A composite endophytic microbial fertilizer and its preparation method and application

Abstract

The invention provides a bacillus, named as Bacilus sp., registered and preserved in Guangdong Province Microbiological Culture Collection Center, and the preservation number is GDMCC NO: 63022. The invention also provides a mixed bacterial agent or microbial fertilizer containing bacillus and banana endofibroblastic microbacteria, and the banana endofibroblastic microbacteria is named as Celulosimicrobium sp., and the preservation number is GDMCC NO: 63020. The bacillus and the mixed bacterial agent of the bacillus and the banana endofibroblastic microbacteria have antagonistic effects on banana wilt pathogenic fungus No. 4, pepper anthracnose fungus, etc., and are potential biological agents for preventing and controlling wilt diseases, etc., and the bacillus and the mixed bacterial agent of the invention also have a good effect of promoting plant growth, and have broad development space and good development and application prospects.

Description

Technical Field

[0001] The invention relates to the biological field, and in particular to a composite endophytic microbial fertilizer and a preparation method and application thereof. Background Art

[0002] Banana wilt (Fusarium oxysporum f.sp.cubense), also known as banana Panama disease and yellow leaf disease, is caused by infection of the Cuban-specific pathogen of Fusarium oxysporum, destroying the banana vascular bundle and causing the death of the plant. The harmfulness of banana wilt has spread to countries around the world. In recent years, there have been many reports on the occurrence of the disease in many banana-producing countries and regions in the world. At present, it is common in tropical and subtropical banana-growing areas such as Asia, Africa, and Latin America, seriously affecting the development of the banana industry around the world. Biological control, as one of the prevention and control measures for banana wilt, is currently recognized as the safest and most effective method for soil-borne disease prevention and control. The main ways to obtain antagonistic microorganisms for the prevention and control of banana wilt reported so far include soil in different habitats and environmental conditions such as the ocean, but in actual applications, due to the problem of colonization ability of the strain, many beneficial microorganisms have poor field adaptability and weak colonization ability, which affects the prevention and control effect. Therefore, it is particularly important to screen microbial strains with strong adaptability and strong colonization ability.

[0003] Plant endophytes are microorganisms that live in mutualistic symbiosis with plants and are natural resources for biological control. Therefore, fully tapping into plant endophytes and utilizing the beneficial endophytic resources of the host plant itself is of great significance for inducing host system resistance and promoting the growth of the host plant. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and to provide a composite endogenous microbial fertilizer and a preparation method and application thereof.

[0005] The first aspect of the present invention provides a bacillus, named as Bacilus sp., which is registered and preserved in Guangdong Microbiological Culture Collection Center with a preservation number of GDMCC NO: 63022.

[0006] The second aspect of the present invention is to provide a fermentation broth of the Bacillus according to the first aspect of the present invention.

[0007] The third aspect of the present invention is to provide a mixed bacterial agent, characterized in that it contains the bacillus described in the first aspect of the present invention and banana endofibroblastic microbacteria, wherein the banana endofibroblastic microbacteria is named: Celulosimicrobiumsp., registered and preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 63020.

[0008] The fourth aspect of the present invention is to provide a microbial fertilizer containing the Bacillus described in the first aspect of the present invention or the mixed bacterial agent described in the third aspect of the present invention.

[0009] The fifth aspect of the present invention is to provide the use of the Bacillus as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the mixed bacterial agent as described in the third aspect of the present invention, or the microbial fertilizer as described in the fourth aspect of the present invention in promoting plant growth.

[0010] The sixth aspect of the present invention is to provide the use of the Bacillus as described in the first aspect of the present invention, or the fermentation broth as described in the second aspect of the present invention, or the mixed bacterial agent as described in the third aspect of the present invention, or the microbial fertilizer as described in the fourth aspect of the present invention in increasing plant height, and / or stem diameter, and / or aboveground biomass, and / or chlorophyll content.

[0011] The seventh aspect of the present invention provides the use of the bacillus as described in the first aspect of the present invention, or the fermentation liquid as described in the second aspect of the present invention, or the mixed bacterial agent as described in the third aspect of the present invention, or the microbial fertilizer as described in the fourth aspect of the present invention in antagonizing banana wilt pathogen race 4, and / or pepper anthracnose pathogen, and / or banana long spot pathogen, and / or cucumber wilt pathogen, and / or mango anthracnose pathogen, and / or rice blast pathogen, and / or gloeosporium anthracnose pathogen, and / or wheat fusarium head blight pathogen, and / or apple ring rot pathogen, and / or banana gray spot pathogen, and / or strawberry anthracnose pathogen.

[0012] The eighth aspect of the present invention provides the use of the bacillus as described in the first aspect of the present invention, or the fermentation liquid as described in the second aspect of the present invention, or the mixed bacterial agent as described in the third aspect of the present invention, or the microbial fertilizer as described in the fourth aspect of the present invention in preventing and controlling diseases caused by banana wilt pathogen race 4, and / or pepper anthracnose pathogen, and / or banana long spot pathogen, and / or cucumber wilt pathogen, and / or mango anthracnose pathogen, and / or rice blast pathogen, and / or gloeosporium anthracnose pathogen, and / or wheat fusarium head blight pathogen, and / or apple ring rot pathogen, and / or banana gray spot pathogen, and / or strawberry anthracnose pathogen.

[0013] The ninth aspect of the present invention is to provide the use of the Bacillus as described in the first aspect of the present invention or the fermentation broth as described in the second aspect of the present invention in the preparation of an IAA-producing preparation.

[0014] The tenth aspect of the present invention is to provide the use of the Bacillus as described in the first aspect of the present invention or the fermentation liquid as described in the second aspect of the present invention in the preparation of biological fertilizer or biological pesticide.

[0015] The bacillus and the mixed bacterial agent of the bacillus and the banana endofibroblastic microorganisms have certain antagonistic effects on banana wilt pathogenic fungi No. 4, pepper anthracnose pathogens, banana long spot pathogens, cucumber wilt pathogens, mango anthracnose pathogens, rice blast pathogens, gloeosporium anthracnose pathogens, wheat fusarium head blight pathogens, apple ring rot pathogens, banana gray spot pathogens, strawberry anthracnose pathogens, etc., and can also synergize with the bacillus to improve the antibacterial property, and the bacillus and the banana endofibroblastic microorganisms in the mixed bacteria synergistically improve the antibacterial property, and are potential biological agents for preventing and controlling wilt diseases, anthracnose diseases, etc., and the bacillus and the mixed bacterial agent of the present invention also have a good effect of promoting plant growth, can improve plant height, stem thickness, aboveground biomass, chlorophyll content, etc., and have broad development space and good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The ability of endophytic bacteria ZD3 strain to produce siderophore.

