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During a survey in 2016, Convolvulus glomeratus showing symptoms resembling those caused by phytoplasmas were observed in Bandar Abbas, Hormozgan province, Iran. These plants were examined for phytoplasma presence by nested-PCR assays using universal primer pair P1/P7 followed by R16F2n/R16R2. All the infected plants indicated positive results using universal primer pairs of P1/P7 followed by R16F2n/R16R2. Results of phylogenetic and virtual RFLP analyses of the 16S rRNA gene sequences revealed that the phytoplasma associated with Convolvulus glomeratus witches' broom (CgWB) was a strain of 'Candidatus Phytoplasma phoenicium'. The phytoplasma was successfully transmitted to healthy plants by leafhopper, Orosius albicinctus (Distant) which account as a vector of this phytoplasma. This is the first report on the presence of ‘Candidatus Phytoplasma phoenicium’ (phytoplasma group 16SrIX-J) in C. glomeratus and its insect vector in Iran.
 

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Some samples were collected from tomato fields in Qazvin from tomato plants with big bud symptoms such as plant droop and purplish vein under the leaf, enlarged and sac-like pistils and malformed buds. DNA was extracted from the veins and vascular tissues of the plant with CTAB-based methods. In symptomatic plants, DNA fragments of 1800 and 1200bp were amplified by PCR using P1/P7, R16F2n/R16R2 primers. Restriction fragment length polymorphism (RFLP) analysis of nested R16F2n/R16R2 primed PCR product (1200bp) showed that the tomato big bud phytoplasma from Qazvin (TOM-Qazvin) is a member of clover proliferation (16SrVI). Phylogenetic analysis of 16SrRNA and putative restriction site analysis of the R16F2n/R16R2 primed sequence classified TOM phytoplasma in clover proliferation (16SrVI) group and belonged to subgroup 16SrVI-A. Virtual RFLP by using 1200bp sequencing of 16SRNA and 17 restriction enzymes confirmed that TOM-Qazvin belonged to the subgroup 16SrVI-A and16SrVI group. To our knowledge, this is the first report of tomato big bud disease in Qazvin province.

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The Xanthomonas citri pv. citri (Xcc) is causal agent of bacterial citrus canker which is major disease of citrus throughout the world. The pthA bacterial effector protein is presented within the infected plants and indispensable of canker. The scFv antibodies are valuable tools for diagnosis and suppression of pathogens within plants. The present article describes developing and characterization of specific recombinant monoclonal scFv antibodies against pthA effector protein. For this aim, the gene encoding pthA protein was heterologously expressed in Escherichia coli and used for screening of Tomlinson phage display antibody library to pinpoint specific single chain variable fragment (scFv). In each round of panning, the affinity of phage towards pthA was checked by enzyme linked immunosorbent assay (ELISA). The data was indicative of about 50% of the monoclonal phages to be reactive strongly against pthA protein. Among the positive clones, 5 samples (A12, B8, C1, H8 and G8) were capable of detecting Xcc-infected plant samples and recombinant pthA protein. Restriction fragment length polymorphism showed similar banding pattern for all 5 scFvs as renamed to pthA-scFG8. HB2151 E. coli cells were infected by the phage bearing pthA-scFG8, and the expression of the peptide was induced by IPTG to produce a 30 kDa recombinant molecule. I-TASSER was used for homology modeling of both scFv and pthA and docking was carried out by Hex program. The latter demonstrated binding energy of −784 kcal/mol in scFv-pthA. 

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Pepper Capsicum annuum is one of the important vegetable crops in Iran, especially north of Iran. Various symptoms of stolbur, including limited growth, small and chlorotic leaves, spoon-shaped leaflets, and sterility or fruit alterations, were detected in samples collected from the pepper field in Qazvin province. DNA was extracted from midribs and petioles of pepper leaves using CTAB-based methods. The phytoplasma in all symptomatic pepper plant parts was detected by direct and nested polymerase chain reactions (PCR) using primer pairs P1/P7 and R16F2n/R16R2. The 16S rDNA sequences of phytoplasma isolate were deposited in GenBank (MN877916). Based on phylogenetic studies of the 16S rDNA region, the results of enzymatic digestion of the fragment obtained by amplification with R16F2n/R16R2 primer and virtual RFLP, phytoplasma agent associated with stolbur pepper disease was detected to belong to 16SrXII group and 16XII-A subgroup. According to our knowledge, this is the first report of pepper stolbur disease in Iran.

