Exploration Of A Rapeseed Bio-priming Technique With The Hypovirulent Strain Sclerotinia Sclerotiorum DT-8 And Its Mechanism Of Resistance Promoting

Sclerotinia stem rot?SSR?caused by Sclerotinia sclerotiorum?Lib.?de Bary has astrong impact on the yield and seed quality of rapeseed?Brassica napus?,one of the major edible plant oil crops in the world.The main methods to control the disease include chemical fungicides and biocontrol and.Hypovirulent strain DT-8 of S.sclerotiorum,infected with Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1?Ss HADV-1?,is potential to control SSR by spraying the hyphal fragment suspension in the field.However,the short shelf life is the bottleneck of the application.In this study,for the first time,mycovirus-mediated hypovirulent strain was applied to bio-prime the rapeseeds,and the control effect on SSR and the mechanism were explored.Through the response surface methodology based on the Box-Behnken design,wedetermined the optimal conditions of rapeseeds bio-priming with S.sclerotiorum DT-8.The ratio of seeds of oilseed rape and the hyphal fragments suspension of DT-8 was 1:2?w/v?.The temperature for seed priming was 20°C and the duration was 18 h.After bio-priming treatment,the germinating ability of rapeseeds was improved and emerging ability of rapeseeds was unaffected.After storage for 9 wk,the carrying rate of Ss HADV-1 of 14-day-old rapeseed seedlings was more than 50%.Thus,the short shelf life of S.sclerotiorum DT-8 application was extended by rapeseeds bio-priming.The detection of SsHADV-1 on rapeseed seedlings suggested that S.sclerotiorumstrain DT-8 could live on rapeseed after rapeseeds bio-priming.To prove it further,the rapeseeds were bio-primed with the S.sclerotiorum strain DT-8^RFP which was labeled with m Cherry fluorophore and had the hygromycin phosphotransferase gene.Fungal hyphae of S.sclerotiorum DT-8^RFP could be observed both on the surface and interior of rootlet by confocal microscopy.Through re-isolating,S.sclerotiorum DT-8^RFPisolates could be recovered from the cotyledon and hypocotyl of bio-primed rapeseed.Moreover,the fungal hyphae of S.sclerotiorum DT-8^RFPalso could be observed in the rapeseed hypocotyl which was inoculated with strain DT-8^RFP by confocal microscopy.The successful colonization of S.sclerotiorum DT-8 created possibilities for using rapeseeds bio-priming with S.sclerotiorum DT-8 to control SSR.Bio-priming with S.sclerotiorum strain DT-8 could effectively control SSR ofrapeseed in the greenhouse.The field experiments were carried out in Huazhong Agricultural University,Wuhan City,and Dongshan Village,Ezhou City,Hubei Province,PR China during 2016-2018.The bio-priming treatment could significantly decrease the occurrence of SSR by 19.83%-31.12%and increase the yield by 13.52%-27.76%.The effects were similar to the chemical control prochloraz.Therefore,it is another successful case of using seed bio-priming to control airborne diseases.The symbiotic mechanism between S.sclerotiorum DT-8 and rapeseed was resolved by dual-UMI RNA-seq.Compared with the S.sclerotiorum strain DT-8 growing on PDA medium,there were 1022 up-regulated and 1069 down-regulated S.sclerotiorum genes when S.sclerotiorum strain DT-8 colonized in rapeseed.Compared with the non-bioprimed rapeseed,there were 1816 up-regulated and 72 down-regulated rapeseed genes when S.sclerotiorum DT-8 colonized in rapeseed.Through the GO enrichment analyses,KEGG enrichment analyses,the interaction pattern between S.sclerotiorum and rapeseed were explored.On the one hand,with the help of plant cell wall-degrading enzymes,oxalic acid,secretory proteins and many activated detoxification mechanism,S.sclerotiorum DT-8 could colonize in rapeseed,and with the action of amylase and sugar transporters,S.sclerotiorum DT-8 could use starch as the nutrient.On the other hand,presumably due to the decrease of the growth and steroid biosynthesis,S.sclerotiorum DT-8 lost its virulence.For rapeseed,the up-regulated expression of pectinesterase genes and pectate lyase genes promoted the colonization of S.sclerotiorum DT-8.At the same time,the activated defense response of rapeseed could not only increase the disease resistance,but also regulate the growth of S.sclerotiorum DT-8.By 16S rRNA gene and ITS sequencing techniques,the microbiome of SSR lesionsof the bio-primed and non-bioprimed rapeseed stem was analyzed.Based on the diversity and composition analysis of bacterial and fungal communities,we found bio-priming and SSR could impact the composition and structure of microbial communities,with SSR the main influence factor.Bio-priming could also affect the composition of core microorganisms and key microorganisms,and decrease the abundance of many possible plant pathogens,especially the genus Sclerotinia.Through analysis of microbial interaction network,bio-priming was found to improve the connectivity and robustness of network at the genus level,which may be one of the key reasons for rapeseed bio-priming with S.sclerotiorum DT-8 to suppress SSR and a new bio-control mechanism.In this study,a novel and effective bio-control method was created to control SSR,namely,rapeseeds bio-priming with S.sclerotiorum DT-8,and found a new orientation for using hypovirulence-associated mycoviruses was established to control plant diseases.Furthermore,the interaction mechanism between S.sclerotiorum DT-8 and rapeseed was explored,and a new bio-control mechanism from the point of view of microbe interactions was discovered.Those findings provide a new clue for controlling plant diseases by hypovirulence-associated mycoviruses.