Bacillus vetzmannii for inhibiting phytopathogen and application of bacillus vetzmannii

Abstract

The invention discloses bacillus Witzmannii for inhibiting phytopathogen and application of the bacillus Witzmannii, relates to the field of biological prevention and control, and aims to solve the problem that chemical prevention and control methods of phytopathogen cause negative effects on ecological environment and human health. The bacillus wiedmannii is bacillus wiedmannii SJZ and is preserved in the China Center for Type Culture Collection, the preservation date is December 23, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 20242884. The bacillus vetzmannii disclosed by the invention can be used for inhibiting sclerotinia sclerotiorum and solanum nigrum. And protease, cellulase and chitinase can be secreted. The bacillus vetzmannii is used for nitrogen fixation and can secrete siderophores and IAA (indoleacetic acid).

Description

Technical Field

[0001] The invention relates to the field of biological control, in particular to a strain of Bacillus wiezmannii capable of inhibiting plant pathogens and application thereof. Background Art

[0002] Plant pathogenic microorganisms are key biological stress factors that restrict the stability and sustainability of global agricultural production systems. The diseases they cause cause a significant decline in global crop yields every year. There are many types of plant pathogens, among which fungal diseases have become the main challenge in agricultural production due to their rapid spread, wide range of damage, and difficulty in prevention and control. Although traditional chemical control strategies are effective in the short term, long-term use not only leads to drug resistance in pathogens, imbalance of soil microbial communities, and pesticide residue pollution, but also has a negative impact on the ecological environment and human health. Therefore, the development of efficient and environmentally friendly disease prevention and control strategies has become an urgent need in the field of plant protection.

[0003] As an environmentally friendly disease management method, biological control has received widespread attention in recent years. Its core is to use beneficial microorganisms or their metabolites to inhibit the growth and reproduction of pathogenic microorganisms. Its mechanism of action mainly includes competition for nutrition and space, secretion of antimicrobial substances, and induction of systemic resistance. Compared with chemical pesticides, biological control agents have multiple advantages: first, they have high target specificity and better safety to non-target organisms and the environment; second, biocontrol microorganisms can work synergistically through multiple mechanisms to reduce the risk of pathogens developing drug resistance. Summary of the invention

[0004] The present invention aims to solve the problem that chemical control methods of plant pathogens have negative impacts on the ecological environment and human health, and provides a strain of Bacillus wiezmannii for inhibiting plant pathogens and its application.

[0005] The Bacillus wiedmannii for inhibiting plant pathogens of the present invention is Bacillus wiedmannii SJZ, which has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is December 23, 2024, and the deposit number is CCTCC NO: M 20242884.

[0006] The colonies of the Wiesmannian Bacillus SJZ of the present invention are round or nearly round, opaque, with neat edges, a raised and dry surface, a hard texture and a certain viscosity, and a milky white or light yellow color. The bacteria are rod-shaped, about 2-5 microns in length, and are often arranged in single, paired or short chain forms.

[0007] The Wiesmannian bacillus SJZ of the present invention is a Gram-positive bacterium, has a positive catalase reaction, a positive methyl red test, a negative VP test, a negative indole test, a positive gelatin liquefaction test, a positive nitrate reduction test, and has the ability to secrete protease, cellulase, and chitinase.

[0008] The 16S rDNA sequencing results of the Wiesmannian Bacillus SJZ of the present invention were submitted to the NCBI database, analyzed and compared using BLAST, and then the phylogenetic tree of Wiesmannian Bacillus SJZ was constructed using MEGA 7 software. The strain was closely related to Wiesmannian Bacillus (Bacillus wiedmannii), and the sequence similarity between the two reached 100%. Based on the morphological observation results and physiological and biochemical results, Wiesmannian Bacillus SJZ was identified as Wiesmannian Bacillus (Bacillus wiedmannii).

[0009] The Bacillus wiezmannii SJZ of the present invention is used for inhibiting Sclerotinia sclerotiorum.

[0010] The Bacillus wismannii SJZ of the present invention is used for inhibiting the black rot fungus.

