Bacillus tequilensis t22, microbial inoculant, microbial pesticide and application

Abstract

This invention discloses a strain of Bacillus tekirae T22, its inoculant, microbial pesticide, and its application, belonging to the field of applied agricultural microbiology and biological control technology. The described Bacillus tekirae (… Bacillus tequilensis T22 was deposited on February 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with the accession number CGMCC No. 33612. The Bacillus tekirae T22 and its inoculum involved in this invention exhibit significant inhibitory effects against various plant pathogenic fungi.

Description

Technical Field

[0001] This invention relates to the field of applied agricultural microbiology and biological control technology, specifically to a strain of Bacillus tektii T22, inoculants, microbial pesticides, and their applications. Background Technology

[0002] Plant fungal diseases are one of the major threats to agricultural production. Diseases caused by pathogenic fungi account for about 70% to 80% of all plant diseases, and almost every crop can be attacked by a variety of fungal diseases.

[0003] Currently, the control of plant fungal diseases still relies primarily on chemical agents. However, the long-term and extensive use of chemical agents has led to a series of problems: on the one hand, chemical residues remain in the soil, water, and atmosphere; on the other hand, pathogens easily develop resistance to chemical agents, resulting in a gradual decline in control effectiveness, necessitating increased dosages and creating a vicious cycle; furthermore, pesticide residues in agricultural products directly threaten human health, contradicting the development concepts of green agriculture and food safety. Therefore, finding environmentally friendly and sustainable alternatives to chemical agents has become an urgent need to promote the green and efficient development of agriculture.

[0004] Among numerous biological control resources, Bacillus (Bacillus) Bacillus Due to their unique biological characteristics and functional advantages, bacteria have become a focus of attention for biocontrol strains. This genus of bacteria is widely distributed in nature, easy to isolate and culture, and characterized by rapid growth and active metabolism, facilitating large-scale production. Furthermore, they possess strong resilience, tolerating extreme environments such as high temperatures, drought, and acidity / alkalinity. They can stably survive and function in complex environments such as soil and plant surfaces, and exhibit high biosafety, posing no harm to humans, livestock, crops, or the ecological environment.

[0005] However, existing commercially available Bacillus strains still have certain limitations in practical applications: some strains have a narrow antibacterial spectrum, resulting in unsatisfactory control effects against specific pathogens; the colonization ability and stress resistance of some strains in complex field environments need to be improved, leading to insufficient stability of control effects; in addition, the production processes of some strains are complex and costly, limiting their large-scale promotion and application. Therefore, screening new Bacillus strains with broader antibacterial activity, stronger environmental adaptability, and higher application efficiency, and optimizing their application technologies, is of great significance for improving the level of biological control and promoting green agricultural development. Summary of the Invention

[0006] This invention provides a strain of Bacillus tekiria T22 and its application in the control of plant fungal diseases, which solves the problems of the current main use of chemical control for plant fungal diseases and the single method.

[0007] On the one hand, a type of Bacillus tekirae ( Bacillus tequilaensis Bacillus tekirae T22, was deposited on February 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33612, and classified as Bacillus tekirae. Bacillus tequilaensis .

[0008] On the other hand, there is the application of Bacillus tekiria T22 in inhibiting plant pathogens.

[0009] Preferably, the plant pathogen includes *Dispora cocovenenans*, the causal agent of sisal leaf rot. Lasiodiplodia Theobroma cassava The pathogen causing cowpea wilt is Fusarium oxysporum ( ), Fusarium oxysporum f.sp. tracheal ), Fusarium graminearum, the causal agent of wheat scab ( Fusarium gramineae Mango anthracnose (Colletotrichum gloeosporioides) Colletotrichum gloeosporioides Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease, *Pseudomonas pumilum* ( Pestalotiopsis microspora Corn leaf spot disease, Curvularia corniculatum ( Curved moonlit Phytophthora blight of pepper ( Phytophthora capsicum ), the pathogen of the leaf spot disease of Jade stamens ( ) Diaporthe biconispora Dragon fruit canker disease, new dark-colored arthropoda ( Neocytalidium halimidium Aspergillus niger, a fungus that causes stem rot in sisal ( Aspergillus niger ) or sisal zebra stripe disease caused by Phytophthora indicum ( Phytophthora Nicotiana ).

