. Duke NC, Bunt JS. 1979. The Genus Rhizophora (Rhizophoraceae) in North-eastern Australia. Aust J Bot 27: 657-78 Duke NC, Ge XJ. 2011. Bruguiera (Rhizophoraceae) in the Indo-West Pacific: a morphometric assessment of hibridization within singleflowered taxa. Blumea 56: 36-48. Duke NC, Lo EYY, Sun M. 2002. Global distribution and genetic discontinuities of mangroves – emerging patterns in the evolution of Rhizophora. Trees 16: 65-79 Duke NC. 2006a. Australia’s Mangroves: The Authoritative Guide to Australia’s Mangrove Plants. University of Queensland, Brisbane. Duke NC. 2006b. Rhizophora apiculata, R. mucronata, R. stylosa, R. x annamalai, R. x lamarckii (Indo-West Pacific stilt mangroves), ver. 2.1. In: Elevitch C.R. (ed.). Species Profiles for Pacific Island Agroforestry. Permanent Agriculture Resources (PAR), Hōlualoa, Hawai‘i. <http://www.traditionaltree.org>. Duke NC. 2010. Overlap of eastern and western mangrove in the Southwestern Pacific: hybridization of all three Rhizophora (Rhizophoraceae) combinations in New Caledonia. Blumea 55: 171188. Gathe J, Watson L. 2008. Rhizophora L. FloraBase the Western Australia Flora. http://florabase.dec.wa.gov.au/browse/profile/21806 Giesen W, Wulffraat S, Zieren M, Scholten L. 2006. Mangrove guidebook of Southeast Asia. FAO and Wetlands International, Rome. Guo M, Zhou R, Huang Y, Ouyang J, Shi S. 2011. Molecular confirmation of natural hybridization between Lumnitzera racemosa and L. littorea. Aquat Bot 95: 59-64. Hou D. 1992.Rhizophoramucronata Poiret. In: Lemmens RHMJ, Wulijarni-Soetjipto N (eds.): Plant Resources of South-East Asia. No. 3: Dye and tannin-producing plants. Prosea Foundation, Bogor. Kathiresan K. 1995. Rhizophora annamalayana: A new species of mangroves. Environ Ecol 13 (1): 240-241. Kathiresan K. 1999. Rhizophora annamalayana Kathir (Rhizophoraceae), a new Nothospecies from Pichavaram mangrove forest in Southeastern peninsular India. Environ Ecol 17(2): 500-501. Kavitha S, Kathiresan K. 2011. Reproductive biology of a natural mangrove hybrid Rhizophora annamalayana and its parent species (R. apiculata and R. mucronata) (Rhizophoraceae). Bot Mar doi. 10.1515/BOT.2011.062 Kavitha S, Kathiresan K. 2012. Reproductive biology of the most-at-risk mangrove species (Rhizophora annamalayana) and its parental species. In: Dahdouh-Guebas F, Satyanarayana B (eds). Proceedings of the International Conference: Meeting on Mangrove Ecology, Functioning and Management (MMM3), 2-6 July 2012, Galle, Sri Lanka. Ko WC. 1983. Rhizophoraceae. In: Fang WP, Chang CY (eds.). Fl Reipubl Popularis Sin 52 (2): 125-143. Kumar BA, Thangaraj M, Kathiresan K. 2011. Cross-species amplification of microsatellite loci in three mangrove species of India. Intl J Advan Biotech Res 2 (2): 240-243. Lakshmanan KK. Rajeswari M. 1983. Distribution of organic contents in the leaves of Rhizophora species. In: Proceedings of the 70th Indian Science Congress, India. Lakshmi M, Parani M, Parida A. 2002. Molecular phylogeny of mangroves IX: Molecular marker assisted intra-specific variation and species relationships in the Indian mangrove tribe Rhizophoreae. Aquat Bot 74: 201-217. Lo EYY, Duke NC, Sun M. 2014. Phylogeographic pattern of Rhizophora (Rhizophoraceae) reveals the importance of both vicariance and longdistance oceanic dispersal to modern mangrove distribution. BMC Evol Biol 14:83, http://www.biomedcentral.com/1471-2148/14/83 Lo EYY. 2003. Phylogenetic relationships and natural hybridization in the mangrove genus Rhizophora from the Indo-West Pacific region. [M.Sc. Thesis]. University of Hong Kong, Hong Kong.

Lo EYY. 2010. Testing hybridization hypotheses and evaluating the evolutionary potential of hybrids in mangrove plant species. J Evol Biol 23: 2249-2261 McNeill J, Barrie FR, Burdet HM, Demoulin V, Hawksworth DL, Marhold K, Nicolson DH, Prado J, Silva PC, Skog JE, Wiersema JH, Turland NJ (eds). 2005. International Code of Botanical Nomenclature (Vienna Code) adopted by the Seventeenth International Botanical Congress Vienna, Austria, July 2005. Publ. 2007. Gantner, Ruggell. (Regnum Vegetabile, 146). XVIII, 568 p. grevo. Cloth. Muller J, Caratini C. 1977. Pollen of Rhizophora (Rhizophoraceae) as A guide fossil. Pollen Spores 19: 361-389. Muniyandi K, Natarajan R. 1985. Incidence of seedling formation in Rhizophora lamarckii Montr. in Pichavaram mangrove, Tamil Nadu, India. J Bombay Nat Hist Soc 82: 441-442. Ng WL, Chan HT, Szmidt AE. 2013. Molecular identification of natural mangrove hybrids of Rhizophora in Peninsular Malaysia. Tree Genet Genomes. doi:10.1007/s11295-0Error! Hyperlink reference not valid. Ng WL,Chan HT. 2012.Furtherobservationson a natural Rhizophorahybrid population inMalaysia.ISME/GLOMIS Elec J10: 1-3 Ng WL, Szmidt AE. 2013. A simple and inexpensive molecular assay for species identification of Indo-West Pacific Rhizophora mangroves for conservation and management. Conserv Genet Resour. doi: 10.1007/s12686-013-9973-x Ong, JE. 2003. Plants of the Merbok Mangrove, Kedah, Malaysia and the urgent need for their conservation. Folia Malaysiana4 (1): 1-18. Parani M, Rao CS, Mathan N, Anuratha CS, Narayanan KK, Parida A. 1997. Molecular phylogeny of mangroves III: Parentage analysis of a Rhizophora hybrid using random amplified polymorphic DNA and restriction fragment length polymorphism markers. Aquat Bot 58: 165-172. Qin H, Boufford DE. 2007. Rhizophoraceae. In: Flora of China, Vol. 13. Science Press, Beijing and Missouri Botanical Garden Press. Ragavan P, RavichandranK, Jayaraj RSC, Mohan PM, Saxena A, Saravanan S, VijayaraghavanA. 2014. Distribution of mangrove species reported as rare in Andaman and Nicobar islands with their taxonomical notes. Biodiversitas 15: 12-23 Ragavan P, Saxena M, Coomar T, SaxenaA. 2011. Preliminary study on natural hybrids of genus Rhizophora in India.ISME/GLOMIS Elec J 9:5. Schwarzbachl AE, Ricklefs RE. 2001. The use of molecular data in mangrove plant research. Wetlands Ecol Manag 9: 195-201. Setyawan AD, Ulumuddin YI. 2012.Species diversity of Rhizophora in Tambelan Islands, Natuna Sea, Indonesia. Biodiversitas 13: 172-177. Setyawan AD, Winarno K, Purnama PC. 2004. Review: Mangrove ecosystem in Java: 2. Restoration. Biodiversitas 5 (2): 105-118. Singh VP, Mall LP, George A, Pathak SM. 1987.A new record of some mangrove species from Andaman and Nicobar Islands and their distribution.Indian For113(3): 214. Spalding M, Kainuma M, Collins L. 2010. World atlas of mangroves. Earthscan, London Subramonian N. 1993. Studies on genetic diversity in some Rhizophora species. [Ph.D Dissertation]. Centre for Advanced Studies in Botany, University of Madras, Madras, India. The Plant List. 2010. Version 1. Published on the Internet; http://www.theplantlist.org/ (accessed 1st January). Tomlinson PB, Primack RB, Bunt JS. 1979. Preliminary observations on floral biology in mangrove Rhizophoraceae. Biotropica 11 (4): 256277 Tomlinson PB, Womersley JG. 1976. A Rhizophora new to Queensland and New Guinea, with notes relevant to the genus. Contrib Herb Austral 19: 1-10. Tomlinson PB.1978. Rhizophora in Australasia, some clarification of taxonomy and distribution. J Arnold Arbor 59: 156-169 Tomlinson PB.1986. The botany of mangroves. Cambridge University Press, London. Zhou R, Gong X, Boufford D, Wu CI, Shi S. 2008. Testing a hypothesis of unidirectional hybridization in plants: Observations on Sonneratia, Bruguiera and Ligularia. BMC Evol Biol 8:149 Zhou R, Shi S, Wu CI. 2005. Molecular criteria for determining new hybrid species—an application to the Sonneratia hybrids. Mol Phylogenet Evol 35: 595-601

N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060113

Vol. 6, No. 1, pp. 82-85 May 2014

Short Communication: Growth of seaweed Eucheuma cottonii in multi trophic sea farming systems at Gerupuk Bay, Central Lombok, Indonesia SUKIMAN, FATURRAHMAN, IMMY SUCI ROHYANI, HILMAN AHYADI Biological Studies Program, Faculty of Mathematics and Natural Sciences, University of Mataram. Jl. Majapahit No. 62 Mataram 83125, West Nusa Tenggara, Indonesia. Tel./Fax. +62-370-646506. â&#x2122;Ľemail: sukimandao@yahoo.co.id Manuscript received: 4 December 2013. Revision accepted: 7 March 2014.

Abstract. Sukiman, Faturrahman, Rohyani IS, Ahyadi H. 2014. Growth of seaweed Eucheuma cottonii in multi trophic sea farming systems at Gerupuk Bay, Central Lombok, Indonesia. Nusantara Bioscience 6: 82-85. Eucheuma cottonii is a seaweed commodity that has a high economic value because it contains compounds used as raw materials for industries. Various methods of seaweed farming have been developed, one of which is a system of cultivation Multi Trophic Sea Farming. This study aimed to analyze the growth of E. cottonii by observing the production of biomass in four trophic combinations in the system Multi Trophic Sea Farming. The study was conducted in the area of the marine aquaculture Gerupuk bay, Central Lombok, Indonesia. Experiments were performed on four plots cages with trophic combination treatment as follows: K1 (E. cottonii-lobster-abalone), K2 (E. cottonii-abalone-red carp), K3 (E. cottonii-abalone-grouper), and K4 (E. cottonii-abalone-pomfret fish). Seedling of E. cottonii weighing 50 g was tied to a rope and placed at a depth of 5 cm, 50 cm, 100 cm and 150 cm. Measurement of biomass production was done every ten days until the thirtieth day. The highest biomass production of E. cottonii was obtained in K3 trophic combination (E. cottonii-abalone-grouper fish) with a depth of seedlings of 5 cm. The combination of K3 trophic is recommended for cultivation of seaweed in the MTSF system. Key words: Eucheuma, Multi Trophic Sea Farming, MTSF, seaweed

INTRODUCTION Seaweed is economically important commodities. Seaweed is widely used as food, medicine, and important materials in the food industry, cosmetics, and pharmaceuticals. Seaweed is traditionally mainly used as vegetables, ice mix, and cookies (Nontji 2007). Seaweed is used as medicine because it has the power of antiviral, antifouling, and anti-lung cancer, tumors and AIDS (Smith 2004). Seaweed is also used as a liquid fertilizer in some plant species (Sunarpi et al. 2011), and the source of agar (Widyastuti 2008). Some of seaweed that has been used in Indonesia are of the genus Porphyra, Acanthophora, Catenella, Eucheuma, Gelidium and Gracilaria (Nontji 2007). Eucheuma cottonii is a seaweed commodity that has a high economic value because it contains compounds used as raw materials for industries. The important chemical constituents of Eucheuma cottonii are agar and carrageenan. Agar is widely used in food, pharmaceutical, and cosmetics. In the food industry, agar is used as a food additive, rehydrating food, a thickening agent, and viscosity controller (Reine and Trono 2002). Carrageenan is a hydrocolloid compound formed in the cell walls of red algae (Angka and Suhartono 2000). Carrageenan is used as a stabilizer, thickener, suspending agent, and a gelling agent in food. Carrageenan is also used in non-food products such as toothpaste, cosmetics, paints, and textile dyes (Angka and Suhartono 2000; Reine and Trono 2002). West Nusa Tenggara is one of the centers of seaweed cultivation in Indonesia. One area that has been developed for marine aquaculture is Gerupuk bay. Various methods of

seaweed farming have been developed in the area. One method of cultivation that is being developed is Multi Trophic Sea Farming (MTSF). Multi Trophic Sea Farming is a mariculture system that combines multiple commodities in the farming unit. Multi Trophic Sea Farming System (MTSF) that combines aquatic animal with marine plants potentially reduces costs and improves efficiency and productivity of a number of species and systems (Neori et al. 2004; Pereira et al. 2010). MTSF system has been successfully applied to the cultivation of seaweed Ulva sp. with Australian abalone (Boarder and Shpigel 2001). The cultivation of Gracilaria with abalone can significantly reduce pathogenic bacteria in abalone (Rebours 2010). This study aimed to analyze the growth of E. cottonii on a combination of different trophic and seed position by observing the production of biomass in the MTSF system. Materials and methods The study was conducted in the area of the marine aquaculture Gerupuk bay, Central Lombok, West Nusa Tenggara, Indonesia. Experiments were performed on four plots floating cages with trophic combination treatment as follows: K1 (E. cottonii-abalone-lobster), K2 (E. cottoniiabalone-red carp), K3 (E. cottonii-abalone-grouper), and K4 (E. cottonii-abalone-pomfret fish). Seedling of E. cottonii weighing 50 g was tied to a rope and placed at a depth of 5 cm, 50 cm, 100 cm and 150 cm. Measurement of biomass production was done every ten days until the thirtieth day. Analyses of the quality of sea water in the Gerupuk bay were conducted to assess the feasibility of these waters as a

SUKIMAN et al. – Multi trophic sea farming systems of Eucheuma cottonii

cultivated area of some commodities such as seaweed, abalone, grouper, red carp, pomfret and lobster. In this study, water quality sampling was conducted at locations that may represent other areas. Physical and chemical qualities of the water measured were pH, temperature, salinity, levels of nitrite and ammonia. Results and discussion Analyses of the quality of sea water in the Gulf Gerupuk were conducted to assess the feasibility of these waters as seaweed farming area with MTSF system. The results of measurements of physical and chemical properties of water were presented in the following table:

Eucheuma cottonii is one of seaweed species that can be cultured in the MTSF system (Figure 1.A, 1.B). Seaweeds are the short term commodity in MTSF system because they can be harvested faster than any other components in the MTSF system The growth of E. cottonii for 30 days of observation can be seen in Table 1. Based on the results of the measurement of biomass conducted every 10 days, the highest biomass production of E. cottonii was obtained in combination trophic K3 (E. cottonii-abalonegroper) with the position of the seeds at a depth of 5 cm. While, the lowest biomass was obtained in combination trophic K4 (E. cottonii-abalone-red crap). Table 2. E. cottonii growth with four trophic combination in MTSF

Table 1. Physical and chemical quality of the water of Gerupuk bay Parameters Temperature Brightness Salinity pH NH3 NO3 DO Note: * MoE (1988)

H27 o

28-29 C 1.82 m 33-37 ppt 7.8-8.4 0.03 mg/L 0.07 mg/L 4.82

Standard * o

28-30 C 1.82m 34-36 ppt 7.5-8.4 0.03-0.05 0.07-0.08 4.82

28-32 3-4m 30-40ppt 6-8 <1 0.06 6-8

At baseline, the pH was 7.8-8.4, temperature 28-29 °C, salinity 33-37 ppt, ammonia 0.03 mg/L and nitrite 0.07 mg/ L. After a month-long study, the data were not very different from that at baseline and inter-basin water quality or between treatments, i.e. pH 7.5-8.4, temperature 2830°C, salinity 34-36 ppt, ammonia 0.03-0.05 mg/L and nitrite 0.07-0.08 mg/L. Physical and chemical parameters that should be considered include flow, temperature, brightness, pH, salinity, dissolved oxygen and nitrogen compounds. Achmad et al. (1991) stated that balk flow velocity for marine fish farming in floating cage is 5-15 cm/sec, pH 6.5 to 9.0 (Boyd and Lichtkoppler 1979) and a brightness of > 3 m (MoE 1988). Furthermore Anon. (1986) and Achmad et al. (1991) stated that good oxygen is 5-8 ppm, while the ammonia concentration is less than 0.1 ppm. Glenn and Dotty (1990) suggested that a range of condition under which the eucheumatoides can be productive in farm setting are: maximum temperature of 24-30oC, minimum temperature of 21-22oC, nitrogen of 2-4 µ-atm/L, phosphate of 0.5-1.0 µ-atm/L, and high solar energy level. In addition, Glenn and Dotty (1992) concluded that culture of eucheumatoids species requires a high level of water motion. To optimize the seaweed component of an integrated aquaculture system, particular attention should be given not only to physical and chemical factors (such as light, temperature, effluent nutrient concentration and flux, water motion, etc.) but also to biological factors such as interplant variability, nutrient prehistory, type of tissue in culture, control of parameters triggering reproduction stages, surface area to volume ratio of thalli, and morphological changes induced by cultivation techniques (Chopin et al. 2001).

