Paenibacillus polymyxa with straw degradation, broad-spectrum bacteriostasis and efficient insecticidal activity and application of paenibacillus polymyxa

Abstract

The invention discloses multifunctional paenibacillus polymyxa with straw degradation, broad-spectrum bacteriostasis and efficient insecticidal activity and application, and belongs to the technical field of agricultural microbiology. The strain provided by the invention is paenibacillus polymyxa BMB-Pp0021, and the preservation number of the strain of the paenibacillus polymyxa BMB-Pp0021 is CCTCC (China Center for Type Culture Collection) NO.M20241951; the bacterial strain not only has broad-spectrum antibacterial activity, but also can efficiently kill insects, also has the capability of efficiently degrading straws, and particularly still has the capability of efficiently degrading straws (the weight loss ratio in 7 days is 50.20%) under a low-temperature condition, so that the bacterial strain has huge application potential in agriculture and industry due to the multifunctional characteristics.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural microbiology, and in particular relates to a multifunctional Paenibacillus polymyxa with straw degradation, broad-spectrum antibacterial and high-efficiency insecticidal activities and an application thereof. Background Art

[0002] Cellulose is the world's largest biomass resource. Although cellulase was first discovered in fungi, bacteria have a unique cellulose decomposition mechanism and high enzyme specificity compared to fungi. Cellulose-degrading bacteria have multiple enzyme systems that work together to provide more functions and efficiency. In addition, bacteria are easier to culture on low-cost culture media than fungi and are easier to genetically engineer. At the same time, bacteria can also produce a large number of secondary metabolites and other plant hormones that antagonize plant pathogens to promote plant growth and biological control. Straw is the largest agricultural residue in my country, with an annual production of up to 700 million tons, and its main component is cellulose. However, my country's straw resources are not fully utilized, especially in the colder north. Due to the low temperature and other factors, the straw decomposes slowly after entering the soil and does not achieve the expected effect. Problems such as pests and diseases, reduced grain production, and decreased soil fertility caused by incomplete straw decomposition cannot be ignored. In order to make the straw decompose quickly under low temperature or normal temperature conditions, fertilize the soil without affecting the next season's farming, it is very necessary to apply low-temperature, high-efficiency and fast straw decomposition bacterial agents at the same time as returning the straw to the field.

[0003] At the same time, crop diseases and insect pests are one of the main agricultural disasters in my country. They are characterized by many types, great impact, and high frequency of outbreaks. my country is a major agricultural country, and the scope and severity of crop diseases and insect pests have seriously affected my country's agricultural production. The following types of diseases and insect pests are common in my country's crops: cotton aphids, fungal diseases such as rice sheath blight, rice blast, wheat scab, and bacterial diseases such as bacterial wilt, which have become major diseases and insect pests that seriously affect my country's agricultural production.

[0004] Paenibacillus polymyxa is widely used in agriculture and forestry because it produces a series of plant growth hormones, polysaccharide degrading enzymes and antibacterial substances. It has the functions of promoting plant growth, straw decomposition, preventing and controlling plant diseases and inducing plant resistance. CN117286048A discloses a Paenibacillus polymyxa X-32, the culture product of which has excellent degradation activity on cellulose. The filter paper strip has been completely degraded into a paste after 60 hours; after culturing for 21 days, the degradation rate of rice straw reaches 48.26%. CN101712941A discloses a Paenibacillus polymyxa ZJU0901, which can inhibit the growth of different biochemical strains of Ralstonia solanacearum under in vitro conditions and can effectively prevent and control plant bacterial wilt. CN106591203A discloses a Bacillus polymyxa KM2501-1, the volatile substances produced by the solid-state fermentation of the strain have a good control effect on the plant pathogenic nematode southern root-knot nematode and the animal pathogenic nematode Haemonchus contortus; the fermentation supernatant of the bacteria also has a good control effect on the southern root-knot nematode and various pathogenic fungi; at the same time, Bacillus polymyxa and its metabolites have a good growth-promoting property for tomato plants, and its fermentation supernatant has a good effect on inhibiting the hatching of root-knot nematode eggs.

