Mycoviruses In Plant Pathogenic Fungi Botrytis Cinerea And Amphobotrys Ricini

Botrytis cinerea Pers.and Amphobotrys ricini(Buchw.)Hennebert are plant pathogenic fungi causing gray mold on numerous economically important crops and ornamental plants.B.cinerea infects more than 1,400 plant species,whereas A.ricini infects plants in Euphorbiaceae.This study was conducted to screen RNA mycoviruses from 119 isolates of B.cinerea and 180 isolates of A.ricini,and to characterize two novel mycoviruses,namely Botrytis cinerea RNA virus 1(BcRV1)infecting B,cinerea and Amphobotrys ricini hypovirus 1(ArHV1)infecting A.ricini.The main results were summarized below.Molecular and biological properties of BcRV 1 were characterized.The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames(ORFs),ORF1 and ORF2,coding for a hypothetical polypeptide(P1)and RNA-dependent RNA polymerase(RdRp),respectively.A-1 frameshifting region(designated the KNOT element),containing a shifty heptamer,a heptanucleotide spacer,and an H-type pseudoknot,was predicted in the junction region of ORF1 and ORF2.The-1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein(RFP)-green fluorescent protein(GFP)by the plasmid pET-28a(+)-RKG containing the construct dsRed-KNOT-eGFP in Escherichia coli.Genomic organization,P1,and RdRp of BcRV1 were similar to seven taxonomically unassigned double-stranded RNA(dsRNA)mycoviruses,including GaTV2,FgV3,FvV1,FvV2,PgV2,PiRV3,and SsNsV-L.BcRV1 was found to be closely related to grapevine-associated totivirus 2(GaTV2)and Sclerotinia sclerotiorum nonsegmented virus L(SsNsV-L).Results of phylogenetic analysis showed that BcRV1 and six of the seven above-mentioned mycoviruses,and DsRV1 formed a distinct clade,indicating that BcRV1 belongs to the unassigned dsRNA mycovirus group,which may represent a new mycovirus family.BcRV1 was found capable of being transmitted vertically through macroconidia and horizontally to vegetatively compatible or incompatible strains of B.cinerea through hyphal contact.The presence of BcRV1 was found to be positively correlated with hypovirulence in B.cinerea,with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1.The probable mechanism of hypovirulence of B.cinerea caused by BcRV1 infection may be correlated to reducing infection cushions produced by the host fungal.Acalypha australis was found to be a new host plant for A.ricini in central China.A.ricini was commonly distributed in Hubei Province.In October 2011,average disease incidence of gray mold caused by A.ricini on Acalypha australis in 14 counties in Hubei was 18.6%.In October 2012,the highest disease incidence of gray mold caused by A.ricini on Acalypha australis in Ezhou is 100%.In October 2013,the highest disease incidence of gray mold caused by A.ricini on Acalypha australis in Hanchuan is 90%.Acalypha australis was verified to be a new host of A.ricini by Koch's postulates.Surveys for A.ricini on Acalypha australis in Ezhou and Hanchuan in 2012 and 2013 showed that occurrence of A.ricini on Acalypha australis in Hubei was at high temperatures in late summer and early fall.Epidemic stages of spore formation,transmission,infection,and sclerotia formation of A.ricini on Acalypha australis occurred at average temperatures of 25 to 28 ? in 2012.Adaptability of the mycelia and the conidia of A.ricini and B.cinerea were compared.The results showed that A.ricini was more tolerant to high temperatures than B.cinerea.The optimal temperatures for mycelial growth by A.ricini and B.cinerea are 20 to 30 ? and 20 ?,individually A.ricini grows faster than B.cinerea at 30 0C.A.ricini could grow at a high temperature of 32 ?,but B.cinerea could not grow at 32 ?.The optimal temperatures for conidial germination of A.ricini(26-30 ?)are higher than those of B.cinerea(20-26 ?).Conidial germination rates of A.ricini and B.cinerea at 32? are 94%and 2.3%,respectively.Conidial germination rate of A.ricini is 5.3%at 36 ?,whereas B.cinerea failed to germinate at 36 ?.Molecular properties of ArHV1 were characterized.The genome of ArHV1 is 9,232 bp long,excluding the poly(A)tail.It contains one large ORF,coding for RdRp,on the positive chain.RdRp of ArHV1 showed high identity to mycoviruses in the genus Betahypovirus(Hypoviridae),including CHV3,CHV4,PlHV1,SsHV1 and VcHV1.There are eight conserved motifs in the RdRp domain of ArHV1.Results of phylogenetic analysis showed that ArHV1 is a novel mycovirus of Betahypovirus.ArHV1 was positively detected in all 167 isolates of A.ricini.The accumulation level of ArHV1 was variable in different isolates of A.ricini,ArHV1 was found capable of being transmitted vertically through conidia.Accumulation level of ArHV1 in mycelia of isolate WHA-1 in healthy-disease border area(young mycelia)was higher than in diseased area(old mycelia)on detached caster bean leaves.Accumulation level of ArHV1 in mycelia of isolate WHA-1 in lesions on caster bean leaves was higher than in mycelia cultured on PDA.ArHVl in isolates CopAr-5 and WHA-1 of A.ricini was detected to have the DNA form.Sequence of the DNA form and the cDNA sequence of ArHV1 were conserved in both isolates.To our knowledge,this is the first report of mycovirus in A.ricini and this is the second report of detection of DNA form of non-retroviral RNA mycovirus.These findings enriched mycovirus species in the gray mold fungi,and also expanded our knowledge about the biological features of mycoviruses.