Salt-tolerant bacillus strain EMM919 and application thereof in preparation of microbial pesticide and seed dressing agent for preventing and treating fungal diseases

Abstract

The invention relates to a salt-tolerant bacillus strain EMM919, which is preserved in the China Center for Type Culture Collection on April 25, 2025, and the preservation number is CCTCC NO: M 2025879. The salt-tolerant bacillus strain EMM919 has the advantages that the salt-tolerant bacillus strain EMM919 is preserved in the China Center for Type Culture Collection on April 25, 2025; the nucleotide sequence of the strain comprises a 16S rRNA (ribosomal Ribonucleic Acid) gene sequence and a gyrB gene sequence. The bacterial strain can be used for preparing microbial pesticides and seed dressing agents for preventing and treating fusarium solani EMF893, neurospora EMF992, cylindrospora robusta EMF882, fusarium laminatum EMF998, alternaria alternata EMF993, botryosphaeria dothidea EMF899, solanum nigrum EMF990, phomopsis EMF898 and fusarium graminearum EMF897. The content of viable bacteria in the liquid pesticide is 2 * 10 < 9 >-5 * 10 < 9 > CFU / mL, and the content of viable bacteria in the solid pesticide is 2 * 10 < 9 >-8 * 10 < 9 > CFU / g. In the liquid seed-dressing agent, the content of viable bacteria is 2 * 10 < 9 >-5 * 10 < 9 > CFU / mL, and in the solid seed-dressing agent, the content of viable bacteria is 2 * 10 < 9 >-8 * 10 < 9 > CFU / g.

Description

Technical Field

[0002] This invention belongs to the field of microbial technology, specifically relating to the salt-tolerant Bacillus strain EMM919 and its applications.

[0004] Since traditional Chinese medicine is mostly used in medicine and health products, the problem of chemical pesticide residues is particularly sensitive. The application of traditional chemical control methods is limited, so there is an urgent need to develop green, efficient and residue-free biological control methods.

[0005] Currently, agricultural production still relies heavily on chemical pesticides to control fungal diseases. However, long-term use not only leads to increased pesticide resistance in pathogens but also causes pesticide residues and environmental pollution. Microbial control, as an environmentally friendly alternative strategy, is receiving increasing attention. Among numerous biocontrol microorganisms, Bacillus bacteria show great application potential due to their strong resistance, rapid reproduction, and ease of large-scale production. Of particular note are strains with broad-spectrum antifungal activity that can simultaneously inhibit multiple pathogenic fungi, reduce the occurrence of co-infections, and effectively colonize in the rhizosphere soil through niche competition, significantly improving the stability and persistence of biocontrol effects.

[0006] On the other hand, soil salinization is a significant environmental factor affecting the yield of crops and medicinal herbs. Ordinary microbial agents are easily inhibited in high-salt environments, while salt-tolerant strains can better adapt to salt stress and maintain their biocontrol and growth-promoting effects. Furthermore, strains that produce plant growth regulators such as indole-3-acetic acid (IAA) can directly stimulate crop growth by promoting root development and enhancing nutrient absorption, achieving a dual effect of "control-promoting."

[0007] Several Bacillus strains have been reported for application in crop disease control. For example, CN117210349A discloses a strain of *Bacillus hygroscopicus* for controlling blueberry gray mold; CN119662443A reports the control effect of a salt-tolerant Bacillus strain on tomato root rot; and studies have shown that some Bacillus strains have inhibitory effects on rice blast. However, current technology lacks multifunctional strains that can simultaneously adapt to the needs of both agricultural crops and medicinal herbs, possess salt tolerance, broad-spectrum antifungal activity, and efficient IAA synthesis capabilities. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the shortcomings of the above-mentioned microbial pesticides and provide a salt-tolerant Bacillus strain EMM919 with good inhibitory and killing effects on plant pathogenic fungi.

[0009] Another technical problem to be solved by the present invention is to provide a new use for the salt-tolerant Bacillus strain EMM919.

[0010] The technical solution adopted to solve the above-mentioned technical problems is as follows: a salt-tolerant Bacillus shalotolerans strain EMM919, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M2025879 and deposit date of April 25, 2025; the nucleotide sequence of this strain includes a 16S rRNA gene sequence and a gyrB gene sequence, the 16S rRNA gene sequence is shown in SEQ ID NO:1, and the gyrB gene sequence is shown in SEQ ID NO:2.

[0011] The 16S rRNA nucleotide sequence (SEQ ID NO:1) of strain EMM919 is as follows:

[0012] ctggggcgggcctaatacttgcaagtcgagcggacagatgggagcttgctccctgatgtt 60

[0013] agcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccggg 120

[0014] aaaccggggctaataccggatgcttgtttgaaccgcatggttcaaacataaaaggtggct 180

[0015] tcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctca 240

[0016] ccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacac 300

[0017] ggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgac 360

[0018] ggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaag 420

[0019] aacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggcta 480

[0020] actacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggc 540

[0021] gtaaagggctcgcaggcggttccttaagtctgatgtgaaagccccgggctcaaccgggga 600

[0022] gggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagc 660

[0023] ggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaa 720

[0024] ctgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacg 780

[0025] ccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgc 840

[0026] attaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggg 900

[0027] gcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccagg 960

[0028] tcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacagg 1020

[0029] tggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcg 1080

[0030] caacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgaca 1140

[0031] aaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacaca 1200

[0032] cgtgctacaatggacagaacaaagggcagcaaaaccgcgaggttaagccaatcccacaaa 1260

[0033] tctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagta 1320

[0034] atcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcac 1380

[0035] accacgagagtttgtaacacccgaagtcggtgaggtaacctttatggagccagccgccga 1440

[0036] aggtggatccg 1451

[0037] The gyrB nucleotide sequence of strain EMM919 (SEQ ID No.2) is as follows:

[0038] cggacttgcagggtgtaggtgcctcggtcgttacgcgttatcaacagagcttgatgtgac 60

[0039] tgttcaccgtgacggaaaaatccatcgccaagtctataaccgcggtatcccggtttctga 120

[0040] tctcgaggttattggcgaaacggatcataccggaacgactacacattttgttccagatcc 180

[0041] [[ID=三十一]]tgaaattttcacggaaacaactgagtatgaatatgatctgcttgctaaccgtgttcgtga 240

[0042] actagcctttttgacaaaaggcgtaaacatcacgattgaagataaacgtgaaggacaaga 300 It should be noted that there is a misspelling in your original text. In line , it should be "31" instead of "三十一". I have translated it according to the corrected content. If there are other specific requirements or corrections, please feel free to let me know.