Trophic W0 W10 W20 W30 combi- Seedling position (g) (g) (g) (g) nation Control Long line 50.6 106.15 151.15 250 K1 Horiculture 5 cm 49.8 84.35 110.5 164.65 Verticulture 50 cm 50.5 69.15 74.3 89 Verticulture 100 cm 50.3 56.65 56.3 66.5 Verticulture 150 cm 50.05 60.75 53 55.75 K2 Horiculture 5 cm 50.3 110.5 159.85 237.9 Verticulture 50 cm 50.15 86.15 151.5 145.75 Verticulture 100 cm 50.5 67.65 102.6 91.35 Verticulture 150 cm 49.75 65.65 76 80 K3 Horiculture 5 cm 50.1 101.7 170 259.15 Verticulture 50 cm 50.3 80.65 82.25 128.4 Verticulture 100 cm 49.75 77.05 99.3 92 Verticulture 150 cm 50 62.75 53.8 32.5 K4 Horiculture 5 cm 50.6 78.2 109.5 122.2 Verticulture 50 cm 50.3 75.5 95.05 80.75 Verticulture 100 cm 50.55 69.15 57.8 38.15 Verticulture 150 cm 49.9 61.55 47.15 33.5 Note: Verticulture: vertical line culture, Horiculture: horizontal line culture. K1: E. cottonii-abalone-lobster, K2: E. cottoniiabalone-red carp, K3: E. cottonii-abalone-grouper, and K4: E. cottonii-abalone-pomfret fish. W0: 0th days, W10: 10th days, W20: 20th days, W30: 30th days.

The biomass growth of E. cottonii on MTSF system showed a different pattern among the trophic combinations and depths of seeds. The biomass growth of E. cottonii in K1 trophic combination can be seen in Figure 2.A The highest biomass growth of E. cottonii was obtained at seedlings at a depth of 5 cm with an average of 164 g. While, the lowest biomass production of seeds was at a depth of 150 cm with 55.7 g of total biomass for 30 days. At combination of K2 trophic growth, the highest biomass was obtained from the seeds that were placed at a depth of 5 cm with an average biomass production of 238 g. While, the lowest biomass production was obtained at the seed position of 150 cm with the biomass production of 80 g (Figure 2.B). The biomass growth of E. cottonii on K3 trophic combination can be seen in Figure 2.C. The highest biomass growth of E. cottonii obtained at seedlings at a depth of 5 cm with an average of 260 g of biomass produced after 30 days of observation. Biomass production on a combination of trophic and depth of the seedling was the largest biomass production of the entire treatment. The lowest biomass production on a combination of K3 was at a depth of 150 cm with a total seed biomass is 32.5 g.

N U SAN TA RA B IOSC IE NC E 6 (1): 82-85, May 2014

B Figure 1. A. Position of E. cottonii seedling in floating cages, B. Growth of E. cottonii in MTSF system

250

200

150

150 100

300

140

250

120

200

100 80

150 100

0 W0 W10 W20 W30

100

0 W0 W10 W20 W30

W0 W10 W20 W30

Figure 2. Growth of E. cottonii in combination of K1 trophic (A), K2 trophic (B), K3 trophic (C), and K4 trophic (D). Note: blue = horiculture 5 cm, red = verticulture 50 cm, green = verticulture 100 cm, purple = verticulture 150 cm

On the combination of trophic K4 the highest biomass growth was obtained from seeds placed at a depth of 5 cm with an average biomass production of 122 g. While, the lowest biomass production was obtained at the seed position of 150 cm with biomass production of 33 g (Figure 2.D). Biomass production of seaweed in the combination of trophic K4 decreased after 20 days. The results showed that the growth of seaweed was different for each treatment. In general, the growth of seaweed grown on shallower position was higher than that at deeper position. The highest increase in biomass was achieved in seedlings at a depth of 5 cm in K2 (seaweedabalone-red snapper), while the lowest occurred in K1 and

K4 150 cm treatment. This indicates that horizontal line has a better growth rate compared with vertical line culture. The growth of seaweed on MTSF abalone-lobster (K1) and MTSF abalone-pomfret fish (K4) was very low. This is thought to be caused by the nature of lobster and pomfret fish that like to eat seaweed, where the tips of seaweed are found cut by pomfret fish and lobster bites. Thus seaweed cultivation is not suitable to be integrated with pomfret fish and lobster. There is a tendency that the deeper the planting position, the lower the growth of biomass obtained. Seaweed cultivated on the horizontal line (5 cm) and the strap length of 50 cm verticulture will grow faster than the

SUKIMAN et al. – Multi trophic sea farming systems of Eucheuma cottonii

length of the rope verticulture of 100 cm and 150 cm. Seaweed can optimally utilize sunlight as an energy source for photosynthesis and to obtain nutrients. In addition to temperature, light plays a major role in conditioning the performance of seaweed. These are usually the most important environmental parameters affecting growth and nutrient uptake of seaweed (Maria et al 2011). At verticulture of 5 cm long strap the currents and wave movements are optimal for the growth of seaweed so it has a big opportunity to absorb nutrients. Increased uptake of nutrients and photosynthetic activity led to increased production of biomass in K3 trophic combination. This is in accordance with the statement of Glenn and Dotty (1990) that a range of condition under which eucheumatoids can be productive in farm is in high solar energy. The results of this study differ from those reported by Syahlun et al. (2013) which stated that the verticulture of 100 cm was better than other treatments. The position of 100 cm verticulture is parallel to abalone baskets and has a greater opportunity to be exposed to the nutrients from the abalone metabolic outcomes, but, it is unable to promote the better growth of seaweed because the brightness level of water at the MTSF location is only 1.82 m and this disrupts photosynthesis. Thus, these results are not in accordance with the results of the study Pereira et al. (2010) which showed that the growth of seaweed in an integrated system with abalone MTSF was better than those in the monoculture method. In general, the growth of seaweed in MTSF system was lower than in longline method in the mouth of the Gulf Gerupuk that can reach 250 g in 30 days. Low yield is presumably due to the low water movement, low water levels of brightness and a lot of muddy water as a result of the destruction of mangrove forests. Instead Gracilaria growth in floating cage was very good and has the potential to be developed with MTSF pattern. Application of method-vertical line (verticulture) at MTSF is a strategy to maximize the function of space and increase efficiency. Aquaculture is an essential activity in the economy of Gerupuk bay, with seaweed farming being the most important commodity. MTSF system may be part of the solution in marine aquaculture at Gerupuk bay, because integrated multi-trophic aquaculture has ecological and socio-economic advantages, relative to single-species aquaculture (Nobre et al. 2010). Maria et al. (2011) has reported clear advantages of growing seaweeds near the fish. Seaweed is efficient biofilters due to their ability to efficiently remove both ammonia and nitrate from the culture. Beside its biofiltration efficacy, Nobre et al. 2010, said that integrated multi-trophic aquaculture indicates a decrease in the aquaculture generated ecological pressures with the incorporation of seaweeds, mainly a reduction in nitrogen discharges into the adjacent coastal and raised farm profits by 1.4 to 5%. Eucheuma cottonii can be cultivated through a combination with other commodities in the Multi Trophic Sea Farming Systems. The highest biomass production of E. cottonii was obtained in K3 trophic combination (E. cottonii-abalone-grouper) with a depth of seedlings of 5 cm. The combination of E. cottonii, abalone and grouper

fish are recommended for cultivation of seaweed in the system MTSF. In conclusion, E. cottonii can be cultivated through a combination with other commodities in the Multi Tropic Sea Farming Systems. The highest Biomass production of E. cottonii obtained in combination tropic K3 (E. cottoniiabalone-grouper) and a depth of seedlings 5 cm. The combination of E. cottonii, abalone and grouper fish is suggested for cultivation of seaweed on the system MTSF.

REFFERENCES Achmad T, Imanto PT, Muchori M, Basyarie A, Sunyoto P, Slamet B, Mayunar R, Purba S, Diani S, Rejeki SA, Murtiningsih S. 1991. Operational enlargement grouper fish in floating net cages. Research Institute for Coastal Aquaculture, Maros. [Indonesia] Angka SL, Suhartono MT. 2000. Marine products biotechnology. Bogor Agricultural University, Bogor. [Indonesia] Boarder SJ, Shpigel M. 2001. Comparative performances of juvenile Haliotis roei fed on enriched Ulva rigida and various artificial diets. J Shellfish Res 20: 653-657. Boyd CE, Licthkoppler L. 1979. Water quality management in pond fish culture. Aubum University, Alabama. Chopin T, Alqandro H, Christina H, Troell M, Kautsky N. 2001. Integrating seaweed into marine aquaculture system: a key toward sustainability. J Phycol 37: 975-986. Glenn EP, Dotty MS. 1990. Growth of seaweeds Kappaphycus alvarezi, K. striatum, and Eucheuma denticulatum as affected by environment in Hawaii. Aquaculture 84: 245-255. Glenn EP, Dotty MS. 1992. Water motion affects the growth rates of Kappaphycus alvarezii and related seaweed. Aquaculture 108: 233-246 MoE [Ministry of Environment]. 1988. Decree of the Minister for Population and Environment No. 02/Men KLH/1988. January 19, 1988. [Indonesia] Maria H, Abreu MH, Pereira R, Yarish C, Buschmann AH, Sousa-Pinto I. 2011. IMTA with Gracilaria vermiculophylla: Productivity and nutrient removal performance of the seaweed in a land-based pilot scale system. Aquaculture 312: 77-87. Neori A, Chopin T, Troell M, Buschmann AH, Kraemer GP, Halling C, Shpigel M, Yarish C. 2004. Integrated aquaculture: rationale, evolution and state of the art emphasizing seaweed biofiltration in modern mariculture. Aquaculture 231: 361-391. Nobre AM, Andersson DR, Neori A, Sankar K. 2010. Ecologicaleconomic assessment of aquaculture options: Comparison between abalone monoculture and integrated multi-trophic aquaculture of abalone and seaweeds. Aquaculture 306: 116-126 Nontji A. 2007. Sea of the Indonesian Archipelago. Djambatan, Jakarta. [Indonesia] Pereira R, Abreu MH, Valente L, Rema P, Sousa-Pinto I. 2010. Production of seaweeds in integrated multi-trophic aquaculture for application as ingredients in fish feed. In: González JAZ, PachechoRuíz I, Lepe GG (eds). Proceeding of 20th International Seaweed Symposium. Ensenada Baja California, México, 22-26 February, 2010 Rebours C, Novoa-Garrido M, Pang T. 2010. Antibacterial activity from seaweeds-A review. In González JAZ, Pachecho-Ruíz I, Lepe GG (eds). Proceeding of 20th International Seaweed Symposium. Ensenada Baja California, México, 22-26 February, 2010. Reine WFPV, Trono GC. 2002. Plant Resources of South-East Asia: Algae. Prosea Foundation, Bogor. [Indonesia] Smith AJ. 2004. Medicinal and pharmaceutical uses of seaweed natural products: A review. Appl Phycol 16: 245-262 Sunarpi, Jupri A, Kurnianingsih R, Julisaniah NI, Nikmatullah A. 2011. Effect of seaweed extract on the growth and yield of rice. Bioteknologi 8: 18-23. [Indonesia] Syahlun, Rahman A, Ruslaini. 2013. Growth of seaweed (Kappaphycus alvarezii) brown strain using verticulture method. Mar Aquacult Indon J 1: 122-132. [Indonesia] Widyastuti S. 2008. Post-harvest processing of local strains of red algae in Lombok into the agar using two extraction methods. Unram Res J 14: 63-72. [Indonesia]

N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060114

Vol. 6, No. 1, pp. 86-93 May 2014

Short Communication: Variation in isozymic pattern of germplasm from three ginger (Zingiber officinale) varieties 1

AHMAD DWI SETYAWAN1,â&#x2122;Ľ, WIRYANTO1, SURANTO1, NURLIANI BERMAWIE2 Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Jl. Ir. Sutami 36a, Surakarta 57126, Central Java, Indonesia. Tel./fax.: +62-271-663375, â&#x2122;Ľe-mail: volatileoils@gmail.com 2 Indonesian Spice and Medicinal Crops Research Institute, Jl. Tentara Pelajar No. 3, Cimanggu, Bogor 16111, West Java, Indonesia. Manuscript received: 10 March 2014. Revision accepted: 27 April 2014.

Abstract. Setyawan AD, Wiryanto, Suranto, Bermawie N. 2014. Variation in isozymic pattern of germplasm from three of ginger (Zingiber officinale) varieties. Nusantara Bioscience 6: 86-93. Ginger (Zingiber officinale Rosc.) has long been as spices, flavoring agent and raw material for herbal medicines. In Indonesia, there were three varieties based on color and size of the rhizome, i.e. gajah (big-white ginger), merah (red ginger), and emprit (small-white ginger). This research was conducted to find out: (i) isozymic pattern of three ginger varieties, and (ii) phylogenetic relationship of those three varieties. The plant materials were gathered from Wonogiri, Surakarta and Kulonprogo, Yogyakarta. Two enzyme systems, namely esterase (EST) and peroxidase (PER, PRX) were used in this study. The relationship among ginger varieties was determined by UPGMA. The result indicated that EST showed two bands (i.e. Rf 0.04 and 0.10), and PRX showed six bands (i.e. Rf 0.04, 0.05, 0.09, 0.10, 0.11, and 0.15). Peroxidase produce more numerous and more diverse isozymic bands than esterase, resulting in a more complex relationship. The data used to compile dendrogram affect the grouping; the more data used, the more obvious clustering of accessions in a population. Dendrogam generated from esterase and peroxidase banding patterns produced distinct clusters based on varieties and location. Key words: Ginger, isozyme, varieties, Zingiber officinale, Zingiberaceae

INTRODUCTION Ginger (Zingiber officinale Rosc.), is one of the most important and oldest spices a well-known plant in the Nusantara archipelago, consumed as a delicacy, food preservation, medicine, or spice (de Padua et al. 1999). Its highest value is given by the pungent and aromatic essential oils that produced throughout the plant, especially the rhizomes. The essential oils are responsible for the aroma while the non-volatile components are responsible for the pungency with gingerol the most pungent component in fresh ginger (Juliani et al. 2007). This herbaceous rhizomatous perennial reed-like plant with annual leafy stems is most useful Zingiberaceae in the tropics, as well as turmeric, cardamom and galangal (Poth and Sauer 2000). The Zingiberaceaeconsist of approximately 47 genera and 1400 species, that are found in all tropical regions, but are mostly concentrated in Southeast Asia (Purseglove 1972; Kress 1990; de Padua et al. 1999). This spice is produced from the rhizome (underground stem) of the plant (Purseglove 1981). Distribution and use of ginger in Indonesia are very widespread (Burkill1935; Heyne1950). The aromatic and spicy rhizome is used as a spice, seasoning, and medicine (Heyne1950; Holttum 1950), and the less spicy rhizome was used for food and beverages. Zingiber officinale has three varieties based on size and color of rhizome, i.e. Z. officinale var. officinale (big white ginger or giant ginger, badak or gajah), Z. officinale var. amarum (small white ginger, emprit), and Z. officinale var. rubrum (small red ginger, merah or berem) (Ochse 1931;

