Bacillus velezensis and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses bacillus velezensis and application thereof. The strain is separated from saline-alkali soil in the Uygur autonomous region in Xinjiang and is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No.65485. By utilizing a response surface optimization method, the cellulase activity reaches 92.376 U / mL and is improved by 95.6% compared with that of a non-optimized cellulase activity; the growth inhibition rate on alternaria solani, alternaria alternate, fusarium oxysporum and botrytis cinerea hyphae is 88.47-91.08%, and the metabolite (especially an ethyl acetate extract) can significantly inhibit Korla pear diseases caused by alternaria alternate. The strain is resistant to high and low temperature, acid and alkali and salt stress, can degrade straw (the degradation rate is 21.9%), and provides a high-quality strain resource for resource utilization of agricultural wastes and biological control of plant fungal diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a bacillus velezensis strain and application thereof. BACKGROUND

[0002] Plant pathogenic fungi such as Alternaria solani Alternaria solani , Alternaria alternata Alternaria alternata , Fusarium oxysporum Fusarium oxysporum and Botrytis cinerea Botrytis cinerea cause soil-borne diseases, which are the core problems threatening the sustainable development of global agriculture. Among them, Alternaria alternata can cause leaf spot and fruit rot of various crops, and its toxin can also reduce the safety of agricultural products. The above-mentioned pathogenic fungi invade the vascular bundle or epidermal tissue of crops (such as vascular bundle browning of tomato wilt, brownish-brown disease spot of grape gray mold, and black necrotic spot of leaf caused by Alternaria alternata), causing 20-50% yield loss and quality decline of agricultural products. Although chemical fungicides are still the mainstream of current prevention and control means, their broad-spectrum toxicity can easily lead to increased drug resistance of pathogenic fungi, imbalance of soil microecology and pollution of ecological environment, and therefore it is urgent to develop green and multi-effect synergistic biological prevention and control technology.

[0003] At the same time, the resource utilization of agricultural waste (such as straw) is a key link of agricultural circular economy, and cellulose, as the most abundant renewable carbon source in nature, its efficient degradation depends on the synergistic action of cellulase (composed of exo- β -glucanase, endo β -glucanase and β -glucosidase). However, the traditional chemical treatment method has high energy consumption and heavy pollution, and the existing microbial degradation strains generally have the defects of low enzyme activity, single function (such as lack of pathogenic fungus resistance) and poor environmental adaptability (such as insufficient salt and alkali tolerance), which are difficult to meet the dual needs of efficient conversion of agricultural waste and synergistic prevention and control of soil-borne diseases.

[0004] Xinjiang Uygur Autonomous Region has rich straw resources and extensive saline-alkali soil. The special microbial resource library is formed due to its unique desert-oasis transition zone and salinization biological environment. Microorganisms may evolve efficient lignocellulose degradation system and anti-stress-antibacterial synergistic metabolic pathway in the long-term evolution process of adapting to extreme pH value and high salt environment, which provides potential conditions for mining multifunctional strains with cellulose degradation and antibacterial functions. SUMMARY

[0005] In view of the above technical bottlenecks, it is an urgent need in the industry to mine microbial resources with high enzyme activity, strong environmental adaptability and multiple functions. Therefore, the present application provides the following technical solutions to meet these needs.

[0006] The application isolates a bacillus velezensis from saline-alkali soil in Xinjiang Uygur Autonomous Region, which has the abilities of degrading cellulose and inhibiting various plant pathogenic fungi, and the preservation information is as follows: Strain name: bacillus velezensis Latin name: Bacillus velezensis Strain number: WL-04 Preservation agency: Guangdong Microbial Culture Collection Center Abbreviation of preservation agency: GDMCC Address of preservation agency: 5th floor, No. 59 building, Institute of Microbiology, Guangdong Academy of Sciences, 100, Xianlie Middle Road, Guangzhou Preservation date: November 15, 2024 Preservation number: GDMCC No. 65485.

[0007] Specifically, the bacillus velezensis WL-04 colony surface has a round wrinkle protrusion, the surface is rough, and presents a milky white color on the LB plate; under a microscope, the bacterial body is a straight rod, the two ends are blunt and round, the single cell length is about 2-4 μm, the width is about 0.5-1 μm, and it belongs to the typical bacillus morphology; gram-positive, the cell wall is thick, and it presents a purple color after staining.

[0008] Further, the 16S rDNA nucleotide sequence of the bacillus velezensis WL-04 is shown in SEQ ID NO. 1.

[0009] Further, the bacillus velezensis WL-04 secretes cellulase. Through the method of response surface optimization, the enzyme activity of the cellulase secreted by the bacillus velezensis is increased by 95.6%, reaching 92.376 U / mL.

[0010] Further, the bacillus velezensis WL-04 inhibits plant pathogenic fungi, and the plant pathogenic fungi include Alternaria solani (Kumata) E.G. Simmons, Aspergillus niger (Ehrenb.) Link, Fusarium oxysporum (Fr.) W.C. Snyder et H.N. Hans, and Botrytis cinerea (Pers.) Shear. Alternaria solani Alternaria alternata Fusarium oxysporum Botrytis cinerea . The application proves that the mycelium growth inhibition rates of the bacillus velezensis WL-04 on Alternaria solani, Aspergillus niger, Fusarium oxysporum and Botrytis cinerea are 91.08%, 89.15%, 88.47% and 88.62% respectively.

[0011] In the second aspect, the application requests to protect the application of the bacillus velezensis WL-04.

[0012] Further, the bacillus velezensis is used for preparing cellulase or for degrading cellulose.