[0017] Figure 2 is the relative content of siderophore produced by endophytes.

[0018] Figure 3 Shown are the culture characteristics and electron microscope scanning images of the ZD3 strain on LB medium.

[0019] Figure 4 This is the phylogenetic tree of the ZD3 strain.

[0020] Figure 5 This is a potted inoculation test of ZD3 strain fermentation broth.

[0021] Figure 6 It is the disease index of banana seedlings under different treatments in potted plant prevention and control of banana wilt.

[0022] Figure 7 These are the culture characteristics and spore morphology of the ZD7 strain.

[0023] Figure 8 This is the phylogenetic tree of the ZD7 strain.

[0024] Fig. 9 This is the coating situation after mixed culture of strains ZD3 and ZD7.

[0025] Fig.10 The control effect of different treatments on banana Fusarium wilt.

[0026] Fig.11 The disease index and prevention and control effects of different treatments.

[0027] The strain ZD3 of the present application is a cellulosimicrobium, named: Celulosimicrobium sp., deposited in the Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC NO: 63020, the deposit date is December 23, 2022, and the deposit address is at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0028] The strain ZD7 of the present application is a Bacillus, named: Bacilus sp., deposited in the Guangdong Microbiological Culture Collection Center (GDMCC), with the deposit number GDMCC NO: 63022, the deposit date is November 30, 2022, and the deposit address is at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Where the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be obtained commercially.

[0030] Example 1 About ZD3 strain

[0031] 1 Experimental Materials

[0032] 1.1 Collection of banana tissue samples

[0033] Banana tissue samples were collected during the banana harvest season in July 2020. Three healthy bananas (with no obvious symptoms of the disease) were randomly selected from the Nanbao Banana Garden in Lingao, Hainan (with an incidence rate of less than 0.5% for more than three consecutive years), and tissues from the root, corm, and pseudostem were collected. Five samples were taken from each part and thoroughly mixed as one test sample.

[0034] 1.2 Test culture medium

[0035] The main culture medium formula used in this experiment is shown in Table 1.

[0036] Table 1 Main culture media and their formulations

[0037]

[0038] CAS detection solution: Solution A: weigh 0.079g CAS and dissolve it in 50mL double distilled water, then add 10mL 1mmol / LFeCl3 solution (prepared with 10mmol / LHCl); Solution B: dissolve 0.069g hexadecyltrimethylammonium bromide (HDTMA) in 40mL double distilled water; Solution C: dissolve 4.307g anhydrous piperazine in 100mL double distilled water; slowly add solution A to solution B along the wall of the beaker, then slowly add solution C, stir and mix to obtain 200mL CAS blue detection solution; for quantitative determination, draw 15mL and dilute to 100mL with anhydrous piperazine.

[0039] CAS detection medium: CAS detection solution 100mL, 15g / L agar 100mL, KMB medium 100mL, sterilize at 121℃ for 20min, when cooled to about 50℃, slowly pour agar into the CAS detection solution along the wall, shake slightly to mix, pour onto the plate, wait for it to solidify, and then pour a layer of KMB medium on it.

[0040] 2 Experimental methods and results

[0041] 2.1 Isolation of banana endophytes

[0042] Sample processing method: Take 10 samples of banana roots, pseudostems, corms and other tissues cut to 5 cm, rinse with sterile water 3 times, transfer to 2% sodium hypochlorite solution and soak for 15 minutes, then rinse with sterile water 3 times. Then transfer the samples to 70% ethanol and soak for 5 minutes, rinse with sterile water 5 times, and finally use sterile absorbent paper to dry the surface moisture on a sterile operating table for later use.

[0043] Prepare sample dilution solution: Use a sterile scalpel to cut the root, stem, and leaf tissues into 1 cm tissue blocks on a sterile operating table, transfer to a sterile mortar and grind, and store the ground plant tissue at 4°C for later use. Weigh 5 g of each ground tissue sample from each treatment and add it to a 150 mL Erlenmeyer flask pre-loaded with 45 mL of sterile deionized water. Incubate at 30°C, 180 r / min. -1 After shaking for 30 min, 10 -1 Concentration of dilution.

[0044] Isolation and purification of endophytic bacteria: 100 μL of the corresponding sample dilution was applied to R2A medium. After culturing at 30°C for 3 days, single colonies with complete morphology and obvious differences were selected for streak purification and culture. 100 single colonies were selected from each tissue sample of the plant. After purification, they were stored in glycerol tubes for later use.

[0045] After separation, purification and molecular biological identification, a total of 265 strains of endophytic bacteria in banana were isolated and identified, of which 98, 86 and 81 strains were screened from root, corm and pseudostem samples, respectively.

[0046] 2.2 Screening of siderophore-producing microorganisms

[0047] The purified strains were inoculated on CAS detection medium with a sterile toothpick and cultured at 25℃ for 3-14 days to observe whether orange and red halos were produced on the CAS blue medium. The results showed that among the 265 bacterial strains, 16 strains secreted iron carriers "+ (weak)", 18 strains "+", 23 strains "+-+-", and 8 strains "+++".

[0048] 2.3 Rescreening of siderophore production capacity

[0049] The relative content of iron carrier secretion in the 8 strains of bacteria that were initially screened to have strong iron carrier secretion ability was further tested. A 7mm pathogen cake was taken and inoculated into the KMB double-layer CAS detection medium, cultured at 25℃ for 15 days to observe whether it produces iron carriers. KMB liquid culture medium was prepared, a 7mm cake was taken and inoculated, 180r / min, 25℃ for 10 days, and centrifuged at 10000r / min for 15min. Take 1mL of the supernatant and mix it with 1mL of CAS detection solution As, add 20μL of sulfosalicylic acid, and measure its absorbance at 630nm after standing for 2h under dark conditions. The uninoculated culture medium was used as the control Ar, the blank culture medium was adjusted to zero, and the relative content of iron carriers Su=(Ar-As) / Ar×100% was calculated, and each treatment was repeated 3 times.