Burkill 1935; Heyne 1950). These three varieties may partly be deferred from their essential oil contents and are used for different purposes. The essential oil content of the big white ginger is the lowest compared with the other varieties (Setyawan 2002). The big white ginger is usually used for fresh (green) ginger products, food and beverages, while the other gingers are mostly used for medicinal purposes. Ginger produces clusters of white and pink flower buds that bloom into yellow flowers (Watt and BreyerBrandwijk 1962). There are no differences in flower characters between varieties of gingers; therefore it is necessary to use other characters. In agriculture, the proper identification and characterization of varieties of the cultivated plant is needed to obtain the right herbal material used to develop products of high economic value. It is also necessary for plant breeding as marker assisted selection. Genetic markers are needed to trace the genetic nature of the parent and its descendants. The assessment of genetic variation may be done by using various techniques such as isozymes or DNA analysis (Mondini et al. 2009). Isozymic pattern is a highly effective genetic marker that can be obtained through the quick and easy laboratory process, and less expensive than the DNA sequences. It can be used to determine genetic variation of populations, even to accession genetic differences (Crawford 1990). Isozyme can be used as a genetic trait to study the diversity of accessions in a population, classification of plant species, varieties and identifying of hybrid (Beer et al. 1993; Murphy and

SETYAWAN et al. –The isozymic pattern of ginger

Phillips 1993), as well as markers of plant resistance to certain diseases (Alcazar et al. 1995). Isozyme is a codominant character; heterozygous accessions can be distinguished with homozygous (McDonald and McDermont 1993). However, the isozyme patterns may be affected by different environments and the stage of plant development. Moreover, only limited numbers of isozymes loci are available for certain taxa (McDonald and McDermont 1993; Mangolin et al. 1997; Garkava et al. 2000). Isozyme variations only reflect difference in protein-coding sequences or intron (Adam 1999; Sharma and Jana 2002). Research on ginger diversity by using isozymic pattern has been done by Shamita (1997) irrespective of varieties, while the same research on the other Zingiberaceae family has been conducted on Curcuma (Ibrahim et al. 1991; Apavatjrut et al. 1999; Chokthaweepanich and Paisooksantivatana 2003; Tang et al. 2008; Deng et al. 2011), Curcuma alismatifolia (Paisooksantivatana 2001), Curcuma longa (Shamina et al. 1998), Curcuma xanthorriza (Azizah 2011), Amomum (Shanmugapriya and Prabha2012), Boesenbergia (Vanijajiva et al. 2003), Hedychium spicatum (Jugran et al. 2011) and several species of Zingiberaceae (Ibrahim et al. 1996). The aim of this study was to determine (i) the variation of isozymic pattern of ginger (Zingiber officinale Rosc.) from three different varieties from two different populations, Wonogiri and Kulonprogo, Indonesia, differentiated by color of rhizomal peels and size (small red, small white, and big white) and (ii) the genetic relationship between populations based isozymic pattern. Materials and methods Plants material. Three different varieties of ginger (Zingiber officinale Rosc.), i.e. big white, small white, and small red gingers (Figure 1), were collected from the dry fields in Eromoko, Wonogiri, Central Java and Kokap, Kulonprogo, Yogyakarta, Indonesia, each location with three varieties and for each variety three accessions were collected. The rhizome was harvested at the end of the dry season at the age of about 9-10 months and stored for 2-3 months before investigated. The plant materials were authenticated at the Department of Biology, Sebelas Maret University, Surakarta, Indonesia. Enzyme dye systems

were esterase (EST) and peroxidase (PER, PRX), and separated on a polyacrylamide gel. Procedures. The electrophoresis procedure refers to Crawford (1990) and Weeden and Wendel (1989), and modified by Suranto (1991). Planting of rhizomes. The rhizome was placed in a plastic tray lined with wet paper or cloth to keep moisture, until the leavesgrowalong2-5mm. Shots were cut and immediately used for examining, or could be stored in a refrigerator at 4°C for maximum 14 days, but effectively used within seven days after cutting. Leaf extracts that stored in a refrigerator at 4°C can survive for 30 days. Buffer. Tank buffer was made by dissolving 14.4g of boric acid and 31.5g of borax (sodium borate), in distilled water to a volume of 2L. Extraction buffer was made by dissolving 0.018 g of cysteine, 0.021 g of ascorbic acid, and 5 g of sucrose (PA) in 20 mL of borax buffer pH 8.4. Running buffer was TAE (Tris-Acetic Acid-EDTA) 50x diluted to a concentration of 1x. Preparation of gel. First stock solution: 27.2 g Tris and 0.6 g sodium dodecyl sulfate (SDS) dissolved in 120 mL of distilled water, adjusted to pH 8.8 by adding HCl, then added distilled water up to 150 mL. Second stock solution: 9.08 g Tris and 0.6 g SDS dissolved in 140 mL of distilled water, adjusted to pH 6.8 to 7.0 by adding HCl, then added distilled water up to 150 mL. Third stock solution: 175.2 g of acrylamide and 4.8 g bis-acrylamide dissolved in 400 mL of distilled water and then make up to 600 mL. Loading dye: 250 uL of glycerol and 50 uL bromphenol blue (BPB) dissolved in 200 uL of distilled water. Separating gel: 3.15 mL of the 1st stock solution and 5.25 mL of the 2nd stock solution, added to 4.15 mL of distilled water, 5 uL of TEMED, and 10 uL of APS 10% (new). The mixed solution was poured into the mold, then added with saturated isobutanol. When, the gel was formed (∼45 minutes), saturated isobutanol was absorbed by blotting paper. Stacking gel: 1.9 mL of the 2nd stock solution and 1.15 mL of the 3rd stock solution, added to 4.5 mL of distilled water, 5 uL TEMED, and 10 uL APS 10% (new). Stacking gel was poured above the separation gel, fitted with a comb to make wells that released after gel formation. The formed gel was transferred into the clamping frame and put in a buffer tank, then filled with running buffer until submerged.

Figure 1.Rhizomeofgingervarieties. A.Small red ginger, B.Small white ginger, C. Big white ginger. (photo: Anna Frodesiak)

N U SAN TA RA B IOSC IE NC E 6 (1): 86-93, May 2014

Extraction. Fresh leaf tissue was put in the extraction buffer, with a ratio of 1:4 (w/v), i.e. 68 ug (0.068 g) of leaf samples were pulverized in 272 uL (0.272 mL) of extraction buffer. Crushed in a porcelain dish that was placed above ice crystals, to keep cold (4°C). Samples were centrifuged at 8500rpm for20minat 4°C, then soaked in ice crystals. Supernatant was put in the wells of the gel. Electrophoresis. 3.5 uL supernatant was pipetted with a micro pipette, added with loading dye and sample loading guide, and then placed in the wells. The electrophoresis were undertaken at 200 volts, 60 mA for 5 minutes to reach the separating gel, and followed by further electrophoresis at 150V, 400 mA, for 60 minutes, until the loading dye reaches ∼56 mm from the wells toward anode. The gel was transferred into a plastic tray and colored with enzyme dyes. Staining. Peroxidase: 0.0125g of O-Dianisidine put into erlenmeyer and dissolved to 2.5 mL of acetone, and then added to 50 mL of 0.2M acetate buffer pH 4.5 and two drops of H2O2. Extras: 0.0125g of α-Naphthyl acetate put into erlenmeyer and dissolved in 2.5 mL of acetone, and then added to 50 mL of 0.2M phosphate buffer pH 6.5 and 0.0125 g Fast Blue BB Salt. Separately, the gel was soaked into that solution for 10 minutes and shaken gently every 2 minutes. Once the banding pattern appears, the enzyme dye was disposed and rinsed with distilled water, then recorded by a camera or scanner. Data analysis. Data were analyzed qualitatively based on the presence or absence of bands. Only clear, unambiguous and reproducible bands were considered for data analysis. Rf (retardation factor) value was calculated based on the relative movement of isozyme and loading dye. The present bands were given a value of 1, while those absence bands were given a value of 0. Data were entered in a spreadsheet to create a binary matrix. The genetic similarity and genetic distance among the accessions was calculated according to Jaccard coefficients (Jaccard 1908). The matrices were computed and the corresponding dendrograms of genetic relatedness were constructed by applying the unweighted pair group method with arithmetic mean (UPGMA) clustering algorithm (Sneath and Sokal 1973), using the Dendro-UPGMA program (Garcia-Vallvé and Puigbo 1999; Garcia-Vallve et al. 2002). Results and discussion Variation of isozymic pattern Ginger samples used in this study had a relatively uniform condition; they were all harvested at maturity (910 months), stored for 2-3 months, and then planted to get the shoot tip. Uniformity stage of plant development is very important to ensure similar types of enzymes produced. Physiological developmental stages of an accession can produce different types of enzymes, as observed in the stages of embryogenesis (Bapat et al. 1992; Rout and Das 1995; Dodeman and Ducreux 1996), fruit development (Loveless 1992; Sadka et al. 2000), and colorizing plants (Barrett and Shore 1989). Isozyme pattern may also change in response to environmental condition, which followed by genetic material changes (Kahler et al. 1980; Mastenbroek et al. 1984; Guse et al. 1988; El-Bendary et al. 1998; Li Z and Rutger 2000; Gämperle and Schneller 2002; Oja 2002;

Staszak et al. 2007; Zhang et al. 2009; Dasgupta et al. 2010; Shah and Nahakpam 2012). Figure 2 showed examples of zimogram that demonstrate the diversity of ginger by esterase and peroxidase banding pattern. A

Figure 2. Zimogram of ginger using enzyme staining systems of esterase (A) and peroxidase (B).

Esterase (EST). Esterases (EC no. 3.1.x) are hydrolyzing enzymes which catalyze the addition or removal of water in biological reactions by splitting esters into acids and alcohols in a chemical reaction with water that called hydrolysis. Variation in esterases banding patterns exists due to differences in substrate specificity, protein structure and biological function. Wide variation in esterases are reported in nature and occurs frequently in plants (IUBMB 1992). In this observation, there were not many variations of esterase banding pattern among different ginger cultivars. Esterase isozyme indicated only two bands EST-1 located at Rf 0.10 and EST-2 located at Rf 0.04 from anodal zone, with purple-blue to red color (Figure 3.A). Some bands are fairly thick, but there are also thin bands or very thin, thus overlooked in the research. The difference of isozyme banding thickness is probably due to the differences in the copy number of the gene. On the other hand, a thick band may also be caused by two bands coincide, which indicates heterozygote for two alleles of the monomer, and a thin band indicating homozygote, however, esterase generally have thick bands. The combination of the presence esterase isozymic banding produced four variations of the 18th accessions tested. In accession 3, a small red ginger of Wonogiri, a band of EST-1 at Rf 0.04 was very thick, while in the other accessions was very thin or absent. The existence of unique band was very valuable as a marker for identification of the accession and useful in conservation biology. Perhaps the difference in staining intensity may be related to certain agronomic important traits, but this must be confirmed. On the other ginger family, esterases are also generating a little variation in isozymic banding patterns. Application of esterase on some species of Curcuma only produced 1-4 bands depending on the species (Apavatjrut et al. 1999), while in some species of Amomum this enzyme raised only one band, and could not show the variation among species (Shanmugapriya and Prabha 2012). The limited number of banding patterns caused low variation among populations.

SETYAWAN et al. –The isozymic pattern of ginger

→ ROH + R'OH. For many of these enzymes the optimal A substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides. Peroxidases can contain a heme cofactor in their active sites, or alternately redoxactive cysteine or seleno-cysteine 0.10 residues (IUBMB 1992). 0.04 In this study, peroxidase indicated six isozymic bands, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 PRX-1 at Rf 0.04, PRX-2 at Rf 0.05, PRX-3 at Rf 0.09, PRX-4 at Kulonprogo Wonogiri Kulonprogo Wonogiri Kulonprogo Wonogiri 0.10, PRX-5 at Rf 0.11, andPRXSmall red ginger Small white ginger Big white ginger 6 at Rf 0.15 with red color (Figure 3.B). Of the 18 + accessions tested, as many as 8 B variations of the isozyme banding pattern appear; hence the character is very valuable as distinguishing evidence. Two unique bands were found in 0.15 accession number 5 (small red ginger of Kulonprogo) at Rf 0.09, 0.10 and 0.11. The band at Rf 0.9 was 0.05 only found in accession number 5, while the band at Rf 0.11 was also detected in accession number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 3, 6 (small red ginger), 11 and 12 Wonogiri Kulonprogo Kulonprogo Kulonprogo Wonogiri Wonogiri (small white ginger). These bands Small red ginger Small white ginger Big white ginger were not detected in the other accessions. The band at Rf 0.9 Figure 3. Isozymic banding pattern of ginger varieties. A. Esterase, B. Peroxidase. Note: can be used as genetic markers 1,2,3. Small red ginger of Wonogiri; 4,5,6. Small red ginger of Kulonprogo; 7,8,9 Small for accession 3. Peroxidase white ginger of Wonogiri; 10,11,12. Small red ginger of Kulonprogo; 13,14,15. Big white created many variations of ginger of Wonogiri; 16,17,18. Big white ginger of Kulonprogo. isozymic banding patterns. In higher plants, peroxidase has a wide distribution and exhibit broad substrate specificity. On The results showed that among the three accessions in other ginger family, namely Amomum, peroxides are one population (the same varieties and locations), two or all generating four banding patterns. It is much more than three have in common esterase isozymic pattern, but the esterase that creating one band only (Shanmugapriya and same pattern can also be found precisely in accessions of Prabha 2012). different varieties and locations. It was shown that there The presence of many peroxides patterns caused more was ginger uniting each other based on the location, i.e. varied relationship. The isozyme can be used to test genetic Wonogiri and Kulonprogo or on the variety, i.e. big white variability between accessions within a species. It caused ginger, small white ginger, and small red ginger. But this accession of the same population, i.e. similar in variety and tendency was disguised by grouping of different location planting location, able to have a different isozymic pattern, and a variety. A small number of band pattern emerged and thus the relationship would be in a different situation, even it supported this complication. This is most likely because though there could be a tendency that the same accessions it was taken from the same location, and ginger is always of a same population tend to have a closer relationship than propagated vegetatively thus genetically similar. It might accession of different population. be caused by the history of evolution of the three gingers. They were made up divergently, thus they changed each Phylogenetic relationship other genetic code, or the emerging morphological Esterase (EST). The esterase only generated two difference might be being coded by genetic material, which isozymic bands at Rf 0.04 and 0.10. Variations between has no relation with esterase enzyme. populations are very little, hanya terdapat dua cluster. In Peroxidase (PER, PRX). Peroxidases (EC no. dendrogram, many accessions join at the similarity index 1.11.1.x) are a large family of enzymes that typically up to 100%, although different varieties and locations of catalyze a reaction of ROOR' + electron donor (2 e-) + 2H+ +

