蔬菜软腐病菌广谱拮抗细菌菌株筛选鉴定及防效研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-1349

摘要/Abstract

摘要：

为获得广谱高效拮抗蔬菜软腐病菌的生防菌株，本研究以白菜软腐病菌Pectobacterium carotovorum BC2、圆葱软腐病菌Burkholderia gladioli YC1、娃娃菜软腐病菌Pseudomonas sp. WWC2为靶标，采用梯度稀释法及抑菌圈法从蔬菜根际土中分离筛选拮抗菌株。通过形态、生理生化和16S rDNA序列分析对菌株进行鉴定，明确生防菌株种属地位，并研究了菌株生长特性，利用牛津杯法测定生防菌株对3株软腐病菌及3株人源性病原细菌抑菌作用，采用平板对峙法测定其对8株植物病原真菌的抑菌作用，采用针刺接种法测定其对蔬菜离体叶片和田间的防效，并利用X-gal显色法测定其对白菜软腐病菌群体感应信号因子的降解活性。通过研究菌株生长特性、对白菜软腐病菌群体感应信号因子的降解活性、抑菌谱以及对白菜软腐病的田间防效。结果表明，从20个土样分离的1 012个细菌中，筛选出18株拮抗菌，其中筛选出1株对3种软腐病菌YC1、BC2、WWC2均有抑菌活性的生防菌株DJ1，其抑菌圈直径分别为（10.60 ± 0.20）mm、（6.92 ± 0.56）mm和（3.92 ± 0.16）mm。经鉴定菌株DJ1为贝莱斯芽孢杆菌Bacillus velezensis。菌株DJ1最适生长温度为30℃，具有较好的耐盐性，能在1%-5% NaCl条件下生长，具有一定降解白菜软腐病菌群体感应信号因子的能力，能够抑制大肠杆菌、金黄色葡萄球菌和8种植物病原真菌生长，1×10⁸ CFU/mL菌液浓度对白菜、圆葱、娃娃菜软腐病离体防效分别为84.30%、60.21%和69.96%，对白菜软腐病田间防效为79.91%。因此，贝莱斯芽孢杆菌DJ1在防治蔬菜软腐病方面具有潜在应用潜能。

关键词: 蔬菜, 软腐病菌, 拮抗菌, 筛选, 防效, 贝莱斯芽孢杆菌, 鉴定, 抑菌作用

Abstract:

To obtain a broad-spectrum biocontrol strain with high efficiency against vegetable soft rot bacteria, in this study, a biocontrol strain DJ1 with good inhibition effect on three pathogens was screened from vegetable rhizosphere soil by the method of bacteriostasis circle, using soft rot pathogen of Chinese cabbage Pectobacterium carotovorum BC2, onion Burkholderia gladioli YC1 and baby cabbage Pseudomonas sp. WWC2 as the targets. Antagonistic strains were isolated and screened from vegetable rhizosphere soil by gradient dilution and bacteriostatic zone method. Through morphological, physiological and biochemical analysis and 16S rDNA sequence analysis, the species of the biocontrol strains were identified, and their the growth characteristics were studied. The Oxford Cup method was used to determine the bacteriostasis of the biocontrol strains on 3 strains of soft rot and 3 strains of human pathogenic bacteria, and the plate confrontation method to determine the bacteriostasis on 8 strains of plant pathogenic fungi. Needling inoculation method was applied to determine the control effect on vegetables leaves in vitro and field, and X-gal chromogenic method to determine the degradation activities of quorum-sensing signal factors against Chinese cabbage soft rot pathogen. The growth characteristics of the strain, its ability to degrade quorum sensing signal factors, its bacteriostatic spectrum and its field control effect on soft rot of Chinese cabbage were studied. The results showed that 18 antagonistic strains were selected from 1 012 bacteria isolated from 20 soil samples, among which 1 biocontrol strain DJ1 had antibacterial activity against 3 soft rot pathogens YC1, BC2 and WWC2. The antibacterial zone diameters were（10.60±0.20）,（6.92±0.56）and（3.92±0.16）mm, respectively. The strain DJ1 was identified as Bacillus velezensis. The optimal growth temperature of strain DJ1 was 30℃, and it had good salt tolerance and grew under 1%-5% NaCl. It had the ability to degrade the quorum-sensing signaling factors of Chinese cabbage, and inhibited the growth of Escherichia coli, Staphylococcus aureus and 8 plant pathogenic fungi. The 1×10⁸ CFU/mL of bacterial solution was 84.30%, 60.21% and 69.96% in vitro against soft rot of Chinese cabbage, onion and baby cabbage, respectively, and 79.91% in field. Therefore, B. velezensis DJ1 presents potential application in the control of vegetable soft rot.