[0011] The Bacillus wieldis SJZ of the present invention can secrete protease, cellulase and chitinase.

[0012] The Bacillus Weismannii SJZ of the present invention is used for nitrogen fixation.

[0013] The Bacillus Weismannii SJZ of the present invention is used to secrete siderophore and IAA.

[0014] Beneficial effects of the present invention:

[0015] 1. The Wiesmannian Bacillus SJZ of the present invention has a strong inhibitory effect on the growth of Sclerotinia sclerotiorum, which can make it almost not produce white cotton-like mycelium, and the antibacterial rate reaches 89.41%. It can secrete metabolites with strong antibacterial activity, effectively inhibit the growth of Sclerotinia sclerotiorum, significantly reduce the diameter of Sclerotinia sclerotiorum hyphae, thin the cell wall and show local permeability, significantly increase the frequency of hyphae branching, irregular branching angle, and often accompanied by local swelling at the branching point. The growth of the top of the hyphae is significantly inhibited, and the top extension area becomes blunt or irregularly swollen.

[0016] 2. Bacillus SJZ can inhibit the secretion of oxalic acid by Sclerotinia sclerotiorum, with an inhibition rate of 18.38%.

[0017] 3. The Weizmannian Bacillus SJZ was cultured with Sclerotinia sclerotiorum, and it was found that it could secrete volatile substances with strong antibacterial properties, with an antibacterial rate of 90.00%.

[0018] 4. The clear zone experiment showed that Bacillus SJZ had strong protease and cellulase secretion ability, had certain chitinase secretion ability, and had no β-glucanase secretion ability.

[0019] 5. Sunflower seeds were treated with Bacillus SJZ and 20-fold diluted bacterial solution, and it was found that it could inhibit the infection of Sclerotinia sclerotiorum on sunflower seeds. Potted experiments showed that 24 hours after the application of Sclerotinia sclerotiorum mycelium suspension, the plants showed slight wilting symptoms and the leaves drooped due to water loss; after 48 hours, the degree of wilting intensified and some plants fell over; after 54 hours, rot symptoms appeared in the rhizomes, accompanied by browning and softening of the tissues. The use of Bacillus SJZ suspension for prevention and control reduced the incidence of the sunflower strain to 43.33%.

[0020] 6. The broad-spectrum antibacterial activity and growth-promoting potential of Bacillus wismannii SJZ were investigated, and it was found that its inhibitory effect on Fusarium graminearum, Alternaria solani, and Alternaria tenuissima was relatively limited, but it had a strong inhibitory effect on Sphaerotheca oleracea, and could cause the mycelium of Sphaerotheca oleracea to change color. Bacillus wismannii SJZ has the ability to fix nitrogen, can secrete a small amount of siderophore and IAA, and has a certain growth-promoting potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a confrontation picture of the Weizmann Bacillus SJZ rescreening plates;

[0022] Figure 2 This is the colony morphology of Bacillus Weizmannii SJZ;

[0023] Figure 3 This is the bacterial morphology of Bacillus Weizmannii SJZ;

[0024] Figure 4 is the hydrolase secretion capacity of Bacillus wiezmannii SJZ;

[0025] Figure 5 is the phylogenetic tree of Bacillus Weizmannii SJZ;

[0026] Figure 6 The effect of the metabolites of Bacillus wiezmannii SJZ on the growth of Sclerotinia sclerotiorum;

[0027] Figure 7 The effect of the metabolites of Bacillus wieldensmannii SJZ on the oxalic acid secretion of Sclerotinia sclerotiorum;

[0028] Figure 8 The antibacterial effect of volatile substances of Bacillus Weizmannii SJZ;

[0029] Fig. 9 The effects of different treatments of Bacillus Weizmannii SJZ fermentation broth on the infection of sunflower seed husk;

[0030] Fig.10 The disease susceptibility and control efficacy of sunflower in potted experiments with Bacillus Weizmannii SJZ;

[0031] Fig.11 The broad-spectrum antibacterial analysis of Bacillus wieldensmannii SJZ;

[0032] Fig.12 Analysis of the growth-promoting potential of Bacillus Weizmannii SJZ. DETAILED DESCRIPTION

[0033] The embodiments of the present invention are described in detail below. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0034] Embodiment 1:

[0035] This example is Bacillus wiedmannii SJZ, which has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is December 23, 2024, and the deposit number is CCTCCNO: M 20242884.

