Bacillus velezensis BVL94 for preventing and treating tobacco mosaic virus as well as fermentation liquor and application of bacillus velezensis BVL94

Abstract

The invention provides bacillus velezensis BVL94 for preventing and treating tobacco mosaic virus as well as fermentation liquor and application of the bacillus velezensis BVL94, and belongs to the technical field of biological prevention and treatment. According to the bacillus velezensis BVL94 provided by the invention, the strain preservation number is CGMCC (China General Microbiological Culture Collection Center) No.29688, the strain can be used for remarkably inhibiting infection of common tobacco mosaic viruses, and a half-leaf method biocontrol experiment result shows that the protection effect is close to 100%. Meanwhile, fermentation broth of the bacillus velezensis BVL94 is provided by optimizing fermentation regulation, the prevention effect of the fermentation broth on tobacco mosaic viruses is further improved, the tobacco mosaic viruses can be effectively prevented from infecting leaves by spraying the BVL94 fermentation broth, and the effect is even superior to that of ningnanmycin.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, specifically relating to Bacillus vesiculus BVL94 and its fermentation broth for controlling tobacco mosaic virus disease and its application. Background Technology

[0002] Tobacco mosaic virus (TMV) is a widely distributed plant virus, affecting 268 species of plants across 38 families, particularly Solanaceae crops such as tobacco, tomato, and pepper, where infection rates are high. TMV primarily infects plants during the seedling stage. Due to the relatively weak resistance of seedlings and suitable temperature and humidity conditions, the seedling stage becomes a peak period for TMV infection. If infection occurs before or after transplanting, abnormal plant growth occurs, leading to symptoms such as mosaic patterns, blisters, deformities, and necrosis in the leaves, severely impacting tobacco quality and yield, with losses reaching approximately 50%. Comparatively, infection during the vigorous growth stage has a smaller impact on yield, while infection during the budding stage has little effect on yield and quality. Statistics show that annual tobacco losses due to TMV infection reach as high as $100 million, making this a significant challenge for the international tobacco industry in virus control.

[0003] Currently, the main control methods for tobacco mosaic virus include purchasing virus-free seedlings, promptly removing diseased plants, and using chemical pesticides to kill virus-transmitting aphids. While these methods are effective to some extent in controlling tobacco mosaic virus, they have some drawbacks. Purchasing virus-free seedlings is costly and cannot guarantee absolute virus-free status; removing diseased plants requires significant manual labor and cannot completely eliminate the virus. Although chemical pesticides can assist in virus control, their irrational use can threaten human and animal safety and the ecological environment, which is inconsistent with the requirements of sustainable development in green ecological agriculture.

[0004] In the pursuit of green and ecological agriculture, low-toxicity and residue-free biological agents have gradually gained attention and occupy a core position in the integrated management of crop diseases and pests. Therefore, there is an urgent need to develop a biological agent targeting tobacco mosaic virus to replace or supplement existing control methods. Domestic research on the biological control of tobacco mosaic virus has yielded some results, but most studies are limited to screening rhizosphere biocontrol strains in soil and pot experiments. Research on biocontrol strains resistant to plant viruses and the optimization of fermentation conditions is relatively limited. Summary of the Invention

[0005] The purpose of this invention is to provide a Bacillus vesiculus BVL94 and its fermentation broth for the prevention and control of tobacco mosaic virus disease, and its application. The Bacillus vesiculus BVL94 and its fermentation broth of this invention can significantly inhibit the infection of tobacco common mosaic virus.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a Bacillus velezensis BVL94 strain, with the strain preservation number CGMCC No. 29688.

[0008] This invention provides a fermentation broth of *Bacillus belye* BVL94, characterized in that the preparation method of the fermentation broth includes the following steps:

[0009] The Bacillus berberis BVL94 was inoculated onto LB fermentation medium and cultured to obtain fermentation broth;

[0010] The LB fermentation medium has lactose as its carbon source, tryptone as its nitrogen source, and magnesium sulfate as its inorganic salt component.

[0011] This invention provides a biological agent, the main components of which include the Bacillus berberis BVL94 and / or the fermentation broth.

[0012] This invention provides the application of the aforementioned microbial agent in inhibiting tobacco mosaic virus.

[0013] This invention provides the application of the aforementioned microbial agent in the prevention and control of plant diseases caused by tobacco mosaic virus.

[0014] This invention provides an application of the aforementioned microbial inoculant in improving plant resistance.

[0015] The present invention discloses the following technical effects:

[0016] The *Bacillus velezensis* strain obtained in this study exhibits significant resistance to tobacco mosaic virus (TMV). Half-leaf experiments showed that the fermentation broth of this strain achieved an inhibition rate of up to 97.55% against TMV. Furthermore, by optimizing the nutrient composition of the fermentation medium, the fermentation supernatant of strain BVL94 showed a protection rate of 99.50% against TMV, essentially reaching 100%. This indicates that the fermentation conditions of this strain also play a significant role in enhancing TMV control and have practical value in production.

