Plant Physiology and Biochemistry 67 (2013) 15e19
Contents lists available at SciVerse ScienceDirect
Plant Physiology and Biochemistry
journal homepage: www.elsevier.com/locate/plaphy
Research article
Distribution of the cardiotoxin pavettamine in the coffee family (Rubiaceae)
and its significance for gousiekte, a fatal poisoning of ruminants
Daan Van Elst a, *, Sarah Nuyens a, Braam van Wyk b, Brecht Verstraete c, Steven Dessein d, Els Prinsen a
a
Plant Growth and Development, University of Antwerp, Antwerp, Belgium
H.G.W.J. Schweickerdt Herbarium, University of Pretoria, Pretoria 0002, South Africa
c
Plant Conservation and Population Biology, KU Leuven, Leuven, Belgium
d
National Botanic Garden of Belgium, Meise, Belgium
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 November 2012
Accepted 26 February 2013
Available online 7 March 2013
Gousiekte, a cardiac syndrome of ruminants in southern Africa, is caused by the ingestion of plants
containing the polyamine pavettamine. All the six known gousiekte-causing plants are members of the
Rubiaceae or coffee family and house endosymbiotic Burkholderia bacteria in their leaves. It was
therefore hypothesized that these bacteria could be involved in the production of the toxin. The
pavettamine level in the leaves of 82 taxa from 14 genera was determined. Included in the analyses were
various nodulated and non-nodulated members of the Rubiaceae. This led to the discovery of other
pavettamine producing Rubiaceae, namely Psychotria kirkii and Psychotria viridiflora. Our analysis showed
that many plant species containing bacterial nodules in their leaves do not produce pavettamine. It is
consequently unlikely that the endosymbiont alone can be accredited for the synthesis of the toxin. Until
now the inconsistent toxicity of the gousiekte-causing plants have hindered studies that aimed at a
better understanding of the disease. In vitro dedifferentiated plant cell cultures are a useful tool for the
study of molecular processes. Plant callus cultures were obtained from pavettamine-positive species.
Mass spectrometric analysis shows that these calli do not produce pavettamine but can produce common
plant polyamines.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Pavettamine
Gousiekte
Rubiaceae
Toxin
Polyamine
1. Introduction
South Africa has a rich and varied flora that includes some 600
poisonous plants [1e3]. Plant poisoning of livestock is responsible
for considerable economic losses in southern Africa (that part of the
African continent south of the Kunene, Okavango and Zambezi
Rivers). One of the six most important plant toxicoses in this region
is gousiekte, causing the death of about 7000 head of livestock,
mainly sheep, goats and cattle, each year [1,4,5]. Gousiekte
Abbreviations: UPLCÔ, ultra performance liquid chromatography; MS/MS, tandem mass spectrometry; IS, internal standard; BR, the National Botanic Garden of
Belgium; PRU, the Manie van der Schijff Botanical Garden, University of Pretoria;
DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; TQD,
triple quadrupole detector; BEH, ethylene bridged hybrid; MRM, Multiple reaction
monitoring.
* Corresponding author. Tel.: þ32 3 2653714; fax: þ32 3 2653417.
E-mail addresses: Daan.VanElst@ua.ac.be (D. Van Elst), Sarah.Nuyens@
student.ua.ac.be (S. Nuyens), Braam.VanWyk@up.ac.za (B. van Wyk),
Brecht.Verstraete@bio.kuleuven.be (B. Verstraete), Steven.Dessein@br.fgov.be
(S. Dessein), Els.Prinsen@ua.ac.be (E. Prinsen).
0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.plaphy.2013.02.022
(Afrikaans for "quick disease") is a cardiac syndrome of domestic
ruminants caused by the ingestion of certain poisonous plants. The
disease is characterized by sudden death four to eight weeks after
the initial intake of toxic plants, usually without obvious prodromal
symptoms. At present six plant species, all belonging to the Rubiaceae, are known with certainty to cause the disease: Vangueria
pygmaea (syn. Pachystigma pygmaeum) [6], Vangueria thamnus (syn.
Pachystigma thamnus) [7], Vangueria latifolia (syn. Pachystigma latifolium), Pavetta schumanniana [8], Pavetta harborii [9] and Fadogia
homblei (syn. Fadogia monticola) [4].