[0043] acgcaaaaatgagtatcattacgaaggcggaataaaaagctatgtagagtatttaaaccg 360

[0044] ctccaaagaagttgtccatgaagagccgatttatattgaaggcgaaaaggacggcattac 420

[0045] ggttgaagtcgctctgcaatacaatgacggctacacaagcaatatttactcatttacaaa 480

[0046] caatatcaacacgtacgaaggcggtactcatgaagccggttttaaaacagggctgactcg 540

[0047] tgtcatcaatgattacgccagaaaaaaaggactcataaaagaaaatgatccaaacttgag 600

[0048] cggagatgatgtgagagaagggcttaccgcgattatctcgatcaaacacccggatccgca 660

[0049] gttcgaaggccaaacgaaaacaaaattaggcaactcagaggcacggactatcacagatac 720

[0050] gttattttctgcggcgttggaaacatttatgctggaaaatccagatgcggccagaaaaat 780

[0051] cgttgacaaaggtttaatggcagcaagagcaagaatggctgcgaaaaaagcacgtgaatt 840

[0052] aacgcgccgcaaaagcgctttggagatttcaaaccttcccggtaaattagcggactgctc 900

[0053] ttcgagagacccgagcatctccgagttatatatcgtagagggtgactctgccggaggatc 960

[0054] tgcaaaacaggggcgtgacagacatttccaggccattttgccgcttagaggtaaaatcct 1020

[0055] gaacgttgaaaaagcaaggcttgataaaattctttctaacaacgaagttcgttctatgat 1080

[0056] tactgcactcggcacaggcatcggggaagattcaactggaaaaagcccccaaatccc 1137

[0057] The use of the salt-tolerant Bacillus strain EMM919 of the present invention in the preparation of microbial pesticides for the prevention and control of fungal diseases, wherein the fungus is any one of Fusarium rotundum EMF893, Neurospora emulsifiable concentrate EMF992, Cyclospora robusta EMF882, Fusarium effusum EMF998, Alternaria alternata EMF993, Staphylococcus aureus EMF899, Heterophyllum rotundum EMF990, Pseudomonas cepacia EMF898, and Fusarium graminearum EMF897.

[0058] The microbial pesticide of the present invention is an agriculturally acceptable carrier on which the halophilic Bacillus strain EMM919 is adsorbed. It is prepared into liquid or solid formulations using conventional methods. In the liquid formulation, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0059] The microbial pesticide of this invention is prepared in liquid or solid form using conventional methods. It is formulated with an agriculturally acceptable carrier on which halophilic Bacillus M919 is adsorbed. In the liquid form, the optimal viable count of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the optimal viable count of strain EMM919 is 4 × 10⁻⁶ CFU / mL. 9 CFU / g.

[0060] The use of the salt-tolerant Bacillus strain EMM919 of the present invention in the preparation of a seed dressing agent, wherein the seed dressing agent is

[0061] The salt-tolerant Bacillus strain EMM919 was adsorbed onto an agriculturally acceptable carrier and prepared into liquid or solid formulations as a microbial seed dressing agent using conventional methods. In the liquid formulation, the viable count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial inoculants, the viable count of strain EMM919 was 2 × 10⁻⁶ CFU / mL. 9 CFU / g ~ 8×10 9 CFU / g.

[0062] The present invention relates to the use of the salt-tolerant Bacillus strain EMM919 in the preparation of seed dressing agents. The salt-tolerant Bacillus strain EMM919 is adsorbed onto an agriculturally acceptable carrier and prepared into liquid or solid formulations of microbial seed dressing agents using conventional methods. In the liquid formulation of the microbial seed dressing agent, the optimal viable count of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial inoculants, the optimal viable count of strain EMM919 is 4 × 10⁻⁶ CFU / mL. 9 CFU / g.

[0063] The identification method for the salt-tolerant Bacillus strain EMM919 is as follows:

[0064] 1. Isolation method of halophilic Bacillus strain EMM919

[0065] Soil samples from the rhizosphere of Astragalus membranaceus, collected from a medicinal herb planting base in Ningxia Hui Autonomous Region, were brought back to the laboratory in a plastic bag and stored at 4℃ for later use. 5g of soil sample was weighed and added to an Erlenmeyer flask containing 3 glass beads and 45mL of sterile water. The mixture was thoroughly shaken, serially diluted with sterile water, and then spread onto LB agar plates. The plates were incubated at 37℃ for 1–2 days to purify and obtain a pure culture of the strain.

[0066] 2. Identification of the halophilic Bacillus strain EMM919

[0067] (1) Screening of salt-tolerant Bacillus strain EMM919

[0068] Prepare LB medium: Place 10g tryptone, 5g yeast extract, 10g NaCl and 20g agar into a shaking container and shake until the solutes dissolve. Adjust the pH to 7.0 with 5mol / L NaOH and bring the volume to 1L with deionized water.

[0069] 1) Screening of biocontrol bacteria

[0070] The isolated and purified strains were inoculated into liquid culture medium and shaken at 30℃ and 180 r / min for 24 hours to obtain the seed culture solution. 3 mL of the seed culture solution was transferred to 100 mL of liquid culture medium and cultured for 48 hours. 500 μL of the seed culture solution was transferred to a 1.5 mL centrifuge tube, and the same volume of Salksowski chromogenic solution was added to perform the color reaction. The tube was placed at room temperature and in the dark for 30 minutes, and the color change was observed. Those that turned red were able to produce IAA.

[0071] 2) Secondary screening for IAA-producing bacteria

[0072] Preparation of IAA standard curve: Prepare standard solutions with concentrations of 0, 10, 20, 40, 60, 80, and 100 μg / mL, mix with Salksowski's colorimetric solution at a volume ratio of 1:1, incubate at room temperature in the dark for 30 min, and dilute with distilled water.

[0073] Using an equal volume mixture of Salksowski's colorimetric solution as a control, the OD535nm of each concentration was measured, with IAA concentration...

[0074] Plotting OD535nm on the x-axis and OD535nm on the y-axis yields the IAA standard curve.

[0075] Determination of IAA production concentration in bacterial culture: One loopful of the test strain was placed in liquid culture medium and incubated at 30℃ and 180 r / min for 24 hours as a seed culture. 3 ml of this seed culture was then placed in liquid culture medium and incubated at 30℃ and 180 r / min for 2 days. The culture was then centrifuged at 12000 r / min for 2 min. 2 mL of the supernatant was mixed with an equal volume of Salksowski's colorimetric solution, shaken well, and incubated at room temperature in the dark for 30 min. The OD535nm value was then measured. An equal volume mixture of uninoculated liquid culture medium and Salksowski's colorimetric solution served as a control group. The corresponding IAA-producing bacterial content was calculated based on the standard curve.

