Endophytic Growth Of Sclerotinia Sclerotiorum Infected With SsHADV-1 And The Foundational Research On The Application To Control Sclerotinia Stem Rot Disease

Sclerotinia sclerotiorum,causing Sclerotinia stem rot,is one of the most devastating necrotrophic fungal pathogens with a wide host range?>400 species?.It secretes oxalic acid and plant cell wall-degrading enzymes to kill host cells and tissues.Recent researches revealed that S.sclerotiorum secretes small proteins as pathogenicity factors at the very early stage of infection.For the non-available of resistant cultivars and difficulty in application of fungicides in the field,Sclerotinia stem rot of rape has not been effectively controlled.Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1?Ss HADV-1?is the first DNA virus found in fungi and has an insect transmission vector.Its virus particles could infect hypha S.sclerotiorum directly.Previous research has proved that the hyphal fragment suspension carrying Ss HADV-1 could reduce disease severity of Sclerotinia stem rot of rape and enhance rapeseed yield significantly under field conditions.However,whether the strain carrying Ss HADV-1 could live in the rapeseed is still unknown.The key to control plant pathogen by using mycovirus is whether the survival of the strain carrying hypovirulence-associated mycoviruses in/on plants for a long time.In this research,we focused on the survival mechanism of S.sclerotiorum carrying Ss HADV-1 in rapeseed and its effects on rapeseed.Based on the findings in this research,a new technique of using Ss HADV-1 to control Sclerotinia stem rot of rape was built.The main results achieved are as follows.1.Strain DT-8 with Ss HADV-1 grew on rapeseed as an endophyte and could transfer Ss HADV-1 to Sclerotinia population.Strain DT-8 grows slowly and is extremely hypovirulent,with almost no virulence to rape.In the previous research,the viral DNA could be detected on the silique of rapeseed 7 weeks after spaying strain DT-8 hyphal fragment suspension on the rapeseed at the bolting stage.These characteristics made strain DT-8 an ideal carrier of Ss HADV-1 to control Sclerotinia stem rot of rape.After inoculated on the leaves of rapes for 7 d without causing any symptoms,strain DT-8 was found to spread on the surface of the leaves about 1 cm away from strain DT-8 mycelial agar by fluorescence microscopy.Meanwhile,strain DT-8 also formed small compound appressoria to invade into leaf tissue to get nutrient.It is shown that strain DT-8 could grow long-term on the surface of rape leaves.The surface-sterilized rape seeds were dip-inoculated with the hyphal fragment suspension of strain DT-8,after germination the seedlings were planted in pots containing autoclaved garden soil and tissue culture bottles containing MS medium grew for 30 days and 20 days.Then,strain DT-8 could be detected on the different sections of rape.Those results suggest that DT-8 could grow in different sections without causing any symptoms.Through fluorescence observation and electron microscopy,we observed the hypha of strain DT-8^RFP?labeled with the m Cherry?crossed through root cells and spread on the intercellular space;meanwhile,strain DT-8^RFP spread with vascular bundles on the hypocotyl.This result indicates that strain DT-8 could grow on rape as an endophyte.We planted rapes treated with strain DT-8 in bottles with MS agar media for 6 months,due to the shortage of nutrient,plants became very weak.Strain DT-8 could break through the limit from some weak plants?8/32 and 6/23?.By the colony morphology observation,viral DNA extraction and PCR amplification,we found that some strains?3/8 and 4/6?isolated from the dead plants were still carrying Ss HADV-1.These results suggest that endophytically growing S.sclerotiorum had a chance to come out from the dead plants in a sterile environment which is important for the spread of Ss HADV-1.After inoculated on leaves of plants treated with strain DT-8 for 14 days,strain 1980-hyg were re-isolated from the lesions and grown on hygromycin-amending PDA plates,and 13 cultures were obtained.Among them,five were successfully determined to carry viral DNA through PCR amplification.The growth of the cultures with the virus was significantly slower than that of the strain 1980-hyg;the colony was fluffy with rich aerial mycelia and the virulence was attenuated significantly.These results suggest that endophytically growing strain DT-8 in rapeseed could transfer the virus to its vegetative incompatible virulent strain.Meanwhile,the application of DT-8 hyphal fragment suspension in the field also could transfer the virus to the wild-type strain of S.sclerotiorum,but the efficiency was lower than 15%.2.Ss HADV-1 changed the expression of virulence-related genes significantly in S.sclerotiorum.To understand the potential mechanism of the endophytic growth,the gene expression in strain DT-8 and a virus-free strain DT-8VF either on rapeseed leaves or on PDA medium was analyzed through RNA-seq.By KEGG pathway enrichment and Fungi Fun,we found the most up-regulated differentially expressed genes?DEGs?in strain DT-8 on rapeseed leaves and PDA were enriched on the pathways related to DNA replication and repair which are possibly involved in the virus replication;most down-regulated genes were involved in metabolic pathways and starch and sucrose metabolism pathway.We made a detailed analysis of the DEGs in strain DT-8 inoculated on rape leaves at a different stage?12 hpi,18 hpi,24 hpi?,and found that genes encoding plant cell wall degrading enzymes and virulence-related secretary proteins were down-regulated,and genes related to