烟草根黑腐病拮抗菌的分离鉴定和生防作用特性研究

doi:10.3969/j.issn.1004-1524.2023.04.14

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (4): 873-883.DOI: 10.3969/j.issn.1004-1524.2023.04.14

烟草根黑腐病拮抗菌的分离鉴定和生防作用特性研究

黄婉媛¹(), 李彩斌², 彭宇², 李章海³, 黄衍章¹, 丁婷¹^,^*()

1.安徽农业大学植物保护学院,植物病虫害生物学与绿色防控安徽普通高校重点实验室,作物有害生物综合治理安徽省重点实验室,安徽合肥 230061
2.贵州省烟草公司毕节市公司,贵州毕节 551700
3.中国科学技术大学烟草与健康研究中心,安徽合肥 230061

收稿日期:2022-01-18 出版日期:2023-04-25 发布日期:2023-05-05
通讯作者: ^*丁婷,E-mail: 18355433@qq.com
作者简介:黄婉媛(1999—),女,安徽滁州人,硕士,研究方向为植物病害生物防治。E-mail: 1332424965@qq.com
基金资助:
贵州省烟草公司科技项目(2020XM09);贵州省烟草公司毕节市公司科技项目(2020520500240016)

Studies on isolation and identification of antagonistic bacteria against tobacco root black rot pathogen, Thielaviopsis basicola and their biocontrol characteristics

HUANG Wanyuan¹(), LI Caibin², PENG Yu², LI Zhanghai³, HUANG Yanzhang¹, DING Ting¹^,^*()

1. Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes, Anhui Province Key Laboratory of Integrated Pest Management on Crops, College of Plant Protection, Anhui Agricultural University, Heifei 230061, China
2. Bijie Branch, Guizhou Provincial Tobacco Company, Bijie 551700, Guizhou, China
3. Tobacco and Health Research Center, University of Science and Technology of China, Hefei 230026, China

Received:2022-01-18 Online:2023-04-25 Published:2023-05-05

摘要/Abstract

摘要：

为挖掘对烟草根黑腐病菌有较强拮抗效果,且可在烟株根部稳定定殖的生防菌资源,以烟草根黑腐病菌为靶标,利用稀释涂布法和平板对峙法分离筛选出高效拮抗活性菌株,对其进行系统发育分析以及粗脂肽抑菌活性检测;并运用PCR技术检测拮抗细菌的脂肽编码基因,利用趋化性及生物膜成膜等试验研究烟草根系分泌物及其产生的各种有机酸对拮抗菌根部定殖的影响。结果表明:从烟草根际土壤中分离得到两株Thielaviopsis basicola高效拮抗菌株S2-1和SNSY15-5,分子鉴定表明,S2-1和SNSY15-5均为芽孢杆菌属细菌;拮抗菌S2-1和SNSY15-5脂肽粗提物对T. basicola毒力较强,EC₅₀值分别为1.93和2.56 mg·mL^-1,SNSY15-5含有参与脂肽类家族细菌素 (bmyB)、生物素操纵子 (bioA) 和假定蛋白 (yngG) 合成的基因;S2-1菌株中含有参与脂肽类家族生物素操纵子 (bioA) 合成的基因;烟草品种K326根系分泌物产生的苹果酸对S2-1和SNSY15-5有较强的吸引作用,S2-1和SNSY15-5在苹果酸中的数量分别达到63.35×10⁴ CFU·mL^-1和42.35×10⁴ CFU·mL^-1,K326烟草根系分泌物及其草酸可促进S2-1生物膜形成,而K326烟草根系分泌物及其柠檬酸则促进SNSY15-5生物膜形成。该研究结果为开发效果稳定的微生物菌剂提供理论基础。

关键词: 烟草根黑腐病, 根际细菌, 脂肽, 趋化性, 生物膜

Abstract:

