Antagonistic bacteria to Fusarium graminearum and its application in preparing feed mildew inhibitor

Abstract

The invention belongs to the field of biotechnology, and specifically discloses an antagonistic bacterium of Fusarium graminearum and its application in preparing a feed mildew inhibitor. The antagonistic bacterium of Fusarium graminearum is Bacillus amyloliquefaciens Bacillus amyloliquefaciens 4‑9‑2, deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, with a deposit number of CGMCC No: 32407, a deposit date of October 30, 2024, and a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Compared with the prior art, the lipopeptide active substances in the metabolites of Bacillus amyloliquefaciens 4‑9‑2 of the present invention can inhibit the spore germination and mycelial growth of Fusarium graminearum; have a significant inhibitory effect on the production of major fungal toxins such as zearalenone and vomitoxin, and can still maintain antifungal activity under extreme conditions such as strong acid, strong alkali, and high temperature; the bacteria can be used as a reserve resource for the development of new mildew inhibitors.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an antagonistic bacterium of Fusarium graminearum and application thereof in the preparation of a feed mildew inhibitor. Background Art

[0002] Fusarium graminearum Fusarium graminearum ) is one of the important pathogens of Poaceae crops and can cause Fusarium Head Blight (FHB) in wheat. Fusarium graminearum is a major factor limiting wheat yield (Dubin, 1997).

[0003] Deoxynivalenol (DON, 3,7,15-trihydroxy-12,13-epoxytrichothecenes-9-ene-8-one) produced by Fusarium graminearum is one of the main natural toxins present in wheat grains infected with Fusarium graminearum. DON has been identified as one of the most dangerous naturally occurring food contaminants and has been listed as a priority for international research (Larsen et al., 2004). DON can cause decreased immunity, anemia, headache, abdominal pain, and nausea in humans; and cause animals to refuse to eat, stunted growth, and miscarriage (Rocha et al., 2005; Sobrova et al., 2010; Pestka, 2010). DON is widely present and contaminates wheat and its products in large quantities, posing a great threat to humans and animals, and seriously threatening my country's food safety.

[0004] Chinese patent publication number CN111534460B discloses a preparation method and application of a strain of Bacillus Velezii that is highly effective in antagonizing Fusarium graminearum, in the field of microbial technology. The Bacillus Velezii NF002 of the present invention can effectively antagonize Fusarium graminearum, and can effectively inhibit the production of DON toxin of Fusarium graminearum and can degrade DON toxin in wheat grains. Bacillus Velezii NF002 can be used as a biological control material for wheat fusarium head blight, and has good application prospects in the development of new biological pesticides or new biological control agents.

[0005] Currently available antifungal agent development reserves are limited, therefore, new antagonistic bacteria to Fusarium graminearum need to be developed. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an antagonistic bacterium of Fusarium graminearum and its application in the preparation of a feed mildew inhibitor.

[0007] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0008] One of the purposes of the present invention is to provide an antagonistic bacterium to Fusarium graminearum, wherein the antagonistic bacterium to Fusarium graminearum is Bacillus amyloliquefaciens Bacillus amyloliquefaciens4-9-2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32407, the deposit date is October 30, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The second object of the present invention is to provide an application of antagonistic bacteria of Fusarium graminearum in the preparation of feed mildew inhibitors, wherein Bacillus amyloliquefaciens Bacillus amyloliquefaciens 4-9-2 After continuous shaking culture in LB broth medium for 72 hours, the fermentation supernatant was collected and used to prepare feed mildew inhibitor.

[0010] Compared with the prior art, the Bacillus amyloliquefaciens of the present invention Bacillus amyloliquefaciens The lipopeptide active substances in the metabolites of 4-9-2 can inhibit the spore germination and hyphae growth of Fusarium graminearum; they have a significant inhibitory effect on the production of major fungal toxins such as zearalenone and vomitoxin, and can still maintain antifungal activity under extreme conditions such as strong acid, strong alkali, and high temperature; this fungus can be used as a reserve resource for the development of new mildew inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Bacillus amyloliquefaciens Bacillus amyloliquefaciens 4-9-2 Screening results: A is the CK control group; B is the strain with better anti-Fusarium graminearum activity.

