高地芽孢杆菌CY1的分离鉴定及其对烟草黑胫病的防治作用

doi:10.3969/j.issn.1004-1524.20240188

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (2): 405-416.DOI: 10.3969/j.issn.1004-1524.20240188

高地芽孢杆菌CY1的分离鉴定及其对烟草黑胫病的防治作用

高强¹(), 王丽丽¹, 张渐隆¹, 杨波¹, 李峰¹, 朱先志¹, 刘爱新², 韩超²^,^*(), 田雷¹^,^*()

1.山东临沂烟草有限公司,山东临沂 276003
2.山东农业大学植物保护学院,山东泰安 271018

收稿日期:2024-03-01 出版日期:2025-02-25 发布日期:2025-03-20
作者简介:*韩超,E-mail: hanch87@163.com;
高强(1988—),男,山东临沂人,硕士,主要研究烟草栽培与绿色防控。E-mail:lyysgq@139.com
通讯作者: 韩超,田雷
基金资助:
临沂市烟草公司科技项目(2022009);山东省自然科学基金面上项目(ZR2022MC015);山东省现代农业产业技术体系项目(SDAIT-04-08)

Screening and identification of Bacillus altitudinis strain CY1 and its control effects against tobacco black shank

GAO Qiang¹(), WANG Lili¹, ZHANG Jianlong¹, YANG Bo¹, LI Feng¹, ZHU Xianzhi¹, LIU Aixin², HAN Chao²^,^*(), TIAN Lei¹^,^*()

1. Linyi Tobacco Company, Linyi 276003, Shandong, China
2. College of Plant Protection, Shandong Agricultural University, Tai’an 271018, Shandong, China

Received:2024-03-01 Online:2025-02-25 Published:2025-03-20
Contact: HAN Chao,TIAN Lei

摘要/Abstract

摘要： 为了获得对烟草黑胫病具有高效拮抗作用的生防菌株,采用对峙培养法从烟草黑胫病病田土壤中筛选对烟草疫霉具有良好拮抗作用的细菌菌株,并对其进行形态学、生理生化和分子生物学鉴定。采用单因素试验优化该菌株的发酵条件,对发酵液使用浓度和抑菌作用机理进行研究,并通过室内盆栽和大田试验评价该菌株对烟草黑胫病的防治效果。结果表明,从采集的烟草根际土样中共分离到135个分离物,其中,细菌菌株CY1对烟草疫霉的抑制效果最好,抑菌率为68.6%,且抑菌谱广。经鉴定,CY1为高地芽孢杆菌(Bacillus altitudinis),其最适发酵条件如下:培养基为YSP培养基,初始pH值为7,发酵温度为30 ℃,转速为220 r·min^-1,发酵时间为48 h。1×10⁴~1×10⁸ CFU·mL^-1的发酵液均能抑制烟草疫霉生长,引起菌丝末段畸形膨大并形成褐色沉淀物。1×10⁶ CFU·mL^-1的发酵液对烟草黑胫病的室内盆栽和大田防效分别为71.7%和74.7%,其防治效果与化学农药代森锰锌没有显著差异,但显著(P<0.05)高于霜霉威,低于氟噻唑吡乙酮。综上,高地芽孢杆菌菌株CY1具有良好的生防潜力与应用前景,可为烟草黑胫病的生物防治提供菌种资源。

关键词: 高地芽孢杆菌(Bacillus altitudinis), 烟草黑胫病, 筛选鉴定, 发酵条件, 防治效果

Abstract:

To obtain efficient antagonist strains against tobacco black shank, the rhizosphere soil samples of healthy tobacco plants were collected from tobacco field with tobacco black shank, and the bacterial strain against Phytophthora parasitica was screened out by confrontation culture method. This strain was identified based on morphological, physiological and molecular biology characteristics. The fermentation condition was optimized by single factor experiment, and the available application concentration and antimicrobial mechanism of fermentation broth were explored. Additionally, the control effect of CY1 on tobacco black shank was determined by both indoor pot and field trials. The results showed that a total of 135 soil microbial strains were isolated from the collected rhizosphere soil samples of tobacco, of which a bacterial strain CY1 displayed excellent antagonistic effect against Phytophthora parasitica with an inhibitory rate of 68.6%, and it had a broad spectrum of antifungal function. Based on morphological, physiological and 16S rRNA and gyrB gene sequences analysis, the strain CY1 was identified as Bacillus altitudinis. Its optimal fermentation conditions were as follows: YSP cultural medium with an initial pH value of 7, rotation speed of 220 r·min^-1, at 30 ℃ for 48 h. The fermentation broth with a concentration range from 1×10⁴ CFU·mL^-1 to 1×10⁸ CFU·mL^-1 inhibited mycelial growth, caused mycelial terminal deformity and swell, and induced granular contents in the mycelial tube of Phytophthora. Parasitica. The indoor pot and field control efficacies of the fermentation broth of CY1 with 1×10⁶ CFU·mL^-1 concentration were 71.7% and 74.7%, respectively, which was comparable to those of mancozed, but significantly (P<0.05) higher than those of propamocarb and lower than those of oxathiapiprolin. To sum up, the Bacillus altitudinis strain CY1 has promising biocontrol potential and application prospect, which provided biological control resources of tobacco black shank.

Key words: Bacillus altitudinis, tobacco black shank, screening and identification, fermentation condition, control effect

中图分类号:

S435.72

高强, 王丽丽, 张渐隆, 杨波, 李峰, 朱先志, 刘爱新, 韩超, 田雷. 高地芽孢杆菌CY1的分离鉴定及其对烟草黑胫病的防治作用[J]. 浙江农业学报, 2025, 37(2): 405-416.