[0010] On the other hand, there is a bacterial agent prepared from Bacillus tekirae T22.

[0011] On the other hand, the method for preparing the above-mentioned bacterial agent involves culturing Bacillus tekirae T22 in LB liquid medium.

[0012] Preferably, the culture is carried out at 180 rpm and 28 ℃ until the concentration reaches 1.0 × 10⁻⁶. 8 CFU / mL.

[0013] On the other hand, the above-mentioned microbial agents are used to suppress plant pathogens.

[0014] Preferably, the plant pathogen includes *Dispora cocovenenans*, the causal agent of sisal leaf rot. Lasiodiplodia Theobroma cassava The pathogen causing cowpea wilt is Fusarium oxysporum ( ), Fusarium oxysporum f.sp. tracheal ), Fusarium graminearum, the causal agent of wheat scab ( Fusarium gramineae Mango anthracnose (Colletotrichum gloeosporioides) Colletotrichum gloeosporioides Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease, *Pseudomonas pumilum* ( Pestalotiopsis microspora Corn leaf spot disease, Curvularia corniculatum ( Curved moonlit Phytophthora blight of pepper ( Phytophthora capsicum ), the pathogen of the leaf spot disease of Jade stamens ( ) Diaporthe biconispora Dragon fruit canker disease, new dark-colored arthropoda ( Neocytalidium halimidium Aspergillus niger, a fungus that causes stem rot in sisal ( Aspergillus niger ) or sisal zebra stripe disease caused by Phytophthora indicum ( Phytophthora Nicotiana ).

[0015] On the other hand, a microbial fertilizer, said fertilizer comprising the aforementioned Bacillus tekirae T22.

[0016] On the other hand, the above-mentioned microbial fertilizers are used to suppress plant pathogens.

[0017] Preferably, the plant pathogen includes *Dispora cocovenenans*, the causal agent of sisal leaf rot. Lasiodiplodia Theobroma cassava The pathogen causing cowpea wilt is Fusarium oxysporum ( ), Fusarium oxysporum f.sp. tracheal ), Fusarium graminearum, the causal agent of wheat scab ( Fusarium gramineae Mango anthracnose (Colletotrichum gloeosporioides) Colletotrichum gloeosporioides Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease, *Pseudomonas pumilum* ( Pestalotiopsis microspora Corn leaf spot disease, Curvularia corniculatum ( Curved moonlit Phytophthora blight of pepper ( Phytophthora capsicum ), the pathogen of the leaf spot disease of Jade stamens ( ) Diaporthe biconispora Dragon fruit canker disease, new dark-colored arthropoda ( Neocytalidium halimidium Aspergillus niger, a fungus that causes stem rot in sisal ( Aspergillus niger ) or sisal zebra stripe disease caused by Phytophthora indicum ( Phytophthora Nicotiana ).

[0018] Beneficial effects

[0019] The *Bacillus tekirii* strain T22 provided by this invention exhibits significant inhibitory effects against various plant pathogenic fungi, demonstrating broad-spectrum antibacterial activity in the plate confrontation method experiment. Specifically, it shows strong inhibitory activity against *Melastoma* species, the pathogen causing leaf spot disease in *Magnolia denudata*. Diaporthe biconispora The inhibition rate of ) was as high as 73.00%, and it was effective against corn leaf spot disease Curvularia zei (Curvularia zei). Curved moonlitThe inhibition rate of ) reached 72.18%, and it was effective against rice blast fungus ( Magnaporthe grisea The inhibition rate of ) reached 69.65%, and it was effective against Phytophthora infestans (a fungus that causes zebra stripes on sisal). Phytophthora nicotianae The inhibition rate of ) reached 68.62%, and it was effective against *Colletotrichum candida*, the causal agent of mango anthracnose (…). Colletotrichum gloeosporioides The inhibition rate reached 67.19%, and it was effective against Phytophthora blight of pepper (…). Phytophthora capsicum The inhibition rate of ) reached 63.24%, and it was effective against Fusarium graminearum, the causal agent of wheat scab. Fusarium grasses The inhibition rate of ) reached 63.98%, and it was effective against coconut gray spot disease caused by *Pseudomonas aeruginosa* (…). Pestalotiopsis microspore The inhibition rate of the disease reached 61.28%, and it also inhibited the growth of *Norophorus glomeratus*, a type of scab, which causes canker in dragon fruit. Neocytalidium halved The inhibition rate reached 59.40%, and it was effective against Diplosporum cocovenenans, the causal agent of sisal leaf rot. Lasiodiplodia Theobroma cassava The inhibition rate reached 55.28%, and it was effective against Fusarium oxysporum, the pathogen causing cowpea wilt. Fusarium oxysporum f.sp. tracheal The inhibition rate reached 49.49%, and it was effective against Aspergillus niger, the causal agent of stem rot in sisal. Aspergillus niger The inhibition rate of the fungus reached 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.

