附子白绢病拮抗细菌CZ1的分离鉴定、发酵条件优化及防效测定

An antagonistic bacterial strain CZ1 against southern blight on Aconitum carmichaelii: isolation, identification, optimization of fermentation conditions, and evaluation of control effect

摘要:

【背景】附子白绢病是齐整小核菌(Sclerotium rolfsii)引起的土传病害，给附子生产带来了巨大损失。【目的】明确对齐整小核菌具有较好拮抗作用的菌株CZ1的分类地位、优化发酵条件及对附子白绢病的防治效果。【方法】采用稀释涂布的方法从叉角厉蝽马氏管内分离细菌，平板对峙法筛选对齐整小核菌有较强拮抗力的菌株，且测定该菌株对其他植物病原真菌的抑制作用；通过形态特征、生理生化特征以及16S rRNA基因序列等方法明确该菌株的分类地位；以细菌发酵液在波长600 nm时的OD值为指标，并以单因素试验和正交试验的方法进行培养基组分及发酵条件优化；通过室内盆栽试验明确防治效果。【结果】分离获得6株细菌，其中菌株CZ1对齐整小核菌的平板对峙抑制率最高，菌株CZ1对烟草疫霉(Phytophthora nicotiana)、尖孢镰刀菌(Fusarium oxysporum)等均有抑制效果。菌株CZ1被初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。其最佳培养基配方为：蔗糖15.00 g/L，蛋白胨25.00 g/L，酵母浸粉7.50 g/L；最佳培养条件：接种量2%，装液量90 mL，转速为210 r/min，温度36 ℃，初始pH 8.0、培养时间120 h。室内盆栽试验结果表明，菌株CZ1对附子白绢病的平均防效可以达到59.26%。【结论】菌株CZ1在附子白绢病的防治上具有较好生防潜力。

Abstract:

[Background] Southern blight is a soil-borne disease caused by Sclerotium rolfsii, causing serve loss to Aconitum carmichaelii production. [Objective] To clarify the taxonomic status of an antagonistic strain CZ1 against S. rolfsii, and optimize the fermentation conditions to maximize the control effect on southern blight of A. carmichaelii. [Methods] The dilution-plate coating method was employed to isolate bacteria from the Malpighian tubules of Cantheconidae furcellata. The plate confrontation assay was employed to screen out the strain with strong resistance to S. rolfsii, and the inhibitory effects of this strain on other plant pathogenic fungi were determined. The taxonomic status of the strain was determined based on morphological characteristics, physiological and biochemical properties, and 16S rRNA gene sequence. With OD₆₀₀ value of the fermentation broth as the indicator, single factor and orthogonal experiments were carried out to optimize the medium components and fermentation conditions. Furthermore, indoor pot experiment was carried out to evaluate the control effect of the strain. [Results] Six strains of bacteria were isolated, among which CZ1 had the highest inhibition rate on S. rolfsii. CZ1 had inhibitory effects on other plant pathogenic fungi such as Phytophthora nicotiana and Fusarium oxysporum and was preliminary identified as Bacillus subtilis. The optimal culture medium was composed of 15.00 g/L sucrose, 25.00 g/L peptone, and 7.50 g/L yeast extract. The strain showed the highest biomass when cultured at the inoculation amount of 2%, liquid loading volume of 90 mL, 210 r/min, 36 ℃, and initial pH 8.0 for 120 h. The results of indoor pot experiment showed that the average control efficiency of CZ1 against the southern blight on A. carmichaelii reached 59.26%. [Conclusion] CZ1 strain has good potential in the biocontrol of southern blight on A. carmichaelii.