Paenibacillus polymyxa strain and application thereof

Abstract

A paenibacillus polymyxa and application thereof, relates to the field of microorganisms, in particular to a paenibacillus polymyxa and application thereof.The existing plant growth promotion and antagonistic pathogenic bacteria strain resource shortage problem is solved.The paenibacillus polymyxa is paenibacillus polymyxa X-11, which is preserved in the China general microbiological culture collection center, the address is No.1, Beichen West Road, Chaoyang District, Beijing, the preservation date is August 3, 2020, and the preservation number is CGMCC No.20476.Paenibacillus polymyxa X-11 has a growth promoting effect on tomatoes and rice, and has strong antibacterial activity on various plant pathogens.The application is applied to the field of rice, tomato growth promotion and biological control of diseases.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a polymyxa bacillus strain and its applications. Background Technology

[0002] Over the past few decades, the use of chemical fertilizers and pesticides in agriculture has ensured crop yields and solved the problem of food security for humanity. However, the continuous excessive and unscientific application of chemical fertilizers and pesticides has also brought about a series of problems, such as damage to soil biological and physicochemical properties, pesticide damage, agricultural non-point source pollution, ecological environment destruction, and food quality and safety issues caused by pesticide residues. Biofertilizers and biopesticides can overcome many of the drawbacks of chemical fertilizers and pesticides and have broad application prospects. However, due to the limitations of technology and environmental conditions in the use of biological agents, they have not yet been effectively promoted, and their efficacy has not been widely recognized. Reducing the cost of use, lowering the technical threshold, and improving and stabilizing efficacy are urgent problems that my country's biofertilizer and biopesticide industry needs to solve.

[0003] In natural ecosystems, many beneficial bacteria live, exerting beneficial effects on plants through various means. These are collectively known as plant rhizosphere growth promoters (PGPRs). Because their characteristics meet the needs of plant growth promotion and biocontrol of diseases, people are constantly exploring the use of PGPRs to regulate plant growth and control plant diseases. Furthermore, compared to chemical fertilizers and pesticides, PGPRs, due to their complex and diverse mechanisms of action and lower susceptibility to pathogen resistance, can be applied to the soil or directly to plants to improve the utilization rate of fertilizers and soil nutrients and elements. They can even fix atmospheric nitrogen for plant use. Simultaneously, some PGPRs also possess biocontrol properties, making them a more effective alternative to chemical pesticides for controlling plant diseases, demonstrating strong application potential. However, currently, the resources of strains that can be used for plant growth promotion and pathogen antagonism are extremely limited. Screening for new strains with significant effects from the environment can effectively solve the problem of strain resource shortage. Summary of the Invention

[0004] The present invention aims to address the shortage of strains of fungi that promote plant growth and antagonize pathogens, and provides a strain of Bacillus polymyxa and its applications.

[0005] This invention provides a strain of *Paenibacillus polymyxa* X-11, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on August 3, 2020, with accession number CGMCC No. 20476.

[0006] The *Bacillus polymyxa* X-11 of this invention can grow on potato sucrose agar (PDA) medium. The colonies are milky white, translucent, with a smooth, moist surface, neat edges, and are shaped like half a glass bead; they are sticky when picked up. Under a microscope at 1000x magnification, the bacterial cells are rod-shaped. In the later stages of cultivation, they produce a large amount of glycocalyx, produce spores, and the sporangia swell into an oval shape.

[0007] The present invention relates to *Bacillus polymyxa* X-11, a Gram-negative bacterium that can utilize citrate, hydrolyzed casein, hydrolyzed starch, hydrolyzed fats, and hydrolyzed gelatin. It is positive for the VP reaction and negative for methyl red. It can reduce nitrates and possesses both oxidase and catalase activity, enabling it to hydrolyze tyrosine and discolor litmus milk. It can utilize various carbon sources, including glucose, sucrose, mannitol, lactose, sorbitol, arabinose, and salicin-laminarin.

[0008] The 16S rDNA sequencing results of *Paenibacillus polymyxa* X-11 of this invention were submitted to the NCBI database for BLAST analysis. The results showed that its full-length 16S rDNA was 1444bp, and it showed the highest homology with the 16S rDNA sequences of strains such as *Paenibacillus polymyxa* CJX518 (NCBI sequence number KF991241.1) and Sb3-1 (NCBI sequence number CP010268.1), with a sequence similarity rate of 99.86%. Based on the combined physiological and biochemical results, *Paenibacillus polymyxa* X-11 was identified as *Paenibacillus polymyxa*.

[0009] This invention provides the application of Bacillus polymyxa X-11 in promoting root elongation in rice.

[0010] This invention provides the application of Bacillus polymyxa X-11 in enhancing the activity of defense enzymes in rice.

[0011] Furthermore, the defensive enzyme is superoxide dismutase (SOD).

[0012] This invention provides the application of Bacillus polymyxa X-11 in promoting tomato seed germination.

[0013] This invention provides the application of Bacillus polymyxa X-11 in promoting the elongation of the radicle of tomato seeds.

[0014] This invention provides the application of Bacillus polymyxa X-11 in enhancing the activity of tomato defense enzymes.

[0015] Furthermore, the defensive enzyme is phenylalanine ammonia-lyase (PAL).

[0016] This invention provides the application of Bacillus polymyxa X-11 in improving the cold resistance of tomatoes.

[0017] This invention provides the application of Bacillus polymyxa X-11 in increasing tomato yield.