[0020] In a pot experiment on the control efficacy of sisal leaf rot, Bacillus tektii T22 showed a control efficacy of 49.15%, which significantly reduced the severity of sisal leaf rot and crop losses caused by the disease, demonstrating good practical application results.

[0021] Furthermore, Bacillus tekirae T22, as a natural microbial strain, possesses high biosafety, posing no harm to the environment, humans, livestock, or crops. This avoids the environmental pollution, pathogen resistance, and pesticide residues in agricultural products associated with chemical pesticide use, aligning with the requirements of green agriculture and sustainable development. Simultaneously, Bacillus tekirae is easy to cultivate and mass-produce, facilitating the development of microbial preparations for widespread application. It has broad application prospects and potential commercial value, providing effective technical support for the green control of plant fungal diseases in agricultural production. Attached Figure Description

[0022] Figure 1 The images show the colony morphology of Bacillus tekirulatus T22 on LB solid medium, where A is a single colony morphology image and B is a streak colony morphology image.

[0023] Figure 2The results represent the physiological and biochemical reactions of Bacillus tekirulatus T22, where A represents the ability to secrete siderophores, B represents the ability to solubilize phosphorus, C represents the ability to solubilize potassium, and D represents the ability to fix nitrogen.

[0024] Figure 3 Phylogenetic tree of 16S rDNA from Bacillus tekirae T22;

[0025] Figure 4 For Bacillus tekiratis T22 gyrA Gene phylogenetic evolutionary tree

[0026] Figure 5 The antibacterial spectrum of Bacillus tegmentata T22, where a represents Diplostomum cocovenenans, the causal agent of sisal leaf rot ( Lasiodiplodia theobromae b represents the rice blast fungus ( Magnaporthe grisea c represents the pathogen of the *Cyclocarya paliurus* genus, which causes leaf spot disease in *Cyclocarya paliurus*. Diaporthe biconispora ), d represents Phytophthora blight, the fungus that causes blight in peppers ( Phytophthora capsicum ), e represents *Colletotrichum candida*, the fungus that causes mango anthracnose (… Colletotrichum gloeosporioides f is the pathogen of cowpea wilt, Fusarium oxysporum ( ), Fusarium oxysporum f. sp. tracheal ), g is a new dark-colored scab for dragon fruit canker ( Neocytalidium halimidium ), h is Phytophthora infestans, a fungus that causes zebra stripes on sisal. Phytophthora Nicotiana ), i represents Fusarium graminearum, the pathogen causing wheat scab ( Fusarium gramineae ), j is the coconut gray spot disease spore ( Pestalotiopsis microspora ), k is the corn leaf spot disease Curvularia zei ( Curvularia lunate ), l is Aspergillus niger, the fungus that causes stem rot in sisal ( Aspergillus niger );

