Paenibacillus polymyxa and application of paenibacillus polymyxa in corn disease and pest control

Abstract

The invention belongs to the technical field of biological prevention and control of plant diseases and insect pests, and provides paenibacillus polymyxa and application thereof in prevention and control of corn diseases and insect pests. The paenibacillus polymyxa CGMCC No.27244 provided by the invention can effectively reduce the occurrence of the corn southern leaf blight, has a good control effect on the corn southern leaf blight, and can effectively reduce the use of chemical bactericides at the same time. After corn seeds are treated by the strain, the strain has no adverse effect on seedling emergence and plant growth and development. The strain can also prevent the spodoptera frugiperda from eating corn and reduce the spawning preference of the spodoptera frugiperda. The invention provides beneficial microbial resources for green prevention and control of corn southern leaf blight and spodoptera frugiperda, and has good application prospect and high application value.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for plant diseases and pests, and specifically relates to a polymyxa bacillus strain that can be used for the control of corn diseases and pests. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Corn leaf blight is caused by the fungal pathogen *Sclerotium chrysogenum* (Cercospora cornensis). Bipolaris maydis Caused by leaf blight, it is one of the most destructive leaf blights threatening global maize production and is widespread. Traditionally, the disease has been controlled through field fungicides and resistant crop varieties. Fungicides such as chlorothalonil, carbendazim, and thiophanate-methyl wettable powder have been proven effective in preventing the disease. However, the application of chemical fungicides can cause environmental pollution. Maize resistance to small leaf blight is a polygenic quantitative trait with a complex genetic mechanism, therefore, breeding varieties with durable and stable resistance is challenging.

[0004] fall armyworm Spodoptera frugiperda (Lepidoptera: Noctuidae) is a global lepidopteran crop pest that has developed resistance to most conventional pesticides. Biocontrol agents are receiving increasing attention due to their environmental friendliness. It has been reported that the endophytic bacterium *Bacillus amyloliquefaciens* can enhance the resistance of ornamental hosts to pests and diseases. Currently, reports on the ability of the same biocontrol strain to simultaneously resist pests and diseases on the same plant are very limited. Summary of the Invention

[0005] To address the lack of microbial agents for controlling corn leaf blight in existing technologies, this invention provides a strain of *Bacillus polymyxa* (…). Paenibacillus polymyxa ), can effectively antagonize Helicobacter pylori ( Bipolaris maydis ), used to prevent and control corn leaf spot disease.

[0006] Another object of the present invention is to provide a composition prepared from the above-mentioned Bacillus polymyxa that can be used for the prevention and control of corn diseases and pests.

[0007] This invention also provides the application of the above-mentioned polymyxa bacillus in the control of corn leaf spot and fall armyworm.

[0008] To achieve the above objectives, the present invention adopts the following technical solution.

[0009] A strain of polymyxin Bacillus ( Paenibacillus polymyxa (The accession number is CGMCC No. 27244).

[0010] The present invention also provides the fermentation product of the above-mentioned Bacillus polymyxa, wherein the fermentation product is the cell-free fermentation broth of the above-mentioned Bacillus polymyxa, the dried product of the cell-free fermentation broth, or the concentrate.

[0011] The aforementioned *Bacillus polymyxa* and / or fermentation products can be used to produce microbial inoculants, the active ingredient of which is at least one of the *Bacillus polymyxa* cells, spores, and fermentation products. For ease of use, the dosage form of the aforementioned microbial inoculants can be selected as liquid or solid formulations depending on the intended use and content. In addition to the active ingredients, the aforementioned microbial inoculants also include inert components, such as nutrients for *Bacillus polymyxa*, solvents for the fermentation products, adsorbents, stabilizers, and surfactants.

[0012] The preparation of the above-mentioned microbial agents can be carried out using methods commonly used in the field, such as culturing Bacillus polymyxa on a culture medium containing carbon and nitrogen sources to obtain fermentation broth; after solid-liquid separation of the fermentation broth, bacterial cells, spores and fermentation products are obtained.

[0013] The aforementioned polymyxa bacillus and its fermentation products, as well as microbial agents, can be used to control plant diseases and fall armyworm.

[0014] The plant disease is caused by Fusarium graminearum (… Fusarium graminearum Dendrobium nobile ( ) Epicoccum dendrobii Fusarium oxysporum ( Fusarium oxysporum ), Verticillium dahliae ( Verticillium dahliae ) or Corn spores ( Bipolaris maydis )cause.

[0015] The present invention has the following advantages: The *Bacillus polymyxa* CGMCC No. 27244 provided in this invention, when sprayed after corn emergence, effectively reduces the occurrence of corn leaf blight and provides good control over the disease. Treatment of corn seeds with this strain has no adverse effects on seedling emergence or plant growth and development. This strain also repels fall armyworm from feeding on corn and reduces its oviposition preference. This invention provides beneficial microbial resources for the green control of corn leaf blight and fall armyworm, and has good application prospects and high application value.

