A fungistatic agent and its use in inhibiting fusarium fungi

Abstract

The application belongs to the technical field of biological pesticides, and particularly relates to application of a bacteriostatic agent in inhibition of Fusarium fungi. The bacteriostatic agent contains at least one component of chromanol or tryptanthrin, and can effectively inhibit the growth of F. proliferatum, and the inhibition rate of F. proliferatum is as high as 100%. Since F. proliferatum is a dominant pathogenic fungus causing rhizome rot of Lanzhou lily, the bacteriostatic agent provided by the application can also be used for prevention and treatment of rhizome rot of lily, and the effective active component in the bacteriostatic agent is a metabolic product of microorganisms, so that the bacteriostatic agent has the advantages of green environmental protection, high efficiency and safety.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticide technology, specifically relating to a bacteriostatic agent and its application in inhibiting Fusarium fungi. Background Technology

[0002] Lanzhou lily (Lilium davidii var. willmottiae), a sweet lily variety unique to China, has extremely high edible and medicinal value and is widely cultivated in Gansu and other regions, serving as an important local economic crop. However, during its growth and storage, it faces serious threats from fungal diseases, especially root rot, which greatly affects the yield and quality of the lily.

[0003] Current methods for controlling fungal diseases in Lanzhou lilies include chemical methods using chemical pesticides and biological control methods. While chemical pesticides can effectively control diseases in the short term, long-term use can lead to drug resistance in pathogens, damage the soil ecosystem, cause pesticide residues, and threaten food safety and human and animal health. Biological control methods have the advantages of safety, high efficiency, and environmental friendliness, but they suffer from problems with ease of application and stability. For example, some biocontrol agents (such as live bacteria preparations) require low-temperature storage, strict control of application time (such as avoiding high temperatures and strong light), and are easily affected by antagonistic microorganisms in the soil, which can reduce their activity. The metabolites of biocontrol strains have rich biological activity. Bacillus paralichrysitiformis, as an important member of the Bacillus genus, can produce a variety of metabolites during its metabolism. Existing studies have revealed that Bacillus paralichrysitiformis can produce polypeptide antibiotics, lipopeptide compounds, volatile organic compounds, lysozymes, and various enzymes such as amylase, cellulase, and chitinase. Therefore, identifying key antibacterial substances in the metabolites of Bacillus paralichrysogenus and developing an antibacterial agent is of great significance for the prevention and control of fungal diseases in Lanzhou lilies. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an antibacterial agent and its application in inhibiting Fusarium fungi. The antibacterial agent provided by this invention can effectively inhibit the growth of Fusarium chrysogenum with an inhibition rate of up to 100%, and can be used to prevent and control root rot in lilies.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] The first aspect of the present invention provides the use of an antibacterial agent in inhibiting Fusarium fungi, said antibacterial agent comprising at least one component of tryptol or tryptamine.

[0007] Furthermore, the antibacterial agent uses tryptol and / or tryptamine as active ingredients.

[0008] Furthermore, the Fusarium fungus is Fusarium latifolium.

[0009] A second aspect of the present invention provides the application of the above-described antibacterial agent in the prevention and control of root rot in lilies.

[0010] Furthermore, the lily mentioned is the Lanzhou lily.

[0011] Furthermore, in the above applications, the application methods include watering the roots of the lily with the antibacterial agent, dipping the roots of the lily in the antibacterial agent, or spraying the lily bulb with the antibacterial agent.

[0012] Furthermore, the antibacterial agent is composed of tryptol and tryptamine, and the mass ratio of tryptamine to tryptol in the antibacterial agent is 4:1 to 3.

[0013] Furthermore, the antibacterial agent is in the form of a suspension, microemulsion, aqueous solution, or wettable powder.

[0014] Furthermore, the antibacterial agent is composed of chromool, tryptamine, and adjuvants; the adjuvants are methanol or water.

[0015] Furthermore, the tryptamine content in each liter of the antibacterial agent is 1.1g to 1.3g.