[0005] By searching existing technologies at home and abroad, there is currently no literature report on a multifunctional Bacillus polymyxa that has straw degradation, broad-spectrum antibacterial and high-efficiency insecticidal activities. Summary of the invention

[0006] In view of the deficiencies in the prior art, the first object of the present invention is to provide a multifunctional Paenibacillus polymyxa with straw degradation, broad-spectrum antibacterial and efficient insecticidal activity and its application.

[0007] In order to achieve the above technical objectives, the inventors isolated a strain of Paenibacillus polymyxa from a diseased rhizosphere soil in Xinglongbao Town, Xinmin City, Shenyang City, Liaoning Province. After many experiments, it was found that the fermentation supernatant of the strain has the ability to degrade cellulose under different temperature conditions, especially at 28°C and 15°C. It has excellent straw composting ability. In addition, the strain can inhibit a variety of bacterial and fungal pathogens. At the same time, its fermentation supernatant has the biological activity of efficiently killing cotton aphids. Therefore, the strain was sent to the China Center for Type Culture Collection for preservation on September 10, 2024, and the classification name was Paenibacillus polymyxa BMB-Pp0021, the preservation number was CCTCCNO.M20241951, and the preservation address was Wuhan University, Luojia Mountain, Wuhan City, Hubei Province, China.

[0008] It should be noted that the nucleotide sequence of 16S rDNA of Paenibacillus polymyxa BMB-Pp0021 isolated by the present invention is shown in SEQ ID NO. 1. It has the following morphological characteristics: after being cultured on potato dextrose agar medium (PDA for short) at 28°C for 48-72 hours, the colonies appear translucent, round, and raised, with relatively complete edges and smooth surfaces, and are sticky when picked up ( Figure 1 ), rod-shaped under microscope ( Figure 2 ) can produce spores with stronger resistance to stress.

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

[0010] (1) Application in inhibiting the growth of fungi and / or bacteria; the fungi are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Pseudomonas graminearum, Fusarium solani, Gibberellic acid, Botrytis cinerea, Cuban variant of Fusarium oxysporum, Pyricularia oryzae, Rhizoctonia solani; the bacteria are selected from at least one of the following: Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas campestris, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia solani;

[0011] (2) Application in preventing and controlling plant fungal diseases and / or plant bacterial diseases; the pathogens of the plant fungal diseases are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Pseudomonas graminearum, Fusarium solani, Gibberellic acid, Botrytis cinerea, Cuban variant of Fusarium oxysporum, Pyricularia oryzae, and Rhizoctonia solani; the pathogens of the plant bacterial diseases are selected from at least one of the following: Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas campestris, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia solani;

[0012] (3) Application in composted straw;

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

[0014] In addition, the third object of the present invention is to provide a fermentation liquid or bacterial suspension containing the above-mentioned Paenibacillus polymyxa BMB-Pp0021. The fermentation liquid or bacterial suspension of the strain can be used to prepare a straw composting agent, and can also be used to prepare a biological agent or fertilizer that has a toxic effect on fungal pathogens of crops, bacterial pathogens and cotton aphids.

[0015] Finally, the fourth object of the present invention is to provide a cellulosic organic material decomposition agent, and at the same time provide a microbial pesticide and fertilizer for preventing and controlling plant fungal diseases and / or plant bacterial diseases, as well as an insecticide that has a toxic effect on cotton aphids; the cellulosic organic material decomposition agent is prepared using the fermentation liquid or bacterial suspension of the above-mentioned Paenibacillus polymyxa BMB-Pp0021 as an active ingredient. Further preferably, the cellulosic organic material is straw; the microbial pesticide, fertilizer or insecticide is prepared using the bacterial suspension, bacterial fermentation liquid or sterilized fermentation supernatant of the above-mentioned Paenibacillus polymyxa BMB-Pp0021 as an active ingredient.