[0050] The results are as follows Figure 1 and Figure 2 As shown, the iron carrier secretion of ZD3 strain was significantly higher than that of the other seven bacterial strains, CK was a strain without the ability to produce iron carriers, and ZD3 strain produced an obvious halo on CAS medium.

[0051] 2.4 Antibacterial rate of siderophore-producing strains against pathogens

[0052] The antagonistic effect of 8 strains of bacteria with strong iron carrier secretion ability was tested on wilt pathogen. A 7mm bacterial cake of the pathogen (F.oxysporum race 4) was inoculated in the center of the PDA culture medium, and a sterile toothpick was used to inoculate the purified bacterial strain 2 cm away from the pathogen. The culture was cultured at 28-30℃ for 7 days. The fungal diameter was measured and the inhibition rate was calculated with the control without bacterial strain. Each strain was repeated 3 times. Inhibition rate = (control group colony diameter - treatment group colony diameter) / (control group colony diameter - cake diameter) × 100%. The results are shown in Table 2.

[0053] Taking into account the relative content of siderophore production and the antagonistic effect, strain ZD3 was selected as the object of further study.

[0054] Table 2 Antagonistic effect of siderophore-producing endophytic bacteria on Fusarium wilt

[0055] Siderophore-producing strains Antibacterial rate (%) ZD2 10.03±0.37a ZD3 11.05±0.17a ZD12 7.81±0.30b ZD17 2.29±0.24c 2GL-1 0.35±0.05f 2QN-5 0 2JJ-3 1.42±0.10e 2QL-8 1.86±0.06d

[0056] Note: The data in the table are mean ± standard deviation. Different small letters in the same column indicate significant differences at the P < 0.05 level tested by Duncan's new multiple range method.

[0057] 2.5 Detection of broad-spectrum antibacterial activity of siderophore-producing endophytic bacteria strain ZD3 The broad-spectrum antibacterial activity of strain ZD3 was determined using 11 pathogenic fungi. The method of antibacterial activity determination was the same as that of "2.4 Antibacterial rate of siderophore-producing strains against pathogens", and its antibacterial activity was calculated. The results are shown in Table 3. Strain ZD3 has certain antibacterial activity against banana wilt pathogen race 4, pepper anthracnose pathogen, banana long spot pathogen, cucumber wilt pathogen, mango anthracnose pathogen, rice blast pathogen, gloeosporium anthracnose pathogen, wheat fusarium head blight pathogen, apple ring spot pathogen, banana gray spot pathogen, strawberry anthracnose pathogen, etc., which shows that strain ZD3 has a broad-spectrum antibacterial activity.

[0058] Table 3 Inhibitory effect of strain ZD3 on 11 pathogenic fungi

[0059] Pathogenic fungi Antibacterial rate (%) Banana Fusarium oxysporum Race 4 11.05±0.17 Pepper Anthracnose Colletotrichum acutatum 11.15±0.21 Curvulatia fallax 10.35±0.75 Cucumber wilt pathogen Fusarium oxysporum (Schl.) F.sp cucumerinum Owen 9.27±0.21 Banana Anthracnose Gloeosporium musarum 9.33±0.13 Pyricularia oryae Cav 10.32±0.67 Mango Anthracnose Pathogen Colletotrichum gloeosporioides 11.05±0.37 Wheat FusaHum grammearum Sehw 5.65±0.43 Apple ring rot pathogen Botryosphaeria dothidea 7.44±0.17 Banana gray spot pathogen Curvularia lunata 5.33±0.24 Strawberry Anthracnose Colletotrichum fragariae 10.33±0.13

[0060] 2.6 Identification of the siderophore-producing endophyte ZD3

[0061] (1) Morphological characteristics

[0062] ① Observation of plate colony morphology: streak inoculation on LB solid medium, incubate at 30℃ for 24h, and observe the colony morphology, including color, texture, roughness, etc. At the same time, perform Gram staining test and observe the staining and morphology with electron microscope.

[0063] ② Scanning electron microscopy observation: Take 2mL of bacterial solution cultured to the logarithmic phase, centrifuge at 6000rpm for 1min, wash three times with 0.1M phosphate buffer, add 2.5% glutaraldehyde, mix well, and stand at 4°C overnight; centrifuge to remove glutaraldehyde, wash twice with PBS for 15min each, and dehydrate the bacteria with 30%, 50%, 70%, 80%, 90%, and 100% ethanol in sequence for 15min each, including 100% dehydration twice for 20min each, then treat with a 1:1 mixture of anhydrous ethanol and isoamyl acetate (v / v) for 30min, finally replace with isoamyl acetate for 2h, then apply on a glass slide, spray gold and observe using a scanning electron microscope.

[0064] ③ Gram staining: drop a drop of sterile deionized water on the slide, pick up a small amount of bacteria with an inoculation loop, mix with water and spread, pass over the flame 2-3 times to fix the bacteria, add crystal violet dye for 30 seconds and then rinse with deionized water, add iodine solution for 1 minute and then rinse with deionized water, drain the water and rinse with anhydrous ethanol until colorless, wash with water immediately, add safranin dye for 2 minutes and then wash with water. Drain the water and observe under a microscope.

[0065] The results showed that the colonies of strain ZD3 were light yellow and opaque, with a slightly raised surface and neat and smooth edges ( Figure 3 Left picture); the bacteria are short rod-shaped and about 0.5-0.8 μm in size ( Figure 3 Right figure); Gram staining test results showed that strain ZD3 was a Gram-positive bacterium.

[0066] (2) Physiological and biochemical characteristics

[0067] The physiological and biochemical characteristics of the selected iron carrier-producing strains were identified according to the Manual of Identification of Common Bacterial Systems compiled by Dong Xiuzhu et al. in 2001.

[0068] 1) Carbon source utilization test

[0069] Prepare the carbon source using the basic culture medium, add different carbon sources at a concentration of 0.5% to the basic culture, inoculate the strain to be tested, and culture at a constant temperature of 30°C for seven days, and observe and record the reproduction of the strain. The types of carbon sources are as follows: inositol, xylan, maltose, sucrose, trehalose, glucose, xylose, anhydrous lactose, α-lactose, rhamnose, melibiose, soluble starch, etc. (see Table 3 for details). Basic culture medium: diammonium hydrogen phosphate 2.64g, potassium dihydrogen phosphate 2.38g, dipotassium hydrogen phosphate 5.65g, copper sulfate pentahydrate 0.0064g, magnesium sulfate crystals 1g, zinc sulfate heptahydrate 0.0015g, manganese tetrachloride 0.0079g, ferrous sulfate crystals 0.0011g, distilled water 1000mL, agar 20g, pH 7.2-7.4.

