Al-Subhi et al. BMC Microbiology (2017) 17:221
DOI 10.1186/s12866-017-1130-3
RESEARCH ARTICLE
Open Access
Classification of a new phytoplasmas
subgroup 16SrII-W associated with
Crotalaria witches’ broom diseases in Oman
based on multigene sequence analysis
Ali Al-Subhi1, Saskia A. Hogenhout2, Rashid A. Al-Yahyai1 and Abdullah M. Al-Sadi1*
Abstract
Background: Crotalaria aegyptiaca, a low shrub is commonly observed in the sandy soils of wadis desert and is found
throughout all regions in Oman. A survey for phytoplasma diseases was conducted. During a survey in a wild area in
the northern regions of Oman in 2015, typical symptoms of phytoplasma infection were observed on C. aegyptiaca
plants. The infected plants showed an excessive proliferation of their shoots and small leaves.
Results: The presence of phytoplasma in the phloem tissue of symptomatic C. aegyptiaca leaf samples was confirmed
by using Transmission Electron Microscopy (TEM). In addition the extracted DNA from symptomatic C. aegyptiaca leaf
samples and Orosius sp. leafhoppers were tested by PCR using phytoplasma specific primers for the 16S rDNA, secA,
tuf and imp, and SAP11 genes. The PCR amplifications from all samples yielded the expected products, but not from
asymptomatic plant samples. Sequence similarity and phylogenetic tree analyses of four genes (16S rDNA, secA, tuf and
imp) showed that Crotalaria witches’ broom phytoplasmas from Oman is placed with the clade of Peanut WB (16SrII)
close to Fava bean phyllody (16SrII-C), Cotton phyllody and phytoplasmas (16SrII-F), and Candidatus Phytoplasma
aurantifolia’ (16SrII-B). However, the Crotalaria’s phytoplasma was in a separate sub-clade from all the other
phytoplasmas belonging to Peanut WB group. The combination of specific primers for the SAP11 gene of 16SrII-A, −B,
and -D subgroup pytoplasmas were tested against Crotalaria witches’ broom phytoplasmas and no PCR product
was amplified, which suggests that the SAP11 of Crotalaria phytoplasma is different from the SAP11 of the other
phytoplasmas.
Conclusion: We propose to assign the Crotalaria witches’ broom from Oman in a new lineage 16SrII-W subgroup
depending on the sequences analysis of 16S rRNA, secA, imp, tuf, and SAP11 genes. To our knowledge, this is the
first report of phytoplasmas of the 16SrII group infecting C. aegyptiaca worldwide.
Keywords: WBD, Phytoplasma phylogeny, Crotalaria
Background
Crotalaria aegyptiaca (Benth), a low shrub that reaches
about 60 cm high, is commonly observed in the sandy
soils of desert wadis [1]. C. aegyptiaca is mostly distributed in the Middle East, including Egypt and the Arabian
Peninsula. Additionally it spreads throughout all regions
in Oman [2, 3]. C. aegyptiaca containing pyrrolizidine
* Correspondence: alsadi@squ.edu.om
1
Department of Crop Sciences, College of Agricultural and Marine Sciences,
Sultan Qaboos University, Al Khod 123, PO Box 34, Seeb, Oman
Full list of author information is available at the end of the article
alkaloids (PAs) [4] is used in traditional medicine [5] and
as an antitumor [6]. Sheep and goats do not graze C.
aegyptiaca because of the Pas’ toxicity, but is grazed by
camels and gazelles [3, 5].
A survey for phytoplasma diseases in wild plants was
conducted in the northern regions of Oman in 2015.
During this survey, a typical symptom of phytoplasma
infection was observed on C. aegyptiaca plants in three
different locations. The infected plants showed an excessive proliferation of their shoots which is indicative of
witch’s broom disease (Fig. 1).
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Al-Subhi et al. BMC Microbiology (2017) 17:221
Page 2 of 14
Fig. 1 a & b. is an infected C. aegyptiaca plant showing witches’ broom symptoms with an excessive number of little leaves and short shoots; (c).
& (d). is a healthy C. aegyptiaca plant; (e). is a Orosius sp. Leafhopper which was collected from the infected C. aegyptiaca sites using yellow sticky
traps visible in Fig. 1a
Phytoplasma belonging to the 16SrII-B and -D subgroups have been reported on several economic plants
in Oman. Typical symptoms of phytoplasma in acid lime
(Citrus aurantifolia) showing witches’-broom (WBDL)
were first reported in Oman during 1970’s [7]. Zreik et
al. [8] identified the 16SrII-B subgroup phytoplasma as a
causal agent of WBDL. The 16SrII-B subgroup phytoplasma has only been recorded as a host specific to citrus crops in Oman [8, 9]. Though, more than 20 host
plants belonging to different families were found infected with the 16SrII-D subgroup phytoplasma in
Oman. Examples include alfalfa [10], sesame (Sesamum
indicum L.) [11] and chickpea [12].
Phytoplasmas (genus ‘Candidatus Phytoplasma’) are
intracellular plant pathogenic bacteria in the class Mollicutes [13, 14]. They are transmitted and disseminated into
healthy plant phloem from the salivary glands of insect
vectors, such as, leafhoppers, planthoppers and psyllids
[15–17] and by vegetative propagation [18, 19]. Symptoms
associated with phytoplasmas infections include virescence, phyllody, yellowing, stunting, excessive proliferation of shoots, the formation of witches’ brooms, and big
bud [20]. Phytoplasmas are obligate parasites so they cannot be cultured on artificial growth media; this makes
their identification and characterization difficult. Different methods have been used for the detection of phytoplasma. The first discovery, visual observation and
description of phytoplasmas were accomplished with
transmission electron microscopy (TEM) in 1967 [21].
TEM was frequently used to provide reliable and accurate methods for diagnosing phytoplasma diseases as
well as to get information on the morphology, size, and
concentration of phytoplasma bodies in sieve tube elements and insect vectors [22, 23].
