Bacillus tequilensis T22, microbial agent, microbial pesticide and application

Abstract

The invention provides bacillus tequilensis T22, a microbial agent, a microbial pesticide and application, and belongs to the technical field of applied agricultural microorganisms and biological control. The bacillus tequilensis T22 is preserved in the China General Microbiological Culture Collection Center on February 21, 2025, and the preservation number of the bacillus tequilensis T22 is CGMCC (China General Microbiological Culture Collection Center) No.33612. The bacillus tequilensis T22 is named as bacillus tequilensis T22. The bacillus tequilensis T22 and the fungicide, which are disclosed by the invention, have a remarkable inhibition effect on various plant pathogenic fungi.

Description

Technical Field

[0001] The present invention relates to the technical field of applied agricultural microorganisms and biological control, and in particular to a strain of Bacillus tergillosis T22, a bacterial agent, a microbial pesticide and applications thereof. Background Art

[0002] Plant fungal diseases are one of the main threats to agricultural production. Diseases caused by pathogenic fungi account for about 70% to 80% of all plant diseases, and almost every crop may be attacked by multiple fungal diseases. Currently, the prevention and control of plant fungal diseases still primarily relies on chemical pesticides. However, the long-term and extensive use of these pesticides has led to a series of problems: First, chemical residues remain in the soil, water, and atmosphere; second, pathogens easily develop resistance to these pesticides, resulting in a year-on-year decline in control effectiveness and the necessitating increased dosage, creating a vicious cycle. Furthermore, pesticide residues in agricultural products pose a direct threat to human health, contradicting the development concepts of green agriculture and food safety. Therefore, finding environmentally friendly and sustainable alternatives to chemical pesticides has become an urgent need to promote the green and efficient development of agriculture. Among the many biological control resources, Bacillus spp. Bacillus ) bacteria have become a highly sought-after biocontrol strain due to their unique biological properties and functional advantages. This genus is widely distributed in nature, easy to isolate and culture, and characterized by rapid growth and active metabolism, making it amenable to large-scale production. Furthermore, they possess strong stress tolerance, withstanding extreme environments such as high temperature, drought, and acid and alkaline conditions. They can survive and function stably in complex environments such as soil and plant surfaces, and possess a high biosafety rating, posing no risk to humans, animals, crops, or the ecological environment. However, existing commercially available Bacillus strains still have limitations in practical applications: some strains have a narrow antimicrobial spectrum, resulting in suboptimal control of specific pathogens; some strains need to improve their colonization and stress resistance in complex field environments, resulting in insufficient stability in their control effects; and some strains have complex production processes and high costs, limiting their large-scale application. Therefore, screening new Bacillus strains with broader antimicrobial activity, stronger environmental adaptability, and higher application efficacy, and optimizing their application technologies, are of great significance for improving biocontrol capabilities and promoting green agricultural development. Summary of the Invention The present invention provides a strain of Bacillus tequila T22 and its application in preventing and controlling plant fungal diseases, which solves the problem that current plant fungal diseases are mainly controlled by chemical means and have a single method.

[0003] In one aspect, a Bacillus tequila ( Bacillus tequilaensis) T22, the tequila Bacillus T22 was deposited on February 21, 2025 at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No. 33612, and was classified as tequila Bacillus Bacillus tequilaensis .

[0004] On the other hand, the application of Bacillus tequila T22 in inhibiting plant pathogens.

[0005] Preferably, the plant pathogenic bacteria include Diplodia spp. ( Lasiodiplodia Theobroma cassava ), Fusarium oxysporum, the pathogen of cowpea wilt ( Fusarium oxysporum f.sp. tracheal graminearum ( Fusarium gramineae )、Mango Anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora )、Corn leaf spot Curvularia zeae ( Curved moonlit )、Phytophthora capsici( Phytophthora capsicum ), Barringtonia leaf spot pathogens ( Diaporthe biconispora )、New dark columnar spores of pitaya canker ( Neocytalidium halimidium ), sisal stem base rot Aspergillus niger ( Aspergillus niger ) or Phytophthora nicotianae ( Phytophthora Nicotiana ).

[0006] On the other hand, a bacterial agent is provided, wherein the bacterial agent is prepared from Bacillus terequila T22.

