生防细菌HX0037对栝楼炭疽病的防病能力及其机制

doi:10.13560/j.cnki.biotech.bull.1985.2023-1172

生物技术通报 ›› 2024, Vol. 40 ›› Issue (4): 228-241.doi: 10.13560/j.cnki.biotech.bull.1985.2023-1172

生防细菌HX0037对栝楼炭疽病的防病能力及其机制

徐伟芳¹^,²(), 李贺宇¹, 张慧¹, 何仔昂¹, 高文恒¹, 谢紫洋¹, 王传文³, 尹登科¹()

1.安徽中医药大学药学院中药研究与开发安徽省重点实验室,合肥 230012
2.安徽中医药大学安徽中药材种植联合研究中心, 合肥 230012
3.潜山市传文瓜子有限公司,安庆 246300

收稿日期:2023-12-13 出版日期:2024-04-26 发布日期:2024-04-30
通讯作者: 尹登科，男，博士，教授，研究方向：微生物次级代谢产物；E-mail: yindengke@ahtcm.edu.cn
作者简介:徐伟芳，女，博士，讲师，研究方向：药用植物病害生物防控；E-mail: xuweifang@ahtcm.edu.cn
基金资助:
安徽省自然科学基金项目(2108085QH374);中药研究与开发安徽省重点实验室开放基金(AKLPDCM202310);大健康研究院安徽中药材种植联合研究中心专项资金(yjzx2023005);安徽中医药大学-潜山市医药健康发展技术研究中心科研攻关研发项目(2021QSHZ01);安徽中医药大学人才支持项目(2020rcyb008);大学生创新创业训练计划省级项目(S202210369005)

Efficacy and Its Mechanism of Bacterial Strain HX0037 on the Control of Anthracnose Disease of Trichosanthes kirilowii Maxim

XU Wei-fang¹^,²(), LI He-yu¹, ZHANG Hui¹, HE Zi-ang¹, GAO Wen-heng¹, XIE Zi-yang¹, WANG Chuan-wen³, YIN Deng-ke¹()

1. School of Pharmacy, Anhui University of Chinese Medicine, Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Hefei 230012
2. Joint Research Center for Chinese Herbal Medicine of Anhui of IHM, Anhui University of Chinese Medicine, Hefei 230012
3. County Qianshan Chuan-Wen Trichosanthes Seeds Technology Co. Ltd, Anqing 246300

Received:2023-12-13 Published:2024-04-26 Online:2024-04-30

摘要/Abstract

摘要：

【目的】研究潜在生防细菌HX0037对栝楼炭疽病的防病能力，并探究其生防机制。【方法】采用平板对峙法测定HX0037对栝楼炭疽菌（Colletotrichum gloeosporioides）、枣炭疽菌（Collettrichum coccodes）、梨炭疽菌（Colletotrichum fructicola）和苹果炭疽菌（Cryptosporiopsis malicorticis）的拮抗活性；离体接种法评价其对栝楼叶片炭疽病和果实炭疽病的防治效果；结合形态学、生理生化特征和全基因组的序列分析结果，确定生防细菌的分类地位，并预测其次级代谢产物；采用酸沉淀法提取脂肽类化合物，并对其进行高效液相色谱串联质谱分析（LC-MS）；最后通过观察生防细菌所产脂肽对栝楼炭疽病菌菌株生长和菌丝形态的影响，以及检测该菌株产酶和铁载体能力，探究可能的生防机制。【结果】HX0037菌株对4种瓜果炭疽病菌均有不同程度的抑菌活性，其中对栝楼炭疽病菌的抑菌效果最好，其能明显改变病菌细胞膜的通透性；与单独接种病菌的栝楼组相比，接种HX0037菌株能显著减小栝楼叶片炭疽病和栝楼果实炭疽病的病斑面积，其防病效果分别为61.50%和52.51%；HX0037菌株被进一步鉴定为解淀粉芽孢杆菌（Bacillus amyloliquefaciens），其基因组中包含12个次级代谢产物基因簇，它还具备产生纤维素酶、蛋白酶、淀粉酶和铁载体的能力；从HX0037代谢物中分离获得的脂肽导致栝楼炭疽病菌的菌丝发生扭曲、膨大等畸变现象，经LC-MS鉴定，该脂肽粗提物中含有表面活性素和丰原素。【结论】解淀粉芽孢杆菌HX0037具有良好的抑制炭疽病菌生长的能力，有望被进一步开发为生物农药用于栝楼炭疽病的绿色防控。

