核桃黑斑病拮抗放线菌WMF106的筛选、鉴定及防效

Screening, identification and biocontrol effect of antagonistic actinomycete WMF106 against walnut blight

摘要:

[背景] 核桃黑斑病是由2种病原菌引起的细菌性病害，目前缺乏有效的生物防治方法。[目的] 从核桃树根际土壤中筛选对核桃黑斑病病原菌具有拮抗效果的放线菌菌株，为该病害生防菌剂的开发提供基础。[方法] 采用稀释涂布法分离放线菌，并以病原菌野油菜黄单胞菌（Xanthomonas campestris pv.campestris）和成团泛菌（Pantoea agglomerans）作为指示菌，利用平板对峙法和改良牛津杯法筛选具有高拮抗活性的菌株，通过形态学特征、生理生化特性和16S rRNA基因序列分析确定其分类地位，并测定其无菌发酵液的抗菌谱和室内防效。[结果] 筛选到一株对野油菜黄单胞菌和成团泛菌均有较强拮抗作用的放线菌菌株WMF106，该菌株对2种病原菌的抑菌圈直径分别为2.38 cm和1.82 cm，无菌发酵液对2种病原菌的抑菌圈直径分别为1.75 cm和1.55 cm。根据菌株形态学、生理生化特性及16S rRNA基因序列分析，将菌株WMF106鉴定为暗蓝色链霉菌（Streptomyces caeruleatus）。该菌株对尖孢镰刀菌、腐皮镰孢菌、辣椒刺盘孢菌、灰葡萄孢菌、胶孢炭疽菌5种植物病原菌及大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌、黑曲霉5种指示菌均有抑制作用，抗菌性能广谱高效，其无菌发酵液原液对离体叶片上由野油菜黄单胞菌和成团泛菌造成的核桃黑斑病防效分别为77.44%和58.33%。[结论] 菌株WMF106可作为防治核桃黑斑病的生防材料，具有良好的开发价值和应用前景。

Abstract:

[Background] Walnut blight is a serious bacterial plant disease caused by two pathogens, it lacks effective biological control method at present. [Objective] To isolate and screen antagonistic actinomycetes strains with inhibitory activity on walnut blight from the rhizosphere soil of walnut tree. [Methods] Actinomycete was isolated by using dilution plate coating. The methods of agar disk and improved Oxford-cup test were applied to screen antagonistic strains against Xanthomonas campestris pv. campestris and Pantoea agglomerans. The morphological, physiological, biochemical characteristics combined with 16S rRNA gene sequences were used for classification and identification. The antibacterial spectrum and the biocontrol effect of the cell-free fermentation broth were evaluated. [Results] Strain WMF106 had antagonistic effects on X. campestris pv. campestris with inhibition zone diameter of 2.38 cm and on P. agglomerans with inhibition zone diameter of 1.82 cm. The inhibition zone diameter of cell-free fermentation broth on X. campestris pv. campestris and P. agglomerans were 1.75 cm and 1.55 cm, respectively. Based on the morphological and physiological characteristics and 16S rRNA gene sequence analysis, strain WMF106 was identified as Streptomyces caeruleatus. The antibacterial spectrum experiment results indicated that Streptomyces caeruleatus WMF106 also showed biological activities against five species of plant pathogenic fungi (Fusarium oxysporum, Fusarium solani, Colletotrichum capsici, Botrytis cinerea and Colletotrichum gloeosporioides), and five species of indicator bacteria (Aspergillus niger, Monilia albican, Escherichia coli, Staphlococcus aureus and Pseudomonas aeruginosa). In addition, the biocontrol experiment in vitro showed that the cell-free fermentation broth of strain WMF106 had worthy prevention of walnut blight caused by X. campestris pv. campestris and P. agglomerans, and the biocontrol effects were 77.44% and 58.33%. [Conclusion] Strain WMF106 could be used for controlling the walnut blight, and have a wonderful potential for application.