[0076] 3) Screening for growth-promoting and biocontrol bacteria

[0077] Using Fusarium solani EMF893, Fusarium solani EMF990, and Fusarium graminearum EMF897 as pathogenic fungi, biocontrol bacteria that inhibit IAA-producing bacteria were screened.

[0078] Eight IAA-producing growth-promoting strains selected from pure cultures were evaluated using a colorimetric method. The biocontrol capabilities of IAA-producing strains numbered TX-6-4B, NX-7-G-22, NX-6-G-23, TX-1-18A, NX-5-G-5, TX-1-24B, TX-7-7, and EMM919 were further screened, resulting in the selection of one growth-promoting biocontrol strain, designated EMM919.

[0079] (2) Identification of strains

[0080] 1) Morphological identification

[0081] The colony characteristics and sporulation of biocontrol bacteria EMM919 on LB agar plates were observed, and Gram staining of the biocontrol bacteria cells was performed. The results are as follows: Figure 1 As shown. By Figure 1 As can be seen, EMM919 colonies on LB medium are opaque, dry and wrinkled, yellowish-white, non-reflective, with irregular circular edges, and produce pigment, further confirming that EMM919 is a Gram-positive bacterium.

[0082] 2) Molecular biological identification

[0083] Total DNA was extracted from strain EMM919 using the CTAB method, and PCR amplification and sequencing of 16S rRNA and gyrB were performed.

[0084] A. PCR amplification and sequencing of 16S rRNA. The primers used for PCR amplification are:

[0085] 27F:5'-AGAGTTTGATCCTGGCTCAG-3'

[0086] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0087] PCR amplification conditions were: 94℃ for 3 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, for 30 cycles; 72℃ for 10 min, for 4℃ cycles.

[0088] The 16S rRNA nucleotide sequence of EMM919 (SEQ ID NO:1) is as follows:

[0089] ctggggcgggcctaatacttgcaagtcgagcggacagatgggagcttgctccctgatgtt 60

[0090] agcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccggg 120

[0091] aaaccggggctaataccggatgcttgtttgaaccgcatggttcaaacataaaaggtggct 180

[0092] tcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctca 240

[0093] ccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacac 300

[0094] ggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgac 360

[0095] ggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaag 420

[0096] aacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggcta 480

[0097] actacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggc 540

[0098] gtaaagggctcgcaggcggttccttaagtctgatgtgaaagccccgggctcaaccgggga 600

[0099] gggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagc 660

[0100] ggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaa 720

[0101] ctgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacg 780

[0102] ccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgc 840

[0103] attaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggg 900

[0104] gcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccagg 960

[0105] tcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacagg 1020

[0106] tggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcg 1080

[0107] caacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgaca 1140

[0108] aaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacaca 1200

[0109] cgtgctacaatggacagaacaaagggcagcaaaaccgcgaggttaagccaatcccacaaa 1260

[0110] tctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagta 1320

[0111] atcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcac 1380

[0112] accacgagagtttgtaacacccgaagtcggtgaggtaacctttatggagccagccgccga 1440

[0113] aggtggatccg 1451

[0114] PCR amplification and sequencing of EMM919 gyrB are as follows:

[0115] The primers used for PCR amplification are:

[0116] gyrB-F:GGAAGCGGATATAAAGTATCCGG

[0117] gyrB-R: CCGTCRACRTCGGCRTCNGTCATAATG

[0118] The PCR amplification conditions were: 94℃ for 3 min, 94℃ for 30 s, 55℃ for 30 s, 72℃ for 90 s, for 30 cycles; 72℃ for 10 min; and 4℃ for the cycle.

[0119] The PCR amplification products were sequenced, and the sequencing results of the gyrB sequence (SEQ ID NO:2) are as follows:

[0120] cggacttgcagggtgtaggtgcctcggtcgttacgcgttatcaacagagcttgatgtgac 60

[0121] tgttcaccgtgacggaaaaatccatcgccaagtctataaccgcggtatcccggtttctga 120

[0122] tctcgaggttattggcgaaacggatcataccggaacgactacacattttgttccagatcc 180

[0123] tgaaattttcacggaaacaactgagtatgaatatgatctgcttgctaaccgtgttcgtga 240

[0124] actagcctttttgacaaaaggcgtaaacatcacgattgaagataaacgtgaaggacaaga 300

[0125] acgcaaaaatgagtatcattacgaaggcggaataaaaagctatgtagagtatttaaaccg 360

[0126] ctccaaagaagttgtccatgaagagccgatttatattgaaggcgaaaaggacggcattac 420

[0127] ggttgaagtcgctctgcaatacaatgacggctacacaagcaatatttactcatttacaaa 480

[0128] caatatcaacacgtacgaaggcggtactcatgaagccggttttaaaacagggctgactcg 540

[0129] tgtcatcaatgattacgccagaaaaaaaggactcataaaagaaaatgatccaaacttgag 600

[0130] cggagatgatgtgagagaagggcttaccgcgattatctcgatcaaacacccggatccgca 660

[0131] gttcgaaggccaaacgaaaacaaaattaggcaactcagaggcacggactatcacagatac 720

[0132] gttattttctgcggcgttggaaacatttatgctggaaaatccagatgcggccagaaaaat 780

[0133] cgttgacaaaggtttaatggcagcaagagcaagaatggctgcgaaaaaagcacgtgaatt 840

[0134] aacgcgccgcaaaagcgctttggagatttcaaaccttcccggtaaattagcggactgctc 900

[0135] ttcgagagacccgagcatctccgagttatatatcgtagagggtgactctgccggaggatc 960

[0136] tgcaaaacaggggcgtgacagacatttccaggccattttgccgcttagaggtaaaatcct 1020

[0137] gaacgttgaaaaagcaaggcttgataaaattctttctaacaacgaagttcgttctatgat 1080

[0138] tactgcactcggcacaggcatcggggaagattcaactggaaaaagcccccaaatccc 1137

[0139] Phylogenetic tree of 16S rRNA as follows Figure 2 As shown, the phylogenetic tree of gyrB is as follows: Figure 3 As shown.

[0140] 3) Identification of extracellular enzyme activity

[0141] The physiological and biochemical properties of the selected strain EMM919 were identified, and the specific identification process is as follows.

[0142] A. Starch hydrolysis test

[0143] Strain EMM919 was inoculated into starch hydrolysis medium and incubated upside down at 30°C for 12 hours. Lurjohn's iodine solution was added to the center of the medium, and the plate was observed after the solution was evenly distributed. A positive result was indicated by a colorless transparent zone around the colony. This test was used to determine whether bacteria could produce extracellular amylase to hydrolyze starch. Each test was repeated three times.

