拮抗植物病原真菌的纤维素/木质素降解菌株的筛选及鉴定

Screening and identification of cellulose/lignin-degrading strains against plant pathogenic fungi

摘要:

【背景】秸秆还田在改善土壤肥力和丰富营养等方面有重要作用，但是也存在秸秆难以快速降解利用和病原真菌病害威胁的问题。【目的】为解决还田秸秆的降解和病原真菌病害问题，从长期秸秆还田地区采集样品，从中筛选出具有降解秸秆和抑菌功能的菌株。【方法】采用稀释分离法、苯胺蓝染色法和刚果红染色法等对秸秆高效降解菌株进行筛选，通过16S rRNA基因测序及构建系统发育树进行菌株鉴定。采用对峙培养法测定筛选到的秸秆降解菌株对玉米大斑病菌(Setostphaeria turcica)、梨黑斑病菌(Alternaria kikuchiana)、马铃薯早疫病菌(Alternaria solani)、链格孢属真菌(Alternaria alternata) ACCC38230和ACCC38231等5种供试植物病原真菌的抑制作用。以玉米大斑病菌(Setostphaeria turcica)为后期供试植物病原真菌，测定拮抗菌株代谢产物的抑菌能力；通过观察拮抗菌株粗提液对玉米大斑病菌分生孢子萌发和菌丝生长的影响，从而测定菌株对病原真菌的抑制作用。【结果】从秸秆还田土壤中分离筛选获得3株高效降解纤维素及木质素菌株并命名为JY122、ZY133和JY215。通过形态学观察和16S rRNA基因测序鉴定上述菌株全部为芽孢杆菌属(Bacillus)。通过系统发育树分析结果表明，JY122与蜡样芽孢杆菌(Bacillus cereus)相似性为99.4%，ZY133与枯草芽孢杆菌(Bacillus subtilis)相似性为100%，JY215与贝莱斯芽孢杆菌(Bacillus velezensis)相似性为99.1%。平板对峙实验结果显示，JY122、ZY133和JY215菌株对不同种属的植物病原真菌均有较强的抑制作用，抑制率高达43.74%-67.54%。此外，上述芽孢杆菌代谢产物具有抑菌活性及很强的热稳定性，经95℃处理后依然具有良好的抑菌效果。【结论】筛选获得的JY122、JY133和JY215菌株具有高效降解纤维素/木质素能力，抑制多种植物病原真菌生长，代谢产物抑菌能力强且热稳定性高。这为玉米秸秆还田提供菌株资源，也为进一步解决秸秆还田难点提供了新方法和思路。

Abstract:

[Background] While improving soil fertility and nutrients, straw returning faces the challenge of slow straw degradation and threat of pathogenic fungi. [Objective] To screen efficient straw-degrading strains against the fungi from samples in areas with long-term straw returning. [Methods] The isolation by dilution in liquid, aniline blue staining, and Congo red staining were used to screen the efficient straw-degrading strains and the strains were identified based on 16S rRNA gene sequencing and phylogeny analysis. The inhibition of the screened straw-degrading strains on Setostphaeria turcica, Alternaria kikuchiana, Alternaria solani, and Alternaria alternate aCCC38230 and aCCC38231 was examined by the confrontation assay. Moreover, the ability of the metabolites of the antagonistic strains against S. turcica was determined, and the influence of the strains on conidial germination and hypha growth of S. turcica was detected with crude extract of antagonistic strains. [Results] Three efficient cellulose/lignin-degrading strains were screened out and named as JY122, ZY133, and JY215, which all belong to Bacillus. Phylogeny analysis revealed 99.4% similarity between JY122 and Bacillus cereus, 100% similarity between ZY133 and Bacillus subtilis, and 99.1% similarity between JY215 and Bacillus velezensis. The three had strong inhibitory effect on pathogenic fungi of different species with the inhibition rate in the range of 43.74%-67.54%. In addition, the metabolites of them showed antifungal activity and strong thermal stability, as the antifungal activity was still high after treatment at 95 ℃. [Conclusion] JY122, ZY133, and JY215 show high efficiency in degrading cellulose/lignin and inhibit the growth of a variety of plant pathogenic fungi. The metabolites of the three have strong antifungal ability and high thermal stability. This study provides strain resources for returning maize straw and a method for tackling the challenges in straw returning.