Bacillus velezensis GX0003742 and biocontrol application thereof

Abstract

The invention discloses bacillus velezensis GX0003742 and biocontrol application thereof, and belongs to the technical field of microorganism application. The invention discloses bacillus velezensis GX0003742 with a broad-spectrum bacteriostatic effect, which is preserved in the China General Microbiological Culture Collection Center, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, the preservation date is September 14, 2024, and the preservation number is CGMCC No.31981. The bacillus velezensis GX0003742 has the advantages that the bacillus velezensis GX0003742 can be used for preparing the bacillus velezensis GX0003742; the strain can obviously inhibit growth of colletotrichum basjoo mycelia and obviously reduce occurrence of postharvest diseases of bananas, has an inhibiting effect on various pathogenic bacteria, has a good insecticidal effect on auricular bean root-knot nematode, and provides a new strain resource for biological prevention and control of plant diseases.

Description

Technical Field

[0001] The invention relates to the technical field of microbial application, in particular to a strain of Bacillus velez GX0003742 and a biocontrol application thereof. Background Art

[0002] Banana (Musa nana) is the main fruit grown in Guangxi, and the region's geographical and climatic factors provide excellent conditions for banana cultivation. However, since bananas are typical respiratory climacteric fruits, they have a short storage time after harvest, and banana anthracnose is prone to occur, which seriously restricts the development of the banana industry. Banana anthracnose, caused by Colletotrichum musae, is an extremely serious disease in the storage process of bananas after harvest. The disease can lurk on banana plants, leaves, and buds, and is dispersed by rainwater. After the disease occurs, round spots are formed. During the fruit ripening process, the released ethylene can accelerate the growth of attached spores, causing the round spots to continue to expand, the peel to sag, and the flesh to rot, which seriously affects the appearance and flesh quality of bananas.

[0003] Root-knot nematode is an animal of the genus Meloidogyne of the family Meloidogyneidae. Its second-instar larvae have the ability to migrate, so they can parasitize plants. When the larvae parasitize plants, their proboscis will pierce the host and absorb the host's nutrients, forming root knots on the crops, thus causing the host's nutrient absorption and affecting the yield of the plants. Meloidogyne enterolobii has a high reproduction level and can overcome resistance genes such as Mi, N and RK compared to other nematodes, making it easier to damage the characteristics of crops, becoming one of the key focus species of root-knot nematodes in China. At present, the elephant ear bean root knot nematode is widely present in Hainan, my country, affecting most of the local crops such as Cucurbitaceae and Solanaceae, and has a trend of spreading from south to north. However, the main means of preventing and controlling banana anthracnose and elephant ear bean root knot nematodes is still chemical control, but the safety of chemical pesticides is questionable, and long-term use will also cause adverse effects such as soil compaction and environmental pollution.

[0004] In recent years, studies have found that Bacillus can secrete and synthesize a variety of secondary metabolites to achieve the inhibition of pathogens, mainly lipopeptide compounds and polypeptide compounds synthesized by nonribosomal peptide synthetase (NRPS), and polyketide compounds synthesized by polyketide synthase (PKS). These bacteriocins and lipopeptides have stable physical and chemical properties, a broad antibacterial spectrum, and are not easy to develop drug resistance. Therefore, the discovery of Bacillus with antibacterial effects and the preparation of microbial agents is one of the research hotspots of biological control, and it is also an urgent problem to be solved for banana anthracnose and elephant ear bean root knot nematodes. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Bacillus Velez GX0003742 and its biological control use, so as to solve the problems existing in the above-mentioned prior art. The present invention discloses a strain of Bacillus Velez GX0003742 with a broad-spectrum antibacterial effect, which can significantly inhibit the mycelial growth of banana anthracnose fungus, significantly reduce the occurrence of banana post-harvest diseases, and has an inhibitory effect on a variety of pathogens. At the same time, the strain has a good insecticidal effect on ear bean root knot nematodes, providing a new strain resource for biological control of plant diseases.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a strain of Bacillus velezensis GX0003742, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date being September 14, 2024, and the deposit number being CGMCC No.31981.

