Molecular identification of phytoplasmas associated with some weeds in West Azarbaijan province , Iran

During field surveys in 2013 and 2014, about 14 weed plants showing phytoplasma diseases symptoms including yellowing and witches’ broom were collected and tested by polymerase chain reaction (PCR) using universal primers for 16SrRNA starting by primer pairs P1/P7 in first round PCR followed by primer pair R16F2n/R16R2 in nested PCR. The detected phytoplasmas were characterized and differentiated through sequence analysis of PCR-amplified rDNA and virtual restriction fragment length polymorphism (RFLP). The phytoplasmas detected in symptomatic horseweed (Erigeron canadensis L.), common madder (Rubia tinctorum L.), Johnson grass (Sorghum halepense [L.] Pers.) and Sophora root (Sophora alopecuroides L.) were identified as members of the clover proliferation group (16SrVI group) by construction of phylogenetic trees. Further analysis by virtual RFLP classified the phytoplasmas of Erigeron canadensis L. and Sorghum halepense L. in subgroup 16SrVI-A and phytoplasmas of Rubia tinctorum L. and Sophora alpecuriodes L. in subgroup 16SrVI-D. This is the first report on the occurrence of phytoplasma diseases of weeds in west Azarbaijan, Iran.


INTRODUCTION
Phytoplasmas are a group of wall-less phloemlimited plant pathogenic bacteria belonging to Mollicutes which have been described relative recently (Lee et al., 1998).They are associated with diseases in several hundreds of plant species, including weeds (Marcone et al., 1997).Typical symptoms include virescence/phyllody, sterility of flowers, proliferation of axillary buds resulting in witches ' broom growth, abnormal internodes elongation and generalized stunting (Bertaccini et al., 1996;2014).Phytoplasmas are introduced by insect vectors (mostly leafhoppers) during feeding activity into plant sieve tube elements, from which they spread systemically through the plants (Bertaccini et al., 2014).Currently, characterization and classification of phytoplasmas are based mainly on restriction fragment length polymorphism (RFLP) and sequence analysis of 16SrDNA or other less-conserved genes, whereas detection is done mainly by polymerase chain reaction (PCR) assay (Bertaccini et al., 2014;Lee et al., 2000;Seemuller et al., 1998).Weeds serve as both a reservoir for phytoplasma infection and as reproductive hosts for the vectors (Singh and Upadhyaya, 2013).Phytoplasmas are known to cause diseases in weeds including field bindweed (Convolvulus arvensis L.), prickly lettuce (Lactuca serriola L.), Johnson grass (Sorghum halepense (L.) Pers.), bermuda grass (Cynodon dactylon (L.) Pers.), horseweed (Conyza canadensis (L.) Cronq.) and some others were reported worldwide (Arocha-Rosete and Jones, 2010; Babaie et al., 2007;Chen et al., 2003;Li et al., 2013;Marcone et al., 1997;Salehi et al., 2006;Shubhrata, 2004;Thereza and Baross, 2002;Vali Sichani et al., 2014).However little is known about phytoplasma diseases of weeds in west Azarbaijan province of Iran.The aim of this study was to verify the presence of phytoplasma diseases in symptomatic weeds in West Azarbaijan province using PCR assay.The detected phytoplasmas were characterized and classified using sequence analysis of PCR-amplified 16SrDNA and virtual gel RFLP.

Plant materials
Fourteen weed plants related to 4 plant species including Johnson grass (Sorghum halepense (L.) Pers.),Canadian horseweed (Conyza canadensis.(L.) Cronquist), Sophora root (Sophora alopecuroides L.) and common madder (Rubia tinctorum L.) showing symptoms typical of phytoplasmal infection were collected during 2013 and 2014 growing seasons (Table 1) from different regions of West Azarbaijan province including Urmia, Salmas, Khoy and Mahabad cities. Asymptomatic plants were also collected and used in molecular analysis as negative controls.

DNA Extraction
Total DNA was extracted from 0.25g of leaves and midribs according to the method described by Zhang et al (1998).Total DNA of healthy plants were extracted and used as negative controls.