[0007] On the other hand, the preparation method of the above-mentioned bacterial agent is to culture Bacillus tequila T22 in LB liquid culture medium.

[0008] Preferably, the culture is carried out at 180 rpm and 28°C until the concentration is 1.0×10 8 CFU / mL.

[0009] On the other hand, the above-mentioned bacterial agent is used to inhibit plant pathogens.

[0010] Preferably, the plant pathogenic bacteria include Diplodia theobrominata ( Lasiodiplodia Theobroma cassava ), Fusarium oxysporum, the pathogen of cowpea wilt ( Fusarium oxysporum f.sp. tracheal ), wheat fusarium graminearum ( Fusarium gramineae)、Mango Anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora )、Corn leaf spot Curvularia zeae ( Curved moonlit )、Phytophthora capsici( Phytophthora capsicum ), Barringtonia leaf spot pathogens ( Diaporthe biconispora )、New dark columnar spores of pitaya canker ( Neocytalidium halimidium ), sisal stem base rot Aspergillus niger ( Aspergillus niger ) or Phytophthora nicotianae ( Phytophthora Nicotiana ).

[0011] On the other hand, a microbial fertilizer is provided, which comprises the above-mentioned Bacillus tequila T22.

[0012] On the other hand, the above-mentioned bacterial fertilizer is used to inhibit plant pathogens.

[0013] Preferably, the plant pathogenic bacteria include Diplodia theobrominata ( Lasiodiplodia Theobroma cassava ), Fusarium oxysporum, the pathogen of cowpea wilt ( Fusarium oxysporum f.sp. tracheal ), wheat fusarium graminearum ( Fusarium gramineae )、Mango Anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora )、Corn leaf spot Curvularia zeae ( Curved moonlit )、Phytophthora capsici( Phytophthora capsicum ), Barringtonia leaf spot pathogens ( Diaporthe biconispora )、New dark columnar spores of pitaya canker ( Neocytalidium halimidium ), sisal stem base rot Aspergillus niger ( Aspergillus niger ) or sisal zebra stripe disease of tobacco ( Phytophthora Nicotiana ).

[0014] Beneficial effects The Bacillus tekila T22 strain provided by the present invention has a significant inhibitory effect on a variety of plant pathogenic fungi and exhibits a broad spectrum of antibacterial activity in a plate standoff experiment. Diaporthe biconispora ) has an inhibition rate of up to 73.00% against corn leaf spot disease Curvularia zeae ( Curved moonlit ) had an inhibition rate of 72.18% against rice blast fungus ( Magnaporthe grisea) had an inhibition rate of 69.65% against sisal zebra pattern tobacco phytophthora ( Phytophthora nicotianae ) reached an inhibition rate of 68.62%, and the inhibition rate of mango anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ) had an inhibition rate of 67.19% against pepper blight fungus ( Phytophthora capsicum ) reached an inhibition rate of 63.24%, and the inhibition rate of wheat fusarium graminearum ( Fusarium grasses ) reached an inhibition rate of 63.98%, and the inhibition rate of Pseudomonas aeruginosa ( Pestalotiopsis microspore ) had an inhibition rate of 61.28%, and had an inhibition rate of 61.28% against the new dark columnar spores of pitaya canker ( Neocytalidium halved ) had an inhibition rate of 59.40% against sisal leaf rot, and theobroma cacao ( Lasiodiplodia Theobroma cassava ) had an inhibition rate of 55.28% against Fusarium oxysporum ( Fusarium oxysporum f.sp. tracheal ) has an inhibition rate of 49.49% against Aspergillus niger ( Aspergillus niger ) has an inhibition rate of 30.6%, which can effectively inhibit plant diseases caused by various fungi and provide a powerful means for the prevention and control of a wide range of plant diseases.

[0015] In the potted plant prevention experiment of sisal leaf rot, the prevention efficiency of Bacillus tekila T22 reached 49.15%, which can significantly reduce the incidence of sisal leaf rot and reduce crop losses caused by the disease, reflecting good practical application effects.

