甘蔗内生解淀粉芽孢杆菌CGB15的分离、鉴定及生防活性

Isolation, identification, and biocontrol activity of an endophytic strain Bacillus amyloliquefaciens CGB15 from sugarcane

摘要:

真菌病害占作物病害种类的一半以上，病原真菌是目前已知种类最多的作物病原菌。从作物根际与/或体内分离筛选具有生防活性的微生物，并应用于病害的防控，是除作物品种改良与化学防治外的另一种高效的病害防控策略。【目的】本研究拟筛选并分离鉴定对重要作物病原真菌具有拮抗作用的甘蔗内生细菌，为开发生物防治作物真菌病害新策略提供理论依据。【方法】采用平板对峙法初步筛选对病原真菌具有拮抗能力的甘蔗叶片内生细菌，通过16S rRNA基因测序鉴定其种属；进一步检测候选拮抗内生细菌对甘蔗鞭孢堆黑粉菌(Sporisorium scitamineum)致病发育过程关键步骤：有性配合/菌丝生长、冬孢子萌发的抑制率，田间试验检测其对甘蔗鞭黑穗病的防治效果；检测候选拮抗内生细菌对稻梨孢菌(Pyricularia oryzae)附着胞形成、离体叶片及盆栽条件下叶片病斑形成的抑制作用。【结果】分离自甘蔗叶片的细菌菌株，编号为CGB15，经分子鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。CGB15菌株能有效抑制甘蔗鞭孢堆黑粉菌有性配合/菌丝生长，对峙培养条件下使真菌菌落呈现光滑；抑制冬孢子萌发，抑制率为(89.01±0.12)%。CGB15对甘蔗鞭黑穗病的田间相对防效达到48.65%。另一方面，CGB15发酵上清液显著抑制稻梨孢菌附着胞形成，抑制率为(93.55±1.11)%，且在离体叶片和盆栽水稻幼苗接种条件下均能抑制叶片病斑形成，推测CGB15能产生并分泌抑制稻梨孢菌细胞分化与致病性的活性物质。【结论】分离自甘蔗叶片的内生解淀粉芽孢杆菌CGB15对于甘蔗鞭黑穗病、稻瘟病的防控具有潜在应用价值。

Abstract:

More than half of the crop diseases are caused by fungal pathogens, which comprise the majority of crop pathogens. In addition to germplasm improvement via breeding and application of agrochemicals, another efficient strategy for disease prevention and control is to isolate and apply the biocontrol rhizospheric and/or endophytic microorganisms of the crop. [Objective] In this study we screened, isolated, and identified the endophytic bacteria with antagonistic effect against fungal pathogens infecting major crops from sugarcane to provide a theoretical basis for developing a new strategy for the biocontrol of crop fungal diseases. [Methods] Plate confrontation assay was used to isolate bacteria against multiple crop pathogenic fungi from sugarcane leaves. We identified the isolates based on 16S rRNA gene sequences, tested the inhibitory effect of the candidate strain on the sexual mating/filamentous growth and teliospore germination of Sporisorium scitamineum causing sugarcane smut, and then carried out field trial to explore the effect of the strain on sugarcane smut under field conditions. Furthermore, we examined the inhibitory effect of the candidate strain on the appressorium formation of Pyriculariaoryzae causing rice blast, as well as on diseased spot formation of detached leaves and rice seedlings cultivated in pots. [Results] The isolated bacterial strain CGB15 was identified as Bacillus amyloliquefaciens. CGB15 suppressed sexual mating/filamentous growth of S.scitamineum, making the fungal colony appear smooth under confrontation culture conditions. CGB15 suppressed the teliospore germination of S.scitamineum, with an inhibitory rate of (89.01±0.12)%. Under field conditions, CGB15 treatment showed a relative disease control rate of 48.65%. The supernatant of the fermented liquid of CGB15 inhibited the appressorium formation of P.oryzae, with an inhibitory rate of (93.55±1.11)%. Moreover, such supernatant suppressed the formation of diseased spots on detached leaves and rice seedlings cultivated in pots. We thus infer that CGB15 can produce and secret bioactive compounds to suppress the cell differentiation and pathogenicity of P.oryzae. [Conclusion] B.amyloliquefaciens CGB15 is an endophytic bacterial strain isolated from sugarcane leaves and has the potential to be applied in the prevention and control of sugarcane smut and rice blast.