Evolution And Pathogenicity Of Two Potyviruses Causing Soybean Mosaic Diseases In China

Potyvirus is one of the largest genera of plant viruses, and many of its members are important pathogens of agricultural crops. Soybean mosaic virus (SMV), a typical Potyvirus member, is widely known as a devastating pathogen of soybean [Glycine max (L.) Merr.], causing significant yield losses and seed quality deterioration in various soybean-growing regions around the world. SMV is seed-borne and efficiently transmitted by aphids in a non-persistent manner. In addition, another seven potyviruses were reported to infect soybean. These includes Bean common mosaic virus (BCMV), Peanut mottle virus (PeMoV), Cowpea aphid-borne mosaic virus (CABMV), (Bean yellow mosaic virus, BYMV), Clover yellow vein virus (CYVV), Passion fruit woodiness virus (PWV) and Watermelon mosaic virus (WMV). Actually, five of them showed closer relationships to SMV according to our potyviruses phylogeny analysis using 155 genomic sequences of 6A potyviruses.Potyvirus is a single-stranded positive sense RNA virus. RNA viruses are characterized by large population sizes, fast replication, high mutation rates and frequent recombination, which results in great evolutionary potentials. In recent years, the selection conferred by continuously growing of resistant soybean materials is likely to alter the population structure and dominant strains of potyviruses quickly. Moreover, if potyviruses occur in complex with other viruses, the impacts on yield quantity and quality will be more severe. Plants infected by potyviruses are also more susceptible to fungal pathogens. Hence, it is necessary to detect and identify the potyviruses and dominant strains in our soybean producing areas, to explore their evolution characteristics and to screen the corresponding resistance resources for preventing the soybean disease caused by potyviruses. In addition, recent studies have suggested that SMV likely originated in South and East Asia, particularly in China, and many SMV strains were also identified in Chinese soybean producing areas. The evolutionary relationships of SMV strains/isolates from different countries (mainly the South Korea and USA) have been studied using genomic sequences, with only two SMV genome sequences or partial genome sequences from China. Therefore, the genetic diversity of SMV strains/isolates of China and their evolutionary relationships with that from other countries at the whole genomic sequences level are still needed to be further studied.This study was carried out based on the above questions. We detected and identified the potyviruses in soybean leaves with typical mosaic symptoms; obtained the genomic sequences of the main potyviruses and studied their evolutionary patterns at the whole genomic sequence level, respectively; examined the pathogenicity of the isolated viruses by mechanical inoculation on soybean cultivars with various genetic backgrounds.Firstly, a total of 370 soybean leaf samples with typical mosaic symptoms were collected from four provinces, representing three major soybean growing areas of China in the summer of 2013, with 92 samples collected from Heilongjiang province that represented Northeast area,156 samples from Jiangsu province that represented Huang-Huai Valley area, and 71 and 51 samples from Hubei and Jiangxi provinces that represented the Southern Yangzi River area. We detected and identified the potyviruses in soybean leaves using transcriptome sequencing and RT-PCR by potyviruses degenerate primers. The results of mixed samples transcriptome sequencing showed that the SMV and BCMV were the most abundant potyviruses. Meanwhile,178 samples were selected for total RNA extraction and RT-PCR detection using potyviruses degenerate primers, and 72 samples were found to be positive. Subsequently, among the 72 samples,33 samples were identified to be SMV, 38 samples were identified to be BCMV and one was identified to be WMV. Therefore, it was speculated that SMV and BCMV were the significant potyviruses inducing soybean mosaic diseases in China soybean producing areas at the present stage.Secondly, based on the sequences identity and clean sequencing chromatograms of the PCR products of potyviruses degenerate primers,14 out of the 33 SMV isolates, 30 out of 38 BCMV isolates, together with 4 previously obtained SMV strains, were further chosen for whole genome sequencing. Subsequently, we utilized the 18 SMV genome sequences or the 30 BCMV genome sequences together with the SMV (65) or BCMV (16) genomes available in Genbank to explore the evolutionary patterns such as the genetic variation, recombination events, selection pressures and evolutionary relationships, of SMV or BCMV, respectively.1. The evolution of SMVBy obtaining 18 new genomic sequences of Chinese SMV strains/isolates and further compiling these with available data, we have explored the evolution of SMV from multiple aspects. As in other potyviruses, recombination has occurred frequently during SMV evolution, and a total of 32 independent events were detected. Using a maximum-likelihood method and removing recombinant fragments, a phylogeny covering 83 SMV sequences sampled from all over the world was reconstructed and the results showed four separate SMV clades, with clade I and II recovered for the first time. The SMV isolates from China were found in all four clades and the fact that many isolates from China were located at the basal positions of the SMV phylogeny gave some support to the hypothesis that this virus originated from China, accompanied with its natural host soybean. Population structure analysis of SMV revealed significant genetic differentiations between China and two other countries (Korea and USA).Selection analysis showed that in general, SMV coding regions are under negative selection, indicating a role of purifying selection in removing detrimental mutations among SMV population. However, certain regions in the PI, HC-Pro, P3, and CI genes showed higher dN/dS ratios and certain amino acid sites in SMV-encoded genes exhibited higher non-synonymous substitution rate (dN) than synonymous substitution rate (dS) values (ω>1), suggesting that these regions may have experienced different evolutionary pressures and these sites have been influenced by positive selection.2. The evolution of BCMVSimilarly to SMV, the evolutionary patterns of BCMV were studied using the obtained 30 genome sequences together with 16 other BCMV genomes available in Genbank. Phylogenetic analysis showed that both soybean-infecting BCMVs (group I) and peanut-infecting BCMVs (group II) are distantly related to other BCMVs, suggesting ancestral differentiation and host adaptation. Genetic variation analysis showed that P1, P3 and 6K2 genes and the beginning portion of CP gene showed higher levels of variation relative to other genes, suggesting that these genes likely suffered less evolutionary constraints. However, selection analyses showed that a number of sites within the P1 and P3 genes have been effected by positive selection. These obtained BCMV sequences also exhibit high intraspecific recombination frequencies, indicating a more dynamic evolutionary history. In addition, the interspecific recombination phenomenon was also detected between SMV and BCMV in the SMV and BCMV population evolutionary analysis at the whole genomic sequences level.Thirdly, four Chinese SMV strains/isolates, XFQ001 (clade I), SX-Z (clade I) SC6-N (clade I), and SC7-N (clade II), were selected for inoculation of both USA and Chinese differential soybean cultivars, with their pathogenic phenotypes significantly different from those of American strains. Similarly, the infective abilities of four representative BCMV isolates, DXH015 (group I-a), HZZB011 (group I-b), HZZB012 (group I-c) and CDXQ003 (group I-c), were tested on 12 differential soybean cultivars, with most of the cultivars successfully infected and induced systemic mosaic or necrosis symptoms. It is suggest that BCMV is a potential threat to soybean production. Additionally, we screened and found that the soybean cultivars Sewuon97, Raiden and V94-5152 possessing excellent BCMV-resistance genes for the first time, and positioned the BCMV-resistance genes in Sewuon97 preliminarily.Overall, these findings aid in expanding our knowledge of the soybean mosaic disease caused by potyviruses, the evolutionary patterns of SMV and BCMV, and can potentially assist researchers in designing detection and management schemes against SMV and BCMV.