[0016] Furthermore, each liter of the antibacterial agent is prepared by the following steps: according to the above-mentioned addition amounts of tryptol and tryptamine, the tryptol and tryptamine are dissolved in methanol solution, and then water is added to make up to 1L.

[0017] Furthermore, based on the volume of the antibacterial agent, the volume percentage of methanol is ≤5%.

[0018] Furthermore, the content of the active ingredient in the tryptol is ≥97% by mass fraction; the content of the active ingredient in the tryptamine is ≥98% by mass fraction.

[0019] Furthermore, the pH value of the antibacterial agent is 7.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention provides an antibacterial agent for inhibiting Fusarium fungi. This antibacterial agent contains at least one component of tryptol or tryptamine, which can effectively inhibit the growth of *Fusarium chrysogenum*, achieving an inhibition rate of up to 100%. Since *Fusarium chrysogenum* is the dominant pathogen causing lily root rot, the antibacterial agent provided by this invention can also be used for the prevention and control of lily root rot. Furthermore, the effective active ingredient in the antibacterial agent is a microbial metabolite, giving it the advantages of being environmentally friendly, highly efficient, and safe to use.

[0022] 1. Highly effective antibacterial: The single component of the antibacterial agent of this invention has antibacterial effect, and the combination of tryptamine and tryptol has a synergistic antibacterial effect, showing a significant inhibitory effect on the root rot fungus of Lanzhou lily and other fungal pathogens.

[0023] 2. Green and environmentally friendly: Compared with chemical pesticides, the antibacterial agent of this invention contains metabolites of Bacillus paralicheniformis, which are easily degraded in the natural environment, leave no pesticide residues, do not pollute the ecological environment such as soil and water, are safe for humans and animals, meet the requirements of green agricultural development, and help protect the ecological balance.

[0024] 3. Not prone to drug resistance: The effective component of the antibacterial agent of this invention is the metabolic product of bacteria. As is well known, the antibacterial mechanism of microbial metabolic products is diverse. They play a role in many ways, such as destroying the cell wall and cell membrane of pathogens and interfering with the metabolic pathways of pathogens. Pathogens are unlikely to develop drug resistance through a single gene mutation, and fungal diseases can be controlled sustainably and effectively.

[0025] 4. Promotes plant growth: In addition to its antibacterial effect, the antibacterial agent provided by this invention contains tryptamine and / or cadmium, which are precursors or intermediates in the biosynthesis pathway of indoleacetic acid (IAA, the core auxin of plants). These substances can indirectly provide raw materials for auxin synthesis, thereby assisting in the regulation of cell division, elongation and other growth processes. This is beneficial for promoting the root growth of Lanzhou lily, enhancing plant immunity, improving the lily's resistance to pathogens, and increasing the yield and quality of lily.

[0026] 5. Broad application prospects: The application method provided by this invention can be widely applied to all aspects of Lanzhou lily cultivation, including bulb treatment, field disease control, and storage preservation. Moreover, the application method and formulation type can be flexibly adjusted according to actual needs, which has good prospects for industrial application and can provide strong support for the healthy development of the Lanzhou lily industry. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The results are from the two-point confrontation method experiment on a flat plate. Figure 1 In the image, A represents the control pathogen (Fusarium solani) plate. Figure 1 B in the figure represents the results of the confrontation experiment. The strain on the left side of the plate is the pathogen (Fusarium moniliforme), and the strain on the right side is Bacillus paralicheniformis.

[0029] Figure 2 The results are from the four-hole plate confrontation experiment. Figure 2 In the image, A represents the control pathogen (Fusarium solani) plate. Figure 2B in the figure represents the results of the confrontation experiment, with the strain in the middle of the plate being the pathogen (Fusarium moniliforme) and the strain around the plate being Bacillus paralicheniformis.

[0030] Figure 3 The results are from orthogonal partial least squares discriminant analysis.

[0031] Figure 4 Main component analysis results

[0032] Figure 5 The results are from a thermogram analysis of the metabolites.