[0013] ​​​​Alternatively, the B. velezensis is used for preventing and treating plant fungal diseases. The pathogens causing plant fungal diseases include Alternaria solani (Kumata) Morik (A.soli), Alternaria alternata (Fr.) Keissler (A.alternata), Fusarium oxysporum (Fr.) W.C. (F.oxysporum) and Botrytis cinerea (Pers.) (B.cinerea). Alternaria solani Alternaria alternata Fusarium oxysporum Botrytis cinerea

[0014] In a third aspect, the present application claims a microbial agent containing the bacterial body of the B. velezensis WL-04 and / or its fermentation product. The use of the microbial agent includes but is not limited to: for preventing and treating plant fungal diseases; for preparing cellulase; for degrading cellulose; for improving soil fertility.

[0015] Compared with the prior art, the present application "a B. velezensis and its application" has the following beneficial effects: The present application screens a B. velezensis WL-04 from the saline-alkali soil in Xinjiang Uygur Autonomous Region. The strain not only has a high cellulase secretion ability (the enzyme activity of cellulase is 92.376 U / mL), but also can inhibit a plurality of plant pathogenic fungi. The mycelial growth inhibition rate of the strain on A.soli and other four kinds of plant pathogenic fungi is 88.47~91.08%. Moreover, the volatile substances produced by the growth and metabolism of the strain also have an inhibitory effect on the growth of A.alternata and A.soli. In addition, the B. velezensis WL-04 grows rapidly and has outstanding characteristics of high temperature resistance (50℃), acid and alkali resistance (pH=5~9), salt resistance (NaCl concentration 1~7%), and can prevent and treat the disease spot of Korla fragrant pear caused by A.alternata. Based on the above characteristics of the strain, the efficient biological degradation of crop straw and the green prevention and control of plant fungal diseases can be realized simultaneously, which provides an innovative solution for constructing a circular technology system of "agricultural waste-biological organic fertilizer-biological prevention and control". BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The transparent circle diagram of the B. velezensis WL-04 degrading cellulose on the CMC-Na solid culture medium plate.

[0017] Figure 2 The screening test results of the cellulose degradation ability of the B. velezensis WL-04.

[0018] Figure 3 The colony morphology of the B. velezensis WL-04.

[0019] Figure 4 The phylogenetic tree diagram of the B. velezensis WL-04.

[0020] Figure 5 The growth curve diagram of the B. velezensis WL-04.​​​​

[0021] Figure 6 The standard curve of glucose.

[0022] Figure 7 The cellulase activity and cell growth of B. berlesiana WL-04 under different fermentation time conditions. 600

[0023] Figure 8 The cellulase activity and cell growth of B. berlesiana WL-04 under different fermentation temperature conditions.

[0024] Figure 9 The cellulase activity and cell growth of B. berlesiana WL-04 under different initial pH conditions.

[0025] Figure 10 The cellulase activity and cell growth of B. berlesiana WL-04 under different inoculation amount conditions.

[0026] Figure 11 The cellulase activity and cell growth of B. berlesiana WL-04 under different NaCl concentration conditions.

[0027] Figure 12 The contour plot (left) and response surface plot (right) of the interaction of culture time (A) and culture temperature (B) on the cellulase activity of B. berlesiana WL-04.

[0028] Figure 13 The contour plot (left) and response surface plot (right) of the interaction of culture time (A) and pH (C) on the cellulase activity of B. berlesiana WL-04.

[0029] Figure 14 The contour plot (left) and response surface plot (right) of the interaction of culture temperature (B) and pH (C) on the cellulase activity of B. berlesiana WL-04.

[0030] Figure 15 The straw degradation of the test group and the control group (CK).

[0031] Figure 16 The electron microscope images of the test group and the control group (CK).

[0032] Figure 17 The B. berlesiana WL-04 respectively with Alternaria solani (A), Alternaria alternata (B), Fusarium oxysporum (C) and Botrytis cinerea (D). Alternaria solani Alternaria alternata Fusarium oxysporum Botrytis cinerea ​​​​The results of the flat confrontation test of B. velezensis WL-04 and Alternaria solani (left) and A. alternata (right).

[0033] Figure 18 The results of the flat confrontation test of B. velezensis WL-04 and Alternaria solani (left) and A. alternata (right).

[0034] Figure 19 The inhibition effect of different concentrations of sterile fermentation filtrate of B. velezensis WL-04 on A. solani.

[0035] Figure 20 The EC50 of the sterile fermentation filtrate of B. velezensis WL-04 on A. solani. 50 The determination results.

[0036] Figure 21 The relative conductivity determination results of A. solani treated with different concentrations of sterile fermentation filtrate of B. velezensis WL-04.

[0037] Figure 22 The protein and nucleic acid leakage determination results of A. solani treated with different concentrations of sterile fermentation filtrate of B. velezensis WL-04.

[0038] Figure 23 The extracellular acidification kinetics detection (proton pump activity evaluation) results of A. solani treated with different concentrations of sterile fermentation filtrate of B. velezensis WL-04.

[0039] Figure 24 The electron microscope comparison chart of A. solani hyphae before and after treatment.

[0040] Figure 25 The prevention and treatment effect chart of B. velezensis WL-04 metabolites on Korla fragrant pear disease spots. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0042] The medium formula used in the embodiments is shown in Table 1.

[0043] Table 1. Medium formula

[0044] Embodiment 1 This example describes the isolation and identification of Bacillus velezensis WL-04.

[0045] Strain source: The Bacillus velezensis WL-04 was isolated from saline-alkali land in Shihezi City, Xinjiang Uygur Autonomous Region.