[0012] Figure 2 Bacillus amyloliquefaciens Bacillus amyloliquefaciens The 16S rDNA sequencing results of 4-9-2 were BLASTed against the NCBI database and a phylogenetic tree was constructed.

[0013] Figure 3 Bacillus amyloliquefaciens Bacillus amyloliquefaciens The antibacterial effect diagram of the metabolites of 4-9-2: A is the CK control group; B is the antibacterial effect of the intracellular substances of the antagonist bacteria; C is the antibacterial effect of the cell-free supernatant.

[0014] Figure 4 The whole genome sequencing and analysis results of Bacillus amyloliquefaciens 4-9-2.

[0015] Figure 5 These are the results of HPLC purification of the fermentation broth of Bacillus amyloliquefaciens 4-9-2.

[0016] Figure 6 This is the result of qualitative analysis of active single peak TLC.

[0017] Figure 7This is the result of the single-peak activity test on the ability to inhibit the germination of Fusarium graminearum spores.

[0018] Figure 8 These are the test results of the ability of lipopeptide B1 to inhibit the growth of Fusarium graminearum hyphae.

[0019] Fig. 9 The IC value of lipopeptide B1 on the growth of Fusarium graminearum 50 Measurement results.

[0020] Fig.10 These are the stability test results of lipopeptide B1: A is pH stability; B is temperature stability; C is metal ion stability; and D is protease stability.

[0021] Fig.11 The results of the biological activity test of lipopeptide B1 against Fusarium graminearum: A: Detection of the inhibitory ability on the biomass of Fusarium graminearum; B: Detection of the inhibitory ability on the synthesis of vomitoxin in Fusarium graminearum; C: Detection of the inhibitory ability on the spore production of Fusarium graminearum; D: Detection of the inhibitory ability on the synthesis of zearalenone in Fusarium graminearum.

[0022] Fig.12 These are the observation results of the protective ability of lipopeptides against corn infection with Fusarium graminearum. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0024] Example 1. Screening and identification of antagonistic bacteria to Fusarium graminearum

[0025] We collected samples of plant roots, weed roots, plant rhizosphere soil, and marine sediments from different regions of China. We weighed 4 g of plant tissue or soil sample, added it to 36 mL of sterile water, and shook it for 20 min. Then, 100 µL of the suspension was serially diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4, and placed on LB agar plates (LB medium includes the following ingredients: 6g yeast extract, 10g tryptone, 10g sodium chloride, 1L deionized water, 20g agar powder) at 28℃ for 16-18 hours. Single colonies were cultured in 1 mL LB liquid medium at 28℃, 200 rpm shaking overnight. Fresh mycelial cakes of Fusarium graminearum (purchased from Beina Biotechnology, No.: 337560) were inoculated in the center of a solid plate made of PDA medium (200.0 g potato, 20.0 g glucose, 20.0 g agar, 1.0 L distilled water; natural pH, sterilized at 115℃ for 30 min), and then 2µL of the strain to be screened was added at a distance of 2cm from the center, and each strain was repeated 3 times. The PDA plate with only mycelial cakes of Fusarium graminearum in the center was used as a control. After culturing at 28℃ for 5 days, the colony diameter of Fusarium graminearum was measured, and the inhibition rate was calculated using the colony diameter of the control group as a control. The strains with an inhibition rate higher than 50% were selected as the preliminary screening strains for subsequent biological control studies. The inhibition rate was calculated as follows:

[0026] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100

[0027] The antagonistic bacteria with antibacterial effect were purified and stored in a -80°C refrigerator with 16% glycerol.