Research on gousiekte commenced in 1908 when Walker
attempted to establish the cause of the disease [6]. After many
earlier authors have failed in their attempts, Fourie and coworkers
[10] succeeded in isolating the causal toxin. They demonstrated the
presence of the gousiekte-inducing compound in Pa. harborii, Pa.
schumanniana, V. pygmaea and F. homblei. The chemical structure of
the toxin was published in 2010 [11]. It is a polyamine and was
named pavettamine after the genus Pavetta, of which two species
have been identified to cause the disease. It was hypothesized that
endosymbiotic bacteria could be involved in the production of the
toxin due to the fact that all six gousiekte-causing plants house
16
D. Van Elst et al. / Plant Physiology and Biochemistry 67 (2013) 15e19
bacteria of the genus Burkholderia in their leaves [12e14]. At present it is not known whether the endosymbiont plays any role in
the production of the poisonous compound. Analysis of in vitro
cultures of the F. homblei endosymbiont, however, did not reveal
production of pavettamine [15].
In the past, studies aimed at proving the link between gousiekte
and suspected plants met with considerable difficulties as a significant number of animal feeding experiments gave negative results
[10,13,16]. The toxicity of the known gousiekte-causing plants is
variable and diminishes during drying. Animals differ in their susceptibility to the toxin and the disease cannot be induced in small
laboratory animals. Moreover, feeding experiments have to deal
with a long latency period and the lack of premonitory signs [16]. An
earlier experiment in which sheep were fed limited quantities of F.
homblei gave negative results. It was assumed that the dose
employed at that time was too low since subsequent studies proved
this plant to cause gousiekte [4]. Therefore it was suggested that any
rubiaceous plant could only be discounted as a possible cause of
gousiekte if subjected to extensive feeding experiments [13].
Many plants closely related to the six known gousiekte-causing
species occur in southern Africa. The Rubiaceae or coffee family is
the fourth most species-rich flowering plant family with more than
13 000 species comprising about 600 genera [17]. The Rubiaceae is
particularly well represented in humid tropical forests, with species
diversity decreasing rapidly from the subtropics through the
temperate regions to the poles [17,18]. In southern Africa alone there
occur more than 30 species of Pavetta [19,20]. It would be helpful to
determine if in any of these plants the toxic principle is present and
in which order of magnitude. Other Rubiaceae, or even species from
other plant families, might contain pavettamine, perhaps in a lower
concentration, insufficient to cause gousiekte. Alternatively such
plants may not be consumed in significant quantities by domestic
ruminants. The isolation procedure for the toxin described by Fourie
et al. [10] made it possible to chemically assay plants for their
toxicity. However, this method does not quantify the concentration
of pavettamine and, as the authors stated, the procedure is tedious.
Recently, a mass spectrometry based method for the analysis of
pavettamine was reported [15]. It allows detection and quantification of pavettamine in biological samples in a fast and sensitive
manner without the need for large sample volumes. Hitherto, plants
or plant fractions could only be assayed for toxicity by using ethically questionable biological trials [10].
The primary objective of the present study is to assess whether
pavettamine is present in other plant species, including species
that lack bacterial endosymbionts. To estimate the role of bacteria
and plants in the production of the toxin, callus cultures of
pavettamine-positive species were tested in their capacity to produce the toxin in the absence of bacteria.
2. Results and discussion
2.1. Pavettamine is present in other plants than the six known
gousiekte-causing species
The potential presence and concentration of pavettamine were
determined through detection by tandem mass spectrometry after
derivatization with benzoyl chloride and separation by ultraperformance liquid chromatography (UPLCÔ) [15]. The selected
plants, 82 taxa from 14 genera, are from the Rubiaceae since gousiekte has invariably been associated with plants of this family
[4,16]. Given the presumed link between gousiekte-causing species
and endosymbiotic bacteria, nodulated species were of particular
interest. Plant taxa, in which pavettamine was detected, are listed
in Table 1. Two additional species were found to be positive for the
toxin, namely Psychotria kirkii and Psychotria viridiflora. The genus
Table 1
List of plant samples in which pavettamine was detected. Value in nmol/g fresh
weight (* nmol/g dry weight) st error (N ¼ 5).