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Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The disease keeps spreading in several citrus-growing areas of southern Iran. The potential of (GTG)₅-rep marker in revealing the genetic diversity of geographic isolates of Candidatus Liberibacter asiaticus was evaluated in the present study. Twenty-one HLB-infected samples collected from the Hormozgan and Sistan and Baluchestan provinces were used in the trial. PCR with the (GTG)₅ primer produced 16 scorable bands, of which nearly 100% were polymorphic among or within the populations. The most observed variation resided within (80.56, P < 0.10) and a substantially less (19.44, P < 0.10) between the populations. The isolates were distributed in two main (A and B) clusters, each consisting of several subgroups. Group A included Sistan and Baluchestan and Hormozgan isolates and the group B embraced Hormozgan isolates. Based on pairwise genetic differences, the Haji Abad and Hashtbandi populations exhibited the highest between-population variation, and the Siahoo, Hashtbandi, Nikshahr, Haji Abad, and Sarbaz showed the greatest within-population variation. The first three coordinates of the principal coordinate analysis explained more than 72.39% of the variation among or within the populations. The first two coordinates explained 58.20% of band variation in plotting, and the first and third coordinates explained 55.54% of band variation. These results may be inferred that the Hormozgan populations might have diverged from the Sistan populations, or both might have originated separately from an initial or parental population possessing a high genetic diversity.

 

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Tomato bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is a destructive tomato disease in the world and Iran, which can seriously affect the quality of the product. An integrated management program using pathogen-free seeds and resistant cultivars is necessary for disease control. In this study, the reaction of 24 cultivars of tomato was assessed against the disease under greenhouse conditions. Inoculation of seedlings at the 4-5 leaf stage was carried out by injecting a bacterial suspension of 1 × 10⁴ CFU ml^-1 at the axil, where the petiole meets the stem. The response of cultivars to the disease was evaluated via three indices, including the time of disease onset, disease severity (DS), and the area under the disease progress curve (AUDPC). The results indicated that AUDPC positively correlated with the time of disease onset (r = 0.85) and disease severity (r = 0.86). Based on the current findings, applying different indexes in response of tomato cultivars to bacterial canker disease provides accurate information about host resistance. The AUDPC index is the most reliable as it has a high positive correlation coefficient with two other indexes. In this study, cultivars ‘Rio Grenade’, ‘King Stone’, ‘Early Urbana Y’, ‘CalJ N3’, and ‘Hyb. Firenze’ showed more resistance against the disease than other studied cultivars. Usage of the mentioned cultivars is recommended in the integrated management program of tomato bacterial canker disease.

 

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Wheat seeds harbor different microbial populations, which can be associated with each other in neutral, positive, or negative interactions. The present study investigated the interaction of fluorescent Pseudomonas and Xanthomonas translucens (Xt) as an important wheat seed-borne pathogen. In addition to P. fluorescens (A7) and P. chloroaphis (A4), which were previously isolated from the rhizosphere of potato as potent biocontrol agents, six more non-pathogenic Pseudomonas strains that were isolated from wheat seeds, were studied. According to the general biochemical tests and partial 16S rRNA sequences alignment, the isolated strains were closely related to the species of P. gessardii, P. orientalis, P. poae, P. koreensis, and P. cedrina. The Pseudomonas strains exhibit different antagonistic activities, such as phosphate solubilization, cellulase, protease, and lipase production. Also, they have an apparent inhibition effect under in vivo conditions against X. translucens. Seed treatment by these strains led to suppressing bacterial leaf streak disease incidence in an early growth stage. However, disease progress enhanced with the seedling growth, resulting in the treated plants' complete death. Only in treated seeds by P. fluorescens (A7), P. chloroaphis(A4), and P. orientalis (Ais119) decrease of AUDPC up to 83%, 74%, and 63% was achieved, respectively, compared with the untreated controls. Our results showed that some fluorescent Pseudomonas strains could cause delay at the beginning of the disease appearance due to competition or producing antimicrobial metabolites during that time. In contrast, some may be considered a threat, enhancing disease development through synergistic effects.
 

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Most plant pathology research has focused on single-host–single pathogen interactions. Here, are the consequences of co-infection of bean seeds with two important seed-borne pathogens, Xanthomonas phaseoli pv. phaseoli (Xpp) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) were investigated in terms of disease severity and bacterial population dynamics. Cff and Xpp isolates were collected from infected bean seeds and were identified by PCR with specific primers. Some physiological, pathogenicity, and antagonistic traits of Cff and Xpp were compared. These pathogens exhibited different characteristics, such as the production of extracellular compounds, including indole acetic acid, biofilm formation, and motility which can potentially affect each other and host plants. The results revealed that simultaneous infection of bean seeds with two pathogens increased the area under disease progress curve (AUDPC) by 1.71 and 2.38 times compared to a single infection of those with Xpp and Cff, respectively. Pathogen populations in stems and leaves were different under co-infection and single conditions. The data exhibited that the ascending Xpp population in the leaves developed from bean seeds co-infected by Cff and Xpp resulted in a descending Cff population. Xpp isolate displayed greater motility, colonized the plant earlier than Cff, and accelerated disease onset. More biofilm production, confirmed in both pathogens, under co-infection conditions caused earlier plant death via water movement restriction. Our results substantiated that the higher pathogenicity abilities of Xpp played a more critical role in the disease progression in plants developed from bean seeds co-infected by Cff and Xpp. This study provides evidence for the co-occurrence of Xpp and Cff in nature, highlighting the importance of co-infection in common bacterial blight (CBB) and bacterial wilt (BW) disease dynamics.