茶轮斑病拮抗细菌kc-16的抑菌性能及田间防效

Antimicrobial activity and field control effect of an antagonistic bacterial strain kc-16 against tea gray blight

摘要:

【背景】茶轮斑病是茶树的主要病害，目前仍以化学防治为主，而化学防治引起的农残与抗药性问题亟待解决，发掘和利用生防微生物资源是保障茶叶安全生产和提高产量的重要举措。【目的】筛选对茶轮斑病菌(Pseudopestalotiopsis theae)具有拮抗能力的芽孢杆菌并初步明确该拮抗菌的抑菌性能及其对茶树的田间防病促生效果，为茶轮斑病菌生防技术开发提供支持。【方法】利用平板对峙法筛选对茶轮斑病菌具有拮抗效果的细菌；通过形态特征、生理生化特征结合16S rRNA基因测序对筛选所得拮抗菌进行种属鉴定；利用不同检测平板分析产真菌细胞壁裂解酶的特性，通过平板对峙试验测定菌株kc-16不同类型代谢产物对茶轮斑病菌的抑制能力，同时测定菌株kc-16对其他9种常见植物病原真菌的拮抗率以确定其广谱抑菌能力，最后进行田间试验评价菌株kc-16对茶轮斑病的防治和对茶树的增产效果。【结果】筛选到一株对茶轮斑病菌有较好抑制效果的菌株kc-16，抑菌率为(79.62±0.82)%。经生理生化特征比对与16S rRNA基因序列同源性分析，将菌株kc-16鉴定为枯草芽孢杆菌沙漠亚种(Bacillus subtilis subsp. inaquosorum)；该菌具有固氮、产铁载体与溶磷能力，但不具有解钾能力；能够分泌蛋白酶、纤维素酶、β-1,3葡聚糖酶和几丁质酶。此外，其对9种病原真菌均有不同程度的抑制效果，抑制率为46.67%−81.00%。同时，其胞外抑菌物质均对茶轮斑病菌有抑制作用，菌株kc-16的无菌滤液、蛋白类代谢物、挥发性代谢产物和非挥发性代谢产物的平板抑制率分别为69.00%、58.00%、47.50%和78.71%；田间试验表明枯草芽孢杆菌沙漠亚种对茶树有良好的防病促生效果，在显著降低茶轮斑病发病率的同时，使茶叶鲜重(1芽1叶)相较空白对照提升了25.0%。【结论】枯草芽孢杆菌沙漠亚种对茶轮斑病菌有较好的抑制效果，同时对茶树也有较好的促生效果，具有潜在的应用价值。本研究结果为该菌株的后续研究和其在防治植物病害方面的应用提供了理论支撑。

关键词:茶轮斑病;枯草芽孢杆菌沙漠亚种;生物防治;促生

Abstract:

[Background] Tea gray blight is a major disease attacking tea plants. Chemical methods are mainly used to control this disease, and the chemical residues and pathogen resistance caused by chemical control need to be solved urgently. Exploring biocontrol microorganisms is an important measure to ensure the safety and increase the yield of tea production. [Objective] To screen the Bacillus strains with inhibitory effects on tea gray blight and clarify the inhibitory activity and field control effect of the strain screened out, providing support for the development of biocontrol methods for tea gray blight. [Methods] A plate confrontation experiment was conducted to screen out the strain with antagonistic effect on Pseudopestalotiopsis theae, the pathogen causing tea gray blight. The strain was identified based on morphological, physiological, and biochemical characteristics and sequence analysis of the 16S rRNA. Various plates were utilized to examine the production of enzymes lysing fungal cell walls by the strain. Plate confrontation tests were conducted to determine the inhibitory activities of different metabolites produced by the strain on Ps. theae. The inhibition rates against nine common species of plant pathogenic fungi were measured to assess the antifungal spectrum of the strain. Finally, field experiments were conducted to evaluate the effects of the strain on tea gray blight and yield of tea plants. [Results] A strain kc-16 with antagonistic effect on Ps. theae was screened out, with the inhibition rate of (79.62±0.82)%, and it was identified as Bacillus subtilis subsp. inaquosorum by morphological, physiological, and biochemical characteristics and 16S rRNA gene sequence analysis. Strain kc-16 demonstrated nitrogen-fixing, iron carriers producing and phosphorus-solubilizing abilities but lacked the potassium-solubilizing ability, and it could secrete protease, cellulase, β-1,3-glucanase, and chitinase. In addition, strain kc-16 inhibited nine species of pathogenic fungi, with inhibition rates ranging from 46.67% to 81.00%. The extracellular substances of strain kc-16 exhibited inhibitory effects on the pathogen of tea gray blight. Specifically, the cell-free filtrate, crude protein extract, volatile substances, and non-volatile substances of this strain showcased the inhibition rates of 69.00%, 58.00%, 47.50%, and 78.71%, respectively. In field experiments, B. subtilis subsp. inaquosorum showed tea gray blight-preventing and growth-promoting effects on tea plants. It reduced the incidence of tea gray blight while increasing the fresh weight of tea leaves (one bud and one leaf) by 25.0% compared with the blank control. [Conclusion] B. subtilis subsp. inaquosorum inhibits tea gray blight and promotes the growth of tea plants, demonstrating the application potential. The results of this study provide theoretical support for the future research and application of this strain in the control of plant diseases.

Key words:tea gray blight;Bacillus subtilissubsp.inaquosorum;biocontrol;growth promotion