枯草芽孢杆菌B579对黄瓜枯萎病的防治及其诱导抗性研究

doi:10.13560/j.cnki.biotech.bull.1985.2024-0160

摘要/Abstract

摘要：

【目的】 探究枯草芽孢杆菌B579对黄瓜枯萎病菌的生防效果及诱导抗性机理。【方法】 采用平板对峙试验和温室盆栽试验，检测病原菌生长及形态变化、植物生长量、叶片防御酶活性、丙二醛含量、防御相关基因表达量等指标。【结果】 枯草芽孢杆菌B579对黄瓜枯萎病菌具有较强的抑制作用，抑菌圈周围菌丝出现粗细不均、膨胀、断裂等现象。盆栽试验表明B579对黄瓜枯萎病的防治效果为78.80％。B579处理组黄瓜幼苗的株高、茎粗、鲜重、干重分别比对照组增加了13.87％、4.17％、15.15％、12.77％。相比单独接种病原菌（FOC）处理，接种 B579 再接种病原菌（B579+FOC）处理在第4天和第7天显著提高了黄瓜叶片苯丙氨酸解氨酶（PAL）、多酚氧化酶（PPO）、过氧化物酶（POD）和过氧化氢酶（CAT）的活性，并减少黄瓜叶片丙二醛（MDA）的含量（P<0.05）。实时荧光定量PCR 检测结果表明，接种 B579 再接种病原菌后，黄瓜叶片防御相关基因LOX、PR1、PR3和CAT表达量在第4天和第7天显著高于其他处理（P<0.05）。【结论】 枯草芽孢杆菌B579能够通过直接抑制病原菌生长和使黄瓜产生诱导抗性来有效防治黄瓜枯萎病。

关键词: 枯草芽孢杆菌B579, 促生, 黄瓜枯萎病, 诱导抗性, 生物防治

Abstract:

【Objective】 To explore the control effect and mechanism of Bacillus subtilis B579 on cucumber Fusarium wilt. 【Method】 Plate confrontation test and greenhouse pot experiment were used to detect the growth and morphological changes of the pathogen, plant growth, activity of defense enzymes in leaves and malondialdehyde content, and expression of defense-related related genes. 【Result】 The B. subtilis B579 had a strong inhibitory effect on cucumber Fusarium wilt, and the mycelium around the inhibition zone showed uneven thickness, swelling and breakage. Pot experiment demonstrated that B579 achieved the disease control efficiency against cucumber wilt, reaching 78.80%. The plant height, stem diameter, fresh weight, and dry weight of cucumber seedlings in B579 treatment increased by 13.87%, 4.17%, 15.15% and 12.77% compared with the control group, respectively. After pretreatment with B579, pathogen inoculation significantly increased the activities of phenylalanine ammonilyase（PAL）, polyphenol oxidase（PPO）, peroxidase（POD）, and catalase（CAT）and reduced the content of malondialdehyde（MDA）in cucumber leaves on day 4 and 7 compared with the control group. The results of real-time fluorescence quantitative PCR showed that the expressions of LOX, PR1, PR3 and CAT genes in cucumber leaves after inoculation with B579 and then challenge with pathogen were significantly higher than those in other treatments at 4 and 7 d. 【Conclusion】 B. subtilis B579 can control cucumber Fusarium wilt by directly inhibiting the growth of pathogen and inducing resistance in cucumber.

Key words: Bacillus subtilis B579, promoting growth, cucumber Fusarium wilt, induced resistance, biological control

范宗强, 冯靖涵, 郑丽雪, 王硕, 彭向前, 陈芳. 枯草芽孢杆菌B579对黄瓜枯萎病的防治及其诱导抗性研究[J]. 生物技术通报, 2024, 40(7): 226-234.

FAN Zong-qiang, FENG Jing-han, ZHENG Li-xue, WANG Shuo, PENG Xiang-qian, CHEN Fang. Study on the Control and Induced Resistance in Cucumber with Bacillus subtilis B579 against Cucumber Fusarium Wilt[J]. Biotechnology Bulletin, 2024, 40(7): 226-234.

图/表 9

表1 实时定量PCR反应的引物信息

Table 1 Primer information for the real-time quantitative PCR reaction

基因Gene	基因序列Sequence（5'-3'）	登录号Accession number	参考文献Reference
ACTIN	F: TGGACTCTGGTGATGGTGTTA	DQ641117	[24]
	R:CAATGAGGGATGGCTGGAAAA	DQ641117	[24]
LOX	F:GAGAGCGTAAGGAATGGGATAGAA	NM_001305708	[8]
	R: CACCGGGTTCGGAAAGG	NM_001305708	[8]
PR1	AACTCTGGCGGACCTTAC	DQ641122	[24]
	GACTTCCTCCACACTACT	DQ641122	[24]
PR3	GCCTTACTCCATAACATCACTCC	DQ641104	[25]
	GATTTCGATATCGAGTCTGGCT	DQ641104	[25]
CAT	AACCCAACCCAAAATCCCA	GU248529	[26]
	TCTAATAGCCTCCTCTTCCAGCA	GU248529	[26]

图1 B579发酵液对病原菌生长的影响数据为平均值±标准偏差，柱形图不同小写字母表示不同处理之间具有显著差异（P<0.05）；下同

Fig. 1 Effect of B579 fermentation broth on the growth of pathogen Data are mean ± SE，different small letters on the bar chart indicate significant differences between each treatment group and the control group（P<0.05）；the same below

图2 光学显微镜下对照组（A）和处理组（B, C）病原菌菌丝的形态 A: 菌丝折叠; B: 菌丝膨胀; C: 菌丝破裂

Fig. 2 Mycelium morphology of pathogens in control group（A）and treatment group（B, C）under optical microscope A: Mycelium folding; B: hyphal expansion; C: hyphal rupture

图3 不同处理下的黄瓜植株

Fig. 3 Cucumber plants under different treatment

表2 B579处理对黄瓜枯萎病的防治效果

Table 2 Control effect of B579 treatment on cucumber Fusarium wilt

组别 Treatment	病情指数 Disease index	防治效果 Control efficiency/％
CK	0c	-
B579	0c	-
B579+FOC	11.12±2.41b	78.80％
FOC	52.78±4.81a	-

表3 不同处理对黄瓜幼苗生长的影响

Table 3 Effects of different treatments on cucumber seedling growth

组别Treatment	株高Plant height/cm	茎粗Stem diameter/mm	鲜重Fresh weight/g	干重Dry weight/g
CK	9.95±1.30b	4.80±0.39b	9.11±1.07b	0.47±0.06b
B579	11.33±0.79a	5.00±0.57a	10.49±0.87a	0.53±0.06a
B579+FOC	9.32±0.62b	4.66±0.45b	7.98±0.76c	0.42±0.07b
FOC	7.42±1.10d	3.97±0.58c	6.41±1.19d	0.33±0.07d

图4 不同处理下黄瓜叶片四种防御性酶活性

Fig. 4 Activities of 4 defensive enzymes in cucumber leaves under different treatments

图5 不同处理下黄瓜叶片丙二醛（MDA）的含量

Fig. 5 MDA content in cucumber leaves under different treatments

图6 不同处理下黄瓜叶片4种防御基因的表达量

Fig. 6 Expressions of four defensive genes in cucumber leaves under different treatments

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