梨火疫病拮抗菌JK2的筛选鉴定及发酵条件优化

doi:10.13560/j.cnki.biotech.bull.1985.2024-0202

摘要/Abstract

摘要：

【目的】 梨火疫病是由解淀粉欧文氏菌（Erwinia amylovora）引起的一种重要细菌病害，严重影响梨、苹果等蔷薇仁果类果树的生产安全。从海棠树（Malus spectabilis）果实及其根际土壤分离得到对解淀粉欧文氏菌具有较强拮抗作用的优良菌株，为梨火疫病生防菌剂的开发与利用提供菌种资源及理论支撑。【方法】 采用平板稀释法分离菌株，以解淀粉欧文氏菌（Erwinia amylovora）为指示菌，利用平板对峙法和牛津杯扩散法初筛和复筛具有拮抗效果的菌株，结合形态学观察、生理生化反应和16S rDNA测序对拮抗菌进行鉴定，确定其分类地位；以单因素试验和正交试验对发酵条件进行优化；将优化的发酵液在新疆野苹果（Malus sieversii）离体果实上进行温室防效测定。【结果】 分离得到4株具有较强拮抗作用的菌株（JK1、JK2、JK3、JK4），其中拮抗效果最强的菌株JK2抑菌效价达到359.7 mm/mL，结合该菌形态学特征、生理生化特性和16S rDNA基因序列分析，将菌株JK2鉴定为贝莱斯芽孢杆菌（Bacillus velezensis）。优化后的最佳培养条件：初始pH为6.66，温度为30.6℃，接种量为3.9%，转速为208 r/min。在该优化条件下，活菌数可达到1.29×10⁹ CFU/mL，相比优化前提高了53.1%；温室防效测定结果表明，接种菌株JK2果实的保护性试验病情指数显著降低，防效达到65.88%，且保护性防效优于治疗性防效。【结论】 菌株JK2对梨火疫病具有较好的防治作用，具有较大的应用潜力，可作为开发梨火疫病生防制剂的候选菌株。

关键词: 梨火疫病, 生物防治, 贝莱斯芽孢杆菌, 发酵条件

Abstract:

【Objective】 Fire blight is a devasting bacterial disease caused by Erwinia amylovora, which seriously affects the production safety of rosaceous plants, such as pears and apples. To explore potential bacterial strains as biocontrol agents for managing fire blight, antagonistic bacteria were isolated from fruits and rhizosphere soil of Malus spectabilis. 【Method】 Biocontrol strains were isolated using plate dilution method, using E. amylovora as the indicator bacterium. Strains with antagonistic effects were screened and rescreened using plate confrontation method and Oxford cup diffusion method. The antagonistic bacteria were identified by morphological observation, physiological and biochemical tests and 16S rDNA sequence homology analysis. The fermentation of antagonistic bacteria were optimized using the single-factor procedure and orthogonal experiments. The fermentation broth of antagonistic bacteria was used to determine the in vitro antagonistic activity against E. amylovora on the fruit of wild apple（Malus sieversii）. 【Result】 Four strains with strong antagonistic effects were isolated（JK1, JK2, JK3 and JK4）, and the strain JK2 showed the strongest antagonistic effect on E. amylovora with a bacteriostatic potency of 359.7 mm/mL. Based on morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain JK2 was identified as Bacillus velezensis. The optimized culture conditions were determined as follows: initial pH of 6.66, culture temperature of 30.6℃, inoculation size of 3.9%, and rotation speed of 208 r/min. Under these conditions, the number of viable counts was 1.29×10⁹ CFU/mL, a significant increase（53.1%）as compared to the control. The control effects in the fruit of wild apple in greenhouse showed that the disease index of the protective test inoculated with strain JK2 significantly reduced, reaching 65.88%, and the protective effect was better than the therapeutic effect. 【Conclusion】 Strain JK2 demonstrates considerable potential for controlling fire blight, which is a promising candidate for use as a biological control agent.

