疮痂链霉菌拮抗菌定向筛选及其功能评价

Targeted screening and functional evaluation of the bacterial antagonistics to Streptomyces scabies

摘要:

近年来由多种致病链霉菌引起的马铃薯疮痂病在我国普遍流行，且危害程度逐年加重，严重影响块茎的品质和商品价值。病原菌土传和种传，难以防控。利用拮抗微生物抑制病菌生长是目前防控疮痂病的重要措施。【目的】从病薯田土样中定向筛选对马铃薯疮痂病具有显著防效的菌种，研究其拮抗机制，评价其环境适应性，为开发可产业化应用的高效复合功能菌剂提供理论依据。【方法】通过平板对峙及盆栽试验研究目标菌株对主要病原菌疮痂链霉菌Streptomyces scabies的抑制效果；采用形态学、生理生化实验及分子生物学方法，确定其分类地位；结合高效液相色谱质谱联用方法分析相关抑菌活性物质。【结果】获得3株对致病链霉菌S.scabies具有显著拮抗功能的菌株HZ11-4、HS-12、HZ13-1，抑菌圈直径分别为34、29、30 mm，对马铃薯微型薯疮痂病的防效分别为68.57%、57.15%和65.96%。菌体革兰氏染色呈阳性，经鉴定均为解淀粉芽孢杆菌Bacillus amylolique-faciens；3株菌皆可扩增出surfactin、iturin和fengycin等脂肽类物质合成酶相关基因片段，检测到上述脂肽类抗生素的存在，其中，仅surfactin对S.scabies有一定抑制效果，但非主要活性物质。3株菌对茄链格孢菌、尖孢镰刀菌、立枯丝核菌、大丽轮枝菌等多种植物病原菌均具有明显的抑制效果；可耐受pH 5–9、NaCl含量1%–7%的盐碱环境和100℃高温；对氟菌·霜霉威、氟硅唑、吡唑醚菌酯、春雷霉素、中生菌素和甲基硫菌灵等生产上常用的杀菌剂不敏感；具有显著促生长特性。【结论】HZ11-4、HS-12和HZ13-1具有良好的环境适应性和广谱抗病性，均可作为防控马铃薯土传病害功能菌剂的候选菌株。文章首次验证了脂肽类抗生素surfactin、iturin A和fengycin均非抑制S.scabies的主要活性物质。

关键词:疮痂链霉菌;马铃薯疮痂病;解淀粉芽孢杆菌;脂肽类抗生素

Abstract:

Potato common scab (CS) caused by pathogenic Streptomycetes has occurred widely in China and led to increasing damage to the quality and commercial value of potato. Since the pathogen is soil-borne and seed-borne, antagonistic microorganisms are regarded as an important method for the prevention and control of this disease. [Objective] We aim to screen out the antagonistic bacterial strains from the soil in the field with serious CS, reveal the mechanism of antagonism against Streptomyces scabies, and evaluate the environmental adaptability of the targeted strains. This study can provide a theoretical basis for developing applicable microbial agents. [Methods] The strains with antagonistic effects were screened out via the plate confrontation method and pot experiment and then identified based on morphological, physiological, biochemical, and molecular characteristics. Their metabolites with antifungal functions were detected via high performance liquid chromatography coupled with mass spectrometry.[Results] Three Gram-positive strains with antagonistic effects were identified as Bacillus amyloliquefaciens and designated HZ11-4, HS-12, and HZ13-1, which showed the inhibition zone diameters of 34, 29 and 30 mm to S.scabies and the control effects of 68.57%, 57.15%, and 65.96%, respectively. The genes coding for synthetases of surfactin, iturin, and fengycin were amplified from these strains, and existence of the lipopeptide antibiotics was detected by high performance liquid chromatography. S.scabies could be inhibited only by surfactin which was not the main active compound of the strains. The three strains exerted inhibitory effects on a variety of pathogens such as Alternaria solani, Fusarium oxysporum, Rhizoctonia solani, and Verticillium dahliae. They could live in a wide pH range of 5-9, tolerate 1%-7% NaCl and high temperature of 100℃, promote potato growth, and were insensitive to fluopicolide + propamocarb, flusilazole, pyraclostrobin, kasugamycin, zhongshengmycin and thiophanate-methyl. [Conclusion] Owing to the good environmental adaptability and broad-spectrum resistance, B.amyloliquefaciens HZ11-4, HS-12, and HZ13-1 can be used to develop compound microbial agents against potato soil-borne diseases. This study verifies for the first time that surfactin, iturin A, and fengycin are not the main active substances to inhibit S.scabies.

Key words:Streptomyces scabies;potato common scab;Bacillus amyloliquefaciens;lipopeptide antibiotics