Screening Of Antagonistic Strains To Clubroot Of Brassica Oleracea And The Study Of Its Mechanisms Of Disease Prevention And Growth Promotion

Clubroot of Brassica oleracea,caused by Plasmodiophora brassicae,which is a worldwide soil-borne diseases,decreasing the yield and quality of Brassica oleracea seriously,thus hindering the sustainable economic development of vegetable industry in China.However,with the excessive use of chemical pesticides,Plasmodiophora brassicae has become resistant,causing the disease to resurgence.Moreover,it has caused a series of problems such as pesticide residues in vegetables and soil in environment pollution.Therefore,it is essential to develop alternative methods,which show low toxicity and are environmentally friendly,for the efficient control of Plasmodiophora brassicae.In this study,the bacteria Pla6 and Juj3 with biocontrol potential were screened,and their biocontrol effect on cabbage clubroot were verified by pot and field experiments.To further illustrate the biocontrol mechanism of two strains,the active components were extracted using the techniques of silica gel column chromatography,high performance liquid chromatography(HPLC)and mass spectrometry(MS)etc.In addition,the expression level of induced systemic resistance related genes in Cabbage were detected by real-time quantitative PCR.The biocontrol mechanisms of Pla6 were elucidated,providing the theoretical basis for its exploitation and application in the biocontrol of cabbage clubroot.The main research results are as follows:1)Isolation,screening and identification of biocontrol bacteria.Six strains of biocontrol bacteria with antifungal activity were isolated from soil.The bacteria Pla6 and Juj3 with broad-spectrum antimicrobial activities and growth promoting effect were screened by the method of dual-cultural and seed germination bag test.The strains Pla6 and Juj3 had a close relationship with Bacillus velezensis and Alcaligenes faecalis,respectively,according to their 16S r DNA sequence.2)Greenhouse and field experiments.In the greenhouse experiment,the cabbage biomass such as fresh weight,dry weight and plant height were significantly higher than the control group,after two strains were treated with soil powder at a concentration of 2.00×10~6CFU/g.The fermentation broth concentration of 2.50×10~6CFU/m L used for root irrigation after transplanted,which could effectively prevent the club root of cabbage in the seedling stage.The photosynthetic intensity of cabbage leaves were significantly higher than the blank control,when two strains treated cabbage with 1.00×10~6CFU/m L fermentation broth concentration under the stress of Plasmodiophora brassicae.In the field experiments,both Pla6 and Juj3 showed the good biocontrol efficacy with 1.25×10~6CFU/m L fermentation broth concentration,the relative control effects were 62.19%and 51.62%,respectively,what's more,two strains not only promoted the growth of cabbage,but also improved the quality of cabbage leaves.3)Biocontrol characteristics of Pla6 and Juj3:The secondary metabolites of Pla6and Juj3 were tested qualitatively for biocontrol and antimicrobial ability.Besides,the ability of two strains to induce disease resistance on the plants were studied.The results showed the two strains were able to secrete siderophore and dissolved phophorus.Pla6 not only secreted protease,lipase,cellulase and amylase,but also was capable of synthesizing lipopeptide antibiotics.Real-time quantitative PCR showed that PR protein-related genes PR1 and PR2and ethylene signaling pathway-related genes EIN3,were significantly up-regulated in the roots and leaves of cabbage after treatment with strain Pla6.Juj3 significantly up-regulated the PR2 and EIN3 expression in the root of cabbage,while both the two strains significantly down-regulated the expression level of phenylalanine ammonia lyase gene PAL,the results indicated that both strains can induce the expression of resistant related genes in plants.4)Identification of Pla6 active substances.In this experiment,the lipopeptide and polyketone antimicrobial substances from the Pla6 fermentation broth were roughly extracted for activity detection respectively.LH-20 gel column chromatography and HPLC were used to purify the crude extracts,the structure of the purified product was determined by UV spectra,MS and NMR.The results showed that the polyketide extracts inhibited the germination of resting spores.A kind of macrolide antibiotic named macrolactin a was obtained by further extraction and purification,which has inhibition effect on Staphylococcus aureus,Bacillus thuringiensis and Xanthom onasoryzae pv.oryzae,impaired the germination and growth of resting spores.The surface of resting spores treated were wrinkled and sunken under the electron microscope.5)Pla6 secondary metabolites analysis in Genomics.The whole genome size were3,927,969 bp and the content of G+C%in genome was 47.23%.Four secondary metabolite biosynthetic gene clusters were forecast with 100%similarity to known gene clusters were predicted to be Bacillibactin?Bacilysin?Bacillaene and Macroactin,which laid a theoretical foundation for the follow-up exploitation and modification of Pla6.