枯草芽胞杆菌NIP3产伊枯草菌素发酵条件优化及对柑橘青霉病的防效

doi:10.11924/j.issn.1000-6850.casb2024-0095

中国农学通报 ›› 2024, Vol. 40 ›› Issue (35): 26-34.doi: 10.11924/j.issn.1000-6850.casb2024-0095

枯草芽胞杆菌NIP3产伊枯草菌素发酵条件优化及对柑橘青霉病的防效

张悦(), 齐晓华, 徐明玉, 杜春梅()

黑龙江大学，生命科学学院，农业微生物技术教育部工程研究中心，黑龙江省寒区植物基因与生物发酵重点实验室，黑龙江省普通高校微生物重点实验室，哈尔滨 150080

收稿日期:2024-02-10 修回日期:2024-05-15 出版日期:2024-12-15 发布日期:2024-12-12
通讯作者:
杜春梅，女，1972年出生，教授，博士，研究方向：微生物资源挖掘与利用。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学生命科学学院501室，Tel：0451-86609134，E-mail：2002092@hlju.edu.cn。
作者简介:
张悦，女，2000年出生，黑龙江哈尔滨人，研究生，研究方向：微生物资源挖掘与利用。通信地址：150080 黑龙江省哈尔滨市南岗区学府路74号黑龙江大学生命科学学院501室，Tel：0451-86609134，E-mail：zhangyue2354@163.com。
基金资助:
国家自然科学基金资助项目“侧孢短芽孢杆菌与枯草芽孢杆菌共培养抗菌活性增强的生态学机理”(31370511)

Optimization of Fermentation Conditions for Production of Iturin by Bacillus subtilis NIP3 and Its Control Effect on Citrus Blue Mold

ZHANG Yue(), QI Xiaohua, XU Mingyu, DU Chunmei()

Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education / Heilongjiang Provincial Key Laboratory of Plant Genetic Engineering and Biological Fermentation Engineering for Cold Region / Key Laboratory of Microbiology, College of Heilongjiang Province / School of Life Sciences, Heilongjiang University, Harbin 150080

Received:2024-02-10 Revised:2024-05-15 Published:2024-12-15 Online:2024-12-12

摘要/Abstract

摘要：

柑橘青霉病是柑橘类水果储运期的常发病害，造成的经济损失严重。伊枯草菌素具有防治该病害的潜力，但是产量较低。本研究对枯草芽胞杆菌NIP3产生伊枯草菌素的3个关键因素进行了响应面优化，并分析了伊枯草菌素在体内和体外对意大利青霉的防治效果。响应面分析结果表明，枯草芽胞杆菌NIP3产生伊枯草菌素的最佳葡萄糖浓度为30 g/L、L-谷氨酸钠浓度为6 g/L、温度为33℃。在此条件下，伊枯草菌素产量达到721.33±3.72 mg/L，较优化前提高了51.59%。检测方法对伊枯草菌素的体外防治效果有显著的影响。利用杯碟法测得伊枯草菌素对意大利青霉生长的最低有效浓度为312.5 μg/mL。而利用凹玻片法和菌丝体干重法测得的伊枯草菌素抑制意大利青霉孢子萌发和菌丝体生长的有效中浓度(EC₅₀)分别为29.11 μg/mL和22.80 μg/mL。果实体内防效试验表明，10 mg/mL的伊枯草菌素对柑橘青霉病的预防效果好于治疗效果，且发病抑制率可以达到100%。本研究为开发柑橘青霉病生物防治剂提供了科学依据。

关键词: 柑橘青霉病, 枯草芽胞杆菌, 伊枯草菌素, 响应面优化, 防效

Abstract:

