Application of sodium alginate in enhancing bacillus subtillis in plant disease control

Abstract

The invention discloses an application of sodium alginate in enhancing bacillus subtillis in plant disease control. According to the application of the sodium alginate and the bacillus subtillis R31 in preventing and treating plant diseases, the bacillus subtillis R31 is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M209261. Sodium alginate is used as a synergist, so that the formation of a biofilm of bacillus subtillis can be enhanced, the athletic ability of bacillus subtillis under different concentrations can be improved, and the planting ability of a plant root system is improved, so that the prevention effect on banana wilt is improved.

Description

Technical Field

[0001] This invention belongs to the field of plant protection technology, specifically relating to a method for enhancing the formation of biofilm by Bacillus subtilis R31 using sodium alginate and improving its control efficacy against banana wilt disease. Background Technology

[0002] banana( Musa Banana wilt (Fusarium wilt) is one of the world's most important tropical fruits, but its production has long been affected by Fusarium wilt (Fusarium wilt). Fusarium The disease is threatened by *Fusarium oxysporum* var. *cubicans* (FWB). Fusarium oxysporum Caused by *F. sp. cubense* (FOC), this fungal infection spreads through the soil and infects the vascular system, leading to plant death. In continuous cropping patterns, declining soil organic matter and rhizosphere microbial imbalance are key triggers for *F. whey* outbreaks: the microbiome, previously dominated by bacterial functional modules, gradually shifts to fungal dominance, further weakening plant resistance and promoting pathogen colonization. Studies have shown that FOC invasion significantly alters the rhizosphere microbial structure, manifested as decreased bacterial and actinomycete abundance, fungal proliferation, and carbon metabolism disorders.

[0003] Currently, FWB control mainly relies on disease-resistant breeding and biological control. Chemical control is only used as a supplementary method due to its low efficiency and high environmental pollution risk; while biocontrol agents (such as Pseudomonas, Trichoderma, and Bacillus) have attracted much attention due to their high environmental compatibility and diverse mechanisms of action. Among them, Bacillus ( Bacillus Bacillus spp. has significant advantages in formulation processing, storage and rhizosphere colonization due to its heat-resistant spore characteristics, but its commercial application is still limited: only 19 Bacillus spp. formulations in existing patents target FWB, and there is a lack of high-efficiency products based on synbiotics (probiotic-prebiotic synergy).

[0004] Bacillus subtilis ( Bacillus subtilis R31 is an endophytic strain isolated from Dendrobium leaves by our team. Its control efficacy against FWB is positively correlated with its biofilm formation ability and rhizosphere colonization level. However, field trials show that the R31 formulation requires a spore concentration of 1×10⁻⁶. 7 The required concentration of CFU / mL is necessary to achieve ideal control efficacy, resulting in high costs. To improve the colonization efficiency of R31 and reduce application costs, our team discovered through preliminary screening that adding sodium alginate can significantly enhance its biofilm formation ability. Among these, plant polysaccharides (such as sodium alginate) have the dual potential to promote probiotic colonization and induce plant stress resistance, providing a theoretical basis for the development of synbiotic Bacillus preparations. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned shortcomings of the prior art by providing the application of sodium alginate combined with Bacillus subtilis R31 in the prevention and control of plant diseases.

[0006] Another objective of this invention is to provide a method for improving the efficacy of Bacillus subtilis R31 against banana wilt using sodium alginate.

[0007] Another objective of this invention is to provide a biological pesticide for controlling banana wilt disease.

[0008] This invention is achieved through the following technical solutions: The application of sodium alginate combined with Bacillus subtilis R31 in the control of plant diseases, Bacillus subtilis ( Bacillus subtilis R31, deposited at the China Center for Type Culture Collection, on November 12, 2009, accession number CCTCCNO:M209261.

[0009] Preferably, the plant disease is banana wilt.

[0010] Application of sodium alginate combined with Bacillus subtilis R31 in the preparation of products for the prevention and control of plant diseases.

[0011] Preferably, the plant disease is banana wilt.

[0012] Preferably, the concentration of sodium alginate in the product is 40-400 µg / mL, more preferably 400 µg / mL.

[0013] A method for enhancing the control efficacy of Bacillus subtilis R31 against banana wilt using sodium alginate involves applying a Bacillus subtilis R31 fermentation broth containing sodium alginate to the rhizosphere of banana plants, with an application rate of 50 mL per banana plant. The Bacillus subtilis R31 fermentation broth containing sodium alginate is prepared by the following method: Bacillus subtilis R31 is inoculated into NB liquid culture medium at an inoculum rate of 6%, sodium alginate stock solution is added, and fermentation is carried out at 37°C with shaking for 48 h to obtain the fermentation broth. The final concentration of sodium alginate in the NB liquid culture medium is 40-400 µg / mL.

[0014] Preferably, the concentration of the sodium alginate mother liquor is 0.5% and the viscosity is 46.632 mPa·s.

