辣椒炭疽病生防菌株TN2的筛选鉴定与抑菌效果

doi:10.13560/j.cnki.biotech.bull.1985.2024-0719

摘要/Abstract

摘要：

【目的】筛选和鉴定一种具有防治辣椒炭疽病能力的生防菌株，评估其防治效果，并探究其对辣椒果实抗病活性物质的影响及其抑菌物质的稳定性。【方法】将从土壤样品中分离出的单一菌株通过平板对峙试验和辣椒离体生防试验进行筛选，最终获得一株拮抗效果显著的细菌菌株。对其进行形态学分析、生理生化特性分析及16S rRNA序列测定分析鉴定，并测定其接种后辣椒内丙二醛（MDA）含量、防御酶活性以及抑菌活性物质的稳定性。【结果】在平板拮抗试验中，TN2菌株对尖孢炭疽菌（Colletotrichum acutatum）有强烈的抑制作用，抑菌率达70%以上。同时，通过离体生防试验验证，TN2处理后辣椒植株的病情指数显著低于阳性对照组。综合菌株TN2的形态学、生理生化特征以及16S rRNA序列系统进化树分析，鉴定TN2为贝莱斯芽胞杆菌。TN2能够增强过氧化物酶（peroxidase, POD）、超氧化物歧化酶（superoxide dismutase, SOD）、丙氨酸解氨酶（phenylalnine ammonialyase, PAL）、过氧化氢酶（catalase, CAT）活性，使MDA含量相对阳性对照处理减少，且在不同处理下，其抑菌物质相对稳定。【结论】贝莱斯芽胞杆菌TN2菌株具有良好的防治辣椒炭疽病的潜力，可作为一种有效的生防菌株应用于辣椒炭疽病的生物防治中，具有重要的实际应用价值。

关键词: 辣椒炭疽病, 贝莱斯芽胞杆菌, 生物防治, 鉴定, 抗菌活性物质

Abstract:

【Objective】To screen and identify a biocontrol strain with the ability to control pepper anthrax, and to evaluate its control effect, and to explore its effect on disease activity and antibacterial stability of pepper fruit.【Method】A single strain isolated from soil samples was screened by plate confrontation test and pepper isolation test, finally a bacterial strain with significant antibacterial activity was obtained. It was identified by morphological observation, physiological and biochemical characteristics and 16S rRNA sequence determination, and the malondialdehyde（MDA）content, defense enzyme activity and stability of antibacterial substances in pepper after inoculation were determined.【Result】In the plate antagonism test, the TN2 strain had a strong inhibitory effect on Colletotrichum acutatum, and the antibacterial rate reached > 70%. Meanwhile, the disease index of the pepper plants after TN2 treatment was significantly lower than that of the positive control group, as verified by in vitro biocontrol test. Combining the morphological, physiological and biochemical characteristics of strain TN2 and the phylogenetic tree analysis of 16S rRNA sequence, we identified TN2 as Bacillus velezensis. TN2 enhanced the activity of peroxidase（POD）, superoxide dismutase（SOD）, phenylalnine ammonialyase（PAL）, catalase（CAT）, but reducing the MDA content to positive control treatment, and its antibacterial substances were relatively stable under different treatments.【Conclusion】B. velezensi TN2 strain has promising potential to control anthrax in capsicum, and can be used as an effective biocontrol strain in the biological control of pepper anthrax, which has important practical application value.

Key words: pepper anthracnose, Bacillus velezensis, biological control, identification, antibiotic active substance

刘倩, 马连杰, 张慧, 王冬, 范茂, 廖敦秀, 赵正武, 卢文才. 辣椒炭疽病生防菌株TN2的筛选鉴定与抑菌效果[J]. 生物技术通报, 2025, 41(1): 287-297.

LIU Qian, MA Lian-jie, ZHANG Hui, WANG Dong, FAN Mao, LIAO Dun-xiu, ZHAO Zheng-wu, LU Wen-cai. Screening, Identification and Control Effects of Biocontrol Strain TN2 against Pepper Anthracnose[J]. Biotechnology Bulletin, 2025, 41(1): 287-297.

图/表 13

表1 拮抗菌株对辣椒尖孢炭疽菌的拮抗作用

Table 1 Antagonism of antagonistic strains against Colle-totrichum acutatum

菌株编号 Strain number	菌落直径 Colony diameter/mm	抑菌率 Antibacterial rate/%
TN2	15.54±0.14e	71.23±0.26a
CB2	16.4±0.21d	67.91±2.36b
BSJ4	18.65±0.11c	64.62±0.96c
BSJ12	19.4±0.16b	61.48±2.22d
YC8	21.36±0.27a	60.44±0.49d