关键词: 解淀粉芽孢杆菌, 生物防治, 栝楼炭疽病, 拮抗, 全基因组, 菌种鉴定, 脂肽

Abstract:

【Objective】The biocontrol ability of bacterium HX0037 against anthracnose disease of Trichosanthes kirilowii Maxim and its biocontrol mechanism were studied. 【Method】The antagonistic activities of HX0037 against four pathogens of fruit anthracnose disease were determined in petri dishes experiments. The biocontrol efficiencies of HX0037 against Trichosanthes leaf anthracnose and Trichosanthes fruit anthracnose were evaluated on detached leaves and fruits in vitro. In addition, HX0037 was further identified based on the analyses on morphological features, physiological and biochemical characteristics and molecular biological identification results of the whole genome, and the secondary metabolites of biocontrol bacteria HX0037 were also predicted. The lipopeptide compounds produced by strain HX0037 were extracted by acid precipitation and analyzed by high-performance liquid chromatography tandem mass spectrometry（LC-MS）. Finally, the possible biocontrol mechanism was explored through two experiments, observing the effects of lipopeptides produced by strain HX0037 on the hyphal growth and morphology of Colletotrichum gloeosporioides, and the abilities of strain HX0037 to produce enzyme and siderophore. 【Result】Strain HX0037 showed varied antimicrobial activity against four pathogens of fruit anthracnose disease, especially the best against C. gloeosporioides, a pathogen of Trichosanthes anthracnose disease, and it obviously changed the cell membrane permeability of the pathogenic fungi. Compared with plant tissues inoculated with the pathogen alone, inoculation with strain HX0037 significantly reduced the incidence of leaf and fruit anthracnose diseases of T. kirilowii Maxim, and the biocontrol efficacy was 61.50% and 52.51%, respectively. Strain HX0037 was further identified as Bacillus amyloliquefaciens, and its genome contained 12 secondary metabolite gene clusters, and it decomposed the protein, cellulose and starch, and also produced siderophore. The hyphae of C. gloeosporioides were distorted, intumescent, broken and more transparent after treatment with the antifungal lipopeptide produced by strain HX0037. Surfactin and fengycin were detected from the lipopeptide extracts of strain HX0037 by LC-MS. 【Conclusion】The B. amyloliquefaciens HX0037 has strong antagonistic activity on C. gloeosporioides, and is expected to be further developed as a biological agent for the biocontrol of Trichosanthes anthracnose diseases.

Key words: Bacillus amyloliquefaciens, biological control, Trichosanthes anthracnose disease, antagonism, whole genome, species identification, lipopeptide

徐伟芳, 李贺宇, 张慧, 何仔昂, 高文恒, 谢紫洋, 王传文, 尹登科. 生防细菌HX0037对栝楼炭疽病的防病能力及其机制[J]. 生物技术通报, 2024, 40(4): 228-241.

XU Wei-fang, LI He-yu, ZHANG Hui, HE Zi-ang, GAO Wen-heng, XIE Zi-yang, WANG Chuan-wen, YIN Deng-ke. Efficacy and Its Mechanism of Bacterial Strain HX0037 on the Control of Anthracnose Disease of Trichosanthes kirilowii Maxim[J]. Biotechnology Bulletin, 2024, 40(4): 228-241.

