Paenibacillus latifolius for inhibiting phytopathogen and application of paenibacillus latifolius

Abstract

The invention relates to the field of biological prevention and control, in particular to bacillus latipetii for inhibiting phytopathogens and application thereof, and provides the bacillus latipetii for inhibiting the phytopathogens, which has the capability of antagonizing sclerotinia sclerotiorum of sunflowers and is a biocontrol strain with certain application prospects. The bacillus latipetii disclosed by the invention can be used for inhibiting sclerotinia sclerotiorum, and also can be used for inhibiting fusarium graminearum, alternaria solani, alternaria tenuissima and solanum nigrum. The bacillus palatii disclosed by the invention can secrete protease, cellulase and chitinase. The bacillus lawtii provided by the invention 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 tuvanii for inhibiting plant pathogens and an application thereof. Background Art

[0002] Biological control mainly refers to methods and measures to control pathogens by utilizing beneficial microorganisms and the secondary metabolites secreted by such microorganisms, so as to reduce the incidence of plants and achieve the prevention and control of plant diseases. Such microorganisms are biocontrol strains or antagonistic strains.

[0003] At present, the most studied biocontrol strains are Actinomycetes, Trichoderma, Bacillus, etc. A large number of biocontrol strains have been developed for the prevention and treatment of plant diseases, but there are problems such as a single variety of applied strains, poor adaptability of strains, and inability to adapt to different species, different origins, and different ecological environments. At the same time, the quality of bacterial agents is also uneven, and there is a problem of low and unstable survival rate of strains. Therefore, it is of great significance to develop biocontrol strains with strong adaptability and strong prevention and control ability.

[0004] A large number of studies have discovered a variety of potential biocontrol bacteria. Compared with agricultural and chemical control, biological control is less harmful to the environment and will become the main technical method for plant disease control in the future. Summary of the invention

[0005] The invention aims to provide a Tuvanilloids bacillus strain capable of inhibiting plant pathogens, which has the ability to antagonize sunflower sclerotinia pathogen and is a biocontrol strain with certain application prospects.

[0006] The present invention provides a strain of Bacillus toyonensis for inhibiting plant pathogens, which is Bacillus toyonensis 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 20242883.

[0007] The colony of the Tuvanyong Bacillus SJZ of the present invention is white in appearance, round, opaque, easy to pick up, raised, with smooth edges, no obvious luster, and can emit an unpleasant volatile odor. The bacterial body is rod-shaped, slender and straight.

[0008] The Tuvanillosis 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.

[0009] The 16S rDNA sequencing results of the Tuwanyong Bacillus SJZ of the present invention were submitted to the NCBI database, analyzed and compared using BLAST, and then the phylogenetic tree of Tuwanyong Bacillus SJZ was constructed using MEGA7 software. The strain was closely related to Bacillus toyonensis, and the sequence similarity between the two reached 100%. Based on the morphological observation results and physiological and biochemical results, Tuwanyong Bacillus SJZ was identified as Bacillus toyonensis.

[0010] The Tuvanian Bacillus SJZ of the present invention is used for inhibiting Sclerotinia sclerotiorum.

[0011] The Tuvanian Bacillus SJZ of the invention is used for inhibiting Fusarium graminearum, Alternaria solani, Alternaria tenuissima and Sordella solani.

[0012] The Tuvanian Bacillus SJZ of the present invention can secrete protease, cellulase and chitinase.

[0013] The Tuvanian Bacillus SJZ of the present invention is used for nitrogen fixation.

[0014] The Tuvanian Bacillus SJZ of the present invention is used for secreting siderophore and IAA.

[0015] Beneficial effects of the present invention:

[0016] 1. The aseptic fermentation liquid of Tuvanyong Bacillus SJZ has a strong inhibitory effect on the growth of Sclerotinia sclerotiorum, with an inhibition rate of 89.41%. It can affect the hyphae morphology of Sclerotinia sclerotiorum to a large extent, inhibit the growth of new hyphae, make the hyphae of Sclerotinia sclerotiorum thinner, more forked, and transparent, and "tumor-like" aggregations will appear in the middle and end of the hyphae.

[0017] 2. The aseptic fermentation broth of Bacillus Tuvanianus SJZ can inhibit the secretion of oxalic acid by Sclerotinia sclerotiorum, with an inhibition rate of 75.22%.

[0018] 3. The Tuvanian 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 79.02%.