[0017] This invention aims to explore the characteristics and advantages of endophytic biocontrol bacteria in the biological control of tobacco mosaic virus (TMV). By screening plant endophytic antagonistic strains with significant control efficacy and optimizing the nutrients required for their fermentation process, this invention seeks to apply these substances to control plant diseases caused by TMV. Furthermore, the yield loss in tobacco production due to TMV depends specifically on the growth stage of the infected tobacco, the type of virus, and its severity. Therefore, the successful application of this invention will directly alleviate the economic burden on the tobacco industry, improve yield and quality, and is expected to significantly reduce annual losses caused by TMV infection. This will provide new ideas and approaches for solving the problem of tobacco mosaic virus control and contribute to the sustainable development of the tobacco industry. Attached Figure Description

[0018] Figure 1 The image shows the inhibitory effect of strain BVL94 on TMV infection.

[0019] Figure 2 This is a diagram showing the protective effect of strain BVL94 against TMV infection in tobacco.

[0020] Figure 3 The effects of nutrient composition on the resistance to TMV in the fermentation medium of strain BVL94 were investigated (AG: different carbon sources: A: maltose, B: yeast extract, C: lactose, D: sucrose, E: glucose, F: soluble starch, G: no carbon source added; HN: different nitrogen sources: H: ammonium chloride, I: urea, J: ammonium sulfate, K: soybean peptone, L: potassium nitrate, M: tryptone, N: no nitrogen source added; OU: different five inorganic salts: O: potassium chloride, P: sodium chloride, Q: ferric chloride, R: zinc sulfate, S: calcium chloride, T: magnesium sulfate, U: no inorganic salt added).

[0021] Figure 4 Normal distribution diagram of the culture medium for strain BVL94 optimized using Box-Behnken response surface methodology;

[0022] Figure 5 Response surface and contour plots showing the effects of lactose, tryptone, and magnesium sulfate on the inhibitory effect of TMV;

[0023] Figure 6 The effect of strain BVL94 on resistance to TMV under different fermentation conditions is shown in the graph (AF: different pH conditions: pH4 B: pH5 C: pH6 D: pH7 E: pH8 F: pH9; GJ: different fermentation times: G: 12h H: 24h I: 48h J: 72h; KN: different fermentation temperatures: K: 20℃ L: 28℃ M: 37℃ N: 45℃; OP: different rotation speeds: O: 140rpm P: 160rpm Q: 180rpm R: 200rpm; S: CK).

[0024] Figure 7 The effect of fermentation conditions on TMV control of strain BVL94 before and after optimization (A: before optimization B: after optimization C: CK);

[0025] Figure 8 The therapeutic effects of different treatments against TMV (BVL94 fermentation supernatant; Nanningmycin 500x solution; CK:LB liquid medium);

[0026] Figure 9 The protective effects of different treatments against TMV (BVL94 fermentation supernatant; Nanningmycin 500x solution; CK: LB liquid medium);

[0027] Figure 10 The effect of treatment of BVL94 strain on the activity of tobacco defense enzymes;

[0028] Figure 11 The colony morphology and Gram staining of strain BVL94 are shown.

[0029] Figure 12 Figure 1 shows the physiological and biochemical characteristics of strain BVL94 (A: carbon source utilization; B: malonate; c: nitrate reduction; D: VP test; E: gelatin biochemistry; F: methyl red staining; G: starch hydrolysis; H: hydrogen sulfide biochemistry; I: bromocresol purple staining; J: phenol red staining).

[0030] Figure 13 This is a phylogenetic tree constructed based on the 16sRNA gene of strain BVL94;

[0031] Figure 14 This is a phylogenetic tree constructed based on the gyrB gene of strain BVL94;

[0032] Figure 15 This is a phylogenetic tree constructed based on the rpoB gene of strain BVL94;

[0033] Figure 16 The functional characteristics of strain BVL94 are shown in the figure (A: protease production; B: cellulase production; C: organic phosphorus solubilization; D: inorganic phosphorus solubilization; E: potassium solubilization; F: nitrogen fixation; G: ironophile production).

[0034] Biological Preservation Information

[0035] Bacillus velezensis was isolated from the rhizosphere soil of apple orchards at the Hebei Agricultural University experimental base and deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 29, 2023. The address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The strain accession number is CGMCC NO. 29688, and the strain number is BVL94. Detailed Implementation

[0036] This invention provides a Bacillus velezensis BVL94 strain, with the strain preservation number CGMCC No. 29688.

