ظهور، خصوصیات مولکولی و ناقل فیتوپلاسمای همراه با بیماری جاروک اطلسی بنفش Petunia violacea در ایران

The disease, Petunia Witches broom (PvWB), characterised by phyllody, virescence, witches' broom, little leaf and yellowing was observed in municipal lands and parks in Bandar Abbas, Hormozgan province, Iran. In addition, the incidence of the disease was calculated in three BLVDs of municipal lands of Bandar Abbas. High incidence of the disease may be suspected to have an insect vector. This study was carried out to find causal agent and the vector of this disease. To do so, DNA was extracted from 0.5 g of the mid-vein of ten symptomatic plants and used as template in nested polymerase chain reactions (PCRs) primed by phytoplasma-universal primer pair P1/P7 followed by primer pair R16F2n/R16R2n for amplification of phytoplasmal 16S rRNA gene sequences (rDNA). The PCR products (two samples) were sequenced bidirectionally by Macrogen Sequencing Service. To find insect vector, P. violacea seeds were sown in pots and maintained in growth chamber (25 ± 1 ºC, 16:8 L:D and 70 ± 5 % RH). Before transmission trials, all plants were tested for phytoplasma presence by Nested-PCR using the same primer pairs. For transmission trial, Orosius albicinctus (the only leafhopper collected from infected plants) adults were collected from infected plants by using a D-Vac aspirator and were placed and caged on five healthy P. violacea (2-3 week old). Each cage contained 5-7 insect individuals. Plants were maintained in the growth chamber (as described above) for monitoring disease symptoms development and sampling for PCR assays. Two month post-inoculation, plants were molecularly tested for phytoplasma presence. Symptoms of disease development also were observed weekly. Five individual plants with no contact with insects kept in the same condition were used as control. BLAST analysis of the 1250 bp of ribosomal RNA gene revealed the phytoplasma is associated with P. violacea witches' broom (PvWT) which belongs to group 16SrII, 'Candidatus Phytoplasma aurantifolia' with highest identity (99%) to that of the Parthenium hysterophorus phytoplasma (accession no. MG748745). In transmission trial, after eight weeks of inoculation, four out of five P. violaceae plants fed on by the O. albicinctus showed phytoplasma disease symptoms such as yellowing, little leaf, stunting, phyllody and witches' broom. Plants that were not exposed to O. albicinctus were grown normally. The sequences obtained from infected plants, insects and experimentally vector challenged P. violacea were aligned (Clustal omega) together and showed 100% similarity. All sequences were deposited in GenBank under acc numbers MH068683-4. Based on these results, Orosius albicinctus can be a vector of this disease on P. violaceae in Bandar Abbas. To our knowledge, this is the first report of detection a Ca. phytoplasma aurantifolia-related strain (16SrII group) affecting P. violacea and insect vector in Iran and most probably in the world.