The latest developments in the last three decades of
molecular-based methods for the detection and identification of phytoplasmas have largely replaced the traditional methods [24]. Furthermore, they have led to a
dramatic increase in the understanding of phytoplasmas
in the fields of classification, genome sequencing and
their interaction with plant hosts and insect vectors. In
the early 1990’s, the design of “universal” primers, helped
in classification of phytoplasmas [24–26]. For the fine
classification of phytoplasmas, to describe subgroups
within the 16Sr groups, the 16S rRNA gene is not sufficient due to being highly conserved and is a non-coding
gene [27–30]. Many studies have used less-conserved or
variable genes as extra molecular markers in conjunction
with the 16S rRNA gene for the finer classification of
closely related phytoplasma species. The tuf gene has
been used for 16SrI and 16SrXII subgroup diversity of
phytoplasmas [28, 31]. Makarova et al. [32] reported that
the tuf gene provides a better phytoplasma identification
than the 16S rRNA gene. The sequences of secA and 23S
rRNA genes were used for finer phylogenetic analyses of
phytoplasma and their efficient use in phytoplasma disease
Al-Subhi et al. BMC Microbiology (2017) 17:221
diagnostics [33]. Three non-homologous protein types including immunodominant membrane protein (Imp),
immunodominant membrane protein A (ImpA), and antigenic membrane protein (Amp) were registered as surface
membrane protein genes and highly variable genes in the
phytoplasmas genome [34–36]. In Iran Siampour et al.
[37] used the imp gene to characterize and study the
phylogenetic trees of several 16SrII-A, −B, and -C subgroups of phytoplasma strains that cause the disease in
various host plants in Iran, East Asia, Africa, and
Australia. The phytoplasmas induce symptoms by secretion of SAP11 effector protein. SAP11 modify the plantgene activity and has a role in genetic regulator of the
changed phenotype produced [38, 39]. The SAP11 gene
(effector proteins) contains eukaryotic nuclear localization
signals (NLS) that localize in plant cell nuclei and interfere
with plant TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTORS 1 and 2),
which are conserved gene among plant species [38, 40].
The SAP11 transgenic Arabidopsis thaliana plants show
crinkled leaves and produce multiple stems; moreover,
these symptom, down regulate Jasmonic acid (JA) synthesis and modulated phosphate (Pi) homeostasis [38, 41].
Phytoplasmas are disseminated into healthy plant
phloem from the salivary glands of phloem-feeding and
sap sucking insect vectors, belonging to hemipteran
order including the families Cicadellidea (leafhoppers),
Fulgoridea (planthoppers) and Psylloidea (psyllids) insect
while feeding [16]. The insect vectors spread phytoplasma
diseases [42], so a successful phytoplasma management is
to reduce and control the insect vectors [43]. Two leafhopper species, Austroagallia avicula and Empoasca sp.,
were registered as putative vectors of alfalfa witches’
broom phytoplasma in Oman [44].
The objective of this study is to diagnose and detect
the causal agent and potential insect vector species for
the phytoplasma-like symptoms (Witches’ Broom) of C.
aegyptiaca in Oman by using electron microscopy and
molecular approaches to define a detailed classification
of the causal agent of the C. aegyptiaca disease. This detailed classification was achieved by comparing multiple
gene regions including 16S rDNA, secA, tuf, imp and
SAP11 genes.
Methods
Page 3 of 14
from the sampled locations and stored at −80 °C until
used. Sampling was done after getting the necessary permissions from plant owners Leafhoppers were collected
from the three sites using yellow sticky traps that were
placed near the infected plants for 5 days in order to investigate the putative insect vectors of the pathogen using
molecular detection techniques.
Transmission electron microscopy (TEM)
Fresh midribs of symptomatic C. aegyptiaca leaf samples
were placed in karnovesky’s fixative (2% Gluteraldehyde
and 4% paraformaldehyde containing 1 M cacodylate
buffer) overnight at 4 °C. After fixation the samples were
washed in two 10-min cycles of a 1 M cacodylate buffer
(pH from 7.2 to 7.4) then left overnight in a 1 M cacodylate buffer. In secondary fixation, the samples were then
placed in 1% osmium tetraoxide (OsO4) (prepared in
distilled water, pH 7.2) for 1 h on a rotary shaker,
followed by washing in distilled water for two 20-min
cycles. The samples were then dehydrated in increasing
gradients of acetone (25%, 75% and 95%) for 15 min,
followed by 3 cycles of absolute acetone in the following
order, 2 cycles for 30 min each and third cycle for 1 h to
ensure the complete removal of water. Specimens were
then infiltrated with an acetone/resin (1:1) mixture at
room temperature on rotators overnight. After that the
samples were then transferred to acetone/resin (1:3)
mixture overnight on a rotator. Further 2 cycles of fresh
100% araldite resin were carried out for 1 h each. Each
piece of mid-rib was placed in a standard mold block
(capsules) with labels, fresh resin was added in the capsules, and they were incubated at 60 °C overnight for
polymerization. Sections of 0.5 μm thicknesses were cut
with glass knives using an Ultra Microtome and stained
with a 1% toluidine blue dye and examined by through
an optical microscope to determine the area for ultrathin sectioning. Ultra-thin sections of 70 nm thicknesses
were cut using an Ultra Microtome fitted with a diamond knife. The ultra-thin sections were placed in
holders called GRIDS (300 mesh) then stained with
aqueous uranyl acetate for 30 min and lead citrate for
25 min. Finally, the stained ultra-thin sections were examined using a Jeol Jem-2100S transmission electron
microscope at the Electron Microscopy Unit in Sultan
Qaboos University.