[0015] A biological pesticide for controlling banana wilt disease is a Bacillus subtilis R31 fermentation broth containing sodium alginate at a final concentration of 40-400 µg / mL.

[0016] Preferably, the biopesticide is prepared by the following method: Bacillus subtilis R31 is inoculated into NB liquid culture medium at an inoculation rate of 6%, sodium alginate stock solution is added, and fermentation is carried out at 37°C with shaking for 48 h to obtain fermentation broth. The final concentration of sodium alginate in NB liquid culture medium is 40-400 µg / mL.

[0017] Preferably, the concentration of sodium alginate stock solution is 0.5%, and the final concentration of sodium alginate in NB liquid culture medium is 400 µg / mL.

[0018] Beneficial effects

[0019] To explore the mechanism by which sodium alginate promotes the formation of Bacillus subtilis R31 biofilm and improves its control effect on banana wilt disease, the concentration-viscosity relationship of sodium alginate was optimized (0.5% stock solution, 46.632 mPa·s) to achieve stable formation of Bacillus subtilis R31 biofilm, overcoming the bottleneck of traditional biofilms being easily affected by environmental conditions and providing structural support for strain colonization. It was found that sodium alginate has a concentration-dependent bidirectional regulatory effect: low concentrations promote biofilm formation and enhance bacterial stability, while high concentrations enhance bacterial motility and root colonization efficiency. Furthermore, it directly stimulates bananas to produce endogenous defense enzyme systems, enhancing the antagonistic activity of R31 against Fusarium oxysporum XJZ2. Through concentration gradient regulation, the problem of colonization decline of biocontrol bacteria was successfully solved. The 400 µg / mL treatment group achieved continuous colonization growth (peaking at 15 days), overcoming the technical bottleneck of short effective period of traditional inoculants. After determining the appropriate concentration, a banana pot experiment was conducted. The results showed that sodium alginate as an synergist can enhance the biofilm formation of Bacillus subtilis and improve its motility and root colonization ability at different concentrations, thereby improving the control efficacy against banana wilt disease. Attached Figure Description

[0020] Figure 1 The study investigated the effects of different concentrations of sodium alginate on the biological phenotype of Bacillus subtilis R31. Specifically: A: Results of sodium alginate at different mother liquor concentrations on the formation of a thin film on R31; B: Quantitative results of crystal violet on the formation of a thin film on R31 using sodium alginate at different mother liquor concentrations; C: Determination of the growth curve of R31 using sodium alginate; D: Quantitative analysis of biofilm amount using crystal violet staining; E: Determination of the formation of a thin film on R31 using sodium alginate; F: Effect of sodium alginate on the motility of R31.

[0021] Figure 2 This study investigated the effects of sodium alginate on the colonization of R31 in banana roots and the determination of enzyme activities in banana roots. Specifically, A represents the colonization status of R31 in banana roots at different time points; B represents the determination of the amount of R31 colonized in banana roots and the determination of various enzyme activity indicators. Figure 3Sodium alginate affects the control effect of R31 on FOC; where A: plate antagonistic activity experiment; B: pot experiment; C: pot experiment control efficacy index. Information on the preservation of biological materials

[0022] Bacillus subtilis R31, classified and named Bacillus subtilis R31 is deposited at the China Center for Type Culture Collection (CCTCC), dated November 12, 2009, at Wuhan University, Wuhan, China, with accession number CCTCCNO:M209261. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0024] Example 1

[0025] 1. Test strains

[0026] Green fluorescently labeled R31 (pHT315-gfp) and wild-type R31 strains were preserved by the Institute of Plant Health Innovation, Zhongkai University of Agriculture and Engineering / Key Laboratory of Green Control of Fruits and Vegetables in South China, Ministry of Agriculture and Rural Affairs / Key Laboratory of Green Control of Fruits and Vegetables in Guangdong Province.

[0027] 2. Test plant materials

[0028] Brazilian banana ( Musa (spe. AAA) Purchased from Zhanjiang Haida Nursery Base. Place the purchased Brazilian bananas outdoors and spray them with a small amount of water regularly to keep the leaves moist. After 7 days, thoroughly wash the agar off the roots of the seedlings with water, soak the roots in 0.3% thiram solution for 10 seconds, then rinse twice with water for disinfection. Plant the seedlings in sterilized quartz sand trays and place them in a plant light incubator at 28℃, watering regularly with Hoagland's nutrient solution. When the seedlings have grown to 3-5 leaves, remove them from the tray, wash away the mud and sand with clean water, and place some of the seedlings in jam jars wrapped in black plastic sheeting. Cultivate them in water, and after a few days, add Hogrange medium. Once the seedlings have grown whiter and tender new roots, they can be inoculated for experiments. The other part is transferred to flower pots containing sterilized substrate soil and yellow clay soil (1:3 v / v mixture), watered regularly, and urea is applied to supplement nutrients. After the bananas have grown 6 leaves, pot experiments can be conducted.