[0027] Figure 6 This study aims to evaluate the control effect of Bacillus tekirii T22 on potted sisal leaf rot. A represents the negative control (PDA treatment group), B represents the positive control (Diospora cacodilobata treatment group), C represents the control effect of the whole potted plant in the Diospora cacodilobata + T22 treatment group, D represents a magnified image of the whole potted plant in the negative control PDA group, E represents a magnified image of the whole potted plant in the positive control Diospora cacodilobata treatment group, F represents a magnified image of the whole potted plant in the Diospora cacodilobata + T22 treatment group, G represents a magnified image of the leaves in the negative control PDA group, H represents a magnified image of the leaves in the positive control Diospora cacodilobata treatment group, and I represents a magnified image of the leaves in the Diospora cacodilobata + T22 treatment group. Detailed Implementation

[0028] Experimental materials

[0029] PDA medium: Boil 200 g potatoes for 15 min, filter, retain the filtrate, add 18 g glucose, 18 g agar, and ddH2O to 1000 mL; sterilize at 121℃ for 20 min.

[0030] LB medium (Luria-Bertani medium): 10.0 g tryptone, 5.0 g yeast extract, 8.0 g sodium chloride (NaCl), 20 g agar, ddH2O to 1000 mL; sterilize at 121℃ for 20 min.

[0031] NBRIP Inorganic Phosphorus Medium: 10.0 g glucose, 0.1 g ammonium sulfate, 0.25 g magnesium sulfate heptahydrate (MgSO4·7H2O), 0.2 g potassium chloride (KCl), 5.0 g magnesium chloride (MgCl2), 5.0 g calcium phosphate (CaCO3), ddH2O to 1000 mL; sterilize at 121℃ for 20 min.

[0032] Alexandrite medium: 5.0 g sucrose, 2.0 g disodium hydrogen phosphate (Na2HPO4), 0.5 g magnesium sulfate heptahydrate (MgSO4·7H2O), 0.005 g ferric chloride (FeCl3), 0.1 g calcium carbonate (CaCO3), 2.0 g potassium feldspar, ddH2O to 1000 mL; sterilize at 115℃ for 20 min.

[0033] Assumption nitrogen-free medium: dipotassium 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) 0.2 g, calcium carbonate (CaCO3) 5.0 g, mannitol 10.0 g, ddH2O to 1000 mL; sterilize at 121℃ for 20 min.

[0034] Chrome azurol (CAS) medium: The basal medium consists of 100.0 g glucose, 0.5 g magnesium sulfate heptahydrate (MgSO4·7H2O), 20.0 g tryptone, 0.5 g calcium chloride (CaCl2), and 10.0 g agar; the CAS assay solution consists of 0.06 g chrome azurol (CAS), 0.0027 g ferric chloride (FeCl3), and 0.073 g hexadecy-1,4-trimethylammonium bromide (HDTMA); 10× buffer PIPES 1 mol / L, pH 7.0; weigh 141 g of the basal medium and add ddH2O to bring the volume to 800 mL; sterilize at 115 ℃ for 20 min; cool the basal medium to 60 ℃ and slowly add 100 g of 10× buffer preheated to 60 ℃. mL, 100 mL of 10×CAS detection solution.

[0035] Example 1 Bacillus tergentii ( Bacillus tequilaensis The acquisition of T22.

[0036] Rhizosphere soil and tissue samples were collected from healthy cowpea plants in areas affected by sisal leaf rot. Soil samples were separated using the dilution-spread method: 10 g of soil sample was placed in a 250 mL Erlenmeyer flask, 90 mL of ddH₂O was added, and the sample was placed on a shaker at 28 ℃ and 180 rpm for 30 min to mix thoroughly. The sample was then placed in an 80 ℃ water bath and allowed to stand for 10 min before being diluted to a final concentration of 10 g. -2 10 -3 10 -4 10 -5 Dilute the medium three times. Add 10-15 mL of preheated LB medium to a petri dish. After cooling, add 0.2 mL of each of the above dilutions to the plates and spread evenly with a glass spreader until dry. Repeat three times. After sealing, incubate at 28 °C under 14 h light and 10 h dark conditions. Observe the colonies after 2 days. Select suspected colonies and streak them on LB plates for purification. Number and store the strains.