[0070] The results are shown in Table 4. Strain ZD3 can utilize inositol, xylan, maltose, mannitol, sucrose, trehalose, glucose, xylose, D-galactose, anhydrous lactose, sorbitol, α-lactose, melibiose, D-fructose, soluble starch, D-mannose, etc., but cannot utilize melezitose, rhamnose, etc.

[0071] Table 4 Biochemical and physical indicators of ZD3

[0072]

[0073] “+” represents a positive test result; “-” represents a negative test result.

[0074] 2) Nitrogen source utilization test

[0075] Prepare nitrogen source using basal culture medium, add different nitrogen sources to basal culture medium at a concentration of 0.5%, inoculate the strain to be tested, culture at a constant temperature of 30°C for seven days, and observe and record the reproduction of the strain. Different nitrogen sources are added to the basal culture at a concentration of 0.5%. The types of nitrogen sources are as follows: asparagine, histidine, serine, tyrosine, arginine, ammonium chloride, ammonium nitrate, methionine, ammonium sulfate, ammonium molybdate tetrahydrate, cystine, valine, methionine, tryptophan, phenylalanine, ammonium oxalate, ammonium acetate, glycine, etc. (see Table 3 for details). Basal culture medium: 10.00g glucose, 0.5g magnesium sulfate crystals, 0.05g sodium chloride, 0.001g ferrous sulfate crystals, 0.01g potassium dihydrogen phosphate, 1000mL distilled water, 20g agar, pH 7.2.

[0076] The results are shown in Table 4. The strain ZD3 can utilize asparagine, serine, tyrosine, arginine, ammonium nitrate, methionine, ammonium sulfate, cysteine, valine, methionine, tryptophan, phenylalanine, glycine, etc., but cannot utilize histidine, ammonium chloride, ammonium molybdate tetrahydrate, ammonium oxalate, ammonium acetate, etc.

[0077] 3) Salt tolerance test

[0078] Bennett agar medium with different NaCl concentrations was prepared, the strain to be tested was inoculated into the medium, and cultured at a constant temperature of 35°C for seven days, and the growth of the strain in the medium with different NaCl concentrations was observed and recorded. The NaCl concentrations were 0, 1%, 3%, 5%, 7%, 9%, 11%, 13%, and 15%. Medium: 10g glucose, 2g enzymatic hydrolyzed casein, 1g beef extract, 1g yeast extract, 20g agar, and 1000ml distilled water. pH 7.0. The results are shown in Table 4. The strain ZD3 grew well when the NaCl concentration was 0-9%, and had a certain salt tolerance.

[0079] Table 5 ZD3 strain salinity tolerance test

[0080]

[0081] “+” represents a positive test result; “-” represents a negative test result.

[0082] 4) pH tolerance test

[0083] The strains to be tested were inoculated into different pH culture medium shakers and cultured at 30°C for seven days, and the growth of the strains in different pH culture medium was observed and recorded. Solution A: 27.218g potassium dihydrogen phosphate, 1000ml distilled water. Solution B: 45.644g potassium dihydrogen phosphate, 1000ml distilled water. The proportions of different pH solutions are shown in Table 6. As shown in Table 6, strain ZD3 grew well at pH 5-10.

[0084] Table 6 pH test different concentration ratios

[0085]

[0086] “+” represents a positive test result; “-” represents a negative test result.

[0087] 5) Nitrate reduction test

[0088] Nitrate reduction medium: KNO3 1g, K2HPO4 0.5g, MgSO4 0.5g, NaCl 0.5g, sucrose 20g, distilled water 1000ml, pH 7.2. After the strain to be tested is inoculated into the nitrate medium and cultured for seven days, the nitrate reduction is measured. Some bacteria can reduce nitrate to nitrite, ammonia and nitrogen, etc. When Griggs reagent is added to the culture solution inoculated with bacteria, the solution will appear pink, rose red, orange, brown, etc. The results are shown in Table 4. Strain ZD3 can reduce nitrate.

[0089] 6) H2S generation test

[0090] Chessner medium: 10g peptone, 0.5g ferric citrate, 20g agar, 1000ml distilled water. pH 7.0. After the strain to be tested is inoculated into Chessner medium and cultured for seven days, if the medium produces melanin, it proves that H2S is produced. Since H2S and ferric citrate react to produce FeS, the medium will turn black. The results are shown in Table 4. Strain ZD3 cannot produce H2S.

[0091] 7) Starch hydrolysis experiment

[0092] Starch hydrolysis medium: soluble starch 10g, MgCO31 g, K2HPO4 0.3g, KNO31 g, NaCl 0.5g, agar 20g, distilled water 1000ml, pH 7.0. Preparation of iodine solution: iodine tablets 1g, potassium iodide 2g, distilled water 1000ml, pH7.2-7.4. The strain to be tested is inoculated on the starch hydrolysis agar plate by dot grafting, and the inoculation diameter does not exceed 5mm. After seven days of constant temperature cultivation at 30℃, iodine solution is sprayed on the culture medium. If the colony produces amylase, a transparent circle will be produced around the colony. The results are shown in Table 4. The strain ZD3 cannot hydrolyze starch.

[0093] 8) Gelatin liquefaction test

[0094] Gelatin liquefaction medium: 5g peptone, 20g glucose, 200g gelatin, 1L distilled water, pH 7.2-7.4. After the strain to be tested is inoculated into the gelatin liquefaction medium and cultured for seven days, the medium is observed. This experiment mainly determines whether the strain can produce protease. Because gelatin at 30°C is liquid, and gelatin below 20°C is solid. If the strain can produce gelatinase, the gelatin medium can also be liquid below 20°C. The results are shown in Table 4, and strain ZD3 can liquefy gelatin.

[0095] 9) Lipase (Tween 20, 40, 80) test

[0096] Lipase basal medium: peptone 1g, CaCl 2· 7H20 0.1g, NaCl 5g, agar 20g, distilled water 100ml. pH7.4. Add 1g of Tween-20, Tween-40, Tween-80 to the substrate. Inoculate the strain to be tested on the culture medium plate by dot-grafting. After one week of constant temperature culture at 30℃, observe whether there is a fuzzy transparent circle around the colony. If there is a fuzzy circle, it is positive, and if there is no fuzzy circle, the test is negative.