[0008] The present invention also provides the use of the above-mentioned Bacillus Velezii GX0003742 in preparing a biocontrol agent for plant pathogens, wherein the plant pathogens include: Fusarium oxysporum, Botrytis cinerea, wheat root rot, wheat crown rot, Asian anthracnose, fruit anthracnose, gloeosporium anthracnose, new dark columnar arthrospora, Fusarium equisetum, Fusarium rubrum, Fusarium chlamydophorum, chestnut blight and banana anthracnose.

[0009] The present invention also provides a plant pathogenic bacteria biocontrol agent, the main component of which is any one or more of the above-mentioned Bacillus Velezii GX0003742, its metabolites and its volatile gases.

[0010] Preferably, the pathogens include: Fusarium oxysporum, Botrytis cinerea, wheat root rot, wheat crown rot, Asian anthracnose, fruit anthracnose, gloeosporioides, new dark columnar arthrospora, Fusarium equisetum, Fusarium rubrum, Fusarium chlamydosporum, chestnut blight and banana anthracnose.

[0011] The present invention also provides application of the Bacillus Velezii GX0003742 in preparing an elephant ear bean root knot nematode insecticide.

[0012] The invention also provides an elephant ear bean root knot nematode insecticide, the main component of which is any one or more of the above-mentioned Bacillus Velezii GX0003742, its metabolites and its volatile gases.

[0013] The present invention also provides application of the Bacillus Velezii GX0003742 in preventing and controlling banana anthracnose.

[0014] The present invention also provides a method for preventing and controlling banana anthracnose, comprising the following steps: spraying the above-mentioned Bacillus Velez GX0003742 agent on bananas; or,

[0015] Inoculate the above-mentioned Bacillus Velez GX0003742 into a culture medium, and use a closed fumigation method to allow the volatile gas of the above-mentioned Bacillus Velez GX0003742 to act on bananas; or,

[0016] The sterile filtrate of the above-mentioned Bacillus Velez GX0003742 was sprayed on bananas.

[0017] The present invention also provides the use of the Bacillus Velez GX0003742 in prolonging the fresh-keeping period of post-harvest bananas.

[0018] The present invention also provides a method for extending the shelf life of post-harvest bananas, comprising the following steps: inoculating the above-mentioned Bacillus Velez GX0003742 into a culture medium, and allowing the volatile gas of the above-mentioned Bacillus Velez GX0003742 to act on the post-harvest bananas through a closed fumigation method; or,

[0019] The sterile filtrate of the above-mentioned Bacillus Velez GX0003742 was sprayed on the surface of the harvested bananas.

[0020] The present invention discloses the following technical effects:

[0021] The invention provides a strain of Bacillus velez GX0003742, which has a broad-spectrum antibacterial property, can significantly inhibit the growth and reproduction of multiple plant pathogenic fungi such as Fusarium oxysporum, Botrytis cinerea, wheat root rot, wheat crown rot, Asian anthracnose, fruit anthracnose, gloeosporium anthracnose, new dark columnar arthrospora, Fusarium equisetifolia, Fusarium rubrum, Fusarium chlamydophorum, chestnut blight, and banana anthracnose, and also has the effect of killing elephant ear bean root knot nematodes; at the same time, the Bacillus velez GX0003742 and its metabolites and volatile gases can significantly inhibit banana anthracnose, significantly reduce the occurrence of banana post-harvest diseases, and extend the shelf life of post-harvest bananas. Therefore, the Bacillus velez GX0003742 provides strain resources for researching and developing new biological fresh-keeping preparations and growth-promoting bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is the morphological observation of Bacillus velez GX0003742, where A is the colony morphology and B is the bacterial morphology under Gram staining optical microscope (100× oil objective);

[0024] Figure 2 This is the phylogenetic tree of Bacillus velez GX0003742 based on the 16S rDNA gene sequence;

[0025] Figure 3 This is the phylogenetic tree of Bacillus velez GX0003742 based on the gyrB gene sequence;

[0026] Figure 4 The growth status of Bacillus velez GX0003742 in different culture media, among which A is PKO inorganic phosphorus medium; B is Montkina organic phosphorus medium; C is CAS detection medium; D: nitrogen fixation medium; E is amylase detection medium; F is protease detection medium; G is phytase detection medium; H is peptone water medium, the left test tube is peptone water medium, and the right test tube is peptone water medium inoculated with Bacillus velez GX0003742;