[0037] Using the sisal leaf rot pathogen as a control strain, the preserved strains were initially screened using the plate confrontation method. A 0.5 cm wilt fungal cake of cowpea was inoculated in the center of a PDA solid medium plate. Four points 2.5 cm away from the center of each medium plate were selected symmetrically in a cross pattern. The same strain was isolated, purified, and preserved at the four points using toothpicks. The strains with antagonistic effects were screened again using the same method and preserved. The colony diameter was measured using the cross cross method. Inhibition rate / % = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100. The experiment was repeated 3 times, with 3 replicates for each treatment. The relevant data were compiled using Excel, and the significance of data differences was analyzed using SPSS (25) software. Multiple comparisons were performed using Tukey's Method.

[0038] The morphology of strain T22 obtained through screening on LB solid medium is as follows: Figure 1 As shown, on LB medium, colonies appear as light yellow spots, which are relatively scattered. The colonies are irregular in shape and vary in size. Some colonies are clearly round or oval, and there are obvious intervals between the colonies, forming small clusters.

[0039] Example 2 Bacillus tergentii ( Bacillus tequilaensis Physiological and biochemical characteristics of T22.

[0040] Determination of phosphorus solubilization ability: After activating strain T22, it was inoculated onto NBRIP inorganic phosphorus medium and placed in a constant temperature incubator at 28 ℃ for 7 days. The presence of transparent oil droplet-like halos around the colonies was observed. If present, it indicated that the strain had phosphorus solubilization ability. The size of the halo was measured.

[0041] Determination of potassium solubilizing ability: After activating strain T22, it was inoculated onto Alexandrite silicate medium and placed in a constant temperature incubator at 28 ℃ for 7 days. The presence of transparent oil droplet-like halos around the colonies was observed. If present, it indicated that the strain had potassium solubilizing ability. The size of the halo was measured.

[0042] Determination of nitrogen fixation function: After activation, strain T22 was inoculated onto Assumption nitrogen-free medium and placed in a constant temperature incubator at 28 ℃ for 7 days. Colony growth was observed. The ability to grow on Assumption nitrogen-free medium was considered to have nitrogen fixation function. The ammonia fixation capacity was judged based on the diameter of the colony growth.

[0043] Siderophore production capacity determination: After activation, strain T22 was inoculated onto chromium azurite (CAS) medium and placed in a constant temperature incubator at 28 ℃ for 7 days. The presence or absence of a yellow halo around the colony was observed, and the size of the halo was measured.

[0044] The results are as follows Figure 2As shown, strain T22 possesses certain abilities in nitrogen fixation, phosphorus solubilization, potassium solubilization, and siderophore secretion. On CAS medium, a clear yellow halo with a diameter of 17.46 mm was observed in T22, indicating its siderophore secretion ability. On Assumption nitrogen-free medium, the growth diameter of strain T22 was 7.5 mm, indicating its nitrogen-fixing ability. On Alexandrite silicate medium, the colony diameter of T22 was 38.03 mm, indicating a certain potassium solubilization ability. On NBRIP inorganic phosphorus bacteria medium, the secretion zone diameter of T22 was 29.58 mm, indicating its ability to decompose organic phosphorus.

[0045] Example 3 Bacillus tergentii ( Bacillus tequilaensis Molecular identification of T22.

[0046] The T22 strain was inoculated into LB liquid medium and incubated at 180 rpm for 16 h. Bacterial DNA extraction was performed using the bacterial DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd., following the manufacturer's instructions. The obtained bacterial DNA was used as a template for PCR amplification. The target gene was selected using universal primers for the conserved bacterial region, specifically the 16S rDNA gene of the ribosomal region, and bacterial-specific primers for the helicase A subunit gene. gyrA The amplification primers were the universal 16S rDNA 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′).

[0047] The PCR reaction system consisted of: 12.5 μL of 2×Tap PCR MasterMix, 9.5 μL of ddH2O, 1 μL each of forward and reverse primers, and 1 μL of DNA. The 16S rDNA amplification program was as follows: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 45 s, extension at 72 ℃ for 1 min, and further extension at 72 ℃ for 10 min, for a total of 35 cycles. circle A The amplification program was as follows: pre-denaturation at 95 ℃ for 4 min, denaturation at 94 ℃ for 40 s, annealing at 60 ℃ for 45 s, extension at 72 ℃ for 1 min, and further extension at 72 ℃ for 10 min, for a total of 35 cycles.