Samples collection
C. aegyptiaca infected samples showing phytoplasma-like
symptoms (witches’ broom) were collected from three
sites in Oman, in Al-Seeb (N: 23.587794, E: 58.317110)
area from the Muscat governorate and two locations are
from Al-Dakhilia governorates which were Samail (N:
23.082359, E: 57.819496) and Izki (N: 22.911003, E:
57.741790). Healthy and infected samples showing typical
witches’ broom symptoms of C. aegyptiaca were collected
DNA extraction and PCR amplification
Four symptomatic and four asymptomatic C. aegyptiaca
samples as well Orosius sp. leafhoppers (Table 1) from
the three sites of this study in Oman were included in
the DNA extraction. Total DNA was extracted from
0.1 g of plant samples and 5 to 10 specimens from each
leafhoppers species using the Doyle and Doyle [45]
method with some modifications. The sample tissue
Al-Subhi et al. BMC Microbiology (2017) 17:221
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Table 1 List of phytoplasma isolates of the 16Sr-II group used in this study with their source and geographical origins, and corresponding 16S rDNA, secA, imp, and tuf gene accession numbers
Isolate number Phytoplasma strain
Isolate
Source
Acronym Location/governorate Accession numbers*
16S rDNA imp
secA
tuf
1
Crotalaria witches’ broom SQU-Sa1
C. aegyptiaca CrWBDL
Samail/Al-Dakhilia
KY872734 KY872727 KY872719 KY872723
2
Crotalaria witches’ broom SQU-Sa2
C. aegyptiaca CrWBDL
Samail/Al-Dakhilia
KY872735 KY872728 KY872720 KY872724
3
Crotalaria witches’ broom SQU-Iz1
C. aegyptiaca CrWBDL
Izki/Al-Dakhilia
KY872736 KY872729 KY872721 KY872725
4
Crotalaria witches’ broom SQU-Se1
C. aegyptiaca CrWBDL
Al-Seeb/Muscat
KY872737 KY872730 KY872722 KY872726
5
Crotalaria witches’ broom SQU-Sa3
Orosius sp.
OrWBDL
Samail/Al-Dakhilia
KY872738 KY872731 –
–
6
Crotalaria witches’ broom SQU-Iz2
Orosius sp.
OrWBDL
Izki/Al-Dakhilia
KY872739 KY872732 –
–
7
Crotalaria witches’ broom SQU-Se2
Orosius sp.
OrWBDL
Al-Seeb/Muscat
KY872740 KY872733 –
–
10
Lime witches’ broom
SQU-LW1 Lime
WBDL
Al-Seeb/Muscat
KX358574 KX358610 KX358586 KX358598
11
Alfalfa witches’ broom
SQU-Al1
AlWB
Al-Najed/Dhofar
KX358564 KX358600 KX358576 KX358588
Alfalfa
*
The deposited accession numbers in GenBank can be accessed by blasting the accession numbers through the following link
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
powder was immediately added in nucleic acid extraction buffer (100 mM Tris- HCl at pH 8.0, 1% cetyltrimethylammonium bromide [CTAB)], 2% PVP-10, 1.4 M
NaCl, 20 mM EDTA, and 0.1% 2-mercaptoethanol), then
1% sodium dodecyl sulfate (SDS) was added. The sample
was incubated for 30 min at 65 °C.Total DNA was extracted with an equal volume of phenol-chloroformisoamyl alcohol (25:24:1). The total DNA was precipitated with 0.6 volumes of isopropanol and 0.3 M sodium
acetate. Then the DNA pellet was washed twice with
70% ethanol, dried, and re-suspended in 100 μL of TE
buffer (10 mM Tris-HCl, 1 mM EDTA) and the DNA
was stored at −20 °C until use. Polymerase Chain Reactions (PCR) for five gene sequences including 16S
rDNA, secA, tuf, imp, and SAP11 genes were performed
for the detection, diagnostic, and phylogenetic studies of
the phytoplasma that is associated with the C. aegyptiaca witches’ broom disease. Phytoplasma DNA extracted from lime witches’ broom (WBDL) (Table 1) and
alfalfa witches’ broom disease (AlWD) (Table 1) were
used as positive control, whereas the negative control
samples had been DNA extracted from asymptomatic
plants and sterile water. Primer pairs P1/P7 [26, 46] and
R16F2n/R16R2 [47] were used, as a direct and nested
PCR respectively, to amplify the entire 16Sr RNA gene
region at annealing temperatures of 55 °C and 60 °C respectively. The 16SrII group phytoplasmas specific
primers sequences (Table 2) were used to amplify secA,
tuf, imp, and SAP11 genes sequences. The secA gene
was partially amplified using the primer pair SecA-II-F1/
SecA-II- R1 as the direct PCR and primer pair SecA-IIF1/SecA-II-R4 as the semi-nested PCR at an annealing
temperature of 53 °C. A portion of the tuf genes were
amplified with primer pairs TUF-II-F1/TUF-II-R1 and
TUF-II-F2/TUF-II-R1, as the direct and the semi-nested
PCR respectively, at an annealing temperature of 53 °C.