[0037] This invention tested the inactivation effect of different endophytic strains on TMV using the half-leaf method. TMV-GFP crude extract and endophytic bacterial suspension were mixed and inoculated onto tobacco leaves. The results showed that the BVL94 bacterial suspension could inhibit TMV infection, with an inhibition rate of approximately 78%, indicating that the BVL94 bacterial suspension has a strong inactivation effect on TMV. This invention also tested the effect of applying BVL94 bacterial suspension to tobacco leaves and inoculating with TMV after 7 days. The results showed that the inhibition rate of TMV infection by BVL94 bacterial suspension remained at approximately 86% after 7 days of treatment, indicating that the BVL94 strain provides effective protection against TMV infection for at least 7 days. Therefore, Bacillus belycei BVL94 has a strong inhibitory effect on TMV.

[0038] This invention provides a fermentation broth of *Bacillus belye* BVL94, characterized in that the preparation method of the fermentation broth includes the following steps:

[0039] The Bacillus berberis BVL94 was inoculated onto LB fermentation medium and cultured to obtain fermentation broth;

[0040] The LB fermentation medium has lactose as its carbon source, tryptone as its nitrogen source, and magnesium sulfate as its inorganic salt component.

[0041] In this invention, the concentration of lactose is preferably 7-7.5 g / L, more preferably 7.28 g / L, the concentration of tryptone is preferably 8-8.5 g / L, more preferably 8.33 g / L, and the concentration of magnesium sulfate is preferably 13-14 g / L, more preferably 13.67 g / L.

[0042] In this invention, the pH of the fermentation broth is 5.8 to 6.2, more preferably 6; the culture time is preferably 45 to 55 hours, more preferably 48 hours; the temperature is 27 to 29°C, more preferably 28°C; and the rotation speed is 170 to 190 rpm, more preferably 180 rpm.

[0043] This invention compared the inhibition rate of TMV by LB medium shake-flask fermentation strains with optimized fermentation conditions and those without. Tobacco was treated with BVL94 fermentation filtrate (before and after optimization) for 24 hours before inoculation. The number of infection spots on tobacco leaves was counted on day 3. The results showed that the average inhibition rate of TMV by BVL94 fermentation filtrate was 97.7% before optimization, and 99.4% after optimization, indicating that the inhibition effect on TMV was improved after optimizing the fermentation conditions. This invention further determined the protective effect of BVL94 fermentation broth on tobacco. Leaves were sprayed with BVL94 fermentation broth and ningnanmycin, respectively, and inoculated with TMV-GFP after 24 hours. The results showed that the control group showed many fluorescent spots on day 3 after inoculation, while ningnanmycin only showed obvious fluorescent spots on day 5. No fluorescent spots were found in BVL94 fermentation filtrate within 15 days, indicating that BVL94 fermentation filtrate prevented TMV infection from the beginning. Therefore, no green fluorescence was observed throughout the experiment, and the tobacco grew well. The above results indicate that the BVL94 fermentation broth has the best protective effect and can effectively prevent TMV infection, even outperforming ningnanmycin.

[0044] This invention provides an application of the aforementioned microbial inoculant in improving plant resistance.

[0045] This invention further determined the effect of BVL94 fermentation broth treatment on the activity of defensive enzymes in tobacco. The results showed that the activity of phenylalanine ammonia-lyase (PAL) in tobacco treated with BVL94 increased significantly, the activity of catalase (CAT) showed a gradual increasing trend, and the activity of peroxidase (POD) was significantly improved. It can be seen that BVL94 fermentation broth can improve the resistance of plants.

[0046] In this embodiment of the invention, the tobacco variety is Bunsen tobacco.

[0047] In this embodiment of the invention, the calculation formulas for TMV inhibition rate, disease index, and prevention and treatment effect are shown in Formulas I to III.

[0048] TMV inhibition rate (%) = (Number of fluorescent spots in control - Number of fluorescent spots in treatment) / Number of fluorescent spots in control × 100% Formula I: Disease index = [∑(Number of diseased leaves at each level × Corresponding disease level) / Total number of leaves surveyed × 9] × 100 Formula II: Control effect (%) = (Disease index of control - Disease index of treatment) / Disease index of control × 100% Formula III:

[0049] The present invention will be further described below with reference to the accompanying drawings, but this is not intended to limit the present invention in any way. Any changes or improvements made based on the teachings of the present invention shall fall within the protection scope of the present invention.