[0046] 1. Soil sample treatment: Collect soil samples from saline-alkali land using a five-point method, mix them, and then place them in sterile sampling bags. Immediately transport them to the laboratory and store them at 4°C. Accurately weigh 5 g of soil sample into a triangular flask containing 45 mL of sterile normal saline (0.9% NaCl) (actual ratio 1:9), and then add 5 sterile glass beads (diameter 3 mm). Shake the flask at 30°C and 160 r / min for 24 h. After the incubation, take the supernatant bacterial solution and dilute it in a gradient of 10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ,10 -6 ,10 -7 on a clean bench. Perform gradient dilution on the bacterial solution at a dilution ratio of 10

[0047] 2. Cellulose degradation ability screening: Inoculate the isolated and purified strain into LB liquid medium for activation, and incubate it at 28°C and 160 rpm for 1-3 days. Re-plate the single colony and inoculate it into CMC-Na solid medium plate. Incubate it at 28°C for 4 days. After the incubation, add 1 mg / mL of Congo red dye to the plate, and let it stand for 20 min. Then, discard the dye, wash it with 1 mol / L NaCl solution for 15 min, and repeat the process twice. Measure the diameter of the colony (d) and the diameter of the transparent cellulose degradation ring around the colony (D), and calculate the D / d value. After washing, the transparent ring of the colony on the CMC-Na solid medium plate is shown in Figure 1 , and the D / d ratio is 4.2.

[0048] 3. Cellulose degradation ability rescreening: Rescreen the WL-04 strain with a larger transparent inhibition ring. Inoculate the purified colony into LB medium, and incubate it at 28°C and 160 r / min for 24 h. Then, take 5 mL of bacterial solution, and inoculate it into 100 mL of filter paper disintegration medium containing sterile and starch-free filter paper strips (1×6 cm, 3 strips). Observe the disintegration of the filter paper strips, and further determine the cellulose degradation ability of the strain. The filter paper disintegration medium without bacterial solution is used as a control (CK). The test results are shown in Figure 2 Figure 2 ​It is evident that the filter paper strips in the experimental group disintegrated significantly, indicating that the WL-04 strain has the ability to produce cellulase and thus degrade cellulose.

[0049] 4. Morphological analysis and molecular identification: The colonies of the WL-04 strain have rounded, wrinkled protrusions on their surface, and the surface is rough, appearing milky white on LB plates. Figure 3 Under a microscope, the bacteria appear as straight rods with blunt, rounded ends. A single cell is approximately 2–4 μm long and 0.5–1 μm wide, typical of Bacillus genus morphology. They are Gram-positive, with thick cell walls that stain purple.

[0050] The nucleotide sequence of the 16S rDNA of strain WL-04 was obtained by sequencing as shown in SEQ ID NO:1, and was compared with... Bacillus velezensis The similarity reached 99.8%, and the phylogenetic tree ( Figure 4 This further confirms its taxonomic position, hence it is named: Bacillus belesiensis ( Bacillus velezensis The *Bacillus belyssus* WL-04 is currently deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 65485.

[0051] 5. Determination of growth curve: Single colonies of *Bacillus belyssioides* WL-04 plates were picked and inoculated into LB liquid medium. The culture was incubated at 37℃ and 160 rpm for 24 h to obtain a seed culture. The prepared seed culture was added to LB liquid medium at a ratio of 1.0% and cultured in a constant temperature shaker at 37℃ and 160 rpm. Bacterial samples were collected every 4 h. When the OD... 600 If the value is greater than 1, dilute 5-fold using LB liquid medium. Measure the OD using a spectrophotometer. 600 The values ​​were calculated, and a growth curve was plotted. The growth curve of the *Bacillus belyssioides* WL-04 is shown below. Figure 5 As shown, the OD is visible around 12 hours. 600 The value was close to 4 and reached a plateau at around 24 hours, indicating that Bacillus belye WL-04 had a relatively fast growth rate.

[0052] Example 2 This embodiment describes the optimization of cellulase activity produced by Bacillus belyssus WL-04.

[0053] As shown in Example 1, the Bacillus belye WL-04 has the ability to produce cellulase. In this example, the enzyme production conditions were further optimized through single-factor experiments and response surface methodology.

[0054] 1. Construction of the glucose standard curve The glucose standard solution was prepared in a test tube according to the concentration gradient shown in Table 2, 1.5 mL of 3, 5-dinitrosalicylic acid (DNS) color developing agent was accurately added into each test tube, vortex mixed immediately, and then transferred into a boiling water bath for color development for 10 min. After the reaction system was cooled to room temperature, deionized water was added to make up to 25 mL, and the mixture was fully shaken and suspended. The absorbance value (OD 540 ) of the color developing solution was measured at 540 nm by a UV-visible spectrophotometer, and each sample was set in triplicate. Finally, the standard curve regression equation was established with the mass of the glucose standard (mg) as the independent variable and the OD 540 value as the dependent variable. As Figure 6 , the glucose standard equation was y = 0.3727x - 0.014, R 2 = 0.9991.

[0055] Table 2. Concentration gradient of glucose standard solution

[0056] 2. Definition of cellulase activity and conversion formula Cellulase activity unit: 1 mL of enzyme solution catalyzing the hydrolysis of CMC-Na to generate 1 μg of glucose in 1 min under the conditions of 50°C and pH = 4.8 is 1 unit of activity, unit U / mL.

[0057] Enzyme activity conversion formula:

[0058] In the formula, M is the glucose content (mg) obtained by the control glucose standard curve; N is the dilution multiple during the reaction; L is the volume of the crude enzyme solution (mL) in the reaction; and T is the reaction time (min).

