تعيين ويژگي هاي مولكولي فايتوپلاسماهاي همراه سيب و گلابي در استان اصفهان

چكيده انگليسي :

112Molecular characterization of Phytoplasmas associated with apple and pear in Isfahan province Mahdis Hashemi Tame mahdis hashemi@yahoo com Date of submission January 15 2013 Department of Plant Protection Isfahan University of Technology Isfahan 84156 83111 Iran Degree M Sc Language Farsi M Bahar Ph D Assoc Professor Supervisor mbahar@cc iut ac irAbstractPhytoplasma caused diseases pose a potentially serious threat to the production of fruit trees worldwideand cause great losses of these products In recent years typical symptoms of phytoplasma induceddisease on apple trees including witches broom leaf stunting and abnormal growth of petioles and alsorolling leaf quick decline and slow decline of pears have been observed in different areas of Isfahanprovince To detect and classify phytoplasmas associated with apple and pear trees in the region totalDNA was extracted from veins and petioles of symptomatic trees First round PCR analysis wasperformed with primer sets P1 P7 and P1A P7A Except for few samples no DNA amplification wasobserved when the primer pair P1 P7 were used in first round of PCR while primer sets P1A P7A couldamplify a 1759 bp fragment in some samples A nested PCR with the first round primer pair P1A P7Afollowed by second round primer pairs designed from a definite region within 16S 23S rRNA R16F2n R16R2 and R16F1 X R16R1 X resulted in amplification of DNA fragments sizedapproximately 1239bp and 1100bp respectively from the symptomatic samples collected in late summeror early spring but not from those collected in spring or early summer Based on the results the nestedPCR using P1A P7A followed by R16F2n R2 was found a sensitive method for detection of pytoplasmasin diseased trees To determine the association of apple proliferation group phytoplasmas in pear andapple trees a nested PCR with P1A P7A followed by the specific primer R16F1 X R16R1 X wascarried out which resulted in amplification of a 1100 bp fragment from pear diseased samples but none ofthe apple samples showed the corresponding band In PCR RFLP analysis the 1239bp PCR productsfrom R16F2n R16R2 were digested with restriction enzymes RsaI HaeIII HpaII TaqI and HhaI CfoI endonucleases and the restriction patterns confirmed a significant genetic difference among thephytoplasmas indicating that divergent strains of phytoplasmas should be associated with diseased applesand pears The representatives of each PCR RFLP patterns were subjected to DNA sequencing Thenucleotide sequence analysis of 16S rRNA region of the samples revealed a high level similarity 99 100 to 16S rRNA sequences of previously reported phytoplasmas According to sequence analysis theassociated phytoplasmas with apple trees were belong to Candidatus phytoplasma asteris and Ca P aurantifolia The causal agent of Apple proliferation disease in Europe Ca P mali was not detected inIsfahan orchards In this study Ca P pyri as the world wide prevalent phytoplasma was found the mostfrequent phytoplasma associated with pear trees although Ca P asteris in few samples and Ca P prunorum European stone fruit yellows disease agent in a single pear tree from Rasht were alsodetected in pear orchards A partial amplification of ribosomal protein rp gene from aster yellowsstrains using rp I F1A rp I R1A primer pair and its sequencing revealed that Aster yellows phytoplasmadetected in apple and pear trees in Isfahan are belong to rp B subgroup Identification of severalphytoplasmas in apple and pear orchards suggests that these phytoplasmas are spreading with a similartransmission manner which threaten further extension of apple and pear production in the Isfahanprovince Key words Apple Pear Phytoplasma Apple proliferation Nested PCR PCR RFLP DNA sequencing

هاشمي طامه، محديث

تعيين ويژگي هاي مولكولي فايتوپلاسماهاي همراه سيب و گلابي در استان اصفهان

گروه شاخه انبوهي سيب , آناليز PCR-RFLP , تعيين توالي نوكلئوتيدي