[0050] Example 1

[0051] Isolation and identification of endophytic bacteria in apples and preparation of fermentation broth

[0052] 1. Apple sample collection

[0053] The sampling site was the apple experimental orchard of Hebei Agricultural University. Using the equidistant sampling method, 10-year-old 'Fuji', 'Zhongqiu Wang', 'Jinxiu Hong' and 'Huang Yuanshuai' apple trees were selected. At a distance of 0-1m from the fruit tree in four different directions on each tree, soil-bearing rootlets were taken from a depth of 10-20cm below the ground. After each cutting, the tools were disinfected. Sufficient samples were placed in self-sealing bags and stored in a refrigerator at 4℃ for later use.

[0054] 2. Isolation and purification of rhizosphere antagonistic microorganisms

[0055] In a clean bench, gently shake off the soil from the fibrous roots. Weigh 10g of rhizosphere soil and add it to 90mL of sterile saline. Weigh 10g of the remaining fibrous roots and add them to a 90mL Erlenmeyer flask containing sterile saline. Label the flasks and place them in a shaker at 37℃ and 200rpm for 20 minutes, then let them stand for 10 minutes. Take 0.5mL of the supernatant from each flask and add it to a 10mL centrifuge tube containing 4.5mL of sterile water for serial dilution. Take 0.1mL of the diluted solution to achieve a 10:10 concentration. -4 , 10 -5 and 10 -6 Soil dilution was spread on PDA, LB and modified Gao's No. 1 medium, sealed and labeled, and incubated in a 28℃ incubator for 48 hours. Single colonies with different morphologies were picked for streak purification.

[0056] The inoculation loop was sterilized by burning it in the outer flame of an alcohol lamp for 10 seconds. After cooling, the rhizosphere and root surface bacteria that grew on the culture medium were purified. Using the streak plate method, the inoculation loop was dipped in a small amount of bacterial mucus and streaked at least three times, with the loop being sterilized after each streaking. Single bacterial colonies were obtained after incubation at 28°C. The fermentation broth was prepared by shaking fermentation of the single colonies, then mixed with a sterilized 50% glycerol solution at a 1:1 ratio and stored at -80°C.

[0057] 3. Preparation of fermentation broth for rhizosphere antagonistic microorganisms

[0058] To obtain the biocontrol bacteria fermentation broth, a single colony of the purified strain was first inoculated into 3 mL of LB liquid medium and cultured for 24 hours at 28°C and 180 rpm with shaking. Subsequently, seed culture medium was inoculated into 250 mL of LB liquid medium at a ratio of 1:100 and fermented under the same conditions for 48 hours. Finally, the fermentation broth was diluted with sterile water to a concentration of 1×10⁻⁶. 8 The concentration of cfu / mL was determined and stored at 4°C for later use.

[0059] Example 2

[0060] Determination of the inhibitory effect of antagonistic bacteria on TMV infection using the half-leaf method

[0061] In our previous work, we constructed an infectious TMV-GFP clone labeled with green fluorescent protein using the method of Shailaja Shivprasad (Viro1ogy, 1999, 255: 312-323). The infectious RNA transcript transcribed in vitro from this clone was used to infect tobacco leaves. One to two days after inoculation, the green fluorescence in the inoculated leaves and the entire plant could be observed using a handheld UV lamp (wavelength 490-500 nm) to determine whether tobacco mosaic virus infection was successful. The severity of tobacco mosaic virus disease could be assessed by comparing the number and size of fluorescent spots in the treatment and control groups. This method, by counting the number of visually visible necrotic spots, is more accurate than traditional methods for evaluating tobacco mosaic virus symptoms. This is because the symptoms caused by tobacco mosaic virus infection are diverse, and necrotic spots are only one of the symptoms in the later stages of infection; furthermore, the formation of necrotic spots cannot rule out cell damage caused by mechanical friction during inoculation. By observing the number and size of fluorescent spots after TMV-GFP infection, we can not only accurately determine the success rate of TMV infection and the severity of TMV infection during follow-up, but also observe the transfer path and disease severity of the virus from infected leaves to other parts after infection.

[0062] 1. Preparation of crude extract of TMV virus inoculation:

[0063] Five fresh tobacco leaves (approximately 2g) infected with the TMV-GFP infectious clone were placed in a sterile mortar and ground thoroughly with an appropriate amount of silicon carbide and phosphate-PBS buffer (135mM NaCl, 2.7mM KCl, 1.5mM KH2PO4 and 8mM K2HPO4, pH 7.2). The mixture was then filtered through sterile double-layered gauze, and finally, phosphate-PBS buffer was added to bring the volume to 100mL to obtain the crude virus extract.

[0064] 2. Half-leaf friction inoculation:

[0065] Healthy tobacco plants were selected. First, the leaf surface was rinsed with sterile water to remove impurities. Then, silicon carbide was evenly sprinkled onto the leaves. A sterile cotton swab was used to apply crude virus extract or endophytic bacteria suspension to the leaves for TMV inoculation. Ten minutes after inoculation, the leaves were rinsed with sterile water to prevent other bacteria from entering the tobacco plant through the micro-wounds created during the inoculation process. On days 3, 5, 7, and 9 post-inoculation, the number of fluorescent infection points was observed under ultraviolet light, and TMV inhibition rate and disease index were statistically analyzed.

