Saccharifying bacillus L11 and application thereof

Abstract

The invention provides saccharified bacillus L11 and application thereof, and belongs to the technical field of microbial control. The bacillus glycinifermentans L11 is preserved in the Guangdong Microbial Culture Collection Center, the address of the Guangdong Microbial Culture Collection Center is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, the preservation date is August 16, 2024, and the preservation number is GDMCC No: 65016. The saccharified bacillus L11 not only has a good effect of inhibiting phytopathogen, but also has a certain growth promoting characteristic.

Description

Technical Field

[0001] The invention relates to the technical field of microbial control, and in particular to a saccharifying Bacillus L11 and an application thereof. Background Art

[0002] Grape white rot attacks the leaves, branches and fruits of grapes, with the fruit being the main target. The pathogen forms a yellow-brown and water-soaked area that quickly expands and covers the entire berry, eventually becoming soft and rotting. White rot is one of the main fungal diseases of grapes, which can significantly reduce grape yield and quality. If not prevented or controlled in time, white rot can cause 20-50% crop losses, or even higher. Fungicides such as chlorothalonil, thiophanate-methyl, carbendazim, thiram, mancozeb and amicida can be used to control white rot. However, the continued use of chemical fungicides not only leads to the emergence of drug-resistant pathogens, but also ultimately causes food safety and environmental pollution problems. Therefore, the development of an eco-friendly and sustainable biological control agent is expected to solve the ecological problems caused by the excessive use of pesticides in agriculture.

[0003] Bacillus is a spherical or rod-shaped bacterium that can survive for a long time in harsh environments due to the formation of endospores. Many Bacillus species have been shown to have the ability to control plant diseases and have been widely used in the control of crop diseases. For example, Bacillus amyloliquefaciens, Bacillus megaceus, Bacillus licheniformis and Bacillus subtilis have been commercially produced as biological control agents. Currently, the Bacillus species that have biological control effects on grape white rot include Bacillus stercoris, Bacillus velezensis, Bacillus subtilis, Bacillus methylotrophicus, Bacillus amyloliquefaciens and Paenibacillus peoriae. Finding a new Bacillus preparation that can control grape white rot is of great significance for the research and development of biological control agents for grape white rot. Summary of the invention

[0004] The purpose of the present invention is to provide a saccharifying Bacillus L11 and an application thereof, so as to provide a new method for preventing and treating plant diseases such as grape white rot.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Bacillus glycinifermentans L11, which is deposited in Guangdong Microbial Culture Collection Center, with the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation date of which is August 16, 2024, and the preservation number of which is GDMCC No: 65016.

[0007] The invention provides a method for preparing the aseptic fermentation liquid of Bacillus saccharifying L11, which comprises culturing Bacillus saccharifying L11 on medium No. 38, separating solid from liquid, taking the liquid part, and subjecting the liquid part to aseptic treatment to obtain the aseptic fermentation liquid of Bacillus saccharifying L11.

[0008] Preferably, the No. 38 culture medium comprises yeast powder, malt extract, glucose and water, the mass ratio of the yeast powder, malt extract and glucose is 1.6-2.4:1.6-2.4:1.6-2.4, and the mass volume ratio of the mixture of the yeast powder, malt extract and glucose to water is 4-8g:0.8-1.2L;

[0009] The culture temperature is 26-30°C and the culture time is 5-10 days;

[0010] The method of aseptic treatment is filtering with a sterile filter.

[0011] The invention provides a sterile fermentation liquid of Bacillus saccharifying L11 prepared according to the method.

[0012] The present invention also provides a method for preparing the crude extract of the fermentation broth of Bacillus saccharifying L11, which comprises culturing Bacillus saccharifying L11 on medium No. 38, separating the solid from the liquid, taking the liquid portion and extracting it with an organic solvent, removing the solvent in the organic phase obtained by the extraction, and obtaining the crude extract of the fermentation broth of Bacillus saccharifying L11.

[0013] Preferably, the No. 38 culture medium comprises yeast powder, malt extract, glucose and water, the mass ratio of the yeast powder, malt extract and glucose is 1.6-2.4:1.6-2.4:1.6-2.4, and the mass volume ratio of the mixture of the yeast powder, malt extract and glucose to water is 4-8g:0.8-1.2L;

[0014] The culture temperature is 26-30°C and the culture time is 5-10 days;

[0015] The organic solvent is ethyl acetate.

