Molecular And Biological Characterization Of A Novel Partitivirus In The Phytopathogenic Fungus Botrytis Cinerea

Gray mold is an important plant disease caused by the necrotrophic plant pathogenic fungus Botrytis cinerea,which has a wide spectrum of host range(?1400 plant species),including many economically important crops,including grape,strawberry and tomato.It causes considerable economic losses at both pre-and post-harvest stages.Resistant varieties against this pathogen are not available in most cultivated fruits and vegetables.As a consequence,control of gray mold largely depends on repeated use of fungicides.Although expected control efficacy can be achieved by optimal use of fungicides,however,use of fungicides is not environmentally friend and repeated use of fungicides can result in generation of resistant pathogen strains.Thus,it is useful to explore other safe and sustainable control measures,including mycovirus-mediated biological control.Mycoviruses and mycoviruses-mediated hypovirulence have been reported in Botrytis cinerea.However,none of the known mycoviruses has been successfully used to control B.cinerea.The limiting factors include:(i)restricted transmission of the mycoviruses from mycoviruses-containing to mycoviruses-free individuals due to hyphal incompatibility;and(ii)low competitiveness of the mycoviruses-containing strains due to poor mycelial growth.Therefore,it is necessary to screen more mycoviruses.In this study,a novel partitivirus infecting an isolate(QT5-19)of B.cinerea was identified and its biocontrol potential was evaluated.Main results achieved were summarized below:1.A hypovirulent strain(QT5-19)of B.cinerea was obtained.Strain QT5-19 produced pink pigment.It grew at 20oC vigorously on many agar media,such as potato dextrose agar(PDA),but failed to produce conidia and sclerotia on this medium.QT5-19 did not infect apple,cucumber,oilseed rape,strawberry,table grape,tobacco and tomato.A double stranded(dsRNA) mycovirus was identified in QT5-19 based on analysis of the genome feature and on observation of the virus particles.Two dsRNAs were detected in the nucleic acid extracts from the mycelia of QT5-19 and the spherical virus particles(?36 nm in diameter) isolated from the mycelia of QT5-19.DsRNA 1(1,909 bp long) and dsRNA 2(1,883 bp long) were deduced to encode two polypeptides with homology to viral RNA-dependent RNA polymerase(RdRp) and coat protein(CP),respectively.Phylogenetic analysis of RdRp and CP indicated that the two dsRNAs represent the genome of a novel partitivirus in the genus Alphapartitivirus,designated as Botrytis cinerea partitivirus 2(BcPV2).BcPV2 in QT5-19 was successfully transmitted to three virulent strains of B.cinerea(08168,B05.10,XN-1)through hyphal contact(possibly through anastomosis).The resulting BcPV2-infected derivatives 08168 T,B05.10 T and XN-1T showed rapid growth on PDA with defects in conidiogenesis and sclerogenesis,and hypovirulence on tobacco.This result suggests that BcPV2 is closely associated with hypovirulence as well as defects in production of conidia and sclerotia.2.Strain QT5-19 has high competitive saprophytic ability.Strain QT5-19 exhibited a higher competitive saprophytic ability(CSA) than strain 08168 of B.cinerea(virulent) and strain EP-1PNA367 of Sclerotinia sclerotiorum.Vigorous mycelial growth and production of antifungal volatile organic compounds(VOCs) are probably two mechanisms involved in improved CSA of QT5-19.The hyphal fragments of QT5-19 were found to be able to suppress infection of leaves of oilseed rape by virulent strains 08168(B.cinerea) and Ep-1PNA367(S.sclerotiorum).3.The VOCs of QT5-19 have a wide-spectrum of antifungal activity.Except for B.cinerea,several other fungi,including B.aclada,B.pyriformis,B.sinoallii,Rhizopus stolonifer,Sclerotinia minor,S.sclerotiorum,and Streptobotrys caulophylli were inhibited for growth by the QT5-19 volatiles.The cultures of autoclaved wheat grains(AWG) of QT5-19 were used as source of the VOCs for testing their efficacy in suppression of strawberry fruit rot caused by B.cinerea,S.sclerotiorum,Mucor hiemalis and Rhizopus stolonifer.The results showed that the QT5-19 volatiles showed high control efficacy against these fungi.GC-MS analysis identified 26 VOCs emitted from the AWG cultures.The compounds include hexanal,heptanal,1-octen-3-ol,(E)-2-octenal,(E)-2-nonenal,2-undecanol,2-decenal,benzothiazole,1-(2-hydroxy-5-methylphenyl)-ethanone,and tetradecane.All these results suggest that the hypovirulent isolate QT5-19 is a promising biocontrol agent of B.cinerea.4.The VOCs of QT5-19 showed a growth-promotion effect on tomato seedlings.We used the VOCs from the PDA cultures of QT5-19 to fumigate seedlings of tomato in closed growth containers.Compared to the tomato seedlings in the control treatment(without VOCs sources),tomato seedlings in the treatments the VOCs of QT5-19 from the PDA cultures were significantly improved for growth.The minimum duration of the treatment of the QT5-19 VOCs was one week.Expression of the genes related to biosynthesis of auxin(SlIAA9),cytokinin(SlCKX1),giberellin(SlGAox1) and ethylene(SlACO1) in tomato plants in response to the QT5-19 VOCs were investigated by quantitative PCR.The results showed that expression of SlIAA9 and SlCKX1 in leaves of tomato seedlings was significantly increased in leaves,compared to that in the tomato seedlings in the control treatment.This result suggests that growth-promotion of tomato seedlings might be caused by increased production of auxin and cytokinin.This study suggests that strain QT5-19 of B.cinerea infested with BcPV2 has a promising biocontrol potential against B.cinerea and S.sclerotiorum.The VOCs produced by QT5-19 has double roles with antifungal activity and plant growth-promotion activity.