[0059] Enzyme activity determination method: 5 mL of fermentation broth was taken into a 50 mL centrifuge tube in a clean bench, and centrifuged at 5000 rpm for 10 min. After centrifugation, 500 μL of crude enzyme solution was taken and placed into another centrifuge tube containing CMC-Na solution (1.5 mL, 1% concentration, pH = 4.8). The centrifuge tube was placed in a 50°C water bath for reaction for 30 min. After the reaction was completed, 1.5 mL of DNS solution was added into the centrifuge tube, and boiled for 10 min. After heating, it was quickly taken out. After cooling and making up to 25 mL, it was shaken and the absorbance value was measured at 540 nm, with the inactivated enzyme solution as a control. The glucose content generated in the reaction was obtained from the glucose standard curve equation, and the enzyme activity value was converted into enzyme activity value by substituting into the enzyme activity equation, and each group was set in triplicate.

[0060] 3. Single factor test The general fermentation culture conditions were as follows: initial pH = 7, temperature 30℃, rotation speed 160 r / min, liquid volume 100 mL (250 mL capacity of triangular flask), inoculation amount 1%.

[0061] Under the general fermentation culture conditions, single factor tests were carried out by selecting different fermentation time (12-72 h, sampling every 12 h), fermentation temperature (25, 30, 35, 40, 45 and 50℃), initial pH value (4.0, 5.0, 6.0, 7.0, 8.0 and 9.0), inoculation amount (1%, 2%, 3%, 4%, 5%, 6% and 7%) and NaCl concentration of culture medium (1%, 2%, 3%, 4%, 5%, 6% and 7%) to determine the cellulase activity and the growth amount (expressed by OD 600 ) of Bacillus velezensis WL-04, with 3 repeats for each treatment.

[0062] 1) Time: The cellulase activity and the growth amount under different fermentation time conditions are shown in Table 1, Figure 7 , which indicates that Bacillus velezensis WL-04 grows rapidly and the enzyme activity is the highest at 24 h, which is 54.85 U / mL. Figure 7 2) Temperature: The cellulase activity and the growth amount under different fermentation temperature conditions are shown in Table 2,

[0063] , which indicates that the cellulase activity of Bacillus velezensis WL-04 is the highest at 40℃, which is 63.91 U / mL; it can still survive and has enzyme activity at 50℃, and has certain high-temperature resistance. Figure 8 Figure 8 3) Initial pH value: The cellulase activity and the growth amount under different initial pH value conditions are shown in Table 3, , which indicates that Bacillus velezensis WL-04 can survive and secrete cellulase in the pH value range of 5-9, and the cellulase activity reaches the peak value (91.58 U / mL) at pH = 6, and has strong acid-alkali environment tolerance.

[0064] Figure 9 4) Inoculation amount: The cellulase activity and the growth amount under different inoculation amount conditions are shown in Table 4, Figure 9 , which indicates that in the inoculation amount range of 1-7%, the optimal inoculation amount is 4%, and the inoculation amount has less effect on the cellulase activity.

[0065] 5) NaCl concentration: The cellulase activity and the growth amount under different NaCl concentration conditions are shown in Table 5, Figure 10 , which indicates that in the NaCl concentration range of 1-7%, the optimal NaCl concentration is 4%, and the NaCl concentration has less effect on the cellulase activity. Figure 10

[0066] Figure 11 Figure 11 ​​​​It showed that B. velezensis WL-04 could survive in the range of 1-7% NaCl concentration, and still secrete cellulase under the condition of 6% NaCl concentration, which had strong tolerance to NaCl stress.

[0067] According to the single factor test results, the optimum enzyme production conditions were determined as follows: fermentation time 24 h, temperature 40℃, pH 6.0.

[0068] 4. Response surface optimization test 1) Response surface optimization test design According to the single factor test results, the three factors with greater influence on enzyme activity were selected: time (12 h, 24 h, 36 h), temperature (35℃, 40℃, 45℃), and initial pH of enzyme production medium (5, 6, 7) for response surface optimization test. The cellulase activity was used as the response value. The response surface optimization test factors and level settings are shown in Table 3. According to Table 3, a three-factor three-level test was designed, and the response surface test design grouping and results are shown in Table 4.

[0069] Table 3. Response surface optimization test factor table

[0070] Table 4. Response surface test design grouping and results

[0071] 2) Variance and significance analysis Multiple fitting was performed using Design Expert to obtain the regression equation: Y = 93.01 + 2.77 A -2.86 B + 7.60 C -0.3577 AB + 2.33 AC + 2.86 BC -12.79 A 2 -13.68 B 2 -24.95 C 2 Variance and significance analysis was performed on the above equation (see Table 5). As shown in Table 5, the P value of the enzyme activity model was less than 0.0001, which was very significant, indicating that the independent variables had high statistical significance on the response value. The lack-of-fit test showed PA significance threshold above 0.05 indicates no significant deviation between the experimental data and the model's predicted values, validating the model's effectiveness. Regarding model fit, both the coefficient of determination (R²) and the corrected coefficient of determination (Adj-R²) are greater than 0.9, with a difference of less than 0.2, demonstrating that the established model can accurately explain over 90% of the response value variation and avoids over-parameterization. A signal-to-noise ratio (S / N) greater than 4 proves that the model has excellent anti-interference capabilities and can effectively distinguish between the true effect and background noise. Overall, considering all validation metrics, the model exhibits excellent predictive accuracy and reliability under experimental conditions.