[0027] Figure 5 is the antifungal activity of Bacillus Velez GX0003742 against different plant pathogenic fungi, wherein A is Botrytis cinerea, B is Fusarium chlamydophorum, C is Colletotrichum gloeosporioides, D is Colletotrichum foetidis, E is Colletotrichum asiaticum, F is Fusarium oxysporum, G is Fusarium graminearum, H is chestnut blight, I is wheat root rot, J is Neochromis fuscipenser, K is Fusarium equisetum, and L is Fusarium rubrum;

[0028] Figure 6 This is a diagram showing the inhibitory effect of Bacillus Velez GX0003742 on Colletotrichum musa, wherein A is the control group and B is the Bacillus Velez GX0003742 treated group;

[0029] Figure 7 The results show the inhibitory effect of the sterile fermentation filtrate of Bacillus Velez GX0003742 on Colletotrichum musa, wherein A is the control group, B is the 1% sterile fermentation filtrate treatment group, C is the 2% sterile fermentation filtrate treatment group, D is the 3% sterile fermentation filtrate treatment group, E is the 5% sterile fermentation filtrate treatment group, and F is the 10% sterile fermentation filtrate treatment group;

[0030] Figure 8 The figure shows the inhibitory effect of volatile gas of Bacillus Velez GX0003742 on Colletotrichum musa, where A is the control group and B is the group treated with Bacillus Velez GX0003742;

[0031] Fig. 9This is a diagram showing the biocontrol effect of Bacillus Velez GX0003742 on postharvest banana anthracnose, where A is the control group and B is the Bacillus Velez GX0003742 treatment group;

[0032] Fig.10 The figure is a picture showing the preservation effect of Bacillus Velez GX0003742 on post-harvest bananas, wherein A is a control group, B is a picture showing the preservation effect after treatment with volatile gas of GX0003742, and C is a picture showing the preservation effect after treatment with sterile filtrate of GX000372;

[0033] Fig.11 These are microscopic observations of the elephant ear bean root-knot nematodes after treatment with Bacillus Velez GX0003742, where A is the elephant ear bean root-knot nematodes under CK-1, B is the elephant ear bean root-knot nematodes under GX0003742 bacterial suspension, C is the elephant ear bean root-knot nematodes under GX0003742 sterile filtrate, D is the elephant ear bean root-knot nematodes under CK-2, and E is the elephant ear bean root-knot nematodes under GX0003742 VOCs. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The sources of the experimental materials used in the examples of the present invention are as follows:

[0040] Montgena organophosphate medium, PKO inorganic phosphorus medium, and CAS detection medium were all purchased from Qingdao Haibo Biological Co., Ltd.;

[0041] Phytase assay medium (g / L): glucose 10 g, NaCl 0.3 g, (NH 4 ) 2 SO 4 0.5 g, KCl 0.3 g, MgSO 4 0.3 g, FeSO 4 0.03 g, MnSO 4 0.03 g, sodium phytate 5.0 g;

[0042] Protease detection medium (g / L): skim milk powder 15g, LB medium 25g, agar 18g;

[0043] Amylase assay medium (g / L): tryptone 10.0 g, yeast extract powder 5.0 g, NaCl 10.0 g, soluble starch 2 g;

[0044] Peptone water medium (g / L): peptone 10 g, NaCl 10 g, pH 7.2;

[0045] Nitrogen fixation medium (g / L): mannitol 10g, KH 2 PO 4 0.2 g, MgSO 4 7H 2 O 0.2g, NaCl 0.2g, CaSO 4 0.1 g, CaCO 3 5 g, agar 18 g, pH 7.2;

[0046] Fusarium oxysporum, Botryosphaeria dothidea, Bipolaris sorokiniana, Fusariumpseudograminearum, Coletotrichum asianum, coletotrichum fructicola, Colletotrichum gloeosporioides, Neoscytalidium dimidiatum, Fusarium equiseti, Fusarium lateritium, Fusarium chlamydosporum, Cryphonectriaparasitica, Colletotrichum musae, and root-knot nematode of elephant-ear bean were provided by the Key Laboratory of Microbial Resources and Utilization, College of Ocean and Biotechnology, Guangxi University for Nationalities, and can also be purchased through conventional channels.