[0048] PCR amplification products were detected by 1% agarose gel electrophoresis. After observing and photographing the target fragment bands in a gel imaging system, the PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were compared and analyzed for homology in the NCBI database, and highly similar gene sequences and their type strain sequences were downloaded. A phylogenetic tree was constructed using the maximum likelihood method with Mega 11 software.

[0049] 16S rDNA of strain T22 and gyrA The gene fragment was amplified by PCR, yielding 1400 bp of 16S rDNA and... gyrA The target fragment was approximately 1000 bp. The PCR amplification product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The obtained sequence was aligned using NCBI. 16S rDNA alignment results showed that T22 is related to *Bacillus tekirae*. Bacillus tequilaensis The similarity of 10b (NR_117611.1) is 99.92%. gyrA The comparison results showed that T22 was related to Bacillus tekirae. Bacillus tequilaensis The similarity of NRRL B-41771 (NZ_EU138625.1) was 98.54%. Highly similar gene sequences and type strain sequences were downloaded, and 16S rDNA of T22 was constructed using MEGA11 software. gyrA Phylogenetic tree, the results are as follows Figure 3 and Figure 4 As shown, T22 and Bacillus tekirae Bacillus from Tequila They clustered within the same branch. Therefore, T22 was identified as Bacillus tekirae. Bacillus tequilaensis Bacillus tekiria ( Bacillus tequilaensis The T22 strain is deposited at the China General Microbiological Culture Collection Center (address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​February 21, 2025, with accession number CGMCC No. 33612.

[0050] Example 4 Bacillus tergentii ( Bacillus tequilaensis T22 antibacterial ability determination.

[0051] The tested plant pathogenic fungi included *Dispora cocovenenans*, the causal agent of sisal leaf rot. Lasiodiplodia Theobroma cassava The pathogen causing cowpea wilt is Fusarium oxysporum ( ), Fusarium oxysporum f. sp. tracheal ) and Fusarium graminearum, the pathogen causing wheat scab ( Fusarium gramineaeMango anthracnose (Colletotrichum gloeosporioides) Colletotrichum gloeosporioides Rice blast fungus ( Magnaporthe grisea ), Coconut gray spot disease, *Pseudomonas pumilum* ( Pestalotiopsis microspora Corn leaf spot disease, Curvularia corniculatum ( Curvularia lunate Phytophthora blight of pepper ( Phytophthora capsicum ), the pathogen of the leaf spot disease of Jade stamens ( ) Diaporthe biconispora Dragon fruit canker disease, new dark-colored arthropoda ( Neocytalidium halimidium Aspergillus niger, a fungus that causes stem rot in sisal ( Aspergillus niger ), Sisal zebra stripe disease, Phytophthora indicum ( Phytophthora nicotianae )wait.

[0052] The antibacterial activity of Bacillus tekirae T22 strain was determined using the plate confrontation method. Bacillus tekirae T22 strain was cultured in LB liquid medium at 180 rpm and 28 ℃ for 16 h to obtain bacterial fermentation broth. Using the "cross-cutting method," sterile 5 mm filter paper discs were affixed to four points 2.5 cm apart on PDA culture plates. Using a sterile 5 mm punch, bacterial discs of the same age were inoculated at the edge of the activated bacterial colonies and placed in the center of the PDA plate. 1 μL of bacterial fermentation broth was added to the center of each of the four filter paper discs. Each treatment was repeated in triplicate. The treated PDA plates were incubated at 28 ℃ for 4–5 days, during which colony growth was observed. When the colonies had covered approximately 3 / 4 of the culture plate, the antibacterial activity was measured, recorded, and photographed. The formula for calculating the antibacterial rate is as follows:

[0053] Inhibition rate / % = [(Diameter of pathogen growth in control group - Diameter of pathogen block) - (Diameter of pathogen growth in treatment group - Diameter of pathogen block)] / (Diameter of pathogen growth in control group - Diameter of pathogen block) × 100.