[0036] The method for obtaining Bacillus wiedmannii SJZ in this example is as follows:

[0037] Preliminary screening: Soil samples were collected from Arctic Village, Mohe City, Daxinganling Region, Heilongjiang Province. Based on the five-point sampling method, 6 soil samples were collected from different sampling points in Mohe area at a depth of 1-3m. First, impurities and large particles were removed, and the disinfected soil samples were sieved to screen out fine soil particle samples. Then 5g of each sample was taken, and 5g of soil sample was added to a triangular flask containing 45mL of water and sterilized glass beads. The suspension was thoroughly mixed and cultured at 150r / min for about 20 hours to disperse the bacterial cells. Heat in an 80℃ water bath for 30 minutes to kill other bacteria and vegetative cells. Then, the ten-fold dilution method was used to transfer 1mL of the extracted supernatant from the solution into a test tube filled with 9mL of distilled water with a pipette, and then diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6Five dilutions. Then, 100 μL of soil dilutions with different contents were taken and spread on LB medium. After each dilution was repeated three times, it was placed in an incubator at 28°C for inverted culture. Several strains were isolated and activated by streaking on the LB medium plate to obtain single colonies. The plate confrontation method was used to inoculate the 8.0 mm diameter Sclerotinia cake in the center of the PDA medium. Bacillus was inoculated 1 cm away from the plate. The plate was inverted at 28°C and the diameter of the Sclerotinia was observed after 3 days. The strains that formed obvious antibacterial zones were selected for rescreening.

[0038] Rescreening: Use the plate confrontation method to inoculate a 8.0mm diameter Sclerotinia cake in the center of a PDA medium plate, and inoculate two points of Bacillus at a distance of 1cm from the plate. Incubate the plate upside down at 28℃, and measure the width of the inhibition zone after 5 days. Screen out antagonistic bacteria with an inhibition zone width of more than 10mm, such as Figure 1 As shown, Figure 1 The left side in the middle is the control, and the right side is the result of the re-screening of Bacillus Weizmannii SJZ. Finally, Bacillus Weizmannii SJZ was screened out.

[0039] Example 2: Identification of Bacillus wiezmannii SJZ

[0040] 1. Morphological observation of Bacillus wieldensmannii SJZ

[0041] The colonies of Bacillus Weizmannii SJZ are round or nearly round, opaque, with neat edges, a dry and convex surface, a hard texture and a certain degree of stickiness, and are milky white or light yellow in color. The bacteria are rod-shaped, about 2-5 microns in length, and are often arranged in single, paired or short chains. They are Gram-positive bacteria. The colony photos of Bacillus Weizmannii SJZ are shown in the figure below. Figure 2 As shown, the bacteria photos are as follows Figure 3 shown.

[0042] 2. Physiological and biochemical characteristics of Bacillus wieldensii SJZ

[0043] The results of physiological and biochemical tests of Bacillus wieldis SJZ are shown in Table 1.

[0044] Table 1 Results of physiological and biochemical indexes of Bacillus wieldis

[0045]

[0046]

[0047]

[0048] Note: “+” indicates positive, “-” indicates negative.

[0049] The results of the hydrolase secretion ability test of Bacillus wieldis SJZ are as follows Figure 4 shown.

[0050] Determination of the protease secretion capacity of Bacillus wismannii SJZ: Inoculate Bacillus wismannii SJZ on a protease screening plate and incubate it upside down at 28°C for 3 days. Figure 4 As shown in a, it can be seen that the opaque milky solid culture medium is hydrolyzed into a transparent state under the action of protease, indicating that it has the ability to secrete protease.