[0144] B. Cellulase detection

[0145] Strain EMM919 was inoculated onto cellulose medium plates and cultured at 30°C for 2 days. It was then stained with 0.5% Congo red dye, allowed to stand for 30 minutes, the dye solution was poured out, and the mixture was eluted with 5% NaCl solution for 1 hour. The solution was then poured out and the presence of a clear zone was observed.

[0146] C. Protease detection

[0147] Inoculate strain EMM919 onto a skim milk powder culture medium plate and incubate at 30°C for 2 days, then observe whether a clear zone is formed.

[0148] D. Detection of dextranase

[0149] Inoculate strain EMM919 onto dextran agar plates and incubate at 30°C for 2 days, then observe whether a clear zone is formed.

[0150] The identification results of the starch hydrolysis test, cellulase detection, protease, and dextranase detection are shown in Table 1.

[0151] Table 1. Results of extracellular enzyme activity identification

[0152]

[0153] Note: - indicates negative, + indicates positive.

[0154] 4) Test for ability to promote fertility

[0155] A. Iron-producing capacity testing

[0156] EMM919 was inoculated onto CAS detection medium plates and incubated at 30°C for 2 days. Afterward, the colonies were observed to see if a yellow or orange halo appeared around them.

[0157] B. Nitrogen Fixation Capacity Test

[0158] EMM919 was inoculated onto Assumption medium plates and cultured at 30°C for 2 days. The strain was then observed to see if it could grow normally.

[0159] C. Potassium solubility test

[0160] EMM919 was inoculated onto potassium feldspar solid medium plates and incubated at 30°C for 2 days. Observe whether a clear zone is formed around the colony.

[0161] D. Phosphorus solubility test

[0162] EMM919 was inoculated onto phosphate-solubilizing solid medium plates and incubated at 30°C for 2 days. Afterwards, clear spores were observed around the colonies.

[0163] A clear transparent ring appears.

[0164] Table 2 Results of the assessment of progesterone-promoting ability

[0165]

[0166] Note: - indicates negative, + indicates positive.

[0167] Morphological identification, molecular biological identification, physiological and biochemical identification, and growth-promoting ability testing confirmed that the growth-promoting and biocontrolling bacteria strain EMM919 isolated from the rhizosphere soil of Astragalus membranaceus was Bacillus halotolerans EMM919.

[0168] The beneficial effects of this invention are:

[0169] This invention relates to a novel salt-tolerant Bacillus halotolerans strain, EMM919. This strain exhibits excellent inhibitory effects against *Fusarium solani* EMF893, *Neurospora* EMF992, *Strombosporium robustum* EMF882, *Fusarium solani* EMF998, *Alternaria* EMF993, *Botrytis cinerea* EMF899, *Heterophyllum hexandrum* EMF990, *Pseudomonas stearans* EMF898, and *Fusarium graminearum* EMF897. It also demonstrates broad-spectrum inhibitory activity against various pathogenic fungi affecting crops and traditional Chinese medicinal herbs, including *Fusarium graminearum* causing wheat scab, *Alternaria* causing early blight of potato, *Fusarium solani* infecting *Astragalus membranaceus*, and *Strombosporium* harming ginseng. This strain possesses significant salt tolerance, maintaining growth activity and antibacterial function in high-salt environments, and adapting to different regional planting environments. In addition, this strain can synthesize indoleacetic acid, which promotes crop root development and nutrient accumulation. This makes strain EMM919 suitable for major crops such as wheat, corn, and tomatoes, as well as for disease control and growth promotion of traditional Chinese medicinal herbs such as astragalus and ginseng. It provides excellent germplasm resources for the development of widely applicable and multifunctional microbial agents.

[0170] The strain EMM919 can disrupt the hyphal and spore structure of plant pathogenic fungi, inhibit the hyphal growth and spore production of plant pathogenic fungi, and cause root rot caused by Fusarium hyphae and spores to vacuolate, shrink and disappear. It is especially effective for the microbial control of root rot of Astragalus membranaceus planted in saline-alkali soil.

[0171] Biological pesticides prepared from strain EMM919 solve the problem of secondary environmental damage caused by chemical pesticides in the control of fungal plant diseases. They are safe and environmentally friendly, do not induce drug resistance, and have no adverse effects on plant quality. They are conducive to pollution-free plant production and increased yield and can be promoted and applied as microbial preparations, pesticides or inoculants. Attached Figure Description

[0172] Figure 1 These are images showing the colony morphology, Gram staining, and scanning electron microscopy results of the salt-tolerant Bacillus strain EMM919 in LB medium.

[0173] Figure 2 This is a phylogenetic tree diagram of the 16S rRNA sequence of the salt-tolerant Bacillus strain EMM919.

[0174] Figure 3 This is a phylogenetic tree diagram of the gyrB sequence of the halophilic Bacillus strain EMM919.

[0175] Figure 4 This is a plate confrontation diagram of the salt-tolerant Bacillus strain EMM919 against Fusarium solani.

[0176] Figure 5This is a diagram showing the destructive effect of the halophilic Bacillus strain EMM919 on the mycelium of Fusarium rotundum.

[0177] Figure 6 This is a diagram showing the destructive effect of the halophilic Bacillus strain EMM919 on Fusarium solani spores. Detailed Implementation

[0178] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the present invention is not limited to the following embodiments.

[0179] Example 1

[0180] A halophilic Bacillus halotolerans strain, EMM919, was deposited on April 25, 2025, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025879, located at Wuhan University, Wuhan, China. Its nucleotide sequence consists of a 16S rRNA sequence and a gyrB sequence. The 16S rRNA sequence is shown in SEQ ID No. 1 of the sequence listing, and the gyrB sequence is shown in SEQ ID No. 2 of the sequence listing.

