Screening， Identification and Optimization of Fermentation Conditions of Antagonistic Bacteria against Potato Early Blight

doi:10.13560/j.cnki.biotech.bull.1985.2025-0264

Abstract

Abstract:

Objective Potato early blight, caused by Alternaria solani, is a global crop disease that severely threatens potato yield and quality. This study is aimed to isolate strains with strong inhibitory effects against A. solani from potato rhizosphere soil and optimize their fermentation conditions, providing a microbial resource for developing biocontrol agents against potato early blight. Method Antagonistic strains against A. solani were screened using gradient dilution and plate confrontation methods. The strains were identified through morphological observation, API 50CH physiological-biochemical tests, 16S rRNA gene sequencing, and gyrB gene sequencing. Single-factor and orthogonal experiments were conducted to optimize the fermentation conditions of the antagonistic strains. Result Six antagonistic strains against A. solani were isolated, and strain LYB08 showing the highest fungistatic rate 54.10%. Strain LYB08 was identified as Bacillus velezensis. After optimization, the optimal carbon and nitrogen sources for fermentation were corn starch and soybean meal. When fermented with corn starch 30 g/L, soybean meal 20 g/L, and initial pH 7.5, the viable spore number reached 1.17×10¹⁰ CFU/mL, total number of viable bacteria was 1.32×10¹⁰ CFU/mL, representing increases of 120.75% and 116.39% respectively, compared to the initial medium. When fermented with corn starch 30 g/L, soybean meal 30 g/L, and initial pH 8.5, the fungistatic rate of the fermentation broth peaked at 81.04%, a 50.49% improvement over the initial medium. Conclusion An antagonistic strain LYB08 is successfully isolated against potato early blight, which may provide a basis for the development of biocontrol agents for this disease.

Key words: biocontrol agent, potato early blight, screening and identification, fermentation conditions, Bacillus velezensis

LYU Ji-min, LIU Wei, SUN Min, LI Hong-shun, PENG Zhen-xing, QIU Peng-fei, ZHU Qi-li. Screening， Identification and Optimization of Fermentation Conditions of Antagonistic Bacteria against Potato Early Blight[J]. Biotechnology Bulletin, 2025, 41(10): 175-185.

