Paenibacillus polymyxa, bacterial inoculum prepared therefrom and use thereof

Abstract

The present application relates to a Paenibacillus polymyxa strain Y31, a microbial inoculum prepared therefrom and applications thereof. The strain Y31 can effectively inhibit the growth of Dothiorella gregaria, and field control tests show that the comprehensive prevention and control effect of the microbial inoculum prepared from the strain Y31 on the disease is as high as 72% or more in the early stage of the disease. Antagonistic experiments show that the strain Y31 also has good bacteriostatic effect on Dothiorella septospora and Dothiorella needle red rot fungus, and is suitable for being used as a broad-spectrum antibacterial agent in the biocontrol practice of pine fungal diseases. Meanwhile, the strain Y31 also has obvious inhibitory effect on bacterial diseases such as willow bacterial wilt fungus, pear fire blight fungus, walnut bacterial black spot fungus and kiwi fruit canker fungus, and can be used for the prevention and control of diseases such as willow wilt. In addition, the strain Y31 also has functions such as nitrogen fixation, phosphorus and potassium solubilization, and has certain salt and alkali tolerance, and can be used for promoting growth and disease resistance of plants in saline-alkali land.

Description

Technical Field

[0001] This invention relates to the field of biological control, and more specifically, to a polymyxa bacillus, a bacterial agent prepared therefrom, and its application. Background Technology

[0002] Pine trees refer to species in the genus *Pinus*, which includes many important economic and commercial forest species, such as Korean pine (*P. koraiensis*), Scots pine (*P. sylvestris* var. *mongolica*), Chinese pine (*P. armandii*), and oil pine (*P. tabuliformis*). However, in the production of commercial pine forests, pine needle diseases are widespread and cause serious damage, leading to massive needle loss, slowed growth, reduced timber yield, and even death, resulting in huge economic losses. Among these, pine needle red spot, pine needle blight, and pine needle red blight are the most serious and widespread.

[0003] There are two pathogens causing pine needle red spot disease. In my country, the disease is caused by *Dothistoma septospora*. Initially, chlorotic spots appear, which then turn brown in the center and yellow at the edges. The spots gradually enlarge, with a reddish-brown center and light brown edges. As the disease progresses, the tips of the needles turn entirely reddish-brown, or reddish-brown bands form in the center of the needles. Severely infected needles fall off prematurely. The disease first affects the lower needles and gradually spreads upwards, weakening the tree and eventually causing it to die. The pathogen causing pine needle red spot disease can infect over 80 species of pine trees worldwide and is listed as a quarantine pathogen in my country and the European Union.

[0004] Pine twig blight is a disease of pine trees caused by *Diplodia pinea*. It typically affects the buds, needles, shoots, and rhizomes of seedlings to mature trees. Initially, young buds, needles, and shoots turn bronze, gradually becoming reddish-brown, giving the entire crown a "red-topped" appearance. This disease is prevalent and harmful worldwide. According to incomplete statistics, nearly 30 countries worldwide have reported the occurrence and damage of pine twig blight, causing significant economic losses.

[0005] Pine needle blight (Pestalotiopsis funerea) primarily affects the needles of pine trees, causing them to gradually turn yellow and wither starting from the leaf tips or edges, eventually leading to the death of the entire branch or tree. Initial symptoms are often subtle, but as the disease progresses, yellowish-brown lesions appear at the tips or edges of the needles. These lesions gradually enlarge, and the needles darken to reddish-brown before finally drying out and falling off. Severely affected pine trees experience massive needle loss, resulting in a sparse crown and significantly impacting the tree's growth and ornamental value.

[0006] Currently, the main control measures for pine needle diseases remain strengthening quarantine, silvicultural measures, and chemical control. Agents such as chlorothalonil, thiram, zineb, and carbendazim are effective against pine needle red spot, pine needle blight, and pine needle red blight. However, conventional chemical control techniques cause environmental pollution; therefore, there is an urgent need to develop broad-spectrum, green control agents for pine needle diseases.

[0007] Bacterial wilt of willow is a disease caused by the bacterium *Brenneria salicis*, which occurs both domestically and internationally. *Brenneria salicis* causes the new leaves at the tips of willow shoots to curl. As the disease progresses, infected branches wither and die. Eventually, the lesions spread from the branch tips to the trunk, leading to the death of the entire willow tree. Commonly used antibiotics for the control of bacterial diseases include agricultural streptomycin and tetracycline. However, the use of these agents poses potential hazards to human and animal health, the environment, and public health safety, including the development of bacterial resistance, drug residues, and food safety risks. Currently, there is a lack of highly effective, green control agents for bacterial wilt of willow. Summary of the Invention

[0008] This invention provides a Paenibacillus polymyxa strain, named Paenibacillus polymyxa Y31, which is deposited at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: April 8, 2025; accession number: CGMCC No. 34130.