[0181] The 16S rRNA nucleotide sequence (SEQ ID NO:1) of strain EMM919 is as follows:

[0182] ctggggcgggcctaatacttgcaagtcgagcggacagatgggagcttgctccctgatgtt 60

[0183] agcggcggacgggtgagtaacacgtgggtaacctgcctgtaagactgggataactccggg 120

[0184] aaaccggggctaataccggatgcttgtttgaaccgcatggttcaaacataaaaggtggct 180

[0185] tcggctaccacttacagatggacccgcggcgcattagctagttggtgaggtaacggctca 240

[0186] ccaaggcaacgatgcgtagccgacctgagagggtgatcggccacactgggactgagacac 300

[0187] ggcccagactcctacgggaggcagcagtagggaatcttccgcaatggacgaaagtctgac 360

[0188] ggagcaacgccgcgtgagtgatgaaggttttcggatcgtaaagctctgttgttagggaag 420

[0189] aacaagtaccgttcgaatagggcggtaccttgacggtacctaaccagaaagccacggcta 480

[0190] actacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggaattattgggc 540

[0191] gtaaagggctcgcaggcggttccttaagtctgatgtgaaagccccgggctcaaccgggga 600

[0192] gggtcattggaaactggggaacttgagtgcagaagaggagagtggaattccacgtgtagc 660

[0193] ggtgaaatgcgtagagatgtggaggaacaccagtggcgaaggcgactctctggtctgtaa 720

[0194] ctgacgctgaggagcgaaagcgtggggagcgaacaggattagataccctggtagtccacg 780

[0195] ccgtaaacgatgagtgctaagtgttagggggtttccgccccttagtgctgcagctaacgc 840

[0196] attaagcactccgcctggggagtacggtcgcaagactgaaactcaaaggaattgacgggg 900

[0197] gcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccagg 960

[0198] tcttgacatcctctgacaatcctagagataggacgtccccttcgggggcagagtgacagg 1020

[0199] tggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcg 1080

[0200] caacccttgatcttagttgccagcattcagttgggcactctaaggtgactgccggtgaca 1140

[0201] aaccggaggaaggtggggatgacgtcaaatcatcatgccccttatgacctgggctacaca 1200

[0202] cgtgctacaatggacagaacaaagggcagcaaaaccgcgaggttaagccaatcccacaaa 1260

[0203] tctgttctcagttcggatcgcagtctgcaactcgactgcgtgaagctggaatcgctagta 1320

[0204] atcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcac 1380

[0205] accacgagagtttgtaacacccgaagtcggtgaggtaacctttatggagccagccgccga 1440

[0206] aggtggatccg 1451

[0207] The gyrB nucleotide sequence list of strain EMM919 (SEQ ID No. 2) is as follows:

[0208] cggacttgcagggtgtaggtgcctcggtcgttacgcgttatcaacagagcttgatgtgac 60 [[ID=II]]

[0209] tgttcaccgtgacggaaaaatccatcgccaagtctataaccgcggtatcccggtttctga 120<C

[0210] tctcgaggttattggcgaaacggatcataccggaacgactacacattttgttccagatcc 180

[0211] tgaaattttcacggaaacaactgagtatgaatatgatctgcttgctaaccgtgttcgtga 240

[0212] actagcctttttgacaaaaggcgtaaacatcacgattgaagataaacgtgaaggacaaga 300

[0213] acgcaaaaatgagtatcattacgaaggcggaataaaaagctatgtagagtatttaaaccg 360

[0214] ctccaaagaagttgtccatgaagagccgatttatattgaaggcgaaaaggacggcattac 420

[0215] ggttgaagtcgctctgcaatacaatgacggctacacaagcaatatttactcatttacaaa 480

[0216] caatatcaacacgtacgaaggcggtactcatgaagccggttttaaaacagggctgactcg 540

[0217] tgtcatcaatgattacgccagaaaaaaaggactcataaaagaaaatgatccaaacttgag 600

[0218] cggagatgatgtgagagaagggcttaccgcgattatctcgatcaaacacccggatccgca 660

[0219] gttcgaaggccaaacgaaaacaaaattaggcaactcagaggcacggactatcacagatac 720

[0220] gttattttctgcggcgttggaaacatttatgctggaaaatccagatgcggccagaaaaat 780

[0221] cgttgacaaaggtttaatggcagcaagagcaagaatggctgcgaaaaaagcacgtgaatt 840

[0222] aacgcgccgcaaaagcgctttggagatttcaaaccttcccggtaaattagcggactgctc 900

[0223] ttcgagagacccgagcatctccgagttatatatcgtagagggtgactctgccggaggatc 960

[0224] tgcaaaacaggggcgtgacagacatttccaggccattttgccgcttagaggtaaaatcct 1020

[0225] gaacgttgaaaaagcaaggcttgataaaattctttctaacaacgaagttcgttctatgat 1080

[0226] tactgcactcggcacaggcatcggggaagattcaactggaaaaagcccccaaatccc 1137

[0227] Use of salt-tolerant Bacillus strain EMM919 in the preparation of microbial pesticide EMF893 for controlling Fusarium rot.

[0228] The aforementioned microbial pesticide and formulation are prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier, and then preparing them into liquid or solid formulations using conventional methods. In the liquid formulation, the viable cell count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0229] The microbial pesticide and formulation in this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the salt-tolerant Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 4 × 10⁻⁶.9 CFU / g.

[0230] Its usage method is as follows:

[0231] When using the halophilic Bacillus strain EMM919 as a biopesticide, dilute it with water at a ratio of 1:20 to 40 and spray it evenly on the roots of crops to inhibit or kill Fusarium rotten bacterium EMF893. Alternatively, the biopesticide can be diluted with water at a ratio of 1:20 to 40 and allowed to enter the soil around the roots to inhibit or kill Fusarium rotten bacterium EMF893. The application rate of microbial pesticide is 2.5 kg per acre.

[0232] Example 2

[0233] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0234] Use of halophilic Bacillus strain EMM919 in the preparation of microbial pesticides for controlling fungal diseases caused by Neurospora EMF992.

[0235] The aforementioned microbial pesticide and formulation are prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier, and then preparing them into liquid or solid formulations using conventional methods. In the liquid formulation, the viable cell count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0236] The microbial pesticide and formulation in this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the salt-tolerant Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0237] Its usage method and dosage are the same as in Example 1.

[0238] Example 3

[0239] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0240] Use of salt-tolerant Bacillus strain EMM919 in the preparation of microbial pesticides for controlling fungal diseases caused by Sterculia lanceolata EMF882.

[0241] The aforementioned microbial pesticides and formulations are prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier, and then preparing them into liquid or solid formulations using conventional methods. In the liquid formulation of the microbial pesticide, the viable cell count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0242] The microbial pesticide and formulation of this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the halophilic Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0243] Its usage method and dosage are the same as in Example 1.

[0244] Example 4

[0245] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0246] Use of halophilic Bacillus strain EMM919 in the preparation of microbial pesticides for controlling fungal diseases caused by Fusarium moniliforme EMF998.

[0247] The aforementioned microbial pesticide and formulation are prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier, and then preparing them into liquid or solid formulations using conventional methods. In the liquid formulation, the viable cell count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0248] The microbial pesticide and formulation in this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the salt-tolerant Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶.9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0249] Its usage method and dosage are the same as in Example 1.

[0250] Example 5

[0251] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0252] Use of salt-tolerant Bacillus strain EMM919 in the preparation of microbial pesticides for controlling Alternaria alternata EMF993 fungal diseases.

