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

doi:10.3969/j.issn.1004-1524.2023.04.14

Acta Agriculturae Zhejiangensis ›› 2023, Vol. 35 ›› Issue (4): 873-883.DOI: 10.3969/j.issn.1004-1524.2023.04.14

• Plant Protection • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S435.72

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.

Figures/Tables 11

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

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.

Fig.2 Phylogenetic tree of GyrB sequence of antagonistic bacteria

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

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

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.

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

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

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

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

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

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