[0066] Table 1. Grading standards for tobacco mosaic disease (GB / T23222-2008):

[0067]

[0068] 3. Test on the inhibitory effect of apple endophytic bacteria on TMV infection:

[0069] The inhibitory effect of the endophytic strains isolated from apple tissue on TMV was tested using the half-leaf method. First, the bacterial suspension of the endophytic strain was evenly spread on the left half of the leaf as the experimental group, and liquid LB medium was evenly spread on the right half of the leaf as the control group. After 24 hours, an appropriate amount of silicon carbide powder was evenly sprinkled on the treated leaves, and a 10-fold diluted crude TMV-GFP extract was evenly spread over the entire leaf. Three days after inoculation, the number of fluorescent spots in the experimental group on the left side and the control group on the right side of the leaf was counted, and the inhibitory effect of the antagonistic bacteria on TMV was calculated. The results are shown in Table 2. Figure 1 As shown, the fermentation broth of strain BVL94 showed an average inhibition rate of 78.8% against TMV infection, while strain PGG2 showed virtually no resistance to TMV infection, indicating that strain BVL94 can effectively prevent TMV infection.

[0070] Table 2. Effects of apple endophytic strains on the number of infection sites produced by tobacco mosaic virus infection in Nicotiana Bunsenata (inhibitory effect)

[0071]

[0072] 4. Test on the protective period of apple endophytic bacteria against TMV in tobacco:

[0073] Similar to the test method for the inhibitory effect of apple endophytic bacteria on TMV, the inhibitory effect of the endophytic strains isolated from apple tissue on TMV was tested using the half-leaf method. The difference was that the TMV inoculation was performed seven days after spraying the biocontrol bacteria to be tested. Three days after inoculation, the number of fluorescent spots in the experimental group on the left side of the leaf and the control group on the right side were counted, and the inhibitory effect of the antagonistic bacteria on TMV was calculated. The results are shown in Table 3. Figure 2 As shown, after 7 days of treatment with the fermentation broth of strain BVL94, the inhibition rate against TMV infection remained above 86%, indicating that the continuous protection period of strain BVL94 against TMV infection is no less than 7 days.

[0074] Table 3. Results of the survey on the number of TMV infection sites 7 days after apple endophytic fungus treatment (protection period evaluation)

[0075]

[0076] Example 3

[0077] Optimization of fermentation conditions for biocontrol strain BVL94 and the anti-TMV effect of fermentation products

[0078] 1. Screening of nutrient components in the culture medium:

[0079] Using LB medium as the basal medium, single-factor experiments were conducted to analyze the carbon source, nitrogen source, and inorganic salt components in LB medium, using the inhibition rate of TMV as the optimization index to screen suitable components. With the other two nutrient components unchanged, yeast extract in the basal medium was replaced with maltose, sucrose, glucose, lactose, and soluble starch, respectively, with the medium without yeast extract serving as the carbon source control. After fixing the optimal carbon source, tryptone was replaced with soybean peptone, ammonium chloride, urea, potassium nitrate, and ammonium sulfate, respectively, with the medium without tryptone serving as the nitrogen source control. After fixing the optimal carbon and nitrogen sources, sodium chloride was replaced with magnesium sulfate, potassium chloride, zinc sulfate, ferric chloride, and calcium chloride, respectively, with the medium without sodium chloride serving as the inorganic salt control. Pot experiments were used to test the inhibitory effect of BVL94 fermentation filtrate cultured with different nutrient components on TMV, to determine the optimal medium formulation, and to... Figure 3 It was found that among the six components—yeast extract, maltose, soluble starch, lactose, glucose, and sucrose—lactose was selected as the carbon source for optimizing the culture medium, resulting in the fewest virus infection points on the treated tobacco leaves. Yeast extract and soluble starch followed. These three components showed significantly higher inhibitory effects on TMV than the control group without a carbon source. Figure 3 It can be seen that tobacco leaves treated with fermentation broth made from tryptone strains had the fewest virus infection points, while the inhibitory effects of urea and ammonium chloride fermentation filtrates were not significantly different from the control group without nitrogen source, indicating that these two substances may be detrimental to the production of antiviral active substances by strain BVL94; Figure 3 It was found that when zinc sulfate, magnesium sulfate, and ferric chloride were used as inorganic salt components in the fermentation medium, the number of infection points on tobacco leaves was lower, showing a significant difference from the control (CK). However, when calcium chloride was used as the inorganic salt component, the number of virus infection points was higher than in the control group without inorganic salts, indicating that using calcium chloride as the inorganic salt component was unfavorable for the growth of strain BVL94. In summary, lactose was the optimal carbon source; tryptone was the optimal nitrogen source; and magnesium sulfate was the optimal inorganic salt component. Therefore, these three substances were selected for response surface methodology optimization.

