Detection And Sequence Analysis Of Viroids From Citrus And Grapevine Trees

Viroids are important pathogens of many plants. In citrus trees Citrus exocortis viroid (CEVd) and Citrus viroidâ…¡(CVd-â…¡,known as Hop stunt viroid, HSVd) can cause the dwarf of citrus trees, decrease growth vigor and fruit production. These two viroids also can infect grapevine. However, they usually do not induce any visible symptoms. In this study, the viroids in citrus and grapevine trees grown in China were detected and their molecular characteristics were analysized. The obtained results will provide some more useful information for realizing the viroid species and understanding their molecular features. The obtained results are as followings:1.Totally 24 citrus samples were collected from Zigui, Wuhan, Hubei province and Ganzhou, Jiangxi province. RT-PCR was carried out for the detection of 7 citrus viroids CEVd, CVd-â… ,CVd-â…¡,CVd-â… -LSS,CVd-â…¢,CVd-â…£and CVd-OS.The results showed there were at least four kinds of citrus viroids,CEVd,CVd-â… ,CVd-â…¡and CVd-â…¢in these citrus samples.Among these viroids, CVd-â…¡had a high infection frequnce and was detected from 9 samples;CEVd, CVd-â… and CVd-â…¢were detected from 7,3 and 5 samples, repectively. Among these viroids positive samples, only 2 samples were single infection by CVd-â…¡and 1 sample was single infection by CEVd, all other samples were infected by two or more than two viroids.2.The full-length genome cDNA of four viroids CEVd, CVd-â… ,CVd-â…¡and CVd-â…¢from the same citrus sample Hb-C1 were amplified by RT-PCR and cloned. Twenty clones from each viroid were randomly selected and sequenced. The population structures of these viroids were analyzed based on their molecular characteristics.Results showed that all these viroids had quosi-species feature and each viroid was composed of several molecular variants. The molecular variants from the same viroid isolate shared the similarity above 90%. Among four viroids, CVd-â… showed a lowest variation and its molecular variants had 96.3%-100% similarities, one predominant molecular variant with a frequency of 20%, two subdominant molecular variants and each with a about 10% frequency, and the non-predominant haplotypes 60%. For CVd-â…¡,the molecular variants shared the similarities 95.1%-100%, one predominant molecular variant with a frequency of 35%, one subdominant with a frequency about 10%, and the non-predominant haplotypes 50%.For CEVd isolate, the molecular variants shared the similarities 92.6%-100%,one predominant molecular variant with a frequency 15%,two subdominan molecular variants and each with a frequency about 10%, and the non-predominant haplotypes 65%.For CVd-â…¢isolate, the molecular variants shared the similarities of 92.4%-100%, two predominant molecular variants and each with a frequency of 15%.3.Totally 40 grapevine samples were collected from Zhengzhou, Henan province and Wuhan, Hubei province. RT-PCR were carried out for the detection of CEVd and HSVd. The results showed that 2 samples were infected with CEVd and this was the first report of CEVd from grapevine in China. The infection rate by HSVd was 35%.The amplified products from CEVd were cloned, and 10 randomly selected clones were sequenced from each isolate. The population structures of the viroid was analyzed based on their molecular characteristics.For both isolates,20% of the clones represented the same variant (CEVd-hn-g-1,GenBank Accession GU592444) and the molecular variants shared the similarity of 98.1% -100%.It shared the similarity 93.8% with the variants from grapes registered in NCBI, showed a max identity 99.0% with the variants from citrus, and showed the similarity 96.6%,95.5% and 90.9% with the variants from,, Gynura, beans and tomatoes, respectively.