Paenibacillus polymyxa jx-1 and application thereof

Abstract

The application discloses Paenibacillus polymyxa JX-1 and application thereof. The Paenibacillus polymyxa JX-1 is preserved in the China General Microbiological Culture Collection Center on March 10, 2025, and the preservation number is CGMCC NO.33781. The Paenibacillus polymyxa JX-1 can significantly prevent and treat the influence of the infection of gnomoniella on a host, and promote the growth of a plant root system. The Paenibacillus polymyxa JX-1 can also significantly prevent and treat the occurrence of a large-sized white fruit disease of mulberry in a field experiment. The Paenibacillus polymyxa JX-1 can produce various extracellular enzymes and biological membranes, and the bacterial body itself and culture supernatant can antagonize various plant pathogenic bacteria including fusarium, sclerotinia, pectobacterium, pseudomonas, and cuphelia mori.

Description

Technical Field

[0001] This invention belongs to the field of biological pesticides and relates to Bacillus polymyxa JX-1 and its applications. Background Technology

[0002] Clubroot disease, caused by Plasmodiophora brassicae, is a global soil-borne disease affecting cruciferous crops. Its dormant spores can survive in the soil for over 20 years. It causes significant yield losses and severe economic damage. Because Plasmodiophora brassicae is an obligate live parasite and difficult to purify, genetic manipulation of the pathogen itself is impossible. Therefore, there are currently no effective control methods for clubroot disease, making the search for green control methods targeting Plasmodiophora brassicae an urgent priority.

[0003] A team from the Yunnan Academy of Agricultural Sciences discovered that azalomycin F3 / F4 significantly inhibits the germination of *Platycorrhizal* spores in rapeseed. Root irrigation treatment can achieve a contact system concentration of 0.5-6 ppm, with a control efficacy of over 70% against crops such as cabbage and Chinese cabbage. This technology obtains antibiotics through microbial fermentation, making it environmentally friendly, but it suffers from high costs for large-scale production (CN106719770A). A team from Huazhong Agricultural University isolated *Bacillus velezensis* strain F85 (accession number CCTCC M2019105) from the rhizosphere of healthy rapeseed. This strain inhibits *Platycorrhizal* spore activity by secreting active metabolites. In pot experiments, it achieved a comprehensive control efficacy of 83.4% against rapeseed clubroot disease and exhibited better adaptability to extreme soil environments than traditional chemical agents (CN110184208A).

[0004] Paenibacillus strains have attracted much attention due to their significant biological activity. These microorganisms can synthesize a variety of antibacterial substances, effectively inhibiting the growth of plant pathogens, while also enhancing plant systemic resistance, improving the rhizosphere microecological environment, and promoting crop growth and development. *Paenibacillus polymyxa* is listed as a species exempt from safety identification, leaving no chemical residues, and can be compounded with organic fertilizers and rooting agents. However, research on the control of clubroot disease in cruciferous plants using *Paenibacillus polymyxa* remains relatively scarce. Controlling clubroot is costly and has a limited function targeting only underground root diseases. Therefore, developing *Paenibacillus polymyxa* strains with broad-spectrum control capabilities, enabling them to simultaneously combat clubroot disease as well as bacterial and fungal diseases, has significant research value and application prospects. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing a polymyxa bacillus that can effectively prevent and control clubroot infection and antagonize various pathogens.

[0006] Another object of the present invention is to provide the application of this polymyxa bacillus.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] Paenibacillus polymyxa JX-1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 10, 2025, with accession number CGMCC NO.33781.

[0009] The bacterial agent prepared by Bacillus polymyxa JX-1 described in this invention.

[0010] The preparation method of the bacterial agent of the present invention involves inoculating a single colony of the polymyxa Bacillus JX-1 into fresh sterile liquid PDA medium and incubating it at 28-30°C and 200-220 rpm for 70-75 hours.

[0011] The application of the polymyxa bacillus JX-1 described in this invention in the prevention and control of clubroot infection in plants.

[0012] As a preferred embodiment of the present invention, the application of the polymyxa bacillus JX-1 in preventing and controlling the effects of clubroot infection on the host and promoting plant root growth.

[0013] The application of the polymyxa bacillus JX-1 described in this invention in the prevention and control of any one or more of Fusarium, Sclerotinia sclerotiorum, Pectinobacterium, Ralstonia solanacearum, and Pleurotus erythrorhizon.