[0019] 4. The clear zone experiment showed that Bacillus Tuvanianus SJZ had strong protease and cellulase secretion ability, had certain chitinase secretion ability, and had no β-glucanase secretion ability.

[0020] 5. Sunflower seeds were treated with Tuvanillosis SJZ bacterial solution 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 the sunflower plants treated with Sclerotinia sclerotiorum mycelium suspension showed slight wilt after 24 hours, and the plants wilted and fell over after 48 hours. Root rot and stem rot were observed in the rhizomes of the plants after 54 hours. The use of Tuvanillosis SJZ for prevention and control reduced the incidence of sunflower strains to 38.33%.

[0021] 6. The broad-spectrum antibacterial activity and growth-promoting potential of Tuvayon Bacillus SJZ were investigated, and it was found that it had weak inhibition on Fusarium graminearum, Alternaria solani, and Alternaria tenuissima, but had strong inhibition on Sphaerotheca solani, and could cause the mycelium of Sphaerotheca solani to change color. Tuvayon Bacillus SJZ has nitrogen fixation ability, can secrete a small amount of siderophore and IAA, and has certain growth-promoting potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of the confrontation of the plates of Tuwanyong Bacillus SJZ rescreening;

[0023] Figure 2 This is the colony morphology of Tuwanyong Bacillus SJZ;

[0024] Figure 3 This is the microscopic morphology of Tuwanyong Bacillus SJZ;

[0025] Figure 4 is the hydrolase secretion capacity of Bacillus tuwanensis SJZ;

[0026] Figure 5 is the phylogenetic tree of Bacillus Tuvayon SJZ;

[0027] Figure 6 The effect of the metabolites of Bacillus tuwanianus SJZ on the growth of Sclerotinia sclerotiorum;

[0028] Figure 7 The effect of the metabolites of Bacillus tuwanensis SJZ on the oxalic acid secretion of Sclerotinia sclerotiorum;

[0029] Figure 8 This is the antibacterial effect of volatile substances of Tuwanyong Bacillus SJZ;

[0030] Fig. 9 The effect of different treatments of Tuvanillon Bacillus SJZ fermentation broth on the infection of sunflower seed husk;

[0031] Fig.10 The disease susceptibility and control efficacy of sunflower in the Tuvayong Bacillus SJZ potted experiment;

[0032] Fig.11 This is the broad-spectrum analysis of the antibacterial activity of Bacillus tuwanensis SJZ;

[0033] Fig.12 Analysis of the growth-promoting potential of Bacillus SJZ in Tuwanyong. DETAILED DESCRIPTION

[0034] 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.

[0035] Embodiment 1:

[0036] The Tuwanian Bacillus that inhibits plant pathogens in this embodiment is Tuwanian Bacillus (Bacillus toyonensis) 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 20242883.

[0037] The separation and screening method of Tuwanyong Bacillus SJZ in this embodiment is as follows:

[0038] Initial screening: Soil samples were taken from Arctic Village, Mohe City, Daxinganling Region, Heilongjiang Province. Impurities and large particles were first removed. The disinfected soil samples were sieved to screen out fine soil particle samples. 5g of soil samples were added to a triangular flask with 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. The suspension was heated in an 80℃ water bath for 30 minutes to kill other bacteria and vegetative cells. The suspension was then diluted to 10 by the ten-fold dilution method. -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Five dilutions. Then, 100 μL of soil dilutions with different contents were taken and spread on LB medium and NA medium. After each dilution was repeated three times, it was placed in an incubator at 28°C for inverted culture to isolate several strains. The strains were activated by streaking on the LB medium plate and a single colony was obtained. The plate confrontation method was used to inoculate the 8.0 mm diameter Sclerotinia cake in the center of the PDA medium. The strains to be screened were inoculated 1 cm away from the edge of the plate. The culture was inverted at 28°C and the diameter of the Sclerotinia was observed after 3 days. The strains that formed obvious inhibition zones were selected for rescreening.

[0039] Second screening: Use the plate confrontation method to inoculate a 8.0mm diameter Sclerotinia cake in the center of the PDA medium plate, and inoculate the primary screening strain 1cm away from the edge of the plate. Incubate 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, strain SJZ was finally screened.

[0040] Example 2: Identification of strain SJZ

[0041] The colony morphology of Bacillus tuwanensis SJZ is as follows Figure 2 The microscopic morphology of strain SJZ is shown in Figure 3 As shown. The colony is white in appearance, round, opaque, easy to pick up, raised, with smooth edges, no obvious luster, and can emit an unpleasant volatile odor. The bacteria are rod-shaped, slender, and straight.