PCR was conducted to amplify the full length of the imp
and SAP11 genes sequences of the phytoplasma that is
associated with the C. aegyptiaca witches’ broom disease
in Oman at an annealing temperature of 53 °C. Primer
Table 2 Primers used for PCR amplification and sequencing of the 16S rRNA, secA, imp, tuf, and SAP11 genes of the phytoplasma
Primer Sets
Location
PCR product size Reaction
Sequence (5′ to 3′)
SecA-II-F1/SecA-II-R1
secA gene
2141 bp
Direct PCR
AAAGATGAAGATTTTCCTAAAGA/TCCATATCATTTATATGACGTTGA
SecA-II-F1/SecA-II-R4
secA gene
1511 bp
Semi-Nested PCR AAAGATGAAGATTTTCCTAAAGA/ACAAAAAATTTAGTATAACCAGGATC
IMP-II-F1/IMP-II-R1
imp gene
786 bp
Direct PCR
IMP-II-F2/IMP-II-R1
imp gene
717 bp
Semi-Nested PCR GATCATATTTGGTTTATAGGAG/ATAGAGGAGAAGAAAAAGTTTCT
TUF-II-F1/TUF-II-R1
tuf gene
1490 bp
Direct PCR
TUF-II-F2/TUF-II-R1
tuf gene
1094 bp
SAP11-IID-F1/SAP11-IID-R1 SAP11 gene 820 bp
GTTATAATTGAAGGCGATATTG/ATAGAGGAGAAGAAAAAGTTTCT
GCTTTTGTTCCTTTAGCAGAA/AGACTATACACTAGTCTTCTT
Semi-Nested PCR CGCAAAGATATTAAAACTTTAG/AGACTATACACTAGTCTTCTT
Direct PCR
CGGCAAAATAAAAGTTCAAATCA/AATCGAAACCAACCAACTTATAG
SAP11-IID-F2/SAP11-IID-R2 SAP11 gene 465 bp
Nested PCR
TTCTCAATTAAACGAACTCTACG/AAAAAGACCCTTCAGAAAGGGTC
SAP11-WBDL-F1/SAP11WBDL-R1
SAP11 gene 1050 bp
Direct PCR
CTTCAGCCACAAATAGAATCTTT/CAAATACAAATCGCTGCATAAA
SAP11-WBDL-F2/SAP11WBDL-R2
SAP11 gene 550 bp
Nested PCR
TTCCTTTTATGAAATCACCTCAG/GCGCATATTATTAAACTCCTTT
Al-Subhi et al. BMC Microbiology (2017) 17:221
pairs IMP-II-F1/IMP-II-R1 and IMP-II-F2/IMP-II-R1, as
the direct and the semi-nested PCR reactions respectively, were used to amplify the imp gene. The specific
primers for the SAP11 genes of 16SrII-D subgroup and
16SrII-B subgroup pytoplasmas were utilized in direct
and nested PCR including the SAP11-IID-F1/SAP11IID-R1 and the SAP11-IID-F2/SAP11-IID-R2 primers
pairs of 16SrII-D subgroup phytoplasma and excluding
the SAP11-WBDL-F1/SAP11-WBDL-R1 (direct PCR)
and the SAP11-WBDL-F2/SAP11-WBDL-R2 (direct
PCR) for the 16SrII-B subgroup phytoplasma. PCR reactions were performed in ‘Ready-To-Go’ PCR beads
(Pharmacia Biotech, Sweden) with 25 μl reaction volumes, which contain 50 ng of genomic DNA, 0.5 μl of
each primer (10 pmoles); the reaction volumes were adjusted with sterile deionized water. The PCR was performed for 40 cycles using the following parameters:
denaturation at 94 °C for 45 s (2 min for the first cycle),
annealing for 1 min at X °C (X = annealing temperature
specified for each set of primers), and primer extension
at 72 °C for 1.5 min with a final extension cycle for
10 min at 72 °C. The resulting PCR products were visualized by electrophoresis in a 1.4% agarose gel, stained
with ethidium bromide, placed under a UV transilluminator, and photographed. The PCR products were purified and sequenced at Macrogen Company (South
Korea).
Sequence analysis and construction of phylogenetic trees
The DNA sequences of both strands, forward and reverse, of each sample were edited, assembled, and
aligned using BioEdit 7.0.4.1 [48]; the sequences were
adjusted manually where it was necessary. The resulting
sequences of the phytoplasma 16S rDNA, secA, tuf, and
imp genes were compared to phytoplasma species available in the National Center for Biotechnology (NCBI)
GenBank database (http://ncbi.nlm.nih.gov/BLAST) by
BLAST searches to identify homologous sequences. The
DNA sequences were deposited in GenBank (NCBI, Bethesda, MD, USA) under the accession numbers in Table 1.
Sequence data of the phytoplasma 16S rDNA, secA,
tuf, and imp genes were obtained from GenBank to
study the genetic relationships of the collected C. aegyptiaca phytoplasma samples to known phytoplasma
groups. Sequences were aligned using CLUSTAL W [49]
then checked and confirmed manually. Sequences from
our study were aligned with 56 16S rDNA, 29 secA, 14
imp, and 38 tuf gene sequences of reference strains from
GenBank (Additional file 1: Table S1 and Table S2). A
partition-homogeneity test (PHT) in the PAUP* 4.0b10
[50] package was implemented to test whether data for
the 16S rDNA, secA, imp, and tuf genes regions could
be combined in a single tree. The combined tree has
phytoplasma sequences of the four genes and is available
Page 5 of 14
in the GenBank (Additional file 1: Table S2). The phylogenetic analysis of the 16S rDNA, secA, imp, and tuf
genes as well as the tree of combined genes were carried
out with MEGA 6 software [51]. The neighbour-joining
method was used to construct the phylogenetic trees
with 1000 replications for bootstrap analysis and a
Kimura-2-parameter model [52]. The DNA sequences of
Bacillus subtilis (AB042061), B. subtilis (D10279) and B.
subtilis strain 168 (GCA_000789275) were used as the
out-groups taxa of the trees of 16S rDNA, secA, and tuf
genes respectively.
Virtual RFLP analysis
Computer-simulated RFLP analysis was performed using
iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) tools [53] for the DNA sequence of 16S rRNA gene (1242 bp) phytoplasma from C.
aegyptiaca samples and Orosius sp. leafhopper samples
compared with all 21 strains of16SrII group phytoplasmas
(Additional file 1: Table S1). The pDRAW32 software
(http://www.acaclone.com) were used to perform virtual
RFLP plotting of 16S rRNA gene sequence from C. aegyptiaca samples and Orosius sp. leafhopper samples and
16SrII-M subgroup phytoplasmas.
Results
Symptomatology and leafhopper identification
During the survey on phytoplasma diseases in wild
plants of Oman in 2015, C. aegyptiaca showed symptoms indicative of a phytoplasma disease from three
sites. Symptoms included the significant proliferation of
shoots, reduced stem height, and an increased number
of leaves compared to healthy plants and at the same
time witches’ broom symptoms were observed with the
progress of the disease symptoms (Fig. 1a to c). All of
the yellow sticky traps which were placed near the infected C. aegyptiaca in the three sites, consistently collected brown leafhopper specimens that were 3.8–
4.2 mm in length size, these were identified as Orosius
sp. (Fig. 1e).
