Biocontrol Effect And The Mechanism Of Isolates On Plant Diseases Of Two Crops

Previous studies have investigated the effects of Actinomycetes biocontrol agents on soil-borne plant diseases（TYLCD）,yet the effect of actinobacterial biocontrol agent on the plant leaf viral disease remains unclear.And the study of the biocontrol of clinical Penicillium griseofulvum on phytopathogens was still lacking.In the present study,we regarded the monkshood（Aconitum carmichaeli Debx.）soil-borne disease and tomato yellow leaf curl virus disease as our model diseases to assess the biocontrol effect of our actinobacteria,pathogenesis of monkshood soil-borne disease,the biocontrol effect of the P.griseofulvum CF3 on Sclerotium rolfsii and the biocontrol effect and the mechanism of actinobacteria on our model diseases.The results showed that the biocontrol isolates could not only directly inhibit the fungal phytopathogens but also active the plant systemic resistance,as a consequence to control TYLCD.The main results were listed below:1.Study on biocontrol effect of our actinobacteria.Plate culture method and microscopy examination method were used to investigate inhibition effect of cell-free culture filtrates on growth of S.rolfsii hyphae,formation and germination of sclerotia and dillution effect on S.rolfsii hyphae.S.flavotricini and S.senoensisisolates were subsequently examined their ability to inhibit sclerotial germination in unsterile soil in Perti dish.The results showed that:each tested actinomyces could inhibit the growth of S.rolfsii hyphae,formation and germination of sclerotia.the inhibition rate of S.flavotricini 5-fold dillution cell-free culture filtrate on growth of S.rolfsii hyphae,formation and germination of sclerotia were 52.8%,99.5%and 100%.The inhibition rate of S.senoensis 10³-fold dillution cell-free culture filtrate on the sub-hyphae growth was 90.3%.Both the biocontrol agents S.flavotricini and S.senoensis could promote the sclerotia decay.When the dosage of biocontrol agent S.senoensis was 1 g per kilogram soil,68.0 percent of sclerotia lost the ability of germination and 48.0 percent of sclerotia rotted.All 5 strains have the ability of producing cell-wall degrading enzymes as S.rolfsii thallus were the unique carbon source.When cultivated for 5 days,the activity of chitinase,β-1,3-glucosidase and FP-cellulase were 4.3U,4.6U and 2.4U separately.The crude enzyme suspensions of S.flavotricini and S.pactum could dissolve the cell-wall of S.rolfsii.2.Study on pathogenesis of monkshood soil-borne disease.The microbial communities in the rhizosphere,rhizoplane soil,and endosphere of healthy and southern blight monkshood were analyzed using culture-based methods.Hyphal growth measurement and petri dish assay were performed to evaluate the effect of cell-free cultures of dominant microbes in these root-associated communities on the hyphal growth of the pathogen Sclerotium rolfsii and seedling growth of melon,respectively.The results showed that there were significant structural and functional differences in the rhizosphere,rhizoplane soil,and endosphere microbial communities between S.rolfsii-infected and healthy monkshood.The ratio of bacteria to fungi decreased in the rhizosphere and rhizoplane soil of diseased monkshood,while the number of actinomycetes significantly higher in the rhizosphere,rhizoplane soil,and endosphere of healthy monkshood.The dominant species in the infected rhizosphere,rhizoplane soil,and endosphere either promoted the growth of S.rolfsii hyphae or inhibited the growth of melon seedlings.The dominant species in the healthy rhizosphere,rhizoplane soil,and endosphere inhibited the hyphal growth of S.rolfsii and promoted the seedling growth of melon.The results suggest that plant health status and growth are controlled by the rhizosphere dominant microbes.3.Biocontrol effect of actinobacteria.Three-year plot experiments were conducted to investigate the ability of two Actinomycetes biocontrol agents（Streptomyces pactum Act12and S.rochei D74）to control soil-borne root diseases and promote plant growth in A.carmichaeli.We also examined the long-term effects after soil application of a mixed Actinomycetes preparation（spore powder）was completed.High-throughput sequencing was used to explore shifts in the structure of the rhizosphere microbiota under different treatment conditions.The biocontrol characters of two Actinomycetes were also tested.Disease severity of southern