茶树炭疽病菌拮抗链霉菌的筛选及其抑菌特性研究

张玉丹; 谭琳; 刘仲华; 肖敦根; 邓玉莲; 李桂花; 黄虹; 杨学宇; 胡秋龙

doi:10.13305/j.cnki.jts.2024.02.008

茶叶科学 >

2024 , Vol. 44 >Issue 2: 283 - 298

DOI: https://doi.org/10.13305/j.cnki.jts.2024.02.008

研究报告

茶树炭疽病菌拮抗链霉菌的筛选及其抑菌特性研究

张玉丹 ,
谭琳 ,
刘仲华 ,
肖敦根 ,
邓玉莲 ,
李桂花 ,
黄虹 ,
杨学宇 ,
胡秋龙

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1.湖南农业大学茶学教育部重点实验室,湖南长沙 410128;
2.湖南农业大学园艺学院,湖南长沙 410128;
3.湖南农业大学植物保护学院,湖南长沙 410128;
4.湖南中茶茶业有限公司,湖南长沙 410218

张玉丹,女,硕士研究生,主要从事茶树病害及其生物防治研究。

收稿日期: 2023-11-18

修回日期: 2023-12-24

网络出版日期: 2024-04-30

基金资助

湖南省重点研发计划项目（2022NK2051）、湖南省科技创新重大项目（2021NK1020）、国家重点研发计划项目（2022YFD16000803-6）

收起

Identification of Antagonistic Streptomycetes Against Anthracnose Pathogen of Tea Plants and Determination of Their Inhibitory Properties

ZHANG Yudan ,
TAN Lin ,
LIU Zhonghua ,
XIAO Dungen ,
DENG Yulian ,
LI Guihua ,
HUANG Hong ,
YANG Xueyu ,
HU Qiulong

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1. Key Lab of Tea Science of Ministry of Education, Hunan Agricultural University, Changsha 410128, China;
2. College of Horticulture, Hunan Agricultural University, Changsha 410128, China;
3. College of Plant Protection, Hunan Agricultural University, Changsha 410128, China;
4. China Tea (Hunan) Co. LTD., Changsha 410128, China

Received date: 2023-11-18

Revised date: 2023-12-24

Online published: 2024-04-30

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摘要

茶炭疽病菌刺盘孢菌（Colletotrichum camelliae）是引起茶树炭疽病的重要致病菌,为获得对C. camelliae具有拮抗作用的链霉菌,采用稀释涂布法和平板对峙法从茶园生境中分离筛选出对其具有明显拮抗作用的链霉菌菌株,并结合形态学观察、生理生化特征和16 S rRNA基因序列分析对其进行种属鉴定;开展基于链霉菌菌株的抗菌谱测定、茶炭疽病菌菌丝生长抑制试验和孢子萌发抑制试验;通过菌丝生长速率法测定其无菌发酵滤液对茶炭疽病菌的抑菌活性及其抑菌活性稳定性,并测定其产胞外降解酶能力、抗菌物质合成基因、挥发性与非挥发性代谢物抑菌活性。结果表明,筛选获得一株对茶炭疽病菌C. camelliae具有良好抑菌效果的菌株XS-4,对茶炭疽病菌的平板抑制效果为76.42%;结合形态学观察、生理生化特征,以及16 S rRNA基因序列分析,将菌株XS-4鉴定为多产色链霉菌（Streptomyces polychromogenes）;菌株XS-4对其他8种植物病原菌均具有较好的抑菌效果,抗菌性能具有广谱性。扫描电镜结果表明,菌株XS-4能抑制茶炭疽病菌菌丝生长。孢子萌发抑制试验发现,拮抗菌XS-4发酵液能抑制茶炭疽病菌的孢子萌发,抑制率为62.48%;菌株XS-4的最佳发酵培养基为KMB培养基,在KMB培养基中培养7 d所产生的抑菌活性物质对茶炭疽病菌的抑菌效果最好;其无菌发酵滤液对温度、酸碱度、紫外照射、蛋白酶均具有较好的稳定性;菌株XS-4的非挥发性代谢物对茶炭疽病菌的抑菌活性较好,抑菌率达81.92%;菌株XS-4具有产生淀粉水解酶、蛋白酶、β-1,3-葡聚糖酶、纤维素酶的能力;菌株XS-4具有产生抗菌物质的pks-Ⅰ及pks-Ⅱ基因。综上所述,菌株XS-4在茶树炭疽病生物防治方面具有巨大的应用潜力。

关键词： 茶炭疽病菌; 链霉菌; 抑菌效果; 稳定性; 抗菌基因

本文引用格式

张玉丹 , 谭琳 , 刘仲华 , 肖敦根 , 邓玉莲 , 李桂花 , 黄虹 , 杨学宇 , 胡秋龙 . 茶树炭疽病菌拮抗链霉菌的筛选及其抑菌特性研究[J]. 茶叶科学, 2024 , 44(2) : 283 -298 . DOI: 10.13305/j.cnki.jts.2024.02.008

Abstract

Colletotrichum camelliae is an important pathogen causing anthracnose of tea plants. In order to obtain Streptomyces strains with better antagonistic effect on C. camelliae, the dilution coating method and plate standoff method were used to isolate and screen Streptomyces strains from the habitat of tea plantations. Morphological observation, physiological and biochemical characteristics and 16 S rRNA gene sequence analysis were combined to identify their species. Antimicrobial spectrum determination, mycelial growth inhibition test and spore germination inhibition test of C. camelliae were carried out for the isolated strains. The antifungal activities and stabilities of aseptic fermentation filtrate of the isolated strains against C. camelliae were determined by using the mycelial growth rate method. The extracellular enzyme-producing capacity, antimicrobial substance synthesis genes, volatile and non-volatile metabolite antifungal activity of the isolated strains were also determined. The results show that a strain XS-4 with better inhibitory effect on tea anthracnose pathogen was obtained and the plate inhibition effect on tea anthracnose pathogen was 76.42%. The strain XS-4 was identified as Streptomyces polychromogenes, which had good inhibitory effect on eight other plant pathogens and the antifungal properties have a broad spectrum. Scanning electron microscopy results show that the strain XS-4 could inhibit the growth of mycelium of tea anthracnose pathogen, and the mycelium was tightly entangled with each other and deformed. The spore germination inhibition test shows that the fermentation solution of strain XS-4 could inhibit the spore germination of C. camelliae, and the inhibition rate was 62.48%. The best fermentation medium for strain XS-4 was KMB medium, and the inhibitory active substance produced by 7 d incubation in KMB medium had the best inhibitory effect on tea anthracnose, the aseptic fermentation filtrate of strain XS-4 had a better stability to temperature, acid and alkali, ultraviolet, and protease. The non-volatile metabolites of strain XS-4 show better antifungal activity against tea anthracnose pathogen with aninhibition rate of 81.92%. The strain XS-4 has the ability to produce amylase, protease, β-1,3-glucanase, and cellulase. The pks-Ⅰ and pks-Ⅱ genes of strain XS-4 were associated with the production of antimicrobial substances. In conclusion, the strain XS-4 has a great potential for application in the biocontrol of anthracnose in tea plants.

Key words： tea anthracnose; Streptomyces; antifungal effect; stability; antimicrobial genes

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