芒果炭疽病拮抗菌分离、鉴定及生防机制研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0908

摘要/Abstract

摘要：

从环境中分离获得芒果炭疽病病原菌的拮抗细菌，明确菌株RL-LL04对病原菌的拮抗机理并探索菌株的生物防治应用潜能。通过稀释法与平板对峙法，分离筛选出胶孢炭疽菌（Colletotrichum gloeosporioides）的拮抗细菌并进行系统鉴定，采用平板对峙法研究菌株对多种常见热带水果病原菌的抑制作用，通过胞外酶检测以及固相微萃取气质联用检测挥发性有机物成分，进行生防机制研究，并在光学显微镜下观察菌株对病原菌菌丝生长的影响，开展离体芒果接种试验进行芒果炭疽病生物防治应用。分离获得73株具有拮抗效果的细菌，5株对胶孢炭疽菌的抑制率达到70%以上，其中以菌株RL-LL04的抑菌率最高（82.2%），经菌落形态特征、生理生化特征与16S rRNA、gyrB和rpoB基因序列分析，鉴定为贝莱斯芽孢杆菌（Bacillus velezensis），该菌对多种常见热带水果病原真菌具有拮抗能力，该菌通过产生含有苯甲醛、3-甲基丁酸和苯酚等具有抑菌活性的挥发性有机物与纤维素酶、蛋白酶和木聚糖酶等胞外酶抑制病原菌生长，通过光学显微镜观察到病原菌菌丝畸形、扭曲及断裂，且离体芒果炭疽病防治效率达52.7%。该结果为芒果炭疽病的生物防治提供了菌种资源，也为阐明菌株RL-LL04对芒果炭疽病的拮抗机理提供了依据。

关键词: 热带水果病害, 芒果炭疽病, 胶孢炭疽菌, 生物防治, 贝莱斯芽孢杆菌, 挥发性有机物, 胞外酶

Abstract:

The antagonistic bacteria of mango anthracnose were isolated from the environment, the antagonistic mechanism of the strain RL-LL04 against the pathogen was clarified and the potential of the strain for biological control applications was investigated. Antagonistic bacteria of Colletotrichum gloeosporioides were isolated and screened via dilution and plate confrontation methods, and then were systematically identified. The plate confrontation method was used to study the inhibitory effect of the strains on a variety of pathogens on common tropical fruit. The extracellular enzyme detection and solid-phase micro extraction gas-mass spectrometry were combined to detect the volatile organic components and to study biocontrol mechanism. Moreover, the influence of the strain on the growth of pathogenic mycelium was observed under the light microscope. In addition, in vitro inoculation trials of mangoes were conducted for biological control applications of mango anthracnose. A total of 73 antagonistic bacterial strains against C. gloeosporioides were isolated and the inhibition rates of five strains were above 70.0%. Among these isolates, strain RL-LL04 showed the highest inhibition rate（82.2%）. Strain RL-LL04 was identified as Bacillus velezensis by morphologial, physiological & biochemical characteristics and 16S rRNA, gyrB and rpoB gene analysis. Strain RL-LL04 demonstrated antagonistic ability toward a variety of pathogenic fungi and strain RL-LL04 inhibited the growths of pathogenic fungi by producing volatile organic compounds containing benzaldehyde, 3-methylbutyric acid and phenol as well as extracellular enzymes such as cellulase, protease and xylanase. The pathogenic mycelium was deformed, twisted and broken, while observed by light microscopy. The inhibition rate of in vitro anthracnose on mango by strain RL-LL04 reached 52.7%. This study may provide a microbial resource for the biological control of mango anthracnose as well as a fundamental insight for elucidating the antagonistic mechanism of strain RL-LL04 against mango anthracnose.

Key words: tropical fruit diseases, mango anthracnose, Colletotrichum gloeosporioides, biological control, Bacillus velezensis, volatile organic compounds, extracellular enzyme

章乐乐, 王冠, 柳凤, 胡汉桥, 任磊. 芒果炭疽病拮抗菌分离、鉴定及生防机制研究[J]. 生物技术通报, 2023, 39(4): 277-287.

ZHANG Le-le, WANG Guan, LIU Feng, HU Han-qiao, REN Lei. Isolation, Identification and Biocontrol Mechanism of an Antagonistic Bacterium Against Anthracnose on Mango Caused by Colletotrichum gloeosporioides[J]. Biotechnology Bulletin, 2023, 39(4): 277-287.