[0009] This invention also provides a Bacillus polymyxa agent, which is prepared from the aforementioned Bacillus polymyxa. Specifically, the number of viable Y31 bacteria in the agent is ≥10. 8 CFU / mL.

[0010] The present invention also provides the application of the aforementioned Bacillus polymyxa agent in the prevention and control of fungal needle diseases on pine trees.

[0011] Specifically, the fungal diseases on pine trees include pine needle red spot (Dothistomaseptospora), pine needle blight (Diplodia pinea), and pine needle red blight (Pestalotiopsis funerea).

[0012] The present invention also provides an application of the aforementioned Bacillus polymyxa agent in the prevention and control of bacterial wilt disease in willow trees.

[0013] Specifically, the microbial agent is applied by spraying.

[0014] The amount of the microbial agent to be sprayed is determined by the amount of liquid droplets dripping from the pine needles.

[0015] The application period is at the initial stage of the disease or 1-2 weeks before the onset of the disease.

[0016] The beneficial effects of this invention include: The inventors isolated the *Bacillus polymyxa* strain Y31 from samples of tree branches, roots, rhizosphere soil, and saline-alkali soil. Strain Y31 effectively inhibits the growth of *Pinus pineneatus*, and field control trials showed that applying an agent prepared from strain Y31 at the early stage of disease development had a significant control effect on pineneatus, with a comprehensive control efficacy exceeding 72%. Antagonistic experiments showed that strain Y31 also had good inhibitory effects on *Pinus pineneatus* and *Pinus pineneatus*, making it suitable as a broad-spectrum antifungal agent for the biocontrol of fungal diseases in pine trees. Simultaneously, strain Y31 also showed significant inhibitory effects on bacterial diseases such as *Fusarium wilt* of willow, *Pseudomonas aeruginosa* of pear, *Pinus walnut*, and *Actinidia chinensis*, and can also be used for the prevention and control of diseases such as *Fusarium wilt* of willow. Furthermore, strain Y31 also possesses nitrogen-fixing, phosphorus- and potassium-solubilizing functions, and has a certain degree of salt and alkali tolerance, making it suitable for promoting growth and disease resistance in saline-alkali soil plants. Attached Figure Description

[0017] Figure 1 The *Bacillus polymyxa* strain Y31 (concentration of 10) in Example 1 of this invention. 8 The graph shows the antibacterial test results (CFU / mL), where A is the blank control; B is the graph showing the inhibition of pine red spot pathogen by Bacillus polymyxa strain Y31. Figure 2 This is a high-throughput test of the inhibitory effect of some of the bacteria to be screened on bacterial wilt of willow in Example 1; Figure 3 The image shows the symptoms of pine needle red spot disease after 4 months in the field control trial of Example 2. In the image, A is the sterile water control; B is the result of applying the inoculant Y31 of this invention; and C is the result of applying a commercial polymyxa spore-forming agent. Figure 4 The experiment in Example 3 showed that strain Y31 inhibited the pine needle blight and pine shoot blight. In this experiment, A was the middle inoculation with Y31, the left side inoculated with pine shoot blight, and the right side inoculated with pine needle blight. B was the pine needle blight control and C was the pine shoot blight control. Figure 5 Example 3 shows the inhibition test of Bacillus polymyxa strain Y31 against damping-off and wheat take-all pathogens. In this test, A and C are pure culture controls, B is Rhizoctonia solani CFCC 50564, and D is Wheat take-all pathogen CFCC 50445. Figure 6The experiment in Example 3 showed that strain Y31 inhibited pear fire blight, walnut bacterial black spot, and kiwifruit canker, where A is kiwifruit canker, B is walnut bacterial black spot, and C is pear fire blight. Detailed Implementation