[0253] The aforementioned microbial pesticide and formulation are prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier, and then preparing them into liquid or solid formulations using conventional methods. In the liquid formulation, the viable cell count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0254] The microbial pesticide and formulation in this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the salt-tolerant Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0255] Its usage method and dosage are the same as in Example 1.

[0256] Example 6

[0257] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0258] Use of salt-tolerant Bacillus strain EMM919 in the preparation of microbial pesticides for controlling fungal diseases caused by Staphylococcus aureus EMF899.

[0259] The aforementioned microbial pesticide and formulation are prepared as a liquid formulation of Bacillus halophilus strain EMM919 adsorbed on an agriculturally acceptable carrier, using conventional methods. The viable cell count of strain EMM919 in the liquid formulation is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0260] The microbial pesticide and formulation in this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the salt-tolerant Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0261] Its usage method and dosage are the same as in Example 1.

[0262] Example 7

[0263] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0264] Use of salt-tolerant Bacillus strain EMM919 in the preparation of microbial pesticides for controlling black rot fungus EMF990.

[0265] The aforementioned microbial pesticide is prepared from the halophilic Bacillus strain EMM919 in liquid or solid form using conventional methods. The halophilic Bacillus strain EMM919 is adsorbed onto an agriculturally acceptable carrier and prepared into liquid or solid formulations using conventional methods. In the liquid formulation, the viable cell count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g. Its usage and dosage are the same as in Example 1.

[0266] The microbial pesticide and formulation of this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the halophilic Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0267] Example 8

[0268] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0269] Use of salt-tolerant Bacillus strain EMM919 in the preparation of microbial pesticides for controlling fungal diseases caused by Pseudomonas thuringiensis EMF898.

[0270] The aforementioned microbial pesticides and formulations are prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier, and then preparing them into liquid or solid formulations using conventional methods. In the liquid microbial seed dressing, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0271] The microbial pesticide and formulation of this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the halophilic Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0272] Its usage method and dosage are the same as in Example 1.

[0273] Example 9

[0274] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0275] Use of halophilic Bacillus strain EMM919 in the preparation of microbial pesticides for controlling Fusarium graminearum EMF897 fungal diseases.

[0276] The aforementioned microbial pesticides and formulations are prepared in liquid or solid form using conventional methods. The microbial pesticides are formulated with an adsorbed salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier. In the liquid form of the microbial seed dressing agent, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form seed dressing, the viable count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0277] The microbial pesticide and formulation of this embodiment are prepared by conventional methods using an agriculturally acceptable carrier on which the halophilic Bacillus strain EMM919 is adsorbed, and in liquid or solid form. In the liquid form of the microbial pesticide, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

[0278] Its usage method and dosage are the same as in Example 1.

[0279] Example 10

[0280] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0281] In Examples 1-9 above, the use of the halophilic Bacillus strain EMM919 in the preparation of microbial pesticides for the prevention and control of fungal diseases is described. The fungus is any one of Fusarium rotundum EMF893, Neurospora EMF992, Sterculia robusta EMF882, Fusarium effusum EMF998, Alternaria alternata EMF993, Staphylococcus aureus EMF899, Heterophyllum hexandrum EMF990, Pseudomonas stoloniferum EMF898, and Fusarium graminearum EMF897.

[0282] The microbial pesticide of this embodiment is a liquid formulation prepared by conventional methods using an agriculturally acceptable carrier on which the salt-tolerant Bacillus strain EMM919 is adsorbed. The liquid formulation contains 2 × 10⁻⁶ viable bacteria of strain EMM919. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0283] In this embodiment, the live bacteria content of strain EMM919 in the liquid formulation microbial pesticide is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable count of strain EMM919 is 4 × 10⁻⁶ CFU / mL. 9 CFU / g.

[0284] Its usage method and dosage are the same as in Example 1.

[0285] Example 11

[0286] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0287] In Examples 1-9 above, the use of the halophilic Bacillus strain EMM919 in the preparation of microbial pesticides for the prevention and control of fungal diseases is described. The fungus is any one of Fusarium rotundum EMF893, Neurospora EMF992, Sterculia robusta EMF882, Fusarium effusum EMF998, Alternaria alternata EMF993, Staphylococcus aureus EMF899, Heterophyllum hexandrum EMF990, Pseudomonas stoloniferum EMF898, and Fusarium graminearum EMF897.

[0288] The microbial pesticide of this embodiment is an agriculturally acceptable carrier on which the salt-tolerant Bacillus strain EMM919 is adsorbed. It is prepared into liquid or solid formulations using conventional methods. In the liquid formulation, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

[0289] In this embodiment, the live bacteria content of strain EMM919 in the liquid formulation microbial pesticide is 5 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 8 × 10⁻⁶. 9 CFU / g. Note that the higher of these two values ​​should be taken.

[0290] Its usage method and dosage are the same as in Example 1.

[0291] Example 12

[0292] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0293] Use of salt-tolerant Bacillus strain EMM919 in the preparation of seed dressing agents.

[0294] The seed dressing agent is a liquid or solid microbial seed dressing agent prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier using conventional methods. In the liquid microbial seed dressing agent formulation, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial inoculants, the viable count of strain EMM919 was 2 × 10⁻⁶ CFU / mL. 9 CFU / g ~ 8×10 9 CFU / g.

[0295] In this embodiment, the liquid-form microbial inoculant contains 3 × 10⁻⁶ viable bacteria of strain EMM919. 9 CFU / mL; In the solid-form microbial inoculant, the viable count of strain EMM919 was 4 × 10⁻⁶ CFU / mL. 9 CFU / g.

[0296] Its usage method and dosage are the same as in Example 1.

[0297] Example 13

[0298] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0299] Use of salt-tolerant Bacillus strain EMM919 in the preparation of seed dressing agents.

[0300] The seed dressing agent is a liquid or solid microbial seed dressing agent prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier using conventional methods. In the liquid microbial seed dressing agent formulation, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial inoculants, the viable count of strain EMM919 was 2 × 10⁻⁶ CFU / mL. 9 CFU / g ~ 8×10 9 CFU / g.

[0301] In this embodiment, the liquid-form microbial inoculant contains 2 × 10⁻⁶ viable bacteria of strain EMM919. 9 CFU / mL; In the solid-form microbial inoculant, the viable count of strain EMM919 was 2×10⁻⁶ CFU / mL. 9 CFU / g.

[0302] Its usage method and dosage are the same as in Example 1.

[0303] Example 14

[0304] The sequence of the salt-tolerant Bacillus strain EMM919 is the same as that in Example 1.

[0305] Use of salt-tolerant Bacillus strain EMM919 in the preparation of seed dressing agents.