[0028] The results showed that one strain had good anti-Fusarium graminearum activity (e.g. Figure 1 As shown in the figure), the colony morphology was observed and it was found that the colony was white and wrinkled, with a rough surface and irregular edges. This strain was used as the core strain for subsequent experiments:

[0029] Identification method: The strain was sequenced by 16S rDNA using molecular biology techniques. The 16S gene sequence is:

[0030]

[0031] The sequence was compared with the NCBI database by BLAST. Figure 2 As shown, the strain was identified as Bacillus amyloliquefaciens Bacillus amyloliquefaciens 4-9-2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32407, the deposit date is October 30, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0032] Example 2: Preparation of metabolites and detection of their antibacterial effects

[0033] Fermentation broth preparation: Bacillus amyloliquefaciens Bacillus amyloliquefaciens4-9-2 was inoculated in LB broth (tryptone 10.0 g, yeast extract 5.0 g, NaCl 10.0 g, distilled water to 1.0 L; pH 7.0, sterilized at 121°C for 20 min) with an inoculum size of 1%. After continuous shaking culture at 37°C for 72 h, the fermentation supernatant and bacterial extract were collected, and the antibacterial activity of the fermentation supernatant and bacterial extract was tested. The results showed that the active substances were mainly distributed in the fermentation supernatant, as shown in Figure 2. Figure 3 , the antibacterial substances are mainly concentrated in the fermentation supernatant.

[0034] Whole-genome sequencing and analysis: Genomic DNA was quantified using Qubit 4 Fluorometer (LifeTechnologies, USA), and libraries were prepared using SQK-LSK109 Ligation Sequencing kit (Oxford Nanopore Technologies, UK). The experimental procedures followed the standard protocol provided by Oxford Nanopore Technologies (ONT), including sample quality testing, library construction, library quality testing, and library sequencing. In terms of genome assembly, Canu v1.5 software was used to assemble the filtered reads, and then circularized using Circlator v1.5.5. In terms of genome annotation, Prodigal v2.6.3 was used to predict coding genes, GenBlastA v1.0.4 program was used to scan the entire genome after predicting functional genes, and RapidAnnotation using Subsystem Technology (RAST) SEED viewer (website: http: / / rast.nmpdr.org / ) was used for gene prediction. AntiSMASH (V6.0.1) was applied to discover and identify secondary metabolite biosynthesis gene clusters. The whole genome sequencing results are as follows Figure 4 As shown, the RAST server displayed 3926 coding sequences, and the pie chart showed the distribution of subsystem feature counts. It revealed 23 genes involved in iron acquisition and metabolism, 36 genes involved in toxicity, disease, and defense, 6 genes involved in secondary metabolism, and 12 genes involved in aromatic compound metabolism. In addition, there were 44 genes related to stress response, including cold and heat shock, oxidative stress, osmotic stress, and detoxification, which helped Bacillus amyloliquefaciens Bacillus amyloliquefaciens4-9-2 Adapt to extremely changing environmental conditions. The antiSMASH website was used to predict the secondary metabolite biosynthesis gene clusters in the genome. The analysis results are shown in Table 1. It was found that there were 12 secondary metabolite biosynthesis gene clusters in the genome, including two lipopeptides. Relevant reports in the literature indicate that lipopeptides are the main antifungal substances synthesized by Bacillus.

[0035] Table 1 Prediction results of gene clusters for biosynthesis of secondary metabolites of Bacillus amyloliquefaciens 4-9-2

[0036] Gene Clusters Types Genome Locations Most Similar Known Clusters Similarity Cluster 1 NRPS 240,673-306,080 surfactin 78% Cluster 2 T1PKS, transAT-PKS 584,968-696,417 myxovirescin A1 21% Cluster 3 PKS-like 915,924-957,168 Butirosin A / butirosin B 7% Cluster 4 terpene 1,039,945-1,060,685 Cluster 5 transAT-PKS 1,339,617-1,427,832 macrolactin H 100% Cluster 6 transAT-PKS, T3PKS, NRPS 1,646,486-1,756,576 bacillaene 100% Cluster 7 NRPS,transAT-PKS, 1,819,072-1,956,885 fengycin 100% Cluster 8 terpene 1,979,830-2,001,713 Cluster 9 T3PKS 2,052,136-2,093,236 Cluster 10 transAT-PKS 2,251,743-2,357,925 difficidin 100% Cluster 11 NRP-metallophore,NRPS,RiPP-like 2,974,396-3,026,186 bacillibactin 100% Cluster 12 other 3,546,822-3,588,240 bacilysin 100%

[0037] Purification and characterization of lipopeptide antibacterial substances:

[0038] First, the fermentation supernatant was treated with 6 M HCl to precipitate the protein substances, and then repeatedly extracted with methanol to obtain the crude lipopeptide. The results of subsequent HPLC purification are as follows: Figure 5 As shown, two single peaks were obtained. The purified samples were qualitatively analyzed by thin layer chromatography combined with literature reports, and the thin layer chromatography plates were stained with iodine vapor and fumigated with concentrated hydrochloric acid. The results are shown in Figure 6 As shown, it was found that the purified substance could be stained yellow by elemental iodine, and after being treated with concentrated hydrochloric acid, it could be stained blue-purple by ninhydrin, and was determined to be a cyclic lipopeptide substance.