[0033] Figure 6 The inhibitory effect of different concentrations of chromol on Fusarium effusum. Figure 6 In the image, A represents the untreated control plate. Figure 6 B in the table represents the control plate treated with methanol. Figure 6 D1, D2, D3, and D4 in the table are plates treated with chromolate at concentrations of 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, and 0.7 mg / mL, respectively.

[0034] Figure 7 The inhibitory effect of different concentrations of tryptophan on Fusarium effusum. Figure 7 In the image, A represents the untreated control plate. Figure 7 B in the table represents the control plate treated with methanol. Figure 7 E1, E2, E3, and E4 in the table are plates treated with tryptamine at concentrations of 0.4 mg / mL, 0.8 mg / mL, 1.2 mg / mL, and 1.6 mg / mL, respectively.

[0035] Figure 8 This is an antibacterial agent that inhibits the growth of Fusarium moniliformes. Figure 8 In the image, A represents the untreated control plate. Figure 8 In the table, B is the control plate treated with methanol, and F1, F2, F3, and F4 are plates treated with antibacterial agents at concentrations of 1.5 mg / mL, 1.7 mg / mL, 1.9 mg / mL, and 2.1 mg / mL, respectively. Detailed Implementation

[0036] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0037] Example 1: Metabolomics analysis of Bacillus paralicheniformis strain culture filtrate

[0038] I. Antagonistic effect of Bacillus paralicheniformis on Fusarium solani, the pathogen causing root rot of lily.

[0039] The *Bacillus paralicheniformis* used in this invention is strain ZYGT1811, deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO. M2020759. *Fusarium proliferatum* was isolated, identified, and preserved in the inventor's laboratory from the bulbs of *Lilium lanzhouense* (accession number: PX389705.1).

[0040] 1. Two-point standoff

[0041] The pathogen causing lily root rot is a mixture of various Fusarium species, but different regions have dominant populations. Previous research in this invention found that *Fusarium moniliforme* is the dominant pathogen causing root rot in Lanzhou lilies. The antagonistic effect of *Bacillus paralicheniformis* on *Fusarium moniliforme* was determined using the two-point plate confrontation method, with *Fusarium moniliforme* cultured alone as a control group, and the experiment was repeated three times. The diameter of the pathogen and the width of the inhibition zone were recorded for each treatment, and the inhibition rate was calculated.

[0042] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0043] The results are as follows Figure 1 As shown in the figure, it can be seen that Bacillus paralichrysogenum has a significant inhibitory effect on Fusarium moniliforme, with an inhibition rate of 32.97%.

[0044] 2. Four-point standoff

[0045] A 6mm mycelial cake of *Bacillus paralichrysogenus* was inoculated in the center of a PDA plate. Four 6mm wells were punched at equidistant intervals of 1.5cm around the center. 30μL of *Bacillus paralichrysogenus* suspension was added to each well. Each treatment was repeated three times, with LB liquid medium as a control. The plates were incubated at 25℃ for 5 days to observe the antibacterial effect, record the inhibition rate, and take photographs.

[0046] Inhibition rate (%) = (Control colony diameter - Treatment colony diameter) / Control colony diameter × 100%.

[0047] The results are as follows Figure 2 As shown in the figure, Bacillus paralichrysogenum has a significant inhibitory effect on Fusarium moniliforme, with an inhibition rate of 54.89%.

[0048] II. Effects of metabolites in Bacillus paralichrysiforme culture filtrate on the mycelial growth of the pathogen.

[0049] 1. Determination of metabolites in Bacillus paralichrysiforme culture filtrate

[0050] 1.1 Experimental Methods

[0051] Preparation of paralichrysanthemum culture filtrate samples: Fresh single colonies of activated antagonistic bacteria (Bacillus paralichrysanthemum) were inoculated into 150 mL Erlenmeyer flasks containing 50 mL LB medium and cultured at 28 °C and 180 r / min for 24 h. Then, the culture was inoculated into fresh LB medium at a volume ratio of 1% and cultured at 32 °C and 210 r / min for 24 h. The culture was then centrifuged at 9000 g for 5 min in centrifuge tubes. The culture medium was filtered through a 0.22 μm bacterial filter, and the supernatant was collected as the paralichrysanthemum culture filtrate sample, with LB liquid medium as a control.