Citrus blue mold is a prevalent disease affecting citrus fruits during storage and transportation, leading to serious economic losses. Iturins exhibit potential for controlling citrus blue mold disease, but its yield remains low. This study conducted the response surface optimization on 3 key factors influencing the production of iturins by Bacillus subtilis NIP3, and analyzed the efficacy of iturins against Penicillium italicum both in vivo and in vitro. The results from response surface analysis showed that the optimal concentrations of glucose and L-glutamate, as well as temperature, were 30 g/L, 6 g/L, and 33℃ respectively for maximizing iturin production by B. subtilis NIP3. Under these optimized conditions, the yield of iturins reached 721.33±3.72 mg/L, an increase of 51.59% compared to pre-optimization levels. The detection method significantly influenced the control efficacy of iturins in vitro. The Oxford cup diffusion method revealed that the lowest effective concentration of iturins inhibiting P. italicum growth was 312.5 μg/mL. Furthermore, the effective concentration (EC₅₀) inhibiting spore germination and mycelium growth were determined to be 29.11 μg/mL and 22.80 μg/mL respectively when measured via hollow slide method and mycelium dry weight method. Results demonstrated that while at a concentration of 10 mg/mL, iturins exhibited superior preventive effects over therapeutic effects against citrus penicilliosis, with an incidence inhibition rate reaching up to 100%. This study provides a scientific foundation for developing biocontrol agents targeting citrus blue mold disease.

Key words: citrus blue mold disease, Bacillus subtilis, iturin, response surface optimization, control effect

张悦, 齐晓华, 徐明玉, 杜春梅. 枯草芽胞杆菌NIP3产伊枯草菌素发酵条件优化及对柑橘青霉病的防效[J]. 中国农学通报, 2024, 40(35): 26-34.

ZHANG Yue, QI Xiaohua, XU Mingyu, DU Chunmei. Optimization of Fermentation Conditions for Production of Iturin by Bacillus subtilis NIP3 and Its Control Effect on Citrus Blue Mold[J]. Chinese Agricultural Science Bulletin, 2024, 40(35): 26-34.

图/表 9

参考文献 39

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方差来源	平方和	自由度Df	均方	F-值	P
模型	23749.86	9	2638.87	122.17	0.0001^**
A	60.50	1	2.80	2.80	0.1381
B	220.50	1	10.21	10.21	0.0152^*
C	180.50	1	8.36	8.36	0.0233^*
AB	90.25	1	4.18	4.18	0.0802
AC	42.25	1	1.96	1.96	0.2406
BC	56.25	1	2.60	2.6	0.1506
A²	12818.02	1	593.43	593.43	0.0001^**
B²	2775.60	1	128.50	128.5	0.0002^**
C²	5358.76	1	248.09	248.09	0.0001^**
Residual	151.20	7	21.06
Lack-of-fit	10	3	3.33	0.0944	0.9592
Pure Error	141.20	4	35.30
Cor Total	23901.06	16
R²	0.9937

方差来源	平方和	自由度Df	均方	F-值	P
模型	23749.86	9	2638.87	122.17	0.0001^**
A	60.50	1	2.80	2.80	0.1381
B	220.50	1	10.21	10.21	0.0152^*
C	180.50	1	8.36	8.36	0.0233^*
AB	90.25	1	4.18	4.18	0.0802
AC	42.25	1	1.96	1.96	0.2406
BC	56.25	1	2.60	2.6	0.1506
A²	12818.02	1	593.43	593.43	0.0001^**
B²	2775.60	1	128.50	128.5	0.0002^**
C²	5358.76	1	248.09	248.09	0.0001^**
Residual	151.20	7	21.06
Lack-of-fit	10	3	3.33	0.0944	0.9592
Pure Error	141.20	4	35.30
Cor Total	23901.06	16
R²	0.9937

伊枯草菌素浓度/(μg/mL)	孢子萌发率/%	相对抑制率/%	菌丝体干重/mg	相对抑制率/%
CK	71.17±1.53g	—	178.49±2.36g	—
6.25	52.17±1.04f	26.70	131.653±3.53f	26.24
12.5	42.83±1.53e	39.82	106.86±2.87e	40.13
25	35.67±1.26d	49.88	86.55±0.81d	51.51
50	31.5±1.8c	55.74	66.33±2.05c	62.84
100	21.67±0.76b	69.55	44.37±4.06b	75.14
200	12.83±3.51a	81.97	24.10±2.69a	86.50
毒力回归方程	y=3.5028x-0.0314		y = 3.8084x-0.1716
EC₅₀/(μg/mL)	29.11		22.80