[0018] The beneficial effects of this invention are:

[0019] This invention screened a spore-producing bacterium from soil, namely *Bacillus polymyxa* X-11. Testing revealed that this strain possesses xylanase and glucanase activities, can fix atmospheric nitrogen, and can also produce indole. *Bacillus polymyxa* X-11 has low nutritional requirements, can utilize multiple carbon sources, exhibits good production performance, can produce highly resistant spores, and has low application technology requirements. The preparation method of *Bacillus polymyxa* X-11 spore solution is simple, low-cost, and has a long shelf life. Genomic sequencing analysis shows that its genome carries multiple nitrogenase genes, xylanase genes, glucanase genes, pectinase genes, peptidase genes, polyketide synthase genes, and antimicrobial peptide (such as polymyxin, biotin, etc.) synthase genes.

[0020] Fermentation broth of Bacillus polymyxa X-11 stored for 6 months, when applied to rice seedlings, significantly promoted root growth. Application of Bacillus polymyxa X-11 also increased the activity of the SOD (superoxide dismutase) defense enzyme in rice seedling leaves. Treatment of tomato seeds with fermentation broth of Bacillus polymyxa X-11 stored for 6 months significantly improved germination rate and promoted radicle elongation. Application of Bacillus polymyxa X-11 spore solution stored for 6 months to greenhouse-grown tomatoes significantly increased PAL (phosphorus aminotransferase) activity in leaves. Compared with carbendazim, it significantly increased both SOD and PAL activities in leaves. This X-11 spore solution also improved tomato cold resistance and increased tomato yield. Polymyxobacterium X-11 exhibits significant antagonistic effects against Fusarium fujikuroi, Fusarium oxysporum, Magnaphalthe oryzae, Botrytis cinerea, and Exserohilum turcicum, the causal agent of rice seedling blight.

[0021] The polymyxa Bacillus X-11 and its fermentation broth of the present invention are easy to preserve and cultivate, have good production performance, low technical requirements, long shelf life of spore liquid, and are safe and non-toxic. They can be used to promote the growth of rice and tomatoes, improve system resistance, increase tomato yield, and can also be used to antagonize plant pathogens and prevent tomato chilling injury.

[0022] Polymyxobacterium X-11 has a growth-promoting effect on tomatoes and rice, and has strong antibacterial activity against a variety of plant pathogens. It has strong application value in promoting growth and biological control of diseases in rice and tomatoes. Attached Figure Description

[0023] Figure 1The colony morphology of Bacillus polymyxa X-11 plate;

[0024] Figure 2 Cell morphology of Bacillus polymyxa X-11 under a microscope;

[0025] Figure 3 Pie chart showing the gene distribution of Bacillus polymyxa X-11 and its homologous species;

[0026] Figure 4 The effect of the application time of Bacillus polymyxa X-11 on rice plant height;

[0027] Figure 5 The effect of the application time of Bacillus polymyxa X-11 on the root length of rice;

[0028] Figure 6 The effect of the timing of application of Bacillus polymyxa X-11 on the activity of the rice defense enzyme CAT;

[0029] Figure 7 The effect of the timing of application of Bacillus polymyxa X-11 on the activity of SOD, a defensive enzyme in rice;

[0030] Figure 8 The effect of the timing of application of Bacillus polymyxa X-11 on the activity of the rice defense enzyme PAL;

[0031] Figure 9 The effect of the timing of application of Bacillus polymyxa X-11 on the activity of the rice defense enzyme GA;

[0032] Figure 10 The seed germination rate of the tomato pink crown variety under different concentrations of Bacillus polymyxa X-11 germination treatment is shown; where 0 represents the blank control, and 1×, 10×, 50×, 100×, and 1000× represent the dilution factors of X-11 bacterial solution, respectively.

[0033] Figure 11 The seed germination rate of the tomato variety Jinpeng No. 1 under different concentrations of Bacillus polymyxa X-11 germination treatment is shown; where 0 represents the blank control, and 1×, 10×, 50×, 100×, and 1000× represent the dilution factor of X-11 bacterial solution, respectively.

[0034] Figure 12 Photographs of the radicle length of Tomato radicle under different concentrations of Bacillus polymyxa X-11 treatment; B, C, D, E, and F show the radicle length of the tomato at dilutions of 1×, 10×, 150×, 300×, and 1000×, respectively.

[0035] Figure 13A bar chart showing the root length of Tomato radicle under different concentrations of Bacillus polymyxa X-11 treatment; where 1×, 10×, 150×, 300×, and 1000× represent the dilution factors of X-11 bacterial solution, respectively.

[0036] Figure 14 Photographs of the radicle length of Jinpeng No. 1 tomato under different concentrations of Bacillus polymyxa X-11 treatment; B, C, D, E, and F show the radicle length of tomato at dilutions of 1×, 10×, 150×, 300×, and 1000×, respectively.

[0037] Figure 15 A bar chart showing the statistical length of the radicle of Jinpeng No. 1 tomato under different concentrations of Bacillus polymyxa X-11 treatment; where 1×, 10×, 150×, 300×, and 1000× represent the dilution factors of X-11 bacterial solution, respectively.