[0072] Table 5. Analysis of Variance and Significance

[0073] The optimal enzyme production conditions for Bacillus belyssus WL-04 can be predicted using model equations. Analysis of variance results show that the main effects of the three factors on enzyme activity are ranked as follows: pH (C) > culture temperature (B) > culture time (A). Among these, the linear parameters A, B, and C... p All values ​​were <0.01, indicating extremely significant results. In the interaction of the two factors, the synergistic effect of the BC combination was extremely significant. P <0.01), while the AC interaction was significant ( P <0.05. The squared terms (A², B², C²) are all highly significant in the model. P <0.01 indicates that there is a non-linear response relationship between enzyme activity and various factors.

[0074] 3) Response Surface Analysis Contour lines and response surface curves illustrating the effect of the interaction between three factors—culture time (A), culture temperature (B), and pH value (C)—on enzyme activity. Figures 12-14 It can be concluded that: The interaction between culture time (A) and culture temperature (B) had little effect on the cellulase activity of Bacillus belye WL-04, with no significant impact. The pairwise binding response surface showed little curvature, and the contour lines were nearly circular. Figure 12 ).

[0075] The interaction between culture time (A) and pH (C) significantly affected the cellulase activity of Bacillus belyssus WL-04, with the response surface exhibiting a high degree of curvature and the contour lines appearing elliptical. Figure 13 ).

[0076] The interaction between culture temperature (B) and pH (C) has a highly significant effect on the cellulase activity of Bacillus belyssus WL-04. The response surface of the pairwise interactions is highly curved, and the contour lines are elliptical. Figure 14 ).

[0077] The optimal enzyme production conditions were obtained by solving the model corresponding to the response value Y using software DesignExpert, and the optimal enzyme production conditions were time of 25.48 h, temperature of 39.55°C, and pH value of 6.153. At this time, the enzyme activity was 93.897 U / mL. To verify the reliability of the response surface optimization, the above conditions were used for testing. Considering the operability of the test, the optimized conditions were set as follows: time of 25 h, temperature of 39.5°C, and pH value of 6.1. The enzyme activity measured under the above conditions was 92.376 U / mL, which was relatively close to the predicted result of the model. Compared with the enzyme activity of 47.223 U / mL before optimization (conditions: time of 12 h, temperature of 30°C, pH value of 7, and inoculum of 1%), the enzyme activity was increased by 95.6%. Therefore, the response surface optimization method for optimizing the enzyme production capacity of Bacillus velezensis WL-04 is feasible and has certain practical application value.

[0078] Example 3 This example describes the straw degradation capacity test of Bacillus velezensis WL-04.

[0079] The straw was dried at 60°C to constant weight, crushed through a 40-mesh sieve, and 90 mL of a straw liquid medium containing straw powder 20 g (mass denoted as m1) as the only carbon source was added to each triangular flask. The straw liquid medium was sterilized at 121°C for 20 min. After cooling to room temperature, 10 mL of bacterial suspension (seed liquid was centrifuged at 7000 rpm for 10 min, and the supernatant was discarded, and the bacterial body was resuspended with an equal volume of sterile water) was inoculated into each flask, and the flask was cultured at 35°C for 15 d on a shaking table. Then, the supernatant was discarded by centrifugation at 5000 r / min for 10 min, and the precipitate was washed repeatedly with distilled water by centrifugation for more than 3 times. The precipitate was dried at 60°C to constant weight (mass denoted as m2). The degradation rate was calculated according to m1 and m2, and the degradation rate (%) = [(m1-m2) / m1] x 100%. The straw liquid medium added with 10 mL of sterile water was used as a control (CK). The test group and the control group were set in triplicate.

[0080] The straw degradation conditions of the test group and the control group are shown in Table 1. Figure 15 Figure 15 It can be seen that the test group has obvious degradation phenomenon, and the straw particles are smaller than those of the control group (CK), and have adhesion and dissolution phenomenon. The calculated degradation rate is 21.9%.

[0081] The straw particles of the test group and the control group were observed by scanning electron microscopy, as shown in FIG. 2. Figure 16 The straw particles of the test group have obvious holes and decomposition and fracture conditions caused by enzyme degradation.

[0082] Example 4 This example describes the inhibition capacity test of Bacillus velezensis WL-04 on plant pathogenic fungi. The plant pathogenic fungi used in this example include Alternaria solani (E.G. Simmons) E.G. Simmons (alternaria solani), Alternaria alternata (Fr.) Keissler (alternaria alternata), Fusarium oxysporum (Schl) S. Ito (fusarium oxysporum), Botrytis cinerea (Pers.) Shear (botrytis cinerea), and Magnaporthe grisea (Hebert) Barron (magnaporthe grisea). Alternaria solani ​​Alternaria alternata Fusarium oxysporum f. sp. vasinfectum Fusarium oxysporum and Botrytis cinerea Botrytis cinerea .

[0083] 1. Plate confrontation method PDA plates were inoculated with the above four pathogenic fungi for more than 5 days, and spores on the surface of the colonies were scraped with a sterile spatula and added to 10 mL of sterile saline to mix thoroughly to form a crude spore suspension, which was filtered through double sterile gauze, and then the spore concentration was adjusted to 1 x 10 6 ~10 7 CFU / mL to prepare a spore suspension; 100 μL of the spore suspension was uniformly coated on a PDA plate, and cultured for 24 h. Bacillus velezensis WL-04 was activated and cultured on an LB plate at 28°C until single colonies were formed.