[0016] Compared with the prior art, the Paenibacillus polymyxa BMB-Pp0021 provided by the present invention not only has the ability to efficiently degrade straw, but also has broad-spectrum antibacterial activity and can also kill insects efficiently. These multifunctional characteristics make it have great application potential in agriculture and industry, which is specifically reflected in the following aspects:

[0017] (1) The sterilized fermentation supernatant of the strain of the present invention has high cellulase activity. After fermentation on LB medium for 3 days, the cellulase activity can reach 41.07U / ml, and the total protein content is 0.155mg / ml. When cultured on a cellulose plate at 28°C, the cellulose hydrolysis zone is 20mm; when cultured on a cellulose plate at 4°C, the cellulose hydrolysis zone is 15mm. This indicates that the cellulase produced by the strain is relatively strong and still has the biological activity of decomposing cellulose at low temperatures.

[0018] (2) The enzyme activity of the sterilized fermentation supernatant of the strain of the present invention after optimization of carbon source, nitrogen source and pH can be as high as 666.95U / ml, and the total protein content is 1.079mg / ml, which is 15.24 times higher than that of the unoptimized culture medium; and the total protein content is increased by 5.96 times on the LB medium containing starch.

[0019] (3) The strain of the present invention was tested for straw maturity at 28°C and 15°C, and was inoculated into the treated wheat straw culture medium at an inoculum size of 5%. After 7 days of culture, the weight loss rates of the Bacillus polymyxa BMB-Pp0021 after wheat straw maturity degradation were 58.72% and 50.20%, respectively.

[0020] (4) The strain of the present invention has a wide range of antibacterial activities, including plant fungal pathogens and bacterial pathogens, among which the fungi include Fusarium oxysporum, Codonopsis pilosula root rot, Pseudomonas graminearum, Fusarium solani, Gibberellic acid, Botrytis cinerea, Cuban variant of Fusarium oxysporum, Pyricularia oryzae, and Rhizoctonia solani; the bacteria include Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas campestris, Xanthomonas oryzae, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia solani.

[0021] (5) The fermentation supernatant of the bacterial strain of the present invention has a highly effective insecticidal activity, and the control efficiency of cotton aphids is above 90%.

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

[0023] (7) The fermentation supernatant produced by the strain of the present invention at low temperature still has the ability to antagonize multiple bacterial pathogens, including Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas campestris, Xanthomonas oryzae, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly describe the technical solution of the present invention, the following briefly describes the drawings involved in the embodiments. At the same time, the drawings listed are only a presentation of part of the contents of the embodiments of the present invention. Figure 8-Figure 16 In the figure, the left side is CK and the right side is the strain of the present invention.

[0025] Figure 1 This is the colony morphology of the Paenibacillus polymyxa BMB-Pp0021 described in the present invention on the PDA solid culture medium.

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

[0027] Figure 3 This is the 16srRNA phylogenetic tree of the Paenibacillus polymyxa BMB-Pp0021 strain of the present invention.

[0028] Figure 4 This is a diagram showing the hydrolysis zone test effect of the Paenibacillus polymyxa BMB-Pp0021 described in the present invention on a cellulose plate (Congo red staining).

[0029] Figure 5This is the appearance of wheat straw after being degraded by the Bacillus polymyxa BMB-Pp0021 of the present invention for 7 days; wherein A is CK and B is the strain of the present invention.

[0030] Figure 6 This is a comparison chart of the weight loss rate of wheat straw after degradation by the Bacillus polymyxa BMB-Pp0021 of the present invention at 15°C and 28°C respectively (cultured for 7 days).

[0031] Figure 7 This is the thermal stability test result of the enzyme activity of Bacillus polymyxa BMB-Pp0021 described in the present invention.

[0032] Figure 8 This is a diagram showing the antagonistic effect of the Bacillus polymyxa BMB-Pp0021 described in the present invention on the pathogen Fusarium oxysporum (ginseng root rot).

[0033] Fig. 9 This is a diagram showing the antagonistic effect of the Paenibacillus polymyxa BMB-Pp0021 described in the present invention on the pathogen of Codonopsis pilosula root rot (Fusarium genus).