[0047] Materials or reagents whose sources are not specified in the examples of the present invention can be purchased through conventional channels unless otherwise specified.

[0048] Example 1 Identification of Bacillus velez GX0003742

[0049] 1. Isolation of strains

[0050] Strain GX0003742 was isolated from an abandoned salt field in Beihai City, Guangxi Zhuang Autonomous Region (E109°28′26″, N21°28′52″) and purified by dilution plate method. The strain was streaked and inoculated into LB solid medium and cultured at 30°C for 24 hours. The morphological characteristics of single colonies, such as color, size, and transparency, were observed. After Gram staining of a single colony, the strain morphology was observed under an optical microscope.

[0051] The results of morphological observation of this strain are shown in Figure 1 The strain A is white on LB solid medium, with wrinkled surface and transparent consistency. The morphological observation results of the strain after Gram staining are shown in Figure 1 B. The bacteria are rod-shaped under an optical microscope and are positive in the Gram staining test.

[0052] 2. Physiological and biochemical characteristics

[0053] With reference to the Manual of Identification of Common Bacterial Systems and Bergey's Manual of Bacterial Identification, the strains were identified for oxidase, starch hydrolysis, gelatin liquefaction, methyl red and other physiological and biochemical indicators.

[0054] The results of physiological and biochemical characteristics analysis are shown in Table 1. The results showed that the strain was an aerobic bacterium, with negative results in oxidase, methyl red test and VP test, and positive results in starch hydrolysis, gelatin hydrolysis, malonate utilization and citrate utilization tests.

[0055] Table 1 Results of physiological and biochemical characteristics analysis

[0056]

[0057]

[0058] 3. Molecular Biology Identification

[0059] The total DNA of strain GX0003742 was extracted after activation and used as a template to perform PCR amplification of the 16S rRNA gene and gyrB gene of strain GX0003742 using bacterial universal primers 27F and 1492R and primers UP-1 and UP-2r of the housekeeping gene gyrB, respectively.

[0060] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 1);

[0061] 1492R: 5'-TACGGCTACCTTGTTACGAGTT-3' (SEQ ID NO. 2);

[0062] UP-1: 5'-ATTTGGCGCTGGCGGTTAT-3' (SEQ ID NO. 3);

[0063] UP-2r: 5'-GGTTTCGGCTGGGCTGGTA-3' (SEQ ID NO. 4);

[0064] PCR amplification system (50 μL): template 1 μL, ddH 2 O 22μL, 2×Taq PCR Mix 25μL, and 1μL each of upstream and downstream primers.

[0065] PCR amplification program: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min, 32 cycles; extension at 72°C for 10 min; storage at 4°C.

[0066] The amplified products were sent to Aoke Biotechnology (Wuhan) Co., Ltd. for sequencing. The sequences were compared with those in the NCBI database for homology, and the phylogenetic tree was constructed using MEGA7.0 software.

[0067] The results of molecular biological identification are as follows Figure 2 and Figure 3 As shown, the 16S rRNA gene sequence of strain GX0003742 was subjected to homology analysis with existing data in the NCBI database and a phylogenetic tree was constructed. It was found that the 16S rRNA sequence of strain GX0003742 had a similarity of 99.65% with Bacillus velezensis. The gyrB sequence alignment results showed that it had the highest similarity with Bacillus velezensis, and the phylogenetic tree was clustered in the same branch.

[0068] Based on the morphological characteristics, physiological and biochemical properties and molecular biological identification results of the strain, it was determined that the strain was Bacillus velezensis. The Bacillus velezensis GX0003742 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on September 14, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31981.

[0069] Example 2 Determination of Growth Promotion and Enzyme Production Capacity of Bacillus Velezii GX0003742

[0070] Preparation of culture medium of Bacillus Velez GX0003742: Inoculate Bacillus Velez GX0003742 into 50 mL LB liquid medium and culture at 30°C and 180 rpm until OD 600 ≈1.0.

[0071] Preparation of single colonies of Bacillus velez GX0003742: Bacillus velez GX0003742 was inoculated on LB solid medium and cultured in three zones for 1 day.

[0072] 5 μL of Bacillus Velezii GX0003742 culture medium (OD 600 ≈1.0) in the middle of Montgena organic phosphorus medium, PKO inorganic phosphorus medium, CAS detection medium, phytase detection medium, protease detection medium, and amylase detection medium, and cultured at 30°C for 5 days. The amylase detection medium was colorized with iodine solution.

