Virulence Differentiation And Genomic Characterization Of Phytophthora Vignae

Mung bean(Vigna radiata(L.)),as one of the main grain crops,has been widely grown in China because of its rich nutrients,nice healthy effect,short growth period,and nitrogen fixation characters.The disease has been an important reason limiting the mung bean yield.In recent years,diseases on mung bean are increasingly aggravated with the climatic change,the regulation of planting structure and culture simplification.Phytophthora stem rot is an emerging disease which first reported in Mingguang city,Anhui Provence,and subsequently discovered in Hubei and Jiangsu Province during field survey.In this study,we firstly confirmed the host-specificity of the pathogen Phytophthora vignae causing Phytophthora stem rot on mung bean by morphological comparison,phylogenetic analysis and host range test.Then,the genomes of the three formae speciales of P.vignae were sequenced and assembled,which expected to understand the difference of the three formae speciales from molecular characters.Subsequently,we developed some SSR markers to analyze genetic diversity of Phytophthora vignae f.sp.mungcola isolates,and meanwhile obtained a specific marker that can distinguish the three formae speciales of P.vignae.Finally,by screening the resistant cultivars,a set of differential hosts was developed to identify the pathotype of the isolates from mung bean.The main results are as follows:1.The pathogen Phytophthora vignae causing Phytophthora stem rot on mung bean was identified as a new forma specialis,P.vignae f.sp.mungcola.20,23,5,and 6 isolates were respectively obtained by pathogen isolation from collecting soil samples of mung bean in Mingguang city,Anhui Province,Hefei city,Anhui Province,Wuhan city,Hubei Province and Nanjing city,Jiangsu Province.These isolates from mung bean were initially identified as P.vignae based on morphological and biological comparison with the P.vignae f.sp.vignae and P.vignae f.sp.adzukicola,pathogenicity test,and the multigene phylogenetic analysis.Then the host range tests on 8 legume crops showed the mung bean isolates of P.vignae were only pathogenic towards mung bean and adzuki bean,while the P.vignae f.sp.vignae and P.vignae f.sp.adzukicola was only pathogenic to their original hosts,cowpea and adzuki bean,thus indicating that the mung bean isolates significantly differ from the P.vignae f.sp.vignae and P.vignae f.sp.adzukicola in host range,designated as P.vignae f.sp.mungcola.2.The genome sequencing and de novo assemble of three formae speciales of P.vignae were performed,and host-specific candidate effectors which related to pathogenicity were identified.By sequencing and de novo assembly,the genome size of P.vignae f.sp.mungcola PVMG4,f.sp.adzukicola Pa V1,f.sp.vignae Pc V2 was 89.9 Mb,84.5 Mb and 84.0 Mb,respectively.Then the effectors associated with pathogenicity were predicted and a total of 207,187 and 194 effector proteins containing RXLR motif were discovered in genomes of PVMG4,Pa V1 and Pc V2,respectively.By classification analysis,we found that 120 unique candidate effectors existed in P.vignae f.sp.mungcola,which indicated relationship with its specific pathogenicity.Gene family analysis showed that 121,34 and 47 specific gene families were identified in PVMG4,Pa V1 and Pc V2.The genomic collinearity analysis indicated that there was a lot of structural variation(chromosomal translocation and chromosomal inversion)and indel difference among the three formae speciales,although the collinearity was better.Combining the difference sites with effector proteins,this can be used to better analyze the formation mechanism of host specificity of P.vignae.Moreover,both the phylogenetic analysis and collinearity showed that the P.vignae f.sp.vignae has a closer relationship with P.vignae f.sp.adzukicola.3.We developed SSR markers from P.vignae f.sp.mungcola and analyzed the genetic variation of P.vignae f.sp.mungcola from different locations.By identifying the microsatellite loci from the genome of P.vignae f.sp.mungcola and screening the polymorphic primers,12 pairs of representative SSR primers showed better polymorphism and were used to conduct the analysis of genetic diversity of P.vignae f.sp.mungcola isolates.The UPGMA analysis showed that the 54 mung bean isolates can be divided into three groups at the genetic similarity coefficient of 0.59,indicating a rich genetic diversity among the mung bean isolates.Interestingly,we developed a marker that can effectively distinguish the P.vignae f.sp.vignae,P.vignae f.sp.adzukicola,and P.vignae f.sp.mungcola isolates.4.This study obtained mung bean cultivars resistance to Phytophthora stem rot by resistant screening,and a set of differential hosts was developed to identify the pathotype of P.vignae f.sp.mungcola isolates.17 resistant materials and 25 intermediate material were obtained from 288 mung bean cultivars and resources.Based on their genetic background and resistance stability,a set of differential hosts containing four resistant mung bean cultivars was developed,namely Weilv 6,Zheng 8-4-6,Jilv 0816,and Elv 1.The study of pathogenicity differentiation among the 54 mung bean isolates was conducted using selected differential hosts and the results showed that three pathotypes were identified.Pathotype 1 contained 29 isolates,of which 17 were isolated from Mingguang,accounting for 58.6%,making it the predominant pathotype of this region.Pathotype 2included 22 isolates,of which 17 from Hefei,accounting for 58.6%,making it the main pathotype of Hefei city.Pathotype 3 only contained three isolates,PVNJ-2,PVNJ-3,and PVNJ-6 from Liuhe,Nanjing city.In summary,in this study we identified the pathogen P.vignae causing Phytophthora stem rot on mung bean as a new forma specialis,P.vignae f.sp.mungcola.Based on the genome sequencing and assembly of different formae speciales of P.vignae,we found that120 unique candidate effector proteins were identified in P.vignae f.sp.mungcola isolate PVMG4,which may be related with the formation of its host specificity.Furthermore,we developed SSR marker for the genome of P.vignae f.sp.mungcola,and screened specific marker that can effectively differentiate the three formae speciales of P.vignae.Finally,we established a set of differential hosts by resistance screening,which could be used in the study of virulence differentiation of P.vignae f.sp.mungcola isolates.Based on the differential hosts,P.vignae f.sp.mungcola was identified existing three pathotypes.These results laid a foundation for the research on host specificity of P.vignae f.sp.mungcola.