[0034] Fig.10 This is a diagram showing the antagonistic effect of the Bacillus polymyxa BMB-Pp0021 described in the present invention on the pathogen of Fusarium graminearum (wheat stem rot).

[0035] Fig.11 This is a diagram showing the antagonistic effect of the Bacillus polymyxa BMB-Pp0021 described in the present invention on the Cuban-specific type of Fusarium oxysporum (banana wilt) pathogen.

[0036] Fig.12 This is a diagram showing the antagonistic effect of the Bacillus polymyxa BMB-Pp0021 described in the present invention on the rice blast pathogen.

[0037] Fig.13 This is a diagram showing the antagonistic effect of the Paenibacillus polymyxa BMB-Pp0021 described in the present invention on the pathogen of Rhizoctonia solani (potato black mole disease).

[0038] Fig.14 This is a diagram showing the antagonistic effect of the Paenibacillus polymyxa BMB-Pp0021 described in the present invention on the pathogen Fusarium solani (garlic red rot).

[0039] Fig.15 This is a diagram showing the antagonistic effect of the Bacillus polymyxa BMB-Pp0021 described in the present invention on the pathogen Gibberella (rice seedling disease).

[0040] Fig.16 This is a diagram showing the antagonistic effect of the Paenibacillus polymyxa BMB-Pp0021 described in the present invention on the pathogen Botrytis cinerea (gray mold).

[0041] Fig.17 The diagram shows the inhibitory effect of the fermentation supernatant produced by the Bacillus polymyxa strain BMB-Pp0021 of the present invention under low temperature on a variety of bacterial pathogens; wherein, 1: the fermentation supernatant of the strain of the present invention under low temperature; 2: the control strain BMB Pp-0040; CK: the control group with the same amount of sterile water.

[0042] Fig.18 This is a diagram showing the effect of the sterile fermentation supernatant produced by the Bacillus polymyxa BMB-Pp0021 of the present invention on killing cotton aphids. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the following examples. Unless otherwise specified, the experimental methods used are conventional operating methods in the field of microbiology; the consumables and reagents used are all from commercial channels.

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

[0045] The strain of the present invention, Bacillus polymyxa BMB-Pp0021, is isolated from a diseased rhizosphere soil in Xinglongbao Town, Xinmin City, Shenyang City, Liaoning Province, and the soil is provided by Wuhan Kono Biotechnology Co., Ltd. The collected soil sample is placed in a 50°C oven for drying, and is ground to remove impurities such as stones for standby use. Take 1g of completely dried soil sample to LB liquid culture medium containing sodium acetate (liquid volume 10ml / 250ml), and shake the flask for 4h at 28°C and 220rpm. Then heat bath in a constant temperature water bath at 80°C for 30min, and shake 3-5 times appropriately during the water bath. Cool to room temperature, take 50ul of the suspension and apply it to PDA solid culture medium, repeat coating three plates in each group, and invert and culture in a constant temperature incubator at 28°C for 48-72h.

[0046] According to the morphological characteristics of Bacillus polymyxa on PDA, select Bacillus polymyxa and streak it on PDA solid medium again. Invert and culture it in a 28℃ constant temperature incubator for 48-72h. Pick a single colony and culture it on LB liquid medium with a liquid volume of 10ml / 30ml for 24h. Centrifuge at 8000rpm for 10min, take the supernatant, and filter it through a 0.22ul sterile filter membrane. The sterilized fermentation supernatant is obtained. .