[0039] Evaluation of the antibacterial activity of lipopeptides:

[0040] Detection of the effect of lipopeptides on spore germination of Fusarium graminearum:

[0041] Preparation of Fusarium graminearum spores: Fusarium graminearum (purchased from Beina Biotechnology, No.: 337560) was inoculated into a 250 mL bottle containing 100 mL CMC medium (CMC-Na 20.0 g; Na2HPO4 2.5 g; KH2PO4 1.5 g; peptone 2.5 g; distilled water to 1.0 L; pH 7.0-7.5, sterilized at 121°C for 20 min), and cultured with shaking at 180 rpm and 25°C for 7 days. The culture was filtered with two layers of sterile gauze to remove the mycelium and prepare a large conidia suspension. The spores were collected after centrifugation at 4°C and 10,000 rpm for 20 min, washed twice with distilled water, and the concentration of the suspension was adjusted to 1×10 using 0.1% (v / v) Tween 20 solution. 5 CFU / mL.

[0042] The minimum inhibitory concentration (MIC) of two lipopeptides A1 and B1 was determined by the microbroth method. First, a 96-well plate was prepared. PDB and lipopeptides A1 and B1 were added to the first row, respectively, with a total volume of 190 μL and an initial concentration of 512 μg / mL. Then, the sample wells on the same column were diluted 2 times. The final concentration of each sample well was 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, and 4 μg / mL. Finally, 10 μL of spore solution was added to make the final spore concentration of 1×10 5 CFU / ml. Place it in a 28°C temperature box for 12 hours, and use enzyme markers to detect the light absorption value of each well when the OD value is 600. The results are as follows Figure 7 As shown, the minimum inhibitory concentration of lipopeptide A1 was 128 μg / mL, and lipopeptide B1 could completely inhibit the germination of Fusarium graminearum spores at a concentration of 64 μg / mL.

[0043] Detection of the inhibitory effect of lipopeptides on the mycelial growth of Fusarium solani:

[0044] Lipopeptides were diluted to different concentrations with methanol, and then 0.2 mL of the diluted lipopeptides was added to 14.8 mL of the prepared PDA medium, cooled to 40-50 °C, and poured into 60 mm culture dishes, 5 ml per plate. The final concentrations of lipopeptides were 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL, respectively. Methanol was used instead of antifungal peptide solution in the control group. A mycelial disk with a diameter of 5 mm was inoculated in the center of the plate in the treatment group and the control group, respectively, and then cultured at 28 °C. When the mycelial colony of the control group grew to the edge of the culture dish (about 96 h), the colony diameter of each treatment group and the control group was determined by the intersection method. The rate of inhibition of mycelial growth was calculated using the following formula: Growth inhibition rate = (mycelial colony diameter control group − mycelial colony diameter treatment group) / mycelial colony diameter control group × 100%. The half-maximal inhibitory concentration (IC50) was calculated using GraphPad 9.5 software. The results showed that the IC50 of lipopeptide B1 on mycelial growth was 26.1 μg / mL ( Figure 8 , Fig. 9 ).

[0045] 2. Stability test of lipopeptide antibacterial substances:

[0046] The antifungal effect of the lipopeptide solution under different treatments was determined using the filter paper method. Briefly, a 6 mm diameter filter paper was placed on the PDA plate, and then 7 μL of 2.5 mg / ml lipopeptide solution was dripped into the center of the filter paper. After it was completely absorbed, 5 μL of Fusarium graminearum spore suspension (final spore concentration was 1×10 5CFU / ml) was dripped into the center of the filter paper, and then placed in a temperature box at 28°C for 24 hours. The colony diameters of the treated and control groups on the filter paper were measured, and the antibacterial rate of each treatment was calculated.