[0016] The present invention also provides a crude extract of fermentation liquid of Bacillus saccharifying L11 prepared according to the method.

[0017] The present invention provides a microbial preparation, comprising the saccharifying Bacillus L11 or the sterile fermentation liquid of the saccharifying Bacillus L11 or the crude extract of the fermentation liquid of the saccharifying Bacillus L11.

[0018] The present invention also provides an application of the microbial preparation in preventing and controlling plant pathogens and / or plant diseases and / or promoting plant growth.

[0019] Preferably, the plant pathogens include white rot shield mold, banana wilt Fusarium oxysporum, pitaya canker, fragrant Fusarium and Fujikura Fusarium;

[0020] The plant diseases include grape white rot;

[0021] The plant growth-promoting effects of the microbial preparation include the ability to produce siderophores, solubilize inorganic phosphorus and produce IAA.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention separates an antagonistic bacterium L11 which has obvious inhibitory effect on white rot shield fungus from deep-sea sediments of seahorse. The antagonistic bacterium is identified as Bacillus glycinifermentans.

[0024] The aseptic fermentation liquid of Bacillus saccharifying L11 has a significant inhibitory effect on the germination of white rot shield mold spores, with an inhibition rate of 73.87% ± 3.95%. It can also inhibit the growth of mycelium by changing the morphology of mycelium, with an inhibition rate of 26.77% ± 1.46%. The crude extract of the fermentation liquid of Bacillus saccharifying L11 also has a significant inhibitory effect on the growth of white rot shield mold mycelium, with an inhibition rate of 84.91% ± 1.54%. Bacillus saccharifying L11 can also reduce the incidence of grape white rot. In addition, Bacillus saccharifying L11 also has a good inhibitory effect on banana wilt Fusarium oxysporum No. 4, pitaya canker pathogen, fragrant Fusarium and Fujikura Fusarium, showing a broad-spectrum antibacterial effect.

[0025] In addition to having a good effect of inhibiting plant pathogens, Bacillus sacchariformis L11 also has certain growth-promoting properties, including producing siderophores, dissolving inorganic phosphorus and producing IAA. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The growth of white rot shield mold on the control plate (A) and the confrontation plate (B) in Example 1;

[0027] Figure 2The colony morphology (A) and the morphology under an optical microscope (B, scale bar is 10 μm) of the antagonistic strain L11 in Example 1;

[0028] Figure 3 16S rRNA phylogenetic analysis results of the antagonistic strain L11 in Example 1;

[0029] Figure 4 The spore germination of the white rot shield mold in the control group (A) and the sterile fermentation liquid group (B) at 24 hours of culture in Example 2 and the morphology of the white rot shield mold spores in the control group (C) and the sterile fermentation liquid group (D) after 48 hours of culture, wherein the scale bars of A and B in the figure are both 200 μm, and the scale bars of C and D in the figure are both 100 μm;

[0030] Figure 5 The mycelial growth of the white rot Shieldia odorata in the blank control group (A) and the sterile fermentation liquid group (B) in Example 2;

[0031] Figure 6 The morphological diagram of the mycelium of the white rot shield mold in the blank control group (A) and the sterile fermentation liquid group (B) in Example 2, the scale bar in the figure is 100 μm;

[0032] Figure 7 The growth of the mycelium of the white rot shield mold in the blank control group (A) and the fermentation broth crude extract group (B) in Example 3;

[0033] Figure 8 The results of the detection of cell wall hydrolase characteristics of Bacillus saccharigenicus L11 in Example 4;

[0034] Fig. 9 This is a graph showing the degree of decay of grape fruits in each treatment group after storage for 5 days in Example 5;

[0035] Fig.10 The results of the plate confrontation between Bacillus saccharifying L11 and Fusarium oxysporum race 4 of banana wilt, pitaya canker pathogen, Fusarium odoratum and Fusarium fujikura in Example 6;

[0036] Fig.11 The calculation results of the inhibition rate of Bacillus saccharifying L11 against banana wilt Fusarium oxysporum race 4, pitaya canker pathogen, Fusarium odoratum and Fusarium fujikura in Example 6;

[0037] Fig.12 This is the test result of the growth-promoting properties of Bacillus saccharigenicus L11 in Example 7.