Plant name
Accession
Pavettamine
Fadogia hombleia,c
Pavetta sp.b
Pa. harboriib
Pa. schumannianab
Pa. schumannianab
Psychotria sp.b
Ps. kirkiib
Ps. kirkiib
Ps. kirkiib
Ps. kirkiib
Ps. kirkiib
Ps. kirkiib
Ps. kirkiib
Ps. kirkiib
Ps. kirkiib
Ps. kirkii var. hirtellab
Ps. kirkii var. nairobiensisb
Ps. kirkii var. tarambassicab
Ps. viridiflorab
Ps. cf. kirkiib
Vangueria pygmaeac
Wild collected
BR-20060123-38
Wild collected
BR-20041430-66
BR-20001942-57
BR-20001933-48
BR-19951273-22
BR-20010513-92
BR-19761893
BR-20070328-58
BR-19750521
BR-20021203-15
BR-20021526-47
BR-20070330-60
BR-20001946-61
BR-20001036-24
BR-19981825-19
BR-19536779
BR-20070138-62
BR-20001943-58
Wild collected
296
4135
1284
230
1381
4084
3116
551
414
1352
3660
3330
1644
2095
2005
8674
322
1396
1256
5172
374
a
b
c
47*
121
68*
18
79
174
101
21
31
156
151
226
118
272
79
1124
26
219
49
513
13*
value adopted from Van Elst et al. [15].
Species with leaf nodules.
Species with non-nodulating bacterial endophytes.
Psychotria was previously not linked to the aetiology of gousiekte.
In fact, the six gousiekte-causing species all belong to the subfamily
Ixoroideae, while the genus Psychotria is of the subfamily Rubioideae [14,21]. Two other accessions, one nodulated Psychotria and
one nodulated Pavetta species also produce pavettamine. Psychotria
is the world’s third largest flowering plant genus and the largest in
the Rubiaceae [17]. We were unable to detect pavettamine in any of
the other genera tested. Considering the concentration of pavettamine detected in these plants, it appears that the Psychotria species produce pavettamine in higher amounts that the traditional
gousiekte-causing plant species. However, it is known that the
toxicity in these plants varies at different times of the year, as well
as from year to year [4,6,8]. Toxicity apparently also varies according to locality, habitat and probably climatic conditions [6,8].
The conditions of the plants grown in the greenhouses of the National Botanic Garden of Belgium might not accurately correspond
to in-field conditions. Furthermore, a threshold concentration of
pavettamine in leaves has not been determined for causing the
onset of the development of gousiekte.
The mass spectrometry method for the quantification of
pavettamine as described by Van Elst et al. [15] allows the detection
of several other important polyamines (diaminopropane, putrescine, cadaverine, spermidine, spermine and agmatine) alongside
pavettamine in biological samples. Pavettamine certainly is an unusual polyamine and of the common plant polyamines most closely
resembles cadaverine (see Fig. 1), both having a carbon chain of five
carbon atoms. Cadaverine is formed by the decarboxylation of lysine
[22]. We did not detect cadaverine in many of the selected plants.
However, we observed that all plants able to produced pavettamine
can also produce cadaverine (see Table A.1, Supplementary files). At
the moment, it is not known how pavettamine is synthesised. Given
their structural similarity, cadaverine might be involved in the
biosynthesis of pavettamine. Further studies should elucidate the
possible relation between cadaverine and pavettamine.
2.2. Gousiekte, a disease of southern Africa?
In 1923, the Director of Veterinary Services in South Africa,
Arnold Theiler, claimed that ‘Gousiekte is a disease of South Africa’
D. Van Elst et al. / Plant Physiology and Biochemistry 67 (2013) 15e19
H2N
OH
OH
2
4
1
H2N
3
2
1
H2N
H2N
NH 2
NH 2
5
4
NH 2
3
2
1
H
N
4
2
OH
5
3
1
OH
4
3
N
H
NH 2
Fig. 1. Comparison of the structures of pavettamine, cadaverine, putrescine and
spermidine (from top to bottom).