Transmission electron microscopy (TEM)
Examination of ultra-thin cross and elongation sections
of C. aegyptiaca leaf midrib, from witches’ broom infected plants, showed numerous phytoplasma bodies in
the sieve tubes (Fig. 2a & b). The observed phytoplasma
cells were spherical to ovoid measuring 200–600 nm in
diameter, enclosed by a single unit outer membrane (Fig.
2c). The distribution of phytoplasma cells was irregular
in the infected phloem. Some phloem elements, mainly
concentrated along sieve plates, showed phytoplasma
bodies in large abundance and were almost clogged (Fig.
2c). No phytoplasma bodies were found in the xylem tissues of infected leaves (Fig. 2d). As a result, the TEM
Al-Subhi et al. BMC Microbiology (2017) 17:221
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Fig. 2 Transmission electron micrograph of phytoplasma cells within the phloem of minor veins of infected C. aegyptiaca witches’ broom leaf.
a elongationsection showing phytoplasma within the sieve tube (phloem) (Ph); b A cross section showing a high concentration of phytoplasma
bodies in sieve tubes P = phytoplasma cell; c elongation section showing the sieve plate (S) of sieve tubes and that phytoplasma cells have
moved through sieve plate pores; d Xylem cell (X) was free from phytoplasma cells
images confirmed that phytoplasma is the causal agent
of the C. aegyptiaca witches’ broom disease in Oman.
PCR amplification and sequence analyses to detect
phytoplasma: 16S rDNA
Symptomatic and asymptomatic C. aegyptiaca samples
and Orosius sp. leafhopper samples were utilized for the
detection of phytoplasma by amplifying the 16S rRNA
gene. The alfalfa witches’ broom (AlWB) and lime
witches’ broom samples (WBDL) were used as the positive control and a water sample as the negative control.
The direct and nested PCR assay were conducted with
P1/P7 and R16F2n/R16R2 primer pairs and yielded the
expected amplification fragment length of about 1.8 kb
and 1.2 kb fragment respectively (data not shown) from
all of the symptomatic C. aegyptiaca and entire Orosius
sp. leafhopper samples tested. The asymptomatic plants
and water samples tested tested negative. PCR tests confirmed the association of phytoplasma diseases with C.
aegyptiaca plants and Orosius sp. leafhoppers in Oman.
The 16S rRNA gene sequence analyses of 4 C. aegyptiaca phytoplasmas and three Orosius sp. leafhopper isolates confirmed the PCR results. The sequences were
deposited in the National Center for Biotechnology
(NCBI) database under the accession numbers listed in
Table 1. The sequence homology of C. aegyptiaca isolates and Orosius sp. leafhopper isolates of 16S rRNA
gene phytoplasma were 100% identical to each other and
>99% and 99% similar with that of the Lime (WBDL)
and alfalfa (AlWB) phytoplasmas respectively, which
served as controls. The BLAST searches at NCBI of the
16S rRNA gene from C. aegyptiaca phytoplasma and Orosius sp. leafhopper isolates showed that these isolates have
99% sequence similarity with Cotton phyllody phytoplasma strain CoP (Accession. No. JQ868439) described
as the 16SrII-F subgroup, and also with Candidatus Phytoplasma aurantifolia strain 37oman (Accession. No.
LN873017) and the Iranian apple phytoplasma (Accession.
No. KC902794) belonging to the 16SrII-B subgroup ‘Candidatus Phytoplasma aurantifolia’. Therefore, the C.
aegyptiaca phytoplasma is a member of the 16SrII group
phytoplasmas and is closely related to ‘Candidatus Phytoplasma aurantifolia’. As shown in Fig. 3, the NeighborJoining phylogenetic tree, derived from partial 16S rDNA
(1151 bp) sequences data, placed all seven phytoplasmas
isolates of Crotalaria witches’ broom phytoplasmas from
C. aegyptiaca and Orosius sp. leafhoppers from Oman in
one separate sub-clade within the 16SrII group (Peanut
Al-Subhi et al. BMC Microbiology (2017) 17:221
Page 7 of 14
Fig. 3 Phylogenetic tree of the 16S rRNA gene sequences from the four C. aegyptiaca phytoplasma and three Orosius sp. Leafhopper isolates plus
the Lime witches broom sample (WBDL) and the Alfalfa witches’ broom (AlWB) from Oman (with circular black shape). The tree also includes 56
phytoplasma strains from previously published sequences, shown in GenBank accession numbers, and 16S groups are indicated in brackets
(Additional file 1: Table S1); it was rooted using Bacillus subtilis (AB042061). The phylogenetic tree was constructed by the neighbour-joining
method and Kimura’s two-parameter model, and is in the units of the number of base substitutions per site. The bootstrap values are expressed
as percentages of 1000 replications
WB) clade with 85% bootstrap support. They were found
to be closely related to the Fava bean phyllody (Accession.
No. X83432), from 16SrII-C subgroup phytoplasmas, and
they were further away from lime witches’ broom ‘Ca. P.
aurantifolia’ (WBDL) (Fig. 3). The virtual RFLP patterns
analysis results using the iPhyClassifier software and the
pDRAW32 software for the 16S rDNA R16F2n/R16R2
fragment sequence phytoplasma from C. aegyptiaca
Al-Subhi et al. BMC Microbiology (2017) 17:221
samples and Orosius sp. leafhopper samples clearly distinguished our phytoplasma from the 21 strains of16SrII
group phytoplasmas (Additional file 2: Figure S1, Additional file 3: Figure S2). The virtual RFLP analysis gave
identical results with the 16S rDNA phylogenetic tree
finding.