[0016] Furthermore, as a natural microbial strain, Bacillus tekila T22 possesses high biosafety, poses no risk to the environment, humans, livestock, or crops. It avoids environmental pollution, pathogen resistance, and pesticide residues in agricultural products caused by the use of chemical agents, thus meeting the requirements of green agriculture and sustainable development. Furthermore, Bacillus tekila is easy to cultivate and mass-produce, making it suitable for the formulation and application of microbial agents. It has broad application prospects and potential commercial value, providing effective technical support for the green prevention and control of plant fungal diseases in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The colony morphology of Bacillus tekila T22 on LB solid medium, where A is a single colony morphology and B is a flat plate line colony morphology; Figure 2 The physiological and biochemical reactions of Bacillus tequilae T22, where A represents the ability to secrete siderophores, B represents the ability to solubilize phosphate, C represents the ability to solubilize potassium, and D represents the ability to fix nitrogen. Figure 3This is the 16S rDNA phylogenetic tree of Bacillus tequila T22; Figure 4 Bacillus tequila T22 gyrA Gene phylogenetic tree Figure 5 The antibacterial spectrum of Bacillus tekila T22, where a is the sisal leaf rot pathogen Diplodia cocoa ( Lasiodiplodia theobromae ), b is rice blast fungus ( Magnaporthe grisea ), c is the pathogen of Barringtonia dasyphylla ( Diaporthe biconispora ), d is the pepper blight fungus ( Phytophthora capsicum ), e is mango anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), f is the pathogen of cowpea wilt Fusarium oxysporum ( Fusarium oxysporum f. sp. tracheal ), g is the new dark columnar spore of pitaya canker ( Neocytalidium halimidium ), h is sisal zebra stripe disease of tobacco Phytophthora ( Phytophthora Nicotiana ), i is Fusarium graminearum ( Fusarium gramineae ), j is the coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora ), k is corn leaf spot disease Curvularia maydis ( Curvularia lunate ), l is sisal stem base rot Aspergillus niger ( Aspergillus niger ); Figure 6 Figure 2 shows the control effect of Bacillus tekila T22 on sisal leaf rot in potted plants, where A is the negative control (PDA treatment group), B is the positive control (Diplodia theobrominum treatment group), C is the control effect of the whole potted plant in the Diplodia theobrominum + T22 treatment group, D is an enlarged image of the whole potted plant in the negative control PDA group, E is an enlarged image of the whole potted plant in the positive control Diplodia theobrominum treatment group, F is an enlarged image of the whole potted plant in the Diplodia theobrominum + T22 treatment group, G is an enlarged image of the leaves in the negative control PDA group, H is an enlarged image of the leaves in the positive control Diplodia theobrominum treatment group, and I is an enlarged image of the leaves in the Diplodia theobrominum + T22 treatment group. DETAILED DESCRIPTION

[0018] Experimental Materials PDA medium: Boil 200 g of potatoes for 15 min, filter, retain the filtrate, add 18 g of glucose, 18 g of agar, and ddH2O to 1000 mL; sterilize at 121°C for 20 min.

[0019] LB medium (Luria-Bertani medium): 10.0 g tryptone, 5.0 g yeast extract, 8.0 g sodium chloride (NaCl), 20 g agar, and ddH2O to 1000 mL; sterilize at 121°C for 20 min.

[0020] NBRIP inorganic phosphate medium: glucose 10.0 g, ammonium sulfate 0.1 g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.25 g, potassium chloride (KCl) 0.2 g, magnesium chloride (MgCl2) 5.0 g, calcium phosphate (CaCO3) 5.0 g, ddH2O to 1000 mL; sterilize at 121°C for 20 min.

[0021] Alexandrite medium: sucrose 5.0 g, disodium hydrogen phosphate (Na2HPO4) 2.0 g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.5 g, ferric chloride (FeCl3) 0.005 g, calcium carbonate (CaCO3) 0.1 g, potassium feldspar 2.0 g, ddH2O to 1000 mL; sterilize at 115°C for 20 min.

[0022] Ashubei nitrogen-free medium: 0.2 g potassium hydrogen phosphate (K2HPO4), 0.2 g magnesium sulfate heptahydrate (MgSO4·7H2O), 0.2 g calcium sulfate dihydrate (CaSO4·2H2O), 0.2 g sodium chloride (NaCl), 5.0 g calcium carbonate (CaCO3), 10.0 g mannitol, ddH2O to 1000 mL; sterilize at 121°C for 20 min.