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origin. Except for accession 3, all accessions of ginger of Wonogiri, both small red ginger (1,2), small white ginger (7, 8, 9), and big white ginger (13,14,15), and also one accession of small white ginger of Kulonprogo (12) and big white ginger of Kulonprogo (16, 17, 18) join with similarity index up to 100%, although these populations are from different varieties and locations of origin. Other accession joins a cluster with a similarity index of up to 100%, i.e. one small red ginger of Wonogiri (3), all accessions of small red ginger of Wonogiri (4,5,6) and two small white ginger of Kulonprogo (10, 11). In this cluster, some varieties the same accessions join, but a different site of origin, i.e. small red ginger of Wonogiri (3) and of Kulonprogro (4,5,6). There is also the accession of the same origin, but different varieties, i.e. all small red ginger (4,5,6) and two small white ginger (10,11) of Kulonprogo. However, ginger that derived from the same varieties and location generally have a very strong relationship with similarity index up to 100%, for example, all populations of the small red ginger of Kulonprogo (4, 5, 6), big white ginger of Wonogiri (13, 14, 15), small white ginger of Wonogiri (7, 8, 9), and big white ginger of Kulonprogo (16,17,18) (Table 1A; Figure 4.A). The dendrogram showed that among the three accessions of ginger in one variety and one location, the two or all of them have a same esterase banding pattern up to 100%, but a same pattern could be found precisely with similarity index up to 100% on accession from different variety and location (Figure 4.A). Since, esterase only generate two isozymic banding patterns, many different varieties and location of ginger joint together in a cluster, that less reflects the grouping based on variety and location. Peroxidase (PER, PRX). The peroxidase isozyme generated six bands with Rf value of 0.04, 0.05, 0.09, 0.10, 0.11, and 0.15. Peroxidase has much more variation of isozymic pattern. Relationship dendrogram shows that there are only two independent ginger accessions, where the similarity index with the other accession less than 100%, i.e. the accession small red ginger of Kulonprogro (5) and the accession small white ginger of Kulonprogro (11). Meanwhile, other accessions have in similarity index up to 100% with one or more other accessions. There is a large cluster with similarity index 100% consisting of various varieties of ginger, i.e. three accessions small red ginger of Wonogiri (1, 2, 3) and one of Kulonprogo (6), one accessions small red ginger of Wonogiri (7) and two accessions small red ginger of Kulonprogo (14, 15). There is also a cluster with % similarity index 100that contains one accession small red ginger of Kulonprogo (4), two accessions small white ginger of Wonogiri (8, 9), one accession small red ginger of Kulonprogo (10), and one accession big accession white ginger of Wonogiri (13). Meanwhile, two ginger accessions of Kulonprogo from different varieties have up to similarity index 100%, i.e. one accession small white ginger (12) and one accession of big white ginger (18). On the other hand, two big white ginger accessions of Kulonprogo (16, 17) form a separate cluster. Those accessions are generally still joined in one group, although one of them is a separate accession. Population ginger that still collects all members in the group with

similarity index 100% are the small red ginger of Wonogiri (1,2,3). Meanwhile, on the other population, there are only two accessions clustered together with similarity index 100%, while the others are separated, for example, small red ginger of Wonogiri (1,2, 3), small white ginger of Wonogiri (8,9), the big white ginger of Wonogiri (14,15), and big white ginger of Kulonprogo (16,17) (Table 1.B; Figure 4.B). Grouping generally occurs in accessions that have same varieties and locations of origin, but it may also occur in accessions that have different varieties and locations of origin. This because, ginger generally propagated vegetatively and have close relationships, although differ on varieties or locations of origin. Meanwhile, the existence of stand alone accessions, i.e. the accession small red ginger of Kulonprogro (5) and the accession small white ginger of Kulonprogro (11), shows the existence of evolutionary process, i.e. changes in the genetic composition of vegetative propagation of ginger. This mutation is important for survival against environmental changes. Combination of esterase and peroxidase. The dendrogram, which based on the combination of esterase and peroxidase markers, is clearer in showing the grouping based on its variety, locations of origin or both. Grouping based on location can be found either in ginger population of Wonogiri or Kulonprogo. In Wonogiri, two accessions red gingers (1, 2), one accession small white gingers clustered with two accessions big white gingers (14, 15) with similarity index 100%. Meanwhile, two small red ginger accessions from different locations, i.e. Wonogiri (3) and Kulonprogo (6) is also clustered with the similarity index 100%. Finally, the two groups joined. In Kulonprogo, the accession small white ginger (12) and big white ginger (18) which has similarity index 100%, and the two accessions big white ginger (16, 17) which also has similarity index 100% joined in one cluster. On the other hand, two accessions of different variety are also clustered with the similarity index 100%, i.e. a small red ginger (4) and a small white ginger (10). In Wonogiri, two accessions small white ginger (8, 9) and one accession big white ginger (13) also integrates with similarity index 100%. Finally, the two groups joined (Table 2; Figure 4.C). Relationship dendrogram shows that the more data that is used as a distinguishing character, the clearer the grouping based on the variety and growing location. On esterase-based dendrogram, it appears that the groupings based on population is less reflected, while on peroxidasebased dendrogram, the presence of grouping in population is more obvious. In the dendrogram based on the combined data of esterase and peroxidase, the population with which the members fall into one group is increasing. Meanwhile, as in peroxidase-based dendrogram, it is shown that in the population, the two members congregate but the other one apart. In the combined dendrogram accession 5 and 11, each of them tends to stand alone. The existence of such accessions is very valuable for plant breeding, because of these differences reflect a typical mutation, making it very useful in adapting to the climate and environment changes, such as global warming and land use change (Table 2).

SETYAWAN et al. â&#x20AC;&#x201C;The isozymic pattern of ginger

Table 1. Matrix of similarity computed with Jaccard coefficient on three varieties of ginger from Wonogiri and Kulonprogo, Indonesia based on esterase and peroxidase isozymic pattern. A. Similarity matrix of esterase banding pattern 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 1 1.000 0.500 0.500 0.500 0.500 1.000 1.000 1.000 0.500 0.500 1.000 1.000 1.000 1.000 1.000 1.000 1.000 2 1.000 1 0.500 0.500 0.500 0.500 1.000 1.000 1.000 0.500 0.500 1.000 1.000 1.000 1.000 1.000 1.000 1.000 3 1.000 1.000 1 1.000 1.000 1.000 0.500 0.500 0.500 1.000 1.000 0.500 0.500 0.500 0.500 0.500 0.500 0.500 1 1.000 1.000 0.500 0.500 0.500 1.000 1.000 0.500 0.500 0.500 0.500 0.500 0.500 0.500 4 0.750 0.750 0.750 5 0.667 0.667 0.667 0.500 1 1.000 0.500 0.500 0.500 1.000 1.000 0.500 0.500 0.500 0.500 0.500 0.500 0.500 1 0.500 0.500 0.500 1.000 1.000 0.500 0.500 0.500 0.500 0.500 0.500 0.500 6 1.000 1.000 1.000 0.750 0.667 7 1.000 1.000 1.000 0.750 0.667 1.000 1 1.000 1.000 0.500 0.500 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1 1.000 0.500 0.500 1.000 1.000 1.000 1.000 1.000 1.000 1.000 8 0.750 0.750 0.750 1.000 0.500 0.750 0.750 9 0.750 0.750 0.750 1.000 0.500 0.750 0.750 1.000 1 0.500 0.500 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1 1.000 0.500 0.500 0.500 0.500 0.500 0.500 0.500 10 0.750 0.750 0.750 1.000 0.500 0.750 0.750 1.000 1.000 11 0.500 0.500 0.500 0.667 0.333 0.500 0.500 0.667 0.667 0.667 1 0.500 0.500 0.500 0.500 0.500 0.500 0.500 1 1.000 1.000 1.000 1.000 1.000 1.000 12 0.500 0.500 0.500 0.667 0.333 0.500 0.500 0.667 0.667 0.667 0.333 13 0.750 0.750 0.750 1.000 0.500 0.750 0.750 1.000 1.000 1.000 0.667 0.667 1 1.000 1.000 1.000 1.000 1.000 1 1.000 1.000 1.000 1.000 14 1.000 1.000 1.000 0.750 0.667 1.000 1.000 0.750 0.750 0.750 0.500 0.500 0.750 15 1.000 1.000 1.000 0.750 0.667 1.000 1.000 0.750 0.750 0.750 0.500 0.500 0.750 1.000 1 1.000 1.000 1.000 1 1.000 1.000 16 0.750 0.750 0.750 0.500 0.500 0.750 0.750 0.500 0.500 0.500 0.250 0.667 0.500 0.750 0.750 17 0.750 0.750 0.750 0.500 0.500 0.750 0.750 0.500 0.500 0.500 0.250 0.667 0.500 0.750 0.750 1.000 1 1.000 1 18 0.500 0.500 0.500 0.667 0.333 0.500 0.500 0.667 0.667 0.667 0.333 1.000 0.667 0.500 0.500 0.667 0.667 B. Similarity matrix of peroxidase banding pattern Note: 1,2,3. Small red ginger of Wonogiri; 4,5,6. Small red ginger of Kulonprogo; 7,8,9 Small white ginger of Wonogiri; 10,11,12. Small white ginger of Kulonprogo; 13,14,15. Big white ginger of Wonogiri; 16,17,18. Big white ginger of Kulonprogo. Table 2. Matrix of similarity and distance computed with Jaccard coefficient on three varieties of ginger from Wonogiri and Kulonprogo, Indonesia based on a combination of isozymic pattern of esterase and peroxidase. Similarity matrix computed with Jaccard coefficient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 1.000 0.833 0.667 0.625 0.833 1.000 0.800 0.800 0.667 0.500 0.600 0.800 1.000 1.000 0.800 0.800 0.600 2 0.000 0.833 0.667 0.625 0.833 1.000 0.800 0.800 0.667 0.500 0.600 0.800 1.000 1.000 0.800 0.800 0.600 3 0.167 0.167 0.833 0.750 1.000 0.833 0.667 0.667 0.833 0.667 0.500 0.667 0.833 0.833 0.667 0.667 0.500 0.625 0.833 0.667 0.800 0.800 1.000 0.800 0.600 0.800 0.667 0.667 0.500 0.500 0.600 4 0.333 0.333 0.167 5 0.375 0.375 0.250 0.375 0.750 0.625 0.500 0.500 0.625 0.500 0.375 0.500 0.625 0.625 0.500 0.500 0.375 0.833 0.667 0.667 0.833 0.667 0.500 0.667 0.833 0.833 0.667 0.667 0.500 6 0.167 0.167 0.000 0.167 0.250 7 0.000 0.000 0.167 0.333 0.375 0.167 0.800 0.800 0.667 0.500 0.600 0.800 1.000 1.000 0.800 0.800 0.600 1.000 0.800 0.600 0.750 1.000 0.800 0.800 0.600 0.600 0.750 8 0.200 0.200 0.333 0.200 0.500 0.333 0.200 9 0.200 0.200 0.333 0.200 0.500 0.333 0.200 0.000 0.800 0.600 0.750 1.000 0.800 0.800 0.600 0.600 0.750 0.800 0.600 0.800 0.667 0.667 0.500 0.500 0.600 10 0.333 0.333 0.167 0.000 0.375 0.167 0.333 0.200 0.200 11 0.500 0.500 0.333 0.200 0.500 0.333 0.500 0.400 0.400 0.200 0.400 0.600 0.500 0.500 0.333 0.333 0.400 0.750 0.600 0.600 0.750 0.750 1.000 12 0.400 0.400 0.500 0.400 0.625 0.500 0.400 0.250 0.250 0.400 0.600 13 0.200 0.200 0.333 0.200 0.500 0.333 0.200 0.000 0.000 0.200 0.400 0.250 0.800 0.800 0.600 0.600 0.750 1.000 0.800 0.800 0.600 14 0.000 0.000 0.167 0.333 0.375 0.167 0.000 0.200 0.200 0.333 0.500 0.400 0.200 15 0.000 0.000 0.167 0.333 0.375 0.167 0.000 0.200 0.200 0.333 0.500 0.400 0.200 0.000 0.800 0.800 0.600 1.000 0.750 16 0.200 0.200 0.333 0.500 0.500 0.333 0.200 0.400 0.400 0.500 0.667 0.250 0.400 0.200 0.200 17 0.200 0.200 0.333 0.500 0.500 0.333 0.200 0.400 0.400 0.500 0.667 0.250 0.400 0.200 0.200 0.000 0.750 18 0.400 0.400 0.500 0.400 0.625 0.500 0.400 0.250 0.250 0.400 0.600 0.000 0.250 0.400 0.400 0.250 0.250 Distance matrix based on Jaccard coefficient Note: 1,2,3. Small red ginger of Wonogiri; 4,5,6. Small red ginger of Kulonprogo; 7,8,9 Small white ginger of Wonogiri; 10,11,12. Small white ginger of Kulonprogo; 13,14,15. Big white ginger of Wonogiri; 16,17,18. Big white ginger of Kulonprogo.

This study still requires further study to obtain a more stable marker accuracy, but the identification result has been added the information on genetic diversity of ginger for the selection and for the improvement of varieties. Further research with other chromosomal data, such as karyotype and molecular cytogenetic will greatly assist the identification of ginger diversity, especially when they are combined with DNA sequence data. Isozymes were only able to detect the genetic diversity in the introns, which was then translated into protein material. This led to the ability of isozyme, as

distinguishing characteristics are relatively limited, given the parts of introns that are not translated are undetectable for its diversity. Some researchers began to leave isozymes as genetic markers and test its genetic diversity right on the DNA. Results showed that ginger undergoes genetic variation due to a wide range of ecological conditions. This investigation was an understanding of genetic variation within the accessions. It will also provide an important input into determining resourceful management strategies and help to breeders for the ginger improvement program.

N U SAN TA RA B IOSC IE NC E 6 (1): 86-93, May 2014

Figure 4. The dendrogram relationship of ginger varieties based on isozymic banding patterns of esterase (A), peroxidase (B) and combination of esterase and peroxidase (C). Note: 1,2,3. Small red ginger of Wonogiri; 4,5,6. Small red ginger of Kulonprogo; 7,8,9 Small white ginger of Wonogiri; 10,11,12. Small white ginger of Kulonprogo; 13,14,15. Big white ginger of Wonogiri; 16,17,18. Big white ginger of Kulonprogo. Note: small red ginger, small white ginger, big white ginger; Kulonprogo ginger, Wonogiri ginger.

In conclusion, variation was observed in esterases and peroxidases in the three varieties of ginger. Esterase generated two isozyme bands, at the Rf value of 0.04 and 0.10, while peroxidase generated six isozyme bands, at the Rf value of 0.04, 0.05, 0.09, 0.10, 0.11, and 0.15. Accessions 3 has specific isozyme banding, on the other hand, accession 5 has the most specific peroxidase banding. Dendrogram on esterase generated 4 groups of 18 accessions; while based on peroxidase, there were 15 groups. Peroxidase produces more numerous and more diverse isozymic bands than esterase, resulting in a more complex relationship. The amount of data that is used to compile dendrogram affects the grouping, the more data used, the more obvious clustering of accessions in a population. ACKNOWLEDGEMENTS The part of the work was supported by the DGHE Republic of Indonesia with research grant no. 022/LIT/BPPK-SDM/IV/2002. The authors thank to Dr. Molide Rizal of Balittro Bogor, Indonesia for valuable discussion on ginger diversity. The authors also thank to Suhar Iriyanto and Delima Susanti Roesyat for his generosity in providing some plant materials, and Setyanto and Asriyati Asih Wardani for the kindly assistance with the laboratory work.

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N U SAN TA RA B IOSC IE NC E

ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060115

Vol. 6, No. 1, pp.94-101 May 2014

Short Communication:Comparisons of isozyme diversity in local Java cardamom (Amomum compactum) and true cardamom (Elettaria cardamomum) 1

AHMAD DWI SETYAWAN1,â&#x2122;Ľ, WIRYANTO1, SURANTO1, NURLIANI BERMAWIE2, SUDARMONO3 Department of Biology, Faculty of Mathematics and Natural Sciences, Sebelas Maret University, Jl. Ir. Sutami 36a, Surakarta 57126, Central Java, Indonesia. Tel./fax.: +62-271-663375,â&#x2122;Ľemail: volatileoils@gmail.com 2 Indonesian Medicinal and Aromatic Crops Research Institute, Jl. Tentara Pelajar No.3, Cimanggu, Bogor 16111, West Java, Indonesia. 3 Bogor Botanic Garden, Indonesian Institute of Sciences, Jl. Ir. H. Juanda No.13, Bogor16122, West Java, Indonesia. Manuscript received: 13 February 2014. Revision accepted: 28 April 2014.