[0014] The application of the polymyxa bacillus JX-1 described in this invention in the preparation of products for the prevention and control of any one or more of clubroot fungi, Fusarium, Sclerotinia sclerotiorum, Pectinobacterium, Ralstonia solanacearum, and Pleurotus erythrorhizon.

[0015] As a preferred embodiment of the present invention, the product is a biological pesticide.

[0016] The application of the microbial agent described in this invention in preventing and controlling clubroot infection in plants.

[0017] The application of the microbial agent described in this invention in the prevention and control of any one or more of Fusarium, Sclerotinia sclerotiorum, Pectinobacillus, Ralstonia solanacearum, and Pleurotus erythrorhizon.

[0018] The application of the aforementioned Bacillus polymyxa JX-1 and the aforementioned bacterial agent in the prevention and control of clubroot disease or mulberry fruit disease.

[0019] The application of the aforementioned Bacillus polymyxa JX-1 and the aforementioned bacterial agent in the preparation of biological pesticides for the prevention and control of clubroot disease or mulberry fruit disease.

[0020] Beneficial effects:

[0021] The *Bacillus polymyxa* JX-1 strain of this invention can significantly prevent and control the effects of clubroot infection on the host plant, and promote plant root growth. It can produce various extracellular enzymes and biofilms, and the bacterial cells themselves and the culture supernatant can antagonize various plant pathogens, including *Fusarium*, *Sclerotinia sclerotiorum*, *Pectinobacterium*, and *Ralstonia solanacearum*. In field experiments, it can effectively control sclerotinia stem rot in mulberry fruit.

[0022] The *Bacillus polymyxa* JX-1 of this invention is simple to use. In pot experiments on clubroot disease, a single root drenching after shaking culture of the bacterial solution effectively prevented the impact of clubroot infection on plant growth. In field experiments targeting large-scale mulberry fruit (mulberry bark), two applications with a 15-day interval were sufficient to effectively control the occurrence of clubroot. Currently, as a biocontrol agent, field experiments generally require more than three applications, while this invention only requires two applications with a 15-day interval. In conclusion, the *Bacillus polymyxa* JX-1 of this invention can be used as a biological pesticide. Attached Figure Description

[0023] Figure 1 Morphology of Polymyxobacterium JX-1 (A), 16S phylogenetic tree analysis (B), and electron microscopy observation of flagellar negative staining (C).

[0024] Figure 2 Detection of biomembranes and extracellular enzymes in JX-1

[0025] Figure 3 JX-1 antagonistic effect against clubroot bacteria in Arabidopsis thaliana live potted plants

[0026] Figure 4 Detection of JX-1 antagonism against clubroot bacteria in live potted cabbage

[0027] Figure 5 JX-1 supernatant, volatile organic compounds (VOCs), and antagonistic effects of bacterial and fungal strains

[0028] Figure 6 Statistics on the number of diseased fruits 15 days after applying JX-1 bacterial solution in the field

[0029] Information on the preservation of biological materials

[0030] JX-1, classified as Paenibacillus polymyxa, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is March 10, 2025, and the accession number is CGMCC NO.33781. Detailed Implementation

[0031] Example 1 JX-1 was obtained by isolating it from eggplant stems using the following method:

[0032] Cut the stems of eggplants with mild sclerotinia disease into small sections, soak them in 75% alcohol for disinfection, and rinse them twice with sterile water. Chop them in sterile water, dip an inoculation loop in the solution, streak the loop, and incubate at 28 degrees Celsius.

[0033] like Figure 1 As shown in Figure A, JX-1 appears as opaque white, circular colonies on PDA plates; it can grow on LB, YPDA, and PDA plates, but grows best on PDA plates; the optimal growth temperature is 28 degrees Celsius; it has peritrichous flagella. Based on morphological observation and 16S sequencing comparison analysis with NCBI, JX-1 was identified as Paenibacillus polymyxa.

[0034] Example 2 Preparation of bacterial agent

[0035] Inoculate a single colony of JX-1 into fresh, sterile liquid PDA and incubate at 28°C and 220 rpm for 72 hours. The resulting fresh bacterial solution is used as the inoculum.

[0036] Example 3 JX-1 can produce a large number of extracellular enzymes and biomembranes.

[0037] 1) The pectinase activity was detected according to the method established by Barnard et al. (Barnard et al., 2010). JX-1 bacterial culture was prepared according to Example 2, and sterile water was adjusted to OD0.05. 600 =1.0; Take 2 μL of the cultured bacterial solution to be tested and drop it onto the surface of the prepared pectinase detection plate, and incubate at 28℃ for 48 h; stain with 7.5% (w / v) copper acetate solution for 1 h, observe the double-layered milky white degradation zone under the blue background, take a picture and measure the diameter of the degradation zone, and repeat the experiment three times.