DNA sequences analysis:- secA, imp, tuf, and SAP11 genes
The secA, tuf, and imp genes sequence with amplicon
size ~1370 bp, ~996 bp, and 516 bp respectively of Crotalaria witches’ broom phytoplasmas from C. aegyptiaca
and just imp gene sequence from Orosius sp. leafhoppers
phytoplamsa from Oman were submitted to the GenBank NCBI database under the accession numbers in
Table 1. Sequences of secA, tuf, and imp genes from C.
aegyptiaca and Orosius sp. leafhoppers from Oman samples were 100% identical to each other. BLAST search of
the secA gene at NCBI showed that Crotalaria witches’
broom phytoplasmas from Oman had a 98% similarity
with the sequence of primula blue yellow phytoplasma
(Accession. No. KJ462018) and a 97% similarity with
witches’ broom disease of lime phytoplasma (‘Candidatus Phytoplasma aurantifolia’) sequence (Accession. No.
Page 8 of 14
KJ462017). Moreover the sequence of the tuf gene
showed that Crotalaria witches’ broom phytoplasmas
had a 98% similarity with a phytoplasma associated with
primula blue yellow disease (Accession. No. JQ824229)
and had a 97% similarity with witches’ broom disease of
lime phytoplasma (Accession. No. JQ824276). Nonetheless, the sequence identity of the imp gene of Crotalaria
witches’ broom phytoplasmas from Oman showed only
a 90% similarity with primula blue yellow disease (Accession. No. JQ745272) and witches’ broom disease of
lime (Accession. No. JQ745278) phytoplasmas. The
combination of specific primers for the SAP11 gene of
16SrII-A, −B, and -D subgroup phytoplasmas were
tested against Crotalaria witches’ broom phytoplasmas
and no PCR product was amplified. However, the positive control samples gave the expected PCR amplicon
size, therefore, there is homology of the SAP11 DNA sequence of Crotalaria witches’ broom phytoplasma unlike the SAP11 DNA sequence of 16SrII-A, −B, and -D
subgroup pytoplasmas. So, the sequences analysis of
secA, imp, tuf, and SAP11 genes confirmed the 16S
rRNA gene result to classify Crotalaria witches’ broom
phytoplasma in the 16SrII-W subgroup.
Fig. 4 Phylogenetic tree of the secA gene sequences from the four C. aegyptiaca phytoplasma isolates plus the Lime witches broom sample (WBDL)
and the Alfalfa witches’ broom (AlWB) from Oman (with circular black shape). The tree also includes 29 phytoplasma strains from previously published
sequences, shown in GenBank accession numbers, and 16S groups that are indicated in brackets (Additional file 1: Table S2); it was rooted using
Bacillus subtilis (D10279). The phylogenetic tree was constructed by the neighbour-joining method and Kimura’s two-parameter model, and is in the
units of the number of base substitutions per site. The bootstrap values are expressed as percentages of 1000 replications
Al-Subhi et al. BMC Microbiology (2017) 17:221
Phylogenetic analysis
The DNA sequences of SecA, IMP, and TUF genetic
markers from this study were used to construct the phylogenetic trees based on partial sequences of the secA and tuf
genes, 536 bp (Fig. 4) and 385 bp (Fig. 5) nucleotides respectively as well as complete sequences of imp gene
Page 9 of 14
516 bp nucleotides (Fig. 6). Moreover, the phylogenetic tree
of the combined dataset was built; this included the 16S
rRNA, secA, tuf, and imp gene sequences (Fig. 7). The
phylogenetic trees of secA, tuf, and imp gene sequences
and combined tree separated the phytoplasma 16Sr group’s
lineage which is similar with that inferred by the 16S rRNA
Fig. 5 Phylogenetic tree of the tuf gene sequences from the four C. aegyptiaca phytoplasma isolates plus the Lime witches broom sample
(WBDL) and the Alfalfa witches’ broom (AlWB) from Oman (with circular black shape). The tree also includes 38 phytoplasma strains from
previously published sequences, shown in GenBank accession numbers, and 16S groups that are indicated in brackets (Additional file 1: Table S2);
it was rooted using Bacillus subtilis strain 168 (GCA_000789275). The phylogenetic tree was constructed by the neighbour-joining method
and Kimura’s two-parameter model, and is in the units of the number of base substitutions per site. The bootstrap values are expressed as
percentages of 1000 replications
Al-Subhi et al. BMC Microbiology (2017) 17:221
Page 10 of 14
Fig. 6 Phylogenetic tree of the imp gene sequences from the four C. aegyptiaca phytoplasma and three Orosius sp. Leafhopper isolates plus the
Lime witches broom sample (WBDL) and the Alfalfa witches’ broom (AlWB) from Oman (with circular black shape). The tree also includes 14
phytoplasma strains from previously published sequences, shown in GenBank accession numbers, and 16S groups that are indicated in brackets
(Additional file 3: Table S2). The phylogenetic tree was constructed by the neighbour-joining method and Kimura’s two-parameter model, and is
in the units of the number of base substitutions per site. The bootstrap values are expressed as percentages of 1000 replications
gene-based phylogeny in this study (Fig. 3). The 16SrII
phytoplasma group was comprised of two sub clades on all
four phylogenetic trees (Figs 4 to 7). In these analyses, all
isolates of Crotalaria witches’ broom phytoplasmas from
Oman were 100% identical and clustered in a subclade in
section 16SrII- B, −C, and –F subgroup pytoplasmas, but
they were clearly separated into an individual subgroup,
supported with a very strong bootstrap analysis.
Discussion
C. aegyptiaca showed symptoms typical of phytoplasma
infection. The symptoms of C. aegyptiaca witches’
broom disease are similar to those of lime witches’
broom disease (WBDL) [54] in Oman, associated with
‘Candidatus Phytoplasma aurantifolia’ (16SrII-B subgroup) [8]. TEM showed numerous phytoplasma bodies
in the sieve tubes of C. aegyptiaca. TEM is frequently
used to provide a reliable and accurate method for diagnosing phytoplasma diseases in sieve tube elements and
insect vectors [22, 23]. González et al. [55] used TEM to
observe maize bushy stunt phytoplasma within its plant
host and insect vector. TEM also was applied to detect
the phytoplasma associated with sunflower phyllody in
India [56] and for elm yellows group phytoplasma that is
associated with camellia in China [57].