[0038] Collection Instructions

[0039] Bacillus glycinifermentans L11, the strain is deposited in Guangdong Microbiological Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is August 16, 2024, and the deposit number is GDMCC No: 65016. DETAILED DESCRIPTION

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1 Isolation and identification of Bacillus saccharigenicus L11

[0042] 1. Isolation of strains

[0043] Weigh 1.0 g of dry seahorse cold seep deep-sea sediment (from the Nansha Sea area in the South China Sea) in 9.0 mL of sterile water, shake evenly, and press 10 -1 Perform gradient dilution. Take 200 μL of each dilution of 10 -2 and 10 -3 The sample solution was spread on ISP3 solid culture medium and cultured at 28°C for 14 days. Single colonies were picked for three-zone streaking to purify the strains. The purified strains were transferred to cryopreservation tubes containing 30% glycerol and stored in a -80°C refrigerator.

[0044] 2. Screening of antagonistic strain L11

[0045] Coniothyrium diplodiella IVF134 (accession number: ACCC 36140) purchased from the China Agricultural Microbiological Culture Collection was used as an indicator bacterium, and strains with antagonistic effects were screened by the plate confrontation method.

[0046] The isolated strains were inoculated 2.6 cm from the center of the PDA plate by the cross-point method. After culturing at 28°C for 24 hours, a white rot shield mold block with a diameter of 8 mm was inverted and inoculated in the center of the PDA plate; the control group plate was only inoculated with white rot shield mold, with 3 replicates in each group. After culturing at 28°C until the control group covered the PDA plate, the colony diameter of the white rot shield mold was measured by the cross-point method, and the inhibition rate was calculated.

[0047] Inhibition rate (%) = (colony diameter of plant pathogens in the control group - colony diameter of plant pathogens in the treatment group) / colony diameter of plant pathogens in the control group × 100%

[0048] The growth of white rot shield mold on the control plate (A) and the confrontation plate (B) is shown in Figure 1As shown, the calculated inhibition rate of the antagonistic strain L11 against Pseudomonas aeruginosa was 54.32%±1.01%.

[0049] 3. Identification of antagonistic strain L11

[0050] 1) Morphological observation

[0051] The antagonistic strain L11 was inoculated into 2216E solid culture medium and cultured at 28°C for 3 days. The color and growth morphology of the colonies were recorded, and the morphology of the bacteria was observed under an optical microscope.

[0052] The colony morphology of the antagonistic strain L11 (A) and the morphology under an optical microscope (B) are shown in Figure 2 As shown, it can be seen that the antagonistic strain L11 grows well in the 2216E solid culture medium. The colonies are white and translucent, with a reflective surface and a circular shape with concentric rings and a depression in the middle. The antagonistic strain is rod-shaped under an optical microscope.

[0053] 2) 16S rRNA phylogenetic analysis of antagonistic strain L11

[0054] Chelex-100 resin was used to extract DNA from the antagonistic strain L11, and the primers for PCR amplification were universal primers 27F and 1522R. The amplified fragments were detected by 1% agarose gel (voltage 110V, time 30min), and the successfully amplified products were sequenced by Shanghai Sangon Biotechnology Co., Ltd. The sequences were spliced ​​using the software SeqMan and submitted to the NCBI database for BLAST homology search. The 16S rRNA gene sequences of strains with high similarity were used as reference objects, and multiple sequence alignments were performed using MEGA 7.0 software. The phylogenetic tree was constructed using the neighbor-joining method (NJ), and the bootstrap value (Bootstrap value) was used to detect the confidence value of each branch 1000 times to analyze the phylogenetic status of the strain.

[0055] The 16S rRNA gene sequence of the antagonistic strain L11 is shown in SEQ ID NO.1, and the results of 16S rRNA phylogenetic analysis are shown in Figure 3 shown.

[0056]

[0057] Depend on Figure 3 It can be seen that the similarity between the antagonistic strain L11 and the Bacillus strain is greater than 98.65%, and the highest homology with Bacillus glycinifermentans GO-13 (T) is 100.00%. Therefore, the antagonistic strain L11 was preliminarily identified as Bacillus glycinifermentans.

