烟草疫霉生防放线菌的分离鉴定、功能探究及培养条件优化

Isolation, identification, functional investigation, and culture condition optimization of biocontrol actinomycetes against Phytophthora nicotianae

摘要:

【背景】烟草疫霉(Phytophthora nicotianae)是引起烟草黑胫病的重要病原菌，严重影响烟草种植业的生产和经济效益。采用病原菌的拮抗微生物进行生物防治，是一种控制植物病害的有效途径。【目的】从不同作物的根际土壤环境中分离具有拮抗烟草疫霉活性的放线菌，评价其生物防治效果和促生能力，并对其培养条件进行优化。【方法】采取稀释涂布平板法从作物根际土壤样本中分离放线菌，结合形态学分析、生理生化检测和16S rRNA基因序列比对分析对菌株进行鉴定；通过菌丝生长抑制、离体叶片和室内盆栽等试验进一步探索放线菌对烟草疫霉的拮抗作用。使用铬天青(chrome azurol sulphonate, CAS)平板检测法、产蛋白酶和烟草种子萌发等试验对菌株进行促生能力的测定；利用单因素试验优化发酵工艺。【结果】分离得到一株既对烟草疫霉产生拮抗作用又具有促生作用的菌株FK-15，经菌落形态和碱基序列比对分析，鉴定菌株FK-15为苍黄拟无枝酸菌(Amycolatopsis lurida)，对烟草疫霉的抑制率达到80.37%，室内盆栽防治效果为61.55%。同时，有嗜铁和产蛋白酶能力，利用该菌株制备的发酵液处理烟草种子后，胚根长度增加53.12%，胚芽长度增加51.20%，活力指数增加73.09%，促生能力显著提升；培养条件优化后菌株生长量增加132.48%，抑菌率增加198.80%。【结论】本研究从不同土壤环境中分离出一株具有强拮抗烟草疫霉能力的促生放线菌FK-15。进一步优化其培养条件，有望在实际应用中发挥作用。

Abstract:

[Background] Phytophthora nicotianae is a key pathogen causing tobacco black shank, severely impacting the cultivation and economic benefits of tobacco. Using antagonistic microorganisms for biocontrol is an effective approach of managing plant diseases. [Objective] To isolate actinomycetes with antagonistic activity against P. nicotianae from the rhizosphere soil of various crops, evaluate their biocontrol and plant growth-promoting effects, and optimize their culture conditions. [Methods] Actinomycetes were isolated from crop rhizosphere soil samples by the dilution-plate method and identified by morphological observation, physiological and biochemical tests, and 16S rRNA gene sequence comparison. The antagonistic effects of the isolates against P. nicotianae were evaluated by the mycelial growth inhibition assay, detached leaf assay, and indoor pot experiments. The plant growth-promoting effects of the strains were assessed by CAS plate culture, protease production, and tobacco seed germination tests. The culture conditions of the strains were optimized by single factor experiments. [Results] A strain, FK-15, exhibiting both antagonistic activity against P. nicotianae and tobacco growth-promoting effects, was isolated and identified as Amycolatopsis lurida. It showed an inhibition rate of 80.37% against P. nicotianae and the indoor control efficacy of 61.55%. Additionally, it demonstrated the abilities of producing siderophores and proteases. Treatment of tobacco seeds with the fermentation broth of FK-15 increased the radicle length, plumule length, and vigor index by 53.12%, 51.20%, and 73.09%, respectively, which indicated significant plant growth-promoting effects. After optimization of the culture conditions, the biomass and inhibition rate of the strain increased by 132.48% and 198.80%, respectively. [Conclusion] This study isolated a plant growth-promoting actinomycete strain FK-15 with strong antagonistic activity against P. nicotianae from different soil environments. Further optimization of its culture conditions may enable its practical application.