[0084] A puncher was used to punch the pathogenic fungal cake (d = 5 mm) and inoculate it in the center of a PDA plate, and a single colony of Bacillus velezensis WL-04 was picked with an inoculation needle and inoculated at four points around the pathogenic fungal cake, about 2.5 cm from the center of the cake, for confrontation culture; as a control (CK), only the pathogenic fungi were inoculated on the PDA plate without Bacillus velezensis WL-04, and each group was set in triplicate. After incubation at 28°C for 5 days, the colony morphology of the test and control groups was observed and photographed (Fig. 1), and the mycelial growth inhibition rate was calculated according to the following formula: Figure 17 Mycelial growth inhibition rate (%) = (net mycelial growth of the CK group - net mycelial growth of the test group) / net mycelial growth of the CK group x 100%; Test results: the mycelial growth inhibition rates of Bacillus velezensis WL-04 on Alternaria solani Alternaria solani , A. alternata Alternaria alternata , F. oxysporum f. sp. vasinfectum Fusarium oxysporum and B. cinerea Botrytis cinerea were 91.08%, 89.15%, 88.47% and 88.62%, respectively.

[0085] 2. Plate confrontation method Alternaria alternata Alternaria alternata , A. solani Alternaria solani ​This method was used for the plate-on-plate test. The pathogenic fungus was cultured on PDA plates until colonies appeared. Separately, a seed culture of *Bacillus belye* WL-04 (prepared by culturing in LB liquid medium at 37°C for 24 hours) was diluted and spread onto LB plates. After complete drying, the plates were inverted onto the PDA plates inoculated with the pathogenic fungus, with the plate inoculated with *Bacillus belye* WL-04 facing upwards. The plates were sealed with sealing film and incubated at 28°C for 3–5 days. A sterile LB plate was used as a control instead of the LB plate inoculated with *Bacillus belye* WL-04, with all other experimental conditions remaining the same.

[0086] Test results 1) Alternaria ( Alternaria alternata ) Control group: Mycelial growth was vigorous, exhibiting typical red mycelial morphology, with uniform colony expansion covering the PDA plate surface; Experimental group: Mycelial growth was inhibited, no red mycelial formation occurred, and the bacterial cells were significantly smaller than those in the control group. Figure 18 (Aa in the middle).

[0087] 2) Alternaria solanacearum ( Alternaria solani ) Control group: Mycelia are densely interwoven, forming a thick layer of grayish-brown mycelia, and the colony diameter is significantly expanded; Experimental group: Mycelia are sparse, the growth rate is reduced, the colony color is significantly lighter than that of the control group, and mycelial breakage or lysis occurs in some areas. Figure 18 As in (the text).

[0088] The above results indicate that the volatile substances of Bacillus belye WL-04 have an effect on Alternaria alterniflora (… Alternaria alternata Alternaria solanacearum ( Alternaria solani The growth of ) has an inhibitory effect, with a more significant inhibitory effect on the former.

[0089] Example 5 This embodiment describes the effect of Bacillus belye WL-04 fermentation filtrate on Alternaria (… Alternaria alternata Inhibition ability test.

[0090] 1. Aseptic fermentation filtrate EC 50 Measurement Preparation of sterile fermentation filtrate: Bacillus belye WL-04 was cultured in LB liquid medium at 37℃ and 160 r / min on a shaker for 24 h to obtain activated bacterial solution; the activated bacterial solution was transferred to fresh NYBD liquid medium at an inoculation rate of 1% and cultured under the same conditions to obtain fermentation broth; the fermentation broth was placed in a centrifuge tube and centrifuged at 8000 r / min for 10 min at 4℃, and the supernatant was collected; the supernatant was filtered through a 0.22 μm sterile filter membrane to obtain sterile fermentation filtrate.

[0091] The antifungal activity of *Bacillus belyssiensis* WL-04 sterile fermentation filtrate at different fermentation times (24 h, 48 h, 72 h) was evaluated. Different volumes of *Bacillus belyssiensis* WL-04 sterile fermentation filtrate were mixed with 20 mL of PDA medium at 45°C to prepare inhibition media at different concentrations (0, 20, 40, 60, 80, 120, 180, 240, 300, 340 μL / mL). After cooling, *Alternaria alternata* mycelial cakes with a diameter of 7 mm were placed in the center of PDA petri dishes and incubated at 28°C. Ordinary PDA medium without sterile fermentation filtrate served as the control group. When the colonies in the control group reached the edge of the petri dish, the diameter of the fungal colonies in each group was measured using a cross-hatching method, and the inhibition rate (%) was calculated using the formula. The experiment was repeated three times, and the half-maximal inhibitory concentration (EC50) was calculated using GraphPadPrism. 50 )value.

[0092] Inhibition rate (%) = [(Control group diameter - Treatment group diameter) / (Control group diameter - 7mm)] × 100% Experimental results: As the concentration of the sterile fermentation filtrate increased, its inhibitory effect on the growth of Alternaria alternata colonies gradually increased. In the control group, colonies grew to the edge of the petri dish. Figure 19 The aseptic fermentation filtrate of Bacillus belyssus WL-04 at different time points (24h, 48h, 72h) showed antifungal activity against Alternaria alternata. GraphPad Prism calculations showed that the EC50 of the aseptic fermentation filtrate against Alternaria alternata was [data missing]. 50 The value was 88.62 μL / mL ( Figure 20 ).

[0093] 2. Determination of the relative conductivity of Alternaria alternata First, *Alternaria alternata* mycelium was inoculated into 100 mL of liquid culture medium and incubated at a constant temperature for 3 days. Mycelia were then collected, and equal amounts (0.2 g) of mycelia were weighed and added to sterile fermentation filtrate at concentrations of 0 and EC, respectively. 50 and 2×EC 50 Conductivity was measured using a conductivity meter in 30 mL of distilled water at 0, 4, 8, 12, 16, 20, and 24 hours to assess the leakage of Alternaria alternata cell contents. After the final measurement, the hyphae were boiled for 10 minutes, and the final conductivity was measured. The relative conductivity of the hyphae under different treatments was calculated.

