水稻细菌性叶枯病病原菌鉴定、耐药性测定及生防菌防效评估

    Pathogen identification, bactericide-resistance determination, and control efficacy evaluation of the biocontrol bacteria for rice bacterial leaf wilt

    • 摘要:
      目的 由稻生黄单胞菌稻致病变种(Xanthomonas oryzae pv. oryzaeXoo)引起的水稻白叶枯病是危害水稻的世界性重要病害。近年来发现菠萝泛菌Pantoea ananatis、肠杆菌Enterobacter spp.等可引起水稻细菌性叶枯病,对水稻生产造成严重影响。本研究旨在分离鉴定近年来广东等地发现的水稻细菌性叶枯病病原菌,明确田间病原菌主要流行种,测定病原菌对常规杀菌剂的耐药性,明确病害频发难控的原因;同时分离健康水稻叶际微生物,筛选具生防效果的拮抗细菌,为病害的田间防控提供有益微生物资源及可替代的绿色防控策略。
      方法 收集广东等地水稻叶片样本,使用组织分离法分离叶际微生物,结合菌落形态、生理生化特征和看家基因序列鉴定微生物种类,通过回接试验确定致病性,验证科赫氏法则。利用梯度稀释培养法确定5种田间常用杀菌剂对病原菌的最小抑菌浓度(Minimal inhibitory concentration,MIC),评估其耐药性。利用抑菌圈法测定细菌的拮抗活性,并通过拮抗菌的预接种和同时接种明确其对病害的生防潜力。
      结果 从多地多批次水稻病叶分离的细菌类群多样,其中菠萝泛菌占比(21.95%)最大;从水稻健康叶片分离的细菌类群较单一,仅芽孢杆菌Bacillus spp.占比就高达52.46%。XooP. ananatis、假罗根肠杆菌Enterobacter pseudoroggenkampii对春雷霉素、氯溴异氰尿酸、噻唑锌和噻菌酮均产生耐药性,中生菌素对3种病原菌的广谱抑菌效果较好。施用解淀粉芽孢杆菌、枯草芽孢杆菌、贝莱斯芽孢杆菌可有效减轻病害的发生。
      结论 菠萝泛菌已成为田间水稻细菌性叶枯病的主要病原,多病原的出现和复合侵染使得对病害的防控变得更加复杂,长期大量使用化学农药造成水稻细菌性叶枯病病原菌的多重耐药性。研究水稻细菌性叶枯病新型致病菌的致病机理并开发新型高效安全的绿色病害防控措施,尤其是筛选和利用叶片可培养有益微生物,对病害有效防控至关重要。

       

      Abstract:
      Objective Bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is a globally important disease that harms rice worldwide. In recent years, Pantoea ananatis and Enterobacter spp. have been discovered to cause rice bacterial leaf wilt and have a serious impact on rice production. This study aims to isolate and identify the pathogens of rice bacterial leaf wilt in Guangdong and other places in recent years to clarify the prevalent pathogenic species in the field, and conduct conventional bactericide resistance tests on the main pathogens to clarify the reasons for the frequent occurrence and difficulty in field disease control. Meanwhile, the study isolates and screens healthy foliar microorganisms to obtain antagonistic bacteria with potential biocontrol efficacy to provide beneficial microbial resources and alternative disease prevention strategies for field prevention and control of the disease.
      Method Rice samples from Guangdong and other places were collected and the leaves were subjected to microbial isolation using tissue isolation method. The bacteria were identified in combination of colony morphology, physiological and biochemical characteristics and housekeeping gene sequences, and their pathogenicity was determined by inoculation tests to verify Koch’s Rule. Gradient dilution culture method was performed to evaluate the resistance of the pathogens to five commonly used bactericides by measuring the minimum inhibitory concentration (MIC). Antibacterial zone method was applied to determine the antagonistic activity of bacteria against the pathogens, and their biocontrol potential against the disease was clarified through pre- and simultaneous inoculation.
      Result The bacterial communities isolated from diseased rice leaves in multiple locations and batches were diverse, with P. ananatis accounting for the largest proportion (21.95%). The bacterial groups from healthy leaves were relatively limited, with Bacillus spp. accounting for 52.46%. Xoo, P. ananatis, and E. pseudoroggenkampii had developed resistance to kasugamycin, chloroisobromine cyanuric acid, thiazole zinc, and thiamethoxam, while zhongshengmycin had a good broad-spectrum antibacterial effect on the three pathogens. The application of B. amyloliquefaciens, B. subtilis, and B. velezensis could effectively reduce the occurrence of diseases.
      Conclusion P. ananatis has become the primary pathogen of rice bacterial leaf wilt in the field. The emergence and co-infection of multiple pathogens make disease prevention and control more complex. Long term and extensive use of the chemical pesticides cause multiple bactericide resistance of the pathogens. Therefore, studying the pathogenic mechanism of newly prevalent pathogens of rice bacterial leaf wilt and developing new efficient and safe measures, especially screening and using beneficial foliar culturable microorganisms, are crucial for effective disease prevention and control.

       

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