اولین گزارش از همراهی یک فایتوپلاسما با جاروک سرو زربین Cuperrus sempervirens var.horizantalis در ایران

Cupressus sempervirens var. Horizontalis is a very important forest tree species for multiple purposes in forestry (Gallis et al., 2006; Xenopoulos et al., 1990). In 2014-2015,diseased trees (Cuperrus sempervirensvar.horizantalis) with unusual symptoms similar to those caused by phytoplasmas were observed in Tehran Province. The characteristic symptom was witch’s broom, dieback on one side of trees and abnormal prolifrations shoot (Fig.1).Total DNA was extracted from 1gof leaves in symptomatic shoots of four diseased trees (two locations) and two symptomless trees (as negative controls) by modified CTAB methods. The presence of phytoplasma in suspended coniferous plants was demonstrated using nested PCR with the primer pair P1⁄P7 (Deng&Hiruki, 1991; Schneider et al., 1995) and a 1.8 kb fragment was amplifiedans then was used as template in second step of PCR by R16F2⁄R16R2 primer pair (Gundersen &Lee, 1996).The expected fragments with 1,246 bp in size was amplified following nested PCR fromall symptom-bear sample, no DNA was amplified from negative control symptomless plants.The PCR products of two trees by R16F2/R16R2 were cloned in pTZ57R/T vctor (Fermentas, Vilnius, Lithuania) and then were sequenced. Blast result analysis of Cupressus sempervirens var. Horizontalis witche’s broom phytoplasma (GenBank Accession No.KU647632) 16SrDNA show highest sequence identity with 16Sr group II (Ca. Phytoplasma aurantifolia) confirmed that is a 16sr group II related strain. Phylogenetic analysis using MEGA software (version 6.0) confirmed that this phytoplasma was clustered within the 16SrII phytoplasma clade (Fig. 2). In silico RFLP patterns of 16S rDNA with restriction endonucleases AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI using iPhyclassifier (Zhao et.al., 2009) (Fig.3) revealed that virtual RFLP patterns of the phytoplasma differs from all previously reported 16SrII sub groups.Also it have most similarity with the references subgroup of the group II, subgroup D (GenBank accession: Y10097), with a similarity coefficient of 0.90, which is less than or equal to 0.97, thus represent a new subgroup within the 16Sr group II. To our knowledge this is the first report of natural occurrence of a phytoplasma associated with Cupressus sempervirens var.Horizontalis disease in Iran.