Key words: vegetables, soft rot pathogens, antagonistic bacteria, screening, control effect, Bacillus velezensis, identification, bacteriostasis

马俊秀, 吴皓琼, 姜威, 闫更轩, 胡基华, 张淑梅. 蔬菜软腐病菌广谱拮抗细菌菌株筛选鉴定及防效研究[J]. 生物技术通报, 2023, 39(7): 228-240.

MA Jun-xiu, WU Hao-qiong, JIANG Wei, YAN Geng-xuan, HU Ji-hua, ZHANG Shu-mei. Screening and Identification of Broad-spectrum Antagonistic Bacterial Strains Against Vegetable Soft Rot Pathogen and Its Control Effects[J]. Biotechnology Bulletin, 2023, 39(7): 228-240.

图/表 12

表1 拮抗菌株对软腐病菌YC1、BC2、WWC2的抑制作用

Table 1 Inhibitions of the antagonistic strains against soft rot pathogens YC1, BC2, and WWC2

菌株 Bacterium	抑菌圈直径 Inhibition zone/mm
菌株 Bacterium	YC1	BC2	WWC2
DJ1	10.60 ± 0.20 a	6.92 ± 0.56 a	3.92 ± 0.16 a
MC1	8.32 ± 0.32 b	6.18 ± 0.24 ab	0.00 ± 0.00 b
SD2	4.72 ± 0.31 c	5.30 ± 0.26 b	0.00 ± 0.00 b
TD1	8.32 ± 0.82 b	5.26 ± 0.36 b	0.00 ±0.00 b
XHS1	9.16 ± 0.17 b	6.96 ± 0.22 ab	0.00 ± 0.00 b
SD1	10.66 ± 0.45 a	5.4 ± 0.21 b	0.00 ± 0.00 b

图1 拮抗菌株对软腐病菌YC1、BC2、WWC2的抑菌圈效果

Fig. 1 Inhibitory zone effect of the antagonistic strains against soft rot pathogens YC1, BC2, and WWC2

图2 菌株DJ1的形态特征（LB培养基） A：革兰氏染色；B：LB培养基上菌落形态

Fig. 2 Morphology of strain DJ1（LB medium） A: Gram staining. B: Colony on LB plate

表2 菌株DJ1的生理生化特性

Table 2 Physiological and biochemical characteristics of strain DJ1

项目 Item	DJ1	文献 Referrence
海藻糖 Trehalose	-	+^[35]
山梨醇 Sorbitol	+	+^[35]
甘露醇 Mannitol	+	+^[35]
纤维素分解 Cellulose decomposition	-	-^[35]
吲哚反应 Indole reaction	-	-^[35]
丙二酸盐利用 Malonate utilization	+	+^[35]
柠檬酸盐利用 Citrate utilization	-	-^[35]
明胶液化 Gelatin liquefaction	+	+^[35]
果糖 Fructose	+	+^[35]
蔗糖 Sucrose	+	+^[35]
葡萄糖氧化发酵 Oxidative fermentation of glucose	+	+^[36]
石蕊牛奶 Litmus milk	+	+^[36]
接触酶 Contact enzyme	+	+^[36]
硝酸盐还原 Nitrate reduction	+	-^[36]
V-P试验 V-P test	+	+^[36]
淀粉水解 Starch hydrolysis	+	+^[36]
硫化氢 Hydrogen sulfide	-	-^[36]
甲基红试验 Methyl red test	-	-^[37]
1%-5% NaCl	+	*
7% NaCl	-	*
10% NaCl	-	*
4℃	-	*
22-42℃	+	*
30℃	最适生长	*