[0038] Figure 16 To evaluate the SOD activity of tomato leaves under different application methods of Bacillus polymyxa X-11;

[0039] Figure 17 Comparison of SOD activity in tomato leaves treated with Bacillus polymyxa X-11 and carbendazim under different application methods;

[0040] Figure 18 The activity of the defense enzyme PA in tomato leaves under different application methods of Bacillus polymyxa X-11;

[0041] Figure 19 Comparison of PAL (a defensive enzyme) activity in tomato leaves treated with Bacillus polymyxa X-11 and carbendazim under different application methods;

[0042] Figure 20 Photographs showing the effect of Bacillus polymyxa X-11 treatment on the chilling injury disease index of tomatoes; where A is the X-11 treatment, B is the control, the leaf between A and B is the top leaf of the compound leaf, and the leaves on both sides are the two lateral leaves adjacent to the top leaf.

[0043] Figure 21 for Figure 20 Bar chart showing the effect of Bacillus polymyxa X-11 treatment on the chilling injury disease index of tomato top leaves;

[0044] Figure 22 for Figure 20 Bar chart showing the effect of Bacillus polymyxa X-11 treatment on the chilling injury disease index of tomato lateral leaves;

[0045] Figure 23 The effect of Bacillus polymyxa X-11 on the number of tomatoes;

[0046] Figure 24The effect of Bacillus polymyxa X-11 on tomato yield;

[0047] Figure 25 The effect of Bacillus polymyxa X-11 on the single fruit weight of tomato;

[0048] Figure 26 The effect of Bacillus polymyxa X-11 on the theoretical yield of tomato;

[0049] Figure 27 To illustrate the antagonistic effect of Bacillus polymyxa X-11 against pathogens of rice, tomato, and corn, the blocks labeled "treatment" with "X-11" are the Bacillus polymyxa X-11 strains described in this invention, while those without labeling are reference strains. Detailed Implementation

[0050] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0051] Example 1:

[0052] In this embodiment, the polymyxa strain is Paenibacillus polymyxa X-11, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is August 3, 2020, and the accession number is CGMCC No. 20476.

[0053] The method for obtaining Bacillus polymyxa X-11 in this embodiment is as follows:

[0054] In August 2010, wheat straw from wheat stubble was collected in farmland in Jiamusi City, Heilongjiang Province (46°36′03.1″N; 130°25′36.4″E). The straw was cut into 1cm pieces, and 1g of each straw piece was placed in a 500mL shake flask containing 100mL of nitrogen-free silicate sucrose medium. After 4 days of enrichment culture at 30℃, the medium was serially diluted. Under aseptic conditions, 200μL of each dilution was inoculated onto nitrogen-free silicate sucrose agar plates. Three plates were spread for each dilution. After 48 hours of incubation at 30℃, single colonies were selected for purification. The uniformity of cells in the colonies was observed under a microscope. Impure cells were further purified until a pure culture was obtained. The pure culture was then inoculated onto potato sucrose agar (PDA) plates or slants for preservation, which is Bacillus polymyxa X-11.

[0055] Example 2: Identification of Bacillus polymyxa X-11 in this example

[0056] *Bacillus polymyxa* X-11 can grow on potato sucrose agar (PDA) medium. Colonies are milky white, translucent, smooth and moist with regular edges, resembling half a glass bead, and are sticky when picked up (e.g., when lifted). Figure 1 Under a microscope at 1000x magnification, the bacterial cells are rod-shaped. In later stages of cultivation, they produce a large amount of glycocalyx, generate spores, and the sporangia swell into oval shapes (e.g., ...). Figure 2 ).

[0057] The present invention relates to *Bacillus polymyxa* X-11, a Gram-negative bacterium that can utilize citrate, hydrolyzed casein, hydrolyzed starch, hydrolyzed fats, and hydrolyzed gelatin. It is positive for the VP reaction and negative for methyl red. It can reduce nitrates and possesses both oxidase and catalase activity, enabling it to hydrolyze tyrosine and discolor litmus milk. It can utilize various carbon sources, including glucose, sucrose, mannitol, lactose, sorbitol, arabinose, and salicin-laminarin.

[0058] The physiological and biochemical characteristics of Polymyxin Bacillus X-11 and the results of glycolysis reaction determination are shown in Table 1.

[0059] Table 1. Physiological and biochemical characteristics and glycolysis test results of Bacillus polymyxa X-11 ("+" indicates positive, "-" indicates negative)

[0060]

[0061]

[0062] Analysis of the 16S rDNA sequence of *Paenibacillus polymyxa* X-11 revealed that its full-length 16S rDNA is 1444 bp, showing the highest homology (99.86%) with *Paenibacillus polymyxa* strains CJX518 (NCBI accession number KF991241.1) and Sb3-1 (NCBI accession number CP010268.1). This confirms that strain X-11 belongs to the taxonomic group *Paenibacillus polymyxa*. Whole-genome sequencing analysis showed that strain X-11 has 4380 genes annotated in the NR species database. Comparison with homologous species revealed that 55.7% of the gene sequences were correlated with *Paenibacillus polymyxa*, 36.7% were correlated with unidentified species within the genus *Paenibacillus*, and a small number were correlated with other strains of *Paenibacillus polymyxa* (e.g., *Paenibacillus polymyxa*). Figure 3Homology analysis of strain X-11's 16S rDNA and whole genome sequences with other strains shows that high homology in the 16S rDNA sequence does not necessarily imply a high matching rate in the genome sequence. This indicates that strain X-11's gene sequence differs significantly from its homologous species, classifying it as a novel *Bacillus polymyxa* strain. With at least 44.3% of its gene sequences not matched with its closest progenitor species, it is foreseeable that strain X-11 and its homologous species will have functional differences in their genes.