[0052] Metabolite extraction: The filtrate sample of *Lichen parasiticus* was slowly thawed on ice. Metabolites were extracted from the sample using a 50% methanol aqueous solution. 100 μL of the metabolite extract was mixed with 400 μL of pre-chilled 50% methanol-acetonitrile mixture (methanol to acetonitrile volume ratio 1:1) and sonicated for 10 min. The mixture was then incubated at -20°C for 1 hour, followed by centrifugation at 20,000 g for 15 min at 4°C. The supernatant was collected and dried overnight. The dried product was reconstituted with a 50% acetonitrile aqueous solution, sonicated for 10 min, and then centrifuged at 20,000 g for 15 min at 4°C. The supernatant was collected and bottled. Additionally, 10 μL of each sample was mixed to form a QC sample, which was also bottled in the same manner.

[0053] Analytical Process: All samples to be tested were sequentially arranged in an ultra-high pressure liquid chromatography (UPLC) instrument and pre-separated using an ACQUITY UPLC T3 column. The column temperature was 50℃, the flow rate was 0.3 mL / min, and the mobile phase A was 0.1% formic acid in water, while the mobile phase B was 0.1% formic acid in acetonitrile. The gradients were as follows: 0 min–0.5 min, 5% B phase; 0.5 min–9.5 min, 5%–100% B phase; 9.5 min–11.5 min, 100% B phase; 11.5 min–12 min, 100% B phase to 5% B phase; 12 min–15 min, 5% B phase. The sample volume was 5 μL.

[0054] The high-resolution mass spectrometer used for data acquisition was a Q-Exactive Plus (Thermo Fisher Scientific). Each sample underwent one acquisition in positive ion mode and one in negative ion mode. The shielding gas pressure of the ion source was 2, the pressure of gas 1 (auxiliary gas) was 40, and the pressure of gas 2 (sheath gas) was 10. The source temperature was 350℃. The voltage was +3800V in positive ion mode and -3100V in negative ion mode.

[0055] Data acquisition was performed in DDA (Information-Dependent Acquisition) mode. In one acquisition cycle, the primary acquisition range was 70 Daltons–1050 Daltons, with a primary resolution of 70 K (m / z 200), an AGC target of 3e6, and a maximum IT of 100 ms. Then, the top three signal ions with a cumulative signal intensity exceeding 100,000 from the primary spectrum were selected for secondary fragmentation scanning. The secondary resolution was 17.5 K (m / z 200), and the maximum IT was 50 ms. During acquisition, QC (Quality Control) samples were scanned every 10 samples. The quality difference between QC samples was used to correct for systematic errors in the entire batch of experiments.

[0056] 1.2. Differential component analysis based on non-targeted metabolomics

[0057] (1) OPLS-DA and PCA

[0058] To investigate the changes in metabolites in *Lichen parasiticus* culture filtrate samples compared to control culture medium, this invention performed metabolic analysis. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to distinguish the metabolic profiles between the *Lichen parasiticus* culture filtrate samples and the control culture medium. Figure 3 The variable importance (VIP) value for each metabolite is obtained based on the OPLS-DA model. Generally, variables (metabolites) with a VIP > 1 are considered statistically significant.

[0059] Principal component analysis showed that the two metabolites exhibited different spatial enrichment patterns. Figure 4 The data showed significant differences between the two groups. Furthermore, the fact that each set of replicates could be combined indicates that the chemical analysis data are reliable and suitable for further investigation. PC1 explained 65.4% of the variation, while PC2 explained 16.5%. The metabolic compounds of FDY(B) and CK(A) differed significantly from those of PC1, indicating differences in metabolic compounds between the paralichrysiform culture filtrate and the culture medium.