[0016] Biological Preservation Information Polymyxin Bacillus ( Paenibacillus polymyxa EP-4 was deposited on May 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 27244. Attached Figure Description

[0017] Figure 1 This section describes the growth of biocontrol bacterium EP-4 in confrontation with different pathogens; among them, a) EP-4 colony morphology; bf) EP-4 in confrontation with maize leaf spot pathogen, Fusarium graminearum, Fusarium oxysporum, Dendrobium nobile, and Verticillium dahliae. Figure 2 This is a phylogenetic tree constructed by EP-4 based on the 16S rDNA sequence; Figure 3 The effect of EP-4 on the mycelium of *Sclerotium cristatum*, the causal agent of corn leaf blight; a: control group; b: treatment group; Figure 4 The inhibitory effect of EP-4 metabolites on the growth of maize leaf spot pathogen; a: control group; b: treatment group; Figure 5 It is the inhibitory effect of EP-4 sterile filtrate on the germination of corn leaf blight spores; Figure 6 This is the EP-4 safety assessment of corn; Figure 7 This shows the control efficacy of EP-4 against maize leaf spot; A, the occurrence of maize leaf spot after EP-4 treatment, with the control group on the left and the treatment group on the right; B, the disease index of maize leaf spot after EP-4 treatment. Figure 8 The effect of EP-4 on the feeding selectivity of fall armyworm; A, feeding behavior of fall armyworm on the plate; B, statistical results of feeding behavior on the plate. Figure 9 The effects of EP-4 on the oviposition of fall armyworm are shown in Figure A, which shows the life cycle of fall armyworm in an insect rearing cage, and Figure B shows the number of eggs laid by fall armyworm on corn. Detailed Implementation

[0018] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0019] Example 1: Isolation, screening and identification of target strains 1. Isolation and screening of target bacteria Endophytic bacteria were isolated from cotton stems using a plant tissue isolation method. The specific steps are as follows: The cotton stems were first rinsed with sterile water, peeled, and then cut into 1 cm long segments. Surface sterilization was performed sequentially with 75% ethanol (immersion for 1 minute) and 10% sodium hypochlorite (immersion for 5 minutes). The sterilized stem segments were then cut into very small pieces (approximately 3 mm) using sterile scissors, placed in a mortar, and ground into a homogenate with 5 mL of sterile distilled water. After standing for 20 minutes, the supernatant was collected and subjected to 10... -1 -10 -4Serial dilutions were performed. 100 μL of each dilution was spread onto beef extract peptone agar (BPA) plates. To verify the surface sterilization effect, the final sterile rinse water was spread on the plates as a negative control. All plates were incubated at 30°C for 5 days. Single colonies with significantly different morphologies were picked and streaked onto fresh BPA plates for purification.

[0020] The antibacterial activity of endophytic bacteria was evaluated using the plate confrontation method. The specific steps are as follows: A perforator was used to extract bacteria from corn leaf blight pathogens (…). Bipolaris maydis Take a 5 mm diameter bacterial disc from the edge of the colony and place it in the center of a PDA plate. Inoculate a 5 mm diameter bacterial block of the test bacteria 3 cm away from the pathogenic bacterial disc. Each experiment was repeated in triplicate. Fusarium graminearum was inoculated using the same method. Fusarium graminearum Dendrobium nobile ( ) E. dendrobii Fusarium oxysporum ( Fusarium oxysporum ) and cotton Verticillium wilt ( Verticillium dahliae All plates were incubated at 25°C. The inhibition rate was calculated using the following formula: Antibacterial rate (%) = × 100%; in, R: Long radius of pathogenic bacterial colony (mm). r: Short radius of pathogenic bacterial colony (mm).

[0021] The bacteria with the best antagonistic activity against the aforementioned pathogens will be selected for further research.

[0022] Five biocontrol bacteria were screened from maize endophytic fungi using a confrontation culture method. Among them, bacterial strain EP-4 showed better antibacterial effect, exhibiting significant inhibitory activity against maize leaf spot pathogens, Fusarium graminearum, Fusarium oxysporum, Dendrobium nobile, and Verticillium dahliae. Figure 1 The mycelial growth inhibition rate of strain EP-4 exceeded 60%. B. maydis , F. graminearum , F. oxysporum , E. dendrobii and V. dahliae The inhibition rates were 85.29%, 75.00%, 81.25%, 61.90%, and 66.67%, respectively.