[0063] Example 3:

[0064] Bacillus polymyxa X-11 was inoculated onto potato sucrose agar and cultured at 28°C for 3 days. After colonies grew, single colonies were collected and inoculated into Erlenmeyer flasks containing potato sucrose liquid medium, filling the flasks to 1 / 4 to 2 / 5 of their volume. The flasks were then incubated at 30°C for 24 hours at 200 rpm until the cells reached the logarithmic growth phase, yielding approximately 1 × 10⁶ nutrient cells. 6 A bacterial suspension of CFU / mL can be used as an inoculum for subsequent propagation. The bacterial suspension is inoculated into the sporulation medium at a rate of 0.5%, and cultured at 30°C for 48 hours at 200 rpm until the cells reach the sporulation stage, yielding approximately 1 × 10⁶ spores. 9 The spore suspension contains cfu / mL of bacteria. The spore suspension can be stored at 4°C for over 6 months. (For large-scale production of bacterial suspension or spore suspension, a fermenter of appropriate volume can be used. The production process is a conventional aerobic fermentation process with a stirrer speed of 300 rpm and an aeration rate of 0.3 m³ / min.) 3 The fermentation rate was 1 / min, and the time required to reach the logarithmic phase was 12 hours. The concentration of vegetative cells in the bacterial suspension could reach 1.0 × 10⁻⁶. 8 The fermentation time to sporulation stage is 48 hours, with cfu / mL, and the spore concentration in the fermentation broth can reach 2.0 × 10⁻⁶. 9 (cfu / mL. Spore culture can be stored at 4°C for more than 6 months.)

[0065] Potato sucrose agar medium: 200g peeled potatoes, 20.0g sucrose, 20g agar powder, bring tap water to a final volume of 1000mL, natural pH, autoclave at 121℃ for 30min before use.

[0066] Potato and sucrose liquid culture medium: 200g peeled potatoes, 20.0g sucrose, and tap water to a final volume of 1000mL. The culture medium is then autoclaved at 121℃ for 30min before use.

[0067] Sporulation medium: 1.2g sucrose, 2.6g corn starch, 1.2g peptone, 0.24g MgSO4·7H2O, 0.6g CaCO3, 1.2g K2HPO4, 6.0g soybean meal, 6.0g cornmeal, bring to a final volume of 1000mL, pH 7.0–7.2, autoclave at 121℃ for 30min before use.

[0068] Example 4: Growth-promoting effect of Bacillus polymyxa X-11 on rice and tomato

[0069] I. Growth-promoting effect of Bacillus polymyxa X-11 on rice

[0070] 1. Test kit

[0071] The catalase (CAT) activity assay kit was produced by Nanjing Jiancheng Bioengineering Institute (catalog number: A007-1-1 Visible light method). The SOD activity assay kit (catalog number: G0101F Spectrophotometric method), PAL activity assay kit (catalog number: G0114F Ultraviolet spectrophotometric method), and dextranase kit (β-1,3-GA) (catalog number: G0526F Spectrophotometric method) were all produced by Suzhou Greens Biotechnology Co., Ltd.

[0072] 2. Processing Settings

[0073] The seedling experiment used Nipponbare (Oryza sativa L. spp. japonica, purchased from Japan Public Co., Ltd.) as the test variety. Different application periods were set (emerging needle stage, one-leaf-one-heart stage, and two-leaf-one-heart stage). Rice was cultivated to the corresponding stages, and Bacillus polymyxa X-11 spores, stored at 4℃ for 6 months, were diluted with tap water to a spore content of 2×10⁻⁶. 7 A spore suspension of CFU / mL, with a spore volume of 10 mL / m 2 Using tap water as a control, six biological replicates were set up. At the three-leaf stage, three replicates of seedlings were randomly selected to measure root length and plant height, while the remaining three replicates of seedlings were used to detect the activity of rice defense enzymes.

[0074] 3. Implementation of rice sowing, seedling cultivation and treatment

[0075] Before sowing, soak and germinate the rice seeds. Sow when 70% of the seeds show signs of germination. The substrate used is a mixture of 60% forest soil, 30% peat moss, and 10% perlite, with 35g / L of organic nutrient soil and 2.5g / L of Stanley compound fertilizer added. Sow 36 seeds per square seedling tray. After sowing, cover with a thin layer of soil and water appropriately to bring the substrate moisture content to 90% of saturation. Place in a light incubator and incubate in the dark at 30℃ for 4 days. After the rice emerges, set the photoperiod to 10 hours of light (temperature 25℃) and 14 hours of darkness (temperature 20℃). When the seedlings reach the appropriate stage, treat them with Bacillus polymyxa X-11 spore solution using a spray method, ensuring even spraying without dripping.

[0076] 4. Determination of biological traits of rice seedlings

[0077] When the rice plants reach the 3-leaf-1-heart stage, pull out the rice plants, take 3 holes, wash the soil off the roots, measure the plant height and root length, separate the above-ground parts from the underground parts, dry them, and weigh them.

[0078] 5. Determination of enzyme activity in rice seedlings

[0079] When measuring plant height and root length, from three additional rice plants, fully expanded green leaves were cut off, washed with distilled water, dried, and the middle half of each leaf was cut into 0.5 cm segments. Samples from each pot were mixed and combined into one sample, flash-frozen in liquid nitrogen, and stored at -80°C for use in measuring physiological indicators. Three replicates were performed for each treatment. CAT, SOD, PAL, and GA activities in rice leaves were determined according to the methods provided in the kit instructions.

