Diversity And Biological Characteristics Of Viruses In Botrytis Cinerea

Botrytis spp.are a group of plant pathogenic fungi with a wide host range and a geographic distribution,and are also have great economic impacts for industries of horticulture and vegetables.The control of B.cinerea mostly relies on chemical fungicides.However,the evolution of fungicide resistance increases the difficulty of only using fungicides for B.cinerea management.Therefore,some alternative methods,such as biological control,were developed for the control of B.cinerea.Mycoviruses,as a biocontrol agent,have been successfully used for the control of chestnut blight in Europe.In addition,mycoviruses are also commonly reported in the population of Botrytis spp.,some were determined to be capable of attenuating virulence of Botrytis.A hypovirulent B.cinerea strain HBtom-372 was co-infected by three mycoviruses,including Botrytis cinerea endornavirus 1?BcEV1?,Botrytis cinerea hypovirus 1?BcHV1?and Botrytis cinerea fusarivirus 1?BcFV1?.The BcEV1 coding strand is 11557 nt long,possessing an open reading frame?ORF?that codes for a polyprotein of 3787 amino acid residues and lacks a site-specific nick.The polyprotein contains viral methyltransferase?MTR?domain,a cysteine-rich region?CRR?,two putative viral helicase?DEXDc-like and Hel-1?domains,and an RNA-dependent RNA polymerase₂?RdRp₂?domain.The genome of BcHV1 is 10214 nt long excluding a poly-A tail,and possesses one large ORF encoding a polyprotein with several conserved domains,including papain-like protease?Prot?domain,a UDP glucose/sterol glucosyltransferase?UGT?domain,an RdRp domain and a Hel domain.The genome of BcFV1 is 8411 nt in length excluding the poly?A?tail and theoretically processes two major ORFs,namely ORF1 and ORF2.The larger ORF,namely ORF11,encodes a polypeptide that contains protein domains of RdRp and a viral helicase,whereas the function of the small ORF,namely ORF2,remains unknown.Both BcHV1 and BcFV1 were capable of being transmitted horizontally through hyphal anastomosis from strain HBtom-372 to strain HBtom-459.The derivative isolates Z26?with BcEV1,BcHV1 and BcFV1?,Z1?with BcHV1 and BcFV1?,Z3?with BcHV1 and BcFV1?and Z33?with BcHV1?showed severely impaired virulence and decreased formation of infection cushions?ICs?.Infection by BcHV1 alone caused attenuated virulence without affecting mycelial growth,significantly inhibited IC formation,and altered expression of several IC-formation-associated genes.However,wound inoculation could fully rescue the virulence phenotype of the BcHV1-infected isolate.These results indicate the BcHV1-associated hypovirulence is caused by the viral influence on IC-formation-associated pathways.The incidence of BcEV1,BcHV1 and BcFV1 in the B.cinerea populations in China were 4.2%,29.6%and 14.3%.In order to investigate the differences between isolate Z33 and strain HBtom-459 at transcriptional level,the transcriptomes of both were analyzed with RNA_Seq technique.The differentially expressed genes?DEGs?of Z33 and HBtom-459 cultured on PDA were 853,of which 342 genes were up-regulated while 511 genes were down-regulated.After inoculation on onion bulb scale 9 h,the DEGs of Z33 and HBtom-459 were 674 DEGs,of which 388 genes were up-regulated and 286 genes were down-regulated.At the stage of BcHV1 infection and infection,293 genes were specifically up-regulated,159 genes were specifically down-regulated,a total of 452 genes were specifically expressed.These 452 genes included cell wall degrading enzymes,toxin synthesis genes,transporters,copper and zinc superoxide dismutase,transcription factors and pathogenesis related genes.According to the enrichment analysis of KEGG pathway,DEGs was mainly enriched in metabolism,secondary metabolism,carbohydrate metabolism and various amino acid metabolism pathways,thus these pathways may be related to the hypovirulence of B.cinerea associated with BcHV1.The metatranscriptomic sequencing technology was used to explore the diversity of viruses in 508 B.cinerea strains.These strains were collected from 12 provinces in China.Through the analysis of viral genome sequences,these viruses are classified into four types,including +ssRNA virus,-ssRNA virus,dsRNA virus and ssDNA virus,which represented partial or complete genome segments of 79 viruses.Phylogenetic analysis showed that these viruses may be mainly belonging to the following 17 viral families?genera?,including +ssRNA viral families: Hypoviridae,Narnaviridae,Tombusviridae,Ourmiavirus,Fusariviridae,Deltaflexiviridae,Gammaflexiviridae and Tymoviridae;-ssRNA viral families:Bunyavirales,Rhabdoviridae,Mymonaviridae and Paramyxoviridae;dsRNA viral families:Endornaviridae,Partitiviridae,Totiviridae and Botybirnavirus;ssDNA viral family Genomoviridae.In addition,there were four unclassified viruses,showing homology to Sclerotinia sclerotiorum tetramycovirus 1,Botrytis cinerea RNA virus 1,Sclerotinia sclerotiorum dsRNA mycovirus L,respectively.One-ssRNA virus,namely Botrytis cinerea mymonavirus 1?BcMyV1?was fully sequenced.The full length of BcMyV1 was 7863 nt,possessing three ORF?ORF1³?.The ORF1 encodes a large polypeptide containing a conserved mononegaviral RNA-dependent RNA polymerase?RdRp?domain showing homology to the protein L of mymonaviruses,whereas the possible functions of the remaining two ORFs are still unknown.The incidence of BcMyV1 in B.cinerea population was relatively low,accounting only 0.8%in the tested strains.Among these viruses,44 viruses have been reported,whereas the remaining 35 viruses have not been reported,indicating the present of diverse viruses in B.cinerea population.This research for the first time detected ssDNA virus in B.cinerea population,namely Botrytis cinerea genomovirus 1?BcGV1?.The genome of BcGV1 is 1710 nt long,possessing two ORFs.The larger ORF encodes a 321 aa putative replication initiation protein?Rep?,which contains seven conserved motifs: Motif I,Motif II,GRS domain,Motif III,Walker A,Walker B and Motif C.The small ORF encodes a 129 aa putative coat protein?CP?.Two ORFs were separated by two intergenic regions,the large intergenic region?LIR?contains 259 nts while the small intergenic region?SIR?contains 95 nts.LIR has an unusual nonanucleotide TAACAGTAC,which is predicted to be the initiation for viral DNA replication.Based on Rep phylogenetic tree analysis,BcGV1 was clustered in the family Genomoviridae,but not clustered with any reported genera,indicating BcGV1 may belong to a new genus.BcGV1 could be detected in 34 strains of B.cinerea,indicating that BcGV1 was widely distributed in the B.cinerea population.