[0073] A single colony of GX0003742 was selected and inoculated in three zones on a nitrogen-fixing medium and cultured at 30°C for 3 days to observe whether it could grow on the nitrogen-fixing medium.

[0074] Take 50 μL of the culture medium of Bacillus velezensis GX0003742 (OD 600 ≈1.0) was inoculated into a test tube containing 4 mL of peptone water culture medium and cultured at 30°C for 48 h. Then, 0.5 mL of Nessler reagent was added. The color of the solution changed from yellow to reddish brown and a precipitate was produced, indicating that it had the ability to produce NH 3 ability.

[0075] The qualitative test results of the growth-promoting function and enzyme production characteristics of Bacillus velez GX0003742 are as follows Figure 4 As shown, Bacillus Velez GX0003742 produced transparent circles on PKO inorganic phosphorus medium (A) and Montkina organic phosphorus medium (B); produced orange-yellow halos on CAS test medium (C); after adding Nessler reagent, the peptone water medium (H) turned reddish brown and precipitated; transparent circles were produced on amylase test medium (E), protease test medium (F), and phytase test medium (G); and it could grow normally on nitrogen fixation medium (D). The above results show that Bacillus Velez GX0003742 has the ability to decompose inorganic phosphorus, decompose organic phosphorus, produce siderophore, fix nitrogen, and produce NH 3 Ability to produce protease, amylase and phytase.

[0076] Example 3 Detection of antibacterial ability of Bacillus velez GX0003742

[0077] The plate confrontation method was used to determine the inhibitory effect of Bacillus Velez GX0003742 on 12 common plant pathogens, including Fusarium oxysporum, Botrytis cinerea, wheat root rot, wheat crown rot, Asian anthracnose, fruit anthracnose, Gloeosporium anthracnose, new dark columnar arthrospora, Fusarium equisetum, Fusarium rubrum, Fusarium chlamydophorum and chestnut blight. The experimental process was as follows: a pathogen cake with a diameter of 6 mm was inoculated in the center of a PDA plate, and 5 μL of the culture solution of Bacillus Velez GX0003742 (prepared in Example 2) was inoculated at 21 mm from the center of the plate. The PDA medium inoculated with only pathogens was used as a control. The experiment was repeated 3 times, and the culture was cultured at 28°C for 5 days. The antibacterial effect of the strain was observed, and the antibacterial rate was calculated.

[0078] Inhibition rate (%) = (colony radius of control group - colony radius of treatment group) / colony radius of control group × 100%.

[0079] The antibacterial effect test results of Bacillus velez GX0003742 are as follows Figure 5As shown, the upper layer is the colony morphology of the control group, and the lower layer is the colony morphology of the treatment group. The results showed that Bacillus Velez GX0003742 had an inhibitory effect on 12 common plant pathogens, including Botrytis cinerea (A), Fusarium chlamydophora (B), Colletotrichum gloeosporioides (C), Colletotrichum foetida (D), Colletotrichum asiaticum (E), Fusarium oxysporum (F), Fusarium graminearum (G), chestnut blight (H), wheat root rot (I), new dark columnar arthrospora (J), Fusarium equisetum (K, Fusarium rubrum (L); the inhibition rate is shown in Table 2, and the results showed that the inhibition rate of Bacillus Velez GX0003742 on 9 pathogenic fungi was greater than 50%.

[0080] Table 2 Antibacterial spectrum determination results of Bacillus velez GX0003742

[0081]

[0082]

[0083] Example 4 Detection of antibacterial activity of Bacillus velezensis GX0003742 against Colletotrichum musa

[0084] 1. Inhibitory effect of Bacillus velez GX0003742 on Colletotrichum musa

[0085] The plate confrontation method was used to test the inhibitory effect of Bacillus Velezii GX0003742 on Colletotrichum musa. The experimental process was the same as that in Example 3.

[0086] The inhibitory effect of Bacillus velez GX0003742 is as follows Figure 6 As shown, A is the control group, and B is the group treated with Bacillus Velez GX0003742. The results showed that Bacillus Velez GX0003742 had a significant inhibitory effect on Colletotrichum musa.

