Analysis Of Genetic Structure Of Chinese Beet Necrotic Yellow Vein Virus Population And Construction Of P25Mutants

Beet necrotic yellow vein virus (BNYVV) is the casual agent of Rhizomania on sugar beet, which was widely distributed in sugar beet growing regions worldwide. The control of the disease mainly depends on resistant cultivars; however resistance-breaking (RB) variants have been occurred in some European and American countries. The67-70amino acids in p25protein (tetrad) were the most variable region among BNYVV genome which is probably associated with RB. To clarify the occurrence and epidemiology of Chinese BNYVV population, RT-RCR detection was performed on sugar beet root and soil samples collected from sugar beet growing regions in China and genetic variation and structure of Chinese BNYVV population were analyzed in this study. Further, infectious cDNA clones of newly found p25tetrad mutants were constructed to provide the basis for researches on the pathogenesity of Chinese BNYW isolates and the mechanisms of virus-host interactions. Finally, BNYVV infection rates of sugar beet cultivars from "Regional trails of sugar beet cultivars in Inner Mongolia" were analyzed to provide valuable information on selection and demonstration of resistant (tolerant) cultivars. Results are as follows:1. During2009-2014,51sugar beet root samples and33soil samples collected from7provinces (Autonomous Regions) were used for the detection of BNYVV by RT-PCR. Results showed that7sugar beet and16soil samples were detected to be positive for BNYW, accounting13.73%and48.48%of the detected samples, respectively. Full-length cDNA clones of CP, RNA3, RNA4and RNA5from15positive samples were sequenced. At least9clones were sequenced from each genomic region of each isolate to analyze within-isolate diversities. Sequence length variations were observed for each RNA component, especially for RNA5, the sequence lengths of which were ranging from1336to1363nucleotides. Sequence analyses revealed several isolates were mixed infections of variants with different genotypes and/or different p25tetrad motifs. Totally, twelve distinct p25tetrads were found in Chinese BNYW population, of which four tetrads were newly identified. Phylogenetic analyses based on the four genes (CP, RNA3-p25, RNA4-p31and RNA5-p26) of worldwide isolates revealed the existence of two to four groups which mostly corresponded to the previously reported phylogenetic groups. Two new subgroups and a new group were identified from the Chinese isolates in p25and p26trees, respectively. Selection pressure analysis indicated that p25of Chinese isolates was under positive selection and the other three proteins were under negative selection. Frequent gene flow was existed between the geographically distant populations, such that BNYW populations of different provinces were not geographically differentiated. These traits indicated that selection and gene flow were the important factors for shaping Chinese BNYW population.2. Based on the p25tetrad variation of Chinese BNYVV isolates, infectious cDNA clones of p25tetrad mutants were constructed. The transcripts of these mutants as well as BNYVV RNA1and RNA2were mixed and mechanically inoculated on leaves of Tetrogonia expansa. After6dpi, phenotypic differences occurred on leaves inoculated with different mutants indicated that the p25mutants may have different degrees of pathogenesity. Further, full-length cDNA clones of these p25mutants were cloned into pSB130vector. These reconstructed plasmids were mixed with the plasmids of BNYVV RNA1and RNA2and were inoculated on N. benthamiana leaves by agro-infiltration. After7dpi, the inoculated leaves inoculated with different mutants showed necrotic symptoms with different degrees. After14dpi, the upper leaves inoculated with different mutants showed yellowing symptoms with different degrees. The results of RT-PCR detection revealed that all of these mutants could induce systematic infection on N. benthamiana.3. During2009-2014, BNYVV infection in sugar beet cultivars from "Regional trails of sugar beet cultivars in Inner Mongolia" was detected by DAS-ELISA. The numbers of detected cultivars in each year were85(2010),103(2011),105(2012),105(2013) and80(2014), respectively. The results showed that the total incidence rates of beet cultivars in rhizomania nursery slightly fluctuated yearly. Whereas the total incidence rates of beet cultivars in open field were on the increase year by year, except for the abnormal high value in2010. Among the cultivars for tests, most were susceptible or moderate susceptible. Among the resistant and moderate resistant cultivars, most were from KWS (Germany) and BETA (USA), and only one from China.