[0029] 3. Test reagents and culture media

[0030] LB medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 18 g agar powder, adjust pH to 7.0, bring volume to 1 L, sterilize and use.

[0031] BGDM medium: 26.631 g tripotassium phosphate trihydrate (pH=7.0), 9.999 g trisodium citrate dihydrate, 0.2465 g magnesium sulfate, 19.821 g ammonium sulfate, 3 g beef extract, 5 g NaCl, 10 g bacteriological peptone, 1 g glucose, add distilled water to 1000 mL, pH=7.0, sterilize and use.

[0032] Msgg medium: Potassium phosphate 5 mmol / L (pH 7.0), MOPs 100 mmol / L (pH 7.0), Magnesium chloride 2 mmol / L, Calcium chloride 700 mmol / L, Manganese chloride 50 µM, Ferric chloride 50 µM, Zinc chloride 1 µM, Vitamin B1 2 µM, Glycerol 0.5%, Glutamic acid 0.5%, Tryptophan 50 µg / mL, Phenylalanine 50 µg / mL, Distilled water 1000 mL, pH 7.0, sterilized for later use.

[0033] NA medium: 3 g beef extract, 10 g bacteriological peptone, 5 g sodium chloride, 2.5 g glucose, 15 g agar powder, bring to a final volume of 1 L, adjust pH to 7.0, and sterilize for later use.

[0034] NB liquid culture medium: 3 g beef extract, 10 g bacteriological peptone, 5 g sodium chloride, 2.5 g glucose, bring to a final volume of 1 L, adjust pH to 7.0, and sterilize for later use.

[0035] 0.7% and 0.5% NA semi-solid culture medium: 3 g beef extract, 10 g bacteriological peptone, 5 g sodium chloride, 2.5 g glucose, agar powder (added according to the required ratio), adjust pH to 7.0, sterilize and use.

[0036] The culture of fluorescently labeled organisms requires the addition of antibiotics: the final concentration of ampicillin in Escherichia coli culture medium is 100 µg / ml, and the final concentration of erythromycin in Bacillus subtilis culture medium is 50 µg / ml.

[0037] Sodium alginate AR 90%: McLean.

[0038] 4. Fermentation broth preparation method

[0039] Activate R31 on LB agar plates and incubate at 37°C for 12 h. Inoculate a single colony into 5 mL of LB liquid medium and incubate at 37°C with shaking for 12 h to obtain a secondary seed culture. Inoculate the secondary seed culture at 1% (v / v) into a fresh 20 mL LB liquid medium and incubate at 37°C with shaking for 4 h to obtain a tertiary seed culture. Inoculate the tertiary seed culture at 6% (v / v) into 200 mL of NB liquid medium and ferment at 37°C with shaking for 48 h to obtain the fermentation broth.

[0040] 5. Screening of the optimal concentration and viscosity of sodium alginate affecting R31 biofilm formation

[0041] To determine the concentration and viscosity of sodium alginate stock solutions, sodium alginate was accurately weighed, and appropriate amounts were added to 50 mL of deionized water. The mixture was gently stirred until the sodium alginate was completely dissolved. After allowing the solutions to stand for several minutes, the temperature was adjusted to 25℃±0.1℃ using a constant temperature water bath to prepare stock solutions with mass fractions of 0.1%, 0.3%, 0.5%, and 0.7%, respectively. The samples were then injected into a viscometer, and the viscosity value of the sodium alginate was determined based on the reading displayed on the viscometer. Specific viscosity measurement conditions are detailed in Table 1 below.

[0042] Table 1

[0043] concentration Time (S) rotational speed (rpm) Viscosity mPa·s 0.1% 60 100 1.343 0.3% 60 100 22.148 0.5% 60 100 46.632 0.7% 60 100 80.144 The effects of different concentrations and viscosities of sodium alginate on biofilm formation of R31 were investigated. Three-stage seed culture of R31 was obtained according to the method in step 4. The three-stage seed culture was diluted with fresh BGDM liquid medium for 4–4.5 h until the OD value at 600 nm was 0.02, yielding R31 dilutions. Different concentrations of sodium alginate (0.1%, 0.3%, 0.5%, 0.7%) were added to 24-well plates containing BGDM liquid medium, with the control group receiving no sodium alginate. The experimental temperature was maintained at 25℃. The final concentration in each well was ensured to reach 40 μg / mL. Then, 9 µL of R31 dilution was added to each well. The experimental system is detailed in Table 1. The plates were incubated at 37℃ for 24 h, and the formation of the thin film was observed and photographed. The bacterial culture, excluding the thin-skinned biofilm, was aspirated from the wells. Residual culture was blotted dry with filter paper strips, and then stained with 1% crystal violet for 20 min. After staining, the unstained crystal violet was washed with double-distilled water until the water in the tube was colorless. The stained biofilm was then eluted with 33% glacial acetic acid. OD was then measured. 570 The reading represents the amount of biofilm.

[0044] Table 2. Experimental system for observing the effect of sodium alginate at different mother liquor concentrations on biofilm formation.

