A paenibacillus with straw degradation, broad-spectrum antibacterial and high-efficiency insecticidal activity and application

Abstract

This invention discloses a multifunctional Paenibacillus polymyxa with straw-degrading, broad-spectrum antibacterial, and highly effective insecticidal activities, and its applications, belonging to the field of agricultural microbiology. The strain provided by this invention is Paenibacillus polymyxa BMB-Pp0021, with the strain preservation number CCTCC NO.M20241951. This strain not only exhibits broad-spectrum antibacterial activity but also highly effective insecticidal properties, and simultaneously possesses the ability to efficiently degrade straw, especially under low-temperature conditions (7-day weight loss rate of 50.20%). These multifunctional characteristics give it enormous application potential in agriculture and industry.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology technology, specifically, it relates to a multifunctional polymyxa bacillus with straw degradation, broad-spectrum antibacterial and highly effective insecticidal activities and its applications. Background Technology

[0002] Cellulose is the world's largest biomass resource. Although cellulase was first discovered in fungi, bacteria possess unique cellulose-degrading mechanisms and high enzyme specificity compared to fungi. Cellulose-degrading bacteria have multiple enzyme systems working synergistically to provide more functions and efficiency. Furthermore, bacteria are easier to culture on low-cost media and are easier to genetically engineer than fungi. Simultaneously, bacteria can produce large amounts of secondary metabolites that antagonize plant pathogens and other plant hormones, thus promoting plant growth and achieving biological control. Straw is my country's largest agricultural residue, with annual production reaching 700 million tons, its main component being cellulose. However, my country's utilization of straw resources is insufficient, especially in the colder north, where low temperatures and other factors slow decomposition of straw in the soil, failing to achieve the desired results. The problems of pests and diseases, reduced grain yields, and decreased soil fertility caused by incomplete straw decomposition are significant and cannot be ignored. In order to enable straw to decompose quickly under low or normal temperature conditions, thereby enriching the soil without affecting the next season's planting, it is essential to apply a low-temperature, high-efficiency, and rapid straw decomposition agent while returning straw to the field.

[0003] Meanwhile, crop diseases and pests are one of the major agricultural disasters in my country, characterized by their wide variety, significant impact, and frequent outbreaks. As a major agricultural country, my country is severely affected by the scope and severity of crop diseases and pests, which significantly impact agricultural production. Common crop diseases and pests in my country include: cotton aphids, fungal diseases such as rice sheath blight, rice blast, and wheat scab, as well as bacterial diseases such as bacterial wilt, which have become major pests and diseases seriously affecting agricultural production in my country.

[0004] Polymyxa (Paenibacillus polymyxa) is widely used in agriculture and forestry due to the production of a series of plant growth hormones, polysaccharide-degrading enzymes, and antibacterial substances. It promotes plant growth, decomposes straw, controls plant diseases, and induces plant resistance. CN117286048A discloses a polymyxa strain X-32, whose culture products exhibit excellent cellulose degradation activity; filter paper strips are completely degraded into a paste after 60 hours; and after 21 days of culture, the degradation rate of rice straw reaches 48.26%. CN101712941A discloses a polymyxa strain ZJU0901, which can inhibit the growth of different biochemical morphologies of Ralstonia solanacearum under in vitro conditions, effectively controlling bacterial wilt in plants. CN106591203A discloses a polymyxin Bacillus KM2501-1 strain. The volatile substances produced by the solid-state fermentation of this strain have a good control effect on the plant pathogenic nematode Southern Root-knot Nematode and the animal pathogenic nematode Haemaphysalis contortus. The fermentation supernatant of this strain also has a good control effect on Southern Root-knot Nematode and various pathogenic fungi. At the same time, polymyxin Bacillus and its metabolites have a good growth-promoting effect on tomato plants, and its fermentation supernatant has a good inhibitory effect on the hatching of root-knot nematode eggs.

[0005] A search of existing technologies both domestically and internationally revealed no literature reports on multifunctional polymyxa bacilli that simultaneously possess straw degradation, broad-spectrum antibacterial, and highly effective insecticidal activities. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the first objective of the present invention is to provide a multifunctional polymyxa bacillus with straw degradation, broad-spectrum antibacterial and highly effective insecticidal activities and its applications.