[0051] Determination of cellulase secretion capacity of Bacillus wismannii SJZ: Use a sterile toothpick to inoculate Bacillus wismannii SJZ on the cellulase screening medium, invert and culture at 28°C for 3 days, stain with Congo red staining solution for 10 minutes, and decolorize with 1 mol / L NaCl decolorizing solution for 10 minutes. Figure 4 As shown in b, Congo red can form a stable red complex with the macromolecular polysaccharide cellulose. When the cellulase secreted by the bacteria degrades the cellulose in the plate into small molecular sugars, the structure of the cellulose is destroyed, making it unable to bind to Congo red, thus forming a transparent hydrolysis circle on the plate. This phenomenon shows that the strain has a strong cellulase secretion ability and can effectively degrade cellulose substrates.

[0052] Determination of chitinase secretion ability of Bacillus wieldingii SJZ: Use a sterile toothpick to inoculate Bacillus wieldingii SJZ on the chitinase screening medium and invert and culture at 28°C for 5 days. Figure 4 As shown in Figure c, a clear transparent hydrolysis zone was observed around Bacillus wieldingii SJZ on the chitinase screening plate, indicating that the strain can secrete chitinase to degrade the chitin substrate in the culture medium. This result confirms that Bacillus wieldingii SJZ has a certain chitinase secretion activity.

[0053] Determination of the β-glucanase secretion capacity of Bacillus wismannii SJZ: Use a sterile toothpick to inoculate Bacillus wismannii SJZ on the β-glucanase screening medium, invert and culture at 28°C for 3 days, stain with Congo red staining solution for 30 minutes, and decolorize with 1 mol / L NaCl decolorizing solution for 10 minutes. Figure 4 As shown in Figure d, no transparent hydrolysis zone was observed around the colonies, indicating that the strain failed to degrade the β-glucan substrate in the culture medium. This result confirmed that Bacillus Weizmannii SJZ did not have the ability to secrete β-glucanase.

[0054] 3. Molecular identification of Bacillus wieldensmannii SJZ

[0055] The genomic DNA of Bacillus wieldensii SJZ was extracted, and the 16S rDNA sequence was amplified by PCR. The PCR amplification product was subjected to agarose gel electrophoresis and sequenced to obtain a 16S rDNA with a length of 1425 bp, as shown in SEQ ID NO: 1 in the sequence table. The sequencing results were uploaded to the NCBI database for BLAS comparison, and a phylogenetic tree was constructed as shown in FIG. Figure 5 As shown. 16S rDNA sequence alignment and phylogenetic analysis showed that the strain was closely related to Bacillus wiedmannii. Based on the morphological observation results and physiological and biochemical results, the strain was identified as Bacillus wiedmannii.

[0056] Example 3: Study on the antibacterial and control ability of Bacillus wieldis SJZ

[0057] 1. Effects of Bacillus wieldis SJZ metabolites on the growth of Sclerotinia sclerotiorum

[0058] The seed liquid of Bacillus wismannii SJZ in the logarithmic growth phase was inoculated into BPY medium at a ratio of 1%, cultured by shaking, the fermentation liquid was aspirated, centrifuged, and the supernatant was filtered using an aqueous microporous filter membrane with a diameter of 0.22nm to obtain a sterile fermentation liquid. The sterile fermentation liquid of Bacillus wismannii SJZ was mixed into PDA medium at a ratio of 5%, the plate was inverted, and a sclerotinia cake with a diameter of 8mm was inoculated on the plate, inverted and cultured at 28℃ for 5 days, and the results were observed. The control group was a sclerotinia cake with a diameter of 8mm inoculated on a PDA plate without adding sterile fermentation liquid.

[0059] The experimental results showed that the growth of Sclerotinia mycelium was significantly inhibited compared with the control group, and almost no white cotton-like mycelium was produced. The inhibition rate of Sclerotinia mycelium reached 89.41%. Figure 6 This result confirmed that Bacillus SJZ could secrete metabolites with strong antibacterial activity and effectively inhibit the growth of Sclerotinia sclerotiorum, indicating that it has a good ability to secrete antibacterial substances.