[0045] Note: The final concentration of sodium alginate is 40 μg / mL.

[0046] Viscosity measurements of sodium alginate mother liquor at different concentrations showed a positive correlation between concentration and viscosity; higher concentrations resulted in higher viscosity. The viscosity of the sodium alginate mother liquor affected the formation of a thin film of R31 in BGDM. Figure 1 A). R31 cannot form a film in BGDM medium. After adding sodium alginate at concentrations of 0.1%, 0.3%, 0.5%, and 0.7% of the stock solution to BGDM and culturing for 24 h, it was found that when the stock solution concentration was above or below 0.5%, the film formation of R31 was incomplete, appearing as a mesh, and some of it precipitated to the bottom of the liquid medium; when the stock solution concentration was 0.5%, the film formation of R31 was relatively complete. Figure 1 A); Crystal violet staining further quantitatively determined that a 0.5% concentration of sodium alginate stock solution was most favorable for the formation of R31 thin skin. Figure 1 B). In summary, the concentration and viscosity of the sodium alginate stock solution are related, and both concentration and viscosity directly affect the morphology of the biofilm. The experimental results confirm that adding a sodium alginate stock solution with a concentration of 0.5% and a viscosity of 46.632 mPa·s can promote the formation of R31 biofilms without altering their morphology.

[0047] 6. Effects of sodium alginate on the growth and biofilm formation of strain R31 Differences in biofilm formation ability can be determined by observing the morphological differences between colonies formed by Bacillus strains on MSgg solid medium and peillicles formed on liquid medium (Branda et al., 2004). Based on the experimental results of step 5, the optimal viscosity for sodium alginate to promote biofilm formation was determined, and the optimal working concentration of sodium alginate was further screened. The obtained data were summarized using Excel 2010, and analysis of variance and multiple comparisons were performed using the bioassay method and LSD in DPS 7.05 software. Growth curves were plotted using GraphPad Prism 9.0.2.

[0048] According to the seed culture preparation method in step 4, R31 tertiary seed culture was obtained. The seed culture was added at a rate of 1% (so that the amount of R31 inoculated is 1% of the volume of MSgg liquid medium) to MSgg liquid medium with different final concentrations of sodium alginate (5 µg / mL, 10 µg / mL, 20 µg / mL, 30 µg / mL, 40 µg / mL, 50 µg / mL, 60 µg / mL, 400 µg / mL; sodium alginate stock solution concentration is 0.5%, temperature 25℃). The medium was cultured at 37℃ and 180 rpm with shaking. Then, the OD value at 600 nm was measured at 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h, and the curve of the relationship between time and OD value was plotted. Sodium alginate was diluted to a concentration of 0.5% at 25°C. Different concentrations of sodium alginate were added to 24-well cell culture plates already filled with fresh MSgg liquid medium and MSgg solid medium, respectively. The experimental system is detailed in Table 2. The cells were incubated statically for 12 h, 24 h, and 48 h, and the formation of the thin film was observed and photographed. The quantitative method for film staining is described in Method 5. Table 3. Experimental system for observing the effect of sodium alginate at different working concentrations on biofilm formation. Final sodium alginate concentration (μg / mL) Sodium alginate addition amount (μL) Grade III seed solution (μL) MSgg medium (μL) Total volume (μL) CK 0 9 1991 2000 5 2 9 1989 2000 10 4 9 1987 2000 20 8 9 1983 2000 30 12 9 1979 2000 40 16 9 1975 2000 50 20 9 1971 2000 60 24 9 1967 2000 400 160 9 1831 2000 Note: The concentration of sodium alginate mother liquor is 0.5%.

[0049] Sodium alginate can increase the growth of Bacillus subtilis R31 strain in MSgg medium. Figure 2 C). The OD values ​​of the R31 fermentation broth after adding different concentrations of sodium alginate were higher than those of the control without sodium alginate. Each working concentration in the experiment increased the bacterial count; the higher the working concentration and the longer the culture time, the greater the OD value of the fermentation broth. Sodium alginate addition shortened the lag phase of R31. R31 was in the logarithmic growth phase at 4-8 h and reached the stationary phase after 20 h, at which point the growth of the strain slowed down. Analysis of variance and multiple comparisons (Table 4) revealed that, after adding different concentrations of sodium alginate, the working concentration of 40 µg / mL had the most significant effect on increasing the R31 bacterial count during the stationary phase. Different concentrations of sodium alginate promoted the formation of a thin skin or submerged film of R31 in the liquid culture medium. With increasing sodium alginate concentration, a small amount of floating thin skin began to appear on the surface of the R31 culture, while bacterial cells precipitated at the bottom of the liquid to form a submerged film. After 24 h, a complete thin skin began to form on the surface of the liquid, and a large number of bacterial cells appeared at the bottom of the liquid. The higher the sodium alginate concentration, the more bacterial cells accumulated at the bottom. Figure 1 E). As determined by crystal violet staining, strain R31 exhibited the highest rate of thin-skin formation when sodium alginate concentration was 40 µg / mL. Figure 1D). The above studies have determined that sodium alginate at a concentration of 40 µg / mL can promote the growth of Bacillus subtilis and the formation of a thin skin, at which the skin structure is most stable.