[0080] 6. Data Processing and Statistical Analysis

[0081] All experimental data were processed using SPSS 19.0 software and subjected to one-way ANOVA. Graphs were generated using Excel software. The significance level was P<0.05.

[0082] 7. Results

[0083] (1) Effects of Bacillus polymyxa X-11 on rice growth

[0084] Spraying rice with Bacillus polymyxa X-11 spores at different stages promoted both plant height and root length. The best results were observed at the one-leaf-one-heart stage, reaching the highest values. Plant height increased by 15.4%, but the difference was not statistically significant (e.g., ...). Figure 4 ), root length increased by 6.8%, reaching a significant level (e.g. Figure 5 It is evident that spraying at the one-leaf-one-heart stage of rice can effectively promote rice plant height and root length.

[0085] (2) Effects of Bacillus polymyxa X-11 on the activity of rice defense enzymes

[0086] There were no significant differences in CAT activity in rice leaves among different application stages of Bacillus polymyxa X-11 spore solution, nor were there significant differences between each application stage and the control (e.g., Figure 6 The earlier *Bacillus polymyxa* X-11 is applied, the greater the increase in SOD activity. Specifically, application at the seedling stamen stage resulted in the highest SOD activity in leaves, reaching 13.5 U / mg, 90.4% higher than the control. The second highest was at the one-leaf-one-heart stage, with SOD activity at 10.7 U / mg, 51.8% higher than the control. Application at the two-leaf-one-heart stage had no effect on SOD activity. Furthermore, application at the stamen stage increased SOD activity by 25.5% compared to the one-leaf-one-heart stage and by 92.8% compared to the two-leaf-one-heart stage. Application at the one-leaf-one-heart stage increased SOD activity by 53.7% compared to the two-leaf-one-heart stage. Figure 7 Unlike SOD, PAL activity showed an increasing trend with the delay in the application of Bacillus polymyxa X-11. Application at the two-leaf-one-heart stage was 1.0 times and 1.1 times higher than at the stamen-emerging stage and the one-leaf-one-heart stage, respectively. Although it was also 49.8% higher than the control, the difference was not statistically significant (e.g., ...). Figure 8 Similar to PAL, β-1,3-GA activity showed an increasing trend with delayed application, reaching its peak at the two-leaf-one-heart stage, which was 45.4% higher than at the budding stage, and the difference was statistically significant. Although application of X-11 at this stage could increase β-1,3-GA by 17.8% compared to the control, the difference was not statistically significant (e.g., ...). Figure 9 Therefore, it can be seen that the effects of different application times on the activity of the aforementioned defensive enzymes are not entirely the same.

[0087] The effects of Bacillus polymyxa X-11 on the growth and defensive enzyme activity of rice seedlings were determined through single-factor experiments. The results showed that spraying Bacillus polymyxa X-11 at the one-leaf-one-heart stage significantly promoted root elongation. However, the timing and frequency of application had varying effects on the activity of different defensive enzymes in the leaves. Notably, earlier application of Bacillus polymyxa X-11 resulted in the greatest increase in SOD enzyme activity. In conclusion, this experiment confirmed that Bacillus polymyxa X-11 primarily exerts its growth-promoting effect by promoting root elongation in rice seedlings, and increasing SOD activity in leaves may be one of its mechanisms for promoting growth and inducing resistance.

[0088] II. Growth-promoting effect of Bacillus polymyxa X-11 on tomatoes

[0089] 1. Germination test

[0090] The tomato varieties tested were Fen Guan and Jin Peng No. 1 (purchased from Shandong Shouhe Seed Industry Co., Ltd.), both of which are stable cultivars. Fen Guan is not resistant to gray mold, while Jin Peng No. 1 is resistant to gray mold.

[0091] The *Bacillus polymyxa* X-11 spore suspension used in the experiment was prepared in advance and stored at 4°C for 6 months before use. A single-factor experimental design was used, with distilled water as a blank control. *Bacillus polymyxa* X-11 spore suspension (1×10⁻⁶) was used. 9 cfu / mL), diluted 10 times (1×10) 8 cfu / mL), 20 times (5×10) 7 cfu / mL), 40 times (2.5×10 7 cfu / mL), 50 times (2×10) 7 cfu / mL), 60 times (1.67×10 7 cfu / mL), 100 times (1×10) 7 cfu / mL), 150 times (6.67×10 6 cfu / mL), 200 times (5×10) 6 cfu / mL), 300 times (3.33×10 6 cfu / mL), 1000 times (1×10) 6 Seeds of two tomato varieties were treated with CFU / mL for soaking and germination. A total of 11 treatments were performed, with 3 biological replicates per treatment. Tomato seeds soaked in distilled water for 6 hours were dried, and 1.0 g of seeds were accurately weighed and evenly placed in Petri dishes lined with sterile filter paper. The Petri dishes were numbered according to dilution group. Then, 10 mL of *Bacillus polymyxa* X-11 bacterial suspension at each dilution was added to the corresponding numbered Petri dish, just enough to saturate the filter paper and moisten the seeds. The seeds were placed in a 28℃ incubator in the dark for germination. Germination rate was observed after 42 hours, and radicle length was measured after 66 hours.

