Investigation And Detection Of Cherry Virus Diseases And Identification Of The Pathogen Viruses In Facilitied Cultivation In Shihezi Of Xinjiang

With the development of agricultural ecomomy transformation in Xinjiang,cherry was firstly selected as the gold trees for the fruit trees in Xinjiang facilities agricultural.Its economical ? ornamental and medicinal value have made a outstanding contribution not only to Xinjiang's economic growth but also farmers' income.However,with the development of cherry' facility cultivation,virus diseases have generally increased.The virus disearses have become a key factor that restricts the quality and yield of cherry and finally leads to economic losses in the region.A fast and efficient detection method was established.The incidence of cherry virus diseases was investigated,and pathogen' varieties were identificated.To provide a scientific basis for preventing and controling cherry virus diseases in the regions of Xinjiang.In the shihezi region six area survey found facilities cherry virus disease symptoms mainly include mosaic and green mottle,roll leaf,yellow,deformity,shrivel,etc.Use eight pairs of specific primers were utilized to detect the 122 symptomatic samples by RT-PCR.RT-PCR amplification showed that amplified products have the expected primer size of Cherry virus A(CVA),Prunus necrotic ring spot virus(PNRSV),Prune dwarf virus(PDV),Cherry green ring mottle virus(CGRMV)in the samples.Through similarity analysis of the nucleotide sequences,the results showed that CVA-SHZ,PNRSV-SHZ,PDV-SHZ and CGRMV-SHZ shared the 99.5%,99.5%,99.0%,99.4% homology with ChTA12(KT310083.1),DJ1-1(JF333586.1),HSY4-1(HQ539657.1),ZZ-Ch-13(KC965106.1)reported in GenBank isolates,respectively.The average rates of infection with CVA,PNRSV,PDV and CGRMV were 67.3%,66.1%,6.5%and4.9%,respectively.The average rates of infection with more than two viruses were 56.5%.CVA and PNRSV were the main viruses in facilitied cherry in Shihezi of Xijiang,and they were widely complex infection.This is the first report on the dectetion of CVA,PNRSV,PDV and CGRMV in cherry in Xinjiang.A multiplex reverse transcription polymerase chain reaction(multiplex RT-PCR)method was developed for the simultaneous detection of three cherry viruses.The crucial factors of multiplex RT-PCR include annealing temperature,number of the PCR cycles,and optimized for the highest sensitivity and specificity.Three specific fragments were simultaneously amplified in uniplex PCR reaction.Their molecular weights were determined to be PNRSV(675bp),CGRMV(363bp)and PDV(304bp),respectively.The sequence analysis indicated that the three viruses shared at least 90% homology with other related viruses.Multiple RT-PCR reaction conditions optimization results showed that optimized conditions is58? for annealing temperature,35 for circulation.But the dNTPs concentration had a little effects on the experimental results,the selection of optimum concentration tendency for 0.6 mmol/L.The sensitivity was shown to be capable of positively identifying the three viruses with ? 59.7ng·?L-1of freesia tissue sample.This method was then applied to detect the incidence of virus disease in Shihezi cherry.The multiplex RT-PCR protocol could be used to simultaneously detect PNRSV,PDV and CGRMV with high stability,accuracy and sensitivity.In order to amplify the CVA-SHZ fragment by RT-PCR,the specific primers were designed for amplification of full-length fragments of CVA-SHZ genome by RT-PCR.And the full-length sequence of CVA-SHZ Xinjiang was finally obtained.The results showed that the size of CVA-SHZ isolate genome is7373 nt(beside Poly(A).It is with the same structure of 4 whole genome sequences in Genebank and Encode two open reading frames(Open,Reading Frame,ORF).The results by sequencing alignment showed that the similarity between CVA-SHZ and ChTA11?ChTA12 were 98.7%;The similarity with nucleotide sequences of India isolate lead to 81.6% and 97.7% with the German isolate nucleotide sequence.The relationship between CVA-SHZ and ChTA12,ChTA11 isolations seems significantly relevant.It was found that CVA-SHZ form a separate branch of evolution.At the same time,CVA-SHZ belongs a large branch with the German isolate,other with,it has no significant relevance with JK(FN691959.1)isolated in India.