[0047] The strain with obvious hydrolysis zone on the sodium cellulose plate was screened from the purified strains and named as Paenibacillus polymyxa BMB-Pp0021; the composition of the sodium cellulose primary screening plate medium used was: CMC-Na (sodium carboxymethyl cellulose) 20.0g / L, dihydrogen phosphate 1.0g / L, sodium chloride 10.0g / L, ammonium sulfate 2.0g / L, magnesium sulfate 0.5g / L, agar 20.0g / L, pH 7.2, sterilized at 121℃ for 30min. A 7mm hole puncher was used to punch holes on the sodium cellulose plate, 100ul of the above-mentioned Paenibacillus polymyxa BMB-Pp0021 sterilized fermentation supernatant was added, and static culture was carried out for 48h (at 28℃ and 4℃, respectively), and a control group was set as the control Paenibacillus polymyxa sterilized fermentation supernatant under the same culture conditions. Then stain with 2% Congo red for 15 min, then wash with 1 mol / L NaCl and measure the diameter of the hydrolysis zone. Figure 4 As shown in the results, it was determined that the diameter of the hydrolysis zone of the BMB-Pp0021 strain on the cellulose plate could reach 20 mm under 28°C culture conditions, and 15 mm under 4°C culture conditions, which were 33.33% and 36.36% higher than those of the control Bacillus polymyxa sterilized fermentation supernatant.

[0048] The strain BMB-Pp0021 was identified by molecular biology. The 16S rRNA library of 394 species and 8 subspecies of the model strains of the genus Paenibacillus was constructed, and the sequencing results were obtained by PCR amplification (such as: BMB-Pp0021 ID NO.1). The sequenced 16S rRNA sequence was compared with the established model strain 16S rRNA library for sequence similarity analysis, and the Neighbor-Joining method was used to construct a phylogenetic tree, as shown in Figure 1. Figure 3 As shown. After sequence alignment, strain BMB-Pp0021 is a strain of Paenibacillus polymyxa. Take 750ul of bacterial solution and 750ul of 50% glycerol, mix well, and store in a -80℃ ultra-low temperature refrigerator.

[0049] Example 2: Determination of enzyme activity of Paenibacillus polymyxa BMB-Pp0021 by DNS reducing sugar method

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

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

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

[0053] 1.3) Boil in water bath for 7 min, then cool rapidly to room temperature under running water.

[0054] 1.4) Fill the volume to 15 ml with water and measure the absorbance at 540 nm.

[0055] 1.5) Take the average value to make a standard curve. The standard curve of reducing sugar concentration is: y = 1.0773x-0.0155, R 2 =0.9991.

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

[0057] 2.1) Inoculate the bacteria from the glycerol tube onto a PDA plate and incubate the plate upside down at 28°C for 72 hours.

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

[0059] 2.3) Inoculate 2% of the seed solution into the fermentation medium, the volume is 50ml / 250ml, 28°C, 220rpm, and culture for 72h.

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

[0061] 2.5) Sterilize with a 0.22 um sterile filter membrane and store at -20°C for future use.

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

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

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

[0065] 2.9) Boil in a boiling water bath for 5 min to stop the reaction, then quickly cool to room temperature under running water.

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

[0067] 2.11) Fill the volume to 15 ml with water and measure the absorbance at OD540nm using a spectrophotometer (use the control group to adjust to zero).

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

[0069] The cellulase activity unit is defined as: under the reaction conditions, 1 ml of original enzyme solution (here refers to the sterilized fermentation supernatant) produces 1 ug of glucose in 1 min, which is positioned as 1 enzyme activity unit (U), expressed as U / ml.

[0070] The enzyme activity was calculated according to the following formula: Where k is the dilution factor and m is the glucose content.