Abstract. Setyawan AD, Wiryanto,Suranto, Bermawie N, Sudarmono. 2014. Comparisons of isozyme diversity in local Java cardamom (Amomum compactum) and true cardamom (Elettaria cardamomum). Nusantara Bioscience 6: 94-101.Fruits of Java cardamoms (Amomum compactum) and true cardamoms (Elettaria cardamomum) had long been used as spices, flavoring agent, garnishing plants etc. This research was conducted to find out: (i) variation of isozymic bands in some population of Java cardamoms and true cardamoms; and (ii) phylogenetic relationship of these cardamoms based on variation of isozymic bands. Plant material (i.e. rhizome) of Java cardamoms was collected from Bogor Botanical Garden, and plant material of true cardamoms was gathered from Indonesian Medicinal and Aromatic Crops Research Institute, Bogor, Indonesia. Ten accessions were assayed in every population. The two isozymic systems was assayed, namely esterase (EST) and peroxidase (PER, PRX). Phylogenetic relationship was determined by UPGMA method. The results showed that esterase gave nine isozymic bands, i.e. Rf 0.15, 0.26, 0.29, 0.33, 0.38, 0.42, 0.45, 0.53, and Rf 0.58., while peroxidase gave 10 isozymic bands, i.e. Rf 0.06, 0.14, 0.18, 0.22, 0.26, 0.29, 0.32, 0.37, 0.41, and Rf 0.46. Relationship dendrogram indicated that the number of data will affect the grouping based on species similarity; and more data was increasingly apparent in groupings; within these groups there were variations among its members. Key words:Amomum compactum, cardamoms,Elettaria cardamomum, isozyme, Zingiberaceae

INTRODUCTION Cardamom is one of the most expensive and most pleasantly scented spices in the world (after saffron and vanilla). Cardamom is used as spices, condiments, perfumes, cosmetics, traditional medicine, pharmaceutical, food and beverage (Heyne 1950).Cardamom produced by three genera of Zingiberaceae, namely Amomum (four of 176 sp.),Aframomum (three of 54 sp.), and Elettaria (one of 11 sp.). World markets provide three types of cardamoms, i.e. green, black and Madagascar cardamoms. Green cardamom (or true cardamom) is produced by Elettaria cardamomum var. cardamomum, which is native to southern Asia. It is the most sought after of all the species and the most expensive, where 80% of the world market fulfilled. Green cardamom of Sri Lanka (Ceylon cardamom) is produced by less quality of E. cardamomum var. major. Black cardamomis produced by Amomum, which has distribution from India, China and Southeast Asia to Australia, includes Amomum aromaticum (Bengali cardamom), A. compactum (Java, round, or Siam cardamom), A. subulatum (Greater Indian orNepal cardamom) and A. testaceum (Cambodian cardamom). Madagascar or Cameroon cardamom is produced by Aframomum angustifolium, which is the most distributed cardamoms of tropical Africa. Aframomum corrarima (Korarima cardamom) and A. melegueta (grains of paradise or Guinea grains) are other cardamoms of

Africa (Heyne 1950; Backer and Bakhuizen v.d. Brink 1968; Purseglove 1972; Purseglove et al. 1981; Wollf and Hartutiningsih 1999; Wardini and Thomas 1999; Peter 2001; Duke et al. 2002). In Indonesia, there are two types of cardamom, i.e. local Java cardamom (Amomum compactum Soland ex. Maton.) and true cardamom (Elettaria cardamomum(L.) Maton.; syn. Amomum cardamomum L.). Java cardamom is an endemic species of West Java and is now cultivated throughout Southeast Asia and South China. Meanwhile, true cardamom comes from Malabar Mountains of western India and introduced to Indonesia since the 1920s, and is cultivated commercially in 1986 (Heyne 1950; Seidemann 2005).Most Indonesian farmers cultivate Java cardamom. True cardamom is less successfully cultivated because of differences in microclimate and soil factors, except in Tasikmalaya of West Java, Kulonprogro of Yogyakarta and West Sumatra (Santoso 1988; Madjo-Indo 1989). True cardamom has better aromatic quality because of higher content of volatile oil (5-8%), and Java cardamom has less oil (2-3.5%) (Santoso 1988). Other literatures stated that volatile oil of true cardamomare 3.5-7% (Guenther1952), 2-8% (Hegnauer 1963; Purseglove 1972), 3-7%(Youngken 1948), or 2.8-6.2% (Trease and Evans 1978). The results were confirmed by Setyawan (2000), where essential oil content of true cardamom is higher (2.25%) than the Java cardamom (1.5%). Genetic,

SETYAWAN et al. – The isozymic diversity of cardamoms

environmental condition, influenced oil yield.

method

oil

extraction

Figure 1. The morphology of A. compactum: A. Clumps, B. Flower, C. Fruits, D. Seeds (pods); and E. cardamomum: E. Clumps, F. Flower, G. Fruits, H. Seeds (pods). (photos from many sources)

The major components of true cardamom seeds are αterpinyl acetate (∼40%), 1,8-cineole (20-25%), and linalool (5-6%) (Marongiu et al. 2004; Kuyumcusavan and Kucukbay2013). The other components include borneol, linalyl acetate, limonene, linalool, α-terpinen, terpinolene, and myrcene (Marongiu et al. 2004). While, the major components of volatile oils of Java cardamom is cineole (60-80%). The other components include α-pinene, βpinene, camphene, limonene, ρ-cymene, α-terpineol and αhumulene (Yu et al. 1982; Feng et al. 2011). True cardamom is contains more α-terpinyl acetate than Java cardamom, a valuable fragrant liquid ester. True cardamom and Java cardamom can be distinguished easily by morphological characteristics. True cardamom plant is higher (1.5-4 m) than Java cardamom (1-2.5 m). The base of true cardamom stem is light green, while Java cardamom is reddish green. True cardamom leaves are lancet-oblong (tapered leaf tip and base); while Java cardamom leaves are lancet. Inflorescence of true cardamom is raceme or botrys, while the Java cardamom is capitulum. Flower stalk of true cardamom has a length of 50-120 cm; spread on the soil surface (Mysore cultivar) or upright (Malabar cultivar). Flower stalk of Java cardamom is very short, as if no stalk at all. The true cardamom pods are green, while the Java cardamom pods are reddish-white and became brownish-black when dry. True cardamom

pods are oblong-triangular, while the Java cardamom pods are round and slightly flattened (Santoso 1988; Madjo-Indo 1989)(Figure 1). Researchofcardamomsvariabilityusingisozymic pattern has not beendone yet, except for Amomum aromaticum, A. cannicarpum, A. kingii, and A. subulatum (Shanmugapriya andPrabha2012), while thesame research on other gingers family had been conducted to Boesenbergia(Vanijajiva et al. 2003), Curcumaalismatifolia(Paisooksantivatana 2001), C. aeruginosa, C. elata, C. rubescens, C. zedoaria (Apavatjrut et al. 1999),C. chuanhuangjiang, C. kwangsiensis,C. phaeocaulis, C. wenyujin (Tang et al. 2008; Deng et al. 2011), C. sichuanenesis (Deng et al. 2011), C.longa(Shamina et al. 1998; Deng et al. 2011), C.xanthorriza(Apavatjrut et al. 1999; Azizah 2011), Hedychiumspicatum(Jugran et al. 2011), Zingiber officinale (Shamita1997; Setyawan et al. 2014 in this issue), and a few other species of Curcuma (ChokthaweepanichandPaisooksantivatana2003) and other species of Zingiberaceae(Ibrahim et al. 1991; 1996). The aim of this studyis to determine(i) the isozymic diversity oflocal Java cardamom (Amomum compactum Soland ex. Maton.) and true cardamom (Elettaria cardamomum(L.) Maton.) based on isozymicpattern of esterase and peroxidase, and (ii) thegenetic

N U SAN TA RA B IOSC IE NC E 6 (1): 94-101, May 2014

96 relationshipbetween isozymicpatterns.

two

speciesbased

the

Materials and methods Plants material. Plant material (i.e. rhizome) of Java cardamoms (Amomum compactum Soland ex. Maton.) was collected from Bogor Botanical Garden, and plant material of true cardamoms (Elettaria cardamomum) was gathered from Indonesian Medicinal and Aromatic Crops Research Institute (IMACRI/Balittro) Bogor, West Java, Indonesia. From each species, 10accessions wereassayed. A series of observations was also conducted on 10 accessions of Java cardamom from the Medicinal Plant and Traditional Medicine Research and Development Center (B2P2TOOT) Tawangmangu, Central Java, Indonesia, but they showed no variation (especially with peroxidase), thus it is ignored in the data analysis. They are probably the sibling of the same accession which is separated vegetatively (Figure 2.E-F). The rhizomewas harvestedfrom the twoyears old mature crops andthen planted. The plant materials were authenticated at the Department of Biology, Sebelas Maret University, Surakarta, Indonesia. Enzyme staining systems wereesterase(EST) andperoxidase(PER, PRX), and separatedon apolyacrylamide gel. Procedures. The electrophoresisprocedurerefers toCrawford(1990) andWeedenandWendel(1989) as modified bySuranto(1991). Planting ofrhizomes. The rhizomewas placedona plastictraylined withwetpaperorclothtokeep moisture, untilthe leaves grow and reach 2-5mm. Shootswere cutandimmediatelyusedfor examination, or were stored in a refrigeratorat 4°Cformaximum 14days, but can only beusedeffectivelywithin sevendaysaftercutting. Leaf extractsthat storedina refrigerator at4°Ccansurvivefor 30days. Buffer. Tank buffer was made by dissolving14.4g of boric acidand31.5g of borax (sodium borate), in distilled water to a volume of2L. Extractionbufferwas madeby dissolving0.018g ofcysteine, 0.021g ofascorbicacid, and5g ofsucrose(PA) in20 mL ofboraxbuffer at pH8.4. Runningbuffer wasTAE(Tris-Acetic Acid-EDTA)

50xdilutedto a concentration of1x. Preparation of gel. First stock solution: 27.2 g Tris and 0.6 g SDS dissolved in 120 mL of distilled water; it is adjusted to pH 8.8 by adding HCl, then is added with distilled water up to 150 mL. Second stock solution: 9.08 g Tris and 0.6 g SDS dissolved in 140 mL of distilled water, adjusted to pH 6.8 to 7.0 by adding HCl, then added distilled water up to 150 mL. Thirdstock solution: 175.2 g of acrylamide and 4.8 g bis-acrylamide are dissolved in 400 mL of distilled water and then make up to 600 mL. Loading dye: 250 uL of glycerol and 50 uL bromphenol blue (BPB) dissolved in 200 uL of distilled water. Separating gel: 3.15 mL of the first stock solution and 5.25 mL of the second stock solution, added with 4.15 mL of distilled water, 5 uL of TEMED, and 10 uL of APS 10% (new). The mixed solution was poured into the mold, then added with saturated isobutanol. When, the gel was formed (∼45 minutes), saturated isobutanol was absorbed by blotting paper. Stacking gel: 1.9 mL of the second stock solution and 1.15 mL of the third stock solution, added with 4.5 mL of distilled water, 5 uL TEMED, and 10 uL APS 10% (new). Stacking gel was poured above the separation gel, fitted with a comb to make wells, that was released after gel formation. The formed gel was transferred into the clamping frame and put in a buffer tank, then filled with running buffer until submerged. Extraction.Freshleaftissuewasput in theextractionbuffer, with a ratioof 1:4(w/v), i.e.68ug(0.068 g) ofleafsamples were pulverizedin272 uL (0.272 mL) of extractionbuffer. Then it is crushedin aporcelaindishthat was placedabove ice crystals, to keep it cold(4°C). Samples werecentrifuged at8500rpm for20minutes at 4°C, thensoaked inice crystals. Supernatant wasputin the wellsof gel. Electrophoresis.3.5 uL supernatant wasaddedwithloadingdyeandsampleloadingguide, and thenplacedinthe wells. Samples wereelectrophoresedat 200volts, 60mAfor 5minutesto reach theseparatinggel, andelectrophoresedat 150V, 400mA, for 60minutes, i.e. loadingdyereaches ∼56mmfromthe wellstowardanode. Gelwas transferred intoa plastictrayandcolored withenzyme dyes. Staining. Peroxidase: 0.0125g of O-Dianisidine was put into Erlenmeyer anddissolvedwith 2.5 mL ofacetone, and then was added with 50 mL of0.2Macetate bufferpH4.5and2drops ofH2O2. B Esterase:0.0125g ofα-Naphthyl acetate was put into Erlenmeyer anddissolvedwith 2.5 mL ofacetone, and then was added with 50 mL of0.2MphosphatebufferpH 6.5and0.0125gFastBlueBBSalt. D Separately, gelwas soakedto those solutionsfor 10minutesand was shakengentlyevery2minutes. F

Figure 2. Zimogram of cardamoms. Java cardamom (A) and true cardamom (B) stained with esterase. Java cardamom (C) and true cardamom (D) stained with esterase. Java cardamom of B2P2TOOT Tawangmangu stained with esterase (E) and peroxidase (F).

SETYAWAN et al. â&#x20AC;&#x201C; The isozymic diversity of cardamoms

Once thebanding patternappeared, the enzymedyewasdisposed and rinsed with distilled water, then it is recordedby a cameraorscanner. Data analysis. Only clear, unambiguous and reproducible bandings were considered for data analysis. Rf (retardantion factor) valuewas calculatedbased onthe relativemovementofisozymeandloadingdye. Present bandswere givena value of 1, while those absenceswere givena value of 0.Data were entered in a spreadsheet to create a binary matrix.The genetic similarity among the accessions was calculated according to Jaccard (1908) coefficients. The matrices were computed and corresponding dendrograms of genetic relatedness were constructed by applying un-weighted pair group method with arithmetic mean (UPGMA) clustering algorithm (Sneath and Sokal 1973), using the Dendro-UPGMA program (Garcia-VallvĂŠ and Puigbo 1999; Garcia-Vallve et al. 2002). Results and discussion Indonesia is home to at least 20 members of the genus ofAmomum, where one of them, Amomum compactum, has a high economic value and is commonly known as Java cardamom (Kasahara and Hemmi 1995). In international trade, the local cardamom got strong competition from true cardamom, Elettaria cardamomum, which originated and widely cultivated in India and Sri Lanka. True cardamom tends to be less suited to the microclimate and soil type in Indonesia. Therefore, it is necessary for genetic improvement of native Indonesian cardamom to compete with true cardamom which now dominates world trade. Identification and characterization of the diversity of cardamom is required to improve thequality and quantity of production.

0.50

Variationof isozymicpattern Esterase (EST). Esterase isozyme indicates the presence ofnine isozymic banding located at Rf 0.15, 0.26, 0.29, 0.33, 0.38, 0.42, 0.45, 0.53, and 0.58 (Figure 2-3). Bands are from pink to brown. The bands are generally very thin, thus it should be repeated 2-3 times to ensure consistency. This is in contrast with observations on the esterase isozyme of ginger, which bands arefrom purpleblue to red, and generally quite thick that they are easily observed (Setyawan et al. 2014). On the other hand, observations of esterase isozymic bands on the sample of Java cardamom taken from the collection of B2P2TOOT Tawangmangu just generate 2-3 clear bands from the total of 10 accessions. No variation between accessions is thought to occur because they are derived from vegetative propagation from the same parent. Vegetative propagation generally results in low diversity (Jia and Sun 2013). Therefore, this material is not used for further data analysis. In this research, the most frequent appeared bands are located at Rf 0.53, this band was not detected in one Java cardamomaccession, and two true cardamomaccessions. Another band appears quite often lies in Rf 0.45. This band appears on all accessions of true cardamom, but only appeared in one Java cardamomaccession. The most distinctive band because it only appears on one accession and quite thick is located at Rf 0.15. The bands belong to accessionno. 11 of Java cardamom. The band distribution pattern of this accessionis very specific that makes it one of the most distinctive accessions because its genetic diversity is relatively different from the other accessions. In this research, it is also found accessions having identical isozymic banding pattern, namely: two accessions of Java cardamom (5, 9) and three accessions of true cardamom (16, 17, 18). However, no two accessions of different species have identical band.

0.30

0.10

0.10 2

0.50

0.30

0.10

0.50

0.30

0.50

0.30

Figure 3. Schematic zimogram of cardamoms. Esterase isozymic pattern of Java cardamom (A) and true cardamom (B). Peroxidase isozymic pattern of Java cardamom (C) and true cardamom (D).