[0092] 2. Field planting experiment

[0093] This experiment was conducted in the ecological greenhouse of the Practice Teaching Base of the School of Life Sciences, Shangrao Normal University. The *Bacillus polymyxa* X-11 spore solution used was prepared in advance and stored at 4℃ for 6 months before use. The tomato variety *Pink Crown* was used as the test plant. A single-factor experimental design was adopted, setting up three treatments: root irrigation, foliar spraying, and a combination of root irrigation and foliar spraying. The *Bacillus polymyxa* X-11 spore solution was diluted 1000 times and applied. A water treatment served as a blank control, and a fungicide, carbendazim wettable powder (produced by Zhongbao Lujia Technology Group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences), was used as a positive control, diluted 500 times according to the instructions. Treatment began when the tomatoes reached the 12-leaf stage, with treatment every 7 days for 3 consecutive treatments. Seven days after the third treatment, three representative tomato plants with consistent growth were selected as three biological replicates. The fifth leaf from the bottom of each plant was taken as a sample, immediately placed on ice, and brought back to the laboratory. After being flash-frozen in liquid nitrogen, the samples were stored at -80°C for the purpose of measuring physiological and biochemical indicators.

[0094] 3. Enzyme solution preparation and assay of defensive enzyme activity

[0095] Tomato leaves were flash-frozen in liquid nitrogen and then ground. The SOD and PAL activities of the samples were measured using a kit (Suzhou Grees Biotechnology Co., Ltd.).

[0096] 4. Data Statistical Analysis

[0097] SPSS 19.0 software was used to perform one-way ANOVA to analyze the significance of the difference between the two variables, and LSD was used to compare the differences among multiple variables. Bar charts were created using WPS software.

[0098] 5. Results

[0099] (1) Effects of Bacillus polymyxa X-11 on tomato seed germination and radicle elongation

[0100] A. Effects of different concentrations of Bacillus polymyxa X-11 bacterial suspension on tomato germination rate

[0101] When using the *Bacillus polymyxa* var. *pinnatifida* as the test subject, use a 100-fold diluted solution of *Bacillus polymyxa* X-11 spores (1×10⁻⁶). 7 After germination for 42 hours with CFU / mL solution, the seed germination rate reached 40.77%, significantly higher than the control germination rate. The former increased the germination rate by 17.74 percentage points, a 77.03% increase. Furthermore, the germination rate under this concentration of spore solution was significantly higher than that of treatments diluted 10 times, 300 times, and 1000 times (e.g., CFU / mL). Figure 10 ).

[0102] When using Jinpeng No. 1 as the test subject, the polymyxa X-11 spore solution was diluted 10 times (1×10).8 The tomato seed germination rate was highest at 36.97% under the X-11 spore solution treatment (cfu / mL), significantly higher than the control, representing an increase of 14.94 percentage points (67.78%). This concentration also showed a significantly higher germination rate than the 1000-fold dilution treatment. Other concentrations of X-11 spore solution did not differ significantly from the control (e.g., Figure 11 It can be seen that the concentration of *Bacillus polymyxa* X-11 spores is 1×10⁻⁶. 7 cfu / mL~1×10 9 At cfu / mL, all concentrations can promote the germination of Jinpeng No. 1 tomato seeds.

[0103] It can be seen that when the polymyxa x 10^11 spore solution is diluted 100 times (1×10^10), the polymyxa x 10^11 spore solution is more effective. 7 The highest concentration of cfu / mL had the strongest promoting effect on the germination of pink crown tomato seeds, with a 10-fold dilution (1×10⁻⁶ CFU / mL) showing the greatest effect. 8 The concentration of cfu / mL had the strongest promoting effect on the germination of Jinpeng No. 1 seeds.

[0104] B. Effects of different concentrations of Bacillus polymyxa X-11 spore solution on tomato seed radicle elongation during germination.

[0105] When using the pink crown variety as the test subject, the use of X-11 stock solution for germination significantly inhibited the elongation of the seed radicle. Using X-11 bacterial solution diluted 300 times (3.33 × 10⁻⁶) further reduced the effect. 6 After germination with cfu / mL, the longest radicle reached 4.5cm, significantly higher than that of other dilutions of the bacterial solution, and compared with the original solution (1×10⁻⁶). 9 The most significant difference was between the two (cfu / mL), with the former having a radicle 5.8 times longer than the latter (e.g., cfu / mL). Figure 12 , 13 It is evident that the X-11 bacterial solution, diluted 300 times, has the strongest effect on promoting the elongation of the radicle of *Pterocarya stenoptera* seeds.

[0106] When using Jinpeng No. 1 as the test subject, X-11 bacterial suspension diluted 50 times (2×10⁻⁶) was used. 7 After germination with cfu / mL solution, the radicle was the longest, reaching 5.3 cm, significantly higher than other dilutions, and compared with the original solution (1×10⁻⁶ CFU / mL). 9 The most significant difference was observed in the cfu / mL ratio, with the former having a root length 5.37 times greater than the latter (e.g., cfu / mL). Figure 14 , 15 It can be seen that X-11 was diluted 50 times (bacterial concentration 2×10⁻⁶). 7 At a concentration of cfu / mL, the effect on promoting the elongation of the radicle of Jinpeng No. 1 tomato seeds was strongest.

