桑疫病病原拮抗菌的分离、鉴定及发酵条件优化

Screening，identification and optimization of fermentation conditions of an antagonistic endophyte to mulberry bacterial blight

摘要:

摘要:【目的】从健康桑树内生菌中分离获得对桑疫病病原菌(Pseudomonas syringae pv. mori)具有显著拮抗作用的菌株，优化其产生抑菌活性物质的发酵条件，为其生防利用奠定基础。【方法】从严格表面消毒的桑树根茎中分离内生菌，采用平板划线法纯化内生菌，用抑菌圈法筛选拮抗菌;根据菌株的形态与培养特征、生理生化特性、16S rDNA 序列分析对其进行鉴定。通过单因素试验和正交设计试验优化培养基组分及发酵条件。【结果】从健康桑树中分离获得内生菌77 株，其中，编号为SWg2 的菌株对桑疫病病原菌具有强而稳定的抑制作用。菌株SWg2的形态与培养特征、生理生化特性和泛菌属(Pantoea sp.)相符，而16S rDNA序列分析结果显示它与成团泛菌(P. agglomerans)的亲缘关系接近。研究表明其最佳发酵配方和培养条件为:甘油(2.00%)、硝酸铵(2.00%)、KH2 PO4(0.10%)和MgSO4·7H2O(0.15%)，起始pH 为7.5，装瓶量20 mL/100 mL，最适培养温度为28℃，转速为170 r/min，种子液接种量为4%，摇瓶培养5 d。【结论】经鉴定，对桑疫病病原具拮抗作用的桑树内生菌SWg2为成团泛菌(P.agglomerans)，命名为成团泛菌SWg2。对其发酵条件进行优化后对桑疫病病原菌显示出更强的拮抗作用。

Abstract:

Abstract:［Objective］Antagonistic endophytic strains with strongly inhibitory activity to mulberry bacterial blight (P．syringae pv．mori) were isolated from mulberry endophytes．we identified the antagonistic endophyte and optimized the fermentation conditions．［Method］Streak plate method was used to separate the endophytes from healthy mulberry tissues after strict surface disinfection．Antagonistic endophytes were screened out through inhibition zone method．Strain SWg2 was identified by morphological，physiological and biochemical characteristics and 16S rDNA sequence analysis．The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment.［Result］In total 77 endophytic strains have been isolated from healthy mulberry. SWg2 showed strong and stable antagonistic activity to mulberry bacterial blight. The morphological，cultural， physiological，and biochemical characteristics assays indicated that SWg2 belongs to Pantoea sp．The 16S rDNA sequence phylogenetic analysis reveals that SWg2 appeared a sister lineage to P．agglomerans．The optimized culture conditions of strain SWg2 were liquid volume 20 mL in 100 mL flask，170 r /min at 28℃，inoculation size of 4% for 5 d with a medium of 2.0% glycerol，2.0% NH4NO3，0.1% KH2 PO4，0.15% MgSO4·7H2O at initial pH of 7.5． ［Conclusion］The antagonistic endophytic strain SWg2 to mulberry bacterial blight was identified as P．agglomerans. SWg2 strain shows stronger antagonistic action to mulberry bacterial blight under optimized fermentation conditions．