[0306] The seed dressing agent is a liquid or solid microbial seed dressing agent prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier using conventional methods. In the liquid microbial seed dressing agent formulation, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial inoculants, the viable count of strain EMM919 was 2 × 10⁻⁶ CFU / mL. 9 CFU / g ~ 8×10 9 CFU / g.

[0307] In this embodiment, the liquid-form microbial inoculant contains 5 × 10⁻⁶ viable bacteria of strain EMM919. 9 CFU / mL; In the solid-form microbial inoculant, the viable count of strain EMM919 was 8 × 10⁻⁶ CFU / mL. 9 CFU / g.

[0308] Its usage method and dosage are the same as in Example 1.

[0309] This invention relates to the application of a microbial inoculant in the agricultural field, specifically to using the microbial inoculant as a mixing agent.

[0310] Astragalus seedlings were used in cultivation to significantly improve the germination rate of Astragalus seeds and promote plant growth, especially the growth of the underground parts.

[0311] The following experiments determined the use of strain EMM919 in the preparation of seed dressing agents.

[0312] 1. Materials and Methods

[0313] 1.1 Test Materials

[0314] Test crop: Astragalus seeds.

[0315] Test inoculum: The microbial inoculum provided in this invention shall be diluted to a concentration of 1×10⁻⁶ before use. 7 CFU / mL (bacterial solution A) and 1×10 6 CFU / mL (bacterial solution B).

[0316] Control (CK): Equal volume of sterile water.

[0317] Cultivation substrate: Soil sourced from the Astragalus membranaceus planting base in Ningxia, sterilized at high temperature before use. Cultivation container: Standard plastic flower pot.

[0318] 1.2 Experimental Design This embodiment sets up 3 treatment groups:

[0319] Treatment group 1: Application concentration was 1×10 7 CFU / mL bacterial culture.

[0320] Treatment group 2: Application concentration was 1×10 6 CFU / mL bacterial culture.

[0321] Control group (CK): Administered an equal volume of sterile water.

[0322] Each processing group is repeated multiple times to ensure data reliability.

[0323] 1.3 Test Methods

[0324] Fill the bottom of each flowerpot with 90 grams of sterilized soil from the Ningxia Astragalus planting base.

[0325] Sow 12 Astragalus seeds evenly on the soil.

[0326] Cover the seeds evenly with 60 grams of the same sterilized soil.

[0327] The following processing is performed based on the grouping:

[0328] Treatment group 1: Apply 2 mL of bacterial solution A.

[0329] Treatment group 2: Apply 2 mL of bacterial solution B.

[0330] Control group (CK): 2 mL of sterile water was applied.

[0331] All flowerpots were cultivated under the same suitable environmental conditions, and observations and records were made regularly.

[0332] 1.4 Measurement Indicators After the test period ends, the following indicators shall be measured:

[0333] Germination rate: Count the number of germinated seeds in each pot and calculate the germination rate (%).

[0334] Plant growth: Carefully remove the plants, wash them with deionized water, and measure the length of the above-ground parts (plant height) and the length of the underground parts (root length). Results are expressed as multiples of the control group (CK).

[0335] 2. Experimental Results

[0336] The experimental results are shown in the table below:

[0337] Table 3. Germination results of Bacillus halophilus strain EMM919 treated with Astragalus membranaceus seed dressing agent

[0338]

[0339] 3. Results Analysis

[0340] As shown in the table above, compared with the control group CK which was treated with sterile water, the germination rate of Astragalus seeds and plant growth of the treatment group treated with the microbial agent of this invention were significantly improved.

[0341] In both concentrations, the concentration is 1×10 6 The bacterial suspension with CFU / mL (treatment group 2) showed better results. Its germination rate was 28.6% higher than that of the control group, and the length of the aboveground part and the length of the underground part were 1.31 times and 1.83 times that of the control group, respectively.

[0342] Of particular note is that the seed dressing agent treatment increased the growth-promoting effect on the underground roots of Astragalus membranaceus by 1.76-1.83 times, which is far superior to the growth-promoting effect on the above-ground parts by 1.17-1.31 times. This has extremely important economic value for Astragalus membranaceus, which is used as medicine for its roots.

[0343] 4. Conclusion

[0344] The results of this embodiment show that diluting the microbial agent provided by the present invention to a suitable concentration (e.g., 1×10⁻⁶) is effective. 6 When used as a seed dressing agent (CFU / mL), it effectively breaks the dormancy of Astragalus seeds, significantly improves the germination rate, and strongly promotes plant growth, especially the development of underground roots, thus laying a solid foundation for high-quality and high-yield Astragalus. This application method is simple, effective, and has good prospects for widespread application.

[0345] Instructions for use: Dissolve the liquid or solid inoculant (both containing 5 × 10⁻⁶ ppm) in water. 9 Mix seeds such as astragalus and soybeans with CFU / mL (g) at a ratio of 1:50-500.

[0346] To verify the beneficial effects of the halophilic Bacillus strain EMM919 in inhibiting or killing Fusarium solani EMF893, the inventors conducted numerous laboratory research experiments, the details of which are as follows:

[0347] 1. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on Fusarium solani EMF893

[0348] (1) Plate confrontation experiment of halophilic Bacillus EMM919 against Fusarium solani EMF893

[0349] The selected biocontrol bacteria were activated on LB medium at 37°C for 24 hours. A straight line passing through the center of a circle was drawn on the back of a 20 mL solid PDA plate using a ruler, and the center of the plate and the 1 / 4 and 1 / 2 points of the line were marked respectively. Fusarium rotundus was punched into a mycelial cake using an 8 mm punch and placed at 1 / 4 of the PDA medium. Strain EMM919 was inoculated at 1 / 2 of the plate using the streak method. Three plates were used as a control without inoculation with strain EMM919. The plates were incubated at 28°C for 7 days, and the antibacterial effects of the experimental and control groups were observed. Figure 4 As shown, the colony radii of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1):

[0350] η=(Rr) / R×100% (1).

[0351] Where R is the fungal colony radius of the control group and r is the fungal colony radius of the experimental group. The inhibition rate η is calculated to be 73.84% by formula (1).

[0352] (2) The inhibitory effect of salt-tolerant Bacillus strain EMM919 on Fusarium hyphae and spores of Fusarium solani.

[0353] 1) The inhibition of the growth of Fusarium solani mycelium by the salt-tolerant Bacillus strain EMM919.