Our findings showed that C. aegyptiaca phytoplasma
is a member of the 16SrII group phytoplasmas and is
closely related to ‘Candidatus Phytoplasma aurantifolia’.
Analysis based on the 16S rDNA placed all seven phytoplasmas isolates of Crotalaria witches’ broom phytoplasmas from C. aegyptiaca and Orosius sp. leafhoppers
from Oman in one separate sub-clade within the 16SrII
group (Peanut WB) clade with 85% bootstrap support.
The four phytoplasma groups, including 16SrII-B & -D,
16SrVI, 16SrIX, and 16SrXXIX, infect wild and economically important plant species in Oman [8, 58–60]. The
virtual RFLP analysis gave identical results with the 16S
rDNA phylogenetic tree finding. The above results
showed that Crotalaria witches’ broom phytoplasmas
from Oman placed with the clade of Peanut WB (16SrII)
including Fava bean phyllody (16SrII-C), Cotton phyllody and phytoplasmas (16SrII-F), and ‘Candidatus Phytoplasma aurantifolia’ (16SrII-B) (Fig. 3) [8, 29, 61]. To
our knowledge, this is the first report of phytoplasmas of
Al-Subhi et al. BMC Microbiology (2017) 17:221
Page 11 of 14
Fig. 7 Phylogenetic tree of the 16S rRNA, secA, tuf, and imp genes sequences from the four C. aegyptiaca phytoplasma isolate plus the Lime
witches broom sample (WBDL) and the Alfalfa witches’ broom (AlWB) from Oman (with circular black shape). The tree also includes 29 16S rDNA,
secA, and tuf phytoplasma sequences and 14 imp phytoplasmas sequences retrieved from the GenBank, shown in phytoplasma disease names,
and 16S groups that are indicated in brackets (Additional file 1: Table S2). The phylogenetic tree was constructed by the neighbour-joining
method and Kimura’s two-parameter model, and is in the units of the number of base substitutions per site. The bootstrap values are expressed
as percentages of 1000 replications
the 16SrII group infecting C. aegyptiaca worldwide. Previously, many studies reported Crotalaria sp. plants infected
with phytoplasma diseases. A phytoplasma belonging to
16SrII-A subgroup has been reported to be associated
with disease in Crotalaria szemaoensis and Crotalaria
zanzibarica plants in China [62]. A 16SrII-A subgroup
phytoplasma was also observed on Crotalaria spp. (sunn
hemp) in Myanmar [63]. Crotalaria juncea (sunn hemp)
plants were found to be infected with the 16SrIX group
phytoplasma in Brazil [64, 65]. Above all, the phytoplasma
associated with Crotalaria witches’ broom in Oman
seemed to be distinguishable from all the other phytoplasmas belonging to Peanut WB group (16SrII). The 16SrII
group has 21 subgroups including 16SrII-A, −B, −C, −D,
−E, and –F [66] therefore, we propose to assign the Crotalaria witches’ broom from Oman in a new lineage 16SrII-
W subgroup. The Orosius sp. was registered as phytoplasmas putative vectors in many studies. Pilkington et al. [67]
reported Orosius argentatus as a vector of the Australian
lucerne yellows phytoplasma. In addition, Orosius albicinctus was the vector insect of the sesame phyllody disease which the 16SrIX-C and 16SrII-D subgroup
phytoplasmas were the causal agent in Turkey, Iran, and
India [68, 69]. Thus, Orosius sp. leafhoppers from Oman
could be responsible for the transmission of inoculum of
Crotalaria witches’ broom phytoplasmas from infected to
healthy C. aegyptiaca plants.
Proposing a new ‘Candidatus Phytoplasma’ species can
be done if the 16S rRNA gene sequence has less than
97.5% similarity according to International Phytoplasma
Working Group (IPWG) [70]. The phytoplasma that
shares more than 97.5% of the 16S rRNA gene sequence
Al-Subhi et al. BMC Microbiology (2017) 17:221
similarity and has unique ecological and biological properties such as a specific plant host or insect vector could be
designated as a separate candidate species [20, 71]. The
finer classification and description of the biology and ecology of phytoplasmas that are closely related but distinct
strains cannot be easily resolved by the highly conserved
16S rRNA gene alone [30]. Therefore, less conserved
markers including secA, imp, tuf, ribosomal protein (rp),
secY, and SAP11 genes, have been utilized for finer classification of closely related phytoplamsas within or between
the existing16S group or subgroup [27, 29, 31–33, 35].
Findings from our study showed that the sequences analysis of secA, imp, tuf, and SAP11 genes confirmed the 16S
rRNA gene result to classify Crotalaria witches’ broom
phytoplasma in the 16SrII-W subgroup. In adition, results
of the phylogenetic trees on secA, tuf, and imp gene sequences and the tree of the four combined genes revealed
that Crotalaria witches’ broom phytoplasma (C. aegyptiaca isolates and Orosius sp. Leafhoppers isolates) from
Oman is a new phytoplasma, having closer relationships
to the phytoplasmas associated with primula blue yellow
disease (16SrII-C) than the Candidatus Phytoplasma aurantifolia’ (16SrII-B) [8, 29, 32, 33, 72].
