赞皇枣黑腐病菌拮抗链霉菌的室内多重筛选及发酵条件优化

Screening in vitro of Antagonistic Streptomyces against the Black Rot Disease(Alternaria tenuissima) of Zanhuang Jujube Fruit and Optimization of Fermentation Conditions

【目的】本研究旨在获得对赞皇枣黑腐病菌具有良好抑制作用的拮抗链霉菌,并对其发酵培养基配方和条件进行优化,以期为进行田间试验和开发生防制剂奠定基础。【方法】以赞皇枣黑腐病优势病原真菌—细极链格孢菌为靶标采用琼脂块法、牛津杯法和含药平板法对从特殊生境分离获得的10株抗真菌链霉菌进行室内多重筛选并采用二次回归正交旋转设计和单因素试验对筛选获得的拮抗链霉菌分别进行发酵培养基优化和最佳发酵条件确立【结果】活体筛选试验中菌株AC03,AC04和AC07表现出了明显的拮抗效果,抑菌圈直径分别为(32.09±1.45),(15.75±1.34),(18.33±0.78)mm;上述3株菌的无菌发酵滤液活性测定表明:菌株ACO3、ACO7的抑菌圈直径分别为(20.94±0.92)和(23.44±1.63)mm,而菌株ACO4仅为(14.5±0.71)mm;对菌株ACO3和ACO7进行无菌发酵滤液的菌丝生长抑制试验结果表明:菌株ACO7代谢产生的拮抗物质抑菌效果、持效性均优于ACO3。综上可知菌株ACO7对赞皇枣黑腐病菌的抑菌效果最优。ACO7的最优发酵培养基配方为:麦芽糖5.7%,大豆粉2.7%,(NH4)2SO40.17%,CaCO30.22%NaCl0.17%,酵母粉0.2%;最佳发酵条件为:种子液浓度1.5×10~7~2×10~8 cfu·mL^(-1)时,接种量2%(V/V)液量40 mL/250 mL三角瓶,原始pH7.0发酵温度28℃发酵时间108 h。【结论】筛选获得的目标拮抗菌ACO7可为开发针对赞皇枣黑腐病的生物农药提供新的原材料,且发酵培养基和条件的优化提高了目标代谢物的产量,为后续的研究和开发奠定基础。

[ Objective] The Zanhuang jujube （ Ziziphus jujuba ＇ Zanhuang＇ ） fruit black rot disease is one of the most serious fungal pathogens in China. Bio-control of the disease is of great significance to the fruit green production and its sustained efficacy as well as being free of fungicide pollution. The objective of the present study is to screen out a Streptomyces strain with good inhibitory effect against the fungal disease pathogen （Ahernaria tenuissima） and optimize the fermentation medium formula, as well as fermentation conditions with the purpose of laying a foundation for field experiment and the development of bio-control agent. [ Method ] The agar block method, the Oxford cup method and the medicated plate method were applied in the multiple screening in vitro of 10 Streptomyces strains against A. tenuissima, which were originally isolated from special habitats. The optimum fermentation medium and conditions for the selected strain were determined by using a quadratic orthogonal rotation combination design and a single-factor experiment design, respectively. [ Result] During in vivo experiment, strains AC03, AC04 and AC07 showed significant antagonistic effect, their diameter of inhibition zone being （32.09 _＋ 1.45 ） ram, （ 15.75 ＋ 1.34） mm and （ 18.33 _＋ 0.78 ）mm respectively. The fermentation filtrate activity test of the three strains demonstrated that the strains AC03 and AC07 had a diameter of inhibition zone of （20.94 ± 0.92） mm and （23.44 ± 1.63 ）mm respectively, while the strain AC04 had a diameter of only （14.5±0.71 ）ram. The further conducted test of the inhibition effect of the fermentation filtrates of the strain AC03 and AC07 on mycelial growth displayed that the antagonistic substance produced by the strain AC07 had a better and longer inhibition effect than that produced by the strain AC03. These tests demonstrated that of the 10 isolates, the strain AC07had the best inhibition effect on A. tenuissima, that is the main cause of the black rot disease on Zanhuang jujube. The optimum fermentation medium for the strain AC07 consists of maltose, 5.7% ; soybean meal, 2.7% ; （ NH4 ）2SO4, 0. 17% ; CaC03, 0.22% ; NaC1, 0. 17% and Yeast Powder, 0.2%. The optimum fermentation conditions were defined as follows： concentration of seed broth, 1.5 × 10^7 -2.0×l0^8 cfu＇mL-1, inoculation amount, 2% （V/V） , the volume of media, 40 mL, in a 250mL flask initial pH at 7.0, fermentation temperature 28℃ and fermentation time 108 h. [ Conclusion] The strain AC07 was selected as a new antagonistic Streptomyces for developing biocontrol agent against the Zanhuang jujube fruit black rot disease. The optimization of its fermentation medium and conditions increased the production of the target metabolites, laying a good foundation for further research and development.