[0097] 10) Urease test

[0098] Urease medium: VP medium: peptone 1g, glucose 1g, KH2PO4 2g, phenol red 0.012g, agar 20g, distilled water 1000ml. PH 6.8-6.9 (slightly yellow), 30g of urea is sterilized separately with ether and medium. When the medium after high temperature sterilization is cooled to 55°C, sterile urea is added to the medium. This experiment is to detect whether the strain has the ability to produce urease. The results are shown in Table 4. The urease test of strain ZD3 is negative.

[0099] 11) VP test

[0100] VP culture medium: 5g peptone, 5g glucose, 5g K2HPO4, 1000ml distilled water. VP reagent: 0.3g creatine, 100ml distilled water. Sodium hydroxide solution: 40g sodium hydroxide, 100ml distilled water. Inoculate the strain to be tested into the VP culture medium and culture it on a shaker for one week. Take 3ml of culture medium and add 3ml sodium hydroxide solution, then add a drop of creatine. If it turns red after 10 minutes, it proves that the experiment is a positive reaction. Otherwise, it is a negative reaction. Some strains decompose the pyruvate produced by glucose, which can be further decarboxylated to produce acetylmethyl carbinol. Acetylmethyl carbinol is oxidized to form diacetyl in alkaline conditions, which can react with arginine in peptone to produce a red compound, indicating that the VP test is positive. The results are shown in Table 4, and the strain ZD3V-P test is negative.

[0101] 12) MR (methyl red) test

[0102] MR culture medium: 5g peptone, 5g glucose, 5g K2HPO4, 1000ml distilled water. Methyl red reagent: 0.1g methyl red, 200ml 95% ethanol, 200ml distilled water. Inoculate the strain to be tested into MR culture medium, culture on a shaker for one week, then add a drop of methyl red reagent to the culture medium. If the culture medium appears red, it is a positive reaction, and if it appears yellow, it is a negative reaction. Bacteria decompose glucose to produce pyruvic acid, which is further decomposed into formic acid, acetic acid, lactic acid and succinic acid, reducing the pH of the culture medium to below 4.5, and the methyl red reagent will appear red. If the bacteria decompose glucose to produce aldehydes, ketones, water, etc., the pH of the culture medium will be above 5.4, and the methyl red reagent will appear orange-yellow. The results are shown in Table 4, and the strain ZD3 MR (methyl red) test is negative.

[0103] (3) Genotype analysis of strains

[0104] The bacterial 16S rDNA universal primers 27F (5-AGAGTTTGATCMTGGCTCAG-3) and 1492R (5

[0105] -TACGGYTACCTTGTTACGACT-3) to establish a PCR amplification system for amplification. Then it was sent to Sangon for sequencing. EzTaxon and GenBank were used to search for sequence similarity, and the model strain sequence with higher similarity was selected. The Neighbor-Joining method in MEGA7.0 was used to compare homology and construct a phylogenetic tree. Figure 4 The phylogenetic tree showed that the strain was clustered with strains of the genus Celulosimicrobium, confirming that the strain belonged to the genus Celulosimicrobium.

[0106] The strain was further compared with the two most similar strains (Celulosimicrobium funkei and Celulosimicrobium celulans) and the ANI values ​​were 87.87% and 88%, respectively, which are far lower than the standard of new species identification <95%. Therefore, the strain was determined to be a new species of Celulosimicrobium.

[0107] Finally, based on the morphological, cultural and physiological and biochemical characteristics, strain ZD3 was identified as a new species of the genus Celulosimicrobium, named: Celulosimicrobium sp., hereinafter referred to as Celulosimicrobium ZD3 or strain ZD3, and deposited in the Guangdong Microbial Culture Collection Center (GDMCC), with the deposit number GDMCC NO: 63020, the deposit date is November 20, 2022, and the deposit address is at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0108] 2.7 Pot inoculation test of siderophore-producing strains

[0109] (1) Banana seedlings for testing

[0110] The banana tissue culture seedlings are all healthy Brazilian banana cup seedlings with relatively uniform growth and 5 to 6 leaves, provided by the Danzhou Tissue Culture Center of the Chinese Academy of Tropical Agricultural Sciences.

[0111] (2) Preparation method of strain fermentation broth

[0112] Soybean meal and brown sugar were purchased from the Comprehensive Grain and Oil Wholesale Market in Haikou City, Hainan Province. Based on the carbon-nitrogen ratio of soybean meal and the carbon content of brown sugar, the carbon-nitrogen ratio of the matrix was adjusted to 20-25:1. The total amount of brown sugar and soybean meal was 250g, that is, 63.7g of soybean meal, 186.3g of brown sugar, and 1000mL of sterile water. The mixture of brown sugar and cake fertilizer was prepared and placed in a 5L triangular bottle for testing.

[0113] Prepare LB liquid culture medium, put it in a 250mL conical flask, sterilize it at 121°C for 20 minutes, inoculate fresh test ZD3 strain after cooling, and culture it in a shaking incubator at 28-30°C and 180rpm / min for 48h. Then, inoculate the LB culture solution of the strain into the mixed fermentation solution of brown sugar and cake fertilizer at a ratio of 1%, and continue to culture it in a shaking incubator at 28-30°C and 180rpm / min for 72h to obtain the fermentation solution of the strain.

[0114] (3) Test soil

[0115] The soil used in the experiment was taken from the experimental field of the two academies in Danzhou, Hainan Province (longitude 109.360252, latitude 19.554628). The soil is sandy loam and red soil. The physical and chemical properties are as follows: organic matter content 17.920 mg / kg, total nitrogen (N) 0.082%, pH 4.31, available potassium (K2O) content 158.88 mg / kg, available phosphorus (P2O5) content 172.2 mg / kg, exchangeable magnesium 130.63 mg / kg, exchangeable calcium 1396.26 mg / kg.

[0116] The pot experiment was conducted in the greenhouse of the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences from July to September 2023. The greenhouse environmental conditions were controlled as follows: humidity of 70%, temperature of about 28°C, and natural light. The experiment set up three treatments: (1) MOCK pathogen untreated: blank (application of clean water); (2) pathogen treatment: CK (application of substrate fermentation broth without adding microbial strain ZD3); (3) ZD3 treatment: inoculation of pathogen + ZD3 fermentation broth treatment.