图3 菌株DJ1的系统发育进化树

Fig. 3 Phylogenetic tree of strain DJ1

图4 拮抗菌DJ1对离体白菜、圆葱和娃娃菜软腐病菌的抑菌活性 1：对照组；2：BC2、YC1和WWC2处理组；3：DJ1处理组.（A为白菜，B为圆葱，C为娃娃菜）

Fig. 4 Antibacterial activities of strain DJ1 against soft rot pathogen of Chinese cabbage（Brassica rapa var. glabra）, onion（Allium cepa）, and baby cabbage（Brassica pekinensis）in vitro 1: Control group. 2: BC2, YC1 and WWC2 treatment group. 3: DJ1 treatment group.（A is Chinese cabbage, B is onion, and C is baby cabbage）

图5 菌株DJ1对离体白菜、圆葱和娃娃菜软腐病菌的防效不同小写字母表示在P < 0.05水平差异显著。下同

Fig. 5 Control efficacy of strain DJ1 against soft rot pathogen of Chinese cabbage, onion, and baby cabbage in vitro Different lower letters indicate significant differences at P < 0.05 level. The same below

图6 拮抗细菌对白菜软腐病菌群体感应信号因子的降解活性 A：只含有5 μmol/L信号因子对照组；B：加入菌株DJ1的处理组；1，2分别为N-己酰基-L-高丝氨酸内酯和N-3-氧-己酰高丝氨酸内酯

Fig. 6 Degradation activity of antagonistic bacteria on the quorum sensing signal factors of soft rot pathogen of Chinese cabbage A: Only 5 μmol/L signal factors control group. B: Treatment group added with strain DJ1. 1 and 2 is N-caproyl-L-homoserine lactone and N-3-ox-hexamyl-homoserine lactone respectively

图7 菌株DJ1对不同病原真菌的抑制作用 z1：刺五加立枯病菌；z2：水稻绵腐病菌；z3：黄瓜枯萎病菌；z4；水稻稻瘟病菌；z5；豆角炭疽病菌；z6：玉米茎基腐病菌；z7；水稻恶苗病菌；z8：番茄灰霉病菌

Fig. 7 Inhibitory effects of strain DJ1 against different pathogenic fungi z1: R. solani. z2: A. oryzae. z3: F. oxysporium. z4: P. oryzae. z5: C. gloeosporioides. z6: F. graminearum. z7: F. moniliforme. z8: B. cinerea

表3 菌株DJ1对不同病原细菌的抑制作用

Table 3 Inhibitory effects of strain DJ1 against different pathogenic bacteria

病原细菌 Pathogenic bacterium	抑菌圈直径 Inhibitive zone/mm
大肠杆菌 E. coli	3.27 ± 0.12 a
金黄色葡萄球菌 S. aureus	1.63 ± 0.26 b
绿脓杆菌 P. aeniginasa	0.00 ± 0.00 c

图8 菌株DJ1对白菜软腐病菌BC2的田间小区防效 A：BC2处理组； B：DJ处理组

Fig. 8 Control effects of strain DJ1 against the soft rot pathogen BC2 of Chinese cabbage in field plots A: BC2 treatment group. B: DJ1 treatment group

表4 菌株DJ1对白菜软腐病菌BC2的田间小区防效

Table 4 Control effects of strain DJ1 against the soft rot pathogen BC2 of Chinese cabbage in field plots

处理 Treatment	病情指数 Disease index	防效 Control efficacy/%
对照CK	66.89 ± 2.38	—
DJ1	13.48 ± 1.68	79.91 ± 2.08

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