生物技术通报 ›› 2023, Vol. 39 ›› Issue (7): 228-240.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1349
马俊秀(), 吴皓琼, 姜威, 闫更轩, 胡基华, 张淑梅()
收稿日期:
2022-11-03
出版日期:
2023-07-26
发布日期:
2023-08-17
通讯作者:
张淑梅,女,博士,研究员,研究方向:微生物农药;E-mail: 1401135157@qq.com作者简介:
马俊秀,女,硕士研究生,研究方向:微生物农药;E-mail: 2634643414@qq.com
基金资助:
MA Jun-xiu(), WU Hao-qiong, JIANG Wei, YAN Geng-xuan, HU Ji-hua, ZHANG Shu-mei()
Received:
2022-11-03
Published:
2023-07-26
Online:
2023-08-17
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。
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.
菌株 Bacterium | 抑菌圈直径 Inhibition zone/mm | |||
---|---|---|---|---|
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 |
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 | |||
---|---|---|---|---|
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 |
项目 Item | DJ1 | 文献 Referrence |
---|---|---|
海藻糖 Trehalose | - | +[ |
山梨醇 Sorbitol | + | +[ |
甘露醇 Mannitol | + | +[ |
纤维素分解 Cellulose decomposition | - | -[ |
吲哚反应 Indole reaction | - | -[ |
丙二酸盐利用 Malonate utilization | + | +[ |
柠檬酸盐利用 Citrate utilization | - | -[ |
明胶液化 Gelatin liquefaction | + | +[ |
果糖 Fructose | + | +[ |
蔗糖 Sucrose | + | +[ |
葡萄糖氧化发酵 Oxidative fermentation of glucose | + | +[ |
石蕊牛奶 Litmus milk | + | +[ |
接触酶 Contact enzyme | + | +[ |
硝酸盐还原 Nitrate reduction | + | -[ |
V-P试验 V-P test | + | +[ |
淀粉水解 Starch hydrolysis | + | +[ |
硫化氢 Hydrogen sulfide | - | -[ |
甲基红试验 Methyl red test | - | -[ |
1%-5% NaCl | + | * |
7% NaCl | - | * |
10% NaCl | - | * |
4℃ | - | * |
22-42℃ | + | * |
30℃ | 最适生长 | * |
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 | - | +[ |
山梨醇 Sorbitol | + | +[ |
甘露醇 Mannitol | + | +[ |
纤维素分解 Cellulose decomposition | - | -[ |
吲哚反应 Indole reaction | - | -[ |
丙二酸盐利用 Malonate utilization | + | +[ |
柠檬酸盐利用 Citrate utilization | - | -[ |
明胶液化 Gelatin liquefaction | + | +[ |
果糖 Fructose | + | +[ |
蔗糖 Sucrose | + | +[ |
葡萄糖氧化发酵 Oxidative fermentation of glucose | + | +[ |
石蕊牛奶 Litmus milk | + | +[ |
接触酶 Contact enzyme | + | +[ |
硝酸盐还原 Nitrate reduction | + | -[ |
V-P试验 V-P test | + | +[ |
淀粉水解 Starch hydrolysis | + | +[ |
硫化氢 Hydrogen sulfide | - | -[ |
甲基红试验 Methyl red test | - | -[ |
1%-5% NaCl | + | * |
7% NaCl | - | * |
10% NaCl | - | * |
4℃ | - | * |
22-42℃ | + | * |
30℃ | 最适生长 | * |
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
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 |
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
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 |
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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