[0117] In this study, FOC4 was selected as the wilt pathogen and inoculated by root injury dipping method. Banana seedlings with uniform growth and 5 to 6 leaves were selected, the second taproot was cut off, and the seedlings were inoculated with a dilution concentration of 10 5 After soaking in a pathogen suspension of CFU / mL for 30 minutes, the banana seedlings were transplanted into a plastic pot with a soil volume of 1kg, and 20mL of pathogen suspension was poured into the rhizosphere soil of the banana seedlings. After the banana seedlings were infected, the ZD3 treatment group was applied with 50 times diluted ZD3 fermentation liquid, 200mL per plant; the MOCK group was applied with an equal amount of clean water; the CK group was applied with an equal amount of substrate fermentation liquid; and each treatment liquid was applied every 7 days, for a total of 6 times. Each treatment was set up with 3 replicates, and each replicate had 10 banana seedlings in a pot. During the experiment, other management measures for each treatment were the same.

[0118] (4) Statistics on disease index and disease prevention effect

[0119] On the 50th day after banana transplanting, the number of normal leaves and yellow leaves of each banana plant in each treatment was recorded, and the disease index and control effect were calculated. Banana wilt disease grading standard: Grade 0, healthy plant; Grade 1, 25% yellow diseased leaves; Grade 3, 25% to 50% yellow diseased leaves; Grade 5, 50% to 90% yellow diseased leaves; Grade 7, all leaves turn yellow and the plant dies. Banana disease index and disease control effect calculation:

[0120]

[0121]

[0122] The results of the pot test are as follows Figure 5 and Figure 6As shown in the figure, the average disease index of the banana seedlings treated with the endophyte ZD3 was 25.99, while the average disease index of the CK treatment was 54.18. It can be concluded that the relative prevention effect (disease prevention effect) of the ZD3 strain on banana wilt in the seedling stage is 52.03%, indicating that the ZD3 strain has a good prevention and control effect on banana wilt and can inhibit the infection of banana wilt pathogens.

[0123] Example 2 About ZD7 strain

[0124] 1. Experimental Materials

[0125] See Example 1.

[0126] 2. Isolation of banana endophytes

[0127] See Example 1.

[0128] 3. Determination of IAA production capacity of strains

[0129] The purified endophytic strains were cultured in King's B medium (peptone 20.0 g, K2HPO4 1.5 g, MgSO4·7H2O 1.5 g, glycerol 10.0 mL, L-tryptophan 0.1 g, distilled water to 1 L) for strain culture and IAA production capacity determination. After culture, the Salkowski colorimetric method was used for color development. If the color changes from yellow to pink, it indicates that IAA can be secreted. The positive strains were inoculated in King's B liquid medium and incubated at 28-30 ° C and 180 r·min -1 Shake culture for 2 days, 13000 r·min -1 Centrifuge for 5 minutes, take the supernatant and add Salkowski colorimetric reagent at a ratio of 1:2, place at 28°C in the dark for 30 minutes, and then measure the OD 530 Using King's B liquid medium without bacteria as control, a standard curve was drawn with IAA (analytical grade) gradient dilutions in the same manner as above, and the IAA content in the fermentation broth was calculated based on the standard curve.

[0130] The Salkowski colorimetric method was used to screen the IAA production ability of the isolated endophytic strains. After screening, 14 strains with IAA production ability were obtained. The relative content of IAA production of the strains was obtained according to the standard curve. The ZD7 strain had the strongest IAA production ability, and the IAA secretion amount reached 37.60±1.57 mg / L.

[0131] 4. Identification of strain ZD7

[0132] (1) Morphological characteristics

[0133] ① Observation of plate colony morphology: streak inoculation on LB solid medium, incubate at 30℃ for 24h and observe colony morphology, including color, texture, roughness, etc. At the same time, Gram staining test was performed and the staining and morphology were observed under electron microscope. The results showed that the colony of strain ZD7 was opaque, with uneven surface and irregular edges; the bacteria were oval and short rod-shaped, with a size of about 0.5-0.8μm×1.0-2.0μm( Figure 7 ). The results of Gram staining test showed that it was a Gram-positive bacterium.

[0134] (2) Physiological and biochemical characteristics

[0135] The physiological and biochemical characteristics of the selected iron carrier-producing strains were identified according to the "Common Bacteria System Identification Manual" compiled by Dong Xiuzhu et al. in 2001. The specific operation is shown in Example 1, and the results are shown in Tables 7-9.

[0136] Table 7 Biochemical and physical indicators of ZD7

[0137]

[0138] “+” represents a positive test result; “-” represents a negative test result.

[0139] Table 8 ZD7 strain salinity tolerance test

[0140]

[0141] “+” indicates that the test result is positive.

[0142] Table 9 pH adaptation index

[0143]

[0144] “+” represents a positive test result; “-” represents a negative test result.

[0145] Through sequencing and genome assembly, the genome size of strain ZD7 was 6000008bp and the G+C content was 37.45%. The sequence was submitted to the Ezbiocloud database for homology comparison, and the results showed that strain ZD7 had the highest similarity with Bacillus. To further determine the species of strain ZD7, the strain sequence with high similarity was downloaded and analyzed together with the sequence of strain ZD7 using BioEdit software. The analysis results were used to construct a phylogenetic tree using the Neighbour-Joining method of MEGA 7.0 software, as shown in the figure. Figure 8As shown. The phylogenetic tree shows that the strain is clustered with the genus Bacillus, confirming that the strain is a Bacillus bacterium. Combining morphological, cultural, physiological and biochemical characteristics, strain ZD7 was identified as a Bacillus bacterium and named: Bacilus sp., deposited in the Guangdong Microbiological Culture Collection Center (GDMCC), with a deposit number of GDMCC NO: 63022, and a deposit date of November 30, 2022. The deposit address is at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0146] 4. Antibacterial activity of strain ZD7

[0147] The broad-spectrum antibacterial activity of strain ZD7 was determined using 11 pathogenic fungi. The method for determining the antibacterial activity was the same as that in Example 1 in "2.4 Antibacterial rate of siderophore-producing strains against pathogens", and the antibacterial activity was calculated. The results are shown in Table 10. Strain ZD7 has certain antibacterial activity against banana wilt pathogen race 4, pepper anthracnose pathogen, banana long spot pathogen, cucumber wilt pathogen, mango anthracnose pathogen, rice blast pathogen, gloeosporium anthracnose pathogen, wheat fusarium head blight pathogen, apple ring spot pathogen, banana gray spot pathogen, strawberry anthracnose pathogen, etc., which shows that strain ZD7 has a broad-spectrum antibacterial activity.