[0007] To achieve the aforementioned technical objectives, the inventors isolated a strain of *Paenibacillus polymyxa* from a diseased rhizosphere soil sample from Xinglongbao Town, Xinmin City, Shenyang, Liaoning Province. Through multiple experiments, it was found that the fermentation supernatant of this strain possesses the ability to degrade cellulose under different temperature conditions, exhibiting excellent straw composting ability, particularly at 28℃ and 15℃. Furthermore, this strain can inhibit various bacterial and fungal pathogens. Simultaneously, its fermentation supernatant possesses highly effective biological activity against cotton aphids. Therefore, this strain was deposited at the China Center for Type Culture Collection (CCTCC) on September 10, 2024, and classified as *Paenibacillus polymyxa* BMB-Pp0021, with accession number CCTCCNO.M20241951, at Wuhan University, Luojia Mountain, Wuhan, Hubei Province, China.

[0008] It should be noted that the 16S rDNA nucleotide sequence of Paenibacillus polymyxa BMB-Pp0021 isolated in this invention is shown in SEQ ID NO.1. It exhibits the following morphological characteristics: after culturing on potato dextrose agar (PDA) medium at 28°C for 48-72 hours, the colonies are translucent, round, raised, with relatively intact edges, smooth surfaces, and adherent when picked up. Figure 1 Under a microscope, it appears rod-shaped. Figure 2 It can produce spores with strong resistance to adverse conditions.

[0009] Secondly, a second objective of the present invention is to provide an application of Paenibacillus polymyxa BMB-Pp0021, wherein the application is selected from any of the following:

[0010] (1) Application in inhibiting the growth of fungi and / or bacteria; wherein the fungi are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Fusarium pseudograss, Fusarium solani, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum cocovenenans, Pyrrosia lingua, Rhizoctonia solani; wherein the bacteria are selected from at least one of the following: Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter spp., Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas spp., Xanthomonas rubrum, Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia;

[0011] (2) Application in the prevention and control of fungal and / or bacterial plant diseases; wherein the pathogens of the fungal plant diseases are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Fusarium oxysporum pseudograss, Fusarium solani, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum cocovenenans, Pyridobacterium oryzae, Rhizoctonia solani; wherein the pathogens of the bacterial plant diseases are selected from at least one of the following: Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas rapa, Xanthomonas caesarea, Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia;

[0012] (3) Application in decomposed straw;

[0013] (4) Application in killing cotton aphids.

[0014] Furthermore, a third objective of this invention is to provide a fermentation broth or bacterial suspension containing the aforementioned Paenibacillus polymyxa BMB-Pp0021. This fermentation broth or bacterial suspension can be used to prepare straw composting agents, as well as biological agents or fertilizers that are toxic to fungal pathogens, bacterial pathogens, and cotton aphids in crops.

[0015] Finally, a fourth objective of this invention is to provide a cellulosic organic material composting agent, and simultaneously provide a microbial pesticide and fertilizer for preventing and controlling fungal and / or bacterial plant diseases, as well as an insecticide effective against cotton aphids; the cellulosic organic material composting agent is prepared using the fermentation broth or suspension of *Paenibacillus polymyxa* BMB-Pp0021 as the active ingredient. More preferably, the cellulosic organic material is straw; the microbial pesticide, fertilizer, or insecticide is prepared using the suspension, fermentation broth containing bacteria, or sterilized fermentation supernatant of *Paenibacillus polymyxa* BMB-Pp0021 as the active ingredient.

[0016] Compared with existing technologies, the *Paenibacillus polymyxa* BMB-Pp0021 provided by this invention not only has the ability to efficiently degrade straw, but also has broad-spectrum antibacterial activity and is highly effective in killing insects. These multifunctional characteristics give it great application potential in agriculture and industry, specifically in the following aspects:

[0017] (1) The supernatant of the sterile fermentation of the strain of the present invention has high cellulase activity. After fermentation on LB medium for 3 days, the cellulase activity can reach 41.07 U / ml and the total protein content is 0.155 mg / ml. When cultured on cellulose plates at 28℃, the cellulose hydrolysis zone is 20 mm. When cultured on cellulose plates at 4℃, the cellulose hydrolysis zone is 15 mm. This shows that the strain produces cellulase with strong ability and still has the biological activity of decomposing cellulose at low temperature.