[0023] 2. Identification of EP-4 strains The EP-4 strain was inoculated onto LB agar plates, and its colony morphology (such as color, edge, and elevation) was observed. Genomic DNA was extracted from the EP-4 strain using a bacterial genomic DNA extraction kit (BioFlux). The 16S rDNA gene was amplified using universal primer 27F as the forward primer and 1492R as the reverse primer. The amplified products were separated by 1.5% agarose gel electrophoresis and purified using a commercial kit. The purified products were then sequenced. Based on the 16S rDNA gene sequence, a phylogenetic tree was constructed using the neighbor-joining method and MEGA 7.0.26 software.

[0024] Morphological observation showed that the colonies of strain EP-4 were white, wrinkled, and irregular. Based on 16S rRNA sequencing and phylogenetic analysis, EP-4 was identified as *Bacillus polymyxa* (…). Paenibacillus polymyxa () Figure 2 ).

[0025] Example 2: Antagonism of *Bacillus polymyxa* EP-4 against maize leaf spot disease 1. Effects of EP-4 treatment on mycelial morphology of *Sclerotium affine* in maize The confrontation culture method was used to evaluate the resistance of strain EP-4 to maize leaf spot pathogen ( ). B. maydis The influence of mycelial morphology. EP-4 mycelial blocks were compared with... B. maydis The bacterial colonies were inoculated onto PDA plates at 3 cm intervals and incubated at 25°C for 12 days. After incubation, the colonies closest to EP-4 were picked using a sterile toothpick. B. maydis The hyphae were placed under an optical microscope to observe changes in hyphal morphology.

[0026] The results are as follows Figure 3 As shown, EP-4 and B. maydis After co-culturing, the fungal hyphae showed signs of shrinkage and deformity.

[0027] 2. Inhibitory effect of EP-4 secondary metabolites on the growth of maize leaf spot pathogen. The antibacterial activity of EP-4 secondary metabolites was evaluated using the double-layer cellophane method. The specific steps were as follows: A double layer of sterile cellophane was laid on one side of a PDA plate. EP-4 bacterial colonies were inoculated into the center of the cellophane. The plate was incubated at 25°C for 3 days until the metabolites permeated into the culture medium. The cellophane and EP-4 colonies were carefully removed, and the area where EP-4 had grown was inoculated with *Sclerotium cristatum* (corn leaf spot pathogen). B. maydis (Inoculate the bacterial block on the other side of the plate) B. maydis The bacterial blocks served as a control group. All plates were incubated at 25°C for 3 days, with each treatment group replicated three times. The difference in colony growth between the treatment group and the control group was measured.

[0028] The results are as follows Figure 4 As shown, the metabolites of EP-4 significantly inhibitedB. maydis The growth of the organism was inhibited by 100%.

[0029] 3. Effects of EP-4 fermentation filtrate on spore germination of *Sclerotium affine* in maize. Corn leaf spot fungus ( B. maydis Inoculate onto sorghum culture medium and incubate at 25°C for 7 days. Once the mycelium has fully grown, pour off the culture medium and dry it. Gently rinse with sterile water to prepare a spore suspension (1×10⁻⁶). 6 CFU / mL). EP-4 was inoculated into LB liquid medium and cultured at 30℃ with shaking at 180 rpm for 2 days. The supernatant was collected by centrifugation at 8000 × g for 10 min and filtered through a 0.22 μm sterile filter to obtain cell-free fermentation filtrate. Experimental group: 400 μL spore suspension (1×10⁻⁶ CFU / mL). 6 (spores / mL) + 400 μL LEP-4 fermentation filtrate; control group: 400 μL spore suspension + 400 μL LB medium; cultured at 25℃ and 150 rpm for 3 h with shaking. Samples were taken at 0, 1, and 3 h, and spore germination was observed under a microscope. At least 100 spores were counted per replicate, and the germination rate and germination inhibition rate were calculated. Germination rate (%) = ×100%; Germination inhibition rate (%) = ×100%.

[0030] The results are as follows Figure 5 As shown, EP-4 metabolites strongly inhibited B. maydis The inhibition rates of spore germination were 62.59% and 64.10% after 1 hour and 3 hours, respectively.

[0031] Example 3: Effects of strain EP-4 on maize emergence and biomass Corn seeds were surface-sterilized with 1.05% sodium hypochlorite for 1 minute, then thoroughly rinsed with sterile water. The treatment group had their seeds soaked in an EP-4 spore suspension (1×10⁻⁶). 7 Seeds were soaked in sterile LB medium for 6 hours (CFU / mL), while control seeds were soaked in sterile LB medium. After aseptic drying, the seeds were planted in autoclaved vermiculite (30 seeds per pot, three replicates per treatment). Plants were grown in a controlled environment at 25°C with a 16 / 8 hour light / dark cycle for 12 days. Germination rate was recorded daily, and plant growth parameters, including plant height, root length, and fresh weight, were measured after 12 days.

[0032] The results are as follows Figure 6 As shown, treatment of maize seeds with strain EP-4 had no significant adverse effects on maize emergence, plant height, root length, or fresh weight.