[0148] Table 10 Inhibitory effect of strain ZD7 on 11 pathogenic fungi

[0149] Pathogenic fungi Antibacterial rate (%) Banana Fusarium oxysporum Race 4 7.05±0.12 Pepper Anthracnose Colletotrichum acutatum 4.25±0.23 Curvulatia fallax 2.15±0.75 Cucumber wilt pathogen Fusarium oxysporum (Schl.) F.sp cucumerinum Owen 6.27±0.18 Banana Anthracnose Gloeosporium musarum 6.21±0.29 Pyricularia oryae Cav 3.39±0.78 Mango Anthracnose Pathogen Colletotrichum gloeosporioides 6.05±0.27 Wheat FusaHum graminearum Sehw 3.15±0.58 Apple ring rot pathogen Botryosphaeria dothidea 3.47±0.47 Banana gray spot pathogen Curvularia lunata 1.33±0.14 Strawberry Anthracnose Colletotrichum fragariae 5.33±0.83

[0150] Example 3

[0151] 1. Symbiosis experiment of banana endophytes ZD3 and ZD7

[0152] Prepare LB nutrient medium, sterilize at 121℃ for 20min and set aside. Inoculate the test strains ZD3 and ZD7, which have been cultured for 24h, into the same solid nutrient medium plate and liquid culture medium at a 5% inoculation rate to perform bacterial flora combination. The plate is inverted and cultured in a constant temperature incubator at 30℃, and the liquid culture medium is cultured in a shaking table at 28-30℃ and 180rpm / min. After 2-3 days, observe whether there is antagonism or inhibition between the strains during their growth on the plate. The liquid culture medium adopts the dilution plate coating method, and the two are combined to analyze the symbiosis between the strains.

[0153] like Fig. 9 As shown, the test strains were inoculated onto nutrient medium plates respectively. The culture results showed that the strain combinations could grow together on the nutrient medium without antagonism and strong symbiosis. The culture fluid of the mixed strains was spread and it was found that both inoculated microorganisms could grow in uniform numbers without mutual inhibition, which was suitable for joint fermentation culture.

[0154] 2. Potted plant test

[0155] ① Preparation of antagonistic bacterial fermentation broth

[0156] The ZD3 and ZD7 strains were cultured in LB liquid medium to obtain the culture fluids of the two bacteria. Peanut cake fertilizer and brown sugar were adjusted to a carbon-nitrogen ratio of 25:1 according to the carbon-nitrogen ratio of peanut cake and the carbon content of brown sugar. The total amount of brown sugar and cake fertilizer was 250g. 1000ml of water was added to make a mixture of brown sugar and cake fertilizer, which was then placed in a 5L triangular flask for testing. The above two strains were mixed in a ratio of 1:1, that is, added in an amount of 5%-10% to the mixture of cake fertilizer and brown sugar. After shaking and culturing at 180rpm and 22-30 degrees Celsius for 7 days, the microorganisms in the fermentation broth were detected for plate counts greater than 1×10 7 CFU / mL, the mixed strain fermentation broth can be obtained.

[0157] The soil used in the experiment was taken from the experimental field of the two academies in Danzhou, Hainan Province (longitude 109.360252, latitude 19.554628). The soil is sandy loam and red soil. The physical and chemical properties are as follows: organic matter content 17.920 mg / kg, total nitrogen (N) 0.082%, pH 4.31, available potassium (K2O) content 158.88 mg / kg, available phosphorus (P2O5) content 172.2 mg / kg, exchangeable magnesium 130.63 mg / kg, exchangeable calcium 1396.26 mg / kg.

[0158] ② Experimental design

[0159] The pot experiment was conducted in the greenhouse of the Chinese Academy of Tropical Agricultural Sciences from June to August 2023. The greenhouse conditions were controlled at 28℃-30℃, 70% humidity, and natural light. Five treatments were set up in the experiment: (1) Pathogen untreated: blank (brown sugar cake fertilizer mixture without added strains) (2) Pathogen treatment: CK (brown sugar cake fertilizer mixture without added strains), BFZD3 (ZD3 strain fermentation liquid), BFZD7 (ZD7 strain fermentation liquid), BFHX (mixed strain fermentation liquid).

[0160] FOC4 was activated and cultured on PDA plates for 5 days. The pathogen spores were washed out with sterile water to prepare a spore suspension. The spore suspension was then diluted to 10 5 CFU / ml concentration; 500g soil / pot; before transplanting, banana seedlings with uniform growth were inoculated with pathogens using the root injury and immersion method, with an inoculation concentration of 10 5CFU / ml suspension. Each treatment was replicated 3 times, with 10 banana seedlings in each replicate. One day after transplanting, 200ml / pot of 50-fold diluted fermentation liquid was applied according to different treatments, and CK and blank were respectively applied with the same amount of uninoculated mixed liquid; the fermentation liquid was applied again every 7 days, for a total of 6 times. During the experiment, other management measures were the same for each treatment.

[0161] ③Statistics of disease index and disease prevention effect

[0162] On the 60th day after banana transplanting, the number of normal leaves and yellow leaves of each banana plant in each treatment was recorded, and the disease index and control effect were calculated. Banana wilt disease grading standard: Grade 0, healthy plant; Grade 1, 25% yellow diseased leaves; Grade 3, 25% to 50% yellow diseased leaves; Grade 5, 50% to 90% yellow diseased leaves; Grade 7, all leaves yellowed and the plant died. The banana disease index and disease control effect calculation formula are as follows:

[0163]

[0164]

[0165] The results are as follows Fig.10 and Fig.11 The control effect of the composite microbial agent of two strains on banana wilt was significantly higher than that of single strain treatment, among which the control effect of ZD7 single strain treatment was 20.12%, and the control effect of ZD3 single strain treatment was 52.01%, but the control effect of the mixed treatment of the two strains reached 60.86%.

