مطالعه فیلوژنیک عامل گال طوقه و ساقه فیکوس و اولین گزارش ازگونه Agrobacterium nepotum در ایران

During 1394 to 1396, Stem and crown gall symptom were observed on Ficus benjamina in the greenhouses in Shiraz and Tehran .samples were collected and transferred to the laboratory to isolation and identification. Pathogenicity tests were conducted on tomato (Lycopersicon esculentum),Christmas kalanchoe (Kalanchoe blossfeldiana) and sunflower (Helianthus annuus cv. Armavirski) plants in greenhous conditions. The generic primers F8360/F8361 and the pTi specific primer pairs virD2A/virD2C , tms2F1/ tms2R2 were used for the identification of the pathogen . Phylogenetic analysis based on gyrB and recA genes used in this study. We used the primer pairs F61/R1093 and F7386/ArecAR for amplification of gyrB and recA genes, respectively and the certificated PCR products were sequenced.There Sequences alignment were made using MEGA 6.06 software and phylogenetic tree using maximum likelihood algorithm ,based on concatenated sequences of gyrB and recA genes. All but two strains (i.e. Fic9 and Fic7-2) were pathogenic on tomato , sunflower and Christmas kalanchoe plants at 15 to 20 days post inoculation. Amplification of 224 bp , 617 bp and 453 bp fragments using virD2A/virD2C and tms2F1/ tms2R2 and F8360/F8361 primer pairs, identified all the weeping fig isolates as the Agrobacterium spp. harboring the tumor inducing (Ti) plasmid. All the nine strains isolated from weeping fig were divided into three distinct clusters , A. larrymoorei , A. nepotum (G14) and atypical group based on the concatenated sequences gyrB and recA genes. the strains fic7-2 and fic9 isolated from weeping fig were clustered as A. nepotum that is first report in Iran. Interestingly, these strains were also non-pathogenic on tomato, Christmas kalanchoe and sunflower plants. Amplification of 224 bp and 617 bp fragment using the primer pairs virD2A/virD2C and tms2F1/ tms2R2 in either fic7-2 or fic9 strains, indicating that both the strains still possess Ti plasmid but the plasmid is no longer functional and not created symptom gall. The A. nepotum strains were isolated from grapevine, raspberry and stone fruits and most of these strains were non-pathogenic on the tested plant species .These results indicate that A. nepotum can colonize more expanded plant species than previously reported. Two strains F.T11 and F.T15 isolated from weeping fig in Tehran were clustered as , A. larrymoorei . More than half of the strains surveyed in this study clustered as an atypical group. based on gyrB and recA genes sequence analysis the atypical group were clearly differentiated from both the A. rubi and A. larrymoorei species and creating a monophyletic group. Further studies using MLSA (multilocus sequence Analysis) approaches are needed to determine the exact phylogenetic position of atypical strains isolated from weeping fig in Iran.