[0080] 2. Optimization of culture medium nutrients using Box-Behnken response surface methodology:

[0081] A Box-Behnken response surface methodology was used, with the independent variables being the contents of carbon source (lactose), nitrogen source (tryptone), and inorganic salt (magnesium sulfate) in the culture medium, and the TMV inhibition rate as the dependent variable. The optimal fermentation conditions were determined through analysis. The virus inhibition rate was used as the indicator. Design-Expert 8.0.6 software was used to design experiments and analyze the results. The response surface methodology factors and levels are shown in Table 4, and the TMV inhibition effect of the corresponding fermentation broth was calculated (Table 5).

[0082] Table 4. Response surface methodology factors and levels for optimizing biocontrol bacteria fermentation conditions.

[0083]

[0084] Table 5. Optimized response surface methodology for BVL94 culture medium and results of TMV inhibition by fermentation broth.

[0085]

[0086]

[0087] The experimental results data were further analyzed using Design-Expert 8.0.6 software to construct a mathematical model. An analysis of variance (Table 6) was then performed on the model to test its significance. The multiple linear regression equation is as follows:

[0088] R=92.72+2.26A+2.84B+1.70C-8.55AB+1.68AC+0.57BC+0.51A 2 -10.84B 2 +2.79C 2 .

[0089] As shown in Table 6, the model F-value is 4.03, and P = 0.0398 < 0.05, indicating that the model is significant. The lack-of-fit term P = 0.2162 > 0.05, indicating that the difference is not significant, which shows that the regression equation has a good fit and can accurately reflect the relationship between lactose, tryptone, magnesium sulfate and virus inhibition rate. The optimal fermentation conditions can be obtained using the regression equation.

[0090] Table 6. Analysis of variance of the regression model for optimizing the culture medium of strain BVL94.

[0091]

[0092] The above experimental results were analyzed using Design-Expert 8.0.6 software. The multi-factor, multi-level experimental data were fitted, and the results were visually displayed using response surface plots and contour plots. Figure 4 and Figure 5The optimal fermentation conditions for this strain to inhibit TMV were deduced to be: lactose 7.28 g / L, tryptone 8.33 g / L, and magnesium sulfate 13.67 g / L, with a theoretical maximum inhibition rate of 99.38%.

[0093] 3. Screening of cultivation environment conditions:

[0094] Optimized culture media were used to screen for optimal pH, fermentation time, fermentation temperature, and fermentation speed. With other environmental factors remaining constant, the culture medium was shaken and cultured under different pH values ​​(4, 5, 6, 7, 8, 9), fermentation times (12h, 24h, 48h, 72h), fermentation temperatures (20℃, 28℃, 37℃, 45℃), and fermentation speeds (140rpm, 160rpm, 180rpm, 200rpm). After fermentation, the optimal fermentation environment for the strain was determined by the inhibition rate of TMV in the sterile filtrate. There was no significant difference in the number of virus infection points between pH 4 and 8, but the lowest number of virus infection points and the best inhibition effect were observed at pH 6. Figure 6 The inhibitory effect was weakest at pH 9, indicating that a highly alkaline environment is unfavorable for the growth of strain BVL94; Figure 6 It was found that the strain exhibited good antiviral effects with no significant difference when fermentation times were 48h and 72h. At fermentation temperatures of 28℃ and 37℃, there was no significant difference in the number of virus infection points, but the fewest infection points were observed at 28℃, resulting in the best inhibitory effect. The antiviral effects were not significantly different between fermentation speeds of 160–200 rpm, with 180 rpm showing the best inhibitory effect. The final determined environmental conditions were: pH = 6, fermentation time 48h, fermentation temperature 28℃, and shaking speed 180 rpm.

[0095] 4. Comparison of TMV suppression effects before and after optimization:

[0096] To verify the accuracy of the results obtained from the response surface methodology (RSM) experiment, the optimal fermentation conditions for strain BVL94 were verified using a half-leaf method to test its resistance to TMV. Figure 7The strain was fermented in shake flasks using both the optimized culture medium and the unoptimized LB medium. The resulting fermentation filtrate was then thoroughly sprayed onto the entire tobacco plant. Following the half-leaf method in Example 2, three leaves from the same leaf position were rubbed with crude TMV extract to test the inhibition rate of TMV and compare the control effects of the strain before and after optimization. Before optimization, tobacco was treated with the fermentation filtrate for 24 hours before inoculation. On the third day, the number of infection points on the tobacco leaves was counted. On average, there were 2 TMV infection points per tobacco leaf, with an average inhibition rate of 97.7%. After optimization, there were 0.4 TMV infection points per tobacco leaf, with an average inhibition rate of 99.4%. The difference from the predicted value was small, indicating that the model obtained by response surface methodology could improve the inhibitory effect of BVL94 fermentation filtrate on TMV, and the optimized fermentation conditions were better than those before optimization.