[23]. So far, gousiekte has been diagnosed in the northeastern part
of South Africa, Botswana and southern Zimbabwe [4,24]. The
geographical ranges of all six gousiekte-inducing plants overlap in
the former Transvaal region, where most of the outbreaks happen
[16]. However, the geographical range of these plants is thus much
wider than the incidence of the poisoning syndrome [25]. We have
detected pavettamine in several plants not collected from the area
where gousiekte occurs. The two analysed accessions of Pa. schumanniana have an origin from Zambia and D.R. Congo. This indicates that the plants do produce the toxin throughout their
geographical distribution range. The natural distribution of the Ps.
kirkii varieties is from Gabon to Ethiopia and southern tropical
Africa. Ps. viridiflora occurs naturally from Indo-China to Malaysia. It
is peculiar that the gousiekte syndrome, which is of such economic
importance in southern Africa, has not been encountered elsewhere in the world [4].
In the present study we have reported pavettamine to be present
in members of the Rubiaceae hitherto not implicated in the disease.
Recently, gousiekte has been diagnosed in wild African buffalo
(Syncerus caffer) in the Mutirikwe Recreational Park southeast of
Masvingo in Zimbabwe. Of the plants known to cause gousiekte, Pa.
schumanniana occurs widely in the area [24]. It was reported that
the buffalo herd, perturbed in their normal behaviour, frequented
the densely wooded areas of the park instead of the more open
vegetation types. It is in these more wooded areas that Pa. schumanniana is quite common. However, our results show that another
plant present in the same area, Ps. kirkii, also produces pavettamine
and consequently could cause gousiekte. It is the only widespread
species of Psychotria in Zimbabwe, occurring in savannah and
various types of woodland, often associated with rocky outcrops or
termite mounds [26,27]. The intoxication of the buffalo reported by
Lawrence et al. [24] could have possibly been caused by or aggravated by Ps. kirkii. It was commented previously that other toxic
rubiaceous species might be ignored in areas where known gousiekte plants have been identified [13]. To date it is only possible to
diagnose gousiekte post-mortem. Therefore, the prevention of
intoxication remains the most important way to protect animals
from gousiekte-causing plants [1,16]. It is therefore advantageous to
know which species of plants contain the toxic pavettamine.
2.3. Possible association between gousiekte and endophytic bacteria
A possible link between endosymbiotic bacteria and gousiekte
was postulated by Van Wyk et al. [13] following the discovery of
non-nodulating bacterial endophytes in the leaves of gousiekteinducing members of the genera Fadogia and Vangueria. In the
17
two gousiekte-causing Pavetta species endosymbiotic bacteria are
confined to distinct nodules in the leaf lamina [28]. Consequently,
all gousiekte-causing plants contain bacterial endosymbionts in
their leaves. DNA analysis of the bacterial endosymbionts in
members of the Rubiaceae revealed that all these bacteria belong to
the same genus, namely Burkholderia [12,14,29]. In addition, Verstraete et al. [14] analysed leaves from members of the genera
Afrocanthium, Canthium, Keetia, Psydrax, Pygmaeothamnus and
Pyrostria and found no presence of endosymbionts. Animal feeding
studies confirmed that two of these non-bacteriophilous Rubiaceae
species (Pygmaeothamnus zeyheri and Pygmaeothamnus chamaedendrum) are unable to cause gousiekte [4,6]. It was thus hypothesized that the endosymbiont might be involved in the production
of the toxic compound [13,14]. For the plant F. homblei, the endosymbiont was shown to be able to grow outside the host. However,
it did not produce pavettamine in vitro when grown axenically [15].
The possibility remained that both partners in this plantebacteria
interaction are needed for the synthesis of the toxin or that unknown signals induce the synthesis of pavettamine in the bacteria.
Our analysis shows that many plant species containing bacterial
nodules in their leaves do not produce pavettamine (see Table A.1,
Supplementary files). This is the case for Pavetta sp. (BR-2012118183), Pavetta radicans, Pavetta bowkeri, Pavetta zeyheri, Pavetta lanceolata, Pavetta gardeniifolia, Psychotria calva, Psychotria humilis,
Psychotria kikwitensis, Psychotria brachyanthoides, Psychotria pumila
var. pumila and Psychotria verschuerenii var. reducta. It is therefore
unlikely that the endosymbiont alone can be accredited for the
production of the toxin. Significantly, all pavettamine positive
plants remain bacteriophilous, since the species shown in this
study to produce pavettamine are also nodulated.
