生物技术通报 ›› 2023, Vol. 39 ›› Issue (4): 277-287.doi: 10.13560/j.cnki.biotech.bull.1985.2022-0908
章乐乐1,2(), 王冠1,2, 柳凤2, 胡汉桥1, 任磊1,2()
收稿日期:
2022-07-22
出版日期:
2023-04-26
发布日期:
2023-05-16
通讯作者:
任磊,男,博士,副教授,研究方向:环境微生物学;E-mail: renlei@gdou.edu.cn作者简介:
章乐乐,女,硕士研究生,研究方向:资源利用与植物保护;E-mail: zhanglele_97@qq.com
基金资助:
ZHANG Le-le1,2(), WANG Guan1,2, LIU Feng2, HU Han-qiao1, REN Lei1,2()
Received:
2022-07-22
Published:
2023-04-26
Online:
2023-05-16
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。
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.
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 |
表1 供试病原真菌信息
Table 1 Information of pathogenic fungi used in this study
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 |
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 |
表2 实验所需培养基配方
Table 2 Medium formulations required for the experiments
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 |
表3 胶孢炭疽菌的拮抗菌筛选
Table 3 Screening of antagonistic bacteria against C. glo-eosporioides
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 |
图1 菌株RL-LL04对胶孢炭疽菌的拮抗效果 A:胶孢炭疽菌正常生长状况;B:菌株RL-LL04对胶孢炭疽菌的拮抗效果
Fig. 1 Effect of strain RL-LL04 against C. gloeosporioides A: Normal growth condition of C. gloeosporioides. B: Antagonistic effect of strain RL-LL04 on C. gloeosporioides
测定内容Measured item | 结果Result |
---|---|
革兰氏染色 Gram stain | + |
接触酶 Contact enzyme | - |
甲基红 Methyl Red | - |
V-P反应 Voges-Proskauer reaction | - |
吲哚 Indole | - |
脂酶 Lipase | - |
淀粉水解 Starch hydrolysis | + |
氨苄青霉素敏感 Ampicillin sensitization | + |
表4 菌株RL-LL04的生理生化特征
Table 4 Physicochemical characteristics of strain RL-LL04
测定内容Measured item | 结果Result |
---|---|
革兰氏染色 Gram stain | + |
接触酶 Contact enzyme | - |
甲基红 Methyl Red | - |
V-P反应 Voges-Proskauer reaction | - |
吲哚 Indole | - |
脂酶 Lipase | - |
淀粉水解 Starch hydrolysis | + |
氨苄青霉素敏感 Ampicillin sensitization | + |
图2 菌株RL-LL04基于16S rRNA(A)、gyrB(B)和rpoB(C)以及联合(D)基因序列的系统发育树
Fig. 2 Phylogenetic tree of strain RL-LL04 based on 16S rRNA(A), gyrB(B), and rpoB(C)gene sequences and a concatenated alignment of 16S rRNA gene, gyrB and rpoB sequences(D)
图3 菌株RL-LL04对不同热带水果病原真菌的拮抗效果 A:暹罗炭疽菌;B:尖孢镰刀菌;C:多主棒孢霉菌;D:可可毛色二孢菌;E:立枯丝核菌;F:厚垣镰刀菌;G:新月弯孢菌;H:芒果镰刀菌;I:新暗色柱节孢
Fig. 3 Antagonistic effect of strain RL-LL04 against patho-genic fungi from different tropical fruit A: Colletotrichum siamense. B: Fusarium oxysporum. C: Corynespora cassiicola. D: Lasiodiplodia theobromae. E: Rhizoctonia solani. F: Fusarium chlamydosporum. G: Curvularia lunata. H: Fusarium mangiferae. I: Neoscytalidium dimidiatum
病原菌 Pathogenic fungi | 抑菌率 Inhibition rate/% |
---|---|
暹罗炭疽菌 C. siamense | 80.96±0.78ab |
尖孢镰刀菌 F. oxysporum | 79.63±1.10b |
多主棒孢霉菌 C. cassiicola | 52.67±4.39d |
可可毛色二孢菌 L. theobromae | 69.33±2.78c |
立枯丝核菌 R. solani | 84.00±1.02a |
厚垣镰刀菌 F. chlamydosporum | 72.28±1.41c |
新月弯孢菌 C. lunata | 79.10±2.50b |
芒果镰刀菌 F. mangiferae | 69.86±1.81c |
新暗色柱节包 N. dimidiatum | 78.67±2.12b |
表5 菌株RL-LL04对不同热带水果病原真菌的抑菌率