Figures/Tables 13

References 34

[1]	崔勇, 李培贵, 杨桂琴, 等. 马铃薯晚疫病防治研究进展 [J]. 中国植保导刊, 2024, 44(12): 21-30.
	Cui Y, Li PG, Yang GQ, et al. Study on the control of potato late blight disease [J]. China Plant Prot, 2024, 44(12): 21-30.
[2]	Nnodu EC, Harrison MD, Workman M. The effect of storage environment on the infection of potato tubers by Alternaria solani [J]. Am Potato J, 1982, 59(7): 313-325.
[3]	杨登路, 雷帮星, 康冀川, 等. 马铃薯早疫病拮抗菌株筛选及其抗菌活性 [J]. 山地农业生物学报, 2016, 35(1): 62-66.
	Yang DL, Lei BX, Kang JC, et al. The screening of antagonistic strains to potato early blight and detection of antimicrobial activity [J]. J Mt Agric Biol, 2016, 35(1): 62-66.
[4]	夏善勇. 马铃薯早疫病及综合防治研究进展 [J]. 农业科技通讯, 2023(4): 152-156.
	Xia SY. Research progress of potato early blight and its integrated control [J]. Bull Agric Sci Technol, 2023(4): 152-156.
[5]	范子耀, 王文桥, 孟润杰, 等. 马铃薯早疫病病原菌鉴定及其对不同药剂的敏感性 [J]. 植物病理学报, 2013, 43(1): 69-74.
	Fan ZY, Wang WQ, Meng RJ, et al. Identification of the pathogens of potato early blight and their sensitivity to different fungicides [J]. Acta Phytopathol Sin, 2013, 43(1): 69-74.
[6]	韩龙, 康冀川, 雷帮星, 等. 马铃薯早疫病拮抗菌及其抑菌作用研究 [J]. 山地农业生物学报, 2018, 37(1): 27-31, 43.
	Han L, Kang JC, Lei BX, et al. A study on antagonistic strains for potato early blight and their antimicrobial activity [J]. J Mt Agric Biol, 2018, 37(1): 27-31, 43.
[7]	王怡凡, 刘巍, 朱其立, 等. 马铃薯早疫病拮抗细菌WK-1的筛选鉴定及其生物学特性分析 [J]. 西南农业学报,2022,35(4): 855-862.
	Wang YF, Liu W, Zhu QL, et al. Screening and identification of potato early blight antagonistic bacteria WK-1 and analysis of its biological characteristics [J]. Southwest China Journal of Agricultural Sciences, 2022, 35(4): 855-862.
[8]	杨继业, 杨帆, 崔冠慧, 等. 马铃薯早疫病拮抗菌的筛选及其代谢产物的抑菌活性 [J]. 中国蔬菜, 2018(5): 62-66.
	Yang JY, Yang F, Cui GH, et al. Screening of antagonistic bacteria against potato early blight and antibacterial activity of its metabolites [J]. China Veg, 2018(5): 62-66.
[9]	于水清, 杨毅清, 张岱, 等. 马铃薯早疫病拮抗细菌的筛选、鉴定及抑菌物质研究 [J]. 西南农业学报, 2021, 34(6): 1234-1241.
	Yu SQ, Yang YQ, Zhang D, et al. Screening, identification and its antifungal substances of antagonistic bacterium against potato early blight [J]. Southwest China J Agric Sci, 2021, 34(6): 1234-1241.
[10]	冯志珍, 陈太春, 段军娜, 等. 烟草黑胫病拮抗根际芽胞杆菌FB-16的筛选鉴定及其抑菌活性 [J]. 植物保护学报, 2012, 39(3): 224-230.
	Feng ZZ, Chen TC, Duan JN, et al. Screening, identification and antifungal activity of antagonistic rhizospheric Bacillus FB-16 against tobacco black shank [J]. J Plant Prot, 2012, 39(3): 224-230.
[11]	Zhao YJ, Selvaraj JN, Xing FG, et al. Antagonistic action of Bacillus subtilis strain SG6 on Fusarium graminearum [J]. PLoS One, 2014, 9(3): e92486.
[12]	Cui XL, Mao PH, Zeng M, et al. Streptimonospora Salina gen. nov., sp. nov., a new member of the family Nocardiopsaceae [J]. Int J Syst Evol Microbiol, 2001, 51(Pt 2): 357-363.
[13]	武志华, 郭维维, 董晔, 等. 抗马铃薯晚疫病菌的粘细菌菌株X6-Ⅱ-1的分离鉴定、拮抗活性及发酵条件的优化 [J]. 农业生物技术学报, 2018, 26(9): 1467-1479.
	Wu ZH, Guo WW, Dong Y, et al. Isolation and identification of myxobacterial strain X6-Ⅱ-1 resistant to Phytophthora infestans and its antibiotic activity and optimal fermentation condition [J]. J Agric Biotechnol, 2018, 26(9): 1467-1479.
[14]	王世信, 杨美娜, 崔友祥, 等. 正交试验设计优选芪参还五胶囊的制备工艺 [J]. 食品与药品, 2022, 24(3): 200-203.
	Wang SX, Yang MN, Cui YX, et al. Optimization of preparation process of qishen Huanwu capsules by orthogonal design [J]. Food Drug, 2022, 24(3): 200-203.
[15]	王钊, 李长滨, 李玉江, 等. 正交法优化超声波辅助枣皮红色素提取工艺研究 [J]. 中国调味品, 2021, 46(11): 161-165.
	Wang Z, Li CB, Li YJ, et al. Study on optimization of ultrasonic-assisted extraction process of red pigment from jujube peel by orthogonal test design [J]. China Condiment, 2021, 46(11): 161-165.
[16]	Chen K, Tian ZH, He H, et al. Bacillus species as potential biocontrol agents against Citrus diseases [J]. Biol Control, 2020, 151: 104419.
[17]	Chandran H, Meena M, Swapnil P. Plant growth-promoting rhizobacteria as a green alternative for sustainable agriculture [J]. Sustainability, 2021, 13(19): 10986.
[18]	Wang BJ, Pu Q, Zhang YH, et al. Secretion and volatile components contribute to the antagonism of Bacillus velezensis 1-10 against fungal pathogens [J]. Biol Control, 2023, 187: 105379.
[19]	马佳, 李颖, 胡栋, 等. 芽胞杆菌生物防治作用机理与应用研究进展 [J]. 中国生物防治学报, 2018, 34(4): 639-648.
	Ma J, Li Y, Hu D, et al. Progress on mechanism and applications of Bacillus as a biocontrol microbe [J]. Chin J Biol Control, 2018, 34(4): 639-648.
[20]	姜旭. 利用微生物防治植物病害研究进展 [J]. 园艺与种苗, 2018(6): 57-58, 62.
	Jiang X. Study on biological control of plant disease with microorganism [J]. Horticulture & Seed, 2018, (6): 57-58, 62.
[21]	黄慧婧, 罗坤. 芽胞杆菌与杀菌剂复配防治植物病害的研究进展 [J]. 微生物学通报, 2021, 48(3): 938-947.
	Huang HJ, Luo K. Research progress in the control of plant diseases by the combination of Bacillus and fungicides [J]. Microbiol China, 2021, 48(3): 938-947.
[22]	Ruiz-García C, Béjar V, Martínez-Checa F, et al. Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain [J]. Int J Syst Evol Microbiol, 2005, 55(Pt 1): 191-195.
[23]	余春芳, 王展鹏, 谭宇航, 等. 白芨根腐病菌腐皮镰孢（Fusarium solani）拮抗菌筛选及抑菌机制 [J]. 微生物学通报, 2025, 52(6): 2666-2678.
	Yu CF, Wang ZP, Tan YH, et al. Screening of antagonistic bacteria against Fusarium solani in Bletilla striata and investigation of their inhibitory mechanisms [J]. Microbiology China, 2025, 52(6): 2666-2678.
[24]	吴国平, 杨利娟, 张春祥, 等. 黄瓜根腐病生防芽胞杆菌的筛选、鉴定及协同效应 [J]. 山东农业科学, 2025, 57(2): 134-140.
	Wu GP, Yang LJ, Zhang CX, et al. Screening, identification and synergistic effects of biocontrol Bacillus for cucumber root rot [J]. Shandong Agricultural Sciences, 2025, 57(2): 134-140.
[25]	丁立孝, 杨增军. 防治烟草赤星病农用抗生素产生菌S-10菌株发酵条件的研究 [J]. 莱阳农学院学报, 1996, 13(3): 6-10.
	Ding LX, Yang ZJ. Study on fermentation conditions of agricultural antibiotic-producing strain S-10 for controlling tobacco alternaria leaf spot [J]. Journal of Laiyang Agricultural College, 1996, 13(3): 6-10.
[26]	王东昌. 链霉菌S-10菌株发酵条件的研究 [J]. 吉林农业大学学报, 2001, 23(1): 35-39.
	Wang DC. Fermenting conditions of S-10 strain of Streptomyces [J]. J Jilin Agric Univ, 2001, 23(1): 35-39.
[27]	何伟, 罗文芳, 周军辉, 等. 贝莱斯芽胞杆菌JTB8-2对加工番茄促生作用及其安全性评价 [J]. 新疆农业科学, 2022, 59(5): 1260-1269.
	He W, Luo WF, Zhou JH, et al. Growth promoting effect and safety evaluation of Bacillus velezensis JTB8-2 on processed tomato [J]. Xinjiang Agric Sci, 2022, 59(5): 1260-1269.
[28]	刘军, 江宇琪, 刘康, 等.贝莱斯芽胞杆菌产抑菌物质培养基发酵优化 [J]. 现代食品科技, 2024, 40(6): 114-123.
	Liu J, Jiang YQ, Liu K, et al. Optimization of fermentation medium for the production of antibacterial substances by Bacillus velezensis [J]. Modern Food Science and Technology, 2024, 40(6): 114-123.
[29]	李紫英. 贝莱斯芽胞杆菌SY01 发酵条件优化及对苹果树腐烂病菌抑菌机理初探 [D]. 阿拉尔:塔里木大学, 2025.
	Li ZY. Optimization of fermentation conditions for Bacillus velezensis SY01 and preliminary exploration of its antibacterial mechanism against apple tree canker pathogen [D]. Aril: Tarim University, 2025.
[30]	邓玉莲, 牛丽, 谭琳, 等.茶树根腐病菌拮抗真菌的筛选及发酵条件优化[J]. 微生物学通报, 2025. DOI: 10.13344/j.microbiol.china.241059 .
	Deng YL, Niu L, Tan L, et al. Screening and fermentation condition optimization of an antagonistic fungus against Fusarium cugenangense [J]. Microbiology China, 2025. DOI: 10.13344/j.microbiol.china.241059 .
[31]	杨然迪, 杨怡妍, 曹灏, 等. 贝莱斯芽胞杆菌TCS001产脂肽类物质抑菌活性及发酵条件优化 [J]. 农药学学报, 2024, 26(3): 504-516.
	Yang RD, Yang YY, Cao H, et al. Antifungal activity and fermentation optimization of lipopeptides produced by Bacillus velezensis TCS001 [J]. Chinese Journal of Pesticide Science, 2024,26(3): 504-516.
[32]	陈尚里, 于福田, 沈圆圆, 等. 高产抗菌脂肽Fengycin 芽胞杆菌的诱变育种和发酵条件优化 [J]. 食品工业科技, 2023, 44(23): 134-143.
	Chen SL, Yu FT, Shen YY, et al. Mutation breeding and optimization of fermentation conditions of Bacillus highly producing antimicrobial lipopeptide fengycin [J]. Sci Technol Food Ind, 2023,44(23): 134-143.
[33]	Schoenborn AA, Yannarell SM, Wallace ED, et al. Defining the expression, production, and signaling roles of specialized metabolites during Bacillus subtilis differentiation [J]. J Bacteriol, 2021, 203(22): e0033721.
[34]	Yi YX, Chen MT, Yang HR, et al. New insights into the role of cellular states, cell-secreted metabolites, and essential nutrients in biofilm formation and menaquinone-7 biosynthesis in Bacillus subtilis natto [J]. Food Res Int, 2025, 206: 116052.