图/表 13

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病原菌种属Species of pathogen	感病水果（物种）Infected fruit（species）	来源Source
胶孢炭疽菌 C. gloeosporioides	芒果 Mangifera indica L.	中国热带农业科学院南亚热带作物研究所，柳凤副研究员惠赠
芒果镰刀菌 Fusarium mangiferae	芒果 M. indica L.
尖孢镰刀菌 F. oxysporum	香蕉 Musa nana Lour.
厚垣镰刀菌 F. chlamydosporum	香蕉 M. nana Lour.	广东海洋大学滨海农业学院，刘月廉教授惠赠
立枯丝核菌 Rhizoctonia solani	芒果 M. indica L.
可可毛色二孢 Lasiodiplodia theobromae	菠萝蜜 Artocarpus heterophyllusLam.
多主棒孢霉 Corynespora cassiicola	番茄 Solanum lycopersicum
暹罗炭疽菌 C. siamensis	香蕉 M. nana Lour.
新暗色柱节孢Neoscytalidiumdimidiatum	火龙果 Hylocereus undulatus Britt	自行分离
新月弯孢菌Curvularia lunata	番茄 Solanum lycopersicum	自行分离

病原菌种属Species of pathogen	感病水果（物种）Infected fruit（species）	来源Source
胶孢炭疽菌 C. gloeosporioides	芒果 Mangifera indica L.	中国热带农业科学院南亚热带作物研究所，柳凤副研究员惠赠
芒果镰刀菌 Fusarium mangiferae	芒果 M. indica L.
尖孢镰刀菌 F. oxysporum	香蕉 Musa nana Lour.
厚垣镰刀菌 F. chlamydosporum	香蕉 M. nana Lour.	广东海洋大学滨海农业学院，刘月廉教授惠赠
立枯丝核菌 Rhizoctonia solani	芒果 M. indica L.
可可毛色二孢 Lasiodiplodia theobromae	菠萝蜜 Artocarpus heterophyllusLam.
多主棒孢霉 Corynespora cassiicola	番茄 Solanum lycopersicum
暹罗炭疽菌 C. siamensis	香蕉 M. nana Lour.
新暗色柱节孢Neoscytalidiumdimidiatum	火龙果 Hylocereus undulatus Britt	自行分离
新月弯孢菌Curvularia lunata	番茄 Solanum lycopersicum	自行分离

培养基名称 Culture medium name	组分 Components /（g·L^-1）
马铃薯葡萄糖琼脂培养基 Potato dextrose agar, PDA	马铃薯200.0，葡萄糖20.0
LB培养基 Luria-Bertani medium	胰蛋白胨 10.0，酵母提取物5.0，NaCl 10.0
纤维素酶检测培养基 Cellulase assay medium	CMC-Na 2.0，（NH₄）₂SO₄ 2.0，KH₂PO₄ 1.0，MgSO₄ 0.5，刚果红0.1
木聚糖酶检测培养基 Xylanase assay medium	木聚糖 5.0，KNO₃2.0，K₂HPO₄ 1.0，KCl 0.5，MgSO₄·7H₂O 1.0，FeSO₄ 0.01，刚果红 0.3
蛋白酶检测培养基 Protease assay medium	脱脂奶粉1.0
几丁质酶检测培养基 Chitinase assay medium	甲壳素 5.0，（NH₄）₂SO₄ 2.0，柠檬酸钠1.0，MgSO₄·7H₂O 0.2，KH₂PO₄ 6.0，K₂HPO₄ 1.0

培养基名称 Culture medium name	组分 Components /（g·L^-1）
马铃薯葡萄糖琼脂培养基 Potato dextrose agar, PDA	马铃薯200.0，葡萄糖20.0
LB培养基 Luria-Bertani medium	胰蛋白胨 10.0，酵母提取物5.0，NaCl 10.0
纤维素酶检测培养基 Cellulase assay medium	CMC-Na 2.0，（NH₄）₂SO₄ 2.0，KH₂PO₄ 1.0，MgSO₄ 0.5，刚果红0.1
木聚糖酶检测培养基 Xylanase assay medium	木聚糖 5.0，KNO₃2.0，K₂HPO₄ 1.0，KCl 0.5，MgSO₄·7H₂O 1.0，FeSO₄ 0.01，刚果红 0.3
蛋白酶检测培养基 Protease assay medium	脱脂奶粉1.0
几丁质酶检测培养基 Chitinase assay medium	甲壳素 5.0，（NH₄）₂SO₄ 2.0，柠檬酸钠1.0，MgSO₄·7H₂O 0.2，KH₂PO₄ 6.0，K₂HPO₄ 1.0

拮抗菌Antagonistic bacteria	抑菌率Inhibition rate/%
RL-BY01	79.70±1.34b
RL-LL04	82.22±0.80a
RL-WG01	80.00±1.94ab
RL-YG01	78.30±1.26b
RL-YX01	69.41±0.46c