生姜根腐病的病原菌鉴定及抗菌脂肽的防治效果

Identification of the pathogen of ginger root rot and the control effeciency of antifungal lipopeptides

[目的]本文旨在分离和鉴定生姜根腐病病原菌,研究芽胞杆菌源抗菌脂肽对病原菌的抑菌作用,并在土壤环境中测定抗菌脂肽对生姜根腐病的防治效果。[方法]采用组织分离法、形态学和分子生物学手段分离、鉴定山东潍坊生姜根腐病病原菌;测定5种芽胞杆菌源抗菌脂肽(fengycin,bacillomycin D,iturin A,brevibacillin V和brevibacillin)对病原菌的抑菌作用;通过生姜切片侵染试验验证抗菌脂肽对根腐病的防治效果。[结果]形态学和分子生物学鉴定结果表明,生姜根腐病病原菌为群结腐霉(Pythium myriotylum)。5种抗菌脂肽对群结腐霉均有抑菌效果,其中分离自侧孢短芽胞杆菌(Brevibacillus laterosporus)的脂肽brevibacillin对群结腐霉的抑制活性最强,其EC50为13.08μg·mL^-1。brevibacillin能抑制群结腐霉孢子囊和卵孢子的形成,改变其细胞膜的通透性,并导致其胞内蛋白质和核酸泄露,引起群结腐霉菌丝形态发生皱缩,从而发挥抑菌作用。生姜切片侵染试验中,26和52μg·mL^-1 brevibacillin处理后,生姜根腐病发病率分别下降了40.0%和56.7%,病情指数分别下降了41.0和58.9。[结论]芽胞杆菌源抗菌脂肽brevibacillin能有效抑制生姜根腐病的发生,在生物防治、保障食品安全和减少土壤污染方面有较好的应用前景。

[Objectives]The paper aimed to isolate and identify the pathogen from diseased ginger,and investigate the activity of different antifungal lipopeptides against the pathogen,and the prevent effect of brevibacillin on the ginger root rot.[Methods]The pathogen strain was isolated from the tissues of diseased ginger root in Weifang,Shandong Province,and identified according to the pathogenicity tests,morphological characters and rDNA-ITS analysis.Five of antifungal lipopeptides(fengycin,bacillomycin D,iturin A,brevibacillin and brevibacillin V)were used to evaluate the antifungal effects on Pythium myriotylum;the antifungal mode of the lipopeptide brevibacillin was investigated by analyzing the leakage of protein and nucleic acid,as well propidium iodide(PI)staining.The control efficiency of antifungal lipopeptide on root rot of ginger was determined by inoculating the pathogen and adding brevibacillin into the ginger slices sterilized using 0.1%NaClO solution with aseptic soil.[Results]The pathogen of ginger root rot was identified as P.myriotylum,and brevibacillin isolatated from Brevibacillus laterosporus was found the largest inhibition effect against P.myriotylum by testing five kinds of antifungal lipopeptides,and the EC50 of brevibacillin was 13.08μg·mL^-1 to P.myriotylum.Moreover,it was found that brevibacillin inhibited the formation of sporangia and oospore of this pathogen,and caused leakage of protein and nucleic acid in fungal cells,changed the permeability of its cell membrane,which made mycelial to shrink,therefore exhibited an antifungal effect.In soil environment,the incidence of root rot in ginger slices decreased by 40.0%,56.7%,and the disease index decreased by 41.0 and 58.9 after treated by brevibacillin at concentrations of 26 and 52μg·mL^-1,respectively.[Conclusions]The antifungal lipopeptide brevibacillin is of certain effect on the control of ginger root rot caused by P.myriotylum,therefore has a good application potential in biocontrol of ginger disease,food safety and reduction of soil pollution.