[0038] 2) Cellulase activity was detected according to Barnard's method (Barnard et al., 2010). JX-1 bacterial culture was prepared according to Example 2, and adjusted to OD200 with sterile water. 600 =1.0; Take 2 μl of the bacterial suspension of the test strain 28 and drop it onto the surface of the cellulase detection plate, and incubate at 28℃ for 48 h; stain with 0.2% (w / v) Congo red for 20 min. Observe the degradation zone, take a picture and measure the diameter of the degradation zone. Repeat the experiment three times. 3) The detection method of protease was carried out according to the method proposed by Barnard et al. (Barnard et al., 2010). Prepare the JX-1 bacterial suspension according to Example 2, and adjust the OD value with sterile water to 1.0. 600 =1.0; Take 2 μl of the bacterial culture of the test strain and drop it onto the surface of the protease detection plate, incubate at 28℃ for 48 h, observe the size of the transparent degradation zone, take a picture and measure the diameter of the degradation zone, and repeat the experiment three times.

[0039] 4) The specific steps for determining biofilm formation capacity were performed according to the literature (O'Toole and Kolter, 1998). JX-1 bacterial culture was prepared according to Example 2, and sterile water was adjusted to OD0.05. 600 =1.0; Pipette 4.0 ml of bacterial culture into a sterile test tube, place the test tube at 28℃ and incubate until OD600 = 2.0; Gently remove the bacterial culture from the test tube, add 4.0 mL of 0.1% crystal violet solution, stain for 30 min, discard the staining solution, wash three times with sterile water, and place the washed test tube in a 37℃ incubator for 2 h; Observe the formation of biofilm and take pictures, and finally dissolve the purple substance on the test tube wall with 1 ml of elution buffer (40% methanol, 10% glacial acetic acid), and use a spectrophotometer to detect the absorbance value of the solution at 575 nm. Each group of experiments is repeated three times.

[0040] The results showed that JX-1 can produce extracellular enzymes such as proteases, pectinases, and cellulases, and these extracellular enzymes are crucial for antagonism; it can also form a large number of biofilms, which are also crucial for antagonism.

[0041] Example 4: Pot Experiment of Chinese Cabbage and Arabidopsis

[0042] Experimental group (only biocontrol bacteria JX-1 applied): The JX-1 bacterial fermentation broth prepared in Example 2 was applied to the roots once, with 1 mL applied to each cruciferous plant. 20 plants were treated in a replicate. No additional bacterial solution was required during the treatment period. The treatment lasted for 3 weeks.

[0043] Experimental group (treated with biocontrol bacteria JX-1 and clubroot bacteria Pb4): The bacterial fermentation broth of JX-1 prepared in Example 2 was used for root irrigation once, with 1 mL per cruciferous plant, and 20 plants were irrigated. 24 hours after JX-1 application, 1 mL of purified clubroot bacteria Pb4 spores (concentration 1 x 10⁻⁶) was applied to each plant. 8 mL / CFU. No additional JX-1 bacterial suspension is required during the treatment, which lasts for 3 weeks.

[0044] Control group (treated with clubroot fungus Pb4): The clubroot tissue of the plants was ground, filtered, and collected to prepare a spore suspension with a concentration of 1×10^8. The prepared clubroot fungus spore suspension was used to drench the roots once, with 1 mL per cruciferous plant, and repeated for 20 plants. No additional fungal solution was required during the treatment period, and the treatment lasted for 3 weeks.

[0045] Control group (treated with sterile PDA medium): The roots of each cruciferous plant were drenched once with sterile PDA liquid medium, with a drenching volume of 1 mL per plant, and 20 plants were replicated. No additional medium was required during the treatment period, and the treatment lasted for 3 weeks.

[0046] Three weeks after all treatments, the roots were removed for inspection and photographic recording. The fresh weight of the plants was also recorded for analysis.

[0047] Depend on Figure 3 and Figure 4 It can be seen that after treatment with biocontrol bacterium JX-1, both Arabidopsis thaliana and Chinese cabbage exhibited increased fresh weight, reduced clubroot size, and improved root growth. In conclusion, JX-1 has significant antibacterial and growth-promoting effects, and can significantly reduce the damage caused by clubroot disease.