项目 Item	检测结果 Result	项目 Item	检测结果 Result
对照Contrast	-	七叶灵Esculin hydrate	+
甘油Glycerol	-	柳醇Salicin	W
赤癣醇Erythritol	-	纤维二糖 Cellobiose	W
D-阿拉伯糖D-arabinose	-	麦芽糖Maltose	W
L-阿拉伯糖L-arabinose	W	乳糖Lactose	W
核糖Ribose	W	蜜二糖Melibiose	-
D-木糖D-Xylose L-Xylose Adong alcohol	W	蔗糖Sucrose	+
L-木糖L-Xylose	-	海藻糖Trehalose	W
阿东醇Adonitoll	-	菊糖Inulin	-
β-甲基-D-木糖甙 β-methyl-D-xyloside	-	松叁糖Melezitose	-
半乳糖Galactose	-	棉子糖Xylosucrose	W
葡萄糖Glucose	+	淀粉Starch	W
果糖Fructose	+	糖原Glycogen	W
甘露糖Mannose	+	木糖醇Xylitol	-
山梨糖Sorbose	-	龙胆二糖Gentiobiose	W
鼠李糖Rhamnopyranose	-	D-松二糖D-turanose	-
卫茅醇Dulcitol	-	D-来苏糖D-lyxose	-
肌醇Inositol	-	D-塔格糖D-tagatose	-
甘露醇Mannitol	W	D-岩糖D-fucose	-
山梨醇Sorbitol	W	L-岩糖L-fucose	-
α-甲基-D-甘露糖甙 α-methyl-D-mannose glycoside	-	D-阿拉伯糖醇D-arabitol	-
α-甲基-D-葡萄糖甙 α-methyl-D-glucoside	W	L-阿拉伯糖醇L-arabitol	-
N-乙酰葡糖胺 N-acetyl glucosamine	W	葡萄糖酸盐Glucose hydrochloride	-
苦杏仁甙Amygdalin	W	2-酮基-葡萄糖酸盐2-keto gluconate	-
熊果甙Arbutin	W	5-酮基-葡萄糖酸盐5-keto gluconate	-