[0050] Table 4. Effects of different concentrations of sodium alginate on the growth of strain R31

[0051] Note: The data in the table are mean ± standard deviation (n=5). Different lowercase letters in the same column represent Duncan's test for significance at r < 0.05.

[0052] 7. Effects of sodium alginate on the movement and cell morphology of strain R31 on solid surfaces A. Preparation of seed culture of strain R31 The experimental method refers to the preparation of Bacillus subtilis seed solution in step 4.

[0053] B. Swarming ability assessment Freshly prepared 0.5% NA semi-solid medium was shaken well, and 0.5% sodium alginate stock solution was added to obtain semi-solid medium containing different final concentrations of sodium alginate. The medium was poured into plates and dried in a clean bench for 30 min. 2 μL of R31 tertiary seed culture was inoculated in the center of the plate, and then dried for another 10 min. After static incubation at 37℃ for 24 h, the bacterial motility was observed and measured. The strength of the colony's ability to swim was determined based on the diffusion of the strain on the plate.

[0054] C. Sliding ability measurement Freshly prepared 0.7% NA semi-solid medium was shaken well, and 0.5% sodium alginate stock solution was added to obtain semi-solid medium containing different final concentrations of sodium alginate. This medium was poured into plates and dried in a clean bench for 30 min. 2 μL of the medium was inoculated in the center of the plate with R31 tertiary seed culture, and then dried for another 10 min. After static incubation at 37℃ for 4 h, the bacterial motility was observed and measured. The strength of the sliding ability was determined based on the diffusion of the strain on the plate.

[0055] In the experiment, we selected the final sodium alginate concentration (40 µg / mL) that had the most stable effect on R31 biofilm formation as the experimental concentration, and further increased this concentration by 10 times as a high-concentration control group. At a sodium alginate concentration of 400 µg / mL, Bacillus subtilis R31 failed to form a complete thin layer on MSgg liquid medium; its cells appeared flocculent and completely sank to the bottom. Colony swimming experiments conducted on 0.5% semi-solid NA medium showed that, compared to the control group, after treatment with 40 µg / mL sodium alginate, R31 could not completely cover the entire plate within 24 h. Figure 1 F), while R31 treated with 400 µg / mL sodium alginate was able to swim across the entire plate. Sliding performance tests on 0.7% NA medium showed that both different concentrations of sodium alginate treatment improved the sliding performance of R31. Our research found that low concentrations of sodium alginate had little effect on the swarming behavior of R31, but did enhance its sliding performance; when the concentration of sodium alginate increased significantly, the swimming and sliding performance of R31 was significantly enhanced. Figure 1 F). The findings mentioned above reveal that the working concentration of sodium alginate has different effects on R31: higher concentrations mainly affect motility, while lower concentrations may alter the ability to form a film.

[0056] By observing the colonies of strain R31 on MSgg solid plates, we found that the control group of R31 cells showed significant wrinkling after 7 days of incubation. However, on the medium supplemented with 40 µg / mL sodium alginate, the colonies showed no significant morphological difference compared to the control group, and their surfaces were smooth without wrinkles.

[0057] D. Effects of sodium alginate on the colonization of R31 in the rhizosphere of banana roots The preparation method for Bacillus subtilis R31 fermentation broth is the same as in step 4. For the co-fermentation broth of sodium alginate and R31, add 1.6 mL and 16 mL of 0.5% sodium alginate stock solution to 200 mL of NB fermentation broth, respectively, to achieve final concentrations of 40 µg / mL and 400 µg / mL. Ferment at 37℃ with shaking for 48 h. Take 0.5 mL of bacterial culture from the fermentation broth for counting; the bacterial count is approximately 1.0 × 10⁻⁶ cells / mL. 9 CFU / mL. Centrifuge the fermentation broth at 4℃ and 6000 rpm for 10 min, discard the supernatant, resuspend the bacteria in sterile water, centrifuge again, and discard the supernatant. Dilute the bacterial suspension to 1.0 × 10⁻⁶ CFU / mL with sterile water. 8 CFU / mL is prepared for later use.

[0058] The colonization effect of Bacillus subtilis strain R31 on the roots of banana seedlings under the influence of sodium alginate was determined using hydroponics. Tissue-cultured banana seedlings were planted in sand pots and allowed to grow to a suitable size. Seven-leaf banana seedlings of similar size were selected, their roots were rinsed with water to remove the culture medium, and then placed in jam jars for further cultivation. An appropriate amount of Hogland nutrient solution was added to each seedling. After one week of growth, the seedlings were used for the experiment. One week later, the culture medium in the jam jars was discarded, and the prepared bacterial solution was added to the jam jars to achieve a final bacterial concentration of 1.0 × 10⁻⁶. 8 CFU / mL; the control group received an equal volume of water. Three banana seedlings were included in each treatment. On days 1, 5, 10, and 15 post-inoculation, the banana seedlings were removed, the roots were rinsed with water, and the epidermis of the root hair zone was manually sectioned from young roots. The prepared root epidermal specimens were observed and photographed under a laser confocal microscope. Based on the observed concentrations of green fluorescent protein (GFP) markers, the colonization ability of the strain in banana roots was preliminarily determined.