[0058] Example 2 Inhibitory effect of sterile fermentation broth of Bacillus sacchariformis L11 on Cercospora albicans

[0059] 1. Preparation of sterile fermentation broth of Bacillus saccharifying L11

[0060] The saccharifying Bacillus L11 was inoculated into liquid culture medium No. 38 (2.0 g of yeast powder, 2.0 g of malt extract, 2.0 g of glucose, and 1.0 L of water). After constant temperature culture at 28°C for 7 days, the culture fermentation broth was centrifuged at 4°C and 12000 rpm for 10 min. The liquid portion was filtered through a 0.22 μm sterile filter to obtain a sterile fermentation broth of the saccharifying Bacillus L11.

[0061] 2. Effect of sterile fermentation broth of Bacillus sacchariformis L11 on spore germination of Pseudomonas aeruginosa

[0062] The white rot shield mold block was inoculated into the mung bean medium and shaken at 28°C and 200 rpm for one month. After sufficient spore production, the spore suspension was collected by filtration with sterile gauze and the spore concentration was adjusted to 2.0×10 5 CFU / mL. The sterile fermentation liquid of Bacillus sacchariformis L11 was mixed with the spore suspension in equal volumes, and then 5% grape juice was added to the mixture. The treatment group with an equal volume of liquid culture medium No. 38 was used as the control. After constant temperature culture at 28℃ for 24h, the spore germination rate and the inhibition rate of spore germination were calculated. The spore germination morphology was observed when the spores were cultured at 28℃ for 48h.

[0063] Spore germination rate (%) = (total number of spores germination / total number of spores examined under microscope) × 100%

[0064] Spore germination inhibition rate (%) = (control spore germination rate - treatment spore germination rate) / control spore germination rate × 100%

[0065] The germination of white rot shield fungus in the control group and the sterile fermentation liquid group after 24 hours of cultivation is as follows Figure 4 As shown in A and B (the scale bars are both 200 μm), the germination of the white rot shield mold in the control group and the sterile fermentation liquid group after 48 hours of cultivation is as follows Figure 4As shown in C and D (scale bars are 100 μm), A and C are control groups, and B and D are sterile fermentation broth groups. Figure 4 It can be seen that the sterile fermentation liquid group can significantly inhibit the spore germination of white rot shield mold. It was calculated that after 24 hours of culture, the germination rate of white rot shield mold spores in the control group was 43.49% ± 1.59%, the germination rate of white rot shield mold spores in the sterile fermentation liquid group was 11.40% ± 2.13%, and the inhibition rate of white rot shield mold spores in the sterile fermentation liquid group was 73.87% ± 3.95%. After 48 hours of culture, it was found that the spores in the control group formed longer germ tubes after germination, and slender hyphae were visible, while the germ tubes in the sterile fermentation liquid group had more branches after germination, and the hyphae growth was significantly restricted, and no slender hyphae were seen. It can be seen that the sterile fermentation liquid of Bacillus sacchariformis L11 has a significant inhibitory effect on the germination of white rot shield mold spores.

[0066] 3. Effect of aseptic fermentation broth of Bacillus sacchariformis L11 on the mycelial growth of Pseudomonas aeruginosa

[0067] The sterile fermentation liquid of Bacillus sacchariformis L11 was mixed with an equal volume of PDA and poured onto a plate. An equal volume of liquid culture medium No. 38 was used as a blank control. A block of white rot shield mold with a diameter of 8 mm was inverted and inoculated in the center of the PDA plate. The plate was cultured at a constant temperature of 28°C. After the blank control group was full of the plate, the mycelial growth diameter was measured by the cross method, and the mycelial growth inhibition rate was calculated. The growth of white rot shield mold mycelium in the blank control group and the sterile fermentation liquid group is shown in Figure 2. Figure 5 As shown, A is the blank control group and B is the sterile fermentation broth group. It can be seen that the sterile fermentation broth of Bacillus saccharigenicus L11 has a significant inhibitory effect on the growth of white rot shield mold hyphae. After calculation, the inhibition rate is 26.77%±1.46%.

[0068] In addition, the mycelium of the white rot shield mold was inoculated into the sterile fermentation liquid of Bacillus sacchariformis L11. After static culture for 3 days, the effect of the sterile fermentation liquid on the mycelium morphology was observed under an optical microscope, and liquid culture medium No. 38 was used as a blank control. Figure 6 As shown in the figure, A is the blank control group, B is the sterile fermentation liquid group, and the scale bar in the figure is 100 μm. Figure 6 It can be seen that the mycelium of the sterile fermentation broth group was deformed, swollen into a spherical or ellipsoidal shape, and had more branches, while the mycelium of the control group was slender and uniform, without any deformity, indicating that the sterile fermentation broth of Bacillus saccharigenicus L11 had a significant effect on the morphology of the mycelium of white rot mold.