[0054] The results are shown in Table 1 and Figure 5 As shown, T22 has good antifungal activity against pathogenic fungi, especially against Diplosporum cocovenenans, the causal agent of sisal leaf rot. Lasiodiplodia theobromae The inhibition rate was 55.28%, which is effective against Fusarium oxysporum, the pathogen causing cowpea wilt. Fusarium oxysporum f. sp. tracheal The inhibition rate was 49.49%, and it inhibited the growth of Fusarium graminearum, the causal agent of wheat scab. Fusarium gramineae The inhibition rate was 63.98%, which is effective against *Colletotrichum candida*, the causal agent of mango anthracnose. Colletotrichum gloeosporioides The inhibition rate was 67.19%, which is effective against rice blast fungus (…). Magnaporth gray The inhibition rate was 69.65%, which is effective against coconut gray spot disease caused by *Pseudomonas aeruginosa* (…). Pestalotiopsis microspora The inhibition rate was 61.28%, which is effective against corn leaf spot disease caused by Curvularia corniculatum (Curvularia corniculatum). Curvularia lunate The inhibition rate was 72.18%, which is effective against Phytophthora blight of pepper (…). Phytophthora capsicum The inhibition rate was 63.24%, which is effective against the pathogen of *Cyclocarya paliurus* causing leaf spot disease. Diaporthe biconispora The inhibition rate was 73.00%, and it inhibited the growth of *Norophorus glomeratus*, a type of scab, in dragon fruit canker. Neocytalidium halved The inhibition rate was 59.40%, and it was effective against Aspergillus niger, the pathogen causing stem rot in sisal. Aspergillus niger The inhibition rate was 30.6%, and it was effective against Phytophthora infestans, the causal agent of zebra stripe disease in sisal. Phytophthora nicotianae The inhibition rate was 68.62%.

[0055] Table 1. Bacillus tekiratis ( Bacillus tequilaensis T22 antibacterial ability

[0056]

[0057] Example 5 Bacillus tergentii ( Bacillus tequilaensis T22's potted plant protection efficacy against sisal leaf rot.

[0058] Preparation of Bacillus tekirae T22 inoculum: Bacillus tekirae T22 strain was cultured in LB liquid medium on a shaker at 180 rpm and 28 ℃ until a concentration of 1.0 × 10⁻⁶ was reached. 8 CFU / mL.

[0059] Eighteen healthy sisal seedlings were selected. Three leaves were chosen from each seedling, and the epidermis of each leaf was carefully punctured with a needle. An inverted 5 mm diameter mycelial cake was inoculated at the puncture site on each leaf. The positive control group used *Dioscorea opposita* mycelial cake, and the negative control group used mycelial cake on PDA medium. The other treatment groups used *Dioscorea opposita* mycelial cake and were sprayed with 10 mL of a 1.0 × 10⁻⁶ solution. 8 Three treatment groups were established, with five replicates per treatment, using CFU / mL *Bacillus tekiri* T22 inoculum. After inoculation, the inoculum was covered with a sterile bag and moistened with sterile water, and the disease development was observed. Disease grading was based on NY / T 1488-2018 "Technical Specifications for Quarantine of Diseases in Sisal Seedlings", as shown in Table 2.

[0060] Table 2 Grading Standards for Leaf Diseases

[0061]

[0062] The formula for calculating the disease index and prevention and control effectiveness is as follows:

[0063]

[0064]

[0065] The results of the pot experiment showed that the PDA control group did not develop the disease, the disease index of the positive control group treated with Diplostomum cocovenenans was 54.63, and the disease index of the Diplostomum cocovenenans + T22 treatment group was 27.78. Compared with the positive control group, the control effect was 49.15%, which means that the T22 bacterial suspension can effectively reduce the occurrence and damage of sisal leaf rot.

[0066] Table 3 Results of potted plant efficacy test

[0067]

[0068] The contents 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, it is not intended to limit the invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A type of Bacillus tekirae ( Bacillus tequilensis T22, characterized in that, The Bacillus tekirae T22 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33612.