[0059] After rinsing the banana roots with sterile water, the root hair zone was cut into approximately 2 cm lengths using scissors. 1 g of root from each plant was randomly selected and mixed thoroughly. 1 mL of PBS solution was added to a mortar and pestle, and the roots were thoroughly ground. The mixture was then poured into a centrifuge tube containing 8 mL of sterile water and vortexed for 15 min. The resulting aqueous solution was used as the stock solution. Serial dilutions were performed with sterile water, and the solutions were spread onto NA plates containing 50 μg / mL erythromycin. Bacterial counts were determined, with three dilution gradients selected for plating, and three replicates for each dilution concentration. The plates were incubated at 37°C for approximately 24 h. A fluorescent excitation flashlight was used to illuminate the plates, and the number of colonies emitting green fluorescence in each plate was recorded. Finally, the bacterial count per gram of root was calculated.

[0060] The labeled strains were detected in banana roots 1 day after inoculation using laser confocal microscopy. Figure 2 A) The colonization rate of banana roots treated with untreated R31 fermentation broth and those treated with two concentrations of R31 fermentation broth was compared, with colonization rates of 0.668 × 10⁻⁶ and 0.668 × 10⁻⁶ respectively. 5 cfu / g (fresh weight) and 1.3×10 5 cfu / g (fresh weight) and 1.334×10 5 CFU / g (fresh weight). Ten days after inoculation, the colonization rate of R31-gfp showed an increasing trend. After 15 days, the colonization rate of untreated R31 fermentation broth in the root system showed a decreasing trend, with a colonization rate of 1.952 × 10⁵ CFU / g (fresh weight). The colonization rate of R31 treated with 40 µg / mL sodium alginate decreased further, reaching 2.118 × 10⁵ CFU / g (fresh weight). 5The bacterial count was higher than that of wild-type R31 when treated with sodium alginate (400 µg / mL), but still higher than that of wild-type R31 when treated alone. Conversely, the colony count of R31 treated with sodium alginate showed an increasing trend, reaching 3.976 × 10⁵ cfu / g (fresh weight). The results indicate that the colony count of R31 in the sodium alginate-treated fermentation broth was significantly higher than that of wild-type R31, and the bacterial count continued to increase over time. After 15-30 days of inoculation, the bacterial count decreased, but the bacterial count of R31 treated with sodium alginate was consistently higher than that of wild-type R31. Figure 2 B). Enzyme activities in banana roots were measured, and the results showed that co-culturing R31 bacterial suspension with sodium alginate + R31 effectively increased NO content after 24 h. Among plant-related defense enzymes, CAT, PAL, and POD were measured. The results indicated that R31 increased the enzyme activities in banana roots to varying degrees, suggesting that sodium alginate can rapidly enhance R31 colonization in banana plants and improve its long-term colonization in the rhizosphere, which is closely related to R31's inhibition of ROS outbreaks in banana roots.

[0061] Example 2: Effect of sodium alginate on the inhibition of banana wilt pathogen by R31 plates The *Fusarium oxysporum* XJZ2 glycerol inoculum was activated onto PDA plates and incubated at 30°C for 7 days until the mycelium completely covered the plates, yielding fresh *Fusarium oxysporum* XJZ2. For the preparation of the R31 seed culture, refer to step 4. An inoculum of 6% was added to 200 mL of NB liquid medium, and 0.5% sodium alginate was added at 25°C to final concentrations of 40 µg / mL and 400 µg / mL, respectively. Fermentation was carried out at 37°C with shaking for 48 h to obtain the fermentation broth. The antagonistic ability against *Fusarium wilt* pathogen XJZ2 was determined using the plate confrontation method. This method was used to determine the antagonistic ability of wild-type *Bacillus subtilis* R31 and two concentrations of sodium alginate + R31 strains against *Fusarium wilt* pathogen XJZ2.

[0062] Method for determining the antagonistic ability of Bacillus subtilis R31 against pathogens: Banana wilt pathogen XJZ2 was inoculated in the center of an NA+PDA plate, while Bacillus subtilis R31 was inoculated at points bisecting the pathogen's mycelial cake and the edge of the plate. Four replicates were set up on the same plate. After incubation at 30℃ upside down for 7 days, the plates were observed, and the antagonistic ability of Bacillus subtilis against the pathogen was measured and statistically analyzed using vernier calipers. Three replicates were used for each strain.