[0087] 2. Antibacterial activity of sterile fermentation filtrate of Bacillus velez GX0003742 against Colletotrichum musa

[0088] Bacillus Velez GX0003742 was inoculated into 50mL LB liquid culture medium at 2%, and cultured at 30℃ and 180r / min for 24h. The fermentation broth was centrifuged at 6000r / min for 10min. The supernatant was filtered and sterilized through a 0.22μm sterile filter membrane, and then added to 65℃ PDA culture medium at a volume ratio of 1%, 2%, 3%, 5% and 10%. The mixture was fully mixed and poured into a plate. PDA culture medium without sterile fermentation filtrate (0%) was used as a control. Each treatment was repeated 3 times. After the culture medium solidified, a banana anthracnose fungus cake with a diameter of 6mm was inoculated in the center. After constant temperature culture at 28℃ for 5d, the colony diameter was measured and the inhibition rate was calculated.

[0089] Inhibitory activity of sterile fermentation filtrate of Bacillus velez GX0003742 against Colletotrichum musa Figure 7 As shown, the results showed that compared with the control group (A), 1% (B), 2% (C), 3% (D), 5% (E) and 10% (F) sterile fermentation filtrates of Bacillus Velezii GX0003742 had inhibitory activity against Colletotrichum musa, and the inhibitory activity increased with the increase of inoculation amount.

[0090] 3. Antibacterial activity of volatile gases from Bacillus velez GX0003742 against Colletotrichum musa

[0091] The inhibitory effect of volatile gases produced by Bacillus Velez GX0003742 on Colletotrichum musa was determined by the double plate buckling method. Bacillus Velez GX0003742 was inoculated into 50mL LB liquid culture medium at 2%, and cultured at 30℃ and 180r / min for 24h. 100μL of fermentation liquid was evenly spread on the LB solid plate, and a 6mm diameter Colletotrichum musa bacteria block was placed in the center of the PDA plate. The two plates were buckled and sealed and cultured at 28℃ for 5d. The control group was a plate coated with blank culture medium instead of the Bacillus Velez GX0003742 plate.

[0092] Antibacterial activity of volatile gases from Bacillus velez GX0003742 against Colletotrichum musa Figure 8 As shown, A is the control group, and B is the group treated with Bacillus Velez GX0003742. The results showed that the volatile gas produced by Bacillus Velez GX0003742 can also significantly inhibit the growth and reproduction of Colletotrichum musa.

[0093] Example 5 Biocontrol Effect of Bacillus Velez GX0003742 on Postharvest Banana Anthracnose

[0094] The in vitro banana inoculation test was used to determine the control effect of antagonistic strains on banana anthracnose:

[0095] Preparation of spore suspension of pathogenic fungus (Coleobacterium musa) : Three fungus blocks with a diameter of 6 mm were taken from the pathogenic fungus cultured on PDA medium for 5 days and inoculated into 50 mL PDB liquid medium. The culture was shaken at 30°C and 180 r / min for 3 days. The filtrate was filtered with sterile gauze and sterile cotton. The filtrate was centrifuged at 4000 r / min for 6 minutes. The precipitate was taken and the concentration of the spore suspension was adjusted to about 1×10 8 CFU / mL is reserved.

[0096] The surface of the collected 50% ripe bananas was wiped with 2% sodium hypochlorite solution, and then repeatedly rinsed with sterile water. After drying, a wound was made on the banana surface with a sterilized bamboo stick, and the above-mentioned pathogenic fungus spore suspension was inoculated. 100 μL of the culture solution of Bacillus Velez GX0003742 (prepared in Example 2) was evenly coated on an LB solid plate, and placed in a transparent box together with the bananas inoculated with pathogenic fungus spores. Sterile water was used instead of the fermentation solution of Bacillus Velez GX0003742 as a control, and the bananas were stored in a constant temperature sealed state at 25°C, and the disease of the bananas was observed periodically.

[0097] The results of Bacillus Velez GX0003742 on the control of postharvest banana anthracnose are as follows Fig. 9 As shown, A is the control group, and B is the group treated with Bacillus Velez GX0003742. The results showed that anthracnose lesions appeared earlier in the control group than in the group treated with Bacillus Velez GX0003742, and the lesion area was significantly larger than that in the group treated with Bacillus Velez GX0003742, indicating that Bacillus Velez GX0003742 can effectively inhibit the infection of anthracnose fungi on post-harvest bananas and reduce the rot of bananas.