N U SAN TA RA B IOSC IE NC E 6 (1): 94-101, May 2014

The number of esterase bands that emerged in this research are quite many (nine bands) compared to some other studies. Esterase in higher plants usually gives only 3-4 isozymic bands. Esterase in some Amomum species raises only one isozymic band, and unable to demonstrate the existence of variation between species (Shanmugapriya and Prabha 2012). Two isozymic bandswere raised by application of esterase in three varieties of ginger, i.e. small white, large white and small red gingers (Setyawan et al. 2014). Application of esterase in several species of Curcuma are also only produces 1-4 isozymic bands depend on the species (Apavatjrut et al. 1999). Peroxidase (PER, PRX). Peroxidase leads to quite a lot of variation in isozymic bandings. Observation on peroxidase isozyme shows 10 bands, namely at Rf 0.06, 0.14, 0.18, 0.22, 0.26, 0.29, 0.32, 0.37, 0.41, and 0.46 (Figure 2-3). It forms reddish brown and relatively thick bandingsothat it is easily observed. However, from the collection of all accessions of Java cardamom from B2P2TOOT Tawangmangu, only two peroxidase bands appear with the same Rf,which indicate monomorphic (Figure 2.F). The application is repeated 2-3 times on these accessions but the resultswas consistently the same,soit is suspected that they derived from the same and homozygote mother plant. Peroxidase isozymic band obtained from samples collected from Tawangmangu is much thicker than the peroxidase isozymic bands obtained on samples from Java cardamomum of IMACRI Bogor or true cardamom from the Bogor Botanical Gardens.The environmental condition where the plants were grown may influence the peroxidase activity. Higher activity was found from samples collected from higher elevation. Tawangmangu located > 1000 m above sea level, while IMACRI and Botanical Garden were both in Bogor with elevation 250 m above sea level. In this research, each accession gives a fair amount of peroxidase isozymic bands, ranging from 3-5 bands per accession. Isozymic banding that appears in all of the accession lies in Rf 0.06, but the quality varies from one accession to another, namely: thin, medium and thick, thus the banding can still be used as distinguishing character in the preparation dendrogram. Other isozymic banding with a fairly high frequency lies in Rf 0.37. The band is present in all accessions of Java cardamom, and seven accessions of true cardamom. Isozymic banding with the lowest frequency is at Rf 0.22, which only presents on one accession of Java cardamom and two accessions of true cardamom. A fairly typical banding pattern is found in accession11 of Java cardamom and the accession16 of true cardamom; whose band pattern is different from other accessions. Meanwhile, some accessions have identical bands, i.e. three groups of accessions of Java cardamom (1, 2), (9, 10) and (3, 5, 7), as well as a group of accessions of true cardamom (16, 18). Similarly with the esterase bands, no two accessions of different species have identical peroxidase bands. In this research, the amount of peroxidase bands detected is 10 isozymic bands. In higher plants, the enzyme is usually only generate 4-6 bands. Research on Amomum suggests that this enzyme only gave 4 isozymic bandings

(Shanmugapriya and Prabha 2012). While, in the research on different varieties of ginger which are distinguished by the color of rhizome, these enzymes can bring 6 isozymic bandings (Setyawan et al. 2014). It was known that peroxidase always produce more isozymic bands than esterase do. This suggests that the reactions catalyzed by the peroxidase enzyme are more diverse than the reactions catalyzed by the esterase enzyme. Phylogenetic relationship Esterase (EST). Esterase enzyme gives a lot isozymic bands i.e. nine bandings which is located at Rf 0.15, 0.26, 0.29, 0.33, 0.38, 0.42, 0.45, 0.53, and 0.58. That large number of isozymic bands causes variety in the formation of dendrogram relationship. However, there are also accessions with identical band, i.e. the fouraccessions of Java cardamom in two groups, i.e. (1, 2) and (5, 9) and three accessions of true cardamom (16, 17, 18), thus that the dendrogram indicates the absence of distance (Figure 4.A; Table 1.A).In this research, the dendrogram has a cascade form, it indicates that the degree of separation by esterase enzymes are not good enough, thus the grouping of accessions based on similarities between species are not quite shown. Nonetheless, some accessions have been grouped by species, for example, five accessions of Java cardamom (1, 2, 3, 4, 10) and four accessions of Java cardamom (5,7,8,9). Besides, almost all accessions of true cardamom clustered in one group. Within this group, sixaccessions of true cardamom (15,16, 17, 18, 19,20) generate a sub-group, while in the other sub-group,its members still mixed between the two species, i.e. three accessions of true cardamom (12, 13, 14) and one accession of Java cardamom (6). Meanwhile, one accession of true cardamom emerge it self (11). Based on zimogram, accession11 has a very distinctive esterase banding pattern (Figure 4.A). Peroxidase (PRX, PER). Peroxidase enzyme brings up 10 isozymic banding i.e. at Rf 0.06, 0.14, 0.18, 0.22, 0.26, 0.29, 0.32, 0.37, 0.41, and Rf 0.46. The amount of isozymic bands leads to inter-accession variability which is reflected properly. Groups of accessions generally gather with the basis of species similarity. There is two group whose members are from the same species, which is Java cardamom (3, 4, 5, 7) and true cardamom (11,12,13). However, in general there are other groups of one to two members of different species; for example, a group of Java cardamom (1, 2, 6, 8) are still mixed with an accession of true cardamom(20). Conversely, there is also a group of true cardamom (14, 15, 16, 17, 18, 19) mixed with two accessions of Java cardamom (9, 10)(Figure 4.B; Table 1.B).In general, the peroxidase enzyme reveals more isozymic banding than esterase enzyme does, thus it gives a better overview of the diversity between accessions and the tendency of clustering among accessions based on species similarity. Esterase enzyme gives lesser amount of isozymic banding than the peroxidase enzyme does, thus dendrogram made by esterase isozymic banding reflects less the grouping based on similarity of species comparedto dendrogram made by peroxidase isozymic banding.

SETYAWAN et al. â&#x20AC;&#x201C; The isozymic diversity of cardamoms

Combination of esterase and peroxidase. Relationship dendrogram formed by the combined characteristics of esterase and peroxidase isozymes utilizes more data genetic diversity, thus dendrogram formed reflects more the grouping based on similarity of species. However, the dendrogram also shows variation between accessions of the same species (Figure 4.C).The combined characters formed three groups. One group is consisted of only Java cardamomum (1, 2, 3, 4, 5, 7, 8), one group is only true

cardamom (14, 15, 16, 17, 18, 19, 20), and a third group consisting of either Java or true cardamoms. The latter group also reflects the presence of another grouping based on similarity of species, namely: a group of Java cardamom (9, 10), and a mixed group consisting of true cardamom (11, 12, 13) and the Java cardamom (6). Meanwhile, accession11 of true cardamom is the most different accession in this last group(Table 2).

Table 1. Matrixs of similarity computed with Jaccard coefficient on species of Java cardamom and true cardamom based on isozymic pattern of esterase andperoxidase. A. Similarity matrix of esterasebanding pattern 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 1 1.000 0.600 0.500 0.600 0.600 0.600 0.500 0.333 0.333 0.143 0.286 0.333 0.250 0.286 0.400 0.400 0.400 0.400 0.750 2 1.000 1 0.600 0.500 0.600 0.600 0.600 0.500 0.333 0.333 0.143 0.286 0.333 0.250 0.286 0.400 0.400 0.400 0.400 0.750 3 0.571 0.571 1 0.800 1.000 0.333 1.000 0.500 0.333 0.333 0.333 0.286 0.333 0.429 0.500 0.400 0.400 0.400 0.400 0.400 4 0.444 0.444 0.750 1 0.800 0.286 0.800 0.429 0.286 0.286 0.286 0.250 0.286 0.375 0.429 0.333 0.333 0.333 0.333 0.333 5 0.571 0.571 0.714 0.556 1 0.333 1.000 0.500 0.333 0.333 0.333 0.286 0.333 0.429 0.500 0.400 0.400 0.400 0.400 0.400 6 0.500 0.500 0.300 0.250 0.300 1 0.333 0.500 0.600 0.600 0.333 0.500 0.600 0.429 0.286 0.400 0.400 0.400 0.400 0.750 7 0.429 0.429 0.571 0.444 0.833 0.200 1 0.500 0.333 0.333 0.333 0.286 0.333 0.429 0.500 0.400 0.400 0.400 0.400 0.400 8 0.444 0.444 0.400 0.333 0.556 0.364 0.444 1 0.286 0.286 0.286 0.429 0.500 0.375 0.429 0.333 0.333 0.333 0.333 0.600 9 0.375 0.375 0.333 0.273 0.500 0.444 0.375 0.400 1 1.000 0.600 0.500 0.333 0.667 0.500 0.750 0.750 0.750 0.750 0.400 10 0.375 0.375 0.333 0.400 0.333 0.444 0.222 0.273 0.714 1 0.600 0.500 0.333 0.667 0.500 0.750 0.750 0.750 0.750 0.400 11 0.091 0.091 0.182 0.250 0.182 0.273 0.200 0.154 0.300 0.444 1 0.800 0.600 0.667 0.500 0.400 0.400 0.400 0.400 0.167 12 0.333 0.333 0.300 0.250 0.300 0.556 0.200 0.364 0.444 0.444 0.556 1 0.800 0.571 0.429 0.333 0.333 0.333 0.333 0.333 13 0.333 0.333 0.300 0.250 0.300 0.556 0.200 0.364 0.300 0.300 0.556 0.750 1 0.429 0.286 0.167 0.167 0.167 0.167 0.400 14 0.250 0.250 0.455 0.385 0.333 0.417 0.250 0.286 0.455 0.455 0.545 0.545 0.545 1 0.833 0.500 0.500 0.500 0.500 0.286 15 0.250 0.250 0.455 0.385 0.333 0.308 0.250 0.385 0.333 0.333 0.308 0.417 0.308 0.667 1 0.600 0.600 0.600 0.600 0.333 16 0.375 0.375 0.500 0.400 0.333 0.444 0.222 0.273 0.500 0.500 0.300 0.444 0.300 0.600 0.600 1 1.000 1.000 1.000 0.500 17 0.375 0.375 0.500 0.400 0.333 0.444 0.222 0.273 0.500 0.500 0.300 0.444 0.300 0.600 0.600 1.000 1 1.000 1.000 0.500 18 0.375 0.375 0.500 0.400 0.333 0.444 0.222 0.273 0.500 0.500 0.300 0.444 0.300 0.600 0.600 1.000 1.000 1 1.000 0.500 19 0.333 0.333 0.444 0.364 0.300 0.400 0.200 0.250 0.444 0.444 0.273 0.400 0.273 0.545 0.700 0.857 0.857 0.857 1 0.500 20 0.429 0.429 0.375 0.300 0.222 0.500 0.250 0.300 0.222 0.222 0.200 0.333 0.333 0.364 0.364 0.571 0.571 0.571 0.500 1 B. Similarity matrix of peroxidasebanding pattern Note: 1-10. Java cardamom, 11-20. True cardamom. Table 2. Matrixs of similarity and distance computed with Jaccard coefficient on species of Java cardamom and true cardamom based on combinationofisozymic pattern of esterase andperoxidase. Similarity matrix of esteraseand peroxidase banding patterns 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 1.000 0.500 0.333 0.500 0.333 0.000 0.333 0.500 0.500 0.000 0.500 0.333 0.250 0.200 0.333 0.333 0.333 0.250 0.000 2 0.000 0.500 0.333 0.500 0.333 0.000 0.333 0.500 0.500 0.000 0.500 0.333 0.250 0.200 0.333 0.333 0.333 0.250 0.000 3 0.500 0.500 0.667 0.333 0.250 0.000 0.250 0.333 0.333 0.000 0.333 0.250 0.500 0.400 0.667 0.667 0.667 0.500 0.333 0.250 0.200 0.000 0.200 0.250 0.667 0.200 0.250 0.200 0.400 0.333 0.500 0.500 0.500 0.400 0.250 4 0.667 0.667 0.333 5 0.500 0.500 0.667 0.750 0.250 0.500 0.667 1.000 0.333 0.000 0.333 0.250 0.200 0.167 0.250 0.250 0.250 0.200 0.000 0.000 0.200 0.250 0.250 0.200 0.667 0.500 0.400 0.333 0.500 0.500 0.500 0.400 0.250 6 0.667 0.667 0.750 0.800 0.750 7 1.000 1.000 1.000 1.000 0.500 1.000 0.333 0.500 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.667 0.250 0.000 0.250 0.200 0.167 0.333 0.200 0.200 0.200 0.167 0.000 8 0.667 0.667 0.750 0.800 0.333 0.800 0.667 9 0.500 0.500 0.667 0.750 0.000 0.750 0.500 0.333 0.333 0.000 0.333 0.250 0.200 0.167 0.250 0.250 0.250 0.200 0.000 0.250 0.333 0.250 0.200 0.167 0.250 0.250 0.250 0.200 0.000 10 0.500 0.500 0.667 0.333 0.667 0.750 1.000 0.750 0.667 11 1.000 1.000 1.000 0.800 1.000 0.800 1.000 1.000 1.000 0.750 0.250 0.500 0.400 0.143 0.200 0.200 0.200 0.167 0.250 0.667 0.500 0.400 0.667 0.667 0.667 0.500 0.333 12 0.500 0.500 0.667 0.750 0.667 0.333 1.000 0.750 0.667 0.667 0.750 13 0.667 0.667 0.750 0.800 0.750 0.500 1.000 0.800 0.750 0.750 0.500 0.333 0.750 0.333 0.500 0.500 0.500 0.400 0.250 0.500 0.750 0.750 0.750 0.600 0.500 14 0.750 0.750 0.500 0.600 0.800 0.600 1.000 0.833 0.800 0.800 0.600 0.500 0.250 15 0.800 0.800 0.600 0.667 0.833 0.667 1.000 0.667 0.833 0.833 0.857 0.600 0.667 0.500 0.600 0.600 0.600 0.800 0.400 1.000 1.000 0.750 0.667 16 0.667 0.667 0.333 0.500 0.750 0.500 1.000 0.800 0.750 0.750 0.800 0.333 0.500 0.250 0.400 17 0.667 0.667 0.333 0.500 0.750 0.500 1.000 0.800 0.750 0.750 0.800 0.333 0.500 0.250 0.400 0.000 1.000 0.750 0.667 0.750 0.667 18 0.667 0.667 0.333 0.500 0.750 0.500 1.000 0.800 0.750 0.750 0.800 0.333 0.500 0.250 0.400 0.000 0.000 19 0.750 0.750 0.500 0.600 0.800 0.600 1.000 0.833 0.800 0.800 0.833 0.500 0.600 0.400 0.200 0.250 0.250 0.250 0.500 20 1.000 1.000 0.667 0.750 1.000 0.750 1.000 1.000 1.000 1.000 0.750 0.667 0.750 0.500 0.600 0.333 0.333 0.333 0.500 Distance matrix of esteraseand peroxidase banding patterns Note: 1-10. Java cardamom, 11-20. True cardamom.

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II II

II III III

III

IV A

IV B

Figure 4. Relationship dendrogram of Java cardamom and true cardamom based on isozymicbanding pattern of esterase (A), peroxidase (B) and combinationofesterase andperoxidase (C). Note: = Java cardamom, = true cardamom.

Accession 11 of Java cardamom has a unique relationship position because it tends to stand alone; it is found both in the dendrogram constructed based on esterase isozymic banding, or a combination of both. Generally, accession which has a distinctive genetic pattern is only able to grow in a particular environment, but if that accession is able to grow in a variety of habitat conditions, it must be very valuable because its distinctive genetic variations enable it to withstand changes in the environment. This accession is much needed in plant breeding programs, either in an effort to increase the quality and quantity of production, as well as in the prevention of pests and diseases and global environmental changes such as land use change and global warming. ACKNOWLEDGEMENTS This work was supported by the DGHE Republic ofIndonesia with research grant no. 031/PUT/DPPM/PDM/ III/2003. The authors thank to the Indonesian Medicinal and Aromatic Crops Research Institute (IMACRI/Balittro) Bogor, the Bogor Botanical Garden, and the Medicinal Plant and Traditional Medicine Research and Development Center (B2P2TOOT) Tawangmangu, Indonesia for providing materials of cardamoms. The authors also thank to Suhar Iriyanto and Delima S. Roesyat for his generosity in providing some plant materials, and Asriyati A. Wardani and Ainur Rohimah for kindly assistance with the laboratory work.

REFERENCES Apavatjrut P, Anuntalabhochai S, Sirirungsa P, Alisi C. 1999. Molecular markers in the identification of some early flowering Curcuma L. (Zingiberaceae) species. Annual of Botany 84: 529-534. Azizah N. 2011. Identification of genetic diversity on Curcuma xanthorriza collection clones in Java and Madura. The 2nd International Symposium on Temulawak, IPB lnternational Convention Center 26-27 May 2011 Backer CA, Bakhuizen van den Brink Jr. RC. 1968. Flora of Java. Vol. 3. Wolters Noordhoff, Groningen. Chokthaweepanich H, Paisooksantivatana Y. 2003. Classification of the genus Curcuma L. (Zingiberaceae) based on morphological characters and isozyme pattern. In: Chantaranothai P (ed). Proceeding of the 3rd Symposium on the Family Zingiberaceae, Khon Kaen, Thailand. Crawford DJ. 1990. Plant Molecular Systematics, Macromolecular Approaces. John Wiley and Sons, New York. Deng JB, CB Ding, L Zhang, YH Zhou, Yang RW. 2011. Relationships among six herbal species (Curcuma) assessed by four isozymes. Pyton 80: 181-188 Duke JA, Bogenschutz-Godwin JC, Duke PAK. 2002. Handbook of Medicinal Herbs. CRC Press, Boca Raton, FL. Feng X, Jiang ZT, Wang Y, Li R. 2011. Composition comparison of essential oils extracted by hydrodistillation and microwave-assisted hydrodistillation from Amomum kravanh and Amomum compactum. J. Essen Oil-bearing Pl 14 (3): 354-359. Garcia-Vallve S, Palau J, Romeu A. 1999. Horizontal gene transfer in glycosyl hydrolases inferred from codon usage in Escherichia coli and Bacillus subtilis. Mol Biol Evol 9: 1125-1134. Garcia-VallvĂŠ S, Puigbo P. 2002. DendroUPGMA: A dendrogram construction utility. Biochemistry and Biotechnology Department. Universitat Rovira i Virgili (URV). Tarragona, Spain. http://genomes.urv.cat/UPGMA/ Guenther E. 1952. The essential oils. Vol. 5. Van Nostrand Co., New York. Hegnauer, R. 1963. Chemotaxonomie der Pflanzen (Monocotyledoneae). Band II. Bassel: Birkhauser Verlag.