[0069] Example 3 Inhibitory effect of crude extract of fermentation broth of Bacillus saccharificans L11 on Cercospora albicans

[0070] 1. Preparation of crude extract of fermentation broth of Bacillus saccharificus L11

[0071] The saccharifying Bacillus L11 was inoculated into liquid culture medium No. 38, and after constant temperature culture at 28°C for 7 days, the fermentation broth was collected by centrifugation at 8500rpm for 10min, and the collected fermentation broth was mixed with an equal volume of ethyl acetate and extracted three times, the extracted organic phases were combined, and the solvent was removed by rotary evaporation to obtain a crude extract of the fermentation broth of Bacillus L11.

[0072] 2. Effect of crude extract of fermentation broth of Bacillus sacchariformis L11 on the mycelial growth of Cercospora sphaerocephala

[0073] The crude extract of fermentation broth of Bacillus sacchariformis L11 was mixed with PDA and poured onto plates to make the final concentration of the crude extract 1 mg / mL; the plate without crude extract was used as a blank control. White rot shield mold blocks with the same diameter were inoculated in the center of the plate. After the mycelium of the blank control group filled the plate, the mycelium growth diameter was determined by the cross method and the inhibition rate was calculated.

[0074] The growth of mycelium of white rot shield mold in the blank control group (A) and the fermentation broth crude extract group (B) is shown in Figure 7 As shown, it can be seen that the crude extract of the fermentation broth of Bacillus sacchariformis L11 has a significant inhibitory effect on the growth of white rot Shield mold hyphae. After calculation, the inhibition rate is 84.91%±1.54%.

[0075] Example 4 Detection of cell wall hydrolase characteristics of Bacillus saccharificans L11

[0076] The cell wall of plant pathogens is composed of cellulose and chitin as the skeleton, and β-1,3-glucan and protein as the main fillers. Therefore, the hydrolases secreted by bacteria, such as cellulase, chitinase, β-1,3-glucanase, protease and lipase, can degrade the cell wall of plant pathogens and inhibit the growth of pathogens. Therefore, detecting the cell wall hydrolase characteristics of Bacillus sacchariformis L11 is conducive to revealing the way in which Bacillus sacchariformis L11 inhibits the growth of pathogens.

[0077] 1. Cellulose production capacity

[0078] The 8 mm diameter Bacillus saccharigenics L11 bacterial block was inoculated into sodium carboxymethyl cellulose medium (10.0 g sodium carboxymethyl cellulose, 10 mL composite salt mother solution, 20.0 g agar, 1000 mL H2O), and cultured at 28°C for 7 days. The colonies were stained with Congo red, and the appearance of an orange-yellow transparent zone around the colonies was positive. The ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d) was calculated.

[0079] 2. Ability to produce β-1,3-glucanase

[0080] The 8 mm diameter Bacillus saccharigenics L11 bacterial block was inoculated on β-1,3-glucan medium (β-1,3-glucan 10.0 g, aniline blue 100 mg, complex salt mother solution 10 mL, agar 20.0 g, H2O 1000 mL), and cultured at 28 ° C for 7 days. The appearance of a transparent zone around the colony was positive, and the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d) was calculated.

[0081] 3. Chitinase production capacity

[0082] The 8 mm diameter Bacillus saccharigenics L11 bacterial block was inoculated on a chitin medium (250 mL 2% chitin colloid, 10 mL complex salt mother solution, 20.0 g agar, 1000 mL H2O), and cultured at 28°C for 7 days. The appearance of a transparent zone around the colony was positive, and the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d) was calculated.

[0083] 4. Protease production capacity

[0084] The 8 mm diameter Bacillus saccharigenics L11 bacterial block was inoculated into skim milk powder medium (10.0 g skim milk powder, 20.0 g agar, 1000 mL H2O) and cultured at 28°C for 5 days. The appearance of a transparent zone around the colony was considered positive, and the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d) was calculated.

[0085] 5. Lipase production capacity

[0086] A bacterial block of Bacillus saccharigenics L11 with a diameter of 8 mm was inoculated into Tween medium (substrate 1% (Tween 20 or Tween 60), tryptone 1.0 g, NaCl 5.0 g, CaCl2·2H2O 0.1 g, agar 20.0 g, H2O 1000 mL), and cultured at 28°C for 5 days. The appearance of a precipitation zone around the colony was positive, and the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d) was calculated.