项目 Item	检测结果 Result	项目 Item	检测结果 Result
对照Contrast	-	七叶灵Esculin hydrate	+
甘油Glycerol	-	柳醇Salicin	W
赤癣醇Erythritol	-	纤维二糖 Cellobiose	W
D-阿拉伯糖D-arabinose	-	麦芽糖Maltose	W
L-阿拉伯糖L-arabinose	W	乳糖Lactose	W
核糖Ribose	W	蜜二糖Melibiose	-
D-木糖D-Xylose L-Xylose Adong alcohol	W	蔗糖Sucrose	+
L-木糖L-Xylose	-	海藻糖Trehalose	W
阿东醇Adonitoll	-	菊糖Inulin	-
β-甲基-D-木糖甙 β-methyl-D-xyloside	-	松叁糖Melezitose	-
半乳糖Galactose	-	棉子糖Xylosucrose	W
葡萄糖Glucose	+	淀粉Starch	W
果糖Fructose	+	糖原Glycogen	W
甘露糖Mannose	+	木糖醇Xylitol	-
山梨糖Sorbose	-	龙胆二糖Gentiobiose	W
鼠李糖Rhamnopyranose	-	D-松二糖D-turanose	-
卫茅醇Dulcitol	-	D-来苏糖D-lyxose	-
肌醇Inositol	-	D-塔格糖D-tagatose	-
甘露醇Mannitol	W	D-岩糖D-fucose	-
山梨醇Sorbitol	W	L-岩糖L-fucose	-
α-甲基-D-甘露糖甙 α-methyl-D-mannose glycoside	-	D-阿拉伯糖醇D-arabitol	-
α-甲基-D-葡萄糖甙 α-methyl-D-glucoside	W	L-阿拉伯糖醇L-arabitol	-
N-乙酰葡糖胺 N-acetyl glucosamine	W	葡萄糖酸盐Glucose hydrochloride	-
苦杏仁甙Amygdalin	W	2-酮基-葡萄糖酸盐2-keto gluconate	-
熊果甙Arbutin	W	5-酮基-葡萄糖酸盐5-keto gluconate	-