[0107] (2) Effects of Bacillus polymyxa X-11 on the activity of tomato defense enzymes

[0108] A. Effects of Bacillus polymyxa strain X-11 on SOD enzyme activity in tomato

[0109] Compared with the control group, the application of Bacillus polymyxa X-11 through root drenching combined with foliar spraying increased the SOD activity of tomato leaves by 34.6%. Although the difference was not statistically significant, this treatment showed a trend towards increasing SOD activity. Meanwhile, the difference in SOD activity was significant between foliar spraying and root drenching combined with foliar spraying, with the latter increasing SOD activity by 79.81% compared to the former (e.g., ...). Figure 16 Under the treatment of Bacillus polymyxa X-11 inoculant by root irrigation combined with foliar spraying, the SOD activity in tomato leaves was 1.34 times higher than that treated with carbendazim (e.g., ...). Figure 17 , Indicates carbendazim, (This refers to Bacillus polymyxa X-11). It is evident that, compared to carbendazim under the same application method, root irrigation combined with foliar spraying of Bacillus polymyxa X-11 inoculant is more effective in enhancing SOD activity in tomato leaves.

[0110] B. Effects of Bacillus polymyxa X-11 on PALase activity in tomato

[0111] Compared with the blank control, the PAL activity in tomato leaves was as high as 40.35 U / g under the Bacillus polymyxa X-11 root drenching treatment, which was 19.02% higher than the control. However, the PAL activity did not differ significantly among the Bacillus polymyxa X-11 treatment methods (root drenching, foliar spraying, and root drenching combined with foliar spraying) (e.g., Figure 18 Under all treatment methods, the PAL activity of *Bacillus polymyxa* X-11 treatment showed a higher trend than that of carbendazim treatment under the same treatment method. Especially under root irrigation treatment, the PAL activity of tomato leaves treated with *Bacillus polymyxa* X-11 was 33.17% higher than that of carbendazim treatment (e.g., ...). Figure 19 , Indicates carbendazim, (This refers to Bacillus polymyxa X-11). It is evident that root drenching with Bacillus polymyxa X-11 has an inducing effect on PAL activity, and the effect is superior to that of carbendazim.

[0112] Polymyxin Bacillus X-11 can promote tomato seed germination and radicle elongation. The optimal concentration for promoting growth varies depending on the tomato variety. Excessive concentration of Polymyxin Bacillus X-11 can inhibit seed germination and radicle elongation. Polymyxin Bacillus X-11 can also induce increased PAL activity in tomato leaves, and the induction effect is better than that of carbendazim.

[0113] III. Effects of Bacillus polymyxa X-11 on cold resistance and yield of tomatoes

[0114] 1. Method

[0115] This experiment was conducted in a glass greenhouse from October 2021 to March 2022. The *Bacillus polymyxa* X-11 spore solution used was prepared in advance and stored at 4°C for 6 months before use. The tomato hybrid variety Jinlinglong (T-3060) [bred by Nongyou Seedling (China) Co., Ltd.] was used as the test plant, and a field planting experiment was conducted with a plot area of ​​20m². 2 Tomatoes were planted in raised beds with a plant spacing of 40cm × 50cm. A combination of root drenching and foliar spraying with *Bacillus polymyxa* X-11 was used to treat tomatoes three times, at 1, 5, and 9 weeks after transplanting. Before treatment, *Bacillus polymyxa* X-11 spores were diluted 500 times with tap water. For root drenching, 200mL of diluted X-11 was used per plant. For foliar spraying, 2-10mL of diluted X-11 was used per plant, depending on the number of leaves. Foliar spraying was continued until the leaves were covered with droplets without dripping. The first spraying treatment used 2mL of diluted X-11 per plant, the second treatment used 5mL, and the third treatment used 10mL. Tap water was used as a blank control. Three biological replicates were set up for each treatment and control, and plots were randomly assigned. Other field management practices were the same as conventional production.

[0116] Due to the low winter temperatures, reaching as low as -5℃ and the greenhouse temperature dropping to 2℃, tomatoes suffered chilling injury. We unexpectedly found that plants treated with Bacillus polymyxa X-11 showed milder symptoms than the control group, and subsequently investigated the severity of the chilling injury. Observations revealed that a compound leaf approximately 1.4m above the ground on the tomato plant was most severely affected by chilling injury. The top leaf and two adjacent leaflets on either side of this leaf showed obvious symptoms, and the degree of damage differed between the top leaf and these two leaflets. Therefore, we selected the most severely affected compound leaf for investigation, recording the disease severity of the top leaf and the adjacent leaflets. Ten plants were randomly selected from each plot for investigation. The disease severity is classified into six levels based on the area of ​​lesions caused by cold damage to the leaflets. Level 0: no discoloration of the entire leaf; Level 1: slight chlorosis at the leaf margins; Level 2: chlorosis of less than 1 / 10 of the leaf margin with spots less than 1 mm in diameter; Level 3: chlorosis of 1 / 10–1 / 5 of the leaf area with spots 1–3 mm in diameter; Level 4: chlorosis of 1 / 5–1 / 3 of the leaf area with spots 3–6 mm in diameter; Level 5: chlorosis of 1 / 3–1 / 2 of the leaf area with spots larger than 6 mm in diameter; Level 6: chlorosis of more than 1 / 2 of the leaf area with spots larger than 6 mm in diameter. The disease index is calculated using the following formula.