[0060] 2. Effects of Bacillus Weizmannii SJZ metabolites on the morphology of Sclerotinia sclerotiorum

[0061] The agar blocks with hyphae of Sclerotinia sclerotiorum treated with the sterile fermentation liquid of Bacillus wismannii SJZ were cut into thin slices with a disposable scalpel, and the hyphae morphology was observed under an optical microscope. The results showed that the group treated with Bacillus wismannii SJZ showed a significant hyphae development inhibition phenotype, while the hyphae of the control group had uniform diameter, intact cell wall structure, high cytoplasm density, and vigorous metabolic activity. There was no obvious swelling or abnormal structure at the branching point. The hyphae were milky white to light gray as a whole, with moderate light transmittance and no obvious transparency. Normal hyphae fusion could be observed between hyphae, forming a complex hyphae network structure. The group treated with Bacillus wismannii SJZ showed a typical fungal growth inhibition phenotype, with a significant decrease in hyphae diameter, thinning of cell wall and local permeability, a significant increase in hyphae branching frequency, irregular branching angles, and local swelling at the branching point. The top growth of hyphae was significantly inhibited, and the top extension area became blunt or irregularly swollen.

[0062] 3. Effects of Bacillus Weizmannii SJZ metabolites on oxalic acid secretion of Sclerotinia sclerotiorum

[0063] The seed liquid of Bacillus wiesemannii SJZ in the logarithmic growth phase was inoculated into 100mL BPY medium at a ratio of 1%, and the active fermentation liquid was obtained by constant temperature shaking culture. The active fermentation liquid was centrifuged at 10000r / min for 10min, the supernatant was carefully aspirated, and the supernatant was filtered using a disposable aqueous microporous filter membrane with a pore size of 0.22nm to obtain a sterile fermentation liquid. The mycelial suspension of Sclerotinia sclerotiorum was inoculated into 100mL PD medium, and the activated mycelial suspension of Sclerotinia sclerotiorum was obtained by shaking culture at 28℃ and 120r / min for 3 days. The sterile fermentation liquid was added to 20mL PD medium at a ratio of 5%, and then 1mL of activated mycelial suspension of Sclerotinia sclerotiorum was added. The mycelial growth was observed and the secretion of oxalic acid by Sclerotinia sclerotiorum was detected by colorimetry. 1mL of mycelial suspension of Sclerotinia sclerotiorum was added to 20mL PD medium as a blank control.

[0064] The experimental results showed that Bacillus SJZ had a significant inhibitory effect on the growth of Sclerotinia sclerotiorum and its oxalic acid synthesis, with the oxalic acid content inhibition rate reaching 18.38%. The effect of Bacillus SJZ on the oxalic acid secretion of Sclerotinia sclerotiorum was measured by colorimetry. Figure 7 As shown in the figure, oxalic acid, as a key pathogenic factor of Sclerotinia sclerotiorum, can destroy the plant cell wall structure, inhibit the host defense response, and provide a suitable acidic environment for the cell wall degrading enzymes secreted by the pathogen. By inhibiting the synthesis of oxalic acid, Bacillus wismannii SJZ can inhibit the pathogenicity of Sclerotinia sclerotiorum to a certain extent.

[0065] 4. Analysis of the inhibitory effect of volatile substances of Bacillus wieldis SJZ on Sclerotinia sclerotiorum

[0066] Bacillus SJZ was inoculated into LB liquid medium and shaken to obtain activated seed solution, the seed solution was spread on LB medium plate, and then the LB medium plate coated with the seed solution and the PDA medium plate inoculated with Sclerotinia sclerotiorum cake were placed side by side for 5 days. It was observed that Bacillus SJZ could produce volatile antibacterial substances that inhibited the growth of Sclerotinia sclerotiorum. The antibacterial effect of volatile substances of Bacillus SJZ is shown in the figure. Figure 8 As shown, the antibacterial rate reached 90.00%, indicating that the strain has a strong ability to synthesize and secrete volatile antibacterial substances.