SETYAWAN et al. â&#x20AC;&#x201C; The isozymic diversity of cardamoms Heyne K. 1950. De Nuttige Planten van Indonesie. Deel I. W. van Hoeve, â&#x20AC;&#x2DC;s-Gravenhage. Ibrahim H, Hussin KH, Sanusi A. 1991. Taxonomic implications of isozyme and anatomical studies on Cucurma species. Botany 2000 Asia. Zingiberaceae workshop, Prince of Songkla University, Hat Yai, Thailand, 15-18 October 1991. Ibrahim H. 1996. Isozyme variations in selected Zingiberaceae spp. In: Wu TL et al. (eds). Proceedings of the 2nd Symposium on the Family Zingiberaceae. Zhongshan University Press, Guangzhou, China. Jaccard P. 1908. Nouvelles recherches sur la distribution florale. Bull Soc Vard Sci Nat 44: 223-270 Jia XP, Sun H. 2013. An intrinsic advantage of sexual reproduction. arXiv:1306.5373 (06/2013). 2013arXiv1306.5373J Jugran A, Bhatt ID, Rawat S, Giri L, Rawal RS, Dhar U. 2011. Genetic diversity and differentiation in Hedychium spicatum Buch. Ham. ex. D. Don - a high value medicinal plant of Indian Himalaya. Biochem Genet (DOI 10.1007/s10528-011-9451-7) KasaharaS, Hemmi S (eds.). 1995. Medicinal Herb Index In Indonesia, 2nd ed. PT. Eisai Indonesia, Jakarta. Kuyumcusavan E, Kucukbay FZ. 2013. Essential oil composition of Elettaria cardamomum Maton. J Appl Biol Sci 7 (3): 42-45, 2013. Madjo-Indo ABD. 1989. Kapulaga: Budidaya, Pengolahan dan Pemasaran. Penebar Swadaya, Jakarta. [Indonesian] Marongiu B, Piras A, Porcedda S. 2004. Comparative analysis of the oil and supercritical CO2 extract of Elettaria cardamomum (L.) Maton. J Agric Food Chem 52 (20): 6278-6282 Paisooksantivatana Y, Kako S, Seko H. 2001. Genetic diversity of Curcuma alismatifolia Gagnep. (Zingiberaceae) in Thailand as revealed by allozyme polymorphism. Genet Res Crop Evol 48 (5): 459-465. Peter KV. 2001. Handbook of Herbs and Spices. Vol. 1. CRC Press, New York. Purseglove JW, Brown EG, Green CL, Robbins SRJ. 1981. Spices, Volume 2. Longman Tropical Agriculture Series, London. Purseglove JW. 1972. Tropical Crops Monocotyledones. Longman, London. Santoso HB. 1988. Kapulaga. Penerbit Kanisius, Yogyakarta [Indonesian] Seidemann J. 2005. World Spice Plants: Economic Usage, Botany, Taxonomy. Springer, New York.

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Setyawan AD, Wiryanto,Suranto, Bermawie N. 2014. Variation in isozymic pattern of three varieties of ginger (Zingiber officinale)germplasm. Nus Biosci 6: 00-00 Setyawan AD. 2002. Chemotaxonomic studies on the genus Amomum based on chemical components of volatile oils. Hayati J Biosci 9 (3): 71-79. Shamina A, Zacharias JT, Sasikumar B, George JK. 1998. Biochemical variation in turmeric (Curcuma longa Linn.) accessions based on isozyme polymorphism. J Horticult Sci Biotechnol 73 (4): 479-483. Shamita A, Zachariah TJ, Sasikumar B, George JK. 1997. Biochemical variability in selected ginger (Zingiber officinale Rosc.) germplasm accessions. J Spic Arom Crops 6 (2): 119-127. Shanmugapriya M, Prabha ML. 2012. Isoenzyme analysis of Amomum species. Intl J Pharm Biol Arch 3 (5): 1091-1094 Sneath PHA, Sokal RR. 1973. Numerical Taxonomy. W.H. Freeman, Co., San Francisco. Suranto. 1991. Studies of Population Variations in Species of Ranunculus. [M.Sc.-Thesis]. Departement of Plant Science University of Tasmania, Hobart. Tang JY, Li QM, Yang RW, Liao JQ, Zhou YH. 2008. Study on isozymes in six species of Curcuma. China J Chin Mat Med 33 (12): 13811386. Trease GE, Evans WC. 1978. Pharmacognasy. 11thed. Bailliere Tindall, London. Vanijajiva O, Suvachittanont W, Sirirugsa P. 2003. Isozyme analysis of relationships among Boesenbergia (Zingiberaceae) and related genera in Southern Thailand. Biochem Syst Ecol 31 (5): 499-511. Wardini TH, Thomas A. 1999. Elettaria cardamomum (L.) Maton. In: de Guzman CC, Siemonsma JS (eds.). Plant Resources of South-East Asia 13: Spices. PROSEA, Bogor. Weeden VF, Wendel JF. 1989. Visualization and interpretation of plant isozymes. In: Soltish DE, Soltish PS (eds.) Isozyme in Plant Biology. Dioscorides Press Porland, Oregon. Wollf XY, Hartutiningsih. 1999. Amomum compactum Soland. ex Maton. In: de Guzman CC, Siemonsma JS (eds.). Plant Resources of SouthEast Asia 13: Spices. PROSEA, Bogor. Youngken HW. 1948. Textbook of Pharmacognosy. 7th ed. The Blakiston Co., Philadelphia, Penn. Yu JG, Feng HJ, Li JT. 1982. Essential oil of fruits and leaves of A. kravanh and A. compactum. Chin Trad Herb Drugs 13: 4-7.

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ISSN: 2087-3948 E-ISSN: 2087-3956 DOI: 10.13057/nusbiosci/n060116

Vol. 6, No. 1, pp. 102-106 May 2014

Short Communication: An insight into protein sequences of PTP-

like cysteine phytases 1

VINOD KUMAR1,2, SANJEEV AGRAWAL1,♥ Department of Biochemistry, College of Basic Sciences and Humanities, G.B. Pant University of Agriculture and Technology, Pantnagar-263145, India. Tel. +91-5944-233310, Fax. +91-5944-233473, ♥email: sanjeevagrawal14@rediffmail.com 2 Akal School of Biotechnology, Eternal University, Baru Sahib, Sirmour-173101, India. Manuscript received: 26 April 2014. Revision accepted: 30 April 2014.

Abstract. Kumar V, Agrawal S. 2014. An insight into protein sequences of PTP-like cysteine phytases. Nusantara Bioscience 6: 102106. Protein tyrosine phosphatase like cysteine phytases (CPhy) are novel phytases reported in the ruminant microbial community and suggested to play major role in phytate-phosphorus hydrolysis in animal feed. These phytases are very promising to be used in animal feed applications for monogastric animals. Present study deals with utilization of sequence information of 40 CPhy reference protein sequences for their sequential characterization for conserved regions, phylogenetic relationship, biochemical features, superfamily and functional motifs therein. The study reveals that CPhy, not well characterized class of phytases, contains conserved sequence feature which may be important catalytic residues. Five major clusters observed in phylogenetic tree with Clostridium sp. as largest cluster. Reported motifs might be used for diversity and expression analysis of CPhy enzymes. Key words. Cysteine phytase, in silico analysis, motifs, phylogenetic tree, phytic acid

INTRODUCTION Phytases (myo-inositol 1,2,3,4,5,6-hexakisphosphate phosphohydrolase) are a special group of phosphatases which catalyzes the stepwise removal of phosphates from phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate; IP6) or its salt phytate (Lei et al. 2013). It is, therefore, useful in various applications, e.g. alleviating antinutritional effect of phytate in animal feed, increase bioavailability of micronutrients to monogastric animals, management of environmental phosphorus pollution (Sapna et al. 2013) and promising for aquafeed application (Kumar et al. 2014b). Phytases are widely distributed among plants and microbial cells (Hegeman and Grabau 2001; Kumar et al. 2013; Singh et al. 2014; Lei et al. 2013). To develop a suitable phytase for above applications and better understand the catalytic mechanism of diverse groups of phytases, large number of such organisms has been studied in detail for their phytase gene sequences and biochemical properties. Based on the specific consensus sequence, catalytic mechanism and three dimensional structures, so far phytases are therefore classified in four classes, i.e. histidine acid phosphatase (HAPhy), cysteine phytase (CPhy), purple acid phosphatase (PAPhy) and betapropeller phytase (BPPhy) (Lei et al. 2007; Mullaney and Ullah 2007; Lei et al. 2013). Further classification based on the site of phytic acid dephosphorylation reveals three groups of phytases i.e. 3-phytase (alternative name, 1phytase; EC 3.1.3.8), 4-phytase (alternative name, 6phytase; EC 3.1.3.26), and 5-phytase (EC 3.1.3.72) (Kumar et al. 2014a).

Although tremendous work has been carried out related to phytase research, new technologies like sequencing advances, enzyme engineering, proteomics and related bioinformatic studies has further given a new life to this old enzyme (Lei et al. 2013). In addition to this, in silico characterization of protein sequences of HAPhy and BPPhy class of phytases has been reported recently (Kumar et al. 2012; Kumar et al. 2014a). These studies have been suggested to be useful in further genetic engineering and classification in important groups of phytases. The use and bioinformatic analysis of resulting DNA and protein sequence information from different studies make it possible to get important predictions and help in the design and success of further studies. Among the different class of phytases, CPhy class is least studied for its biochemical and important sequence catalytic features. Several CPhy have been reported by rumen bacterial isolates, including Megasphaera elsdenii, Clostridium perfringens, and Clostridium botulinum (Yanke et al. 1999). Biochemical characteristic of CPhy has not been studied in detail and very little literature is available on characterization of CPhy. In one such study, CPhy from Selenomonas ruminantium has been studied and characterized extensively by Puhl et al. (2007). In the present study, the 40 reference protein sequences of CPhy from protein databases were retrieved and analyzed ‘in silico’ for their biochemical features, multiple sequence alignment and identity search, phylogenetic tree construction, distribution of motifs and superfamily using various bioinformatics tools.

KUMAR & AGRAWAL – Protein sequences of PTP-like cysteine phytases

Materials and methods The 40 reference protein sequences representing CPhy from NCBI protein database (http: //www.ncbi.nlm.nih.gov) were retrieved in FASTA format for use in this study (Table 1). The protein sequences, which were shown to exhibit phytase activities, were selected for in silico study. Their characterization for homology, phylogenetic relationship, functional domain and other biochemical properties was carried out using freely available bioinformatic tools following the methodology of Kumar et al. (2012). For domain search, the Pfam site (http: //www.sanger.ac.uk/software/pfam/search.html) was used. Functional domains and motifs analysis was done using MEME (http: //meme.sdsc.edu/meme/meme.html).

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acanthamoebae (ZP_06300753.1). Cluster ‘3’ also contains 2 sequences from Desulfovibrio sp. (YP_002953065.1, ZP_07334842.1). Cluster ‘4’ consists of 13 sequences with majority from Selenomonas sp. and Acidaminococcus sp. Cluster ‘5’ consisted by Mitsuokella multacida (ZP_05405390.2) and Bdellovibrio bacteriovorus (NP_968118.1) (Figure 1.A and 1.B). The variations in biochemical features of representing CPhy protein sequences are given in Table 2. The length of protein sequences was found in the range of 283-347 amino acid residues, except 5 sequences from Clostridium sp. were 820 amino acid residues long. The theoretical pI value of CPhy sequences was observed to be highest among four classes of phytase and was in the range of 7-10. The instability index is used to measure in vivo half life of a protein (Guruprasad et al. 1990). The instability index of 7 CPhy protein sequences was above 40 indicating their low in vivo stability (Table 2), while the rest of the sequences with their instability index value below 40 have in vivo stability of more than 16 h (Rogers et al. 1986). Aliphatic index of protein measure the relative volume occupied by aliphatic side chains of the amino acids: alanine, valine, leucine and isoleucine. Globular proteins with high aliphatic index have high thermostability and an increase in

Result and discussion The 40 reference sequences representing CPhy with GenBank accession number are listed in Table 1. Two conserved regions ‘DLR[E/Q]E[S/T]HG[F/L]’ and ‘WxHFHCxxGxGRT’ were obtained in all representative sequences, when analyzed by multiple sequence alignment using ClustalW by MEGA5 (Tamura et al. 2011). High distinctiveness was observed in cysteine phytase protein sequences during alignment. The primary sequence of this enzyme contains a PTP-like signature sequence (C(X)5R), which is ubiquitous among members of the PTP superfamily Table 1. List of source organism of retrieved CPhy protein sequences (with accession number) (Zhang et al. 2002). All PTPs have a phosphate-binding loop (P-loop) at the base Total of their active site which contains the Source organism Accession no. sequences characteristic PTP signature sequence Acidaminococcus fermentans YP_003399467.1 1 C(X)5R (Denu and Dixon 1998; Zhang Acidaminococcus intestine YP_004897589.1 1 2003). Site-directed mutagenesis studies Acidaminococcus sp. ZP_03929107.1 1 have determined that the cysteine residue Bdellovibrio bacteriovorus NP_968118.1 1 present in the P-loop is absolutely required Centipeda periodontii ZP_08501473.1 1 for PTP activity (Puhl et al. 2007). It is a Clostridium acetobutylicum NP_149178.1 1 Clostridium botulinum YP_001787593.1,ZP_02951610.1, 7 strong nucleophile, and is easily modified YP_002863193.1, ZP_02614565.1, by thiol reagents (Sechi and Chait 1998). YP_001391515.1, YP_001781827.1, Chemical modification experiments with YP_001254710.1 alkylating agents also indicate that the PClostridium kluyveri YP_001394001.1 1 loop cysteine is required for PTP activity Clostridium ljungdahlii YP_003781358.1, YP_003781970.1 2 (Zhou et al. 1994). PTPs use the Clostridium perfringens YP_696211.1, ZP_02633371.1, 7 nucleophilic cysteine residue to bind the NP_562440.1, ZP_02635029.1, phosphate monoester of the substrate, ZP_02952453.1, ZP_02642534.1, forming a thiol-phosphate intermediate ZP_02863824.1 Clostridium sp. ZP_09204362.1 1 (Pannifer et al. 1998). S. ruminantium Clostridium sporogenes ZP_02995608.1 1 phytase neither contains the conserved Clostridium tetani NP_782216.1 1 RHGXRXP motif nor is affected by Desulfovibrio fructosovorans ZP_07334842.1 1 divalent metal ions. The active site is Desulfovibrio magneticus YP_002953065.1 1 located near a conserved cysteineDialister invisus ZP_05734150.1 1 containing (Cys241) P loop (Chu et al. Megamonas funiformis ZP_09733511.1 1 2004). Megasphaera elsdenii YP_004767129.1 1 A total of 5 clusters were observed in Mitsuokella multacida ZP_09733511.1, ZP_05405390.2 2 phylogenetic tree constructed by the Parachlamydia acanthamoebae ZP_06300753.1 1 Protochlamydia sp. YP_008827.1 1 Neighbor-Joining method. The largest Selenomonas flueggei ZP_04658998.1 1 cluster ‘1’ contains sequences from Selenomonas infelix ZP_09119488.1 1 Clostridium sp. (21 sequences). Cluster ‘2’ Selenomonas noxia ZP_06603000.1 1 was composed of Protochlamydia Selenomonas sp. ZP_07397197.1 1 (YP_008827.1) and Parachlamydia Selenomonas sputigena

