马铃薯早疫病拮抗菌的筛选鉴定和发酵条件优化

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

摘要/Abstract

摘要：

目的马铃薯早疫病是由茄链格孢菌（Alternaria solani）引起的全球性作物病害，对马铃薯产量和品质造成严重威胁。从马铃薯根际土中分离得到对茄链格孢菌具有较强抑制作用的菌株并对其发酵条件进行优化，为马铃薯早疫病生防菌的开发和应用提供菌种来源。方法采用梯度稀释法和平板对峙法筛选早疫病茄链格孢菌的拮抗菌，并进行形态学观察、API 50CH生理生化检测、16S rRNA基因序列及gyrB 基因序列检测，鉴定拮抗菌株的种属。利用单因素试验和正交试验对拮抗菌株的发酵条件进行优化。结果筛选得到6株对茄链格孢菌具有拮抗作用的菌株，其中菌株LYB08的抑菌率最高，达到54.10%。经鉴定，菌株LYB08为贝莱斯芽胞杆菌（Bacillus velezensis）。经发酵条件优化，菌株LYB08的优势发酵碳、氮源分别为玉米淀粉和豆粕，最适发酵条件为玉米淀粉浓度30 g/L，豆粕浓度20 g/L，初始pH值7.5，在该条件下，活芽孢数为1.17×10¹⁰ CFU/mL，活菌总数为1.32×10¹⁰ CFU/mL，较初始培养基分别提高120.75%、116.39%。当玉米淀粉浓度为30 g/L，豆粕浓度为30 g/L，初始pH值为8.5时，发酵液的抑菌率最高，为81.04%，较初始培养基发酵液的抑菌率提高50.49%。结论筛选得到一株马铃薯早疫病拮抗菌株LYB08，为马铃薯早疫病生防菌剂的开发提供了依据。

关键词: 生防菌, 马铃薯早疫, 筛选鉴定, 发酵条件, 贝莱斯芽胞杆菌

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

吕济敏, 刘巍, 孙敏, 李洪顺, 彭振兴, 邱鹏飞, 朱其立. 马铃薯早疫病拮抗菌的筛选鉴定和发酵条件优化[J]. 生物技术通报, 2025, 41(10): 175-185.

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.

图/表 13

参考文献 34

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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	*