[0033] Example 4: Control effect of strain EP-4 on maize leaf spot disease Corn seeds were sown in sterile vermiculite-filled plastic cups (7 cm in diameter), with 5 seeds per cup (6 cups per replicate), and 3 biological replicates per treatment. Five days after emergence, EP-4 spore suspension (1×10⁻⁶) was sprayed onto the leaves using a nebulizer at a rate of 2 mL / cup. 6 CFU / mL) or sterile LB medium (control). Two hours after treatment, all plants were... B.maydis Spore suspension (1×10 4 Infection was carried out using CFU / mL. To maintain high humidity for infection, plastic cups were covered with transparent polyethylene film for 24 hours. Five days after inoculation, the incidence of maize leaf spot was investigated, and the disease index was calculated.

[0034] The results are as follows Figure 7 As shown, when used as a foliar spray, EP-4 significantly reduced the incidence and disease index of corn leaf spot 5 days after pathogen treatment, compared with the control group.

[0035] Example 5: Effects of strain EP-4 on fall armyworm Maize seedlings were grown under controlled conditions (25±1℃, 70% relative humidity, 16 / 8 hour light / dark cycle) in sterile vermiculite-containing plastic cups (7 cm in diameter). Five days after emergence, EP-4 spore suspension (1×10⁻⁶) was added. 6 The EP-4 spore suspension (CFU / mL) was sprayed onto corn leaves using a handheld atomizer, 2 ml per plant. The control group used LB medium instead of EP-4 spore suspension. The following experiments were then conducted.

[0036] 1. Effects of EP-4 on the feeding of fall armyworm Ten minutes after spraying, uniform leaves (of equal area) were cut from the second fully expanded leaf and placed in sterile petri dishes (9 cm in diameter). Control leaves were placed in the left half, and treated leaves in the right half. Five third-instar fall armyworms were placed in each petri dish (30 petri dishes per replicate). Feeding activity was monitored for 6 hours under controlled conditions (25°C, 60% relative humidity), and the moths were divided into three groups (a, b, and c) based on leaf area consumption. Group A: Leaf loss area in the control group > Leaf loss area in the treatment group; Group b: Leaf area consumed in the control group < Leaf area consumed in the treatment group; Group C: Leaf area consumed in the control group = Leaf area consumed in the treatment group.

[0037] The results are as follows Figure 8As shown, after treating maize leaves with EP-4 spray, they were fed to fall armyworms, and the feeding area was compared. The results showed that group a had 15.7 petri dishes, group b had 7.0 petri dishes, and group c had 7.3 petri dishes. The number of petri dishes in group a was significantly higher than in groups b and c. After EP-4 treatment, the fall armyworm's feeding preference for maize was significantly reduced.

[0038] 2. Effects of EP-4 on oviposition of fall armyworm Ten minutes after spraying, corn seedlings were placed in insect cages, each cage containing one control group and one treatment group. Five pairs of fall armyworm adults were introduced into each cage. Four days later, the number of fall armyworm eggs in the control and treatment groups were recorded.

[0039] The results are as follows Figure 9 As shown, after spraying EP-4 on greenhouse corn leaves, the number of eggs laid by fall armyworm in the treatment group was significantly lower than that in the control group. This indicates that EP-4 can repel fall armyworm from laying eggs on corn.

[0040] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A strain of Polymyxin Bacillus ( Paenibacillus polymyxa (The accession number is CGMCC No. 27244).

2. The fermentation product of Paenibacillus polymyxa as claimed in claim 1, characterized in that, The fermentation product is a cell-free fermentation broth of Paenibacillus polymyxa, a dry product or a concentrated liquid of the cell-free fermentation broth.

3. Use of the Paenibacillus polymyxa of claim 1, the fermentation product of claim 2 in the production of a microbial inoculant and the microbial inoculant produced thereby.

4. Use according to claim 3, characterized in that, The active ingredient of the microbial inoculant is at least one of the cell mass, spores and fermentation product of Paenibacillus polymyxa.

5. Use according to claim 3, characterized in that, The microbial inoculant further comprises inert ingredients such as a nutrient agent for Paenibacillus polymyxa, a solvent for the fermentation product, an adsorbent, a stabilizer, a surfactant.

6. Use of the Paenibacillus polymyxa of claim 1, the fermentation product of claim 2, the microbial inoculant of any one of claims 3-5 in the control of plant diseases and Spodoptera frugiperda.

7. Use according to claim 6, characterized in that, The plant disease is caused by Fusarium graminearum Fusarium graminearum , Claviceps fusiformis Epicoccum dendrobii , Fusarium oxysporum Fusarium oxysporum , Verticillium dahliae Verticillium dahliae or Dictyosporium zeae Bipolaris maydis .

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