GAO Qiang, WANG Lili, ZHANG Jianlong, YANG Bo, LI Feng, ZHU Xianzhi, LIU Aixin, HAN Chao, TIAN Lei. Screening and identification of Bacillus altitudinis strain CY1 and its control effects against tobacco black shank[J]. Acta Agriculturae Zhejiangensis, 2025, 37(2): 405-416.

图/表 8

图1 CY1对烟草疫霉菌的拮抗效果与菌落形态 A,烟草疫霉菌对照;B,CY1对烟草疫霉菌的拮抗效果;C,CY1在LB培养基上的菌落形态。

Fig.1 Antagonistic effect of bacterial strain CY1 on Phytophthora nicotianae and its colonial morphology A, Control; B, Antagonistic effect of CY1 on P. nicotianae; C, Colonial morphology of CY1 on LB medium.

表1 拮抗菌株CY1的抑菌谱

Table 1 Antifungal spectrum of antagonistic bacterium strain CY1

病原菌 Phytopathogen	抑菌率 Inhibition rate/%
烟草疫霉菌Phytophthora parasitica	68.6±1.3a
尖孢镰刀菌Fusarium oxysporum	65.1±2.1ab
茄病镰刀菌Fusarium solani	64.8±1.6ab
立枯丝核菌Rhizoctonia solani	58.9±1.7b
链格孢菌Alternaria alternate	56.3±2.3c
群结腐霉Pythium myriotylum	54.1±1.4c
烟草炭疽菌Collectotrichum nicotianae	52.7±0.9d

表2 拮抗菌株CY1的生理生化特性

Table 2 Physiological and biochemical characteristics of antagonistic strain CY1

指标 Items	CY1菌株 CY1 strain	Bacillus altitudinis	指标 Items	CY1菌株 CY1 strain	Bacillus altitudinis
葡萄糖Glucose	+	+	革兰氏染色Gram stain	+	+
葡聚糖Dextrin	-	-	V-P试验V-P test	+	+
半乳糖Galactose	-	-	ONPG试验ONPG test	+	+
蔗糖Saccharose	+	+	接触酶反应Contactase reaction	+	+
甘露醇Mannitol	+	+	甲基红反应Methyl red reaction	+	+
丙三醇Glycerine	+	+	明胶液化Gelatin liquefaction	+	+

图2 基于16S rRNA和gyrB序列的CY1系统发育树

Fig.2 Phylogenetic tree of strain CY1 based on 16S rRNA and gyrB sequence

图3 CY1发酵条件优化柱上无相同字母的表示差异显著(P<0.05)。

Fig.3 Optimization of fermentation conditions of strain CY1 Bars marked without the same letters indicate significant difference at P<0.05.

图4 不同浓度的CY1发酵液对烟草疫霉菌的抑制作用 A,YSP培养基(对照);B~D中菌液的浓度依次为1×104、1×106、1×108 CFU·mL-1。

Fig.4 Inhibition effects of different concentrations of CY1 on growth of Phytophthora parasitica A, YSP medium (control); B-D, the strain concentration in fermentation liquid is 1×104, 1×106, 1×108 CFU·mL-1.

图5 CY1发酵液对烟草疫霉菌菌丝生长的抑制作用 A,YSP培养基(对照);B~D,CY1发酵液引起烟草疫霉菌丝变化,图中箭头表示CY1引起的烟草疫霉菌菌丝末段畸形膨大,并在菌丝中形成褐色沉淀物。

Fig.5 Inhibition effects of fermentation broth of CY1 on mycelial growth of Phytophthora parasitica A, YSP medium (control); B-D, The fermentation broth of CY1 caused mycelial morphological changes of Phytophthora parasitica, the arrows in the figure represent that CY1 caused mycelial terminal deformity and swell, and induced to produce the granular contents in the mycelial tube of Phytophthora parasitica.

表3 菌株CY1对烟草黑胫病的盆栽和大田防效

Table 3 Control effects of strain CY1 against tobacco black shank in pot and field experiments

处理 Treatment	盆栽试验结果Pot test result			大田试验Field test result
处理 Treatment	发病率 Incidence of disease/%	病情指数 Disease index	防效 Control efficacy/%	发病率 Incidence of disease/%	病情指数 Disease index	防效 Control efficacy/%
CY1发酵液	32.0±6.0 bc	23.8±3.8 bc	71.7±5.2 b	26.0±2.0 d	18.4±1.8 d	74.7±3.9 b
Fermentation broth of CY1
10%氟噻唑吡乙酮OD	28.0±5.0 c	16.2±2.7 d	80.7±4.5 a	24.0±4.0 d	13.8±2.1 d	81.0±4.2 a
oxathiapiprolin 10% OD
80%代森锰锌WP	37.0±6.0 bc	26.8±2.1 bc	68.2±4.2 b	31.0±4.0 c	20.8±2.3 c	71.4±4.3 b
mancozeb 80% WP
72.2%霜霉威WG	43.0±4.0 b	31.8±3.9 b	61.6±3.4 c	38.0±4.0 b	27.2±3.1 b	62.6±2.8 c
propamocarb 72.2% WG
对照Control	93.0±2.0 a	81.4±3.2 a	—	81.0±5.0 a	72.8±4.6 a	—

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高地芽孢杆菌CY1的分离鉴定及其对烟草黑胫病的防治作用

Screening and identification of Bacillus altitudinis strain CY1 and its control effects against tobacco black shank

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