[0087] Among them, the components of the composite salt mother liquor used above are: KNO3 1.0 g, K2HPO4 0.5 g, MgSO4·7H2O 0.5 g, NaCl 0.5 g, NH4NO3 0.1 g, FeSO4 0.01 g, ZnSO4·7H2O 0.001 g, MnCl2·H2O 0.001 g, H2O 10 mL.

[0088] The results of the cell wall hydrolase characteristics test of Bacillus sacchariformis L11 are as follows Figure 8As shown, it can be seen that Bacillus saccharifying L11 can produce transparent circles on sodium carboxycellulose medium and skim milk medium, and an obvious precipitation circle appears on Tween medium, but no transparent circle appears on β-1,3-glucan medium and chitin medium. This shows that Bacillus saccharifying L11 can secrete cellulase, protease and lipase, but cannot secrete β-1,3-glucanase and chitinase. Among them, the D / d value of protease is the largest at 3.47±0.16, followed by the D / d values ​​of lipase, which are 2.5±0.10 (Tween 20) and 2.9±0.10 (Tween 60), and the smallest is the D / d value of cellulase, which is 2.20±0.07. It can be seen that Bacillus saccharifying L11 has the strongest ability to degrade proteins, followed by the ability to degrade lipids.

[0089] Example 5 Control of grape white rot by Bacillus sacchariformis L11

[0090] In vitro grape berries were used as the research object. The disease level was determined by the degree of grape rot, and the biological control effect of Bacillus saccharigenicus L11 on white rot Scleropsis was evaluated.

[0091] Select grapes without external injuries, uniform in size, and relatively uniform in maturity, soak them in 70% (v / v) alcohol for 30 seconds, soak them in 2% (v / v) sodium hypochlorite for 2 minutes, wash them with sterile water 3 times, place them in a clean bench for 1 hour, and use them after drying the surface moisture. Use a puncher to punch a hole with a diameter of 2 mm and a depth of 8 mm at the equator of the grapes.

[0092] Four treatment groups were set up. Each well of treatment group A was injected with 20 μL of medium No. 38, each well of treatment group B was injected with 20 μL of fermentation broth of Bacillus saccharifying L11 (Bacillus saccharifying L11 was inoculated into liquid medium No. 38, cultured at 28°C for 7 days, and centrifuged at 8500 rpm for 10 min to obtain the fermentation broth of Bacillus saccharifying L11), and each well of treatment group C was injected with 10 μL of medium No. 38 and 10 μL of 2.0×10 5 CFU / mL of white rot spores of C. cerevisiae, and each well of treatment group D was injected with 10 μL of fermentation broth of Bacillus sacchariformis L11 and 10 μL of 2.0×10 5 CFU / mL of white rot spores of Shieldia odorifera. Three replicates were set for each treatment, with 10 grapes in each group. The treated grapes were stored at room temperature for 5 days.

[0093] The grading standards for grape white rot are as follows: Grade 0, all fruits are disease-free; Grade 1, less than 25% of the fruits are rotten; Grade 2, 26% to 50% of the fruits are rotten; Grade 3, 51% to 75% of the fruits are rotten; Grade 4, more than 75% of the fruits are rotten. The disease index and biocontrol effect are calculated according to the formula.

[0094] Disease index = ∑ (damage level × number of diseased fruits at that level) / (maximum disease level × total number of fruits) × 100

[0095] Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100% The degree of rot of grapes in each group after 5 days of storage is as follows: Fig. 9 As shown in the figure, it can be seen that the grape fruits inoculated only with medium No. 38 and fermentation liquid of Bacillus sacchariformis L11 were intact, indicating that medium No. 38 and Bacillus sacchariformis L11 would not cause grape fruit rot; the grape fruits of treatment group C and treatment group D both had lesions, but the rot of grape fruits in treatment group C was more obvious. The morbidity rate (disease index) of grape fruits in treatment group C and treatment group D were 100% (69.17) and 36.67% (36.67), respectively, and the control effect was 47.09% ( Fig. 9 C and D). It can be seen that Bacillus saccharigenicus L11 can significantly reduce the incidence of grape white rot.

