内生细菌ZN-S10的鉴定及其对番茄青枯病菌的抑菌作用

doi:10.3969/j.issn.1004-1524.20230864

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (11): 2636-2644.DOI: 10.3969/j.issn.1004-1524.20230864

内生细菌ZN-S10的鉴定及其对番茄青枯病菌的抑菌作用

王晓楠¹(), 冯晓晓², 施斌¹, 陈恩磊¹, 陈梦丽¹, 郑永利³^,^*(), 吴慧明¹^,^*()

1.浙江农林大学现代农学院,浙江杭州 311300
2.浙江大学农业试验站,浙江杭州 310058
3.浙江省农产品绿色发展中心,浙江杭州 310003

收稿日期:2023-07-12 出版日期:2023-11-25 发布日期:2023-12-04
作者简介:王晓楠(1997—),女,内蒙古乌兰察布人,硕士研究生,研究方向为农药毒理与绿色防控。E-mail:1084220367@qq.com
通讯作者: * 吴慧明,E-mail: Wuhm@zafu.edu.cn;郑永利,E-mail: yonglizheng@yeah.net
基金资助:
浙江省重点研发计划(2020C02027)

Identification of Bacillus velezensis ZN-S10 and its antification effect on tomato bacterial wilt

WANG Xiaonan¹(), FENG Xiaoxiao², SHI Bin¹, CHEN Enlei¹, CHEN Mengli¹, ZHENG Yongli³^,^*(), WU Huiming¹^,^*()

1. College of Advanced Agricultural Science, Zhejiang A & F University, Hangzhou 311300, China
2. Agricultural Test Station, Zhejiang University, Hangzhou 310058, China
3. Zhejiang Agricultural Products Green Development Center, Hangzhou 310003, China

Received:2023-07-12 Online:2023-11-25 Published:2023-12-04

摘要/Abstract

摘要：

为鉴定一株来自番茄根部的内生细菌及其抑菌作用,检验番茄内生细菌对不同病原菌的拮抗作用,并为常见细菌性病害的生物防治提供思路,从番茄根分离得到内生细菌,通过形态特征、脂肪酸、生理生化特征测定及建立gyrA和rpoB双基因系统发育树的方法鉴定。采用平板法测试了内生细菌对5种常见植物病原细菌的拮抗作用,并用盆栽试验测试该菌对番茄青枯病的防效。经过鉴定菌株ZN-S10为贝莱斯芽孢杆菌(Bacillus velezensis)。抑菌试验显示,B. velezensis ZN-S10对番茄细菌性髓部坏死病菌(Pseudomonas viridifiava)、甘薯茎腐病菌(Dickeya dadantii)、花椰菜细菌性软腐病菌(Erwinia carotovora)、番茄青枯病菌(Ralstonia solanacearum)、李细菌性穿孔病菌(Xanthomonas campestris)均有不同程度的抑菌活性。温室盆栽试验表明,B. velezensis ZN-S10对番茄青枯病的防治效果为57.14%,与3%中生菌素可湿性粉剂的防治效果无显著差异,可有效降低番茄青枯病的发展。研究结果可以为有效利用内生细菌防治常见细菌性病害提供理论依据,B. velezensis ZN-S10对番茄细菌性病害的防治具有较大的应用前景。

关键词: 贝莱斯芽孢杆菌, 拮抗活性, 青枯菌, 防治效果, 生物防治

Abstract:

In order to identify an endophytic bacterium from tomato root and its antibacterial effect, the antagonistic activity of tomato endophytic bacteria against different pathogens was tested, providing insights for the biological control of common bacterial diseases. Endophytic bacteria were isolated from tomato roots and identified by morphological, fatty acid, physiological characteristics determination as well as a dual-gene system of gyrA and rpoB phylogenetic trees. The antagonistic effect of endophytic bacteria on five common plant pathogenic bacteria was tested using the plate method, and the efficacy of the bacterium against tomato bacterial wilt disease was evaluated using the pot culture experiment. The strain ZN-S10 was identified as Bacillus velezensis. The antibacterial assay showed that B.velezensis ZN-S10 exhibited different degrees of bacteroistatic activity against Pseudomonas viridifiava, Dickeya dadantii, Erwinia carotovora, Ralstonia solanacearum and Xanthomonas campestris. The greenhouse pot culture experiment demonstrated that the control effect of B.velezensis ZN-S10 on tomato bacterial wilt was 57.14%, which was not significantly different from that of 3% Zhongshengmycin WP, and could effectively reduce the development of tomato bacterial wilt. These research findings provided a theoretical basis for the effective use utilization of endophytic bacteria in the control of common bacterial diseases and highlighted the promising application prospects of B. velezensis ZN-S10 in the management of bacterial diseases in tomatoes.

Key words: Bacillus velezensis, antagonistic activity, Ralstonia solanacearum, control efficiency, biological control

中图分类号:

S436.412

王晓楠, 冯晓晓, 施斌, 陈恩磊, 陈梦丽, 郑永利, 吴慧明. 内生细菌ZN-S10的鉴定及其对番茄青枯病菌的抑菌作用[J]. 浙江农业学报, 2023, 35(11): 2636-2644.