[0166] ④ Pot test on growth promotion effect

[0167] The pot experiment was conducted in the greenhouse of the Chinese Academy of Tropical Agricultural Sciences from July to August 2023. The greenhouse conditions were controlled at 28℃~30℃, 70% humidity, and natural light. The experiment set up three treatments: 1) CK (brown sugar cake fertilizer mixture without added strains); 2) ZD7 (ZD7 strain fermentation liquid treatment); 3) ZD3+ZD7 (mixed strain fermentation liquid treatment); 4) ZD3 (ZD3 strain fermentation liquid treatment); each treatment set up 3 replicates, each replicate 10 banana seedlings. One day after transplanting, 200mL / pot of 50-fold diluted treatment solution was applied according to different treatments, and then each treatment solution was repeatedly applied every 7 days, for 6 applications; including the first application, a total of 7 times. During the experiment, other management measures for each treatment were consistent. 45 days after the banana seedlings were transplanted, the chlorophyll content of the second fully expanded leaf at the top of each treatment was measured, and the plant height, pseudostem girth, aboveground biomass, and chlorophyll content were recorded. The chlorophyll was determined by ethanol extraction method and the OD value was determined by UV1000 visible spectrophotometer.

[0168] The results are shown in Table 11. Compared with the control CK, ZD3 had no growth-promoting effect, while ZD7 and mixed strain treatments had different degrees of growth-promoting effects on the growth of banana seedlings. In terms of plant height, stem diameter, aboveground biomass, chlorophyll content and other indicators, the strain fermentation liquid treatment was significantly higher than the control. However, there was no significant difference in stem diameter and plant height between ZD7 single strain and mixed strain treatment, but in terms of aboveground biomass and chlorophyll content, the mixed strain treatment was significantly better than the ZD7 single strain treatment, indicating that ZD3 can improve the growth-promoting effect of ZD7.

[0169] Table 11 Effects of different treatments on the growth of banana seedlings

[0170] deal with Plant height (cm) Stem diameter (cm) Aboveground biomass (g / plant) Chlorophyll content (mg / g) CK 45.13±1.98b 4.14±0.31b 81.90±6.19c 0.92±0.05c ZD3 46.26±1.35b 4.28±0.33b 83.38±4.12c 0.93±0.13c ZD7 57.21±1.25a 5.18±0.20a 105.26±4.11b 1.28±0.06b ZD3+ZD7 58.26±1.63a 5.14±0.16a 113.39±5.65a 1.83±0.10a

[0171] 4. Antibacterial test of mixed bacteria

[0172] The mixed bacteria of strains ZD3 and ZD7 were tested for broad-spectrum antibacterial activity using 11 pathogenic fungi. The method for testing the antibacterial activity was the same as that of "2.4 Antibacterial rate of siderophore-producing strains against pathogens", and the antibacterial activity was calculated. The results are shown in Table 12. Compared with the individual strains ZD3 (Table 3) and ZD7 (Table 10), the mixed bacteria had further improved antibacterial activity against banana wilt pathogen race 4, pepper anthracnose pathogen, banana long spot pathogen, cucumber wilt pathogen, mango anthracnose pathogen, rice blast pathogen, gloeosporium anthracnose pathogen, wheat fusarium head blight pathogen, apple ring spot pathogen, banana gray spot pathogen, strawberry anthracnose pathogen, etc., which shows that the mixed bacteria also have broad-spectrum antibacterial activity, and the antibacterial activity is higher than that of individual strains ZD3 and ZD7.

[0173] Table 12 Inhibitory effect of mixed bacteria on 11 pathogenic fungi

[0174] Pathogenic fungi Antibacterial rate (%) Banana Fusarium oxysporum Race 4 28.28±0.78 Pepper Anthracnose Colletotrichum acutatum 14.75±1.35 Curvulatia fallax 14.15±0.69 Cucumber wilt pathogen Fusarium oxysporum (Schl.) F.sp cucumerinum Owen 10.42±0.98 Banana Anthracnose Gloeosporium musarum 9.98±0.18 Pyricularia oryae Cav 12.65±1.12 Mango Anthracnose Pathogen Colletotrichum gloeosporioides 16.85±1.33 Wheat FusaHum graminearum Sehw 10.15±1.35 Apple ring rot pathogen Botryosphaeria dothidea 12.47±1.21 Banana gray spot pathogen Curvularia lunata 8.45±0.54 Strawberry Anthracnose Colletotrichum fragariae 16.33±1.27

[0175] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the practical is also within the scope of the present invention. Therefore, the equalization transformation and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A Bacillus, characterized in that Named Bacillus sp., registered and deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC NO: 63022.

2. The fermentation liquid of bacillus as claimed in claim 1.

3. A mixed bacterial agent, characterized in that: Contains the bacillus of claim 1 and banana endofibroblastic microorganisms, wherein the banana endofibroblastic microorganisms are named Cellulosimicrobium sp., registered and deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC NO: 63020.

4. A microbial fertilizer containing the Bacillus according to claim 1 or the mixed bacterial agent according to claim 3.

5. The application of bacillus as claimed in claim 1, or the fermented liquid as claimed in claim 2, or the mixed bacterial agent as claimed in claim 3, or the microbial fertilizer as claimed in claim 4 in improving plant height, and / or stem thickness, and / or aboveground biomass, and / or chlorophyll content, wherein the plant is banana.

6. application of bacillus as claimed in claim 1, or fermented liquid as claimed in claim 2, or mixed bacterial agent as claimed in claim 3, or microbial fertilizer as claimed in claim 4 in antagonism against No. 4 races of banana wilt pathogen, and / or pepper anthracnose pathogen, and / or banana long spot pathogen, and / or cucumber wilt pathogen, and / or mango anthracnose pathogen, and / or rice blast pathogen, and / or gloeosporium anthracnose pathogen, and / or wheat fusarium head blight pathogen, and / or apple ring rot pathogen, and / or banana gray spot pathogen, and / or strawberry anthracnose pathogen.

7. application of bacillus as claimed in claim 1, or fermented liquid as claimed in claim 2, or mixed bacterial agent as claimed in claim 3, or microbial fertilizer as claimed in claim 4 in preventing and treating diseases caused by banana wilt pathogenic bacteria No. 4, and / or pepper anthracnose pathogen, and / or banana long spot pathogen, and / or cucumber wilt pathogen, and / or mango anthracnose pathogen, and / or rice blast pathogen, and / or gloeosporium anthracnose pathogen, and / or wheat fusarium head blight pathogen, and / or apple ring spot pathogen, and / or banana gray spot pathogen, and / or strawberry anthracnose pathogen.

8. Use of the Bacillus according to claim 1 or the fermentation broth according to claim 2 in the preparation of an IAA-producing preparation.

9. Use of the bacillus according to claim 1 or the fermentation liquid according to claim 2 in the preparation of biofertilizer or biopesticide.

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