[0018] (2) The supernatant of the sterile fermentation of the strain of the present invention after optimization of carbon source, nitrogen source and pH has an enzyme activity of up to 666.95 U / ml and a total protein content of 1.079 mg / ml, which is 15.24 times higher than that of the unoptimized medium; and the total protein content is 5.96 times higher on LB medium containing starch.

[0019] (3) The strain of the present invention was tested for straw decomposition at 28℃ and 15℃ respectively. It was inoculated into the treated wheat straw culture medium at an inoculation amount of 5%. After 7 days of culture, the weight loss rate of the polymyxa Bacillus BMB-Pp0021 after decomposition and degradation of wheat straw was 58.72% and 50.20% respectively.

[0020] (4) The strains of this invention have broad antibacterial capabilities, including plant fungal pathogens and bacterial pathogens. Among the fungi, Fusarium oxysporum, Codonopsis pilosula root rot, Fusarium oxysporum pseudograecum, Fusarium solani, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum cucumeris, Pyrrosia lingua, and Rhizoctonia solani are included. Among the bacteria, Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter spp., Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas tarda, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia.

[0021] (5) The supernatant of the sterilizing bacteria of the strain of the present invention has high insecticidal activity and the control efficiency against cotton aphids is over 90%.

[0022] (6) The supernatant of the sterile fermentation of the strain of the present invention has strong thermal stability. The results of the enzyme activity thermal stability test show that it retains high enzyme activity at 10℃-50℃.

[0023] (7) The fermentation supernatant produced by the strain of the present invention at low temperature still has the ability to antagonize a variety of bacterial pathogens, including Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas tarda, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia. Attached Figure Description

[0024] To more clearly describe the technical solution of the present invention, the accompanying drawings involved in the embodiments are briefly described below. At the same time, the listed drawings are only a partial presentation of the embodiments of the present invention. Furthermore, Figures 8-16 In the image, the left side represents the CK strain, and the right side represents the strain of this invention.

[0025] Figure 1 The colony morphology of Bacillus polymyxa BMB-Pp0021 described in this invention on PDA solid medium.

[0026] Figure 2 The cell morphology of Bacillus polymyxa BMB-Pp0021 described in this invention is shown under an optical microscope (eyepiece 10×, objective lens 100×).

[0027] Figure 3 This is a phylogenetic tree of the 16S rRNA of the Bacillus polymyxa strain BMB-Pp0021 described in this invention.

[0028] Figure 4 This image shows the hydrolysis zone test results of the *Bacillus polymyxa* BMB-Pp0021 described in this invention on a cellulose plate (Stained with Congo red).

[0029] Figure 5The image shows the appearance of wheat straw after 7 days of degradation by the Bacillus polymyxa BMB-Pp0021 of this invention; where A is CK and B is the strain of this invention.

[0030] Figure 6 This is a comparison of the weight loss rate of wheat straw after degradation by the polymyxa Bacillus BMB-Pp0021 described in this invention at 15℃ and 28℃ (after 7 days of culture).

[0031] Figure 7 The results of the thermal stability test of the enzyme activity of Bacillus polymyxa BMB-Pp0021 described in this invention are as follows.

[0032] Figure 8 This image shows the antagonistic effect of the polymyxa bacillus BMB-Pp0021 described in this invention against Fusarium oxysporum (the pathogen causing ginseng root rot).

[0033] Figure 9 This image shows the antagonistic effect of the polymyxa Bacillus BMB-Pp0021 described in this invention against the pathogen of root rot (Fusarium) in Codonopsis pilosula.

[0034] Figure 10 This image shows the antagonistic effect of the polymyxa bacillus BMB-Pp0021 described in this invention against Fusarium graminearum (wheat stem rot) pathogen.

[0035] Figure 11 This image shows the antagonistic effect of the polymyxa bacillus BMB-Pp0021 described in this invention against the Fusarium oxysporum Cuban-specific type (banana wilt) pathogen.

[0036] Figure 12 This image shows the antagonistic effect of the polymyxa bacillus BMB-Pp0021 described in this invention against the pathogen of *Pyrrosia lingua* (rice blast fungus).

[0037] Figure 13 This image shows the antagonistic effect of the polymyxa bacillus BMB-Pp0021 described in this invention against Rhizoctonia solani (potato black scurf) pathogen.