[0042] The results of physiological and biochemical tests of Bacillus tuyonana SJZ are shown in Table 1.

[0043] Table 1 Physiological and biochemical index test results of Tuwanyong Bacillus SJZ

[0044]

[0045]

[0046]

[0047] Note: “+” indicates positive, “-” indicates negative.

[0048] The hydrolase secretion capacity of Tuvayong Bacillus SJZ was determined, and the results were as follows: Figure 4 As shown, the details are as follows:

[0049] Determination of the protease secretion capacity of Bacillus tuyonnais SJZ: Bacillus tuyonnais SJZ was inoculated on the protease screening medium and incubated at 28°C for 3 days. Figure 4 As shown in a, a very obvious transparent zone was found on the plate, which had the ability to secrete protease.

[0050] Determination of the cellulase secretion capacity of Bacillus tuwayongi SJZ: Use a sterile toothpick to inoculate Bacillus tuwayongi 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, an obvious transparent zone was observed on the plate, which indicated that it had a strong ability to secrete cellulase.

[0051] Determination of chitinase secretion ability of Bacillus tuwayongana SJZ: Use a sterile toothpick to inoculate Bacillus tuwayongana SJZ on the chitinase screening medium and incubate at 28°C for 5 days. Figure 4 As shown in c, it was observed that Bacillus Tuvayon SJZ produced certain transparent zones on the chitinase screening plate, indicating that it had certain chitinase secretion activity.

[0052] Determination of the β-glucanase secretion capacity of Tuwayong Bacillus SJZ: Use a sterile toothpick to inoculate Tuwayong Bacillus 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 d, there is no transparent circle outside the colony, indicating that Tuvanian Bacillus SJZ does not have the ability to produce β-glucanase.

[0053] The 16S rDNA of strain SJZ was amplified by PCR, and the product was recovered and purified to obtain a specific band with a length of 1404 bp. The band was sequenced, and the 16S rDNA sequence is shown in SEQ ID NO: 1 in the sequence table.

[0054] The 16S rDNA sequencing results of the Tuwanyong Bacillus SJZ of the present invention were submitted to the NCBI database, analyzed and compared using BLAST, and then the phylogenetic tree of Tuwanyong Bacillus SJZ was constructed using MEGA7 software, as shown in Figure 5 As shown, the strain is closely related to Bacillus toyonensis, and the sequence similarity between the two reaches 100%. Combining the morphological observation results and physiological and biochemical results, Bacillus toyonensis SJZ was identified as Bacillus toyonensis.

[0055] Example 3: Study on the antibacterial and control ability of Tuwanyong Bacillus SJZ against Sclerotinia sclerotiorum

[0056] 1. Effects of the metabolites of Bacillus tuwanensis SJZ on the growth of Sclerotinia sclerotiorum

[0057] The seed liquid of Tuvanyong Bacillus 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 through an aqueous microporous filter membrane with a diameter of 0.22nm to obtain a sterile fermentation liquid. The sterile fermentation liquid was mixed with PDA medium at a ratio of 5%, and the plate was inverted. The Sclerotinia sclerotiorum cake with a diameter of 8mm was inoculated on the plate, and cultured inverted at 28℃ for 5 days. It was observed that the growth of Sclerotinia sclerotiorum hyphae was greatly inhibited, and the inhibition rate was 89.41%. The experimental results are as follows Figure 6This indicates that the metabolites secreted by Tuwanyong Bacillus SJZ have strong antibacterial ability and have a good ability to secrete antibacterial substances that inhibit the growth of Sclerotinia sclerotiorum.

[0058] 2. Effects of the metabolites of Bacillus tuwanensis SJZ on the morphology of Sclerotinia sclerotiorum

[0059] The agar block with hyphae of Sclerotinia sclerotiorum treated with the above-mentioned Tuvanillosis Bacillus SJZ sterile fermentation liquid was cut off with a disposable scalpel, and the agar block was cut as thin as possible. The hyphae morphology was observed using an optical microscope, and it was found that it had a great influence on the hyphae growth and morphology of Sclerotinia sclerotiorum. The hyphae of the control group were thicker, with obvious branches and bifurcations, and had strong vitality; Tuvanillosis Bacillus SJZ could inhibit the formation of new hyphae of Sclerotinia sclerotiorum, making the hyphae of Sclerotinia sclerotiorum thinner, with more branches, and the hyphae became transparent, and "tumor-like" aggregations would appear in the middle and end of the hyphae.