ZP_05898176.1

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Table 2. Biochemical characteristics of CPhy protein sequences determined by ProtParam server Accession number

Source organisms

YP_003399467.1 YP_004897589.1 ZP_03929107.1 NP_968118.1 ZP_08501473.1 NP_149178.1 YP_001787593.1 YP_002863193.1 ZP_02614565.1 YP_001391515.1 YP_001781827.1 YP_001254710.1 ZP_02951610.1 YP_001394001.1 YP_003781358.1 YP_003781970.1 YP_696211.1 ZP_02633371.1 NP_562440.1 ZP_02635029.1 ZP_02952453.1 ZP_02642534.1 ZP_02863824.1 ZP_09204362.1 ZP_02995608.1 NP_782216.1 ZP_07334842.1 YP_002953065.1 ZP_05734150.1 ZP_09733511.1 YP_004767129.1 ZP_05405389.1 ZP_05405390.2 ZP_06300753.1 YP_008827.1 ZP_04658998.1 ZP_09119488.1 ZP_06603000.1 ZP_07397197.1 ZP_05898176.1

Acidaminococcus fermentans Acidaminococcus intestini Acidaminococcus sp. Bdellovibrio bacteriovorus Centipeda periodontii Clostridium acetobutylicum Clostridium botulinum Clostridium botulinum Clostridium botulinum Clostridium botulinum Clostridium botulinum Clostridium botulinum Clostridium butyricum Clostridium kluyveri Clostridium ljungdahlii Clostridium ljungdahlii Clostridium perfringens Clostridium perfringens Clostridium perfringens Clostridium perfringens Clostridium perfringens Clostridium perfringens Clostridium perfringens Clostridium sp. Clostridium sporogenes Clostridium tetani Desulfovibrio fructosovorans Desulfovibrio magneticus Dialister invisus Megamonas funiformis Megasphaera elsdenii Mitsuokella multacida Mitsuokella multacida Parachlamydia acanthamoebae Protochlamydia sp. Selenomonas flueggei Selenomonas infelix Selenomonas noxia Selenomonas sp. oral taxon Selenomonas sputigena

No. of amino acids 302 326 332 293 328 319 820 820 820 820 820 820 309 312 343 316 308 308 308 308 308 308 308 820 820 307 283 331 408 341 347 640 323 320 311 328 328 328 328 334

Molecular weight 34098.8 36785.4 37381.2 33549.3 37260.4 36199.5 94112.9 93906.9 93926 93922.8 93882.9 94022.1 35527 36206.7 40226.4 36271.8 35643 35662.9 35628.9 35610.9 35540.8 35555.8 35554.8 93753.2 93522.1 35509.7 31305.2 35004 46674.4 39558.9 39486.4 73136.4 36779.8 37249.7 35620.6 36888.7 37258.2 37329.3 36818.5 37515

Theoretical pI 9.3 9.57 9.62 8.65 7.72 9.68 7.32 8.67 8.88 8.78 8.72 8.83 5.35 9.01 9.21 8.96 8.74 7.76 7.76 7.76 7.76 7.76 7.76 8.09 8.27 9.16 5.73 10.19 9.1 8.21 4.79 9.06 9.16 6.46 7.16 7.25 7.75 8.68 6.99 4.76

Instability index 31.84 37.13 37.34 43.78 29.01 27.79 39.07 37.87 39.86 39.08 39.73 39.34 44.46 40.77 39.58 38.07 23.97 24.37 24.37 22.85 22.45 22.85 22.45 32.57 41.85 29.91 46.2 44.77 38.51 28.77 42.89 29.94 35.9 39.84 38.55 28.98 33.12 26.82 31.55 38.84

Aliphatic index 79.26 80.25 82.02 77.2 71.77 82.51 79.26 81.26 79.84 78.88 80.2 80.44 84.24 76.15 84.64 81.65 94.87 94.25 95.32 96.79 95.19 95.84 95.19 82.96 81.5 89.8 82.79 88.34 76.08 81.55 69.14 74.22 70.99 90.5 89.29 72.38 69.7 69.39 70.64 75.48

Table 3. Distribution of Superfamily among CPhy protein sequences determined using superfam server Motif present Motifs in no. of sequence 1 40 2 40 3 40 4 40 5 16 6 8 7 8 8 8 9 8 10 8

Motif Amino acid sequence width

Domain

50 29 29 29 41 50 50 50 50 50

PTPc superfamily No putative conserved domains No putative conserved domains No putative conserved domains No putative conserved domains GxGYxYP superfamily GxGYxYP superfamily GxGYxYP superfamily GxGYxYP superfamily GxGYxYP superfamily

TDHKWPTDEMVDYFVQFVKSMPKDTWLHFHCQAGIGRTTTFMIMYDMMKN ICIVDLRQESHGFINGYPVSWYGEHNWAN PNREGLDTLNISGSQQFSPQNLPLLVKSI PPQTIIPTKVMTEEQLVEHNGMRYVRIPV ADEIINRQLALAGFDEKHMKSFPNKERHDFFQKFYEYVKEQ HYVTFIMSDGDNQQWNLGTNYGSPKWYGSPYRGNFNLGWSLSPSLYYLAP RDKVFSSMDPNSICLGWGPDEFINVSTSSKHGVSMIAADWSYNLTVLSAF KIPTHLYVISQNKMTSSERTMIATLQGIVNNHCSHQIYTLNSSQPDYQIW FYNNKLWDKFTVKPNIQGLFYLDYRKHNNYHGEIIWSNNKPIVSCRDLLW GDCRNTDKDWAYNNLWNSGLNHSIVIQLSPEKETALRDYAIMTKSLIFYE

KUMAR & AGRAWAL â&#x20AC;&#x201C; Protein sequences of PTP-like cysteine phytases

34 ZP 02635029.1| Clostridium perfringens B 62 ZP 02952453.1|Clostridium perfringens

ZP 02642534.1|Clostridium perfringens 83 63 ZP 02863824.1|Clostridium perfringens 100

ZP 02633371.1|Clostridium perfringens

70 NP 562440.1|Clostridium perfringens

YP 696211.1|Clostridium perfringens ZP 09204362.1|Clostridium sp. DL-VIII

NP 782216.1|Clostridium tetani ZP 02995608.1|Clostridium sporogenes

YP 002863193.1|Clostridium botulinum

YP 001254710.1|Clostridium botulinum

ZP 02614565.1|Clostridium botulinum

57 69

Cluster 1: Clostridium species

YP 001787593.1|Clostridium botulinum

100 91

YP 001391515.1|Clostridium botulinum YP 001781827.1|Clostridium botulinum ZP 02951610.1|Clostridium butyricum YP 003781358.1|Clostridium ljungdahlii

YP 001394001.1|Clostridium kluyveri

YP 003781970.1|Clostridium ljungdahlii

100

NP 149178.1|Clostridium acetobutylicum YP 008827.1|Protochlamydia amoebophila ZP 06300753.1|Parachlamydia acanthamoebae

Cluster II

YP 002953065.1|Desulfovibrio magneticus

ZP 07334842.1|Desulfovibrio fructosovorans 31

Cluster III

Cluster IV

Cluster V

0.1

Figure 1.A. Phylogenetic tree constructed by NJ method based on CPhy protein sequences

Cluster 1: Clostridium species

YP 008827.1|Protochlamydia amoebophila ZP 06300753.1|Parachlamydia acanthamoebae

Cluster II

YP 002953065.1|Desulfovibrio magneticus

ZP 07334842.1|Desulfovibrio fructosovorans

Cluster III

100 YP 004897589.1|Acidaminococcus intestini 70 31

ZP 03929107.1|Acidaminococcus sp. D21

YP 004767129.1|Megasphaera elsdenii YP 003399467.1|Acidaminococcus fermentans ZP 09733511.1|Megamonas funiformis

ZP 05405389.1|Mitsuokella multacida

100 68

ZP 05734150.1|Dialister invisus

23 100 77

ZP 04658998.1|Selenomonas flueggei ZP 07397197.1|Selenomonas sp.67H29BP

100 48 56

Cluster IV

ZP 05898176.1|Selenomonas sputigena

0.1

Figure 1.B. Phylogenetic tree constructed by NJ method based on CPhy protein sequences

Cluster V

105

106

N U SAN TA RA B IOSC IE NC E 6 (1): 102-106, May 2014

aliphatic index increases protein thermostability (Atsushi 1980; Rawlings et al. 2006). Aliphatic index of CPhy protein sequences was observed in the range of 70-95, suggesting sequences were varied in their thermostability (Table 2). Superfam analysis revealed the presence of ‘phosphotyrosine protein phosphatase II superfamily’ and ‘myo-inositol hexaphosphate phosphohydrolase PhyA family’ in all 40 protein sequences. Protein tyrosine phosphatases (PTP) catalyze the dephosphorylation of phosphotyrosine peptides; they regulate phosphotyrosine levels in signal transduction pathways. The depth of the active site cleft renders the enzyme specific for phosphorylated Tyr (pTyr) residues, instead of pSer or pThr. Analysis of 10 motifs by MEME suite with provided parameters revealed 50 amino acids long motif ‘1’ ‘TDHKWPTDEMVDYFVQFVKSMPKDTWLHFHCQA GIGRTTTFMI MYDMMKN’ was present in all cysteine phytase protein sequences. The functional domain found in this motif was similar to PTPc superfamily. This family has a distinctive active site signature motif, HCSAGxGRxG, characterized as either transmembrane, receptor-like or non-transmembrane (soluble) PTPs. Receptor-like PTP domains tend to occur in two copies in the cytoplasmic region of the transmembrane proteins, only one copy may be active. Other motifs (6 to 10) were found similar to GxGYxYP superfamily. This family carries a characteristic sequence motif, GxGYxYP, but is of unknown function. Associated families are sugar-processing domains. Complete list of motifs with their characteristics is given in Table 3. In conclusion, this in silico study for phylogenetic clustering, conserved motifs sequences and biochemical features of phytases from class CPhy, could be key information for their further classification and genetic modification within key sequence features for the development of novel phytase with desired properties. Conserved motif sequences are important for conserved primer design and diversity study thereafter. ACKNOWLEDGEMENTS The authors gratefully acknowledge Department of Science and Technology for providing infrastructural facility in the form of DST-FIST grant to Department of Biochemistry, G.B. Pant University of Agriculture and Technology, India. Lead author is grateful to Indian Council of Agriculture Research (ICAR), for providing financial assistance in the form of ICAR-SRF. REFERENCES Atsushi I. 1980. Thermostability and aliphatic index of globular proteins. J Biochem 88: 1895-1898. Chu HM, Guo RT, Lin TW, Chou CC, Shr HL, Lai HL, Tang TY, Cheng KJ, Selinger BL, Wang AHJ. 2004. Structures of Selenomonas

ruminantium Phytase in Complex with Persulfated Phytate: DSP Phytase Fold and Mechanism for Sequential Substrate Hydrolysis. Structure 12: 2015-2024. Denu JM, Dixon JE. 1998. Protein tyrosine phosphatases: mechanisms of catalysis and regulation. Curr Opin Chem Biol 2: 633-641. Guruprasad K, Reddy BVB, Pandit MW. 1990. Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Engineer 4: 155-161. Hegeman CE, Grabau EA. 2001. A novel phytase with sequence similarity to purple acid phosphatases is expressed in cotyledons of germinating soybean seedlings. Plant Physiol. 126: 1598-1608 Kumar V, Sangwan P, Singh G, Verma AK, Agrawal S. 2014a. Cloning, sequencing, expression and in silico analysis of β-propeller phytase from Bacillus licheniformis strain PB-13. Biotech Res Internat 2014: 1-11, Article ID 841353, doi: 10.1155/2014/841353 Kumar V, Sangwan P, Verma AK, Agrawal S. 2014b. Molecular and Biochemical Characteristics of Recombinant β-Propeller Phytase from Bacillus licheniformis Strain PB-13 with Potential Application in Aquafeed. Appl Biochem Biotechnol DOI 10.1007/s12010-0140871-9. Kumar V, Singh G, Verma AK, Agrawal S. 2012. In silico characterization of histidine acid phytase sequences. Enzyme Res 2012: 1-8, 845465, doi: 10.1155/2012/845465 Kumar V, Singh P, Jorquera M, Sangwan P, Kumar P, Verma AK, Agrawal S. 2013. Isolation of phytase-producing bacteria from Himalayan soils and their effect on growth and phosphorus uptake of Indian mustard (Brassica juncea). World J Microbiol Biotech 29: 1361-1369. Lei XG, Weaver JD, Mullaney E, Ullah AH, Azain MJ. 2013. Phytase, a new life for an “old” enzyme. Annu Rev Anim Biosci 1(1): 283-309. Mullaney EJ, Ullah AH. 2003. The term phytase comprise several different classes of enzymes. Biochem Bioph Res Co 312: 179-184. Pannifer ADB, Flint AJ, Tonks NK, Barford D. 1998. Visualization of the cysteinyl-phosphate intermediate of a protein-tyrosine phosphatase by X-ray crystallography. J Biol Chem 273: 10454-10462. Puhl AA, Gruninger RJ, Greiner R, Janzen TW, Mosimann SC, Selinger LB. 2007. Kinetic and structural analysis of a bacterial protein tyrosine phosphatase-like myo-inositol polyphosphatase. Protein Sci 16: 1368-1378. Rawlings ND, Morton FR, Barrett AJ. 2006. MEROPS: the peptidase database. Nucleic Acids Res 34: 270-272. Rogers S, Wells R, Rechsteiner M. 1986. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Sci 234: 364. Sapna, Singh B, Singh D, Sharma KK. 2013. Microbial Phytases in Skirmishing and Management of Environmental Phosphorus Pollution. In: Biotechnology for Environmental Management and Resource Recovery. Springer India. Sechi S, Chait BT. 1998. Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification. Anal Chem 70: 5150-5158. Singh P, Kumar P, Agrawal S. 2014. Evaluation of phytase producing bacteria for their plant growth promoting activities. Internat J Microbiol 2014: 1-7, Article ID: 426483, doi: 10.1155/2014/426483 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28: 2731-2739. Yanke L, Selinger L, Cheng KJ. 1999. Phytase activity of Selenomonas ruminantium: a preliminary characterization. Lett Appl Microbiol 29: 20-25. Zhang ZY, Zhou B, Xie L. 2002. Modulation of protein kinase signaling by protein phosphatases and inhibitors. Pharmacol Therapeut 93: 307317. Zhang ZY. 2003. Mechanistic studies on protein tyrosine phosphatases. Prog Nucl acid Res Mol Biol 73: 171-220. Zhou MM, Logan TM, Theriault Y, Van Etten RL, Fesik SW. 1994. Backbone 1H, 13C, and 15N assignments and secondary structure of bovine low molecular weight phosphotyrosyl protein phosphatase. Biochem 33: 5221-5229.

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Leaching and heating process as alternative to produce fish protein powder from Kilka (Clupeonella cultiventris caspia) Optimization of in vitro sterilization protocol for obtaining contamination free cultures of Tilia platyphyllos Hydrophysical, chemical and microbial properties of imported green waste composts The mycobiota associated with paper archives and their potential control Microbial water quality of coastal recreational water in the Gaza Strip, Palestine Six unrecorded species of Russula (Russulales) from Nagaland, India and their nutrient composition Uniconazole effect on endogenous hormones, proteins and proline contents of barley plants (Hordium vulgare L) under salinity stress (NaCl) Effect of fungicides and bioagents on number of microorganisms in soil and yield of soybean (Glycine max) Effect of nitrogen fertilizers on productivity of Urtica pilulifera plant Using and comparing two nonparametric methods (CART and RF) and SPOT-HRG satellite data to predictive tree diversity distribution Review: The potency of carotenoids production from Neurospora Review: Mangrove hybrid of Rhizophora and its parental species in Indo-Malayan region Short Communication: Growth of seaweed Eucheuma cottonii in multi tropic sea farming systems at Gerupuk Bay, Central Lombok, Indonesia Short Communication: Variation in isozymic pattern of germplasm from three ginger (Zingiber officinale) varieties Short Communication: Comparisons of isozyme diversity in local Java cardamom (Amomum compactum) and true cardamom (Elettaria cardamomum) Short Communication: An insight into protein sequences of PTP-like cysteine phytases

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