Mycoviruses In Botrytis Cinerea Strain IBc-230 And The Interaction With Their Host

Botrytis cinerea,a necrotrophic pathogenic fungus,has a wide host range and causes serious damage to crops.B.cinerea has developed strong resistance to many fungicides that brings high cost of chemical control,thus,it is important to explore efficient and environmentally friendly control strategies.Hypovirulence associated mycoviruses are important agents that can be used to control fungal diseases.In this study,mycoviruses in the hypovirulent strain IBc-230 of B.cinerea from Isreal were identified and the interaction between mycoviruses and B.cinerea was investigated by using RNA-Seq.Strain IBc-230 was co-infected by ten viruses(nine are +ss RNA viruses and one is ds RNA virus)and a satellite RNA.The +ss RNA viruses were two tombusviruses(Bc ULV1/IBc-230 and Bc ULV2,Bc ULV2 was a novel virus of family Tombusviridae),one hypovirus(Bc HV1/IBc-230)and its satellite RNA,four mitoviruses(Bc MV1/IBc-230,Bc MV3/IBc-230,Bc MV9/IBc-230 and Ss MV4/IBc-230),one botourmiavirus(PVLAOLV80/IBc-230)and one deltaflexivirus(Ss DFV2/IBc-230).The ds RNA virus was a novel virus of family Curvulaviridae,tentatively named Botrytis cinerea partitivirus-like virus 1(Bc PLV1).The full-length sequence of the Bc PLV1 was cloned by RACE.RNA1 is 2026 bp,containing one ORF encoding a protein of 606 aa with a conserved Rd Rp domain;RNA2 is 1618 bp,containing two ORFs encoding two proteins of 306 aa and 122 aa,respectively.The first 22 nucleotides of the 5’ termini of the two RNA strands are identical.Bc PLV1 is closely phylogenetically related to Cryphonectria parasitica bipartite mycovirus 1(Cp BV1),and their Rd Rp identity is 59.83%.Phylogenetic analysis of Rd Rp revealed that Bc PLV1 belonged to the family Curvulaviridae.The viral particle of Bc PLV1 was spherical and about 50 nm in diameter.Bc PLV1 was not easily transmitted vertically through conidium.The presence of Bc PLV1 was also detected in other strains,such as strain IBc-207.Mycoviruses,including Bc ULV1/IBc-230,Bc ULV2,Bc MV3/IBc-230,Bc MV9/IBc-230,Bc HV1/IBc-230,PVLAOLV80/IBc-230 and Ss DFV2/IBc-230,can transfect B.cinerea strain B05.10 by using ds RNA or total RNA extracted from strain IBc-230.Bc ULV1/IBc-230,Bc ULV2,Bc MV3/IBc-230 and PVLAOLV80/IBc-230 had no significant impact on the growth of strain B05.10,but they can affect sclerotial formation and maturation of their host,while,Bc ULV1/IBc-230 can reduce the virulence of strain B05.10.An abnormal strain IBc-230-26 which had an abnormal morphology,slow growth rate and weak virulence was obtained using single-condium-isolation from strain IBc-230.It carried nine viruses but lost the ds RNA virus,Bc PLV1.q RT-PCR test showed that the accumulations of viruses Bc ULV1/IBc-230,Bc MV1/IBc-230,Bc MV3/IBc-230,Ss MV4/IBc-230,Bc MV9/IBc-230 and Bc HV1/IBc-230 in strain IBc-230-26 were significantly higher than these in strain IBc-230.From strain IBc-230-26,a single-conidium-isolation strain,IBc-230-26-16,was obtained.Strain IBc-230-26-16 lost viruses Bc ULV1/IBc-230,Bc ULV2,Ss MV4/IBc-230,Ss DFV2/IBc-230 and PVLAOLV80/IBc-230.Unlike,strain IBc-230 and IBc-230-26,strain IBc-230-26-16 grew faster than strain IBc-230-26 and could produce sclerotia,and there was no significant difference in virulence between strain IBc-230 and IBc-230-26-16.The mycelial suspension of the strain IBc-230,IBc-230-26 and IBc-230-26-16 were inoculated on tomato leaves,and the mycelia on the leaves were collected at 0hpi,12 hpi,24 hpi and 36 hpi and the RNA samples were extracted and used to perform RNA-Seq analysis.Compared with strain IBc-230,higher accumulation of four mitoviruses,Bc ULV1/IBc-230 and Bc HV1/IBc-230 leaded to 1705 genes up-regulated and 1500 genes down-regulated in strain IBc-230-26 during infection stage of B.cinerea,among them,298 genes were up-regulated and 164 were down-regulated at all time points.Compared with strain IBc-230-26-16,strain IBc-230-26 carries more virus species caused 2153 genes were up-regulated and 2027 were down-regulated in strain IBc-230-26,among which 499 genes were up-regulated and 453 genes were down-regulated at all time points.Through GO annotation analysis,it was found that the up-regulated genes in strain IBc-230-26 were mainly related to catalytic activity and binding-related functions,and the down-regulated genes were mainly related to transport activity;KEGG enrichment showed that the viruses caused changes in host starch(glycogen)and sucrose metabolic pathways,stimulated the biosynthesis of secondary metabolites and inhibited some amino acids involved in protein synthesis.To understand the effect of viruses on the growth,conidiation and pathogenicity of B.cinerea,genes for these processes which have been identified in B.cinerea B05.10 or T4 strain were selected to analyze the gene expression levels in strain IBc-230,IBc-230-26 and IBc-230-26-16.Transcriptome data showed that the increase of viral accumulation in the strain IBc-230-26 mainly down-regulated the expression of many genes related to growth and development,conidiation and cell wall degrading enzymes.The expression patterns of most secreted proteins and secondary metabolite synthesis related genes were also changed.These results suggest that high accumulation of viruses in host may lead to global change of host’s biological characteristics,including pathogenicity,growth and conidiation.In conclusion,ten mycoviruses,including two novel mycoviruses,were detected in strain IBc-230;strains IBc-230-26 and IBc-230-26-16 were obtained by using single-conidium-isolation and their transcriptomes were analyzed by using RNA＿Seq.Our findings may enhance the understanding of diversity and ecology of mycoviruses,may shed light on understanding the interaction between multiple mycoviruses and fungi and from there it may deepen the understanding of the growth,conidiation and pathogenic mechanism of B.cinerea on molecular level,and may provide potential mycoviruses to control grey mold.