[0063] Method for determining the antagonistic ability of sodium alginate against pathogens: Inoculate the center of an NA+PDA plate with *Fusarium wilt* fungus XJZ2. Simultaneously, place 5 mm diameter filter paper discs at points bisecting the fungal cake and the edge of the plate. Add 10 μL of sodium alginate + R31 fermentation broth to the filter paper discs. Perform four replicates on the same plate. Place the plates in a 4°C refrigerator and allow the solution to fully diffuse. Then, incubate inverted mode at 30°C for 7 days. Observe the plates and measure the antagonistic ability of *Bacillus subtilis* against the pathogen using calipers. Perform three replicates for each strain.

[0064]

[0065] Note: D0—Coronavirus colony diameter (mm), DS—Coronavirus diameter of the corresponding Bacillus after inhibition (mm) F. The efficacy of different combinations of sodium alginate and R31 in controlling Fusarium wilt in potted bananas.

[0066] After obtaining the fermentation broth according to step 4, the viable cell count was performed, and the cell count was approximately 1.0 × 10⁻⁶. 9 CFU / mL. Dilute the bacterial suspension to 1.0 × 10⁻⁶ CFU / mL with sterile water. 8 CFU / mL is prepared for later use.

[0067] Preparation of XJZ2 spore suspension: The XJZ2 strain, the fourth physiological race of banana wilt disease, was removed from a 4°C freezer. A small amount of mycelium was inoculated onto PDA medium using an inoculation loop and cultured for one week until the fungus had completely covered the plate. In a clean bench, a 100 μL pipette tip was used to create a fungal cake, which was then inoculated onto PDA medium and cultured at 30°C with shaking for one week. The pathogenic spores were washed off the medium using four layers of gauze, and the filtrate was collected. After suspending and stirring the filtrate, 1 mL of the collected XJZ2 spore suspension was quickly taken, diluted 2-3 times with sterile water, and 10-20 μL was dropped onto a hemocytometer for counting. This process was repeated three times. Finally, the spore suspension was diluted to 1.0 × 10⁻⁶ with sterile water. 6 After determining the concentration of cfu / mL, it is ready for use.

[0068] Inoculation method for Brazilian banana potted plants; Using the root irrigation inoculation method, 50 mL of the fermentation broth of wild-type R31 strain, sodium alginate, and sodium alginate + R31 strain (refer to the preparation method in step E) was irrigated around the roots of each banana seedling, and the corresponding volume of tap water was applied by drenching as a clear water control. After 5 days of potted cultivation, the pathogen spore suspension inoculation was carried out using the root injury inoculation method. For each banana seedling, 3 lateral roots were wounded in a "pin" shape with a sterilized scissors, and the spore suspension of pathogen XJZ2 was irrigated at the wound. 30 seedlings were set for each treatment. After 50 days of inoculating the pathogen, the disease incidence of the potted plants was investigated. Combining the wilting of the above-ground leaves and the degree of browning of the cross-section of the corm and the base of the pseudostem, photos were taken and recorded, and the disease index and control effect were calculated. And samples of banana roots, rhizosphere soil, and potted soil were collected for microbiome analysis.

[0069] Disease grading standard for banana wilt disease and evaluation standard for disease index Grade 0 - Healthy plants without browning or fracture of corm or pseudostem tissue.

[0070] Grade 1 - 25% of the leaves turn yellow or the stem tissue turns brown, accounting for less than 1 / 4 of the stem area.

[0071] Grade 3 - 25% - 50% of the leaves turn yellow or the stem tissue turns brown, accounting for 1 / 4 - 1 / 2 of the stem area.

[0072] Grade 5 - 50% to 90% of the leaves turn yellow or the stem tissue turns brown, accounting for more than 1 / 2 of the stem area, and the plant shows mild wilting symptoms.

[0073] Grade 7 - All leaves turn yellow, the plant dies or the entire stem tissue turns brown or rots.

[0074]

[0075] Disease index (DS) = 100×∑(number of diseased leaves at each level × representative value of each level) / (total number of leaves investigated × representative value of the highest level) Control effect (BE) = (disease index of pathogen control - disease index of treatment) / disease index of pathogen control × 100% The calculated data was subjected to one-way analysis of variance and multiple comparisons (Duncan) using DPS, with a significance level of r <0.05, and then plotted using GraphPad Prism 9.0.2.