[0023] Chrome azurol (CAS) medium: The basal medium consists of glucose 100.0 g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.5 g, tryptone 20.0 g, calcium chloride (CaCl2) 0.5 g, and agar 10.0 g. The CAS detection solution consists of chrome azurol sulphonate (CAS) 0.06 g, ferric chloride (FeCl3) 0.0027 g, and hexadecy-ltrimethyl-ammonium bromide (HDTMA) 0.073 g. 10× PIPES buffer 1 mol / L, pH 7.0. 141 g of basal medium was weighed and made up to 800 mL with ddH2O. The medium was sterilized at 115°C for 20 min. The basal medium was cooled to 60°C, and 100 g of 10× buffer preheated at 60°C was slowly added to the medium. mL, 10×CAS detection solution 100 mL.

[0024] Example 1 Bacillus tequila ( Bacillus tequilaensis ) Acquisition of T22.

[0025] Rhizosphere soil and tissue samples were collected from healthy cowpea plants in the sisal leaf rot-infected plots. The soil was separated using the dilution and spreading method: 10 g of soil sample was placed in a 250 mL Erlenmeyer flask, 90 mL of ddH2O was added, and the flask was placed in a shaker at 28°C and 180 rpm for 30 min. The flask was placed in an 80°C water bath and allowed to stand for 10 min, and then diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 Add 10-15 mL of preheated LB medium to a Petri dish. After cooling, pipette 0.2 mL of each dilution at different concentrations onto the plate. Use a glass applicator to spread evenly until dry. Repeat three times. Seal the plate and incubate at 28°C under 14 hours of light and 10 hours of darkness. After two days, observe colonies and select suspected colonies for streak purification on LB plates. Identify and store the strains.

[0026] The pathogen of sisal leaf rot was used as the control strain. The above-mentioned preserved strains were initially screened using the plate confrontation method. A 0.5 cm thick cake of cowpea wilt pathogen was inoculated in the center of a PDA solid culture medium plate. Four points 2.5 cm from the center of each culture medium plate were selected symmetrically using a cross. The same isolated and purified preserved strain was picked at these four points with a toothpick. The strains with antagonistic effects were rescreened and preserved using the same method. The colony diameter was measured using the cross method. Inhibition rate / % = (control group colony diameter - treatment group colony diameter) / control group colony diameter × 100. The experiment was repeated three times, with three replicates for each treatment. The relevant data were sorted in Excel, and the significance of the data differences was analyzed using SPSS (25) software. Tukey's method was used for multiple comparisons.

[0027] The morphology of the strain T22 obtained by screening on LB solid medium is as follows Figure 1 As shown, the colonies on the LB medium appear as light yellow spots, which are dispersed. The colonies have irregular morphology and vary in size. Some colonies appear to be more obviously round or oval. There are obvious intervals between the colonies, forming a small cluster structure.

[0028] Example 2 Bacillus tequila ( Bacillus tequilaensis ) Physiological and biochemical characteristics of T22.

[0029] Determination of phosphate solubilization ability: After activation, strain T22 was inoculated onto NBRIP inorganic phosphate medium and placed in a 28°C constant temperature incubator for 7 days. The colonies were observed for the presence of a transparent oil droplet-like halo around them. If so, it indicated that the strain had the ability to solubilize phosphate. The size of the halo was measured.

[0030] Determination of potassium-solubilizing ability: After activation, strain T22 was inoculated onto Alexander silicate medium and placed in a 28°C constant temperature incubator for 7 days. The colonies were observed to see whether there was a transparent oil droplet-like halo around them. If so, it indicated that the strain had potassium-solubilizing ability. The size of the halo was measured.

[0031] Determination of nitrogen-fixing function: After activation, strain T22 was inoculated onto Axubei nitrogen-free medium and placed in a constant temperature incubator at 28°C for 7 days. The growth of the colonies was observed. The strains that could grow on Axubei nitrogen-free medium were considered to have nitrogen-fixing function. The ammonia-fixing ability was determined based on the diameter of the colony growth.