2. The application of Bacillus tekirii T22 as described in claim 1 in inhibiting plant pathogens, characterized in that, The plant pathogen is *Dioscorea opposita*, which causes sisal leaf rot. Lasiodiplodia theobromae The pathogen that causes cowpea wilt is Fusarium oxysporum. Fusarium oxysporum f . sp . tracheiphilum Fusarium graminearum, which causes wheat scab. Fusarium graminearum Colletotrichum gloeosporioides, which causes mango anthracnose. Colletotrichum gloeosporioides The pathogen that causes rice blast Magnaporthe grisea *Plasmodium spp.*, which causes coconut gray spot disease Pestalotiopsis microspora Curvularia corniculatum, which causes corn leaf spot disease Curvularia lunata Phytophthora blight, the fungus that causes blight in peppers Phytophthora capsici Pathogens that cause leaf spot disease in Jade Plants Diaporthe biconispora A new type of dark-colored arthropod spore that causes dragon fruit canker. Neoscytalidium dimidiatum Aspergillus niger, which causes stem rot in sisal. Aspergillus niger Phytophthora indicum, which may cause zebra stripe disease in sisal. Phytophthora nicotianae .

3. A microbial agent, characterized in that, The bacterial agent comprises Bacillus tekirulatus T22 as described in claim 1.

4. The method for preparing the microbial agent according to claim 3, characterized in that, The method involves culturing the Bacillus tekirius T22 of claim 1 in LB liquid medium.

5. The method according to claim 4, characterized in that, The culture was carried out at 180 rpm and 28℃ until the concentration reached 1.0 × 10⁻⁶. 8 CFU / mL.

6. The application of the microbial agent according to claim 3 in inhibiting plant pathogens, characterized in that, The plant pathogen is *Dioscorea opposita*, which causes sisal leaf rot. Lasiodiplodia theobromae The pathogen that causes cowpea wilt is Fusarium oxysporum. Fusarium oxysporum f . sp . tracheiphilum Fusarium graminearum, which causes wheat scab. Fusarium graminearum Colletotrichum gloeosporioides, which causes mango anthracnose. Colletotrichum gloeosporioides The pathogen that causes rice blast Magnaporthe grisea *Plasmodium spp.*, which causes coconut gray spot disease Pestalotiopsis microspora Curvularia corniculatum, which causes corn leaf spot disease Curvularia lunata Phytophthora blight, the fungus that causes blight in peppers Phytophthora capsici Pathogens that cause leaf spot disease in Jade Plants Diaporthe biconispora A new type of dark-colored arthropod spore that causes dragon fruit canker. Neoscytalidium dimidiatum Aspergillus niger, which causes stem rot in sisal. Aspergillus niger Phytophthora indicum, which may cause zebra stripe disease in sisal. Phytophthora nicotianae .

7. A microbial fertilizer, characterized in that, The microbial fertilizer includes Bacillus tekirae T22 as described in claim 1.

8. The application of the microbial fertilizer according to claim 7 in inhibiting plant pathogens, characterized in that, The plant pathogen is *Dioscorea opposita*, which causes sisal leaf rot. Lasiodiplodia theobromae Fusarium oxysporum, the pathogen that causes cowpea wilt. Fusarium oxysporum f . sp . tracheiphilum Fusarium graminearum, which causes wheat scab. Fusarium graminearum Colletotrichum gloeosporioides, which causes mango anthracnose. Colletotrichum gloeosporioides The pathogen that causes rice blast Magnaporthe grisea *Plasmodium spp.*, which causes coconut gray spot disease Pestalotiopsis microspora Curvularia corniculatum, which causes corn leaf spot disease Curvularia lunata Phytophthora blight, the fungus that causes blight in peppers Phytophthora capsici Pathogens that cause leaf spot disease in Jade Plants Diaporthe biconispora A new type of dark-colored arthropod spore that causes dragon fruit canker. Neoscytalidium dimidiatum Aspergillus niger, which causes stem rot in sisal. Aspergillus niger Phytophthora indicum, which may cause zebra stripe disease in sisal. Phytophthora nicotianae .

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