[0098] Example 6 Effect of Bacillus Velez GX0003742 on the preservation of postharvest bananas

[0099] The surface of the 80% ripe bananas collected was wiped with 2% sodium hypochlorite solution, then rinsed repeatedly with sterile water, and dried for later use. The volatile gas of Bacillus Velez GX0003742 and sterile filtrate were used to preserve the harvested bananas:

[0100] Volatile gas treatment: Take 100 μL of the culture solution of Bacillus Velez GX0003742 and spread it evenly on the LB solid plate, place it in a transparent box together with the bananas after the above disinfection treatment, store it in a closed box at a constant temperature of 25°C, and observe the condition of the bananas periodically.

[0101] Sterile filtrate treatment: Spray the sterile filtrate of Bacillus Velez GX0003742 onto the surface of the bananas after the above disinfection treatment until droplets flow down. Repeat the spraying operation 3 times after drying. Place the treated bananas in a transparent box and store them in a closed state at a constant temperature of 25°C, and observe the condition of the bananas periodically.

[0102] In both experiments, bananas that had been disinfected were used as the control group.

[0103] The results are as follows Fig.10As shown, A is the control group, B is the banana preservation effect diagram after volatile gas treatment, and C is the banana preservation effect diagram after sterile filtrate treatment. The results show that the control group began to turn yellow on the 4th day, and C-type spots began to appear on the 7th day. On the 14th day, the area of ​​C-type spots was greater than 50%, while the volatile gas treatment group began to turn yellow on the 7th day, and lesions appeared on the 9th day. The sterile filtrate treatment group began to turn yellow on the 9th day, and spots appeared on the surface on the 14th day. This shows that the volatile gas and sterile filtrate of GX0003742 have a good preservation effect on post-harvest bananas, can delay the time of banana ripening and decay, and the preservation effect of the sterile filtrate is better than that of the volatile gas.

[0104] Example 7 Insecticidal effect of Bacillus velezensis GX0003742 on root-knot nematodes of ear beans

[0105] 1. Insecticidal activity of Bacillus velezensis GX0003742 and its sterile filtrate against root-knot nematodes of ear bean

[0106] Preparation of bacterial suspension: Inoculate 2% of Bacillus Velezii GX0003742 into 50 mL LB medium and culture at 30°C with shaking at 180 rpm for about 24 hours. Centrifuge at 8000 rpm for 4 minutes, remove the supernatant, and resuspend the bacteria in sterile water until the OD 600 nm≈1.0 for standby use.

[0107] Preparation of sterile filtrate: Inoculate 2% of Bacillus Velezii GX0003742 into 50 mL LB medium, and culture at 30°C with shaking at 180 r / min for about 24 hours. Centrifuge at 8000 r / min for 4 minutes, take the supernatant, filter and sterilize it with a 0.22 μm sterile filter membrane, and store it at 4°C for later use.

[0108] Take 200 μL of liquid in a six-well plate, add 600 μL of sterile water, and put in 30 J2 larvae of the root-knot nematode of the ear bean (200 μL of insect suspension), dilute 5 times for the experiment, so that the total volume of the experimental liquid is 1 mL. Sterile water is used as the control (CK-1), repeated 3 times, cultured at 28 ° C for 72 hours, and the death of the root-knot nematode of the ear bean was observed under a microscope, and the mortality rate and corrected mortality rate were calculated.

[0109] Mortality rate (%) = number of dead elephant ear bean root-knot nematodes / total number of elephant ear bean root-knot nematodes × 100%.

[0110] Corrected mortality rate (%) = [(mortality rate of treatment group - average mortality rate of control group) / (1 - average mortality rate of control group)] × 100%.

[0111] 2. Insecticidal activity of volatile substances of Bacillus velezensis GX0003742 against root-knot nematodes of ear bean

[0112] Volatile substances (VOCs) collection: Bacillus Velezii GX0003742 was inoculated into 50 mL LB medium at 2% volume, and cultured at 30°C, 180 rpm for about 24 h until the bacterial solution OD 600 ≈1.0. Pour LB solid culture medium into the center of a sterile six-well plate and sterilize it with ultraviolet light in a clean bench for later use.