2.4. Callus cultures unable to produce pavettamine
Until now the inconsistent toxicity of the gousiekte-causing
plants have hindered studies that aimed at a better understanding
of the disease. Basic knowledge on the plant physiological aspects of
pavettamine is lacking [15]. Plant cell cultures are being widely used
in scientific studies on the physiology, biochemistry and molecular
biology of primary and secondary metabolism [30e32]. In vitro
dedifferentiated plant cell cultures are more convenient for the
study of cellular and molecular processes as they offer the advantage of a simplified model system for the study of plants and are
more easily controlled compared to whole plant systems [32,33].
Callus was successfully initiated from sterilized leaf explants (not
containing visible bacterial nodules) of two pavettamine positive
species (Pa. schumanniana, Ps. kirkii var. tarambassica). Calli were
maintained on growth medium and subcultured every 4 weeks.
Sample of these callus cultures where analysed for their polyamine
content and found to be unable to produce pavettamine. Mass
spectrometric analysis shows that these calli can produce diaminopropane, putrescine, spermidine, spermine and agmatine on
unsupplemented growth medium (see Table 2). Only the callus of
Pa. schumanniana produced cadaverine, yet in very small amounts.
At the moment there is no information on how pavettamine is being
synthesized in the plant. Therefore, the growth media was supplemented with the common polyamine precursors: arginine, ornithine and lysine. Under these conditions, we could also not detect
pavettamine in any of the calli cultures (see Table 2). Despite the low
detection limit of the mass spectrometry method (reported
0.3 pmol in 6 ml injected volume [15]), we could not detect any trace
of pavettamine in the different callus samples. Addition of the
polyamine precursors did have an effect on the concentrations of
other polyamines analysed in the callus samples. For instance, the
amount of cadaverine in the callus was higher when lysine was
supplemented. A number of chemical and physical factors (such as
18
D. Van Elst et al. / Plant Physiology and Biochemistry 67 (2013) 15e19
Table 2
Polyamine concentrations determined in callus samples of Pavetta schumanniana and Psychotria kirkii var. tarambassica grown on unsupplemented and on polyamine-precursors supplemented medium. Value in nmol/g fresh weight st error (N ¼ 4) for putrescine (PUT), spermidine (SPD) and spermine (SPM); value in pmol/g fresh weight st
error (N ¼ 4) for diaminopropane (DAP), cadaverine (CAD), agmatine (AGM) and pavettamine (PAV).
PUT
Pa. schumanniana
Unsupplemented
þ Arginine
þ Lysine
þ Ornithine
Ps. kirkii var. tarambassica
Unsupplemented
þ Arginine
þ Lysine
þ Ornithine
SPD
SPM
DAP
CAD
AGM
PAV
73.1
98.4
68.9
81.9
5.5
12.2
10.2
13.3
24.3
34.5
26.6
28.9
1.2
2.2
1.8
3.4
3.04
3.15
3.19
2.85
0.18
0.36
0.18
0.14
347
273
218
227
10
16
19
24
23 2
ND
240 18
ND
486
828
563
414
47
117
85
65
ND
ND
ND
ND
64.7
81.9
68.5
67.2
4.8
3.2
6.1
6.6
42.9
68.7
41.6
54.5
2.6
3.3
5.0
3.9
8.71
9.70
8.92
9.90
0.70
0.35
0.29
0.58
207
280
240
216
30
24
30
34
ND
ND
217 17
ND
372
548
409
415
41
93
65
42
ND
ND
ND
ND
ND not detected.
media constitution, pH, temperature and light) affect production of
secondary metabolites in plant cell cultures. Manipulation of cell
culture conditions is one of the most fundamental approaches for
optimization of culture productivity [31,32,34]. A yet unknown
stimulus is probably responsible for the onset of pavettamine synthesis. Dedifferentiated cell cultures (i.e. callus or suspension) often
produce low levels of secondary metabolites compared to differentiated cell cultures (i.e. roots or shoots) [32]. Future research into
calli from pavettamine producing plants holds promise for a
simplified model system in which different environmental factors,
as well as the presence or absence of bacterial endophytes, can be
tested for their influence in the production of the toxin.