[0018] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0020] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0021] Example 1: Isolation, screening and identification of antagonistic strain Y31 (1) Isolation of bacteria Since 2018, the applicant's research team has been collecting samples of branches, roots, rhizosphere soil, and saline-alkali soil from trees such as tamarisk, sea buckthorn, walnut, pine, and poplar in Shandong, Hebei, Xinjiang Uygur Autonomous Region, Jilin, and Inner Mongolia Autonomous Region for microbial isolation. 1.0 g of soil sample was weighed and poured into a 9 ml sterile water-filled test tube with a cap. The tube was shaken at 220 rpm for 5 minutes and allowed to stand for 5 minutes. The supernatant was then diluted 10 times. 1 -10 3 After dilution, the samples were plated on NA plates and incubated upside down at 30°C for 2 days, with 3 replicates for each dilution gradient. Then, different single colonies were selected from the plates, streaked to purify the target bacteria, and subsequently stored at -70°C for later use. For bark and root samples, small tissue pieces (1-2 mm * 1-2 mm) were cut into small pieces using sterile scissors, then pulverized using a tissue homogenizer. 1.0 g of the sample was weighed for bacterial isolation; other steps were the same as for soil sample isolation. More than 2000 bacterial strains were obtained.

[0022] (2) Molecular identification of bacterial strains Bacterial identification: DNA was extracted from the bacterial strains using the CTAB method, and PCR amplification was performed using the universal primers 27F / 1492R. The amplified sequences were then sent to a sequencing company for sequencing. The obtained sequences were entered into an online identification tool (https: / / eztaxon-e.ezbiocloud.net / identify) for comparison and identification. Based on the identification results, more than 300 strains, including Bacillus, Bacillus-like organisms, Pseudomonas, Pantotheca, and Streptomyces, were selected for antagonistic bacteria screening.

[0023] (3) Screening of antagonistic bacteria against pine needle red spot pathogen Biocontrol strains of *Pinus pineneatus* were screened using a plate confrontation culture method: First, more than 300 bacterial strains screened in step (2) were prepared into 10... 8 Cell suspensions of *Dothistomaseptospora* mycelial blocks were inoculated onto the center of PDA plates using CFU / mL cell suspensions. Then, 100 μL of cell suspensions of the bacterial strains to be screened were spread around the periphery of the *Dothistomaseptospora* mycelial blocks. Three replicates were performed for each bacterial strain, with a pure culture inoculated only with *Dothistomaseptospora* mycelial blocks serving as a blank control. The plates were incubated upside down at 26°C for four weeks, and the inhibition zones were observed and measured. Through confrontation culture, 36 bacterial strains with antagonistic effects against *Dothistomaseptospora* were screened, among which 12 strains of *Paenibacillus polymyxa* showed the best antibacterial effect. Strain Y31 exhibited the best antibacterial effect, completely inhibiting the growth of *Dothistomaseptospora* in the confrontation experiment (e.g., *Dothistomaseptospora* growth was completely suppressed). Figure 1 As shown in Figure B), the other 11 polymyxin Bacillus strains failed to completely inhibit the growth of this bacterium.

[0024] (4) Screening of antagonistic bacteria against bacterial wilt of willow Antagonistic bacteria of the pathogen causing willow bacterial wilt (Brenneria salicis) were screened using a plate confrontation culture method: First, the pathogen causing willow bacterial wilt was prepared into 10... 8 A CFU / mL cell suspension was prepared by evenly spreading 100 μL of the willow bacterial wilt pathogen suspension onto a square high-throughput screening plate. The bacterial strains to be screened were then spotted at the center of each square on the plate. The plate was then incubated upside down at 30°C for 3 days. The antibacterial activity was observed and measured. Some antibacterial results are shown below. Figure 2 As shown. More than 60 antagonistic bacteria were screened out through this experiment, including 12 strains of Bacillus polymyxa screened out in step (3), and these 12 strains all showed very good antagonistic effects against the bacterial wilt pathogen of willow.

[0025] Subsequently, the antagonistic ability of these 12 *Bacillus polymyxa* strains against the pathogen of bacterial wilt of willow was further screened using the confrontation method: a suspension of the pathogen of bacterial wilt of willow (10... 8 The *Bacillus polymyxa* strains to be screened were evenly spread on 9 cm agar plates (CFU / mL). Three replicates were performed for each treatment. The rescreening results showed that strain Y31 exhibited the best antagonistic effect against *Fusarium wilt*, the causal agent of willow bacterial wilt, with the largest inhibition zone and rapid growth. The results are shown in Table 1.