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

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

[0073] This embodiment provides an experiment on the degradation of wheat straw by Bacillus polymyxa BMB-Pp0021. First, prepare a straw culture medium: cut the wheat straw into 3-5 cm, soak it in 2 mol / L NaOH solution overnight, take out the straw, and rinse it repeatedly with running water until it is neutral. Dry it at 60°C to constant weight, take 5.0g of the treated straw into a 250ml Erlenmeyer flask, add 100ml of pure water, and sterilize it at 121°C for 30min. The strain of the present invention was fermented and cultured on LB culture medium containing starch for 72h, and 5% of the inoculum was taken into the wheat straw culture medium, and its ability to degrade straw at 28°C and 15°C was tested respectively, with three replicates in each group. A 5% inoculum of the culture medium was added to the wheat straw culture medium as a blank control. Observe the straw cutting and degradation effects, such as Figure 5 As shown, it is obvious that the experimental group to which the fermentation broth of the strain of the present invention is added has a better degradation effect than the control group. Pass through a 40-mesh sieve and gently rinse with flowing pure water until it turns to a flowing color. Dry in a 60°C oven to constant weight, and measure the weight loss rate (the mass difference before and after the CK blank control group can be calculated as system loss and natural loss, and the constant weight of the blank control group after drying is 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 results of the wheat straw degradation experiment are as follows Figure 6 As shown, at 15°C, after 7 days of cultivation, the weight loss rate can be as high as 50.20%. At 28°C, the weight loss rate of the straw is 58.72%. This shows that the ability of the strain of the present invention to degrade straw is not significantly reduced under low temperature environment, and it also has good straw decomposition ability.

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

[0076] This example provides a test for the thermostability of enzyme activity of the sterilized fermentation supernatant of Paenibacillus polymyxa BMB-Pp0021. Take 1 ml of the sterilized fermentation supernatant of Paenibacillus polymyxa BMB-Pp0021 (prepared in Example 1) and incubate in a water bath at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 1 hour, and then determine its cellulase activity by the DNS reducing sugar test method under standard reaction conditions (50°C reaction for 30 minutes). Take the highest enzyme activity as 100%, and calculate the enzyme activity percentages under other temperature conditions.

[0077] The results of the thermal stability test of the enzyme activity of Paenibacillus polymyxa BMB-Pp0021 are as follows Figure 7 As shown, the sterilized fermentation supernatant still has the highest enzyme activity stability and the highest enzyme activity under the condition of incubation in a 10°C water bath for 1 hour. However, under the condition of incubation in a 60°C water bath for 1 hour, the enzyme activity decreased the most. The results show that the strain of the present invention retains a high enzyme activity at 10°C-50°C.

[0078] Example 5: Test on the Antagonism of Paenibacillus polymyxa BMB-Pp0021 against Fungal Pathogens

[0079] This embodiment provides a test on the antagonism of fungal pathogens by the suspension of Paenibacillus polymyxa BMB-Pp0021, wherein the fungi include: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Pseudomonas graminearum, Fusarium solani, Gibberellic acid, Botrytis cinerea, Cuban variant of Fusarium oxysporum, Oxysporium oryzae, and Rhizoctonia solani.

[0080] A single colony of the polymyxa bacillus BMB-Pp0021 of the present invention was inoculated into LB liquid culture medium and cultured overnight at 28°C and 220rpm to prepare a fermentation broth. A 5mm fungus cake was inoculated in the middle of a PDA solid culture medium. 5ul of the fermentation broth of the strain of the present invention was evenly inoculated on both sides of the fungus, with two lines parallel and 2-3cm apart. A negative control was set up, and 5ul of LB culture medium was inoculated on both sides in the same way.

[0081] After streaking, wait for the fermentation liquid to dry in a sterile clean bench, and then seal the plate with a sealing film. Incubate the plate upside down in a 28°C constant temperature incubator for 3-4 days. Observe the fungal growth of the experimental group compared with the control group and take photos for record.

[0082] The results of the test of the suspension of Bacillus polymyxa BMB-Pp0021 against fungal pathogens are as follows Figure 8-Figure 16 As shown in the results, it has excellent antagonism to all tested fungi and a wide range of antibacterial activity. Therefore, Paenibacillus polymyxa BMB-Pp0021 has the potential to be used in the preparation of antifungal microbial pesticides.

[0083] Example 6: Test on the Antagonism of Paenibacillus polymyxa BMB-Pp0021 against Bacterial Pathogens

[0084] This embodiment provides a test on the antagonism of the fermentation supernatant of Bacillus polymyxa BMB-Pp0021 against bacterial pathogens, wherein the bacterial pathogens include: Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas campestris, Xanthomonas oryzae, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia.