In order to explore the biocontrol bacteria resources which had the strong antagonistic effect on tobacco black root rot and could stably colonize the tobacco roots, taking Thielaviopsis basicola as the target, the highly antagonistic strains were isolated and obtained by dilution-plate method and plate confrontation method, and the phylogenetic analysis and antibacterial activity of crude lipopeptide of antagonistic strains were carried out; then the genes coding lipopeptide of antagonistic strains were analyzed by PCR, the effects of K326 tobacco root exudates and various organic acids on root colonization of antagonistic bacteria were studied by chemotaxis and biofilm formation tests. Results indicated that the effective antagonistic strains S2-1 and SNSY15-5 were isolated from tobacco rhizosphere soil, and they were Bacillus microorganisms. The crude lipopeptides of the S2-1 and SNSY15-5 were high virulent to Thielaviopsis basicola, and the EC₅₀ of them was 1.93 and 2.56 mg·mL^-1, respectively. SNSY15-5 had the biocontrol marker genes involved in the synthesis of bmyB, bioA and yngG, while S2-1 had the biocontrol marker gene involved in the synthesis of bioA. Malic acid produced by K326 root exudates had better attraction to S2-1 and SNSY15-5, and the amount of S2-1 and SNSY15-5 in malic acid reached 63.35×10⁴ CFU·mL^-1 and 42.35×10⁴ CFU·mL^-1, respectively. The biofilm formation of S2-1 could be promoted by the K326 root exudates and oxalic acid, while the biofilm formation of SNSY15-5 could be promoted by the K326 root exudates and citric acid. The research results provide a theoretical basis for developing microbial agent with stable effect.

Key words: tobacco black root rot, rhizosphere bacteria, lipopeptide, chemotaxis, biofilm

中图分类号:

S435.72

黄婉媛, 李彩斌, 彭宇, 李章海, 黄衍章, 丁婷. 烟草根黑腐病拮抗菌的分离鉴定和生防作用特性研究[J]. 浙江农业学报, 2023, 35(4): 873-883.

HUANG Wanyuan, LI Caibin, PENG Yu, LI Zhanghai, HUANG Yanzhang, DING Ting. Studies on isolation and identification of antagonistic bacteria against tobacco root black rot pathogen, Thielaviopsis basicola and their biocontrol characteristics[J]. Acta Agriculturae Zhejiangensis, 2023, 35(4): 873-883.

图/表 11

表1 用于鉴定脂肽合成基因的引物

Table 1 Primer sequences for amplification of lipopeptide functional genes

序号 Number	引物名称 Primer name	序列 Sequence(5'→3')
1	yndJ-F yndJ-R	CAGAGCGACAGCAATCACAT TGAATTTCGGTCCGCTTATC
2	bioA-F bioA-R	TTCCACGGCCATTCCTATAC TTTGTCCCCTTATCCTGCAC
3	srfAA-F srfAA-R	TTCCACGGCCATTCCTATAC TTTGTCCCCTTATCCTGCAC
4	fenD-F fenD-R	CCTGCAGAAGGAGAAGTGAAG TGCTCATCGTCTTCCGTTTC
5	srfAB-F srfAB-R	GTTCTCGCAGTCCAGCAGAAG GCCGAGCGTATCCGTACCGAG
6	yngG-F yngG-R	GAACTGTCCGAAACATGTCCG CTGAGCTCTTGAACGGTCCGG
7	bmyB-F bmyB-R	TGAAACAAAGGCATATGCTC AAAAATGCATCTGCCGTTCC
8	bmyB-F bmyB-R	TTCACTTTTGATCTGGCGAT CGTCCGGTACATTTTCAC

图1 拮抗菌对烟草根黑腐病菌的抑制作用 1,SNSY15-5与烟草根黑腐病菌对峙培养7 d;2,S2-1生防菌与烟草根黑腐病菌对峙培养7 d;3,烟草根黑腐病菌培养10 d;a,烟草根黑腐病原菌正常菌丝形态;b,接种拮抗菌对峙培养7 d后的烟草根黑腐菌丝形态。

Fig.1 Inhibitory effect of antagonistic bacteria on Thielaviopsis basicola 1, SNSY15-5 and Thielaviopsis basicola were cultured in confrontation for 7 days; 2, S2-1 and Thielaviopsis basicola were cultured in confrontation for 7 days; 3, Thielaviopsis basicola were cultured separately for 10 days respectively; a, Normal mycelium morphology of Thielaviopsis basicola; b, Mycelial morphology of Thielaviopsis basicola treated with antagonistic bacteria for 7 days.

