The Etiology And Integrated Control Of White Rot Of American Ginseng Caused By Sclerotinia Nivalis

American ginseng(Panax quinquefolius L.),a member of the Araliaceae family,is a perennial herbaceous plant.It is an important ingredient of traditional Chinese medicines and high value-added economic crop with a variety of effects such as anti-cancer,improving immunity,anti-fatigue and anti-Aging.Sclerotinia rot caused by Sclerotinia spp.seriously threatens the yield and quality of American ginseng.In 2017-2020,Sclerotinia rot occurred in Liuba and Taibai Counties in the Qinba Mountains and led to huge economic losses with the incidence of the most severely affected fields up to 60%.Therefore,Sclerotinia rot of American ginseng was took as a research object.Disease investigation,pathogen isolation and identification,host range determination,whole genome sequencing analysis,and comprehensive disease prevention and control strategy were carried out to lay a theoretical foundation for the in-depth research and disease control of Sclerotinia rot of American ginseng.The following main results were achieved:1.Sclerotinia nivalis,the pathogen causing Sclerotinia rot of American ginseng,was reported for the first time in the world.The pathogen belongs to Ascomycotina,Discomycetes,Helotiales,Sclerotiniaceae,Sclerotinia.It could infect 43 common plants of10 families including Leguminous,Compositae,Solanaceae,Cucurbitaceae,Cruciferae,Umbelliferae,Rosaceae,Liliaceae,Chenopodiaceae and Orchidaceae.The leaves of diseased plants were unevenly chlorotic in the early stages of disease development and then the whole plant wilted and died within a few days.The diseased roots were yellow-brownish with a water-soaked appearance of softened/decaying internal tissues.White fluffy mycelia grew on the surface of infected roots,and then entangled into clusters to form black rat feces-like irregular sclerotia.2.The influencing factors of mycelial growth,sclerotia production and germination,and apothecium production of S.nivalis were clarified.The optimum temperature,p H,medium,carbon source and nitrogen source for mycelial growth was 20°C,6.0,PDA,maltose and ammonium sulfate,respectively;the optimum temperature,p H,medium,carbon source and nitrogen source for sclerotia production was respectively 15°C,6.0,PDA,polysaccharides(starch and cellulose)and ammonium sulfate;the darkness was more conducive to sclerotia production.Immature sclerotia was easier to germinate than mature sclerotia;the optimal temperature,p H and soil moisture for sclerotia germination was20-25°C,3.0-4.0,and 20-45%respectively;low temperature(4°C)induction for 6-8 weeks was necessary for apothecium production with a low production rate less than 10%;sclerotia maturity and UV treatment had no significant effect on apothecium formation.3.Genome sequencing,functional annotation and comparative genomics analysis for S.nivalis were completed.For the second-generation sequencing data,the total number of reads was 45,453,768 bp.For the third-generation sequencing data,the total number of sequencing data was 2,000,001,716 bp,with reads N50 and N90 16,725 bp and 4,175 bp respectively.The length of the assembled genomic DNA was 50,369,070 bp with the genome integrity of 99.6%.It was annotated with 14,779 genes,14,166 protein-coding genes,40,196 exons,and 26,030 introns.The sequence of S.nivalis SS-TB strain were compared with the KOG protein database,PHI database,anti SMASH database and CAZy database respectively,3989 proteins,3105 genes,25 gene clusters and 6 types of 481 enzymes were successfully annotated.Comparative genomic analysis showed that there are 40244 proteins from five strains including S.nivalis,S.trifoliorum,S.sclerotiorum,Botrytis cinerea and S.borealis involved in clustering with 7748 common protein clusters,and 37,3,63,255 and 13unique protein clusters respectively.Phylogenetic analysis based on single copy orthologous protein showed S.nivalis had a highest similarity with S.sclerotiorum,S.trifoliorum and S.cepivorum.4.Comprehensive prevention and control strategy for Sclerotinia rot of American ginseng were established.The sclerotia were completely dead under 85°C in 5 min,80°C in10 min,75°C in 10 min,70°C in 30 min,65°C in 120 min and 60°C in 180 min.The sclerotia completely lost vitality under sioil temperature treatments of 30-35°C for 5 weeks and 40-45°C for 4 weeks.Long-term soak in water was unfavorable to the survival of sclerotia.The survival rate of sclerotia significantly decreased since soaking in water for 30d,and the sclerotia completely lostity vital after soaking for 47d.P100(700 w)10 min or P80(560 w)microwave treatment for 15-20 min could inactivate all sclerotia at the soil depth of0-15 cm.40%dimetachlone and 15%procymidone had the best inhibitory effects on the mycelial growth of S.nivalis,with EC₅₀of 3.779?g/m L and 2.051?g/m L,respectively,and the two agents had a strong inhibitory effect on sclerotia germination.The sclerotia were all inactivated after treatment with soil fumigants of Mianlong or Junqingxianjing of 30 kg/acre for 15 days,and methamphetamine,horseradishin or lime nitrogen of 30 kg/acre for 30 days.Therefore,the comprehensive prevention and control strategy for Sclerotinia rot of American ginseng are:1)Seed sterilization:800-1000 times dilution of 40%dimetachlone should be used to treat the seeds of American ginseng;2)Soil sterilization:covering the soil with plastic film in the summer to ensure that the soil temperature exceeds 40°C,and treating the soil through high temperature airtight for 4 weeks;or when the soil temperature is above12°C and humidity above 40%,sprinkling the soil fumigant of Mianlong evenly on the soil at an amount of 30 kg/acre,mixing with a rotary tiller,and then mulching and fumigating for15 days;3)Adding microbial agents and decomposed organic fertilizers to the soil to increase soil microbial diversity and fertility;4)Using 800-1000 times solution of 40%dimetachlone to spray the American ginseng;5)In winter,cleaning up the ground residues of American ginseng in time to reduce or eliminate the overwintering fungus source.