Table 5 Inhibition rate of strain RL-LL04 against path-ogenic fungi from different tropical fruit
病原菌 Pathogenic fungi | 抑菌率 Inhibition rate/% |
---|---|
暹罗炭疽菌 C. siamense | 80.96±0.78ab |
尖孢镰刀菌 F. oxysporum | 79.63±1.10b |
多主棒孢霉菌 C. cassiicola | 52.67±4.39d |
可可毛色二孢菌 L. theobromae | 69.33±2.78c |
立枯丝核菌 R. solani | 84.00±1.02a |
厚垣镰刀菌 F. chlamydosporum | 72.28±1.41c |
新月弯孢菌 C. lunata | 79.10±2.50b |
芒果镰刀菌 F. mangiferae | 69.86±1.81c |
新暗色柱节包 N. dimidiatum | 78.67±2.12b |
图5 菌株RL-LL04产生的挥发性有机化合物对胶孢炭疽菌的抑菌效果 A:胶孢炭疽菌正常生长状况;B:挥发性有机化合物对胶孢炭疽菌的抑菌效果
Fig. 5 Antibacterial effect of volatile organic compounds of strain RL-LL04 on C. gloeosporioides A: Normal growth condition of C. gloeosporioides. B: Antibacterial effect of volatile organic compounds on C. gloeosporioides
保留时间 Retention time/min | 化合物名称 Compound name | CAS号 CAS number | 参考文献 Reference |
---|---|---|---|
2.12 | 2,4-二甲基庚烷 | 002213-23-2 | N.R. |
2.15 | 4,4-二甲基庚烷 | 001068-19-5 | N.R. |
2.26 | 丙酮 | 000067-64-1 | N.R. |
2.49 | 六甲基环三硅氧烷 | 000541-05-9 | N.R. |
2.82 | 醋酸乙酯 | 000141-78-6 | N.R. |
3.35 | 三氯甲烷 | 000075-09-2 | N.R. |
3.41 | 乙醇 | 000064-17-5 | N.R. |
3.92 | 正丁醚 | 000142-96-1 | N.R. |
5.72 | 甲苯 | 000108-88-3 | N.R. |
5.88 | 1,2-二氯丙烷 | 000078-87-5 | N.R. |
8.81 | 1,3-二甲苯 | 000108-38-3 | N.R. |
9.02 | 丙酸丁酯 | 000590-01-2 | N.R. |
9.19 | 甲酸丁酯 | 000592-84-7 | N.R. |
18.28 | 乙酸 | 000064-19-7 | N.R. |
20.10 | 苯甲醛 | 000100-52-7 | [ |
20.54 | 丙酸 | 000079-09-4 | N.R. |
21.92 | 二甘醇二乙醚 | 000112-36-7 | N.R. |
23.70 | 3-甲基丁酸 | 000503-74-2 | [ |
24.27 | 3,4-二羟基苯基二醇 | 056114-62-6 | N.R. |
30.82 | 苯酚 | 000108-95-2 | [ |
38.56 | 苯甲酸 | 000065-85-0 | N.R. |
表6 固相微萃取气质联用检测菌株RL-LL04的挥发性有机物主要组分
Table 6 Main components of volatile organic compounds of strain RL-LL04 detected by solid-phase micr-oextraction gas chromatography
保留时间 Retention time/min | 化合物名称 Compound name | CAS号 CAS number | 参考文献 Reference |
---|---|---|---|
2.12 | 2,4-二甲基庚烷 | 002213-23-2 | N.R. |
2.15 | 4,4-二甲基庚烷 | 001068-19-5 | N.R. |
2.26 | 丙酮 | 000067-64-1 | N.R. |
2.49 | 六甲基环三硅氧烷 | 000541-05-9 | N.R. |
2.82 | 醋酸乙酯 | 000141-78-6 | N.R. |
3.35 | 三氯甲烷 | 000075-09-2 | N.R. |
3.41 | 乙醇 | 000064-17-5 | N.R. |
3.92 | 正丁醚 | 000142-96-1 | N.R. |
5.72 | 甲苯 | 000108-88-3 | N.R. |
5.88 | 1,2-二氯丙烷 | 000078-87-5 | N.R. |
8.81 | 1,3-二甲苯 | 000108-38-3 | N.R. |
9.02 | 丙酸丁酯 | 000590-01-2 | N.R. |
9.19 | 甲酸丁酯 | 000592-84-7 | N.R. |
18.28 | 乙酸 | 000064-19-7 | N.R. |
20.10 | 苯甲醛 | 000100-52-7 | [ |
20.54 | 丙酸 | 000079-09-4 | N.R. |
21.92 | 二甘醇二乙醚 | 000112-36-7 | N.R. |
23.70 | 3-甲基丁酸 | 000503-74-2 | [ |
24.27 | 3,4-二羟基苯基二醇 | 056114-62-6 | N.R. |
30.82 | 苯酚 | 000108-95-2 | [ |
38.56 | 苯甲酸 | 000065-85-0 | N.R. |
图6 菌株RL-LL04对胶孢炭疽菌形态变化的影响(比例尺=20 μm) A:正常形态菌丝;B:菌丝畸形;C:菌丝折叠;D:菌丝断裂
Fig. 6 Morphological changes of strain RL-LL04 on C. gloeosporioides(Bars=20 μm) A: Normal form hyphae. B: Deformed hyphae. C: Folded hyphae. D: Broken hyphae
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