[0038] Figure 14 This image shows the antagonistic effect of the polymyxa bacillus BMB-Pp0021 described in this invention against Fusarium solani (garlic red rot) pathogen.

[0039] Figure 15 This image shows the antagonistic effect of the polymyxa Bacillus BMB-Pp0021 described in this invention against Fusarium graminearum (rice seedling blight) pathogen.

[0040] Figure 16 This is a diagram illustrating the antagonistic effect of the polymyxa bacillus BMB-Pp0021 described in this invention against the pathogen Botrytis cinerea (gray mold).

[0041] Figure 17 This image shows the inhibition zone effect of the fermentation supernatant produced by the polymyxa Bacillus strain BMB-Pp0021 of the present invention at low temperature on various bacterial pathogens; wherein, 1: fermentation supernatant of the strain of the present invention at low temperature; 2: control strain BMB Pp-0040; CK: control group with the same amount of sterile water.

[0042] Figure 18 This image shows the effect of the sterile fermentation supernatant produced by Bacillus polymyxa BMB-Pp0021 of the present invention on killing cotton aphids. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the experimental methods used are all conventional operating methods in the field of microbiology; the consumables and reagents used are all from commercial sources.

[0044] Example 1: Isolation, identification and preservation of Bacillus polymyxa BMB-Pp0021

[0045] The *Bacillus polymyxa* strain BMB-Pp0021 of this invention was isolated from a diseased rhizosphere soil sample from Xinglongbao Town, Xinmin City, Shenyang, Liaoning Province, provided by Wuhan Kenuo Biotechnology Co., Ltd. The collected soil sample was dried in a 50℃ oven and then ground to remove stones and other impurities before use. 1g of the completely dried soil sample was transferred to LB liquid medium containing sodium acetate (10ml / 250ml) and incubated at 28℃ and 220rpm for 4 hours. Then, it was heated in an 80℃ water bath for 30 minutes, shaking 3-5 times as needed during the water bath. After cooling to room temperature, 50µl of the suspension was spread onto PDA solid medium, with three replicates per group, and incubated upside down in a 28℃ incubator for 48-72 hours.

[0046] Based on the morphological characteristics of *Bacillus polymyxa* on PDA, *Bacillus polymyxa* was selected and re-streaked onto PDA solid medium. It was then incubated upside down at 28℃ for 48-72 hours. Single colonies were picked and incubated on 10ml / 30ml LB liquid medium for 24 hours. After centrifugation at 8000rpm for 10 minutes, the supernatant was collected and filtered through a 0.22µl sterile filter membrane. This yields the sterilized fermentation supernatant. .

[0047] A strain exhibiting a distinct hydrolysis zone on sodium cellulose plates was screened from purified strains and named *Paenibacillus polymyxa* BMB-Pp0021. The sodium cellulose screening medium used consisted of: CMC-Na (sodium carboxymethyl cellulose) 20.0 g / L, methyl dihydrogen phosphate 1.0 g / L, sodium chloride 10.0 g / L, ammonium sulfate 2.0 g / L, magnesium sulfate 0.5 g / L, agar 20.0 g / L, pH 7.2, and sterilized at 121℃ for 30 min. 7 mm holes were punched in sodium cellulose plates, and 100 μL of the sterilized fermentation supernatant of *Paenibacillus polymyxa* BMB-Pp0021 was added. The plates were incubated statically for 48 h (at 28℃ and 4℃, respectively). A control group was established using sterilized fermentation supernatant of *Paenibacillus polymyxa* under the same culture conditions. Then stain with 2% Congo red for 15 min, followed by elution with 1 mol / L NaCl, and measure the diameter of the hydrolysis zone. Figure 4 As shown, measurements revealed that under 28℃ culture conditions, the diameter of the hydrolysis zone of strain BMB-Pp0021 on cellulose plates reached 20 mm, while under 4℃ culture conditions, the diameter reached 15 mm. This represents an increase of 33.33% and 36.36% respectively compared to the hydrolysis zone produced by the sterile fermentation supernatant of the control *Bacillus polymyxa*.

