Screening and Identification of Antagonistic Bacterium JK2 Against Fire Blight Disease and the Optimization of Its Fermentation Conditions

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

Abstract

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

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.

Figures/Tables 14

References 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