compound appressoria and oxalic acid synthesis had not been found to change significantly.Those differential expressions may be the reason of strain DT-8 living in rape as an endophyte.3.Strain DT-8 promoted plant growth and enhanced plant resistance.To probe whether the endophytic growth of strain DT-8 could be a benefit for the health of rapeseed,strain DT-8-inoculated rapeseed plants were allowed to grow in pots with sterilized garden soil for 36 days.We found that the average fresh weight of the aerial part of treated rapeseed was 17.15±2.0 g/plant,which was significantly higher than that of the non-inoculated plants?14.55±1.1 g/plant?.A similar result was obtained when rapeseed growing in bottles with MS agar media for 21 days.Thus,strain DT-8could promote rapeseed growth significantly.After growing in pots with sterilized garden soil for 36 days,the leaves of rapeseed were inoculated with strain 1980,the diameter of average lesion size on leaves of inoculated plants was 1.22±0.08 mm at48 hpi,which was significantly smaller than that on non-inoculated plants?1.51±0.12mm at 48 hpi?.Inoculated with strain B05.10 of Botrytis cinerea,strong resistance was also observed in the treated plants?1.54±0.24 cm on inoculated plants and 1.99±0.10 cm on non-inoculated plants?.4.Strain DT-8 regulated the resistance and growth related genes.To understand the potential mechanism that strain DT-8 promotes plant growth and enhances resistance,we sampled the shoots of strain DT-8-inoculated plants growing in pots with sterilized soil for 36 days for RNA-Seq analysis.Compared to the non-inoculated plants,strain DT-8 slightly changed the gene expression of rapeseed.There were about 348 DEGs,accounting for 3.4‰of all 101041 genes;among which,258 genes were up-regulated and 90 genes were down-regulated.The most up-regulated DEGs were related to resistance,including plant pathogen signaling and resistance signaling transduction related genes,such as CDPK,CML,WRKY33,MKK9.The resistance related genes regulated by JA and ET and JA and ET synthesis related genes,such as AOC,ACS,ERF etc were also up-regulated.The most down-regulated DEGs were involved in circadian rhythm mediated by CCA1 and LHY.Previous research had shown that repression of circadian rhythm mediated by CCA1 and LHY led to vigour growth and increasing biomass of plants.Those results showed that the resistance promotion of rapes may come from the induced expression of resistance related genes by strain DT-8,and the biomass increasing may come from the repression of circadian rhythm mediated by CCA1 and LHY.5.Application of strain DT-8 in the field could promote rapeseed yield.Since strain DT-8 could grow long-term on the surface of rape leaves,we applied strain DT-8 hyphal fragment suspension to control Sclerotinia stem rot of rape through plot experiments and field experiments in Wuhan,Ezhou,Suizhou,Xiangyang of Hubei Province from 2013 to 2015.In plot experiment,DT-8 hyphal fragment suspension was diluted to 1×10⁵ cfu/m L and 1×10⁴ cfu/m L,and was applied by spraying with a hand sprayer onto the aerial parts of rapeseed at seedling stage,bolting stage,blossom stage or silique stage.In field experiments,DT-8 hyphal fragment suspension??1×104 cfu/m L?was applied at the blossom stage.During harvest season,the stem rot severity,seed yield and seed oil content of each treatment were calculated.The field results showed that all the treatments could suppress the stem rot severity by about50%,improve the seed yield significantly by 10%?20%,and improve oil content of seed by up to 3.8%.Those results suggested that strain DT-8 could be applied as plant vaccine in the field to control Sclerotinia stem rot of rape and promote the yield.6.The new DNA mycovirus found in Botrytis cinerea is associated with hypovirulence.Bc HADV-1 has an isometric virion with a size of 34 nm,its genome consists of four circular ss DNA components with similar sizes around 1700 nt.The four components share a 300 nt common region and a stem-loop with unique nonanucleotide"TAAAATTTT"with a small stem-loop.Phylogenetic analysis indicated t horizontal gene transfer between Bc HADV-1-like viruses or its ancestors and Botrytis spp.occurred after the speciation of genus Botrytis.Bc HADV-1 CP is not phylogenetically related to any CP of known viruses.Hence,Bc HADV-1represents a novel CRESS DNA virus which is phylogenetically related to Genomoviridae.Meanwhile,Bc HADV-1 is associated with hypovirulence of B.cinerea.In this research we found that strain DT-8 could live on rapes as an endophyte and promote the resistance and growth of rapeseed for the first time.Moreover,the endophytically growing strain DT-8 could be a new type of transmission vector for Ss HADV-1.Those findings reveal a new evolution origin of endophytes and a new way of endophyte mediating plant growth,explore new genes resource related to plant resistance and growth,and find a new orientation to search spread way of hypovirulence-associated mycoviruses.Furthermore,based on the tripartite symbiosis system of Ss HADV-1,S.sclerotiorum and rape,we used strain DT-8 as plant vaccine applied in the field to control Sclerotinia stem rot of rape and promote yield successfully.Our research provides a new clue for controlling plant disease and promoting agricultural production.Meanwhile,the discovery of the new DNA mycovirus Bc HADV-1 in Botrytis cinerea revealed that this new biocontrol model may be also available for control of grey mold.There may be abundant resources of hypovirulence-associated mycovirus in Genomoviridae,that are worth to explore to be applied in biocontrol of plant fungal diseases.