试号 No.		A玉米淀粉浓度 A corn starch concentration （g/L）	B豆粕浓度 B soybean meal concentration （g/L）	C初始pH值 C initial pH value	D空白 D blank	活芽孢数 Number of viable spores （10¹⁰ CFU/mL）	活菌总数 Total number of viable bacteria （10¹⁰ CFU/mL）	产孢率 Sporulation rate （%）	抑菌率 Fungistatic rate （%）
1		20	20	6.5	1	0.81	0.88	92.05	71.23
2		20	30	7.5	2	0.79	0.84	94.05	66.37
3		20	40	8.5	3	0.72	0.74	97.30	62.11
4		30	20	7.5	3	1.17	1.32	88.64	76.35
5		30	30	8.5	1	1.09	1.15	94.78	81.04
6		30	40	6.5	2	0.94	1.04	90.38	73.78
7		40	20	8.5	2	0.86	0.94	91.49	70.49
8		40	30	6.5	3	0.70	0.79	88.61	67.35
9		40	40	7.5	1	0.74	0.80	92.50	69.02
初始培养基发酵液 Initial fermentation broth						0.53	0.61	86.89	53.85
活芽孢数 Number of viable spore	k1	0.77	0.95	0.82	0.88
	k2	1.07	0.86	0.90	0.86
	k3	0.77	0.80	0.89	0.86
	R	0.30	0.15	0.08	0.00
活菌总数 Total number of viable bacteria	k1	0.82	1.05	0.90	0.94
	k2	1.17	0.93	0.99	0.94
	k3	0.84	0.86	0.94	0.95
	R	0.35	0.19	0.08	0.00

试号 No.		A玉米淀粉浓度 A corn starch concentration （g/L）	B豆粕浓度 B soybean meal concentration （g/L）	C初始pH值 C initial pH value	D空白 D blank	活芽孢数 Number of viable spores （10¹⁰ CFU/mL）	活菌总数 Total number of viable bacteria （10¹⁰ CFU/mL）	产孢率 Sporulation rate （%）	抑菌率 Fungistatic rate （%）
1		20	20	6.5	1	0.81	0.88	92.05	71.23
2		20	30	7.5	2	0.79	0.84	94.05	66.37
3		20	40	8.5	3	0.72	0.74	97.30	62.11
4		30	20	7.5	3	1.17	1.32	88.64	76.35
5		30	30	8.5	1	1.09	1.15	94.78	81.04
6		30	40	6.5	2	0.94	1.04	90.38	73.78
7		40	20	8.5	2	0.86	0.94	91.49	70.49
8		40	30	6.5	3	0.70	0.79	88.61	67.35
9		40	40	7.5	1	0.74	0.80	92.50	69.02
初始培养基发酵液 Initial fermentation broth						0.53	0.61	86.89	53.85
活芽孢数 Number of viable spore	k1	0.77	0.95	0.82	0.88
	k2	1.07	0.86	0.90	0.86
	k3	0.77	0.80	0.89	0.86
	R	0.30	0.15	0.08	0.00
活菌总数 Total number of viable bacteria	k1	0.82	1.05	0.90	0.94
	k2	1.17	0.93	0.99	0.94
	k3	0.84	0.86	0.94	0.95
	R	0.35	0.19	0.08	0.00

指标 Target	因素 Factor	Ⅲ 类平方和 Type Ⅲ sum of squares	自由度 df	均方 Mean square	F值 F value	显著性 Significance
活芽孢数 Number of viable spores	玉米淀粉浓度 Corn starch concentration	0.176	2	0.088	316.960	**
	豆粕浓度 Soybean meal concentration	0.033	2	0.016	58.720	*
	初始pH值 Initial pH value	0.012	2	0.006	22.360	*
活菌总数 Total number of viable bacteria	玉米淀粉浓度 Corn starch concentration	0.230	2	0.115	1 477.000	**
	豆粕浓度 Soybean meal concentration	0.054	2	0.027	345.143	**
	初始pH值 Initial pH value	0.010	2	0.005	67.000	*