[0060] 3. Effects of the metabolites of Bacillus tuwanensis SJZ on oxalic acid secretion of Sclerotinia sclerotiorum

[0061] The seed liquid of Tuvanyong Bacillus 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°C 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 the activated mycelial suspension of Sclerotinia sclerotiorum was added, and the mycelial growth was observed and the secretion of oxalic acid by Sclerotinia sclerotiorum was detected by colorimetry.

[0062] The effect of Bacillus tuwanensis SJZ on the secretion of oxalic acid from Sclerotinia sclerotiorum was measured by colorimetry. Figure 7 As shown. It was found that Tuvayon Bacillus SJZ can inhibit the growth of Sclerotinia sclerotiorum and the production of oxalic acid, and the oxalic acid content inhibition rate reached 75.22%. Oxalic acid is one of the main pathogenic factors of Sclerotinia sclerotiorum, which can reduce the resistance of plants and provide a suitable environment for the hydrolytic enzymes of pathogenic bacteria. The Tuvayon Bacillus SJZ of the present invention has a good ability to inhibit the secretion of oxalic acid and can inhibit the pathogenicity of Sclerotinia sclerotiorum to a certain extent.

[0063] 4. Analysis of the inhibitory effect of volatile substances of Bacillus tuwanensis SJZ on Sclerotinia sclerotiorum

[0064] Tuvayon Bacillus SJZ was inoculated into LB liquid medium and shaken to obtain activated seed solution, which was then spread on an LB medium plate. The LB medium plate was then placed on a PDA medium plate inoculated with Sclerotinia sclerotiorum cake and cultured for 5 days. It was observed that Tuvayon Bacillus SJZ could produce volatile antibacterial substances that inhibited the growth of Sclerotinia sclerotiorum. The antibacterial effects of the volatile substances were shown in Figure 2. Figure 8 As shown, the inhibition rate reached 79.02%, indicating that Tuvanyong Bacillus SJZ has a strong ability to secrete antibacterial volatile substances.

[0065] 5. Shell infection experiment

[0066] Preparation of Tuvanillon Bacillus SJZ bacterial solution: Inoculate Tuvanillon Bacillus SJZ seed solution in logarithmic growth phase into BPY medium at an inoculum amount of 1%, and culture at 28° C. and 180 r / min constant temperature shaking for 3 days to obtain the original bacterial solution.

[0067] Disinfection and washing of sunflower seeds: soak the sunflower seeds in sterile water for 2 minutes; then soak the sunflower seeds in 75% alcohol for 1 minute, repeat twice; soak the sunflower seeds in 2% sodium hypochlorite solution for 2 minutes; then soak the sunflower seeds in 2% Na2S2O3 solution twice to remove residual chlorine in the seeds; then soak the sunflower seeds in sterile water for 3 times; finally, use sterile filter paper to absorb the moisture on the surface of the seeds.

[0068] 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.

[0069] 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.

[0070] 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:

[0071]

[0072] Where M represents the incidence rate (%), I represents the number of infected seeds (grains), and T represents the total number of seeds (grains).

[0073]

[0074] 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.

[0075] The disease index, incidence rate and husk infection of sunflower seeds in each treatment group are shown in Fig. 9 , Fig. 9 CK1 and CK2 represent the blank control without treatment, SJZ represents the original bacterial solution, and 1 / 20 represents the bacterial solution diluted 20 times. After 4 days of observation and calculation, it was found that the infection incidence of seed husk treated with the original bacterial solution and the infection incidence of seed husk treated with the 20-fold diluted bacterial solution were reduced. The specific analysis is shown in Table 2.

[0076] Table 2 Effects of different treatments of Bacillus Tuvanianus SJZ on sunflower seed husk infection

[0077]

[0078]

[0079] 6. Sunflower pot experiment

[0080] 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.

[0081] 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 activated mycelial suspension of Sclerotinia sclerotiorum. Experimental group: 30mL of mycelial suspension of Sclerotinia sclerotiorum was used 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.

[0082] Potted control experiment: The seed liquid of Tuvanyong Bacillus SJZ was inoculated into BPY medium at a ratio of 1%, and cultured at 28℃ and 180r / min for 3 days to obtain Tuvanyong Bacillus SJZ bacterial liquid. Experimental group: 50mL of Tuvanyong Bacillus SJZ bacterial 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 a total of 20 pots were treated; 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 a total of 20 pots were treated; 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 a total of 20 pots were treated. 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.