[0048] Molecular biological identification of strain BMB-Pp0021 was performed. A 16S rRNA library was constructed from 394 species and 8 subspecies of *Bacillus*, and the sequencing results (e.g., BMB-Pp0021 ID NO.1) were obtained by PCR amplification. The 16S rRNA sequences obtained from sequencing were compared with the established 16S rRNA library of the type strains for sequence similarity analysis. A phylogenetic tree was constructed using the Neighbor-Joining method, as shown below. Figure 3 As shown. After sequence alignment, strain BMB-Pp0021 was identified as a Paenibacillus polymyxa strain. 750 μL of bacterial culture was mixed with 750 μL of 50% glycerol and stored at -80°C.

[0049] Example 2: Determination of enzyme activity in Bacillus polymyxa BMB-Pp0021 using the DNS reducing sugar method

[0050] 1. Determination of standard curve for reducing sugar concentration

[0051] 1.1) Prepare a glucose standard solution (1 mg / ml). Weigh 0.5 g of glucose and dissolve it in 500 ml of pure water for later use.

[0052] 1.2) Take a 15ml centrifuge tube and add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 ml of glucose standard solution respectively. Make up the volume to 1 ml with pure water, and then add 2 ml of DNS (dinitrosalicylic acid method). Repeat each group three times.

[0053] 1.3) Boil in water for 7 minutes, then quickly cool to room temperature with running water.

[0054] 1.4) Add water to the 15ml mark and measure the absorbance at a wavelength of 540nm.

[0055] 1.5) Use the average value to plot a standard curve. The standard curve for reducing sugar concentration is: y = 1.0773x - 0.0155, R0 2 =0.9991.

[0056] 2. Determination of cellulase activity in the supernatant of BMB-Pp0021 sterilized fermentation broth

[0057] 2.1) Inoculate bacteria from glycerol tubes onto PDA plates and incubate at 28°C with the plates inverted for 72 hours.

[0058] 2.2) Pick a single colony and put it into LB liquid medium (10ml / 30ml) to prepare seed culture. Incubate at 28℃ and 220rpm for 24h.

[0059] 2.3) Inoculate 2% seed culture onto the fermentation medium, with a volume of 50ml / 250ml, and incubate at 28℃ and 220rpm for 72h.

[0060] 2.4) Collect the fermentation broth, centrifuge at 8000 rpm and 4℃ for 10 minutes, and collect the supernatant.

[0061] 2.5) Sterilize with a 0.22µm sterile filter membrane and store at -20℃ for later use.

[0062] 2.6) Boil 1 ml of sterile supernatant (control group) in a boiling water bath for 10 min to completely inactivate it, and then cool it to room temperature with running water.

[0063] 2.7) Take 2 ml of 2% sodium carboxymethyl cellulose into a 15 ml centrifuge tube and incubate it together with 1 ml of sterile supernatant (experimental group and control group) in a 37℃ water bath for 5 min.

[0064] 2.8) After 5 minutes, add 1 ml of sterile supernatant (experimental group and control group) to 2 ml of sodium carboxymethyl cellulose and quickly place it in a 50°C water bath for 30 minutes.

[0065] 2.9) Stop the reaction by boiling in a water bath for 5 minutes, then quickly cool to room temperature with running water.

[0066] 2.10) Take 1 ml of the well-mixed saccharified solution, add 2 ml of DNS, and boil in a boiling water bath for 7 minutes. Quickly cool to room temperature under running water.

[0067] 2.11) Add water to the 15ml mark and measure the absorbance at OD540nm using a spectrophotometer (zero the control group).

[0068] 2.12) Calculate the reducing sugar content based on the standard curve, and then calculate the cellulase activity.

[0069] Cellulase activity unit is defined as follows: under these reaction conditions, 1 ml of original enzyme solution (referring to sterile fermentation supernatant) produces 1 μg of glucose in 1 min, which is defined as 1 enzyme activity unit (U), expressed as U / ml.

[0070] Calculate enzyme activity using the following formula: Where k is the dilution factor and m is the glucose content.

[0071] Tests showed that Bacillus polymyxa BMB-Pp0021 can secrete cellulase with an enzyme activity of 41.07 U / ml. In LB liquid medium with 2% starch added, the cellulase activity increased to 666.95 U / ml.