Key words: fire blight disease, biological control, Bacillus velezensis, fermentation conditions

马云涛, 胡丽娜, 孙文婧, 唐莲庚, 孙思远, 邓欣雨, 孙黎. 梨火疫病拮抗菌JK2的筛选鉴定及发酵条件优化[J]. 生物技术通报, 2024, 40(11): 202-213.

MA Yun-tao, HU Li-na, SUN Wen-jing, TANG Lian-geng, SUN Si-yuan, DENG Xin-yu, SUN Li. Screening and Identification of Antagonistic Bacterium JK2 Against Fire Blight Disease and the Optimization of Its Fermentation Conditions[J]. Biotechnology Bulletin, 2024, 40(11): 202-213.

图/表 14

参考文献 36

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拮抗菌 Antagonistic bacteria	JK1	JK2	JK3	JK4
抑菌圈直径Diameter of bacteriostatic ring/mm	4.0±0.1^c	17.9±0.2^a	4.0±0.1^c	13.1±0.2^b
抑菌效价Antibacterial potency/（mm·mL^-1）	79.6±6.6^c	359.7±3.9^a	80.3±4.3^c	266.7±9.5^b

拮抗菌 Antagonistic bacteria	JK1	JK2	JK3	JK4
抑菌圈直径Diameter of bacteriostatic ring/mm	4.0±0.1^c	17.9±0.2^a	4.0±0.1^c	13.1±0.2^b
抑菌效价Antibacterial potency/（mm·mL^-1）	79.6±6.6^c	359.7±3.9^a	80.3±4.3^c	266.7±9.5^b

测定内容Measured item	菌株JK2 Strain JK2
VP	-
MR	+
明胶液化Gelaune liquefaction	+
淀粉水解Starch hydrolysis	+
葡萄糖Glucose	+
甘露醇Mannitol	+
蔗糖Sucrose	+
阿拉伯糖Arabinose	+
丙二酸盐利用Propanedioic acid utilization	-
西蒙氏柠檬酸盐利用Simmons citrate utilization	-
溶菌酶 Lysozyme	-

测定内容Measured item	菌株JK2 Strain JK2
VP	-
MR	+
明胶液化Gelaune liquefaction	+
淀粉水解Starch hydrolysis	+
葡萄糖Glucose	+
甘露醇Mannitol	+
蔗糖Sucrose	+
阿拉伯糖Arabinose	+
丙二酸盐利用Propanedioic acid utilization	-
西蒙氏柠檬酸盐利用Simmons citrate utilization	-
溶菌酶 Lysozyme	-

No.	A	B/%	C/℃	D/（r·min^-1）	OD₆₀₀	No.	A	B/%	C/℃	D/（r·min^-1）	OD₆₀₀
1	5.5	1	30	200	2.018	16	6.5	5	32	200	3.789
2	7.5	1	30	200	2.609	17	5.5	3	28	200	2.153
3	5.5	5	30	200	2.336	18	7.5	3	28	200	2.619
4	7.5	5	30	200	3.086	19	5.5	3	32	200	2.018
5	6.5	3	28	180	2.585	20	7.5	3	32	200	2.846
6	6.5	3	32	180	2.886	21	6.5	1	30	180	2.729
7	6.5	3	28	220	2.706	22	6.5	5	30	180	3.112
8	6.5	3	32	220	3.495	23	6.5	1	30	220	3.161
9	5.5	3	30	180	1.735	24	6.5	5	30	220	3.629
10	7.5	3	30	180	1.839	25	6.5	3	30	200	4.159
11	5.5	3	30	220	2.552	26	6.5	3	30	200	3.861
12	7.5	3	30	220	3.077	27	6.5	3	30	200	3.827
13	6.5	1	28	200	2.683	28	6.5	3	30	200	3.692
14	6.5	5	28	200	3.021	29	6.5	3	30	200	3.945
15	6.5	1	32	200	3.229