利迪链霉菌K2对灰霉病菌的抑菌效果及抑菌物质鉴定

Inhibition of Streptomyces lydicus K2 on Botrytis cinerea and identification of its inhibitory substances

摘要:

灰霉病是多种经济作物生产过程以及果蔬储藏运输中常见的病害，链霉菌能产生丰富的次级代谢产物，对灰霉病菌具有较好的抑制效果。【目的】筛选出更高效、功能更多的链霉菌，为针对灰霉病的生防菌剂的研发提供优良菌种。【方法】采用管碟法对菌株K2进行液体培养基的筛选及培养液活性的测定；双皿对峙等2种方法进行产挥发性物质对灰霉病菌的抑菌活性测定；通过16S rRNA基因序列分析进行菌株K2鉴定；高效液相色谱及液相色谱-质谱2种方法对培养液活性成分进行定性验证；顶空固相微萃取-气质联用对菌株K2产生的挥发性物质成分进行检测及鉴定。【结果】菌株K2在液体培养基A中产生的次级代谢物对苹果腐烂病菌、苹果轮纹病菌、核盘菌、杨树溃疡病菌和烟草赤星病菌等多种植物病原真菌均具有较强的抑制作用；K2产生的挥发性物质对灰霉病菌的抑制率达100%，且抑制效果与挥发性物质的量有关；菌株K2与利迪链霉菌(Streptomyces lydicus)亲缘关系非常接近，相似性为99%；培养液活性成分中含有谷氏菌素、丰加霉素和纳他霉素；在挥发性物质成分中发现了烯类、醇类、酯类及烷烃类等30种挥发性物质，其中含量较多的物质分别是2-methylisoborneol、1-undecene、p-menth-8-ene和3-methylene，同时还检测到已报道的2种抑菌物质benzothiazole和β-pinene。【结论】经鉴定K2菌株为利迪链霉菌，培养液具有广谱抑菌活性，其产生的挥发性物质对灰霉病菌也有较好的抑制效果，可作为优质生防菌种用于开发防治果蔬生产中灰霉病的生物农药。

Abstract:

Gray mold is a common disease in the production process of a variety of cash crops, and in the storage and transportation of fruits and vegetables. Streptomyces spp. can produce many secondary metabolites with strong inhibition effects on Botrytis cinerea. [Objective] The present study focused on screening Streptomyces sp. with more efficient functions and provides excellent strains for the research and development of biocontrol agents for gray mold. [Methods] Tube dish method was applied for the selecting culture media and for determining bioactivities of culture broth of strain K2. The antifungal activity of volatile substances against B. cinerea was determined by double dish confrontation. 16S rRNA gene sequence analysis was used for identification of strain K2. High performance liquid chromatography and liquid chromatography-mass spectrometry methods were applied for qualitatively verification of the active components in the culture broth. Volatile components were determined using headspace solid phase micro-extraction gas chromatography-mass spectrometry. [Results] The secondary metabolites produced by strain K2 in culture medium A had strong inhibitory effects on a variety of plant pathogenic fungi such as Valsa mali, Physalospora piricola, Sclerotinia sclerotiorum, Botryosphaeria dothidea and Alternaria alternata. The inhibition rate of volatile substances produced by K2 on B. cinerea reached 100%, and the inhibition effect was correlated with the amount of volatile substances in the culture broth. K2 was identified as Streptomyces lydicus through the 16S rRNA gene sequence (maximum identity of 99%), glutamicin, fengamycin and natamycin were detected in the culture broth with high performance liquid chromatography. A total of 30 volatile substances including olefins, alcohols, esters and alkenes were detected using headspace solid phase micro-extraction gas chromatography and mass spectroscopy, in which 2-methylisoborneol, 1-undecene, p-menth-8-ene and 3-methylene were the major biochemicals. Two substances with fungicidal activity, benzothiazole and β-pinene, were detected as well. [Conclusion] The strain K2 was identified as Streptomyces lydicus and its culture broth had a broad spectrum of bioactivities against different plant pathogens, and its volatile substances also had good inhibitory effects on B. cinerea. As a result, K2 can be used as a high-quality biocontrol agent to develop bio-fungicides for controlling gray mold on some fruit and vegetable crops.