[0085] The single colonies of the polymyxa bacillus BMB-Pp0021 of the present invention and the control polymyxa bacillus were inoculated into LB liquid culture medium and cultured overnight at 28°C and 220rpm to prepare fermentation broth. The fermentation broth was collected, centrifuged at 8000rpm and 4°C for 10min, and the supernatant was taken. The collected supernatant was sterilized with a 0.22um sterile sterilization filter membrane and stored at -20°C for future use.

[0086] Pick a single bacterial pathogen colony and culture it in LB liquid medium overnight. Take 500ul of pathogen suspension and add it to 20ml LB medium that has not yet solidified, mix well, pour it into a plate and cool it to solidify. Use a 7mm puncher to punch 4 holes on the medium plate containing bacterial pathogens, take 70ul of sterilized supernatant and add it to the wells, and add 70ul of sterile water to the control group.

[0087] Place it in a constant temperature incubator at 28°C, culture it upright for 24 hours, and observe whether it has a transparent inhibition zone.

[0088] The antibacterial spectrum of Paenibacillus polymyxa BMB-Pp0021 is shown in Table 1, and it has an inhibitory effect on all 25 bacterial pathogens tested. This experimental result shows that Paenibacillus polymyxa BMB-Pp0021 of the present invention has the potential to be developed into an antibacterial pathogenic microbial pesticide and bactericide.

[0089] Table 1 shows the antibacterial / insecticidal spectrum of Paenibacillus polymyxa BMB-Pp0021 against various bacterial pathogens

[0090]

[0091] Example 7: Antagonistic bacterial pathogen test of the supernatant of Paenibacillus polymyxa BMB-Pp0021 fermentation under low temperature conditions

[0092] This example provides a test on the antagonism of the sterile supernatant produced by fermentation of Paenibacillus polymyxa BMB-Pp0021 under low temperature conditions against bacterial pathogens, wherein the bacterial pathogens include Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas campestris, Xanthomonas oryzae, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia, etc.

[0093] The single colonies of the polymyxa bacillus BMB-Pp0021 of the present invention and the control polymyxa bacillus were inoculated into LB liquid culture medium containing starch, and cultured at 15°C, 200 rpm, and 72 hours to prepare fermentation liquid. The fermentation liquid was collected, centrifuged at 8000 rpm, 4°C for 10 minutes, and the supernatant was taken. The collected supernatant was sterilized with a 0.22 um sterile sterilization filter membrane and stored at -20°C for future use.

[0094] Pick a single colony of bacterial pathogens and culture it in LB liquid medium overnight. Take 500ul of pathogen suspension and add it to 20ml LB medium that has not yet solidified, mix well, pour it into a plate and cool it to solidify. Take 10ul of fermentation supernatant and drop it on the plate, blow it in the clean bench for 10 minutes, place it in a 28℃ constant temperature incubator, culture it upright for 24 hours, and observe whether it has a transparent antibacterial zone.

[0095] The results of the test of Paenibacillus polymyxa BMB-Pp0021 against bacterial pathogens are as follows Fig.17 As shown, it has an inhibitory effect on all 14 strains of bacterial pathogens tested. However, the control Bacillus polymyxa did not show antibacterial activity. This experimental result shows that the Bacillus polymyxa BMB-Pp0021 of the present invention has the potential to be developed into a microbial bactericide that can be used in low temperature environments.

[0096] Example 8: Test on the toxicity of the fermentation supernatant of Paenibacillus polymyxa BMB-Pp0021 to cotton aphids

[0097] This embodiment provides a test on the poisoning of cotton aphids by the sterilized fermentation supernatant of Paenibacillus polymyxa BMB-Pp0021. Prepare liquid feed for cotton aphids and sterilize it with a 0.45um filter membrane. UV sterilize a glass two-way tube and a sealing film cut into 3cm×3cm for more than 30 minutes. Mix the sterilized fermentation supernatant of the strain Paenibacillus polymyxa BMB-Pp0021 of the present invention (prepared in Example 1) into the liquid feed at a volume ratio of 1:10 and mix well. Seal the mixed liquid feed in the double-layer membrane at one end of the glass two-way tube (be careful not to break the membrane, otherwise it will cause contamination of the feed). Put the second-instar aphids into this two-way tube, 25-35 per aphid, seal the other end of the two-way tube with black cloth, do 3 replicates for each, and set a blank control group (liquid feed only). Invert and culture in an LED artificial climate box, count and calculate the mortality rate at 24h, 48h, 72h, and 96h respectively.