blight and root rot decreased by 75.5%⁷7.0%（P<0.05）,biomass yield of tubers increased by 62.1%⁷4.1%（P<0.05）.Significant disease-control（20.7%,P<0.05）and plant growth-promotion（20.6%,P<0.05）effects were also observed after soil application of the Actinomycetes preparation was stopped.Compared with the controls,there were marked increases in the number of beneficial microbes and decreases in the number of harmful microbes in rhizosphere soil treated with the Actinomycetes preparation.Cell-free culture filtrates of both strains（Act12 and D74）effectively inhibited the growth of fungal pathogens capable of causing southern blight（Sclerotium rolfsii）and root rot（Fusarium oxysporum）in A.carmichaeli.D74 could colonize on the surface of monkshood root.A preparation of two Actinomycetes biocontrol agents can control soil-borne root diseases and promote plant growth in A.carmichaeli by inhibiting the growth of target pathogens and altering the structure of the rhizosphere microbiota.4.Study on disease control and promotes growth of P.griseofulvum and the prediction of sclerotia-formation related gene of S.rolfsii.The effects of P.griseofulvum CF3 not only upon the hyphal growth of two pathogens（Sclerotium rolfsii and Fusarium oxysporum）,the sclerotial formation and germination of S.rolfsii but also the rhizosphere culturable microbial community.A field experiment was conducted to investigate the role of CF3 disease control and plant growth promotion on A.carmichaelii plants.Besides,genes related to sclerotial formation of S.rolfsii were predicted and tested using bioinformatics analysis,molecular biological technique and culture method.The result showed that:Treatment with CF3considerably inhibited the hyphal growth of both S.rolfsii and F.oxysporum,in addition to limiting the former’s sclerotial formation and germination.applying the CF3 agent in a powder form reduced soil-borne disease severity（decreased by 29.8%,P<0.05）_and promoted the growth of A.carmichaelii（increased by 81.5%,P<0.05）as the biocontrol strain massively colonised the rhizosphere soil.The CF3 treatment also markedly reduced the density of some known species harmful to plants while increasing that of some beneficial species in rhizosphere soil.Three genes（ArsclR,ArnsdD1,and ArnsdD2）were predicted and preliminary deemed to be related to sclerotial formation.Our result suggested that CF3 act well in disease control and plant growth promotion on A.carmichaelii plants and could suppress the sclerotia formation of S.rolfsii through down-regulating the expression of genes related to sclerotial formation.5.Study of biocontrol isolates on the control of TYLCD and the activation of plant systemic resistance.The controlling effect of Act12 on TYLCD and its growth-promoting effects on tomato plants were tested and verified through field and pot experiments.The underpinning mechanisms were explored through pot experiments combined with field experiments by measuring virus quantity,defense enzyme activities,endogenous hormone levels,reactive oxygen species accumulation,and systemic resistance-and RNA interference-related gene expression in the leaves,and analyzing microbial community structure in the rhizosphere.Two subsequent field experiments were conducted to verify the biocontrol effect of Act12.Results showed that in field experiments:（1）Act12 agent decreased the disease severity and index by 26.0%³7.9%and 20.0%⁴4.3%（P<0.05）,respectively and increased the fruit mass by 7.2%¹75.3%（P<0.05）.（2）The application of Act12 also increased the diversity of bacteria and abundance of beneficial microbes in root-zone and root-surface soils,resulting in modular differentiation of microbial co-occurrence networks.In pot experiments:（1）Applying Act12 agent was found to enhance the activities of peroxidase,polyphenoloxidase,and phenylalanine ammonia lyase,reduce the accumulation of H₂O₂ and O₂^·-,and elevate the levels of salicylic acid and jasmonic acid in tomato leaves.（2）Act12upregulated the expression of genes related to systemic acquired resistance（PR-1）,induced systemic resistance（SIPI-II）,specific TYLCD resistance（SIPer1 and SIVRSLip）,and RNA interference in tomato leaves,thus improving the TYLCD resistance.In addition,In conclusion,the S.pactum Act12 agent induced/enhanced plant resistance against TYLCD via multiple pathways and regulated microbial community structure in the rhizosphere,thereby controlling virus disease and promoting plant growth in tomato.