Isolation And Identification Of Mycoviruses From Chinese Lentinula Edodes Germplasm Resources And Molecular Variation Of Lentinula Edodes Partitivirus Virus 1(LePV1)

Lentinula edodes(Berk.)Pegler is one of the most widely cultivated edible mushrooms in the world.Virus is a serious potential danger in production.In this study,the dsRNA/virus carrying situation in the wild and cultivated species of Chinese L.edodes were comprehensively analyzed for the first time by using dsRNA and RT-PCR detection technologies,and the new-found dsRNA/viruses were cloned and their molecular characteristics were analyzed.on this basis,the molecular variation and colony structure of these main viruses were analyzed.The concrete results are listed as follow:The 215 Chinese L.edodes germplasm resources,including 90 cultivated strains and 125 wild strains,were selected as experimental materials,the dsRNA/virus carrying situation were investigated by using dsRNA detection technology.The results showed that,the dsRNA band was detected in 57 cultivated strains and 84 wild strains,and the detection rate was 63.33%(57/90)and 67.20%(84/125),respectively.The detected dsRNA contained 10 banding patterns,its size was 12.0kb(dsRNA-1),10.0 kb(dsRNA-2),2.4 kb(dsRNA-3),2.3 kb(dsRNA-4),2.1 kb(dsRNA-5),2.0 kb(dsRNA-6),1.9 kb(dsRNA-7),1.8 kb(dsRNA-8),1.6 kb(dsRNA-9),and 0.6 kb(dsRNA-10),respectively.These bands were further separated and partially cloned,with the exception of dsRNA-7,the partial or full length genome sequence of the other 9 dsRNA were acquired.The sequence analysis determined that,the dsRNA-1 was Lentinula edodes mycovirus HKB(LeV-HKB)reported before,the dsRNA-2 may be one variant or defective virus of LeV-HKB,the dsRNA-3 and dsRNA-4 were Lentinula edodes partitivirus 1(LePV1),the dsRNA-6 was new partitivirus,the new partitivirus were composed by dsRNA-5 and dsRNA-9 was a bipartite virus,dsRNA-7 and dsRNA-8 was a partitivirus.The specific properties of the dsRNA-10 remained unknown.The 10 dsRNA bands totally made up 12 kinds of banding patterns(banding pattern I-XII)in the 215 tested strains,among them,the detection rate of banding pattern I(only RNA-1),banding pattern II(dsRNA-1,dsRNA-3and dsRNA-4),and banding pattern XI(dsRNA-3 and dsRNA-4)were the most highest,which was 26.5%(57/215),16.3%(35/215),and 17.7%(38/215),respectively,and the detection rate of the others were all lower than 0.1%.The above results suggested that,the LeV-HKB and LePV1 were the two main viruses in Chinese L.edodes germplasm resources,which existed in the manner of single infection or mixed infection.In order to verify the accuracy of the above results,furthermore,the two main viruses in L.edodes germplasm resources were detected by RT-PCR,and the results proved that,except for inconsistent results of dsRNA detection and RT-PCR detection in a few strains carrying LePV1 and some strains carrying LeV-HKB,the results of the other strains detected by the above two methods were basically the same.In comparison,the detection ate of LeV-HKB in cultivated strains was higher than that in wild strains,but LePV1 was on the contrary.Moreover,there was no significant correlation between the carrying situation of dsRNA in Chinese L.edodes germplasm resources and the geographical origin and the genetic background of strains.Using the 27 L.edodes strain carrying LePV1 virus identified from L.edodes core germplasm resources as materials,the primers capable of amplifying the full-length genome sequences of dsRNA-3 and dsRNA-4 of LePV1 were designed according to the LePV1 genome sequence,respectively,and the 27 LePV1 isolates were performed the whole genome cloning and sequencing.The results indicated that,all the full-length of dsRNA-3 is 2380 bp,but the cloned dsRNA-4 sequence existed differences due to the adenine number difference of 3’-terminal poly(A)tail,and its size was 2225 bp-2231 bp.The similarity comparison was further carried out between the 27 LePV1 isolates and one LePV1 isolate separated from L.edodes strain SX12 with abnormal production by using sequence analysis software Clustal X2.0,the results showed that,the similarity of the 28 LePV1 was very high,wherein,the nucleotide similarity of the dsRNA-3 and dsRNA-4 was 98.84% and 98.57%,and their amino acid similarity was 99.61% and 99.46%,respectively.The nucleotide variation of dsRNA-3and dsRNA-4 were all presented not uniformly in the genome,by contrast,the untranslated region was more conservative than the coding region;the variation between the 28 isolates was mainly same synonymous mutation,the incidence of nonsynonymous mutation was lower,the nonsynonymous mutation of RdRp and CP was 6.7% and 5.3%,respectively;only one amino acid difference existed between the LePV1 isolate derived from SX12 and the other 27 isolates.The phylogenetic analysis results suggested that,the 28 strains are clustered into 2 types,wherein,the most LePV1 isolates and the isolate derived from SX12,totally 24 isolates,were clustered into subtype I,and the other 4 strains(EFISAAS0351,YAASM3334,L12,and Hunong-1)were clustered into subtype II.In comparison,the genetic diversity of subtype II was more abundant than that of subtype I.In addition,the molecular clustering results of these isolates had no correlation with the geographic origin of the host.This research provides important information for controlling the occurrence of L.edodes viral disease from the source(strain),and also lay important theoretical basis for tracking the occurrence and monitoring the epidemic situation of similar viral diseases.