[0060] Furthermore, by combining FC with VIP in the OPLS-DA model, it was found that 61 metabolites in the filtrate of paralichen culture were altered, a proportion significantly higher than that in the control.

[0061] (2) Metabolite hierarchical clustering and significance analysis

[0062] By detecting and screening metabolites in the culture filtrate of *Lichen parasiticus* using non-targeted metabolomics, a total of 154 negative ions and 431 positive ions were detected between the CK and FDY groups.

[0063] Based on the criteria of P-value < 0.05, FC ≥ 2 or FC ≤ 0.5, and VIP > 1.0, 61 metabolites were screened, with 15 upregulated and 46 downregulated. Hierarchical cluster analysis identified six categories of metabolites: heterocyclic compounds and aromatic amines, dipeptides and oligopeptides, amino acids and their derivatives, fatty acids and their derivatives, organic acids, and natural products. A total of 57 metabolic pathways were enriched. These pathways involve the synthesis, metabolism, and transport of various substances, such as amino acid biosynthesis, and primarily involve organic acids and amino acids. Figure 5 ).

[0064] 2. Inhibitory effect of differential metabolites on the mycelial growth of Fusarium effusum

[0065] Indoor antibacterial assays were performed on differentially expressed metabolites that showed inhibitory activity against Fusarium oxysporum. A stock solution of the metabolites (tryptamine and / or tryptol) at a concentration of 200 mg / mL and a pH of 7 was prepared using methanol. Different concentration gradients of tryptamine, tryptol, and mixtures of tryptamine and tryptol were prepared in PDA medium. After incubation at a suitable temperature, the plates were inoculated with pathogenic bacteria cultured for 5 days at the center. The original bacteria, methanol, and water were used as controls. The antibacterial activity was observed, and the inhibition rate was measured.

[0066] Based on the screening results, nine upregulated metabolites (6-methylquinoline, tryptamine, tryptophol, acetylphenylalanine, acetylleucine, and α-phenylethylamine) and three downregulated metabolites (dimethyl malonate, L-arginine, and D-gluconic acid) were identified. Figures 6-8 As shown, the inhibition rate of tryptamine against Fusarium effusum was 100% at concentrations of 0.7 mg / mL and 1.6 mg / mL. Moreover, the mixture of the two substances at different concentrations had a significant synergistic effect on Fusarium effusum. The inhibition rates against Fusarium effusum reached 95%, 98%, 100%, and 100%, respectively, when the mixture concentrations were 1.5 mg / mL (final tryptamine concentration 1.2 mg / mL + final tryptamine concentration 0.3 mg / mL), 1.7 mg / mL (final tryptamine concentration 1.2 mg / mL + final tryptamine concentration 0.5 mg / mL), 1.9 mg / mL (final tryptamine concentration 1.2 mg / mL + final tryptamine concentration 0.7 mg / mL), and 2.1 mg / mL (final tryptamine concentration 1.2 mg / mL + final tryptamine concentration 0.9 mg / mL).

[0067] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0068] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of an antibacterial agent in inhibiting Fusarium fungi, characterized in that, The active ingredient of the antibacterial agent is at least one of chromol or tryptamine; the Fusarium fungus is Fusarium latifolium.

2. The application of an antibacterial agent in the prevention and control of lily root rot, characterized in that, The active ingredient of the antibacterial agent is at least one of tryptol or tryptamine; the antibacterial agent is applied to the roots of the lily, or the roots of the lily are dipped in the antibacterial agent, or the antibacterial agent is sprayed on the lily bulb to prevent lily root rot; the lily root rot is caused by Fusarium moniliforme.

3. The application according to claim 2, characterized in that, The active ingredient of the antibacterial agent is composed of tryptol and tryptamine, and the mass ratio of tryptamine to tryptol in the antibacterial agent is 4:1~3.

4. The application according to claim 3, characterized in that, The antibacterial agent is composed of chromool, tryptamine and an adjuvant; the adjuvant is methanol.

5. The application according to claim 4, characterized in that, The content of tryptamine in each liter of the antibacterial agent is 1.1g to 1.3g.

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