[0094] Relative conductivity = (R / R0) × 100%; R represents the conductivity at different times, and R0 represents the final conductivity after boiling.

[0095] Test results: such as Figure 21 Alternaria hyphae via 0, EC 50 2×EC 50The relative conductivity of the sterile fermentation filtrate treated Alternaria alternata mycelium at different time (0, 4, 8, 12, 16, 20, 24h) was different. The relative conductivity of the treatment group (EC 50 , 2×EC 50 ) was significantly higher than that of the control group (0 μL / mL), and the relative conductivity of the 2×EC 50 group was higher than that of the EC 50 group, indicating that the cell membrane leakage rate increased and the cell content excretion increased.

[0096] 3. Determination of protein and nucleic acid leakage of Alternaria alternata 1 g (fresh weight) of Alternaria alternata mycelium was weighed and suspended in 20 mL of PBS buffer containing different concentrations (the concentration was set as in "1") of sterile fermentation filtrate. The ordinary PDA medium without the addition of sterile fermentation filtrate was used as the control group, and each experiment was repeated three times. After 24h of shaking culture at 28°C and 180r / min, samples were taken at 0, 6, 12, 18, and 24h, and then centrifuged at 8000r / min and 4°C for 10min to obtain the supernatant. Subsequently, the absorbance of the supernatant was measured at a wavelength of 260nm.

[0097] Test results: as shown in Figure 22 , the absorbance of the supernatant of the Alternaria alternata mycelium treated with the sterile fermentation filtrate of EC 50 and 2×EC 50 was significantly higher than that of the control group (without the addition of sterile fermentation filtrate) at 0, 6, 12, 18, and 24h, and the absorbance showed an upward trend as the culture time prolonged, indicating that the treatment led to protein and nucleic acid leakage of the Alternaria alternata mycelium.

[0098] 4. Detection of Alternaria alternata extracellular acidification kinetics (evaluation of proton pump activity) To explore the effect of the sterile fermentation filtrate of Bacillus velezensis WL-04 on the function of the proton pump of the cell membrane of Alternaria alternata, the H + -ATPase activity was evaluated by monitoring the extracellular pH change induced by glucose. The specific method was as follows: 1.0 g of fresh Alternaria alternata mycelium was taken and suspended in 40 mL of KCl solution (50 mmol / L) at 4°C for 18 hours. The sterile fermentation filtrate of Bacillus velezensis WL-04 was added to make the final concentration of the mixture 0 μL / mL (without addition, as the control group), EC 50 (88.62 μL / mL), and 2×EC 50(177.24 μL / mL). The mixture was pre-incubated in a 25°C constant temperature water bath for 10 min, and then 20 mL of 10% glucose solution was quickly added to initiate the acidification reaction. The pH value of each treatment group was measured using a pH meter at 0, 30, 60, 90, 120, 150, 180, 210 and 240 min after the addition of glucose.

[0099] Test results: such as Figure 23 After adding glucose to initiate the acidification reaction, the pH value of the control group (0 μL / mL) culture medium changed significantly within 0–240 min, and EC 50 (88.62 μL / mL) and 2×EC 50 The pH change in the (177.24 μL / mL) treatment group was smaller than that in the control group, and the 2×EC 50 The group showed a more gradual change, indicating that the WL-04 aseptic fermentation filtrate inhibited the H2O2 of Alternaria cell membranes. + -ATPase activity affects proton pump function.

[0100] 5. Observation of changes in Alternaria hyphae Referring to the method in "1", use EC 50 Alternaria mycelia were treated with aseptic fermentation filtrate of Bacillus belye WL-04. After treatment, Alternaria mycelia were observed by scanning electron microscopy (SEM). The SEM observation results of normal Alternaria mycelia were used as a control.

[0101] Test results: such as Figure 24 Under electron microscopy, the hyphae of Alternaria alternata treated with Bacillus belye WL-04 showed obvious distortion, deformation and breakage, while the hyphae of the control group had normal morphology.

[0102] Experimental conclusion: Bacillus belyssus WL-04 can act on Alternaria alterniflora through its sterile fermentation filtrate, disrupting the cell membrane integrity of Alternaria alterniflora, leading to increased cell membrane leakage, protein and nucleic acid leakage, and inhibiting the cell membrane proton pump (H2). + The activity of ATPase ultimately causes hyphal distortion and breakage, thereby inhibiting Alternaria and clarifying its mechanism of action in inhibiting Alternaria.

[0103] Example 6 This embodiment describes the inhibitory effect of Bacillus vesiculosus WL-04 on Alternaria alternata infection of Korla fragrant pear.

[0104] Korla fragrant pears of uniform size and without damage were selected for testing and randomly divided into 5 groups, with 3 biological replicates per group (1 fruit / replica). After rinsing with sterile distilled water, the surface was wiped with 80% (v / v) ethanol for disinfection and air-dried in a laminar flow hood. A 4mm deep hole was drilled at the equatorial center using a sterile punch (7mm diameter). After cleaning away fruit pulp debris, 500μL of the corresponding treatment solution was added to each hole. Negative control group (CK): Sterile water was added; Fermentation broth group: Add Bacillus belyssus WL-04 fermentation stock broth (containing bacterial cells and fermentation products); Bacterial culture group: Add 1×10 8 CFU / mL Bacillus vesiculosus WL-04 bacterial suspension (containing only bacterial cells); Aseptic supernatant group: Aseptic fermentation filtrate of Bacillus belyssus WL-04 (containing only fermentation products); Extraction solution group: Add ethyl acetate extract of Bacillus belyssus WL-04 fermentation product (extract of fermentation product).