[0047] Inhibition rate (%) = (control colony diameter - treated colony diameter) / control colony diameter × 100.

[0048] Acid-base stability: The lipopeptide was prepared into a solution with a concentration of 2.5 mg / mL, and its pH value was adjusted to 2-14 with sodium hydroxide and hydrochloric acid, respectively, and the antibacterial effect of each treatment group was tested.

[0049] Metal ion stability: Na+, K + , Ca 2+ Mg 2+ , Cu 2+ , Fe 2+ , Fe 3+ The final concentration of the ionic solution was 10 μM, and the lipopeptide powder was dissolved in the metal solution to a final concentration of 2.5 mg / mL, and the antibacterial effect of each treatment group was tested.

[0050] Temperature stability: The lipopeptide solution was prepared at 2.5 mg / ml and treated at 25 ℃, 50 ℃ and 100 ℃ for 30 min respectively to detect the antibacterial effect.

[0051] Protease stability: Lipopeptide was treated with different proteases, the enzyme concentration was 5 mg / ml, the lipopeptide concentration was 2.5 mg / mL, and the reaction was terminated by boiling for 10 min after 1 hour. Then the changes in the antibacterial effect of lipopeptide under the treatment of different proteases were detected.

[0052] The results are as follows Fig.10 It was found that lipopeptide A1 can maintain antifungal activity in a wide pH range, and its activity is not interfered by metal ions. In addition, the lipopeptide has high temperature stability and can still maintain antifungal activity against Fusarium graminearum after treatment at 100°C. In addition, the lipopeptide can also resist the hydrolysis of multiple proteases.

[0053] Application Example 1: Detection of the actual inhibitory ability of lipopeptide B1 on Fusarium graminearum:

[0054] Toxin content, mycelium dry weight and spore production detection:

[0055] Prepare PDB, put it into test tubes, 5 mL per tube, and add Fusarium graminearum spores to a final concentration of 1×10 5CFU / mL. The final concentrations of lipopeptides added to the test tubes were 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, and 4μg / mL, respectively. The same volume of methanol was added as a negative control, and the same volume of culture medium was added as a blank control. After 14 days of constant temperature and shaking culture at 28°C, the toxin content under each treatment was detected using DON and ZEN detection kits, and the detection value in the blank culture medium without any treatment was used as zero correction. The mycelium was dried and weighed to detect the inhibitory ability of lipopeptides on the biomass of Fusarium graminearum. The number of spores in the culture medium was calculated using a hemocytometer to evaluate the effect of lipopeptides on the spore yield of Fusarium graminearum. The results are shown in Fig.11 As shown, 64 μg / mL lipopeptide B1 had a significant inhibitory effect on the spore production, toxin synthesis, and biomass of Fusarium graminearum.

[0056] Detection of lipopeptide's ability to protect corn seeds:

[0057] In order to evaluate the protective effect of lipopeptides on grains in actual use, lipopeptides were dissolved in 10% methanol solution with a concentration of 64 μg / mL to prepare a spray preparation. The corn seeds were treated with spray to evenly cover the seed surface with lipopeptides. After the seeds were naturally dried, the final concentration of 1×10 5 The spore solution of Fusarium graminearum with CFU / mL was sprayed on the surface of the seeds, and the seeds were placed in a culture dish and cultured at room temperature to observe the growth of Fusarium graminearum in each group. The results showed that the lipopeptide spray had a good protective effect on corn and could completely inhibit the infection of Fusarium graminearum on corn seeds ( Fig.12 ).

[0058] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An antagonistic bacterium to Fusarium graminearum, characterized in that The antagonistic bacteria to Fusarium graminearum is Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )4-9-2, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No: 32407, the deposit date is October 30, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the antagonistic bacteria of Fusarium graminearum as claimed in claim 1 in the preparation of a feed mildew inhibitor.

3. The use of the antagonistic bacteria of Fusarium graminearum in the preparation of a feed mildew inhibitor according to claim 2, characterized in that: Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens )4-9-2 After continuous shaking culture in LB broth medium for 72 hours, the fermentation supernatant was collected and used to prepare a feed mildew inhibitor.

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