[0076] The pathogen XJZ2 was inoculated in the center of an agar plate, and the biocontrol bacterium R31 was inoculated around the perimeter. After 7 days of incubation, the antagonistic effect of R31 on the pathogen XJZ2 was observed. When *Fusarium oxysporum* XJZ2 grows naturally on the plate, its hyphae cover the entire plate. Neither of the two individual sodium alginate concentrations (40 µg / mL and 400 µg / mL) showed any antagonistic effect on *Fusarium oxysporum* XJZ2. Within a radius of approximately 2 cm centered on R31, the hyphae of *Fusarium oxysporum* XJZ2 were thin and sparse, mostly aerial hyphae. This indicates that the metabolites secreted by R31 antagonize *Fusarium oxysporum* XJZ2. The results show that R31 with added sodium alginate exhibits strong antagonistic activity against *Fusarium oxysporum* XJZ2, indicating that sodium alginate affects the biofilm formation of *Bacillus subtilis* R31, producing a large amount of metabolites that enhance the antagonistic effect of the strain. Figure 3 By measuring the diameter of pathogen XJZ2 7 days after plate inoculation with pathogen and biocontrol bacteria, it was demonstrated that sodium alginate can affect the antagonistic activity of Bacillus subtilis R31 against pathogen. Figure 3 A). Approximately 40 days after inoculating Brazilian banana potted plants with XJZ2 spore solution, yellowing began to appear on the lower leaf margins. Around 50 days later, the yellowing worsened from the base upwards, and some plants even died. A longitudinal section was cut open at the base of the corm and pseudostem, and the degree of wilting of the above-ground leaves and the degree of browning in the longitudinal section were analyzed (…). Figure 3 B) Classify the disease severity of each plant according to the standard. Longitudinal sections along the central axis of the banana seedling stem revealed that plants with severe yellowing and wilting of leaves were infected with banana wilt disease. The banana seedlings began to rot from the base of the bulb, and purplish-red streaks appeared in the vascular bundles. Calculation results ( Figure 3 C) It was found that the incidence rate of Fusarium oxysporum XJZ2 treatment group (CK) was as high as 73%, with a disease index of 0.489; the incidence rate of wild-type R31 treatment group was 70%, with a disease index of 0.219. Although the incidence rate was close to that of the CK treatment group, when observing the disease grade through cross-section, most of the diseases were grade 1 diseases, indicating that over time, the R31 strain alone could not colonize the banana root system for a long time, and its disease control ability decreased. The incidence rate of R31-40 µg / mL treatment group was 60%, with a disease index of 0.28, and the incidence rate of R31-400 µg / mL treatment group was 33.33%, with a disease index of 0.12. The disease control ability of both groups was more significant than that of R31 alone. The disease control effects of sodium alginate alone for the two groups were: the incidence rate of 40 µg / mL treatment group was 70%, and the incidence rate of 400 µg / mL treatment group was 70%, and the disease control effect of 400 µg / mL treatment group was 400 µg / mL. At a concentration of 63% µg / mL, sodium alginate significantly improved the control of Fusarium wilt. The results indicate that when sodium alginate reaches a certain concentration, it can directly stimulate bananas to produce various defensive enzymes, thereby enhancing the bananas' ability to resist diseases.

Claims

1. The use of sodium alginate combined with Bacillus subtilis R31 in the prevention and treatment of plant diseases, wherein the Bacillus subtilis (Bacillus subtilis) R31 is preserved in the China Center for Type Culture Collection on Nov. 12, 2009, and the preservation number is CCTCC NO: M209261. Bacillus subtilis ​ 2. Use according to claim 1, characterized in that, The plant disease mentioned is banana wilt.

3. Application of sodium alginate combined with Bacillus subtilis R31 in the preparation of products for the prevention and control of plant diseases.

4. Use according to claim 3, characterized in that, The plant disease mentioned is banana wilt.

5. Use according to claim 3, characterized in that, The final concentration of sodium alginate in the product is 40-400 µg / mL.

6. A method for improving the efficiency of Bacillus subtilis R31 in preventing banana fusarium wilt by using sodium alginate, characterized in that, The fermentation broth of Bacillus subtilis R31 containing sodium alginate was applied to the rhizosphere of banana plants at a rate of 50 mL per plant. The fermentation broth of Bacillus subtilis R31 containing sodium alginate was prepared by the following method: Bacillus subtilis R31 was inoculated into NB liquid culture medium at an inoculum of 6%, sodium alginate stock solution was added, and fermentation was carried out at 37°C with shaking for 48 h to obtain the fermentation broth. The final concentration of sodium alginate in NB liquid culture medium was 40-400 µg / mL.

7. The method of claim 6, wherein, The concentration of the sodium alginate mother liquor is 0.5%, and the viscosity is 46.632 mPa·s.

8. A biopesticide for controlling Panama disease of banana, characterized in that, The fermentation broth of Bacillus subtilis R31 containing sodium alginate at a final concentration of 40-400 µg / mL is used.

9. The biopesticide according to claim 8, characterized in that, The biopesticide is prepared by the following method: Bacillus subtilis R31 is inoculated into NB liquid culture medium at an inoculation rate of 6%, sodium alginate stock solution is added, and fermentation is carried out at 37℃ with shaking for 48 h to obtain fermentation broth. The final concentration of sodium alginate in NB liquid culture medium is 40-400µg / mL.

10. The biopesticide as claimed in claim 8, wherein, The concentration of sodium alginate stock solution was 0.5%, and the final concentration of sodium alginate in NB liquid medium was 400 µg / mL.

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