3. Experimental
3.1. Sample material
Leaf sample material of the selected plants was collected from
the living collections of the National Botanic Garden of Belgium
(BR), the Manie van der Schijff Botanical Garden at the University of
Pretoria (PRU) and during a field expedition to South Africa in
February 2010. The most apical leaves on actively growing shoots
were removed and immediately and individually frozen in liquid
nitrogen. Leaf samples were stored at 80 C until processed for
mass spectrometric analysis. Polyamines are generally abundant in
young, non-senescent organs, and decline to a lower concentration
as organs age and senesce [35]. Previous analysis of the gousiektecausing plant F. homblei had shown that young leaves contain the
highest concentration of pavettamine [15].
3.2. Initiation of callus cultures
Two plants, able to produce pavettamine, were selected for the
creation of plant cell cultures: the known gousiekte-causing Pa.
schumanniana (BR-20001942-57) and Ps. kirkii var. tarambassica
(BR-19536779), shown in this study to produce pavettamine. Callus
was initiated from pieces of leaf tissue cut from surface sterilized
plants. Sterilization was achieved by immersing the explants in 70%
ethanol for 1 min followed by 10 min of 1% hypochlorite solution.
Sterilized explants were washed repeatedly with sterilized deionized water to remove the hypochlorite solution and subsequently
placed on growth medium plates containing mediumP (http://
www.dsmz.de/home.html). MediumP contains 20 g/l sucrose,
picloram 0.1 mg/l and Gamborg B5 medium. The explants were
incubated in the dark at 23 C. Calli were formed readily and were
subcultured every 4 weeks. Basic growth medium was supplemented with 0.1 mM of the polyamine precursors L-arginine, Llysine and L-ornithine to elicit pavettamine production. Callus
samples from unsupplemented and polyamine precursor supplemented medium were harvested after 21 days of growth at 23 C in
the dark. All chemicals used were obtained from Sigma Aldrich
(Schnelldorf, Germany).
3.3. Extraction and derivatization of polyamines
The derivatization procedure was adopted from Van Elst et al.
[15]. Briefly, polyamines were extracted by adding 1 ml perchloric
acid (5%) per 100 mg of powdered tissue. After incubation on ice for
60 min, the homogenate was centrifuged (20 min, 20000 g, 4 C).
250 ml of this extract was mixed with 1.5 ml 2 N NaOH and
200 pmol IS. The internal standard 1,7-diaminoheptane was obtained from Sigma Aldrich (Schnelldorf, Germany). The extracts
were derivatized using 20 ml benzoyl chloride (20 min, room
temperature). Benzoyl chloride was of reagent grade, >99% purity
A.C.S. (Sigma Aldrich). Benzoylated polyamines were extracted in
4 ml diethyl ether. The aqueous phase was discarded; the ether
phase was washed with distilled water, collected and evaporated
under a stream of nitrogen. Samples were stored at 20 C until
being redissolved in 80% ACN and transferred to inserts before injection in a UPLCÔ MS/MS system. Acetonitrile (HPLC grade), water
(HPLC grade) and ether were of VWR prolabo (Leuven, Belgium).
3.4. Analysis of benzoylated polyamines by UPLCeMS/MS
Chromatography and detection by mass spectrometry was performed using an ACQUITY UPLCÔ TQD system (Waters, Micromass,
Ltd., Manchester, United Kingdom) equipped with electrospray
ionization. Of the redissolved sample, 6 ml (partial loop) was injected
in an ACQUITY UPLCÔ BEH C18 column (1.7 mm 2.1 mm 50 mm,
Waters) fitted with a VanGuardÔ Pre-Column (2.1 mm 5 mm,
Waters). The mass spectrometer was used in multiple reactionmonitoring mode (MRM). MRM transitions, cone, collision energy
settings and chromatographic parameters are adopted from Van
Elst et al. [15]. Masslynx NT version 4.1 (Waters) software was used
to analyse the data.
