一株烟草疫霉拮抗菌MC4-2的鉴定、发酵条件优化及防效测定

Identification, fermentation condition optimization and control effect of an antagonistic strain MC4-2 against Phytophthora parasitica var. nicotianae

摘要:

[背景] 烟草黑胫病是由烟草疫霉（Phytophthora parasitica var.nicotianae）引起的真菌性病害，给我国烟叶生产带来了巨大损失。[目的] 在美洲大蠊肠道中分离并获得一株对烟草疫霉具有拮抗作用的菌株MC4-2，拟进一步明确该菌株的分类地位、优化发酵条件及对真菌性病害的防治效果。[方法] 通过形态特征、生理生化特征和16S rRNA基因序列等方法对菌株MC4-2进行鉴定；以细菌发酵液在波长为600 nm时的OD值为指标，采用单因素和正交试验的方法对菌株MC4-2的发酵培养基和发酵条件进行优化；通过在温室内进行盆栽防效试验，明确了该菌株对烟草黑胫病的防治效果。[结果] 通过平板对峙试验发现菌株MC4-2对烟草疫霉有较好的抑制效果，抑制率可达到64.04%；16S rRNA基因序列分析表明菌株MC4-2与枯草芽孢杆菌（Bacillus subtilis）相似度达到99%，形态特征和生理生化特征与该菌也基本符合；其优化培养基配方为：蛋白胨1.0 g、酵母浸粉1.0 g、蔗糖1.5 g、蒸馏水100 mL；最佳发酵条件为接种量8%、装液量30 mL、初始pH 6.0、转速210 r/min、温度32 ℃、培养时间60 h、光照时间12 h/d；室内盆栽试验结果表明，菌株MC4-2对烟草黑胫病的平均防效可达到63.86%。[结论] 为利用枯草芽孢杆菌MC4-2进行生物防治提供了重要参考依据。

关键词:美洲大蠊肠道内生菌;枯草芽孢杆菌;烟草疫霉;发酵条件;防效

Abstract:

[Background] Tobacco black shank is a fungal disease caused by Phytophthora parasitica var. nicotianae, which has brought great losses to tobacco production in China. [Objective] In this study, a strain MC4-2 was isolated from Periplaneta americana intestinal tract, which had antagonistic effect on P. parasitica var. nicotianae. [Methods] The strain MC4-2 was identified by morphological characteristics, physiological and biochemical methods and 16S rRNA gene sequencing. The fermentation medium and fermentation conditions of strain MC4-2 were optimized by single factor and orthogonal experiment using the OD value of bacterial fermentation broth at the wavelength of 600 nm as the index. The control effect of this strain on tobacco black shank was confirmed by potted experiment in greenhouse. [Results] Through the plate confrontation test, it was found that strain MC4-2 had a good inhibitory effect on P. parasitica var. nicotianae, and the inhibition rate could reach 64.04%; 16S rRNA gene sequence analysis showed that the similarity between strain MC4-2 and Bacillus subtilis reached 99%, and the morphological and physiological and biochemical characteristics were basically consistent with that of strain MC4-2; The optimal medium formula was peptone 1.0 g, yeast extract 1.0 g, sucrose 1.5 g, distilled water 100 mL. The results showed that the optimal fermentation conditions were inoculation amount of 8%, liquid volume of 30 mL, initial pH of 6.0, rotation speed of 210 r/min, temperature of 32 ℃, culture time of 60 h and light time of 12 h/d. The results of indoor pot experiment showed that the average control effect of strain MC4-2 on tobacco black shank could reach 63.86%. [Conclusion] This study provides an important reference for the biological control of B. subtilis MC4-2.

Key words:intestinal bacteria of Periplaneta americana;Bacillus subtilis;Phytophthora parasitica var. nicotianae;fermentation conditions;control effect