[0096] Example 6 Antagonistic effect of Bacillus saccharigenicus L11 on other plant pathogens

[0097] The banana wilt Fusarium oxysporum race 4 (preservation number ACCC 38875), pitaya canker pathogen Neoscytalidium dimidiatum (preservation number ACCC39211), and Fusarium fujikuroi D69-2 (preservation number ACCC 39250) used in this embodiment are all from the China Agricultural Microbiological Culture Collection Center; Fusarium redolens TR-11 (preservation number CGMCC 3.17397) is from the China General Microbiological Culture Collection Management Center.

[0098] The plate confrontation method was used to detect the antagonistic effect of Bacillus saccharifying L11 on the above four plant pathogens. Bacillus saccharifying L11 was inoculated at 2.6 cm from the center of the PDA plate. After culturing at 28°C for 1 day, pathogen blocks with a diameter of 8 mm were inverted and inoculated in the center of the PDA plate. The plate inoculated only with pathogen blocks was used as the control group, with 3 replicates in each group. After culturing at 28°C until the control group covered the PDA plate, the diameter of the pathogen colony was measured by the cross method to calculate the inhibition rate.

[0099] The results of plate confrontation between Bacillus sacchariformis L11 and Fusarium oxysporum race 4 of banana wilt, pitaya canker, Fusarium odoratum and Fusarium fujikura are as follows: Fig.10 The inhibition rate calculation results are shown in Fig.11It can be seen that Bacillus sacchariformis L11 has different degrees of antagonism against the above four plant pathogens, showing a broad-spectrum antibacterial effect. Among them, the inhibitory effect on Fusarium odoratum is the strongest, with an inhibition rate of 51.20±3.30%, followed by Fusarium odoratum, with an inhibition rate of 51.00±4.29%.

[0100] Example 7 Growth-promoting properties of Bacillus saccharigenicus L11

[0101] 1. Siderophore production capacity

[0102] The 8 mm diameter Bacillus sacchariformis L11 bacterial block was inoculated into CAS detection medium (chrome azurol (CAS) 0.0605 g, hexadecyltrimethylammonium bromide (HDTMA) 0.0729 g, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 0.295 g, sodium hydrogen phosphate dodecahydrate 1.214 g, ammonium chloride 0.125 g, potassium dihydrogen phosphate 0.0375 g, sodium chloride 0.0625 g, agar 20.0 g, H2O 1000 mL) and incubated at 28 ° C for 7 days. The appearance of an orange or yellow halo around the colony was positive, and the ratio of the hydrolysis zone diameter (D) to the colony diameter (d) (D / d) was calculated.

[0103] 2. Potassium-dissolving ability

[0104] The 8 mm diameter Bacillus saccharigenics L11 bacterial block was inoculated into a silicate bacterial culture medium (5.0 g sucrose, 5.0 g glucose, 0.5 g ammonium sulfate, 0.5 g yeast powder, 0.3 g magnesium sulfate, 2.0 g disodium hydrogen phosphate, 0.03 g ferrous sulfate, 0.03 g manganese sulfate, 2.0 g potassium feldspar, 20.0 g agar, 1000 mL H2O), and cultured at 28°C for 7 days. The appearance of a transparent zone around the colony was positive, and the ratio (D / d) of the hydrolysis zone diameter (D) to the colony diameter (d) was calculated.

[0105] 3. Phosphate dissolving capacity

[0106] The 8 mm diameter Bacillus saccharigenics L11 bacterial blocks were inoculated into inorganic phosphorus bacterial culture medium (glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, magnesium sulfate 0.3 g, manganese sulfate 0.03 g, potassium sulfate 0.3 g, ferrous sulfate 0.03 g, calcium phosphate 5.0 g, BPB 0.025 g, agar 20.0 g, H2O 1000 mL) and organophosphorus bacterial culture medium (glucose 10.0 g, ammonium sulfate 0.5 g, sodium chloride 0.3 g, magnesium sulfate 0.3 g, manganese sulfate 0.03 g, potassium sulfate 0.3 g, ferrous sulfate 0.03 g, calcium phosphate 5.0 g, lecithin 0.2 g, BPB 0.025 g, agar 20.0 g, H2O 1000mL), cultured at 28℃ for 7 days, the appearance of transparent zone around the colony was positive, and the ratio of hydrolysis zone diameter (D) to colony diameter (d) (D / d) was calculated.