Conclusions
On the basis of disease symptoms and the molecular analysis of 16S rRNA, secA, tuf, and imp genes, phytoplasma
isolates from the C. aegyptiaca plant and Orosius sp. leafhopper isolates in Oman were found to be associated with
the Crotalaria witches’ broom disease and is a member of
the 16SrII group phytoplasma. No PCR amplification
came from the SAP11 primer sets, which shows the
SAP11 gene of Crotalaria witches’ broom phytoplasma
has different DNA sequences than 16SrII-B and -D subgroup phytoplasmas. These results support the conclusion
that Crotalaria witches’ broom phytoplasmas from Oman
are closely related to phytoplasmas belonging to the
16SrII-C and -B group, but that it represents a distinct
subgroup of phytoplasma. Therefore, we suggested classifying it as the new subgroup 16SrII-W. The results from
this study are supported by the usage of multiple genetic
markers, which is useful in the fine differentiation and
analysis of closely related phytoplasma strain lineages, and
might be extremely important for phytoplasma disease
epidemiological studies or for disease control and quarantine guidelines. The Orosius sp. leafhopper is a putative
vector for Crotalaria witches’ broom phytoplasma, but
the specific transmission tests need to be conducted to
confirm that it is in fact a vector. A field survey will be
helpful to define the economic crops and alternative plant
hosts which are also visited by this leafhopper. Such studies will provide methods toward disease control that could
prevent the spread of Crotalaria witches’ broom phytoplasma to economic crops.
Page 12 of 14
Additional files
Additional file 1: Table S1. 16S rDNA sequences of different
phytoplasma strains obtained from GenBank used for phylogenetic
analysis. Table S2. Phytoplasma 16S rRNA, tuf, secA, and imp genes
sequences used for phylogenetic analysis, obtained from GenBank.
(DOCX 23 kb)
Additional file 2: Figure S1. Virtual RFLP patterns by the iPhyClassifier
software of the 16S rRNA gene phytoplasmas from C. aegyptiaca samples,
Orosius sp. leafhopper samples and all 21 16SrII group strains using AluI,
BgaI, BstVI, EcoRI, HaeIII, HhaI, HinfI, HpaII, MseI, Sau3AI, SspI and TaqI
restriction endonuclease enzymes. (PPTX 184 kb)
Additional file 3: Figure S2. Virtual RFLP comparative analysis with
different restriction enzymes of 16S DNA sequences of phytoplasma from
C. aegyptiaca samples and Orosius sp. leafhopper samples and 16SrII-M
subgroup phytoplasmas using the pDRAW32 software. (PPTX 200 kb)
Abbreviations
AlWB: Almond witches’ broom ‘Ca. P. phoenicium’; AP: Apple proliferation
‘Ca. P. mali’; AshY: Ash yellows ‘Ca. P. fraxini’; AUSGY: Australian grapevine
yellows ‘Ca. P. australiense’; AYWB: Aster yellows witches’ broom (AYWB);
BGWL: Bermudagrass white leaf ‘Ca. P. cynodontis’; CaWB: Cassia witches’
broom (CaWB) ‘Ca. P. omanense’; COAH10: Mexican potato purple top
phytoplasma strain COAH10; CP: Clover proliferation ‘Ca. P. trifolii’;
CPh: Clover phyllody (CPh); CTAB: Cetyltrimethylammonium bromide;
CYE: Clover yellow edge; EDTA: Ethylenediaminetetraacetic acid;
ErWB: Erigeron witches’ broom; ESFY: European stone fruit yellows ‘Ca. P.
prunorum’; EY: Elm yellows ‘Ca. P. ulmi’; HibWB: Hibiscus witches’ broom ‘Ca.
P. brasiliense’; imp: Immunodominant membrane protein; JWB-G1: Jujube
witches’ broom ‘Ca. P. ziziphi’; LufWB: Loofah witches’ broom; LYDM: Lethal
yellow disease Mozambique ‘Ca. P. palmicola’; MaPV: Malaysian p. virescence
(MaPV) ‘Ca. P. malaysianum’; MC: Strawberry multiplier disease; MPV: Mexican
periwinkle virescence; OY-M: Onion yellows mild strain (OY-M); PB: Pecan
bunch; PCR: polymerase chair reaction; PinP: Pinus phytoplasma ‘Ca. P. pini’;
PnWB: Peanut witches’ broom; PnWB: Peanut WB phytoplasma;
PPWB: Pigeon pea witches’ broom; PX11CT1: Peach X-disease ‘Ca. P. pruni’;
RYD: Rice yellow dwarf ‘Ca. P. oryzae’; SAP11: Stress associated protein 11;
secA: preprotein translocase subunit SecA; SoyST1c1: Soybean stunt isolate
SoyST1c1 ‘Ca. P. costaricanum’; SQU: Sultan Qaboos University;
TEM: Transmission electron microscopy; TexPp: Texas Phoenix palm
phytoplasma; tuf: Elongation factor Tu
Acknowledgments
Authors would like to acknowledge SQU TEM unit and Abdul Rahman
Al-Nabhai their help TEM, and Dr. Michael Wilson, Mr. Ali Al-Jahthami and Ali
Al-Raeesi for their help in insect collection and identification.
Funding
Authors would like to acknowledge Sultan Qaboos University for financial
support of the study through projects SR/AGR/CROP/13/01 and SR/AGR/
CROP/17/01. The funding body had no role in the design of the study and
collection, analysis, and interpretation of data and in writing the manuscript.
Availability of data and materials
All data are available and sequences were deposited in GenBank. The
datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request. GenBank accession
numbers can be found in Additional file 1: Table S1.
Authors’ contributions
AS, SH, RA and AM planned the study; AS carried out the work; AS and AM
analyzed data; AS, SH, RA and AM wrote the manuscript; All authors revised
and approved the final version of the paper.
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable.
Al-Subhi et al. BMC Microbiology (2017) 17:221
Competing interests
The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential
conflict of interest. The authors have no competing interests.
Page 13 of 14
22.
23.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Crop Sciences, College of Agricultural and Marine Sciences,
Sultan Qaboos University, Al Khod 123, PO Box 34, Seeb, Oman. 2Department
of Crop Genetics, John Innes Centre, Norwich NR4 7UH, UK.
Received: 18 June 2017 Accepted: 16 November 2017
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