[0026] Table 1. Sources of 12 *Bacillus polymyxa* strains and their inhibitory effects on *Fusarium wilt*, the causal agent of bacterial wilt in willow. (5) Identification and preservation of strain Y31 Using the identification method in step (2) of this embodiment, it was found that the strain Y31 of the present invention is similar to the type strain ATCC 842 of Paenibacillus polymyxa. T The homology was highest, at 99.72%. The 16S rRNA gene sequence showed greater than 98.5% homology with the model bacterial species, which is considered to be of the same species. Therefore, strain Y31 of this invention was identified as Paenibacillus polymyxa. This Paenibacillus polymyxa strain Y31 is deposited at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: April 8, 2025; accession number: CGMCC No. 34130.

[0027] Example 2: Field control experiment of strain Y31 against pine needle blotch. (1) Preparation of strain Y31 inoculum and formulation of commercial polymyxa spore-forming inoculum The preserved strain Y31 was streaked and cultured for three generations via single-colony transfer. Single colonies were then transferred to TSB liquid medium for propagation and cultured at 30°C with shaking at 220 rpm for 36 h. The cultured strain was then centrifuged at 10000 rpm for 5 min to precipitate the culture. The precipitate was diluted with sterile water to a viable count of 10-1 Y31. 8 Prepare a bacterial solution of approximately CFU / mL.

[0028] Purchase one commercially available polymyxa spore-forming agent for this comparative experiment, and prepare it according to the instructions to a 10:1 ratio. 8 Prepare a bacterial solution of approximately CFU / mL.

[0029] (2) Test on the efficacy of pine needle red spot disease control The efficacy experiment was conducted in pine forests within the Chinese Academy of Forestry. In early June 2024, diseased pine trees were sprayed once with the fungal agent of this invention and once with a commercially available polymyxa-based spore-forming agent at the initial stage of disease in the pine needles. A second spraying was performed two weeks later. The number of live bacteria used was 10... 8Spray the fungal agent at approximately CFU / mL until droplets drip from the pine needles, with sterile water used as a control. The control effect was observed and recorded in October, primarily focusing on the number of diseased pine needles. This experiment selected 15 pine branches with consistent disease incidence and similar needle counts as experimental subjects. Five branches were used for the efficacy test of the fungal agent of this invention, five branches for the efficacy test of a commercial polymyxa spore-forming agent, and five branches as a sterile water control (CK). Due to the large number of pine needles and the small size of the lesions, recording and measurement were inconvenient; therefore, the control effect in this experiment was statistically calculated based on the number of diseased pine needles and the incidence rate.

[0030] ×100%=1- ×100% A1 represents the number of diseased pine needles on pine branches in the control group after treatment, and A2 represents the number of diseased pine needles on pine branches in the control group before treatment; B1 represents the number of diseased pine needles on pine branches in the treatment group after treatment, and B2 represents the number of diseased pine needles on pine branches in the treatment group before treatment; C represents the total number of pine needles in each treatment. Since the number of pine needles in each treatment in this application is similar, the value of C is considered to be the same.

[0031] The incidence of disease recorded in the experimental observation of the prevention and control effect is as follows: Figure 3 As shown in Table 2, when the biocontrol strain Y31 of the present invention is applied in the early stage of pine needle red spot disease, the overall control efficacy can reach 72.2%, which is much higher than the control efficacy of commercial polymyxa spore-forming agents (58.6%).

[0032] Table 2 Field control effect Example 3: Antibacterial spectrum test of strain Y31 (1) Test on the inhibition of plant pathogenic fungi by strain Y31 To simultaneously address multiple pine needle diseases, this case study first selected two other highly damaging pine pathogens—Pestalotiapsis funerea and Diplodia pinea—to conduct a plate confrontation experiment with strain Y31. Strain Y31 was first inoculated in the center of a petri dish, with Pestalotiapsis funerea and Diplodia pinea funer ... Figure 4 As shown in the figure, strain Y31 exhibits strong inhibitory effects against both *Pinus pineus* and *Pinus pineus*.

[0033] In addition to pine pathogens, two other important agricultural plant pathogens—*Rhizoctonia solani* strain (strain number CFCC 50564) and *Gaeumannomyces graminis* strain (strain number CFCC 50445)—were selected for a confrontation experiment with strain Y31. *Rhizoctonia solani* and *Gaeumannomyces graminis* pathogens were inoculated into the center of petri dishes, respectively. Y31 was then streaked to both sides of the pathogens. Each treatment was repeated in triplicate, with *Rhizoctonia solani* and *Gaeumannomyces graminis* pathogens inoculated into the center of the petri dishes as controls. The petri dishes were incubated upside down at 26°C for one week, after which the inhibition zones were observed and measured.