[0048] Example 5: Antagonism of JX-1 supernatant against Pectinobacterium and Ralstonia solanacearum

[0049] Here, *Pectobacterium carotovorum* subsp. *carotovorum* is specifically referred to as *Pectobacterium carotovorum*. *Ralstonia solanacearum* is specifically referred to as *Ralstonia solanacearum*. The fresh bacterial culture prepared according to the method in Example 2 was incubated at 6000 rpm for 3 minutes, and the supernatant was collected. The *Pectobacterium carotovorum* and *Ralstonia solanacearum* were adjusted to OD using water. 600 =1.0, then evenly spread it on the dried PDA medium surface, removing excess bacterial solution. Make a hole in the center of the medium using a punch, and take 10 μL of the JX-1 supernatant after culture into the hole. Incubate at 28°C for 48 hours, observing for the appearance of inhibition zones. Results are as follows. Figure 5 As shown in Figure A, the supernatant of JX-1 contains antibacterial substances that can significantly inhibit the growth of Ralstonia solanacearum and Pectinobacterium.

[0050] Example 6: Plate antagonism of JX-1 against Fusarium and Sclerotinia.

[0051] The inhibition rate of JX-1 against Fusarium and Sclerotinia sclerotiorum was determined using the plate confrontation method. Fusarium here refers specifically to *Fusarium fujikuroi*, and Sclerotinia sclerotiorum. The experimental method is as follows: a 5 mm diameter fungal cake or a 1 mm diameter sclerotium was inoculated in the center of a PDA plate, and simultaneously 5 μL of OD was inoculated 3.5 cm from the center of the plate. 600 The test strain (number 1) was used as a control, with no antagonistic strain inoculated, and cultured at 28℃ for 5 days. Each treatment was repeated three times. The experimental results are as follows: Figure 5 As shown in DG, JX-1 can significantly antagonize Fusarium and Sclerotinia.

[0052] Example 7JX-1 Field experiment on the control of large white fruit of mulberry trees.

[0053] Mulberry malformation with large, white fruit is a common disease that severely damages mulberries. In this field experiment, the pathogen of mulberry malformation with large, white fruit was identified as *Ciboria shiraiana*. Fresh bacterial suspension prepared according to the method in Example 2 was used at a rate of 3 L per acre, diluted 100 times with water, and sprayed evenly onto the surface of mulberry leaves and branches using a sprayer. Two applications were made at 15-day intervals, with water used as a control. Diseased fruit was counted 15 days after treatment. Results are as follows... Figure 6 As shown, the rate of white mulberry fruit decreased significantly after the application of JX-1, indicating that JX-1 can effectively inhibit the occurrence of white mulberry fruit in actual production, effectively antagonize the growth and infection of mulberry fruit cup fungus, and has good potential as a biocontrol agent.

Claims

1. Polymyxin Bacillus ( Paenibacillus polymyxa JX-1, deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 10, 2025, with accession number CGMCC NO.33781.

2. An inoculum containing Bacillus polymyxa JX-1 as described in claim 1.

3. The *Bacillus polymyxa* JX-1 as described in claim 1 or the bacterial agent as described in claim 2 in controlling *Cladosporium brassicae* (… Plasmodiophora brassicae Applications in infecting plants.

4. The *Bacillus polymyxa* JX-1 as described in claim 1 or the bacterial agent as described in claim 2 in controlling *Fusarium oxysporum* (Fusarium oxysporum) Fusarium fujikuroi ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Carotene soft rot pectinobacterium carotenoides subsp. carotenoides ( Pectobacterium carotovorum subsp. carotovorum Ralstonia solanacearum (Ralstonia solanacearum) Ralstonia solanacearum ) and mulberry cup plate fungus ( Ciboria shiraiana Applications in ).

5. The use of the polymyxa JX-1 as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of products for the prevention and control of clubroot fungus, Fusarium oxysporum, Sclerotinia sclerotiorum, Bacillus carotenoides subsp. carotenoides, Ralstonia solanacearum, and Pleurotus ostreatus.

6. The application according to claim 5, characterized in that, The product in question is a biological pesticide.

7. The application of the *Bacillus polymyxa* JX-1 as described in claim 1 or the inoculum as described in claim 2 in the prevention and control of clubroot disease caused by *Cladophora brassicae* or large white fruit disease of mulberry caused by *Morchella esculenta*.

8. The application of the *Bacillus polymyxa* JX-1 as described in claim 1 or the bacterial agent as described in claim 2 in the preparation of a biological pesticide for controlling clubroot disease caused by *Cladophora brassicae* or large white fruit disease of mulberry caused by *Mulberry calyx*.

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