[0072] Example 3: Straw degradation test of Bacillus polymyxa BMB-Pp0021

[0073] This embodiment provides an experiment on the degradation of wheat straw by Bacillus polymyxa BMB-Pp0021. First, a straw culture medium was prepared: wheat straw was cut into 3-5 cm pieces, soaked overnight in a 2 mol / L NaOH solution, and then rinsed repeatedly with running water until neutral. It was dried at 60℃ to constant weight, and 5.0 g of the treated straw was placed in a 250 ml Erlenmeyer flask, 100 ml of pure water was added, and the mixture was sterilized at 121℃ for 30 min. The strain of this invention was fermented on LB medium containing starch for 72 h. A 5% inoculum was added to the wheat straw culture medium, and its ability to degrade straw at 28℃ and 15℃ was tested, with three replicates for each group. A 5% inoculum was added to the wheat straw culture medium as a blank control. The straw cutting and degradation effects were observed. Figure 5 As shown, it is obvious that the experimental group with the fermentation broth of the strain of this invention has a better degradation effect than the control group. The solution was passed through a 40-mesh sieve and gently rinsed with running pure water until the solution was clear. It was then dried in a 60°C oven to constant weight, and the weight loss rate was measured (the mass difference before and after drying in the CK blank control group can be considered as system loss and natural loss, with the constant weight of the blank control group after drying as the initial weight).

[0074] Calculate the weight loss rate: Weight loss rate = (CK drying constant weight - experimental group drying constant weight) / CK drying constant weight × 100%. The wheat straw degradation experiment results are as follows... Figure 6 As shown, at 15℃, the weight loss rate can reach as high as 50.20% after 7 days of cultivation. The weight loss rate of straw at 28℃ is 58.72%. This indicates that low temperature does not significantly reduce the ability of the strain of this invention to degrade straw, and it also has good straw decomposition ability.

[0075] Example 4: Thermal stability test of enzyme activity of Bacillus polymyxa BMB-Pp0021

[0076] This embodiment provides a thermostability test for enzyme activity in the sterile fermentation supernatant of *Bacillus polymyxa* BMB-Pp0021. 1 ml of sterile fermentation supernatant of *Bacillus polymyxa* BMB-Pp0021 (prepared in Example 1) was taken and incubated for 1 h in water baths at 0℃, 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, 70℃, 80℃, and 90℃, respectively. Then, under standard reaction conditions (50℃ for 30 min), the cellulase activity was determined using the DNS reducing sugar assay. The highest enzyme activity was taken as 100%, and the percentage of enzyme activity at other temperature conditions was calculated.

[0077] The results of the enzyme activity and thermostability test of Bacillus polymyxa BMB-Pp0021 are as follows: Figure 7 As shown, the supernatant from the sterilized fermentation still exhibited the highest enzyme activity stability and the highest enzyme activity after incubation in a 10℃ water bath for 1 hour. Conversely, the enzyme activity decreased most significantly after incubation in a 60℃ water bath for 1 hour. The results indicate that the strain of this invention retains high enzyme activity across a temperature range of 10℃-50℃.

[0078] Example 5: Test for antagonistic fungal pathogens by Bacillus polymyxa BMB-Pp0021

[0079] This embodiment provides a test for the antagonistic effect of Bacillus polymyxa BMB-Pp0021 bacterial suspension on fungal pathogens, including: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Fusarium pseudograss, Fusarium solani, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum cocovenenans, Rhizoctonia solani, and Rhizoctonia solani.

[0080] A single colony of *Bacillus polymyxa* BMB-Pp0021 of this invention was inoculated into LB liquid medium and cultured overnight at 28°C and 220 rpm to prepare fermentation broth. A 5 mm fungal disc was inoculated into the center of PDA solid medium. 5 μL of the fermentation broth of this invention was evenly inoculated on both sides of the fungus, with the two lines parallel and spaced 2-3 cm apart. A negative control was set up, with 5 μL of LB medium inoculated on both sides using the same method.

[0081] After streaking, allow the fermentation broth to dry in a sterile laminar flow hood, then seal the plates with sealing film. Incubate upside down in a 28°C incubator for 3-4 days. Observe and photograph the fungal growth in the experimental group compared to the control group.

[0082] The results of the antagonistic fungal pathogen test of Bacillus polymyxa BMB-Pp0021 bacterial suspension are as follows: Figures 8-16 As shown, it exhibits excellent antagonistic activity against all tested fungi, with a broad spectrum of inhibition. Therefore, *Bacillus polymyxa* BMB-Pp0021 has the potential to be used in the preparation of antifungal microbial pesticides.