[0067] 5. Shell infection experiment

[0068] Preparation of Bacillus Weizmannii SJZ bacterial solution: The strain seed solution was inoculated into BPY medium at an inoculum volume of 1%, and cultured at 28° C. and 180 r / min for 3 days.

[0069] Disinfection and washing of sunflower seeds: Soak the sunflower seeds in sterile water for 2 minutes; then soak the seeds in 75% alcohol for 1 minute, repeat twice; soak the seeds in 2% sodium hypochlorite solution for 2 minutes; then soak the seeds in 2% sodium hypochlorite solution for 1 minute. 2 S 2 O 3 The solution was used to soak the sunflower seeds twice to remove the residual chlorine in the seeds; then the sunflower seeds were soaked in sterile water for 3 times; finally, the moisture on the surface of the seeds was absorbed with sterile filter paper.

[0070] Reprocessing of sunflower seeds: Take the seeds treated as above and divide them into four portions, 15 seeds in each portion. Add the original bacterial solution that covers the seeds to one portion, add an equal volume of 20-fold diluted bacterial solution to one portion, add an equal volume of distilled water to one portion, and add an equal volume of BPY culture solution to one portion. Soak each portion of seeds for 30 minutes, and set up 3 replicates for each group.

[0071] Husk infection experiment: The four groups of sunflower seeds treated above were evenly placed on plates that were incubated at 28°C for 5 days and were covered with mycelium of Sclerotinia sclerotiorum. Fifteen treated sunflower seeds were placed on each plate, and three replicates were set for each group.

[0072] Observe the susceptibility of sunflower seeds after 2 days, and turn the sunflower seeds over with tweezers, and observe again after 2 days. The susceptibility of sunflowers is divided into 3 levels, namely: Level 0: no hyphae are seen on the surface of sunflower seeds; Level 1: a small amount of hyphae can be seen on the surface of sunflower seeds; Level 2: the surface of sunflower seeds is obviously covered with hyphae. The disease index is used to describe the susceptibility of sunflower seeds, and the results of the experimental group and the control group are recorded separately. The formulas for the incidence rate and disease index are as follows:

[0073]

[0074] Where M represents the incidence rate (%), I represents the number of infected seeds (grains), and T represents the total number of seeds (grains).

[0075]

[0076] Where D represents the disease index, N represents the number of diseased seeds at each level, R represents the representative value at each level, T represents the total number of seeds surveyed, and H represents the highest representative value.

[0077] The disease index, incidence rate and husk infection of sunflower seeds in each treatment group are shown in Fig. 9 . Fig. 9 CK1 is treated with sterile water, CK2 is treated with BPY culture medium, SJZ is treated with original bacterial solution, and 1 / 20 means treated with twenty-fold diluted bacterial solution. After 4 days of observation and calculation, it was found that the incidence of seed husk infection treated with the original bacterial solution and the twenty-fold diluted bacterial solution was reduced. The specific analysis is shown in Table 2.

[0078] Table 2 Effects of different treatments of Bacillus wismannii on sunflower seed husk infection

[0079]

[0080] 6. Sunflower pot experiment

[0081] This experiment adopted the potted root irrigation method, using commercially available nutrient soil, mixed in a ratio of nutrient soil: vermiculite = 4:1, and put into a disposable seedling pot (12cm×10.5cm), sow 1-2 sunflower seeds in the seedling pot, water 100mL, cover with 1cm of soil, germinate indoors, and move the seeds outdoors after germination. Water 100mL every 2 days until the sunflower grows four true leaves. Keep one sunflower plant in each pot for potted infection and potted prevention and control experiments.