[0032] Determination of siderophore production capacity: After activation, strain T22 was inoculated onto chrome azurol (CAS) medium and placed in a 28°C constant temperature incubator for 7 days. The formation of yellow halos around the colonies was observed and the size of the halos was measured.

[0033] The results are as follows Figure 2As shown, strain T22 has a certain degree of nitrogen fixation, phosphate and potassium solubilization, and siderophore secretion. On CAS medium, a fine yellow halo with a diameter of 17.46 mm was observed for strain T22, indicating that strain T22 can secrete siderophores. On Axubei nitrogen-free medium, the diameter of the T22 colony was 7.5 mm, indicating that strain T22 has nitrogen fixation ability. On Alexander silicate medium, the diameter of the T22 colony was 38.03 mm, indicating that strain T22 has a certain potassium solubilization ability. On NBRIP inorganic phosphorus bacteria medium, the diameter of the T22 secretion zone was 29.58 mm, indicating that strain T22 can decompose organic phosphorus.

[0034] Example 3 Bacillus tequila ( Bacillus tequilaensis ) Molecular identification of T22.

[0035] The T22 strain was inoculated into LB liquid medium and shaken at 180 rpm for 16 hours. Biocontrol bacterial DNA was extracted using a bacterial DNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. according to the manufacturer's instructions. The resulting bacterial DNA was used as a template for PCR amplification. The target gene was selected using universal primers for the conserved region of the ribosomal 16S rDNA gene and bacterial-specific primers for the gyrase A subunit gene ( gyrA ), the amplification primers were 16S rDNA universal primers SEQ ID NO.1-27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and SEQ ID NO.2-1492r (5′-TACGGCTACCTTGTTACGACTT-3′), gyrA Primer SEQ ID NO.3- gyrA F (5′-CAGTCAGGAAATGCGTACGTCCTT-3′) and SEQ ID NO.4- gyrA R (5′-CAAGGTAATGCTCCAGGCATTGCT-3′).

[0036] The PCR reaction system consisted of 12.5 μL of 2×Tap PCR MasterMix, 9.5 μL of ddH₂O, 1 μL of each forward and reverse primer, and 1 μL of DNA. The 16S rDNA amplification program included 35 cycles of initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and a further extension at 72°C for 10 min. circle A The amplification program was as follows: pre-denaturation at 95°C for 4 min, denaturation at 94°C for 40 s, annealing at 60°C for 45 s, extension at 72°C for 1 min, and extension at 72°C for 10 min, for a total of 35 cycles.

[0037] PCR amplification products were detected by 1% agarose gel electrophoresis. Targeted fragment bands were visualized and photographed using a gel imager. The PCR products were then sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were compared and analyzed for homology using the NCBI database. Highly similar gene sequences and their model strain sequences were downloaded. A phylogenetic tree was constructed using the maximum likelihood method using Mega 11 software.

[0038] The 16S rDNA and gyrA The gene fragments were amplified by PCR to obtain 16S rDNA 1400 bp and gyrA The target fragment was about 1000 bp. The PCR amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequence was compared on the NCBI website. The 16S rDNA comparison results showed that T22 and Bacillus tequila Bacillus tequilaensis 10b (NR_117611.1) has a similarity of 99.92%, gyrA The comparison results showed that T22 and Bacillus tequila Bacillus tequilaensis The similarity to NRRL B-41771 (NZ_EU138625.1) was 98.54%. The gene sequences with high similarity and the model strain sequence were downloaded and the 16S rDNA and gyrA The phylogenetic tree is as follows Figure 3 and Figure 4 As shown, T22 and Bacillus tequila Bacillus from Tequila Therefore, T22 was identified as Bacillus tequilae. Bacillus tequilaensis Bacillus tequila ( Bacillus tequilaensis ) The T22 strain was deposited in the General Microbiology Center of China Culture Collection Administration (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit date of February 21, 2025, and the deposit number is CGMCC No. 33612.

[0039] Example 4 Bacillus tequila ( Bacillus tequilaensis ) T22 antibacterial ability determination.