[0107] 4. IAA production capacity

[0108] The seed liquid (OD 600 =1.0) was inoculated into LB liquid medium containing 0.5 mg / mL L-tryptophan at a 1% inoculum, and cultured at 28°C at 200 r / min for 6 days. The fermentation broth obtained by culture was centrifuged at 12000 r / min for 10 min, and the fermentation supernatant was mixed with an equal volume of Salkowski reagent (50 mL 35% HClO4 and 1 mL 0.5 mol / L FeCl3 solution) and incubated at room temperature in the dark for 30 min. The color turned red, indicating that IAA could be secreted. Finally, the absorbance at 530 nm (OD530) was measured, and the concentration of IAA in the supernatant was calculated according to the standard curve regression equation.

[0109] The results of the growth-promoting properties test of Bacillus saccharigenicus L11 are as follows Fig.12 As shown, it can be seen that Bacillus sacchariformis L11 can produce orange transparent circles on CAS medium and transparent circles on inorganic phosphorus bacterial medium, and their D / d values ​​are 2.50±0.13 and 1.97±0.09 respectively, but no transparent circles appear on silicate bacterial medium and organophosphorus bacterial medium. It can be seen that Bacillus sacchariformis L11 has the ability to produce siderophores and dissolve inorganic phosphorus, but cannot dissolve potassium and dissolve organic phosphorus. In addition, the color of Salkowski reagent turns red, indicating that Bacillus sacchariformis L11 can secrete the plant hormone IAA, and the IAA produced by quantification using the IAA standard curve is 3.27±0.07μg / mL.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Bacillus glycinifermentans L11, which is deposited in Guangdong Microbiological Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is August 16, 2024, and the deposit number is GDMCC No: 65016.

2. A method for preparing the sterile fermentation liquid of Bacillus saccharifying L11 according to claim 1, characterized in that: After culturing Bacillus saccharifying L11 on medium No. 38, the solid and liquid are separated, and the liquid portion is taken and sterile treatment is performed to obtain a sterile fermentation liquid of Bacillus saccharifying L11.

3. The method according to claim 2, characterized in that The No. 38 culture medium includes yeast powder, malt extract, glucose and water, wherein the mass ratio of the yeast powder, malt extract and glucose is 1.6-2.4:1.6-2.4:1.6-2.4, and the mass volume ratio of the mixture of the yeast powder, malt extract and glucose to water is 4-8g: 0.8～1.2L； The culture temperature is 26-30°C and the culture time is 5-10 days; The method of aseptic treatment is filtering with a sterile filter.

4. A sterile fermentation liquid of Bacillus saccharifying L11 prepared according to the method of claim 2 or 3.

5. A method for preparing a crude extract of fermentation broth of Bacillus saccharifying L11 according to claim 1, characterized in that: After culturing Bacillus saccharifying L11 on medium No. 38, solid-liquid separation is performed, the liquid portion is taken and extracted with an organic solvent, and the solvent in the organic phase obtained by extraction is removed to obtain a crude extract of fermentation broth of Bacillus saccharifying L11.

6. The method according to claim 5, characterized in that The No. 38 culture medium includes yeast powder, malt extract, glucose and water, wherein the mass ratio of the yeast powder, malt extract and glucose is 1.6-2.4:1.6-2.4:1.6-2.4, and the mass volume ratio of the mixture of the yeast powder, malt extract and glucose to water is 4-8g: 0.8～1.2L； The culture temperature is 26-30°C and the culture time is 5-10 days; The organic solvent is ethyl acetate.

7. A crude extract of fermentation broth of Bacillus saccharifying L11 prepared according to the method of claim 5 or 6.

8. A microbial preparation, characterized in that: The invention comprises the sterile fermentation broth of Bacillus saccharifying L11 according to claim 1 or the sterile fermentation broth of Bacillus saccharifying L11 according to claim 4 or the crude extract of the fermentation broth of Bacillus saccharifying L11 according to claim 7.

9. Use of the microbial preparation according to claim 8 in controlling plant pathogens and / or controlling plant diseases and / or promoting plant growth.

10. The use according to claim 9, characterized in that The plant pathogens include white rot shield fungus, banana wilt Fusarium oxysporum, pitaya canker, fragrant Fusarium and Fujikura Fusarium; The plant diseases include grape white rot; The plant growth-promoting effects of the microbial preparation include the ability to produce siderophores, solubilize inorganic phosphorus and produce IAA.

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