图/表 15

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培养基名称Name of culture medium	组分Component/（g·L^-1）
马铃薯葡萄糖培养基Potato dextrose medium, PDB	马铃薯200.0，葡萄糖20.0
LB培养基Luria-Bertani medium	胰蛋白胨10.0，酵母粉5.0，氯化钠10.0
PDB改良培养基Modified potato dextrose broth	马铃薯200.0，麦芽糖20.0，蛋白胨10.0，硫酸铵5.0，磷酸氢二钠1.5
纤维素酶检测培养基Cellulase assay medium	羧甲基纤维素钠 2.0，硫酸铵 2.0，磷酸二氢钾 1.0，硫酸镁 0.5，刚果红 0.1
蛋白酶检测培养基Protease assay medium	葡萄糖15.0，脱脂奶粉20.0，氯化钾 0.5，硫酸镁 0.5
淀粉酶检测培养基Amylase assay medium	牛肉膏5.0，蛋白胨10.0，氯化钠 5.0，可溶性淀粉10.0
铁载体检测培养基主要成分Siderophore assay medium	葡萄糖4.0，硝酸钾0.2，硫酸镁 0.1，氯化钠0.1，磷酸氢二钾 0.1，硫酸亚铁 0.002
Landy发酵培养基Landy fermentation medium	葡萄糖20.0，谷氨酸钠 5.0, 酵母粉1.0, 磷酸二氢钾 1.0，硫酸镁 0.5，氯化钾 0.5，硫酸锰 0.000 05，硫酸铜 0.000 16，硫酸亚铁 0.000 15，苯丙氨酸 0.02

培养基名称Name of culture medium	组分Component/（g·L^-1）
马铃薯葡萄糖培养基Potato dextrose medium, PDB	马铃薯200.0，葡萄糖20.0
LB培养基Luria-Bertani medium	胰蛋白胨10.0，酵母粉5.0，氯化钠10.0
PDB改良培养基Modified potato dextrose broth	马铃薯200.0，麦芽糖20.0，蛋白胨10.0，硫酸铵5.0，磷酸氢二钠1.5
纤维素酶检测培养基Cellulase assay medium	羧甲基纤维素钠 2.0，硫酸铵 2.0，磷酸二氢钾 1.0，硫酸镁 0.5，刚果红 0.1
蛋白酶检测培养基Protease assay medium	葡萄糖15.0，脱脂奶粉20.0，氯化钾 0.5，硫酸镁 0.5
淀粉酶检测培养基Amylase assay medium	牛肉膏5.0，蛋白胨10.0，氯化钠 5.0，可溶性淀粉10.0
铁载体检测培养基主要成分Siderophore assay medium	葡萄糖4.0，硝酸钾0.2，硫酸镁 0.1，氯化钠0.1，磷酸氢二钾 0.1，硫酸亚铁 0.002
Landy发酵培养基Landy fermentation medium	葡萄糖20.0，谷氨酸钠 5.0, 酵母粉1.0, 磷酸二氢钾 1.0，硫酸镁 0.5，氯化钾 0.5，硫酸锰 0.000 05，硫酸铜 0.000 16，硫酸亚铁 0.000 15，苯丙氨酸 0.02

病原菌 Pathogenic bacterium	抑菌率 Inhibition rate/%（x±s, n=3）
栝楼炭疽病菌C. gloeosporioides	57.59±0.00a
梨炭疽病菌C. fructicola	38.97±0.01c
枣炭疽病菌C. coccodes	48.54±0.01b
苹果炭疽病菌C. malicorticis	34.27±0.01d

病原菌 Pathogenic bacterium	抑菌率 Inhibition rate/%（x±s, n=3）
栝楼炭疽病菌C. gloeosporioides	57.59±0.00a
梨炭疽病菌C. fructicola	38.97±0.01c
枣炭疽病菌C. coccodes	48.54±0.01b
苹果炭疽病菌C. malicorticis	34.27±0.01d

检测项目 Test item	检测结果 Test result
葡萄糖（产酸产气）Glucose（Producing acid and gas）	+
乳糖 Lactose	+
麦芽糖 Maltose	-
甘露醇 Mannitol	-
蔗糖 Sucrose	+
蛋白胨水 Peptone water	-
V-P试验Voges-Proskauer reaction	+
西蒙氏柠檬酸盐 Symon's phosphate	+
硫化氢 H₂S production	-
尿素 Urea	-
半固体琼脂Semi-solid agar	-