[0354] The confrontation area between strain EMM919 and Fusarium solani EMF893 was observed using the slide method and examined under a light microscope. The results are as follows: Figure 5 As shown, where, Figure 5 a represents the control group, Fusarium rotundiformes EMF893 mycelium; Figure 5 bf represents the hyphae in the confrontation region between strain EMM919 and Fusarium solani EMF893. From Figure 5 As can be seen from a, the hyphae of the control group *Fusarium solani* EMF893 were full, with smooth edges, and the contents were clearly visible. From... Figure 5 As can be seen from bf, compared with the control group, the mycelia of Fusarium rotundus EMF893 treated with strain EMM919 showed shrinkage, cavitation and rupture.

[0355] 2) The inhibitory effect of the salt-tolerant Bacillus strain EMM919 on the growth of Fusarium solani spores.

[0356] The confrontation area between strain EMM919 and Fusarium solani EMF893 was observed using the slide method and examined under a light microscope. The results are as follows: Figure 6 As shown, where: Figure 6 a represents the control group spores of Fusarium solani EMF893. Figure 6 bf consists of spores of EMM919 and Fusarium solani EMF893. From Figure 6As can be seen from a, the spores of *Fusarium solani* EMF893 in the control group were plump, with smooth edges, and the contents were clearly visible. Compared with the control group (… Figure 6 a) Compared to strain EMM919, the spores of Fusarium rotundus EMF893 treated with strain EMM919 showed signs of shrinkage and rupture. Figure 6 bf).

[0357] 2. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on Neurospora EMF992

[0358] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against Neurospora EMF992 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1). The inhibition rate η was found to be 94.63%.

[0359] 3. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on robust columnar spores EMF882

[0360] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against robust Columnar Spores EMF882 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1), and the inhibition rate η was found to be 92.06%.

[0361] 4. The inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on Fusarium effusum EMF998.

[0362] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against Fusarium EMF998 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1). The inhibition rate η was 66.29%.

[0363] 5. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on Alternaria alterniflora EMF993

[0364] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against Alternaria alterniflora EMF993 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1), and the inhibition rate η was found to be 77.90%.

[0365] 6. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on Staphylococcus aureus EMF899

[0366] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against Staphylococcus aureus EMF899 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1), and the inhibition rate η was found to be 74.96%.

[0367] 7. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on black rot scabies EMF990

[0368] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against Black Rot Bacterium EMF990 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1), and the inhibition rate η was found to be 85.65%.

[0369] 8. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on Stem-like Pterygospermum EMF898

[0370] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against Stem-like Pterygospermia EMF898 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1), and the inhibition rate η was found to be 85.56%.

[0371] 9. Inhibitory or bactericidal effect of halophilic Bacillus strain EMM919 on Fusarium graminearum strain EMF897

[0372] The culture medium and experimental method used in the plate confrontation experiment of halophilic Bacillus EMM919 against Fusarium graminearum EMF897 were the same as in Experiment 1. The colony radius of the experimental group and the control group were measured with a ruler, and the inhibition rate η was calculated according to formula (1). The inhibition rate η was 69.35%.

[0373] 10. Effect of halophilic Bacillus strain EMM919 on the germination of Astragalus membranaceus.

[0374] The salt-tolerant Bacillus strain EMM919 liquid or solid bacterial agent of Example 1 of the present invention was diluted with water to a concentration of 1×10⁻⁶. 6 A CFU / mL seed dressing agent is used. Plant seeds are placed in a container, and the seed dressing agent is added to the container at a ratio of 1:50 to 500 by weight of the seed dressing agent before sowing. This seed dressing agent effectively breaks seed dormancy, increases germination rate, and promotes plant growth, especially prioritizing the development of underground root systems, laying a solid foundation for high-quality and high-yield plant production.

Claims

1. A strain of salt-tolerant Bacillus ( Bacillus halotolerans The strain EMM919 is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2025879 and deposit date of April 25, 2025. The nucleotide sequence of this strain includes the 16S rRNA gene sequence and the gyrB gene sequence. The 16S rRNA gene sequence is shown in SEQ ID NO:1, and the gyrB gene sequence is shown in SEQ ID NO:

2.

2. The use of the salt-tolerant Bacillus strain EMM919 of claim 1 in the preparation of microbial pesticides for the prevention and control of fungal diseases, wherein the fungus is any one of Fusarium solani EMF893, Neurospora EMF992, Sterculia salina EMF882, Fusarium solani EMF998, Alternaria EMF993, Staphylococcus aureus EMF899, Heterophyllum hexandrum EMF990, Pseudomonas stoloniferum EMF898, and Fusarium graminearum EMF897.

3. The use of the halophilic Bacillus strain EMM919 according to claim 2 in the preparation of microbial pesticides for controlling fungal diseases, characterized in that: The aforementioned microbial pesticide is an agriculturally acceptable carrier on which the halophilic Bacillus strain EMM919 is adsorbed. It is prepared into liquid or solid formulations using conventional methods. In the liquid formulation, the viable cell count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial pesticides and formulations, the viable cell count of strain EMM919 was 2 × 10⁻⁶. 9 CFU / g ~ 8×10 9 CFU / g.

4. The use of the salt-tolerant Bacillus strain EMM919 according to claim 2 or 3 in the preparation of microbial pesticides for controlling fungal diseases, characterized in that: The aforementioned microbial pesticide is an agriculturally acceptable carrier on which the halophilic Bacillus strain EMM919 is adsorbed. It is prepared into liquid or solid formulations using conventional methods. In the liquid formulation, the viable count of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial pesticides, the viable cell count of strain EMM919 was 4 × 10⁻⁶. 9 CFU / g.

5. The use of the halophilic Bacillus strain EMM919 of claim 1 in the preparation of seed dressing agents, characterized in that... The seed dressing formulation is prepared by adsorbing the salt-tolerant Bacillus strain EMM919 onto an agriculturally acceptable carrier and using conventional methods to produce a liquid or solid form of microbial seed dressing. In the liquid form of the microbial seed dressing, the viable count of strain EMM919 is 2 × 10⁻⁶. 9 CFU / mL ~5×10 9 CFU / mL; In solid-form microbial inoculants, the viable count of strain EMM919 was 2 × 10⁻⁶ CFU / mL. 9 CFU / g ~ 8×10 9 CFU / g.

6. The use of the halophilic Bacillus strain EMM919 according to claim 5 in the preparation of seed dressing agents, characterized in that: The seed dressing agent is a liquid or solid microbial seed dressing agent prepared by conventional methods, on an agriculturally acceptable carrier with adsorbed salt-tolerant Bacillus strain EMM919. In the liquid microbial seed dressing agent, the viable content of strain EMM919 is 3 × 10⁻⁶. 9 CFU / mL; In solid-form microbial inoculants, the viable count of strain EMM919 was 4 × 10⁻⁶ CFU / mL. 9 CFU / g.

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