PCR Analysis and Primer Pairs
The universal primer pair P1/P7 (Schneider et al., 1995) was employed in first round PCR to amplify a 1.8kbp fragment of 16SrDNA.A 30-fold dilution of the first round PCR product used as template for nested PCR using primer pair R16f2n/R16R2 which amplified an internal fragment of 1.2kbp from the 16SrDNA (Lee et al., 1993).The total volume of 20µl PCR reaction mixtures contained 20ng DNA, 0.2mM of each dNTP (Cinnagen, Iran), 1.6mM MgCl 2 , 1U of Taq DNA polymerase (Cinnagen, Iran), 0.5µl of each primer pair (20pmol/µl) and 1X polymerase buffer.The reaction mixtures were subjected to 35 cycles at the following conditions: First round PCR (35 cycles): 1 min (3 min for the first cycle) for denaturation step at 94ºC, 1 min for annealing at 57ºC and 1.5 min (10 min for the last cycle) for primer extension step at 72ºC.Second round nested PCR (35 cycles): 2 min (5 min for the first cycle) for denaturation step at 94ºC, 1 min for annealing at 57ºC and 2 min (10 min for the last cycle) for primer extension step at 72ºC.The PCR products were analyzed by electrophoresis in a 1 % agarose gel and stained with ethidium bromide.An ultraviolet (UV) transilluminator was used to visualize DNA band.

Sequencing and Phylogenetic Analysis
PCR products of nested PCR were sequenced directly.Sequencing was performed by Macrogen (South Korea) on both strands.Nucleotide sequence similarity, multiple alignment and phylogenetic tree construction using the neighborjoining (NJ) method were done with MEGA5 software (Tamura et al., 2011) and subjected to bootstrap analysis using 500 replicates.The Acholeplasma laidlawii isolate was used as outgroup.

RESULTS AND DISCUSSION
During growing seasons of 2013 and 2014, fourteen weed samples with phytoplasma symptoms were collected from different parts of West Azarbaijan province, Iran.The symptoms varied with the host plant and the most characteristic symptoms were witches' broom, leaf malformation, little leaf and yellowing (the symptoms are summarized in Table 1 and some symptomatic plants were shown in Fig. 1).BamHI,BfaI,BstUI,DraI,EcoRI,HaeIII,HhaI,HinfI,HpaI,HpaII,KpnI,Sau3AI,MseI,RsaI,SspI and TaqI.Two phytoplasmas which were previously reported from Canadian horseweed exhibiting yellowing and witches' broom symptoms were classified in 16SrI (SrI-A) (Lee et al., 2000) and 16SrVII groups (Thereza and Baross, 2002), respectively.This is the first report of little leaf symptom on Canadian horseweed that is classified in phytoplasma 16SrVI group.(Li et al., 2013).There is no data on common madder phytoplasma infection and to our knowledge it is the first report of phytoplasma (16SrVI group) infection of common madder.

Figure 1 :
Figure 1: Weeds with phytoplasma-like symptoms in West Azarbaijan province.A-Common madder showing little leaf, B-Johnson grass with little leaf symptoms, C-Sophora root with yellowing and little leaf symptoms, D-Canadian horseweed showing leaf malformation and witches ' broom.

Figure 3 :
Figure 3: Phylogenetic tree constructed by the neighbor joining method of 16SrRNA gene sequences from 19 phytoplasma and phytoplasmas identified from Sophora root (Tph), common madder (Ruph), Canadian horseweed (Pph), and Johnson grass (Nph) and Acholeplasma laidlawii as outgroup.Numbers at the nodes are bootstrap values based on 500 repetitions.GenBank accession numbers for sequences are given in parentheses.4CONCLUSIONSTo date, phytoplasmas have been documented in more than 100 weed plant species.Phytoplasmas cause diseases on several weeds which result in serious threat as a source of alternative natural host for the spread of phytoplasma pathogen to other economically important plants, thereby creating a chance of causing severe losses.Detection of phytoplasma associated with diseases of weed

Table 1 :
Weeds showing phytoplasma-like symptoms The phytoplasma which causes yellowing on Johnson grass, detected in our survey, was classified in 16SrVI group.Previously, on Johnson grass exhibiting yellowing Arocha-Rosete and Jones (2010) and Singh and Upadhyaya (2013) found phytoplasmas of 16SrXXIV-A and 16SrXII groups, respectively.
Sophora root with little leaf and yellowing symptoms was classified in 16SrVI group in this study.Previous reports of phytoplasmas affecting Sophora species include a 16SrXII phytoplasma associated with S. japonica L (Styphnolobium japonicum (L.) Schott.)yellows in China (Duduk et al., 2010), ʻCa.Phytoplasma ziziphiʼ in China associated with witches ' broom and a 16SrI ʻCa.Phytoplasma asterisʼ associated with Sophora root yellows (Yu et al., 2012; Chen et al., 2013).Recently Allahverdi et al. (2014) reported a 16SrXII phytoplasma association with S. alopecuroides from Firooz-koh (Tehran Province, Iran) with leaf yellowing, little leaf and stunting symptoms.The association of a phytoplasma belonging to 16SrVI was previously established in Sophora root (Sophora alopecuroides L.) exhibiting yellowing and witches ' broom symptoms from China