图1 菌株TN2对辣椒尖孢炭疽菌的抑制作用（标尺：5 mm）

Fig. 1 Inhibition of strain TN2 on C. acutatum（scale: 5 mm）

图2 菌株TN2的菌落形态和扫描电镜下观察形态

Fig. 2 Colony morphology and the morphology of strain TN2 observed by SEM

表2 菌株TN2的生理生化特征

Table 2 Physiological and biochemical characteristics of strain TN2

测试项目 Test item	特征 Characteristic	测试项目 Test item	特征 Characteristic
接触酶	+	MR	-
氧化酶	+	葡萄糖产酸	+
明胶液化	+	葡萄糖产气	-
V-P	+	蔗糖产酸	+
pH 5.5	+	蔗糖产气	-
20% NaCl	+	吲哚乙酸	+
50℃生长温度	+	固氮	+
淀粉水解	+	解磷	-

图3 基于16S rRNA序列构建菌株TN2与其他菌株的系统发育树

Fig. 3 A phylogenetic tree of strain TN2 and other strains constructed based on the 16S rRNA sequence

图4 菌株TN2挥发性有机物及无菌滤液对炭疽病原菌的抑制（标尺：5 mm） A：菌株TN2挥发性有机物对炭疽菌的抑制作用；C：菌株TN2无细胞滤液对炭疽菌的抑制作用；B、D：炭疽菌菌落（CK）

Fig. 4 Inhibition of VOCs and cell-free filtrate of strain TN2 to C. capsici（scale: 5 mm） A: Inhibition of strain TN2 VOCs against anthracnose. C: Inhibition of strain TN2 cell-free filtrate against anthracnose. B, D: Anthrax colonies（CK）

图5 菌株TN2在PDA平板上对7种植物病原菌的拮抗作用（标尺：5 mm） A1-G1为处理组，A2-G2为对照组；A：尖孢镰刀菌；B：辣椒枯萎病菌；C：茄镰刀菌；D：灰葡萄孢菌；E：大丽轮枝菌；F：核盘菌；G：芒果囊孢菌

Fig. 5 Inhibitory effect of strain TN2 on 7 plant pathogens on PDA plate（scale: 5 mm） A1-G1 is the treatment group, and A2-G2 is the control group. A: Fusarium oxysporum; B: Fusarium annuum; C: Haematonectria; D: Botrytis cinerea; E: Verticillium dahliae;F: Sclerotinia sclerotiorum; G: Guignardia

表3 菌株TN2对不同病原真菌的抑制效果

Table 3 Inhibitory effects of strain TN2 on various fungal pathogens

病原菌 Pathogen	菌落直径 Colony diameter/mm	抑菌率 Antibacterial rate/%
尖孢镰刀菌Fusarium oxysporum	34.47±0.77a	53.92±1.26e
辣椒枯萎病菌Fusarium annuum	29.05±0.27c	64.14±0.3c
茄镰刀菌Haematonectria	31.37±0.83b	58.17±1.08d
灰葡萄孢菌Botrytis cinerea	32.26±2.96b	61.48±2.22d
大丽轮枝菌Verticillium dahliae	7.92±0.37f	89.29±0.47a
核盘菌Sclerotinia sclerotiorum	27.36±0.75d	63.99±0.97c
芒果囊孢菌Guignardia	21.42±0.69e	73.87±0.81b

图6 菌株TN2处理对辣椒果实MDA含量的影响

Fig. 6 Effects of strain TN2 treatment on the MDA contents of pepper fruits

图7 菌株TN2处理对辣椒果实抗氧化酶活性的影响

Fig. 7 Effects of strain TN2 treatment on antioxidant enzyme activity in pepper fruits

图8 在不同温度（A）、pH（B）、超声波时长（C）和紫外线照射时长（D）处理下菌株TN2发酵滤液对辣椒尖孢炭疽菌抑制作用的稳定性不同字母表示差异性显著（P<0.05）

Fig. 8 Effect of different temperatures（A）, pH（B）, ultrasonic duration（C）, and UV irradiation duration（D）on the inhibitory stability of TN2 fermentation filtrate against C. acutatum Different letters indicate significant difference（P <0.05）

图9 菌株TN2对辣椒炭疽病的防治效果（标尺：10 mm） CK：无菌水+炭疽菌（阳性对照）；TN2：TN2+炭疽菌

Fig. 9 Biocontrol effects of strain TN2 on pepper anthracnose（scale: 10 mm） CK: Sterile water + pepper anthrax（positive control）; TN2: TN2 + anthrax

表4 菌株TN2对辣椒果实炭疽病的防治效果

Table 4 Efficacy of strain TN2 against anthrax in pepper fruits

处理 Treatment	第1天 Day 1		第3天 Day 3		第7天 Day 7
处理 Treatment	病情指数 Disease index	相对防效 Relative control effect/%	病情指数 Disease index	相对防效 Relative control effect/%	病情指数 Disease index	相对防效 Relative control effect/%
对照 CK	9.44±8.28d	-	47.59±9.67b	-	72.22±3.79a	-
TN2	0.56±1.36e	94.12±0.14a	8.06±4.52d	83.07±0.10ab	20.19±4.93c	72.05±0.07b

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