Studies On Diversity,Mechanisms Responsible For Pathogenicity Debilitation Of Sclerotinia Minor And Its Risk In Infecting Oilseed Rape

Sclerotinia minor Jagger is a cosmopolitan plant-pathogenic fungus.It can aggressively infect many economically important crops,including lettuce,peanut,pea and sunflower.The pathogen mainly infects the base of the plants and causes stem rot,resulting in serious economic loss.In this study,we analyzed genetic diversity of S.minor populations in Hubei Province,elaborated the mechanisms responsible for pathogenicity debilitation of two isolates of S.minor,and investigated the risk of S.minor in infecting oilseed rape plants.Main results are summarized below.1.Ninety-five single-sclerotium isolates were obtained from lettuce and weeds in three counties(Xianning,Dangyang and Lichuan)in central China.They were identified belonging to Sclerotinia minor based on colony morphology and the S.minor-specific DNA marker.We determined the growth rate,sclerotia production,pathogenicity,mycelial compatibility groups(MCGs),the mating type(MAT)alleles and genetic diversity of these isolates based on microsatellite molecular marker(SSR).Ninety-five isolates of S.minor were obtained from 11 plant species,among the 11 sampled plant species,five weed plants including Capsella bursa-pastoris,Oenanthe javanica,Fragaria gracilis,Ranunculus ternatus and Salvia plebeia were identified as the new hosts of S.minor.Pathogenicity differentiation was observed among the 95 S.minor isolates.A significant positive correlation was found between pathogenicity and growth rate in these S.minor isolates(r = 0.372,P<0.05).The 95 isolates were divided into 8 mycelium compatibility groups(MCGs).MCG1 and MCG2 were the most prevalent,comprising 29 and 28 isolates,respectively,the remaining five MCGs(MCG3 to MCG7)were the moderately prevalent,comprising 1 to 10 isolates.Shannon's H index(H)and Simpson index(S)based on the MCG data were 1.748 and 0.786,respectively.Result of molecular variance(ANOVA)based on SSR analysis showed that 97.75%of genetic variation was found among isolates within populations(P= 0.03324),2.25%of genetic variation was found among populations(P = 0.02151).Therefore,the genetic variation within populations was greater than that between the populations.The Bayesian clustering analysis showed that the 95 S.minor isolates were divided into two clusters,which showed extremely high genetic similarity.Phylogenetic tree analysis showed that there was no apparent relationship between the population structure and host,geographic origins and MCGs of S.minor.These results suggest that.minor isolates in Hubei have lower diversity.MAT inversion was detected in S.minor isolates in Hubei.This trait appears very stable in nature.The 95 S.minor isolates were divided into three types,namely Inv-,Inv+and heterokaryon(Inv+ plus Inv-),accounting for 4.2%,58.9%and 36.9%,respectively.Additionally,the S.minor isolates of different MCG groups and the S.minor isolates of different MAT groups were compared for differences in mycelial growth rate and sclerotial yield on PDA,and lesion diameter on lettuce leaves.Results showed that the seven MCG groups did not significantly differ in mycelial growth rates(P = 0.3112)and lesion diameters(P = 0.0427),but significantly differed in sclerotial yield(P = 0.0039).MCG3,MCG5 and MCG7 had significantly higher sclerotial yield than that of MCG6,whereas the remaining three MCGs did not significantly differ.Results showed that the three MAT groups did not significantly differ in mycelial growth rate(P = 0.3328)and lesion diameter(P = 0.4532),but significantly differed in sclerotial yield(P = 0.0008).The Inv-isolates had significantly higher sclerotia yield than the isolates in Inv+ and heterokaryon.2.The full-length cDNA sequence of Sclerotinia minor isolate LC22 debilitation-related virus(12-kb dsRNA)was obtained,it is 12,626 bp in length and belongs to endomavirus,called Sclerotinia minor endornavirus1(SmEV1).SmEV1 encodes a 4020-aa-long polypeptide.The genome of SmEV1 contains four conserved domains,including the MTR domain,CRRs,the Helicase 1 domain and RdRp domain.SmEV1 could be vertically and horizontally transmitted though sclerotia and by hyphal anastomosis.S.minor isolates that were successfully infected by SmEV1 were severely debilitated in several aspects,including colony morphology,growth rate,sclerotial production and pathogenicity,compared to the original virulent S.minor isolates.These results suggest that hypovirulent isolate LC22 of S.minor can be potentiall used to control virulent isolates of S.minor.3.We also analyzed the mechanisms responsible for debilitation of hypovirulent isolate LC38 of S.minor.The mycelial growth rate of this isolate was slow,sclerotial production was low,its hyphae were thin,and the tip of the hyphae became bent under light microscope.Quantitative determination results of oxalic acid showed that the ability to secrete oxalic acid in isolate LC38 was weak.The total amount of oxalic acid secreted by isolate LC38 was significantly lower than isolate LC41(P<0.05).4.Pathogenicity test results indicated that S.minor could infect leaves,stems and pods of detached oilseed rape.Pot experiment in field showed that the minimum inoculation concentration causing disease was ten sclerotia per plant,the disease incidence was higher with increase in size of inoculum.The S.minor sclerotia close to the stem base of oilseed rape(<1 cm)could cause infection.We investigated oilseed rape sclerotinia rot in Hubei(Xianning,Lichuan,Dangyang,Jingmen,Jingzhou and Zhijiang)both at the seedling stage and at the adult-plant stage during April to May of 2015 and December of 2015 to Janurary of 2016,according to a survey of 91,000 oilseed rape plants in 182 fields,results showed the pathogen of oilseed rape sclerotinia rot in fields was Sclerotinia sclerotiorum,rather than S.minor.Therefore,S.minor had a low risk in infecting oilseed rape in Hubei.