[0040] The plant pathogenic fungi tested included Lasiodiplodia theobromina ( Lasiodiplodia Theobroma cassava ), Fusarium oxysporum, the pathogen of cowpea wilt ( Fusarium oxysporum f. sp. tracheal ) and Fusarium graminearum ( Fusarium gramineae)、Mango Anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora )、Corn leaf spot Curvularia zeae ( Curvularia lunate )、Phytophthora capsici( Phytophthora capsicum ), Barringtonia leaf spot pathogens ( Diaporthe biconispora )、New dark columnar spores of pitaya canker ( Neocytalidium halimidium ), sisal stem base rot Aspergillus niger ( Aspergillus niger )、sisal zebra stripe disease tobacco phytophthora ( Phytophthora nicotianae )wait.

[0041] The antibacterial activity of Bacillus tekila T22 was determined using the plate standoff method. Bacillus tekila T22 was cultured in LB liquid medium at 28°C on a shaker at 180 rpm for 16 hours to obtain a bacterial fermentation broth. Using the "cross-cross method," sterile 5 mm filter paper was placed at four points 2.5 cm from the center of a PDA culture plate. Using a sterile 5 mm punch, a bacterial cake of uniform age was punched from the edge of the activated colony of the test pathogenic fungus and inoculated in the center of the PDA plate. 1 μL of the bacterial fermentation broth was dripped onto the center of each of the four filter paper pieces. Three replicates were used for each treatment. The treated PDA plates were incubated in a 28°C incubator for 4–5 days. Colony growth was observed during this time. When the colonies approximately covered three-quarters of the plate, the antibacterial activity was measured, recorded, and photographed. The antibacterial rate was calculated as follows: Inhibition rate / % = [(pathogen growth diameter of control group - pathogen block diameter) - (pathogen growth diameter of treatment group - pathogen block diameter)] / (pathogen growth diameter of control group - pathogen block diameter) × 100.

[0042] The results are shown in Table 1 and Figure 5 As shown in the results, T22 has good antibacterial effect on pathogenic fungi, especially against Diplodia cocoides, the pathogen of sisal leaf rot. Lasiodiplodia theobromae ) inhibition rate was 55.28%, and the pathogen of cowpea wilt, Fusarium oxysporum ( Fusarium oxysporum f. sp. tracheal ) inhibition rate was 49.49%, and the inhibition rate of wheat fusarium graminearum ( Fusarium gramineae ) inhibition rate was 63.98%, and the inhibition rate against mango anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ) inhibition rate was 67.19%, and the inhibition rate of rice blast fungus ( Magnaporth gray ) inhibition rate was 69.65%, and the inhibition rate of Pseudomonas aeruginosa ( Pestalotiopsis microspora ) had an inhibition rate of 61.28% against corn leaf spot disease Curvularia zeae ( Curvularia lunate) inhibition rate was 72.18%, and the inhibition rate of pepper blight fungus ( Phytophthora capsicum ) inhibition rate was 63.24%, and the pathogen of Barringtonia spp. ( Diaporthe biconispora ) inhibition rate was 73.00%, and the new dark columnar spores of pitaya canker ( Neocytalidium halved ) inhibition rate was 59.40%, and the inhibition rate of Aspergillus niger ( Aspergillus niger ) inhibition rate was 30.6%, and the inhibition rate against sisal zebra pattern tobacco phytophthora ( Phytophthora nicotianae ) The inhibition rate was 68.62%.

[0043] Table 1 Bacillus tequila ( Bacillus tequilaensis )T22 antibacterial ability

[0044] Example 5 Bacillus tequila ( Bacillus tequilaensis ) The potted control effect of T22 on sisal leaf rot.

[0045] Preparation of Bacillus tekila T22 inoculum: Bacillus tekila T22 was cultured in LB liquid medium with a shaker at 180 rpm and 28 °C to a concentration of 1.0 × 10 8 CFU / mL.

[0046] Eighteen healthy sisal seedlings were selected, and three leaves were selected from each seedling. The epidermis of the leaves was carefully pierced with a needle. Each leaf was inoculated with an inverted bacterial cake with a diameter of 5 mm at the place where the needle pierced the epidermis. The positive control group was the bacterial cake of Diplodia theobrominum, and the bacterial cake of PDA culture medium was the negative control group. The other treatment groups were the bacterial cake of Diplodia theobrominum and sprayed with 10 mL of 1.0×10 8 Three treatment groups were inoculated with Bacillus tequilae T22 at a concentration of 100 CFU / mL, each with five replicates. After inoculation, the plants were wrapped in sterile bags and sprayed with sterile water to keep them moist. Disease progression was observed. Disease grading was based on NY / T 1488-2018, "Technical Specifications for Quarantine of Sisal Seedling Diseases," as shown in Table 2.