[0082] Potted infection experiment: The mycelial suspension of Sclerotinia sclerotiorum was inoculated into PD medium and cultured at 28℃ and 120r / min for 5 days to obtain the mycelial suspension of Sclerotinia sclerotiorum. Experimental group: 30mL of the prepared mycelial suspension of Sclerotinia sclerotiorum was taken for root irrigation and covered with 1cm of soil, and a total of 20 pots were treated; control group 1: the same amount of sterile water was used for root irrigation and covered with 1cm of soil, and a total of 20 pots were treated; control group 2: the same amount of PD medium was used for root irrigation and covered with 1cm of soil, and a total of 20 pots were treated. The disease susceptibility and symptoms of sunflower plants were observed every 12h and the experimental results of each group were recorded.

[0083] Potted control experiment: The seed liquid of Bacillus wismannii SJZ was inoculated into BPY medium at a ratio of 1%, and cultured at 28℃ and 180r / min for 3 days to obtain Bacillus liquid. Treatment group: 50mL of Bacillus wismannii SJZ liquid was used for root irrigation, and 3 days later, 30mL of Sclerotinia mycelium suspension was used for root irrigation, and 1cm of soil was covered, and 20 pots were treated in total; treatment control group 1: 50mL of sterile water was used for root irrigation, and 3 days later, 30mL of Sclerotinia mycelium suspension was used for root irrigation, and 1cm of soil was covered, and 20 pots were treated in total; treatment control group 2: 50mL of BPY medium was used for root irrigation, and 3 days later, 30mL of Sclerotinia mycelium suspension was used for root irrigation, and 1cm of soil was covered, and 20 pots were treated in total. Each group of experiments was repeated 3 times. The disease susceptibility and symptoms of sunflower plants were observed every 12 hours and the experimental results of each group were recorded. The disease index was used to describe the disease susceptibility of sunflowers, with level 0 indicating no disease, level 1 indicating slight disease, and level 2 indicating plant disease. The results for the experimental group and the control group were recorded separately.

[0084]

[0085] Where D represents the disease index, N represents the number of diseased plants at each level, R represents the representative value at each level, T represents the total number of plants surveyed, and H represents the highest representative value.

[0086] The pathogenicity of Sclerotinia sclerotiorum and the biocontrol effect of Bacillus wismannii SJZ were evaluated by the sunflower pot infection experiment. The susceptibility of sunflower pots in the treatment groups in the pot control experiment is shown in Fig.10 . The results showed that compared with the water blank control group and the PD medium control group, the Sclerotinia infection group showed obvious disease symptoms. After inoculation with Sclerotinia, the sunflower seedlings showed a typical disease development process in a short period of time: 24 hours after the mycelium was irrigated, the plants showed slight wilting symptoms, and the leaves drooped due to water loss; 48 hours later, the degree of wilting intensified, and some plants fell over; 54 hours later, the roots and stems showed rot symptoms, accompanied by tissue browning and softening. In contrast, the disease index and incidence rate of sunflower plants treated with Bacillus wismannii SJZ were 43.33%.

[0087] Example 4: Analysis of the broad-spectrum antibacterial activity of Bacillus wieldis SJZ

[0088] Plant pathogenic bacteria Fusarium graminearum (F.graminearum), Alternaria dauci (A.dauci), Alternaria nees (A.Nees), and V.sordida (plant pathogens purchased from Mingzhou Biotechnology Co., Ltd.) were selected to prepare plant pathogen cakes. The plant pathogen cake with a diameter of 8.0 mm was inoculated in the center of the PDA culture medium using the plate confrontation method, and Bacillus Wiesmannii SJZ was inoculated 1 cm away from the plate. The culture was inverted at 28°C, and the diameter of the pathogen was observed after 5 days. The pure culture of the pathogen without inoculation of Bacillus Wiesmannii SJZ was used as a control. The experimental results are as follows. Fig.11 As shown, Fig.11 A is Fusarium graminearum, B is Solanum solani, C is Alternaria solani, and D is Alternaria tenuissima. The results show that Bacillus wismannii SJZ exhibits differentiated antibacterial activity against a variety of plant pathogenic fungi. Its inhibitory effect on the growth of Fusarium graminearum, Alternaria tenuissima and Alternaria tenuissima is relatively limited, and the colony expansion only shows a slight slowdown. However, the bacterium exhibits a significant inhibitory effect on Solanum solani. The hyphae of Solanum solani near Bacillus wismannii SJZ undergo obvious phenotypic changes: the hyphae gradually change from normal white to yellow-brown, and the extension zone at the top of the hyphae becomes blunt. This color change may be related to the leakage of intracellular substances and oxidation reactions after the integrity of the hyphae cell wall is damaged, indicating that Bacillus wismannii SJZ may interfere with the normal growth and development of pathogens by secreting secondary metabolites. These phenomena suggest that Bacillus wismannii SJZ has certain application potential in the biological control of agricultural and forestry diseases.