Acknowledgements
The authors would like to thank Mr. H. Lekeux, Mr. J., Stallaert,
Mr. J. Van Eeckhoudt, Ms. E. Bellefroid and Mr. F. Van Caekenberghe,
who are involved in the maintenance of the Rubiaceae collection of
the National Botanic Garden of Belgium and Mr. J. Sampson, curator
of the Manie van der Schijff Botanical Garden, University of Pretoria.
This research was supported financially by the Fund for Scientific
Research, Flanders (FWO, G.0343.09N). D.V.E. holds a PhD research
grant from the Fund for Scientific Research, Flanders (FWO, no.
D. Van Elst et al. / Plant Physiology and Biochemistry 67 (2013) 15e19
1.1.722.10.N.00). B.V. holds a PhD research grant from the Institute
for the Promotion of Innovation by Science and Technology in
Flanders (IWT, no. 91158). The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of
the manuscript.
[15]
[16]
Appendix A. Supplementary data
[17]
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.plaphy.2013.02.022.
[18]
[19]
References
[20]
[1] C.J. Botha, M.L. Penrith, Poisonous plants of veterinary and human importance
in southern Africa, J. Ethnopharm. 119 (2008) 549e558.
[2] T.S. Kellerman, Poisonous plants, Onderstepoort J. Vet. Res. 76 (2009) 19e23.
[3] J. Vahrmeyer, Poisonous Plants of Southern Africa that Cause Stock Losses,
Tafelberg, Cape Town, 1981.
[4] L.R. Hurter, T.W. Naudé, T.F. Adelaar, J.D. Smit, L.E. Codd, Ingestion of the plant
Fadogia monticola Robyns as an additional cause of gousiekte in ruminants,
Onderstepoort J. Vet. Res. 39 (1972) 71e82.
[5] T.S. Kellerman, T.W. Naudé, N. Fourie, The distribution, diagnosis and estimated economic impact of plant poisonings and mycotoxicosis in South Africa, Onderstepoort J. Vet. Res. 63 (1996) 65e90.
[6] A. Theiler, P.J. Du Toit, D.T. Mitchell, Gousiekte in Sheep, in: 9th and 10th
Reports of the Director of Veterinary Education and Research, The Government Printing and Stationary Office, Onderstepoort, Pretoria, 1923.
[7] T.F. Adelaar, M. Terblanche, A note on the toxicity of the plant Pachystigma
thamnus Robyns, J. S. Afr. Vet. Med. Assoc. 38 (1967) 25e26.
[8] T.S. Kellerman, J.A.W. Coetzer, T.W. Naudé, Plant Poisonings and Mycotoxicoses
of Livestock in Southern Africa, Oxford University Press, Cape Town, 1988.
[9] P.L. Uys, T.F. Adelaar, A new poisonous plant, J. S. Afr. Vet. Med. Assoc. 28
(1957) 5e8.
[10] N. Fourie, G.L. Erasmus, R.A. Schultz, L. Prozesky, Isolation of the toxin
responsible for gousiekte, a plant-induced cardiomyopathy of ruminants in
southern Africa, Onderstepoort J. Vet. Res. 62 (1995) 77e87.
[11] M.L. Bode, P.J. Gates, S.Y. Gebretnsae, R. Vleggaar, Structure elucidation and
stereoselective total synthesis of pavettamine, the causal agent of gousiekte,
Tetrahedron 66 (2010) 2026e2036.
[12] B. Lemaire, S. Van Oevelen, P. De Block, B. Verstraete, E. Smets, E. Prinsen,
S. Dessein, Identification of the bacterial endosymbionts in leaf nodules of
Pavetta (Rubiaceae), Int. J. Syst. Evol. Microbiol. 62 (2012) 202e209.
[13] A.E. Van Wyk, P.D.F. Kok, N.L. Van Bers, C.F. Van der Merwe, Non-pathological
bacterial symbiosis in Pachystigma and Fadogia (Rubiaceae): its evolutionary
significance and possible involvement in the aetiology of gousiekte in domestic ruminants, S. Afr. J. Sci. 86 (1990) 93e96.
[14] B. Verstraete, D. Van Elst, H. Steyn, B. Van Wyk, B. Lemaire, E. Smets,
S. Dessein, Endophytic bacteria in toxic South African plants: identification,
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
19
phylogeny and possible involvement in gousiekte, PLoS One 6 (4) (2011)
e19265, http://dx.doi.org/10.1371/journal.pone.0019265.
