地黄根圈土壤拮抗放线菌筛选、鉴定及发酵条件优化

Screening,identification and optimized fermentation condition of antagonistic actinomycetes from Rehminnae glutinoso rhizosphere

为研究中药地黄根圈土壤拮抗性放线菌在植物病害生物防治中的应用潜力,用常规方法从地黄根圈土壤中分离获得42株放线菌。采用琼脂块法进行初筛,用纸片扩散法和生长速率法对抑菌效果最好的菌株DHR11进行抑菌活性复筛,通过形态、培养特征及16S rDNA序列分析研究其分类地位,并采用正交设计对从6种培养基中筛选的最佳培养基进行优化。结果表明:4株放线菌对供试植物病原真菌有较好的拮抗作用,其中菌株DHR11对苹果炭疽病菌Colletotrichum gloeospori-oides的抑菌活性最高,抑菌圈直径达30mm,抑菌率达52.5%;菌株DHR11发酵9天时抑菌活性最高;最优培养基组成为:蔗糖15 g、可溶性淀粉25 g、大豆粉10 g、酵母膏7.5 g、K2HPO40.5 g、自来水1000 mL;最佳发酵条件为:培养基初始pH7.5,接种量15%(v/v),摇床转数220 r/min,装瓶量80mL/250 mL,28℃发酵9天。根据形态、培养特征及16S rDNA序列分析,初步鉴定DHR11为加利利链霉菌Streptomyces galilaeus。

In order to confirm the potential appliance of an antagonistic aetinomyeetes for bioeontrol of plant diseases, 42 strains of actinomycetes were isolated from rhizosphere soil of Rehminnae glutinoso through conventional methods. Agar block method was used as preliminary screening, and disk diffusion method and growth speed rate method were used as secondary screening of the inhibition effect of strain DHR11. The taxonomic identification of strain DHR11 was studied through its morphological, cultural characteristics and 16S rDNA sequence alignment. The best liquid fermentation medium screening out six media was optimized by orthogonal array experiments. Results showed that 4 strains of aetinomyeetes exhibited better inhibition activity to plant pathogenic fungi tested than other strains. Among these 4 strains, strain DHR11 exhibited the strongest. The biggest inhibition zone was up to 30 mm in diameter, with an inhibition rate was 52.5% on Colletotrichum gloeosporioides. Strain DHR11 had the strongest antibacterial activity after being fermented for 9 days. Liquid fermentation medium for strain DHR11 was screened out through optimization experiment, with ingredients of sucrose 15 g, soluble starch 25 g, soy- bean meal 10 g, yeast extract 7.5 g, K2HPO4 0.5 g, tap water 1000mL, the optimal fermentation condition being initial pH 7.5, inoculum volume 15% （v/v） , stirring speed 220 r/rain, media volume 80 mL/250 mL, fermented for 9 days at 28 ℃. Studies on its morphology, cultural characteristics and 16S rDNA sequence showed that strain DHR11 belongs to Streptomyces galilaeus.