烟草靶斑病拮抗菌的鉴定及其防治特性

doi:10.13560/j.cnki.biotech.bull.1985.2025-0678

摘要/Abstract

摘要：

目的分离筛选对烟草靶斑病（tobacco target spot disease）有拮抗作用的生防菌。方法从杜鹃（Rhododendron simsii Planch.）根际利用平板涂布法和平板对峙法分离筛选拮抗菌，经生理生化与16S rDNA基因序列分析鉴定，构建系统发育树，同时测定发酵液和乙酸乙酯粗提物的抗菌活性；采用盆栽法测定拮抗菌对烟草靶斑病的防效，以及对丙二醛（MDA）、抗氧化酶活性及病程相关蛋白基因表达的影响。结果获得1株对立枯丝核菌（Rhizoctonia solani）有拮抗作用的唐菖蒲伯克霍尔德氏菌（Burkholderia gladioli）QLDJ-1；平板拮抗96 h，QLDJ-1对烟草靶斑病菌（Rhizoctonia solani）的相对抑菌率为（72.107±2.788）%；与对照组相比，经QLDJ-1处理的烟株靶斑病病情指数降低32.02%；超氧化物歧化酶（SOD）、过氧化氢酶（CAT）和过氧化物酶（POD）活性分别提高33.72%、51.45%和17.49%；丙二醛（MDA）含量下降62.44%；病程相关蛋白基因PR1b、NmIMSP、P450-1、GST及hrs203J表达量与对照有显著差异。结论 QLDJ-1对烟草靶斑病菌有较好的抑制效果，具有开发为生防制剂的潜在应用价值。

关键词: 根际细菌, 伯克霍尔德氏菌, 立枯丝核菌, 烟草靶斑病, 生物防治

Abstract:

Objective To isolate and screen biocontrol bacteria against tobacco target spot disease. Method The antagonists were isolated and screened by plate coating method and plate confrontation method from Rhododendron simsii Planch. rhizosphere. After identification by physiological and biochemical analysis and 16S rDNA gene sequence analysis, the phylogenetic tree was constructed, and the antibacterial activities of the fermented broth and the crude extract of ethyl acetate were determined concurrently. Pot culture method was used to determine the control effect of antagonists on tobacco target spot disease, as well as the effects on the activities of malondialdehyde (MDA), antioxidant enzymes and the regulation and expression of pathogenesis related protein genes. Result A strain of Burkholderia gladioli QLDJ-1 with antagonistic effect against Rhizoctonia solani was obtained. The relative inhibition rate of QLDJ-1 against R. solani was (72.107 ± 2.788) %. Compared with the control group, the disease index of target spot of tobacco plants treated with QLDJ-1 decreased by 32.02%. The activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) increased by 33.72%, 51.45% and 17.49% respectively. The content of malondialdehyde (MDA) decreased by 62.44%. The expressions of pathogenesis-related genes such as PR1b, NmIMSP, P450-1, GST and hrs203J in the tobacco plants treated with QLDJ-1 were different from those of the control. Conclusion QLDJ-1 can effectively control tobacco target spot disease and has the potential to be developed as a biocontrol agent.

Key words: rhizosphere bacteria, Burkholderia, Rhizoctonia solani, tobacco target spot disease, biological control

朱志炎, 宋芷薇, 何珊, 张德清, 何勇, 田志宏. 烟草靶斑病拮抗菌的鉴定及其防治特性[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0678.

ZHU Zhi-yan, SONG Zhi-wei, HE Shan, ZHANG De-qing, HE Yong, TIAN Zhi-hong. Identification and Control Characteristics of Antagonistic Bacteria against Tobacco Target Spot Disease[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0678.

图/表 11

图1 QLDJ-1和QLDJ-2发酵液对立枯丝核菌的平板对峙

Fig. 1 Plate confrontation of bacterial strain QLDJ-1 and QLDJ-2 in fermentation broth against Rhizoctonia solani

表1 QLDJ-1的生理生化特征

Table 1 Physiological and biochemical characteristics of strain QLDJ-1

项目 Item	阳性/阴性 Positive （+）/ negative （-）
生化反应 Biochemical test	+
淀粉水解 Starch hydrolysis	+
接触酶 Catalase production	+
尿素酶 Urease production	+
蔗糖利用 Sucrose utilization	-
生化反应 Biochemical test	+
脂酶 Esterase production	-
硝酸盐还原 Nitrate reduction	-
甲基红试验 Methyl red test	-
VP反应 VP test	-
柠檬酸盐利用 Citrate utilization	+

