Analysis of Antimicrobial Active Metabolites from Antagonistic Strains Against Fusarium solani

doi:10.13560/j.cnki.biotech.bull.1985.2021-0337

Abstract

Abstract:

Previous studies revealed that two stains Bacillus amyloliquefaciens md8 and md9 have strong antagonism to Fusarium solani causing tree peony root rot;however,the components of antimicrobial substances are not clear yet. First,the biosynthesis gene fragments of 3 lipopeptides were identified. Acid precipitation and the Sephadex gel chromatography column were used to separate and purify the antimicrobial substances. Then Oxford Cup method was adapted to detect the inhibitory activity of the crude lipopeptides and separation components. RT-qPCR was employed to detect the expression variations of lipopetide biosynthesis genes in the strains md8 and md9 during inhibiting F. solani process in the dual culture. And finally MALDI-TOF-MS technique was used to analyze the types of antimicrobial substances. The results showed that the crude lipopeptide extracts and separation components via Sephadex gel chromatography had favorable effect against the pathogen of root rot. The RT-qPCR results indicated that gene ituD of md8 and md9 for iturin biosynthesis was constantly expressed in high amounts throughout the antagonism. The gene fenA for fengycin biosynthesis were up-regulated when md8 and md9 were confronted with F. solani. The MALDI-TOF-MS results further verified that iturin B and bacillomycin D were the main antimicrobial components of md8 and md9 against F. solani. Combined with RT-qPCR results,it was speculated that iturin produced by the strains md8 and md9 played an important role in the inhibitory effect on F. solani.

Key words: Paeonia suffruticosa, Fusarium solani, Bacillus amyloliquefaciens, lipopeptide biosynthesis genes, antimicrobial active metabolites

YANG Rui-xian, LIU Ping, WANG Zu-hua, RUAN Bao-shuo, WANG Zhi-da. Analysis of Antimicrobial Active Metabolites from Antagonistic Strains Against Fusarium solani[J]. Biotechnology Bulletin, 2022, 38(2): 57-66.

Figures/Tables 11

Table 1 Primer sequence for lipopeptide biosynthesis genes fragments

序号 No.	基因 Gene	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物大小 Product size/ bp	产生的脂肽类物质 Lipopeptide
1	fenA	FenAa	AAGAGATTCAGTAAGTGGCCCATCCAG	1 500	Fengycin
1	fenA	FenAb	CGCCCTTTGGGAAGAGGTGC	1 500	Fengycin
2	srfAA	Srfkn-1	AGCCGTCCTGTCTGACGACG	1 500	Surfactin
2	srfAA	Srfkn-2	TCTGCTGCCATACCGCATAGTC	1 500	Surfactin
3	ituD	ItuD-f ItuD-r	ATGAACAATCTTGCCTTTTTA TTATTTTAAATTCCGCAATT	1 300	Iturin

Table 2 Primer sequences for lipopeptide biosynthesis genes in RT-qPCR

序号 No.	引物名称 Primer name	引物序列 Primer sequence（5'-3'）	基因 Gene
1	F1-F	CAGGTGCCGTTACAGGACAT	fenA
1	F2-R	TCGCAGGCTGAATCGCTTC	fenA
2	S1-F	CGGTCAGCAATACGGAAGTCT	srfAA
2	S2-R	TCAGTTCAGGACGGTTGTAGTAG	srfAA
3	I1-F	ATTTCCTGGACAAGGGTCTCAAT	ituD
3	I2-R	TCGCATCGCTCGCTTCTTC	ituD
4	rpsJ-F	GAAACGGCAAAACGTTCTGG	rpsJ
4	rpsJ-R	GTGTTGGGTTCACAATGTCG	rpsJ

Fig.1 Colonies and morphological characteristics of anta-gonistic strain md8 and md9（10×100）

Fig.2 Phylogenetic tree of antagonistic strain md8 and md9 based on 16 S rDNA sequences

Fig. 3 Electrophoresis of PCR amplified lipopeptide biosy-nthesis gene fragments of strain md8 and md9 A：Amplified results of the lipopeptide biosynthesis gene fragments of md8. B：Amplified results of the lipopeptide biosynthesis gene fragments of md9. M：DL2000 marker

Fig.4 Inhibitory effect of strain md8 and md9 inhibiting F. solani on the plate（plate confrontation cultured 6 d） A：strain md8;B：strain md9

Fig.5 Relative expressions of the lipopeptide biosynthesis genes of strain md8 inhibiting F. solani on the plate

Fig.6 Relative expressions of the lipopeptide biosynthesis genes of strain md9 inhibiting F. solani on the plate

Fig.7 Inhibition effect of lipopeptide extract and separation components via gel chromatography of strain md8 and md9 inhibiting F. solani A：Inhibition effect of lipopeptide extract from strain md8 and md9. B：Plate inhibition effect of separation components of strain md8 via gel chromatography. C：Inhibition effect of separation components of strain md9 via gel chromatography

Fig.8 MALDI-TOF-MS analysis of separation component A4（A）and A5（B）produced by the strain md8

Fig.9 MALDI-TOF-MS analysis of separation component B2（A）,B3（B）and B4（C）produced by the strain md9

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