[0105] After the treatment solution was air-dried in a sterile environment, it was inoculated with Alternaria alternata mycelial cakes (0.7 cm in diameter, taken from the edge of fresh PDA colonies, with the hyphae facing down and adhering to the pores) and incubated at 25°C and 80-85% relative humidity for 7 days. The diameter of the lesions was determined using the cross-cross method to analyze the inhibitory effect. Sterilization of tools was ensured throughout the experiment (autoclaving at 121°C for 30 min), the treatment solution was prepared fresh for use, and the activity of the pathogenic mycelial cakes was consistent.

[0106] Test results: such as Figure 25 As shown, on day 4, the lesion diameter in the negative control group (CK) was (2.07±0.25) cm, in the fermentation broth group it was (2.00±0.05) cm, in the bacterial solution group it was (1.77±0.13) cm, in the sterile supernatant group it was (2.03±0.26) cm, and in the extract group it was (0.93±0.13) cm. Among them, the extract group showed the most significant antibacterial effect (P<0.05). On day 7, the lesion diameter in the CK group increased to (3.90±0.21) cm, while the diameters in the fermentation broth group (2.37±0.13) cm, the bacterial solution group (1.80±0.17) cm, the sterile supernatant group (2.63±0.26) cm, and the extract group (1.17±0.13) cm were significantly smaller than those in the other groups (P<0.05). Furthermore, the lesion expansion rate was the lowest, and the fruit phenotype remained intact.

[0107] Test conclusion: Bacillus velezensis WL-04 and its metabolites (fermentation products and bacteria, bacteria, fermentation products, fermentation product extract) have inhibitory effect on Alternaria alternata caused by Korla fragrant pear disease, among which the ethyl acetate extract has the best inhibitory effect, can effectively reduce the expansion of disease spots, keep the fruit intact, and can be used as an effective resource for biological control of fungal diseases of Korla fragrant pear.

[0108] In summary, the Bacillus velezensis (i.e. Bacillus velezensis WL-04) with the ability of degrading cellulose and inhibiting a plurality of plant pathogenic fungi is isolated from saline-alkali soil in Xinjiang Uygur Autonomous Region, and the Bacillus velezensis is preserved in Guangdong Microbial Culture Collection Center with a preservation number of GDMCC No. 65485. Firstly, the optimal enzyme production conditions (25 h, 39.5 DEG C, pH = 6.1) are determined by a single factor test + response surface optimization method, and the cellulase activity reaches 92.376 U / mL, which is increased by 95.6% compared with that before optimization; further test proves that the straw degradation rate of the Bacillus velezensis is 21.9%, and the straw degradation trace can be observed under an electron microscope. Secondly, the mycelial growth inhibition rates of the Bacillus velezensis on Alternaria solani, Alternaria alternata, Fusarium oxysporum and Botrytis cinerea are 91.08%, 89.15%, 88.47% and 88.62% respectively, and the volatile substances can also inhibit the bacteria with a significant effect. Finally, the fermentation filtrate of the Bacillus velezensis has an EC 50 of 88.62 muL / mL, can destroy the fungal cell membrane and inhibit the activity of proton pump; the metabolites (especially the ethyl acetate extract) of the Bacillus velezensis can significantly inhibit the Korla fragrant pear disease caused by Alternaria alternata, and the lesion of the extract group is the smallest (1.17+ / -0.13 cm) when inoculated for 7 days. The strain proves to have high-efficiency cellulose degradation and broad-spectrum antifungal ability, and can also resist high and low temperature, acid and alkali and salt stress, which provides high-quality strains for agricultural waste resource utilization and green control of plant fungal diseases, and is of great significance to agricultural circular economy and ecological protection.

[0109] The above-described embodiments are only some of the embodiments of the present application, not all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only to represent selected embodiments of the application. All other embodiments obtained by related deduction and replacement made by those skilled in the art under the condition of the concept of the present application, without making creative efforts, belong to the scope of protection of the present application.

Claims

1. A strain of Bacillus belesii, characterized in that, The Bacillus belyssus is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No. 65485.

2. The Bacillus belye according to claim 1, characterized in that, The nucleotide sequence of the 16S rDNA of the Bacillus belyssus is shown in SEQ ID NO.

1.

3. The Bacillus belesiensis according to claim 1, characterized in that, The Bacillus belyssus secretes cellulase.

4. The Bacillus belesiensis according to claim 1, characterized in that, The Bacillus belye inhibits plant pathogenic fungi.

5. The Bacillus belye according to claim 4, characterized in that, The plant pathogenic fungi include: Alternariae spp. Alternaria solani Alternaria Alternaria alternata Fusarium oxysporum Fusarium oxysporum and Botrytis cinerea Botrytis cinerea .

6. The application of Bacillus belyssus as described in claim 1.

7. The application according to claim 6, characterized in that, The Bacillus belyes is used to prepare cellulase or to degrade cellulose.

8. The application according to claim 6, characterized in that, The Bacillus berleis is used to control plant fungal diseases.

9. The application according to claim 8, characterized in that, Pathogens that cause fungal diseases in plants include: Alternaria solanacea Alternaria solani Alternaria Alternaria alternata Fusarium oxysporum Fusarium oxysporum and Botrytis cinerea Botrytis cinerea .

10. A microbial inoculant, characterized in that, The microbial agent contains the cells of Bacillus belysus as described in any one of claims 1 to 5 and / or its fermentation products.

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