[0117] Disease severity index = 100 × [∑(number of cases at each level × corresponding level)] / (total number of cases surveyed × highest level)

[0118] 2. Results

[0119] The results of the chilling injury investigation showed that the disease indices of the terminal and lateral leaves of the compound leaves in the control group were 86.7 and 53.9, respectively, while those in the Bacillus polymyxa X-11 treatment group were 37.8 and 29.2, respectively. The disease indices of the Bacillus polymyxa X-11 treatment group were significantly lower than those of the control group in both the terminal and lateral leaves (e.g., [missing data]). Figure 20 , 21 ,twenty two).

[0120] Yield measurement results showed that the number of fruits per tomato plant and the weight of individual fruits treated with *Bacillus polymyxa* X-11 were both higher than those of the control, but neither was statistically significant. However, *Bacillus polymyxa* X-11 treatment significantly increased the yield per plant and the theoretical yield compared to the control, by 23.8% (e.g., ...). Figure 23-26 It is evident that Bacillus polymyxa X-11 has a significant effect on increasing tomato yield.

[0121] IV. Antagonistic Effects of Bacillus polymyxa X-11 on Rice and Tomato Pathogens

[0122] 1. Method

[0123] The antibacterial activity of antagonistic bacteria against pathogens was determined using the plate confrontation method, with each treatment replicated in triplicate. *Bacillus polymyxa* X-11 was activated on PDA plates for 2 days. The tested plant pathogens, *Fusarium fujikuroi*, *Fusarium oxysporum*, *Magnaporthe oryzae*, *Botrytis cinerea*, and *Exserohilum*, were then tested. (Turcicum) were activated on PDA plates for 4 days. Bacterial blocks were punched from the edge of the pathogen colony using a punch or cut with an inoculation needle and inoculated into the center of a 90mm PDA-covered petri dish. Except for corn leaf blight and Bacillus polymyxa X-11, which were inoculated simultaneously, all other pathogens were cultured for 48 hours. Then, Bacillus polymyxa X-11 was inoculated at a point 22.5mm from the center of the pathogen block using a spot inoculation method. PDA plates inoculated only with pathogens served as a control. The plates were incubated at 28℃ for another 48 hours. The antibacterial effect of Bacillus polymyxa X-11 on the pathogens was observed, the width of the inhibition zone was measured, and the inhibition rate was calculated using the following formula:

[0124] Inhibition rate % = (Control colony radius - Treated colony radius) / Control colony radius × 100%.

[0125] 2. Results

[0126] *Bacillus polymyxa* X-11 exhibited significant inhibitory effects against *Bakanae diseased*, *Rhizoctonia solani*, *Magnaporta oryzae*, *Botrytis cinerea*, and *Helicobacter pylori*, with average inhibition band widths of 8.1 mm, 7.1 mm, 12.1 mm, 18.1 mm, and 14.5 mm, respectively, and inhibition rates of 50%, 51.1%, 41.7%, 72.7%, and 100% (e.g., *Bacillus polymyxa* X-11). Figure 27 It is evident that *Bacillus polymyxa* X-11 exhibits antagonistic activity against the aforementioned plant pathogens and can be used to inhibit their growth. Notably, in this invention, *Bacillus polymyxa* X-11 achieved a 100% inhibition rate against *Cephalotaxus fortunei*, meaning that *Cephalotaxus fortunei* colonies could not form on the side inhibited by *Bacillus polymyxa* X-11, and only half a colony formed on the side unrestricted by *Bacillus polymyxa* X-11. This inhibitory effect was achieved when *Bacillus polymyxa* X-11 and *Cephalotaxus fortunei* were inoculated simultaneously. If *Bacillus polymyxa* X-11 was inoculated at three points around *Cephalotaxus fortunei*, the pathogen would exhibit an inability to grow. For other pathogens, *Bacillus polymyxa* X-11 was inoculated 48 hours later than the pathogen, achieving the desired effect. Figure 27 The antagonistic effects are shown in A, B, C, and D. If X-11 is inoculated simultaneously with the pathogen, because the pathogen multiplies more slowly, the antibacterial substances secreted by *Bacillus polymyxa* X-11 will diffuse to a greater extent in the surrounding culture medium before the pathogen colony expands, thus confining the original bacterial colony to the inoculation site. This will also result in the observed phenomenon. Figure 14 The pathogen shown in Figure E failed to form colonies on the side inoculated with Bacillus polymyxa X-11. This demonstrates that the antibacterial effect of Bacillus polymyxa X-11 is positively correlated with its growth time; the longer the growth time, the more antibacterial active substances it produces and diffuses into the environment, resulting in a wider area of ​​influence. Therefore, accurately timing the use of Bacillus polymyxa X-11 is crucial for achieving its optimal inhibitory effect.

Claims

1. A polymyxin Bacillus strain, characterized in that... The polymyxobin is a polymyxobin (Bacillus polymyxobin) Paenibacillus polymyxa X-11 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is August 3, 2020, and the accession number is CGMCC No. 20476.

2. The application of Bacillus polymyxa X-11 as described in claim 1 in promoting root elongation in rice.

3. The application of Bacillus polymyxa X-11 as described in claim 1 in improving the activity of superoxide dismutase in rice.

4. The application of Bacillus polymyxa X-11 as described in claim 1 in promoting the elongation of tomato radicles.

5. The application of Bacillus polymyxa X-11 as described in claim 1 in improving the cold resistance of tomatoes.

6. The application of Bacillus polymyxa X-11 as described in claim 1 in increasing tomato yield.

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