[0047] Table 2 Leaf disease classification standards

[0048] The formula for calculating the disease index and prevention and treatment effect is as follows:

[0049]

[0050] The results of the potted test showed that the PDA control group did not develop the disease, the disease index of the positive control group treated with Diplodia cocoa was 54.63, and the disease index of the group treated with Diplodia cocoa + T22 was 27.78. Compared with the positive control group, the prevention and control effect was 49.15%, that is, the T22 bacterial suspension can effectively reduce the occurrence and harm of sisal leaf rot.

[0051] Table 3 Results of potted plant protection test

[0052] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A strain of Bacillus tequila ( Bacillus tequilensis ) T22, characterized in that, The Bacillus tequila T22 is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 33612.

2. The use of the Bacillus tergillosis T22 according to claim 1 in inhibiting plant pathogens, characterized in that: The plant pathogens include Lasiodiplodia theobromina ( Lasiodiplodia theobromae ), Fusarium oxysporum, the pathogen of cowpea wilt ( Fusarium oxysporum f. sp. tracheiphilum ), wheat fusarium graminearum ( Fusarium graminearum )、Mango Anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora )、Corn leaf spot Curvularia zeae ( Curvularia lunata )、Phytophthora capsici( Phytophthora capsici ), Barringtonia leaf spot pathogens ( Diaporthe biconispora )、New dark columnar spores of pitaya canker ( Neoscytalidium dimidiatum ), sisal stem base rot Aspergillus niger ( Aspergillus niger ) or Phytophthora nicotianae ( Phytophthora nicotianae ).

3. A bacterial agent, characterized in that The bacterial agent is prepared from the Bacillus tergillosis T22 described in claim 1.

4. The method for preparing the microbial agent according to claim 3, characterized in that: The method comprises culturing the Bacillus subtilis T22 according to claim 1 in LB liquid culture medium.

5. The method according to claim 4, characterized in that: The culture was carried out at 180 rpm and 28°C until the concentration reached 1.0×10 8 CFU / mL.

6. Use of the microbial agent according to claim 3 in inhibiting plant pathogens, characterized in that: The plant pathogens include Lasiodiplodia theobroma cacao ( Lasiodiplodia theobromae ), Fusarium oxysporum, the pathogen of cowpea wilt ( Fusarium oxysporum f. sp. tracheiphilum ), wheat fusarium graminearum ( Fusarium graminearum )、Mango Anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora )、Corn leaf spot Curvularia zeae ( Curvularia lunata )、Phytophthora capsici( Phytophthora capsici ), Barringtonia leaf spot pathogens ( Diaporthe biconispora )、New dark columnar spores of pitaya canker ( Neoscytalidium dimidiatum ), sisal stem base rot Aspergillus niger ( Aspergillus niger ) or sisal zebra stripe disease of tobacco ( Phytophthora nicotianae ).

7. A microbial fertilizer, characterized in that: The bacterial fertilizer includes the Tequila Bacillus T22 described in claim 1.

8. The use of the bacterial fertilizer according to claim 7 in inhibiting plant pathogens, characterized in that: The plant pathogens include Lasiodiplodia theobromina ( Lasiodiplodia theobromae ), Fusarium oxysporum, the pathogen of cowpea wilt ( Fusarium oxysporum f. sp. tracheiphilum ), wheat fusarium graminearum ( Fusarium graminearum )、Mango Anthracnose Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease Pseudomonas aeruginosa ( Pestalotiopsis microspora )、Corn leaf spot Curvularia zeae ( Curvularia lunata )、Phytophthora capsici( Phytophthora capsici ), Barringtonia leaf spot pathogens ( Diaporthe biconispora )、New dark columnar spores of pitaya canker ( Neoscytalidium dimidiatum ), sisal stem base rot Aspergillus niger ( Aspergillus niger ) or Phytophthora nicotianae ( Phytophthora nicotianae ).

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