WANG Xiaonan, FENG Xiaoxiao, SHI Bin, CHEN Enlei, CHEN Mengli, ZHENG Yongli, WU Huiming. Identification of Bacillus velezensis ZN-S10 and its antification effect on tomato bacterial wilt[J]. Acta Agriculturae Zhejiangensis, 2023, 35(11): 2636-2644.

图/表 9

图1 ZN-S10菌株的菌落形态和革兰氏染色结果

Fig.1 Colony morphology and Gram staining of ZN-S10

表1 ZN-S10的生理生化结果

Table 1 Physiological and biochemical identification results of endophytic bacterium ZN-S10

生理生化指标测试 Physiological and biochemical index	ZN-S10菌株 ZN-S10 strain
氧化酶 Oxidase	+
卵磷脂酶 Lecithinase	+
过氧化氢酶 Catalase	+
淀粉水解 Amylohydrolysis	+
V-P 测定 V-P test	+
甲基红 Methyl red	-
吲哚 Indole	+
氯霉素 Chloromycetin	-
链霉素 Streptomycin	-
卡那霉素 Kanamycin	-
氨苄青霉素 Ampicillin	-

图2 基于rpoB、gyrA基因序列构建的ZN-S10系统发育树分支处的数值为Bootstrap法重复 1 000次评估得到的各节点支持率,标尺0.050为进化距离。

Fig.2 ZN-S10 phylogenetic tree based on rpoB and gyrA gene sequences The value at the branch is the support rate of each node obtained by repeating the Bootstrap method 1 000 times, with a scale of 0.050 as the evolution distance.

图3 Bacillus velezensis ZN-S10对不同病原菌的抑菌效果图中平板左侧滤纸片为CK,右侧为ZN-S10。从左到右依次为花椰菜细菌性软腐病菌ZJUP0154-1、番茄细菌性髓部坏死病菌ZJUP0398-2、甘薯茎腐病菌ZAFU0898、番茄青枯病菌ZAFU0005、李细菌性穿孔病菌ZJUP0463-1。

Fig.3 Antibacterial effect of Bacillus velezensis ZN-S10 against different pathogens The filter paper on the left side of the plate in the picture is CK, and the filter paper on the right side is ZN-S10. From left to right are Erwinia carotovora ZJUP0154-1, Pseudomonas viridifiava ZJUP0398-2, Dickeya dadantii ZAFU0898, Ralstonia solanacearum ZAFU0005, and Xanthomonas campestris ZJUP0463-1.

图4 Bacillus velezensis ZN-S10对5种病原菌的抑菌率数据为平均值±标准误,柱上没有相同小写字母表示差异显著(P<0.05)。下同。

Fig.4 Inhibition rate of Bacillus velezensis ZN-S10 against five pathogens The data is mean ± standard error, and the different lowercase letters on the column indicate significant differences (P<0.05). The same as below.

表2 Bacillus velezensis ZN-S10与3%中生菌素对番茄青枯病的防治效果

Table 2 Effect of Bacillus velezensis ZN-S10 and 3% Zhongshengmycin WP on control of Ralstonia solanacearum

处理 Treatment	病情指数 Disease index	相对防治效果 Relative control effect/%
ZN-S10	31.25	57.14
3%中生菌素 WP	31.25	57.14
3% Zhongshengmycin WP
MOCK2	72.92

图5 ZN-S10对番茄青枯病菌ZAFU0005的防治效果 A, 清水;B,接种番茄青枯病菌ZAFU0005;C,3%中生菌素WP灌根7 d后接种番茄青枯病菌ZAFU0005;D,ZN-S10灌根7 d后接种番茄青枯病菌ZAFU0005。

Fig.5 Control effect of ZN-S10 on Ralstonia solanacearum ZAFU0005 A, Clear water; B, Inoculate Ralstonia solanacearum ZAFU0005; C, Inoculate Ralstonia solanacearum ZAFU0005 after 7 days of root irrigation with 3% mycorrhizal WP; D, After 7 days of root irrigation with ZN-S10, Ralstonia solanacearum ZAFU0005 was inoculated.

图6 番茄的MDA含量变化 MOCK,清水;T1,ZN-S10;T2,ZAFU0005;T3,ZN-S10+ZAFU0005 。同一处理时间下不同处理间没有相同小写字母表示差异显著(P<0.05)。下同。

Fig.6 MDA content changes of tomato MOCK, Clean water; T1, ZN-S10; T2, ZAFU0005; T3, ZN-S10+ZAFU0005. Under the same treatment time, the bars of different treatments marked with different lowercase letters showed significant difference (P<0.05).The same as below.

图7 番茄的GSH含量及PAL、SOD、CAT、POD活性的变化

Fig.7 GSH content and PAL, SOD, CAT and POD activity changes of tomato

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内生细菌ZN-S10的鉴定及其对番茄青枯病菌的抑菌作用

Identification of Bacillus velezensis ZN-S10 and its antification effect on tomato bacterial wilt

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