图2 拮抗菌的 GyrB基因系统发育树

Fig.2 Phylogenetic tree of GyrB sequence of antagonistic bacteria

图3 拮抗菌粗脂肽提取物对烟草根黑腐病菌生长的抑制情况

Fig.3 Effects of crude lipopeptide extracts of antagonistic bacteria on Thielaviopsis basicola A, SNSY15-5; B, S2-1.

表2 拮抗菌脂肽粗提取物对烟草根黑腐病菌生长的影响

Table 2 Antagonistic effect of crude lipopeptide extracts of antagonistic bacteria on Thielaviopsis basicola

处理 Treatments	毒力回归方程 Virulence equation	决定系数 Coefficient of determination	EC₅₀/(mg·mL^-1)	EC₉₀/(mg·mL^-1)
S2-1	y=0.16x+0.192 3	0.987 5	1.93	4.43
SNSY15-5	y=0.217 3x-0.058 3	0.987 4	2.56	4.41

图4 拮抗菌S2-1和SNSY15-5脂肽相关基因扩增结果 M, DL 2000 marker; 1表示S2-1的功能基因bioA;2、3、4分别表示SNSY15-5的功能基因bioA、bmyB、yngG。

Fig.4 Amplification results of functional genes coding lipopeptides of the S2-1 and SNSY15-5 M, DL 2000 marker; 1 represent the functional gene bioA of S2-1; 2, 3, 4 represent the functional genes bioA,bmyB and yngG of SNSY15-5, respectively.

图5 拮抗菌S2-1和SNSY15-5脂肽相关基因的进化分析

Fig.5 Evolutionary analysis of functional genes coding lipopeptides of S2-1and SNSY15-5

表3 拮抗菌对烟草根系分泌物和有机酸的趋化反应

Table 3 The chemotactic response of antagonistic bacteria to the K326 tobacco root exudates and organic acid

处理组 Treatment	拮抗菌细菌数量 Amount of antagonistic bacteria/ (10⁴ CFU·mL^-1)
处理组 Treatment	S2-1	SNSY15-5
草酸 Oxalic acid 苹果酸Malic acid 柠檬酸Citric acid 琥珀酸Succinic acid K326根系分泌物 K326 root exudates CK	58.20 b 63.35 a 46.55 c 12.30 e 16.75 d 11.00 f	22.40 d 42.35 a 17.35 e 27.45 c 30.40 b 14.35 f

图6 K326根系分泌物及有机酸对拮抗菌生物膜成膜能力的影响

Fig.6 Effect of the K326 tobacco root exudates and organic acid on the biofilm formation of antagonistic bacteria

表4 K326根系分泌物及有机酸对拮抗菌生物膜成膜能力的定量检测

Table 4 Quantitative detection of the effect of K326 root exudates and organic acids on the biofilm formation of antagonistic bacteria

处理组 Treatment	拮抗菌生物膜量 Biofilm quantity of antagonistic bacteria(D₅₇₀)
处理组 Treatment	S2-1	SNSY15-5
苹果酸Malic acid	2.21f	2.34 e
柠檬酸Citric acid	2.32 e	2.57 b
琥珀酸Succinic acid	2.58 c	2.46 d
草酸Oxalic acid	2.63 b	2.31 f
K326根系分泌物 K326 root exudates	2.78 a	2.64 a
CK	2.55 d	2.51 c

图7 K326根系分泌物及有机酸对拮抗菌生物膜形成相关基因表达的影响 CK,对照组;W1,拮抗菌添加K326根系分泌物处理组;W2,拮抗菌添加柠檬酸处理组;W3,拮抗菌添加苹果酸处理组;W4,拮抗菌添加草酸处理组;W5,拮抗菌添加琥珀酸处理组。

Fig.7 Effects of K326 root exudates and organic acids on gene expression related to antagonistic biofilm formation CK, Control group; W1, Antagonistic bacteria treated with K326 root exudates; W2, Antagonistic bacteria treated with citric acid; W3, Antagonistic bacteria treated with malic acid; W4, Antagonistic bacteria treated with oxalic acid; W5, Antagonistic bacteria treated with succinic acid.

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烟草根黑腐病拮抗菌的分离鉴定和生防作用特性研究

Studies on isolation and identification of antagonistic bacteria against tobacco root black rot pathogen, Thielaviopsis basicola and their biocontrol characteristics

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