[0097] 5. The control effect and mechanism of action of strain BVL94 against TMV

[0098] (1) Treatment effect: First, a leaf at the same position on the upper part of each Nicotiana benthamiana plant was rubbed and inoculated with crude TMV-GFP virus extract. 24 hours later, the fermentation filtrate of the strain with optimized fermentation conditions was put into a sterile small spray bottle and sprayed all Nicotiana benthamiana plants under test until runoff formed on the leaf surface. About 15 mL was sprayed on each tobacco plant. The control group was sprayed only with sterile optimized liquid culture medium. Tobacco plants sprayed with 800 times dilution of Ningnanmycin served as the drug control group. Each group had 3 replicates, with 3 tobacco plants in each replicate. The disease incidence of tobacco plants was observed and recorded every two days for 15 days. Then, the disease index and control effect were calculated according to Table 1. Results are shown in Table 1. Figure 8 As shown in Table 7, fluorescent spots appeared on tobacco treated with ningnanmycin and strain BVL94 on day 3, simultaneously with the control group (CK). These spots continued to spread, and by day 7, the fluorescence of all three tobacco groups had extended to the heart leaves. Statistically, the therapeutic effects of BVL94 and ningnanmycin were best between days 5 and 7, with a success rate of 13%–15%. On day 5, the disease index of BVL94 was significantly lower than that of the control group. As time progressed, the therapeutic effect gradually decreased, and symptoms such as leaf deformity appeared on the tobacco plants. The therapeutic effects of both treatments were not obvious.

[0099] Table 7. The therapeutic effect of strain BVL94 on TMV in Nicotiana benthamiana.

[0100]

[0101] (2) Protective effect: The culture medium with optimized fermentation conditions was fermented in shake flasks. The fermentation filtrate of the test strain was placed in a sterile spray bottle and sprayed thoroughly on the tobacco plants until runoff formed on the leaf surface. Approximately 15 mL was sprayed per tobacco plant. The control group was sprayed only with sterile optimized liquid culture medium. Tobacco plants sprayed with ningnanmycin (800×) served as the control group. After 24 hours, one true leaf at the same position at the top of each tobacco plant was rubbed and inoculated with TMV-GFP. Each group had 3 replicates, with 3 tobacco plants per replicate. The disease incidence was observed and recorded every two days for 15 days. The disease index and control effect were then calculated according to Table 1. Results are shown in Table 1. Figure 9 The disease index of BVL94 and the positive control ningnanmycin was lower than that of the negative control CK within 15 days. The control group showed numerous fluorescent spots on day 3 after inoculation, while ningnanmycin only showed obvious fluorescent spots on day 5. No fluorescent spots were observed in the BVL94 fermentation filtrate within 15 days. By day 7 after inoculation, CK fluorescence had diffused extensively to the heart leaves, while ningnanmycin did not show green fluorescence in the heart leaves. From day 9 to 15 after inoculation, the inoculated leaves and heart leaves of CK were almost completely covered with fluorescence, while the fluorescence diffusion rate of ningnanmycin was relatively slow. Statistical analysis showed that the protective effect of ningnanmycin was best after 7 days of treatment, with an inhibition rate of 81.53%. Subsequently, as the heart leaves were continuously infected, the control effect gradually decreased. In contrast, the BVL94 fermentation filtrate prevented TMV infection from the beginning, so no green fluorescence was observed throughout the experiment, and the tobacco growth was good. These results indicate that BVL94 has the best protective effect, even superior to the positive control ningnanmycin.

[0102] Tobacco leaves treated with sterile filtrate in the above protective effect experiment were harvested at 12h, 24h, 48h, and 72h. The activities of some defensive enzymes, such as polyphenol oxidase (PPO), superoxide dismutase (SOD), peroxidase (POD), phenylalanine ammonia-lyase (PAL), and catalase (CAT), were detected using a kit produced by Beijing Solarbio Science & Technology Co., Ltd. Figure 10 As shown, PAL activity in tobacco treated with BVL94 increased significantly, peaking at 24 h; CAT activity in tobacco treated with BVL94 showed a gradual increasing trend, peaking at 72 h. From 24 h to 72 h after inoculation, compared with the CK group where CAT activity was continuously decreasing, the activities of BVL94 and the positive control ningnanmycin were significantly increased. Similarly, from 24 h to 72 h after inoculation, POD activity in tobacco treated with both ningnanmycin and BVL94 was significantly increased compared with CK. In tobacco treated with BVL94, PPO reached its peak at 72 h, while SOD did not show a significant peak.

