Application of a strain of fusarium derived from barley in the prevention and control of bacterial diseases of barley

Abstract

The present application discloses a fusarium sp. BH-23-5 (the preservation number PX512781) derived from a healthy barley plant, and application of a fermentation product thereof in preventing and controlling barley bacterial diseases. The fermentation product (fermentation liquor, bacterial body, crude extract or monomer compound) has significant inhibitory activity on barley pathogenic bacteria (Xanthomonas translucens pv. hordei, Pseudomonas syringae, Acidovorax avenae), and can be used for preparing a biocontrol agent. The present application develops endophytic fungal resources, provides an efficient biological control method for green prevention and control of barley bacterial diseases, reduces the use of chemical agents, and meets the development direction of green agriculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology and biological control of plant diseases, specifically to a Fusarium strain derived from barley, its fermentation products, and its application in the control of bacterial diseases in barley. Background Technology

[0002] Barley is an important crop for food, feed, and brewing, but its production is often threatened by bacterial diseases, including barley black luster, bacterial leaf spot, and bacterial streak, caused by *Xanthomonas hygroscopicus*, *Pseudomonas syringae*, and *Oat acidophilus*, respectively. Current control methods rely on chemical agents and seed disinfection, which easily leads to antibiotic resistance in pathogens, grain residues, and damage to soil microbial communities, which is inconsistent with the development direction of green agriculture.

[0003] Endophytic fungi are an important source of antibacterial active substances. This invention isolated Fusarium strain BH-23-5 from healthy barley tissue. Its fermentation products have antagonistic activity against barley pathogens, providing a new resource for the biological control of diseases. Summary of the Invention

[0004] 1. Isolation and Identification of Strains Strain origin: Isolated from healthy barley plant tissue, and identified as Fusarium sp. by morphological and molecular identification (ITS gene sequence comparison), named BH-23-5, with accession number PX512781; Strain characteristics: After 3 days of growth on PDA medium, the colonies are white and fluffy, with no color on the reverse side; after 2 weeks of growth, the colonies are 20 mm in diameter, orange-red on the reverse side, do not produce pigments, and have no exudate.

[0005] 2. Preparation of fermentation products Liquid fermentation: cultured at 28℃ and 180 rpm for 48-72 h with shaking. The fermentation broth was centrifuged and filtered to remove bacteria to obtain the fermentation liquid. The bacterial cells were dried and pulverized to obtain the bacterial cell product. Solid-state fermentation: The fermentation product was cultured at 28℃ for 28 days. The fermentation product was extracted with ethyl acetate and concentrated by rotary evaporation to obtain a crude extract. Further purification by silica gel column chromatography, gel separation and semi-preparative liquid chromatography was performed to obtain monomeric active compounds.

[0006] 3. Scope of Application The fermentation products have broad-spectrum inhibitory activity against barley pathogens and can be used to prepare preparations that inhibit Xanthomonas hyalinus, Pseudomonas syringae, and agaric acid-eating bacteria, or to make biocontrol agents (suspension, powder, granules) for disease control through seed treatment, soil application, and other methods. Attached Figure Description

[0007] Figure 1This is a technical roadmap for strain isolation and fermentation product preparation. Figure 2 The inhibition zone (32 mm in diameter) of the BH-23-5 bacterial suspension against Xanthomonas barleyii is shown. Figure 3 The inhibition zones of BH-23-5 fermentation broth against different pathogens are shown (19 mm for Xanthomonas barleyii, 17 mm for Pseudomonas syringae, and 22 mm for Bacillus acidophilus in oats). Figure 4-6 The results show the antibacterial activity screening of crude extract and isolated components of BH-23-5 against Xanthomonas barleyii. Figure 7 Component H4 was separated into H4-1 and H4-2 via semi-preparative liquid phase separation. Figure 8 The inhibition zone (32 mm in diameter) of the monomeric compound H4-1 against Xanthomonas barleyii is shown. Figure 9 It is one of the ¹H-NMR proton and ¹³C-NMR spectra of H4-1. Figure 10 This is the second of the ¹H-NMR hydrogen spectrum and ¹³C-NMR carbon spectrum of H4-1. Figure 11 The image shows the EI-MS mass spectrum of H4-1. Detailed Implementation

[0008] Example 1: Isolation and Identification of Strains In November 2024, tissues from healthy barley plants were collected, disinfected, and inoculated onto PDA medium. The cultures were incubated at 28°C for 48 h, and single colonies were picked and purified to obtain BH-23-5. Genomic DNA was extracted and amplified using ITS4 / ITS5 primers to obtain a 548 bp sequence (PX512781). NCBI BLAST alignment confirmed it as *Fusarium* sp.

[0009] Example 2: Inhibitory activity of fermentation products against pathogens Liquid fermentation: BH-23-5 was inoculated with PDA liquid medium and cultured at 28℃ and 180 rpm for 2 days. After sterilization, the diameter of the inhibition zone against Xanthomonas barley VP-HL was 32 mm. Inhibition of different pathogens: The diameters of the inhibition zones of the fermentation broth against Pseudomonas syringae and Acidophilus ovalis were 17 mm and 22 mm, respectively.

[0010] Example 3: Separation and purification of crude extract and monomeric compounds Silica gel column chromatography: The crude fermentation extract was separated into 12 fractions by silica gel column chromatography, among which the inhibition zone of fraction K had a diameter of 29 mm; Gel column chromatography: K fraction was separated into K8 fraction by Sephadex LH-20, with an inhibition zone diameter of 44 mm; Semi-preparative liquid phase: The K8 fraction was separated by HPLC to obtain the monomer compound H4-1, which was identified by NMR and MS as Woodwardinsaure methylester (molecular weight 234.0927), and the inhibition zone diameter against xanthophyte was 32 mm.

Claims

1. The application of the fermentation product of a Fusarium strain derived from barley in the control of bacterial diseases in barley, i.e., a Fusarium strain, characterized in that... The strain is Fusarium sp. BH-23-5, with accession number PX512781. The fermentation product is the fermentation broth, bacterial cells, crude fermentation extract, or monomeric active compound obtained by liquid or solid fermentation of the strain. The strain or the fermentation product is used in the preparation of an inhibitory agent against barley pathogens. The barley pathogen is at least one of Xanthomonas translucens, Pseudomonas syringae, and Acidovorax avenae.

2. A biocontrol agent for combating bacterial diseases in barley, characterized in that, It comprises the Fusarium strain of claim 1 and agriculturally acceptable excipients, and is formulated as a suspension, powder or granule.

3. The method for preparing the biocontrol agent according to claim 4, characterized in that, include: (1) Inoculate the strain described in claim 1 into a culture medium and ferment it at 26-28℃ and 120-180 rpm for 48-72 h, and collect the fermentation product; (2) The fermentation product is mixed with agriculturally acceptable auxiliary materials to obtain a biocontrol agent.

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