[0098] The test results of Paenibacillus polymyxa BMB-Pp0021 killing cotton aphids are as follows Fig.18 As shown, the anti-aphid effect of the fermentation supernatant of Paenibacillus polymyxa BMB-Pp0021 in liquid feed can reach more than 90%. This experimental result shows that the Paenibacillus polymyxa BMB-Pp0021 of the present invention has the potential to be developed into a microbial insecticide.

Claims

1. A Paenibacillus polymyxa BMB-Pp0021, whose strain collection number is CCTCC NO.M20241951.

2. The Paenibacillus polymyxa BMB-Pp0021 according to claim 1, characterized in that The nucleotide sequence of its 16S rDNA is shown in SEQ ID NO.

1.

3. The use of Paenibacillus polymyxa BMB-Pp0021 according to claim 1, wherein the use is selected from any one of the following: (1) Application in inhibiting the growth of fungi and / or bacteria; the fungi are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Pseudomonas graminearum, Fusarium solani, Gibberellic acid, Botrytis cinerea, Cuban variant of Fusarium oxysporum, Pyricularia oryzae, Rhizoctonia solani; the bacteria are selected from at least one of the following: Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas campestris, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, Erwinia solani; (2) Application in preventing and controlling plant fungal diseases and / or plant bacterial diseases; the pathogens of the plant fungal diseases are selected from at least one of the following: Fusarium oxysporum, Codonopsis pilosula root rot pathogen, Pseudomonas graminearum, Fusarium solani, Gibberellic acid, Botrytis cinerea, Cuban variant of Fusarium oxysporum, Pyricularia oryzae, and Rhizoctonia solani; the pathogens of the plant bacterial diseases are selected from at least one of the following: Ralstonia solanacearum, Pseudomonas solanacearum, Enterobacter solanacearum, Xanthomonas oryzae, Xanthomonas campestris, Xanthomonas carpetii, Staphylococcus aureus, Salmonella, Escherichia coli, and Erwinia solani; (3) Application in composted straw; (4) Application in killing cotton aphids.

4. A fermentation broth or bacterial suspension containing the Paenibacillus polymyxa BMB-Pp0021 according to claim 1.

5. A cellulosic organic material decomposition agent, characterized in that: The decomposition agent contains the Paenibacillus polymyxa BMB-Pp0021 according to claim 1.

6. The cellulosic organic material decomposition agent according to claim 5, characterized in that: The invention is prepared by using the fermentation liquid or bacterial suspension of Paenibacillus polymyxa BMB-Pp0021 as the active ingredient; the cellulosic organic material is preferably straw.

7. A microbial pesticide or fertilizer for preventing and controlling plant fungal diseases and / or plant bacterial diseases, characterized in that: The microbial pesticide or fertilizer contains the Paenibacillus polymyxa BMB-Pp0021 according to claim 1 or / and its fermented metabolites.

8. The microbial pesticide or fertilizer for preventing and controlling plant fungal diseases and / or plant bacterial diseases according to claim 7, characterized in that: The active ingredient is prepared from the bacterial fermentation liquid or sterilized fermentation supernatant of Paenibacillus polymyxa BMB-Pp0021 as claimed in claim 1.

9. An insecticide having a toxic effect on cotton aphids, characterized in that: The pesticide contains the Paenibacillus polymyxa BMB-Pp0021 according to claim 1 or / and its fermented metabolites.

10. The insecticide according to claim 9, characterized in that The active ingredient is prepared from the bacterial fermentation liquid or sterilized fermentation supernatant of Paenibacillus polymyxa BMB-Pp0021 as claimed in claim 1.

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