表2 QLDJ-1对抗生素的敏感性

Table 2 Antibiotic sensitivity analysis of strain QLDJ-1

抗生素 Antibiotics	浓度 Concentration （μg/mL）	敏感性 Sensibility
抗生素 Antibiotics	浓度 Concentration （μg/mL）	24 h	72 h
氨苄青霉素 Ampicillin	5	+	+
	50	+	+
	100	+	+
卡那霉素 Kanamycin	5	-	+
	50	-	-
	100	-	-
潮霉素 Hygromycin	5	+	+
	50	+	+
	100	+	+
链霉素 Streptomycin	5	+	+
	50	+	+
	100	-	+

图2 基于16S rDNA基因序列相似性构建QLDJ-1的基因进化树

Fig. 2 QLDJ-1 gene evolutionary tree based on 16S rDNA gene sequence similarity

图3 QLDJ-1对9种不同植物病原菌的拮抗效果测定

Fig. 3 Antagonistic effects of biocontrol strain QLDJ-1 against 9 different plant pathogens

表3 QLDJ-1的抗菌能力测定

Table 3 Determination of the broad-spectrum antimicrobial activity of strain QLDJ-1

病原菌 Pathogenic bacteria	病害名称 Disease	最长培养时间 Maximum culture time （h）	最终抑菌圈直径 Final inhibition zone diameter （mm）	最终相对抑菌率 Final inhibition rate （%）
立枯丝核菌 Rhizoctonia solani	烟草靶斑病 Target spot disease	96	63.667±2.268aA	72.107±2.788bcB
交链格孢菌 Alternaria alternata	烟草赤星病 Brown spot	120	36.083±2.504bB	81.490±1.614abAB
茄科雷尔氏菌 Ralstonia solanacearum	烟草青枯病 Bacterial wilt	72	3.333±1.258eD	32.273±9.796eD
寄生疫霉菌 Phytophthora parasitica var. Nicotianae	烟草黑胫病 Black shank	168	31.500±1.000bcBC	79.740±0.515abcAB
立枯丝核菌 Rhizoctonia solani	水稻纹枯病 Sheath blight	72	64.438±3.710aA	71.598±4.123bcB
黄单胞菌 Xanthomonas oryzae pv	水稻白叶枯病 Bacterial leaf blight	96	5.167±1.041eD	70.637±6.870cB
炭疽病菌 Colletotrichum gloeosporioides	芒果炭疽病 Anthracnose	120	36.813±8.017bB	74.527±9.500bcAB
葡萄座腔菌 Botryosphaeria dothidea	芒果果腐病 Fruit rot	84	67.000±1.146aA	88.370±1.026aA
大斑突脐孢菌 Exserohilum turcicum	玉米大斑病 Northern leaf blight	120	29.038±1.338cdBC	78.380±0.759abcAB

图4 QLDJ-1乙酸乙酯粗提物及气体挥发物拮抗效果A：烟草靶斑病24 h化合物拮抗，B：烟草靶斑病72 h化合物拮抗，C：芒果炭疽病96 h化合物拮抗，D：芒果炭疽病120 h化合物拮抗，E：立枯丝核菌12 h化合物拮抗，F：立枯丝核菌48 h化合物拮抗，G、H：48 h后立枯丝核菌气体化合物拮抗

Fig. 4 Antagonistic effects of ethyl acetate compounds and volatile substances from QLDJ-1A: Compounds antagonizing tobacco target spot disease for 24 h. B: Compounds antagonizing tobacco target spot disease for 72 h. C: Compounds antagonizing mango anthracnose for 96 h. D: Compounds antagonizing mango anthracnose for 120 h. E: Compounds antagonizing Rhizoctonia solani for12 h. F: Compounds antagonizing Rhizoctonia solani for 48 h. G, H: Compounds antagonizing Rhizoctonia solani for 48 h

图5 温室盆栽条件下QLDJ-1防治烟草靶斑病效果（72 h）

Fig. 5 Effects of QLDJ-1 on controlling tobacco target spot disease under potted cultivation in greenhouse (72 h)

表4 QLDJ-1对烟草靶斑病病情指数的影响

Table 4 Effects of strain QLDJ-1 on tobacco target spot disease indexes

处理

Treatment

最低病级

The lowest grade disease

最高病级

The highest grade disease

病情指数

Disease index

相对防效

Control effect （%）

图6 QLDJ-1对烟草叶片接种靶斑病原菌后SOD、POD、CAT及MDA含量的影响不同小写字母表示同列差异显著（P<0.05），下同

Fig. 6 Effects of QLDJ-1 on SOD, POD, CAT and MDA contents in tobacco leaves after inoculation with target spot disease pathogensDifferent lowercase letters indicate significant differences in the same column (P<0.05). The same below

图7 病害胁迫下QLDJ-1处理的烟草中相关基因表达水平RS：立枯丝核菌，DJ-1：QLDJ-1，+RS -DJ-1：CK

Fig. 7 Expressions of related genes in tobacco leaves treated with QLDJ-1 under disease stressRS: Rhizoctonia solani. DJ-1: QLDJ-1, +RS -DJ-1: CK

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