[0103] Example 4: Identification of apple endophytic fungus strain BVL94

[0104] 1. Colony morphology identification

[0105] Strawberry strain BVL94 was streaked onto LB agar using a sterile inoculation loop and incubated at 28°C for 3 days. Morphological observation and Gram staining were then performed. Figure 11 The colony morphology and staining results are as follows: Gram staining result is positive.

[0106] 2. Physiological and biochemical identification

[0107] The physiological and biochemical parameters of the selected strains were determined according to the "Handbook of Bacterial Identification". The results are shown in Table 9 and 2019. Figure 12 Consulting the "Handbook of Bacterial Identification" reveals that these physiological and biochemical characteristics are similar to those of Bacillus.

[0108] Table 9. Physiological and biochemical characteristics of strain BVL94

[0109]

[0110] Note: "+" indicates a positive reaction or that the product can be used; "-" indicates a negative reaction or that the product cannot be used.

[0111] 3. Molecular biological identification

[0112] Identification of 16S rDNA, gyrB, and rpoB gene sequences. DNA was extracted from the strain using a rapid DNA extraction kit. PCR amplification was performed using specific primers according to the systems and procedures in Tables 10 and 11. The amplified products showed a bright target band and no non-specific bands on 1% agarose gel electrophoresis. The PCR products were sequenced by Shanghai Sangon Biotech Co., Ltd. The sequence results were compared using BLAST on NCBI (16S rDNA sequence as shown in SEQ ID NO: 1, gyrB sequence as shown in SEQ ID NO: 2, and rpoB sequence as shown in SEQ ID NO: 3). Strain BVL94 showed the highest similarity to B. halotolerans. A phylogenetic tree was constructed using the NJ method (…). Figure 13 , Figure 14 and Figure 15 Based on morphological and physiological and biochemical test results, it was preliminarily identified as Bacillus halotolerans.

[0113] Table 10 PCR amplification system for BVL94 species identification

[0114]

[0115] Table 11 PCR amplification procedures for identifying strains of BVL94

[0116]

[0117] 4. Functional characteristics of strain BVL94

[0118] Simultaneously, seven functional characteristics of strain BVL94 were determined, and the results are shown in Table 12 and... Figure 1 Three days later, observations revealed that strain BVL94 did not produce degradation zones on cellulose, organic phosphorus, inorganic phosphorus, potassium feldspar, or iron-loving media, indicating that strain BVL94 does not possess cellulase, iron-loving, phosphorus-solubilizing, or potassium-solubilizing capabilities. The presence of clear degradation zones on protein media indicates that strain BVL94 can produce proteases. The formation of smaller degradation zones on nitrogen-fixing media suggests that strain BVL94 has some nitrogen-fixing capacity.

[0119] Table 10 Functional characteristics of strain BVL94

[0120]

[0121] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Bacillus belesii ( Bacillus velezensis BVL94, strain preservation number CGMCCNO.29688.

2. A fermentation broth of Bacillus belye BVL94 as described in claim 1, characterized in that, The method for preparing the fermentation broth includes the following steps: The Bacillus berberis BVL94 of claim 1 was inoculated onto LB fermentation medium and cultured to obtain fermentation broth; The LB fermentation medium has lactose as its carbon source, tryptone as its nitrogen source, and magnesium sulfate as its inorganic salt component.

3. The fermentation broth according to claim 2, characterized in that, The concentration of lactose is 7-7.5 g / L, the concentration of tryptone is 8-8.5 g / L, and the concentration of magnesium sulfate is 13-14 g / L.

4. The fermentation broth according to claim 2, characterized in that, The pH of the fermentation broth is 5.8 to 6.

2.

5. The fermentation broth according to any one of claims 2 to 4, characterized in that, The culture time is 45-55 h, the temperature is 27-29℃, and the rotation speed is 170-190 rpm.

6. A biological agent, characterized in that, The main components include Bacillus belye BVL94 as described in claim 1 and / or the fermentation broth as described in any one of claims 2 to 5.

7. The use of the microbial agent according to claim 6 in inhibiting tobacco mosaic virus.

8. The application of the microbial agent according to claim 6 in the prevention and control of plant diseases caused by tobacco mosaic virus.

9. The application of the microbial agent according to claim 6 in improving plant resistance.

10. The application according to claim 9, characterized in that, The application described is in enhancing the activity of plant defense enzymes.

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