[0089] Example 5: Analysis of the Growth-Promoting Potential of Bacillus Weizmannii SJZ

[0090] The growth-promoting ability of the growth-promoting strain mainly includes five aspects: nitrogen fixation, phosphorus solubilization, potassium solubilization, secretion of siderophore, and secretion of IAA. The single colony of Bacillus wismannii SJZ was inoculated into Ashubei nitrogen fixation screening medium, Montkina organic phosphorus phosphorus solubilization screening medium, inorganic phosphorus phosphorus solubilization screening medium, potassium solubilization screening medium, and CAS medium, respectively, and inverted cultured at 28°C for 3 days. If Bacillus wismannii SJZ produces a transparent circle on Ashubei nitrogen fixation screening medium, Montkina phosphorus solubilization screening medium, and potassium solubilization screening medium, the strain has the ability to fix nitrogen, solubilize phosphorus, and solubilize potassium, respectively. If Bacillus wismannii SJZ produces an orange-yellow transparent circle on CAS medium, the strain has the ability to secrete siderophore. Inoculate Bacillus wieldens SJZ into LB medium containing 0.1% L-Try, culture at 37°C and 180r / min for 2 days, aspirate the bacterial solution, centrifuge at 8000r / min for 5min, aspirate 100μL of the supernatant, add 200μL of Salkowski reagent, and color for 30min in the dark. If it turns pink, it is positive. If it is difficult to distinguish, measure its OD 530nm.

[0091] The experimental results are as follows Fig.12 As shown, Fig.12 In the figure, a is the nitrogen fixation, b is the siderophore production, and c is the IAA production. Bacillus Weizmannii SJZ exhibits certain plant growth promotion-related characteristics. The strain has the ability to fix nitrogen and can grow normally on nitrogen-free medium, indicating that it may be able to provide nitrogen source nutrition for plants through nitrogen fixation. However, in the test of phosphorus and potassium solubilization ability, no obvious phosphorus solubility circle or potassium solubility circle formation was observed, indicating that it does not have significant phosphorus solubilization or potassium solubilization ability. Further testing found that the strain can secrete siderophores and form obvious color changes on the CAS detection plate, indicating that it has the ability to chelate iron ions. In addition, colorimetric detection confirmed that Bacillus Weizmannii SJZ can produce the plant growth hormone indole-3-acetic acid (IAA), showing certain growth promotion potential. These characteristics indicate that Bacillus Weizmannii SJZ has certain application value in biological control of agricultural diseases and plant growth promotion, and can be further studied as a potential biocontrol bacterium or rhizosphere growth-promoting bacterium.

Claims

1. A strain of Bacillus wiezmannii for inhibiting plant pathogens, characterized in that: The Bacillus wiedmannii is Bacillus wiedmannii SJZ, which has been deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, the deposit date is December 23, 2024, and the deposit number is CCTCC NO: M 20242884.

2. The Bacillus Wiesmannii as claimed in claim 1 is used to inhibit Sclerotinia sclerotiorum.

3. The bacillus Wiesmannii as claimed in claim 1 is used to inhibit the black rot fungus.

4. The Bacillus Wiesmannii according to claim 1 is capable of secreting protease, cellulase and chitinase.

5. The Bacillus Wiesmannii as claimed in claim 1 is used for nitrogen fixation.

6. The Bacillus Weismannii as claimed in claim 1 is used for secreting siderophores and IAA.

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