[0083] Example 6: Test for antagonistic bacterial pathogens of Bacillus polymyxa BMB-Pp0021

[0084] This embodiment provides a test for antagonistic bacterial pathogens in the fermentation supernatant of Bacillus polymyxa BMB-Pp0021. The bacterial pathogens include: Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas tarda, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia.

[0085] Single colonies of *Bacillus polymyxa* BMB-Pp0021 (the present invention) and control *Bacillus polymyxa* were inoculated into LB liquid medium and cultured overnight at 28°C and 220 rpm to prepare fermentation broth. The fermentation broth was collected, centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was collected. The collected supernatant was sterilized using a 0.22 μm sterile filter membrane and stored at -20°C for later use.

[0086] Pick a single colony of the bacterial pathogen and incubate it overnight in LB broth. Add 500 μL of the pathogen suspension to 20 mL of LB broth before it solidifies, mix well, and pour into agar plates to cool and solidify. Use a 7 mm punch to make 4 wells on each agar plate containing the bacterial pathogen. Add 70 μL of sterile supernatant to each well. For the control group, add 70 μL of sterile water.

[0087] Place it in a 28℃ constant temperature incubator and incubate upright for 24 hours. Observe whether it has a transparent inhibition zone.

[0088] The antibacterial spectrum of Bacillus polymyxa BMB-Pp0021 is shown in Table 1. It showed inhibitory effects on all 25 tested bacterial pathogens. These experimental results indicate that Bacillus polymyxa BMB-Pp0021 of this invention has the potential to be developed into an antibacterial pathogenic microbial pesticide and fungicide.

[0089] Table 1 shows the antibacterial / insectic spectrum of Bacillus polymyxa BMB-Pp0021 against various bacterial pathogens.

[0090]

[0091] Example 7: Test of antagonistic bacterial pathogens in the fermentation supernatant of Bacillus polymyxa BMB-Pp0021 under low-temperature conditions

[0092] This embodiment provides a test for the antagonistic bacterial pathogens in the sterile supernatant produced by fermentation of Bacillus polymyxa BMB-Pp0021 under low-temperature conditions. The bacterial pathogens include: Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas tarda, Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia, etc.

[0093] Single colonies of *Bacillus polymyxa* BMB-Pp0021 (the present invention) and control *Bacillus polymyxa* were inoculated into LB liquid medium containing starch and cultured at 15°C, 200 rpm, for 72 h to prepare fermentation broth. The fermentation broth was collected, centrifuged at 8000 rpm, 4°C for 10 min, and the supernatant was collected. The collected supernatant was sterilized using a 0.22 μm sterile filter membrane and stored at -20°C for later use.

[0094] Single colonies of bacterial pathogens were picked and cultured overnight in LB broth. 500 μL of the pathogen suspension was added to 20 mL of LB broth before it solidified, mixed thoroughly, and poured into agar plates to cool and solidify. 10 μL of the fermentation supernatant was dropped onto the plate, aerated in a clean bench for 10 minutes, and then placed in a 28°C incubator for 24 hours. The presence of a clear inhibition zone was observed.

[0095] The results of the antagonistic bacterial pathogen test for Bacillus polymyxa BMB-Pp0021 are as follows: Figure 17 As shown, it exhibits inhibitory effects against all 14 tested bacterial pathogens. However, the control bacillus polymyxa showed no antibacterial activity. These experimental results demonstrate that the Bacillus polymyxa BMB-Pp0021 of this invention has the potential to be developed into a microbial bactericide applicable in low-temperature environments.

[0096] Example 8: Test of the toxicity of cotton aphids by fermentation supernatant of Bacillus polymyxa BMB-Pp0021

[0097] This embodiment provides a test of the toxicity of *Bacillus polymyxa* BMB-Pp0021 sterilized fermentation supernatant against cotton aphids. A liquid feed for cotton aphids was prepared and sterilized using a 0.45µm filter membrane. A glass double-ended tube and a 3cm×3cm sealing film were subjected to UV sterilization for at least 30 minutes. The sterilized fermentation supernatant of *Bacillus polymyxa* strain BMB-Pp0021 (prepared in Example 1) was mixed into the liquid feed at a volume ratio of 1:10 and thoroughly mixed. The mixed liquid feed was sealed in a double-layered membrane at one end of a glass double-ended tube (care should be taken not to break the membrane, otherwise it will cause feed contamination). Second-instar aphids were placed in this double-ended tube, 25-35 aphids per tube. The other end of the double-ended tube was sealed with a black cloth. Three replicates were performed for each aphid, and a blank control group (containing only liquid feed) was set up. The aphids were incubated upside down in an LED artificial climate chamber, and the aphids were counted and the mortality rate was calculated at 24h, 48h, 72h, and 96h.