生防细菌HX0037对栝楼炭疽病的防病能力及其机制

Efficacy and Its Mechanism of Bacterial Strain HX0037 on the Control of Anthracnose Disease of Trichosanthes kirilowii Maxim

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参考文献 57

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基因类型 Type	数目 Number	长度 Length/bp	占基因组大小 Proportion in genome/%
CDS	3 712	3 436 344	88.38
tRNA	82	6 330	0.16
16S rRNA	9	13 929	0.36
23S rRNA	9	26 370	0.68
5S rRNA	9	1 044	0.03

基因簇Cluster	类型Type	开始From	结束To	最相似的基因簇 The most similar gene cluster	相似度Similarity/%
Cluster1	NRPS, betalactone	1	101 972	Most similar known cluster	86
Cluster2	TransAT-PKS	165 428	266 126	Fengycin	100
Cluster3	TransAT-PKS	490 930	577 278	Bacillaene	100
Cluster4	Terpene	897 298	913 914	Macrolactin	-
Cluster5	PKS-like	998 755	1 039 999	-	7
Cluster6	NRPS	1 570 321	1 635 119	ButirosinAB	82
Cluster7	Lanthipeptide-class-iii	1 730 700	1 753 315	Surfactin	35
Cluster8	Other	2 237 949	2 279 367	Locillomycin	100
Cluster9	Pipp-like,NRPS	2 809 532	2 860 040	Bacilysin	100
Cluster10	TransAT-PKS	3 480 099	3 573 848	Bacillibactin	100
Cluster11	T3PKS	3 701 691	3 742 791	Difficidin	-
Cluster12	Terpene	3 806 279	3 828 162	-	-

菌株Strain	GenBank登录号GenBank No.	平均核苷酸一致性ANI/%	GC含量GC conten t /%	大小Size/bp
B. amyloliquefaciens L-H15	CP010556	98.56	46.60	3 905 973
B. amyloliquefaciens S499	CP014700	98.55	46.60	3 927 922
B. amyloliquefaciens EA19	CP079834	98.55	40.20	3 964 177
B. amyloliquefaciens FZB42	CP000560	97.78	46.48	3 918 596
B. subtilis BSD-2	CP013654	79.91	43.88	4 030 837
B. subtilis 7PJ-16	CP023409	79.87	43.28	4 209 045
B. licheniformis T5	CP124852	76.48	46.16	4 247 430
B. licheniformis TAB7	CP027789	76.46	45.82	4 367 367
B. megaterium HGS7	CP065213	74.48	38.27	5 035 031
B. megaterium QM B1551	CP001983	74.44	38.20	5 097 129
B. cereus ATCC 14579	NC004722	74.23	35.30	5 097 129

质量峰 Mass peak(m/z)	碎片离子 Assignment	参考文献 Reference
1 030.64	Surfactin A C₁₃[M+Na]⁺	[33]
	Surfactin B C₁₄[M+Na]⁺	[33]
1 044.64	Surfactin A C₁₄[M+Na]⁺	[34]
	Surfactin B C₁₅[M+Na]⁺	[34]
1 058.67	Surfactin A C₁₅[M+Na]⁺	[34]
1 072.69	Surfactin A C₁₆[M+Na]⁺	[35]
1 008.66	Surfactin A C₁₃[M+H]⁺	[35]
1 022.67	Surfactin A C₁₄[M+H]⁺	[35]
1 036.69	Surfactin A C₁₅[M+H]⁺	[35]
1 050.71	Surfactin A C₁₆[M+H]⁺	[35]
1 463.81	Fengycin B C₁₄[M+H]⁺	[35]
	Fengycin A C₁₄[M+H]⁺	[35]
1 477.82	Fengycin B C₁₅[M+H]⁺	[35]
	Fengycin A C₁₅[M+H]⁺	[35]
1 491.84	Fengycin B C₁₆[M+H]⁺	[35]
	Fengycin A C₁₆[M+H]⁺	[35]

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