D. Van Elst, B. van Wyk, A. Schultz, E. Prinsen, Production of toxic pavettamine
and pavettamine conjugates in the gousiekte-causing Fadogia homblei
plant and its relation to the bacterial endosymbiont, Phytochemistry 85
(2013) 92e98.
T.S. Kellerman, J.A.W. Coetzer, T.W. Naudé, C.J. Botha, Plant Poisonings and
Mycotoxicoses of Livestock in Southern Africa, second ed., Oxford University
Press, Cape Town, 2005.
A.P. Davis, R. Govaerts, D.M. Bridson, M. Ruhsam, J. Moat, N.A. Brummitt,
A global assessment of distribution, diversity, endemism and taxonomic effort
in the Rubiaceae, Ann. Mo. Bot. Gard. 96 (2009) 68e78.
E. Robbrecht, Tropical woody Rubiaceae, Opera Bot. Belg. 1 (1988) 1e271.
D.M. Bridson, Pavetta L, in: G.V. Pope (Ed.), Flora Zambesiaca, vol. 5, Royal
Botanic Gardens, Kew for Flora Zambesiaca Managing Committee, London,
2003, pp. 544e598 (3).
P.D.F. Kok, N. Grobbelaar, Studies on Pavetta (Rubiaceae) II. Enumeration of
species and synonymy, S. Afr. J. Bot. 3 (1984) 185e187.
B. Bremer, T. Eriksson, Time tree of Rubiaceae: phylogeny and dating the
family, subfamilies, and tribes, Int. J. Plant Sci. 170 (2009) 766e793.
T. Kusano, T. Berberich, C. Tateda, Y. Takahashi, Polyamines: essential factors
for growth and survival, Planta 228 (2008) 367e381.
K. Brown, Poisonous plants, pastoral knowledge and perceptions of environmental change in South Africa, c. 1880-1940, Environ. Hist. 13 (2007)
307e332.
J.A. Lawrence, C.M. Foggin, L. Prozesky, Gousiekte in African buffalo (Syncerus
caffer), J. S. Afr. Vet. Assoc. 81 (2010) 170e171.
T.W. Naudé, T.S. Kellerman, J.A.W. Coetzer, Plant poisonings and mycotoxicoses as constraints in livestock production in East Africa: the southern African experience, J. S. Afr. Vet. Assoc. 67 (1996) 8e11.
M. Coates Palgrave, Keith Coates Palgrave Trees of Southern Africa, third ed.,
second impression, Struik Publishers, Cape Town, 2005.
B. Van Wyk, E. Van den Berg, M. Coates Palgrave, M. Jordaan, Dictionary of
Names for Southern African Trees, Briza Publications, Pretoria, 2011.
I.M. Miller, Bacterial leaf nodule symbiosis, Adv. Bot. Res. 17 (1990) 163e243.
B. Lemaire, E. Robbrecht, B. Van Wyk, S. Van Oevelen, B. Verstraete, E. Prinsen,
E. Smets, S. Dessein, Identification, origin and evolution of leaf nodulating
symbionts of Sericanthe (Rubiaceae), J. Microbiol. 49 (2011) 935e941.
H.P. Mühlbach, Use of plant cell cultures in biotechnology, Biotechnol. Annu.
Rev. 4 (1998) 113e176.
I. Smetanska, Production of secondary metabolites using plant cell cultures,
Adv. Biochem. Eng. Biotechnol. 111 (2008) 187e228.
S.A. Wilson, S.C. Roberts, Recent advances towards development and
commercialization of plant cell culture processes for the synthesis of biomolecules, Plant Biotechnol. J. 10 (2012) 249e268.
N.R. Mustafa, W. de Winter, F. van Iren, R. Verpoorte, Initiation, growth and
cryopreservation of plant cell suspension cultures, Nat. Protoc. 6 (2011)
715e742.
V. Mulabagal, H. Tsay, Plant cell cultures as a source for the production of
biologically important secondary metabolites, Int. J. Appl. Sci. Eng. 2 (2004)
29e48.
A.W. Galston, R.K. Sahwney, Polyamines in plant physiology, Plant Physiol. 94
(1990) 406e410.