[0083]

[0084] Where D represents the disease index, N represents the number of diseased plants at each level, R represents the representative value of each level, T represents the total number of plants surveyed, and H represents the highest representative value.

[0085] The disease susceptibility of sunflower potted plants in the experimental group in the potted plant control experiment is shown in Fig.10 Compared with the water blank control group and the PD medium experimental control group, the Sclerotinia infection group was significantly susceptible to the disease. The sunflower pot infection experiment showed that Sclerotinia could cause sunflower seedlings to become ill in a short period of time. According to observations, the symptoms of sunflower seedlings were slight wilting 24 hours after the mycelium was irrigated, wilting and lodging of the plants after 48 hours, and root rot and stem rot were observed at 54 hours. Compared with the water control group and the PDB control group, the disease index and incidence rate of the sunflower strain treated with Tuvanillosis SJZ were 38.33%.

[0086] Example 4: Analysis of the broad-spectrum antibacterial activity of Tuvanyong Bacillus SJZ

[0087] Plant pathogenic bacteria Fusarium graminearum (F.graminearum), Alternaria dauci (A.dauci), Alternaria nees (A.Nees), and V.sordida (plant pathogenic bacteria purchased from Mingzhou Biotechnology Co., Ltd.) were selected to prepare plant pathogenic bacteria cakes. The plant pathogenic bacteria 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 was inoculated 1 cm away from the edge of the plate. The culture was inverted at 28°C, and the diameter of the pathogen was observed after 5 days. The experimental results are as follows Fig.11 As shown, Fig.11A is Fusarium graminearum, B is Solanum solani, C is Alternaria solani, and D is Alternaria tenuissima. The results show that Tuwanyong Bacillus SJZ can inhibit the growth of Fusarium graminearum, Alternaria tenuissima and Alternaria solani to a small extent; Tuwanyong Bacillus SJZ can inhibit the growth of Solanum solani, and the hyphae of Solanum solani near Tuwanyong Bacillus SJZ turn yellow to brown. The possible reason is that the antibacterial substances secreted by Tuwanyong Bacillus SJZ break the hyphae of pathogens, causing the hyphae contents to leak out and oxidize and turn brown. This shows that Tuwanyong Bacillus SJZ has certain application potential in the field of agricultural and forestry disease prevention and control.

[0088] Example 5: Analysis of the Growth-Promoting Potential of Tuvania sp. SJZ

[0089] The growth-promoting ability of the growth-promoting strain mainly includes five aspects: nitrogen fixation, phosphorus solubilization, potassium solubilization, secretion of iron carriers, and secretion of IAA. The single colony of Tuwayong Bacillus 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, and inverted cultured at 28°C for 3 days. If Tuwayong Bacillus 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 the strain produces an orange-yellow transparent circle on the CAS medium, the strain has the ability to secrete iron carriers. Inoculate Bacillus Tuvanianus SJZ into LB medium containing 0.1% L-Try, culture at 37°C and 180 r / min for 2 days, aspirate the bacterial solution, centrifuge at 8000 r / min for 5 min, aspirate 100 μL of the supernatant, add 200 μL of Salkowski reagent, and color for 30 min in the dark. If it turns pink, it is positive. If it is difficult to distinguish, measure its OD530nm.

[0090] The results are as follows Fig.12 As shown, Fig.12 In the figure, a is nitrogen fixation, b is siderophore production, and c is IAA production. It can be seen that Tuvania sp. SJZ has a certain nitrogen fixation ability, does not have the ability to solubilize phosphorus and potassium, has the ability to secrete siderophores, and can produce IAA, indicating that it has a certain growth-promoting potential and has a certain application prospect in agricultural disease control and biocontrol growth-promoting bacteria development.

Claims

1. A strain of Bacillus tuvanii for inhibiting plant pathogens, characterized in that: The Tuwanensis Bacillus is Tuwanensis Bacillus (Bacillus toyonensis) 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 20242883.

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

3. The Tuvania sp. as claimed in claim 1 is used to inhibit Fusarium graminearum, Alternaria solani, Alternaria tenuissima and Sordella solani.

4. The Tuvanian Bacillus as claimed in claim 1 is capable of secreting protease, cellulase and chitinase.

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

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

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