شناسايي ورده بندي عوامل فايتوپلاسمايي درختان ميوه هسته دار با استفاده از روش هاي مولكولي

چكيده انگليسي :

Abstract Phytoplasma caused diseases pose a potentially serious threat to the production of fruittrees worldwide In recent years stone fruit trees with characteristic symptoms ofphytoplasma associated disease were noticed in Isfahan and Chaharmahal o Bakhtiariprovinces Symptoms included witches broom chlorosis leaf stunting rosette leaf rollingand dieback of branches in almond and yellowing rosette and rolling leaves of plum peachand cherry trees During 2003 2005 symptomatic and apparently healthy leaves of affectedtrees were sampled from different sites in Isfahan Chaharmahal o Bakhtiari EastAzerbaijan Shabestar and Tehran Sahriar karadj The midribs and peduncle of thespecies were retained for DNA extraction First round PCR analysis was performed with allsamples with primer sets P1 P7 P1 Tint and PA2F R Three additional primer pairs16F2 R fU5 rU3 and NPA2F R which prime the amplification of definite regions within16Sr 23S rRNA were utilized for Nested PCR reaction PCR products were detected andtheir sizes estimated by electrophoresis on 1 2 agarose gel in TBE buffer Except fewDNA samples no DNA amplification was observed when the primer pairs P1 P7 P1 Tintand PA2F R were used in first round of PCR However employing Nested PCR usingR16F2 R2 primed a DNA fragment of 1239bp from products of first round PCR by P1 P7and P1 Tint Also primer pair NPA2F R primed amplification of a DNA band ofapproximately 485bp from products of first round PCR with PA2F R Taken together theresults of detection of phytoplasma from symptomatic stone fruit trees withoutamplification of DNA fragment in healthy plants confirmed the capability of Nested PCRtechnique to identify phytoplasma associated trees in the region It is worthwhile to mentionthat Nested PCR with combination of PA2F R in first round and NPA2F R in the secondrun of PCR was evaluated as best method for the detection of phytoplasma agents indiseased trees Restriction enzymes of a part of 16S rRNA gene sequence of the isolatesamplified using R16F2 R2 and then NPA2F R primer sets in Nested PCR indicatedconsiderable differences in RFLP patterns of isolates obtaine from peach cherry and plumtrees Using restriction enzyme TaqI for PCR RFLP analyses of phytoplasmas detectedfrom almond trees showing witches broom symptom exhibited the same pattern which wasdifferent from those associated with branching and die bach syndromes Although HpaIIdigestion showed noticeable divergance within each group Based on restriction analyses five subgroup were distinguished within phytoplasmas isolated from various stone fruits Few strains from each subgroup were subjected for Diedeoxy chain reaction sequencing Asearch of sequence databases indicated that DNA sequences of the representative isolates ofphytoplasma from local stone fruits have striking similarities 97 99 to 16S rRNAsequences of previously sequenced phytoplasmas Taking together all the sequencealignment results it can be concluded that phytoplasma species Ca Phytoplasmaaurantifolia Ca Phytoplasma phoenicium Ca Phytoplasma asteris Ca Phytoplasmatrifolii and Ca Phytoplasma solani are prevalent in stone fruits trees of the region Ca Phytoplasma aurantifolia and Ca Phytoplasma phoenicium were found the most dominantagents associated with dieback disease and witches broom symptoms Also Ca Phytoplasma trifolii was detected in a single almond tree in Najafabad showing dieback andleaf shoestring symptoms which indicated that probably several phytoplasmas are involvedin almond decline syndrome in this area Identification of Ca Phytoplasma solani in peach Ca Phytoplasma trifolii in peach cherry and plum trees and Ca Phytoplasma asteris inpeach and plum samples imply that likely these pathogens are spread with a similartransmission manner which is threaten for further extention of stone fruits production in theregion

زيرك ، ليلا

شناسايي ورده بندي عوامل فايتوپلاسمايي درختان ميوه هسته دار با استفاده از روش هاي مولكولي

مشخصات عمومي ، سلولي ، ژنتيكي , فرآيندهاي متابوليكي , توكسين ها , ميكروسكوپ فلئورسني , الكترو فورزژل آگاروز , ژل پلي آكريل آمايه