[0098] The test results of Bacillus polymyxa BMB-Pp0021 for killing cotton aphids are as follows: Figure 18 As shown, the supernatant from the sterilization fermentation of Bacillus polymyxa BMB-Pp0021 in liquid feed exhibits a cotton aphid control efficacy of over 90%. This experimental result demonstrates that Bacillus polymyxa BMB-Pp0021 of this invention has the potential to be developed into a microbial insecticide.

Claims

1. A type of polymyxin Bacillus ( Paenibacillus polymyxa BMB-Pp0021, whose strain preservation number is CCTCC NO. M20241951.

2. The Paenibacillus polymyxa (BMB-Pp0021) according to claim 1, characterized by, Paenibacillus polymyxa ) BMB-Pp0021, characterized by, Its 16S rDNA nucleotide sequence is shown in SEQ ID NO.

1.

3. The polymyxa bacillus described in claim 1 ( Paenibacillus polymyxa The application of BMB-Pp0021 is selected from any of the following: (1) Application in inhibiting the growth of fungi and / or bacteria; wherein the fungi are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Fusarium oxysporum pseudograecum, Fusarium solani, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum cocovenenans, Pyrrosia lingua, Rhizoctonia solani; wherein the bacteria are selected from at least one of the following: Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter spp., Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas spp., Xanthomonas spp., Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia; (2) Application in the prevention and control of fungal and / or bacterial diseases of plants; wherein the pathogens of the fungal diseases are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Fusarium oxysporum pseudograss, Fusarium solani, Fusarium graminearum, Botrytis cinerea, Fusarium oxysporum cocovenenans, Pyridobacillus oryzae, Rhizoctonia solani; wherein the pathogens of the bacterial diseases are selected from at least one of the following: Ralstonia solanacearum, Ralstonia solanacearum, Enterobacter spp., Xanthomonas oryzae, Xanthomonas oryzae, Xanthomonas lancifolium, Xanthomonas lancifolium, Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia spp. (3) Application in composted straw; (4) Application in killing cotton aphids.

4. A type of *Bacillus polymyxa* containing the content of claim 1 (… Paenibacillus polymyxa Fermentation broth or bacterial suspension of BMB-Pp0021.

5. A straw composting agent, characterized in that, The straw composting agent contains the polymyxin Bacillus as described in claim 1 (… Paenibacillus polymyxa )BMB-Pp0021.

6. A straw composting agent, characterized in that, The straw composting agent is the *Bacillus polymyxa* strain described in claim 4. Paenibacillus polymyxa It is prepared using fermentation broth or bacterial suspension of BMB-Pp0021 as the active ingredient.

7. A microbial pesticide or fertilizer for controlling fungal and / or bacterial plant diseases, characterized in that, The microbial pesticide or fertilizer contains the polymyxa bacillus described in claim 1 (…). Paenibacillus polymyxa )BMB-Pp0021.

8. A microbial pesticide or fertilizer for controlling fungal and / or bacterial plant diseases, characterized in that, The microbial pesticide or fertilizer is based on the *Bacillus polymyxa* strain described in claim 1 (…). Paenibacillus polymyxa The active ingredient is prepared from the bacterial fermentation broth or sterile fermentation supernatant of BMB-Pp0021.

9. An insecticide effective against cotton aphids, characterized in that, The insecticide contains the polymyxin Bacillus as described in claim 1 (… Paenibacillus polymyxa )BMB-Pp0021.

10. An insecticide effective against cotton aphids, characterized in that, The insecticide uses the polymyxin Bacillus (Bacillus polymyxa) as described in claim 1. Paenibacillus polymyxa The active ingredient is prepared from the bacterial fermentation broth or sterile fermentation supernatant of BMB-Pp0021.

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