Background"Candidatus Phytoplasma aurantifolia", is the causative agent of witches' broom disease in Mexican lime trees (Citrus aurantifolia L.), and is responsible for major losses of Mexican lime trees in Southern Iran and Oman. The pathogen is strictly biotrophic, and thus is completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. Therefore, we have applied a cDNA- amplified fragment length polymorphism (AFLP) approach to analyze gene expression in Mexican lime trees infected by "Ca. Phytoplasma aurantifolia".ResultsWe carried out cDNA-AFLP analysis on grafted infected Mexican lime trees of the susceptible cultivar at the representative symptoms stage. Selective amplifications with 43 primer combinations allowed the visualisation of 55 transcript-derived fragments that were expressed differentially between infected and non-infected leaves. We sequenced 51 fragments, 36 of which were identified as lime tree transcripts after homology searching. Of the 36 genes, 70.5% were down-regulated during infection and could be classified into various functional groups. We showed that Mexican lime tree genes that were homologous to known resistance genes tended to be repressed in response to infection. These included the genes for modifier of snc1 and autophagy protein 5. Furthermore, down-regulation of genes involved in metabolism, transcription, transport and cytoskeleton was observed, which included the genes for formin, importin β 3, transducin, L-asparaginase, glycerophosphoryl diester phosphodiesterase, and RNA polymerase β. In contrast, genes that encoded a proline-rich protein, ubiquitin-protein ligase, phosphatidyl glycerol specific phospholipase C-like, and serine/threonine-protein kinase were up-regulated during the infection.ConclusionThe present study identifies a number of candidate genes that might be involved in the interaction of Mexican lime trees with "Candidatus Phytoplasma aurantifolia". These results should help to elucidate the molecular basis of the infection process and to identify genes that could be targeted to increase plant resistance and inhibit the growth and reproduction of the pathogen.

"Candidatus Phytoplasma aurantifolia" is the causative agent of witches' broom disease in the Mexican lime tree (Citrus aurantifolia L.), and is responsible for major tree losses in Southern Iran and Oman. The pathogen is strictly biotrophic, and, therefore, completely dependent on living host cells for its survival. The molecular basis of compatibility and disease development in this system is poorly understood. We applied a proteomics approach to analyse gene expression in Mexican limes infected with "Ca. Phytoplasma aurantifolia". Leaf samples were collected from healthy and infected plants and were analysed using 2-DE coupled with MS. Among 800 leaf proteins that were detected reproducibly in eight biological replicates of healthy and eight biological replicates of infected plants, 55 showed a significant response to the disease. MS resulted in identification of 39 regulated proteins, which included proteins that were involved in oxidative stress defence, photosynthesis, metabolism, and the stress response. Our results provide the first proteomic view of the molecular basis of the infection process and identify genes that could help inhibit the effects of the pathogen.

Grapevine (Vitis vinifera) is one of the most important fruits in Iran where the provinces of Qazvin, Lorestan and Markazi are main producers. During 2013–2015, vineyards located in these provinces were surveyed to verify the presence of phytoplasma. The sample collection was based on symptomatology including decline, leaf yellowing and shortening of internodes. Total DNA was extracted from symptomatic and symptomless grapevine samples and used in nested‐polymerase chain reaction (PCR) assays with phytoplasma ribosomal primers (P1/Tint followed by R16F2n/R2, R16mF1/mR1, R16(I)F1/R1 or 6R758f/16R1232r). Nested‐PCR products were obtained only for symptomatic samples while samples from symptomless plants yielded no PCR products. Restriction fragment length polymorphism (RFLP) analyses with Tru1I, TaqI and Tsp509I and direct sequencing of amplicons followed by phylogenetic analyses indicated the presence of ‘Candidatus Phytoplasma fraxini’, ‘Ca. P. aurantifolia’, ‘Ca. P. solani’ and ‘Ca. P. phoenicium’‐related strains. In Marzaki province, there ‘Ca. P. aurantifolia’ strains were mainly detected, while in the other two provinces, all the four ‘Candidatus species’ were identified with the prevalence of ‘Ca. P. solani’‐related strains. In both provinces in one case, mixed phytoplasma infection was also detected by RFLP analyses. The presence of different phytoplasmas in positive samples indicates great phytosanitary significance due to grapevine economic importance for country. Grapevine phytoplasma infection represents a threat for other crops suggesting grapevine as alternative host species for the phytoplasmas already reported in Iran, while the ‘Ca. P. fraxini’ is for the first time identified in Iran.

A disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.

A large‐scale survey was conducted on pistachio plants exhibiting foliar symptoms including scorch, little leaf, yellows and reddish in pistachio growing areas in the Qom, Yazd and Qazvin provinces of Iran. Total DNA was extracted from symptomatic and symptomless pistachio and used in nested PCR assays with phytoplasma universal primers. Nested PCR products were obtained for symptomatic plant samples while the symptomless plants yielded no PCR products. Virtual restriction fragment length polymorphism, phylogenetic and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to two ribosomal groups; in particular, “Candidatus Phytoplasma solani” and “Ca. P. phoenicium” were identified. The presence of these phytoplasmas in pistachio is of great phytosanitary significance due to its commercial interest.

No abstract

During a survey in an orange (Citrus sinensis) garden in the south of the Kerman province of Iran, some trees were observed showing symptoms of yellowing and decline. Nested PCR analysis using phytoplasma universal primers identified symptomatic samples as phytoplasma-positive, whereas the symptomless samples gave no PCR products. Sequence similarity and phylogenetic studies using 16S rRNA gene sequences demonstrated that the sweet orange decline associated phytoplasma belongs to the 16SrIX 'Candidatus Phytoplasma phoenicium' group. To the best of the authors' knowledge this is the first report of association of a phytoplasma belonging to the 'Candidatus Phytoplasma phoenicium' group with decline symptoms in sweet orange in Iran.

In recent years, a disease has been reported to affect peach trees in Kurdistan province of Iran causing serious losses to the production. Main symptoms of disease include leaf stunting and yellowing, which lead to failure in fruit production at harvest. For diagnosis of disease and identification of the causal agent, symptomatic leaf samples were collected in Kurdistan orchards during summer 2010 and were carried to the laboratory. Total DNA was extracted from plant samples according to the standard procedures and indexed by grafting and nested PCR using phytoplasma generic primers, P1/P7 and R16F2n/R2. PCR products were characterized by RFLP technique and direct sequencing. The 16S rDNA sequences were compared with those of other phytoplasmas in GenBank. Phytoplasma rDNA was amplified from 20 out 35 samples. The 16S rDNA sequences of the phytoplasma were identified in Peach samples which showed 98% similarity to that of "Candidatus Phytoplasma phoenicium" which is considered to be the causal agent of Almond witches' broom. Phylogenetic analysis of 16S rDNA sequences placed peach strains in Almond witches broom isolate as a member of pigeon pea witches broom (PPWB) group. Further studies on the epidemiology of "Ca. Phytoplasma phoenicium" and its vector(s) in Iran are recommended in order to identify new natural hosts and develop successful disease management programs.