[0113] 100 μL of bacterial solution was spread on the culture medium in a six-well plate, and 30 J2 larvae of the elephant ear bean root-knot nematode were placed in adjacent wells. Sterile water was used as the control (CK-2). The process was repeated three times and cultured at 28°C for 72 hours. The death of the elephant ear bean root-knot nematode was observed, and the mortality rate and corrected mortality rate were calculated.

[0114] The results of the study on the growth status of the root-knot nematode of R. edulis by using Bacillus velezensis GX0003742 and its metabolites and volatile substances are as follows: Fig.11 As shown, A is the root-knot nematode of elephant ear bean under CK-1, B is the root-knot nematode of elephant ear bean under GX0003742 bacterial suspension, C is the root-knot nematode of elephant ear bean under GX0003742 sterile filtrate, D is the root-knot nematode of elephant ear bean under CK-2, and E is the root-knot nematode of elephant ear bean under GX0003742 VOCs. The results showed that the body of the root-knot nematode of elephant ear bean in the control group was curled and had obvious activity, while under the treatment of Bacillus Velez GX0003742 and its metabolites and volatile substances, the body of the root-knot nematode of elephant ear bean was rigid and lost its activity.

[0115] The mortality rate and the corrected mortality rate are shown in Table 3. The results showed that the insecticidal activity of Bacillus velezensis GX0003742 and its metabolites and volatile substances against the root-knot nematode of ear bean was above 80%.

[0116] Table 3 Test results of Bacillus velezensis GX0003742 against root-knot nematodes of ear beans

[0117]

[0118] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Bacillus velezensis GX0003742, characterized in that: It is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is September 14, 2024, and the deposit number is CGMCC No.31981.

2. The use of the Bacillus Velezii GX0003742 according to claim 1 in the preparation of a biocontrol agent for plant pathogens, characterized in that: The plant pathogens include: Fusarium oxysporum, Botrytis cinerea, wheat root rot, wheat crown rot, Asian anthracnose, fruit anthracnose, gloeosporium anthracnose, new dark columnar arthrospora, Fusarium equisetum, Fusarium rubrum, Fusarium chlamydophorum, chestnut blight and banana anthracnose.

3. A plant pathogenic bacteria biocontrol agent, characterized in that: The main component is any one or more of the Bacillus Velezii GX0003742, its metabolites and its volatile gases as described in claim 1.

4. The plant pathogenic bacteria biocontrol agent according to claim 3, characterized in that The pathogens include: Fusarium oxysporum, Botrytis cinerea, wheat root rot, wheat crown rot, Asian anthracnose, fruit anthracnose, gloeosporium anthracnose, new dark columnar arthrospora, Fusarium equisetum, Fusarium rubrum, Fusarium chlamydophorum, chestnut blight and banana anthracnose.

5. Use of the Bacillus Velez GX0003742 according to claim 1 in the preparation of an insecticide for root-knot nematodes of elephant ear beans.

6. An elephant ear bean root knot nematode insecticide, characterized in that: The main component is any one or more of the Bacillus Velezii GX0003742, its metabolites and its volatile gases as described in claim 1.

7. Use of the Bacillus Velez GX0003742 according to claim 1 in preventing and treating banana anthracnose.

8. A method for preventing and treating banana anthracnose, characterized in that: The method comprises the following steps: spraying the bacterial agent of Bacillus Velez GX0003742 of claim 1 on bananas; or, Inoculating the Bacillus Velez GX0003742 into a culture medium, and allowing the volatile gas of the Bacillus Velez GX0003742 to act on bananas through a closed fumigation method; or, The sterile filtrate of Bacillus Velez GX0003742 was sprayed on bananas.

9. Use of the Bacillus Velez GX0003742 according to claim 1 in extending the shelf life of post-harvest bananas.

10. A method for extending the shelf life of harvested bananas, characterized in that: The method comprises the following steps: inoculating the Bacillus Velez GX0003742 described in claim 1 into a culture medium, and allowing the volatile gas of the Bacillus Velez GX0003742 to act on post-harvest bananas through a closed fumigation method; or, The sterile filtrate of Bacillus Velez GX0003742 is sprayed on the surface of harvested bananas.

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