[0034] like Figure 5 As shown in Figures B and D, strain Y31 also exhibited good inhibitory effects against *Rhizoctonia solani* and *Tricholoma materia velutipes* in the confrontation experiment. The inhibition rates of strain Y31 against *Rhizoctonia solani* and *Tricholoma materia velutipes* are shown.

[0035] (2) Y31 inhibition test of plant pathogenic bacteria The plant pathogens used in the experiment included three important forest tree pathogens: *Erwinia amylovora* (strain number CFCC17213B), *Xanthomonas arboricola* (strain number CFCC 17470), and *Pseudomonas syringae pv. Actinidiae* (strain number CFCC 17281). Strain Y31 was subjected to confrontation tests against *Erwinia amylovora*, *Xanthomonas arboricola*, and *Pseudomonas syringae*, respectively. The test methods were the same as the confrontation test for the rescreening of *Bacillus polymyxa* in step (4) of Example 1, such as... Figure 6 As shown in A, B, and C. Confrontational experiments revealed that Y31 exhibits antagonistic effects against *Pyrus pyrifolia*, *Bacillus walnutus*, and *Actinidia kiwifruit*.

[0036] Example 4: Tests on nitrogen fixation, phosphorus solubilization, potassium solubilization, and salt and alkali tolerance of Y31 The nitrogen-fixing capacity of the strain was tested by streaking strain Y31 onto modified Assumption agar plates, incubating the plates upside down at 30°C for 7 days, and determining the nitrogen-fixing capacity based on colony growth. The potassium-solubilizing capacity was tested by spot-inoculating strain Y31 onto M1997 agar plates containing 15 g / L agar, and determining its potassium-solubilizing capacity based on growth and the formation of potassium-solubilizing zones. The inorganic phosphorus-solubilizing capacity was tested by observing the formation of clear zones by the strain in phosphate-solubilizing bacterial culture media.

[0037] Salt and alkali tolerance tests were conducted using LB liquid medium as the base medium, with NaCl added to achieve NaCl concentrations of 5% and 8%, respectively. NaOH was added to adjust the pH of the medium to 9 and 10, respectively. Equal volumes of Y31 bacterial culture were transferred to LB medium with different NaCl concentrations and pH values, and cultured at 28 ℃ and 180 r / min for 24 h with shaking. The OD600 absorbance was then measured.

[0038] Studies have found that strain Y31 grows well on modified Assumption solid medium for nitrogen-fixing bacteria, indicating its nitrogen-fixing ability. Strain Y31 can also grow on medium M1997 and produce potassium-solubilizing zones, indicating its potassium-solubilizing ability. Furthermore, strain Y31 can also grow on phosphate-solubilizing bacteria medium and produce clear zones, indicating its ability to solubilize inorganic phosphorus. In addition, strain Y31 grows well on media with pH 10.0 and NaCl concentrations of 5% and 8%. Therefore, strain Y31 possesses nitrogen-fixing, phosphorus-solubilizing, potassium-solubilizing, and salt-alkali tolerant abilities.

Claims

1. A Paenibacillus polymyxa characterized in that, The polymyxobacterium strain is named polymyxobacterium Y31 and is deposited at the China General Microbiological Culture Collection Center (CGMCC); address of the depository: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; deposit date: April 8, 2025; accession number: CGMCC No. 34130.

2. A Paenibacillus polymyxa inoculant characterized in that, The bacterial agent is prepared from the polymyxa bacillus described in claim 1.

3. The Bacillus polymyxa inoculant of claim 2, wherein, The number of viable Y31 in the bacterial agent is ≥10 8 CFU / mL.

4. The application of the fungal agent according to claim 2 or 3 in the prevention and control of fungal needle diseases on pine trees.

5. Use according to claim 4, characterized in that, The fungal diseases affecting pine needles include Dothistoma septospora, Diplodia pinea, and Pestalotiopsis funerea.

6. The application of the fungal agent according to claim 2 or 3 in the control of bacterial wilt disease of willow.

7. Use according to any one of claims 4 to 6, characterized in that, The microbial agent is applied by spraying.

8. Use according to claim 7, characterized in that, The amount of the microbial agent to be sprayed is determined by the amount of liquid droplets dripping from the pine needles.

9. Use according to claim 7, characterized in that, The application period is at the initial stage of the disease or 1-2 weeks before the onset of the disease.

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