伊枯草菌素浓度/(μg/mL)	孢子萌发率/%	相对抑制率/%	菌丝体干重/mg	相对抑制率/%
CK	71.17±1.53g	—	178.49±2.36g	—
6.25	52.17±1.04f	26.70	131.653±3.53f	26.24
12.5	42.83±1.53e	39.82	106.86±2.87e	40.13
25	35.67±1.26d	49.88	86.55±0.81d	51.51
50	31.5±1.8c	55.74	66.33±2.05c	62.84
100	21.67±0.76b	69.55	44.37±4.06b	75.14
200	12.83±3.51a	81.97	24.10±2.69a	86.50
毒力回归方程	y=3.5028x-0.0314		y = 3.8084x-0.1716
EC₅₀/(μg/mL)	29.11		22.80

枯草芽胞杆菌NIP3产伊枯草菌素发酵条件优化及对柑橘青霉病的防效

Optimization of Fermentation Conditions for Production of Iturin by Bacillus subtilis NIP3 and Its Control Effect on Citrus Blue Mold

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因素	样本	水平
因素	样本	-1	0	1
谷氨酸/(g/L)	A	0.15	0.19	0.24
葡萄糖/(g/L)	B	20	30	40
温度/℃	C	30	33	36

试验号	A谷氨酸	B葡萄糖	C温度	Y
1	0	-1	-1	641
2	1	0	-1	612
3	-1	0	-1	611
4	0	0	0	699
5	0	1	-1	645
6	0	1	0	641
7	1	-1	0	615
8	1	1	0	615
9	0	0	0	689
10	0	0	0	698
11	-1	0	1	610
12	0	-1	1	622
13	1	0	1	598
14	-1	-1	0	611
15	0	0	0	704
16	0	0	0	703
17	-1	1	0	630

试验号	A谷氨酸	B葡萄糖	C温度	Y
1	0	-1	-1	641
2	1	0	-1	612
3	-1	0	-1	611
4	0	0	0	699
5	0	1	-1	645
6	0	1	0	641
7	1	-1	0	615
8	1	1	0	615
9	0	0	0	689
10	0	0	0	698
11	-1	0	1	610
12	0	-1	1	622
13	1	0	1	598
14	-1	-1	0	611
15	0	0	0	704
16	0	0	0	703
17	-1	1	0	630

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试验号	A谷氨酸	B葡萄糖	C温度	Y
1	0	-1	-1	641
2	1	0	-1	612
3	-1	0	-1	611
4	0	0	0	699
5	0	1	-1	645
6	0	1	0	641
7	1	-1	0	615
8	1	1	0	615
9	0	0	0	689
10	0	0	0	698
11	-1	0	1	610
12	0	-1	1	622
13	1	0	1	598
14	-1	-1	0	611
15	0	0	0	704
16	0	0	0	703
17	-1	1	0	630

试验号	A谷氨酸	B葡萄糖	C温度	Y
1	0	-1	-1	641
2	1	0	-1	612
3	-1	0	-1	611
4	0	0	0	699
5	0	1	-1	645
6	0	1	0	641
7	1	-1	0	615
8	1	1	0	615
9	0	0	0	689
10	0	0	0	698
11	-1	0	1	610
12	0	-1	1	622
13	1	0	1	598
14	-1	-1	0	611
15	0	0	0	704
16	0	0	0	703
17	-1	1	0	630

试验号	A谷氨酸	B葡萄糖	C温度	Y
1	0	-1	-1	641
2	1	0	-1	612
3	-1	0	-1	611
4	0	0	0	699
5	0	1	-1	645
6	0	1	0	641
7	1	-1	0	615
8	1	1	0	615
9	0	0	0	689
10	0	0	0	698
11	-1	0	1	610
12	0	-1	1	622
13	1	0	1	598
14	-1	-1	0	611
15	0	0	0	704
16	0	0	0	703
17	-1	1	0	630

试验号	A谷氨酸	B葡萄糖	C温度	Y
1	0	-1	-1	641
2	1	0	-1	612
3	-1	0	-1	611
4	0	0	0	699
5	0	1	-1	645
6	0	1	0	641
7	1	-1	0	615
8	1	1	0	615
9	0	0	0	689
10	0	0	0	698
11	-1	0	1	610
12	0	-1	1	622
13	1	0	1	598
14	-1	-1	0	611
15	0	0	0	704
16	0	0	0	703
17	-1	1	0	630