Bacillus velezensis and application thereof in prevention and treatment of wheat leaf blight and inhibition of plant pathogenic fungi

Abstract

The invention relates to bacillus velezensis and application thereof in prevention and treatment of wheat leaf blight and inhibition of plant pathogenic fungi, and belongs to the field of biological prevention and treatment. The bacillus velezensis HXYT7 is separated and screened from tobacco leaves growing in a tobacco plantation (the longitude is 113.73 and the latitude is 33.91) in Xiangchang County in elm city, Henan province, has a remarkable inhibition effect on mophila, fusarium moniliforme, fusarium graminearum, rhizoctonia cerealis, aspergillus flavus and aspergillus niger, can damage the hypha structure of the mophila, and can be used for preparing the bacillus velezensis HXYT7. The bacillus subtilis can inhibit germination of spores of the helminthosporium, has an obvious prevention and treatment effect on the wheat leaf blight caused by the helminthosporium, and provides a new microbial resource for prevention and treatment of the wheat leaf blight.

Description

Technical Field

[0001] The invention relates to a Velez bacillus and application thereof in preventing and controlling wheat leaf blight and inhibiting plant pathogenic fungi, belonging to the field of biological control. Background Art

[0002] Bipolaris fungi are the most destructive wheat pathogens, especially Bipolaris sorokiniana, which can cause wheat root rot and leaf blight, resulting in a reduction in wheat seedlings, yellowing of plants, browning of leaf sheaths, stems, and root surfaces, and the gradual expansion of brown spots leading to plant rot and death, which seriously affects wheat yield. Currently, chemical fungicides such as triazoles, benzimidazoles, and sulfur compounds are commonly used on the market to control wheat root rot and leaf blight, which leads to environmental pollution and easily makes Bipolaris fungi resistant to such chemical fungicides, posing a great threat to human health. Therefore, it is urgent to explore new environmentally friendly methods for wheat disease control. Biological control has the characteristics of being environmentally friendly, green and safe, and not easy to develop drug resistance, which meets the requirements of sustainable agricultural development and has broad prospects.

[0003] Bacillus bacteria are widely used in the field of plant disease control due to their easy reproduction, strong stress resistance and ability to produce secondary metabolites with antibacterial activity. Since Bacillus velezensis was first discovered by Ruiz-García et al. in 2005, it has been confirmed to have certain biocontrol effects in many plants. The Chinese invention patent application with a publication date of 20190205 and publication number CN109306334A verified that the antibacterial substances such as lipopeptides and polyketides produced by Bacillus Velez can kill pathogenic fungi and bacteria, and have the effect of preventing and treating citrus Huanglongbing disease; the Chinese invention patent application with a publication date of 20230704 and publication number CN116369375A verified that Bacillus Velez can prevent and control post-harvest soft rot of eggplant and extend the storage and freshness time of eggplant; the Chinese invention patent application with a publication date of 20231128 and publication number CN117126768A found that Bacillus Velez can prevent and control Polygonatum cyrtonema leaf spot disease caused by Pseudomonas fungi.

[0004] However, there is still a lack of effective biological control measures for wheat leaf blight caused by Bipolaris fungi, which also poses a great challenge to increasing production and income in wheat production areas that are frequently infected with Bipolaris fungi. Summary of the invention

[0005] The first object of the present invention is to provide a strain of Bacillus Velezii to solve the problem of lack of effective biological control measures for wheat leaf blight caused by Bipolaris fungi.

[0006] The second object of the present invention is to provide the use of the above-mentioned Bacillus Velezii in preventing and controlling wheat leaf blight caused by Helminthosporium, which can be applied to the field of biological control of wheat leaf blight.

[0007] The third object of the present invention is to provide a use of the Bacillus Velezii in inhibiting plant pathogenic fungi such as Helminthosporium.

[0008] In order to achieve the above first purpose, the technical solution adopted by the present invention is:

[0009] A strain of Bacillus Velez, which is Bacillus Velez HXYT7 and has a deposit number of CGMCC No.31016.

[0010] The Velez subspore screened by the present invention has been experimentally confirmed to have a fermentation liquid which can cause oxidative stress damage to the cell membrane of the helminth spore, destroy the integrity of the cell membrane, lead to leakage of cell contents, abnormal, deformed and swollen hyphae morphology; meanwhile, it can also increase intracellular reactive oxygen species (ROS) and reduce mitochondrial membrane potential, thereby causing the death of the helminth spore; and the control effect of the Velez subspore on wheat leaf blight caused by the helminth spore is equivalent to that of the typical chemical reagent triadimenol, showing an excellent biocontrol effect.

[0011] To achieve the above second purpose, the technical solution adopted by the present invention is:

[0012] The application of the above-mentioned Bacillus Velezii in preventing and treating wheat leaf blight caused by Helminthosporium.

[0013] When wheat is infected with Helminthosporium and develops leaf blight, using the above-mentioned Bacillus Velezii for biological control can effectively reduce the area of ​​lesions, increase root length and plant height, and increase the fresh weight and dry weight of wheat. It has a significant preventive and therapeutic effect on wheat leaf blight and is conducive to increasing wheat production and income.

[0014] Preferably, the control comprises using the cell-free supernatant (Cell-Free Supernatant, CFS) of the fermentation broth of Bacillus Velez HXYT7 for root irrigation or foliar spraying.

[0015] In order to achieve the third object, the technical solution adopted by the present invention is:

[0016] The application of the above-mentioned Bacillus Velezii in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi include Helminthosporium.

[0017] Through the analysis of the antibacterial activity of Bacillus Velez subtilis, it was found that Bacillus Velez subtilis HXYT7 had the most obvious inhibitory effect on Helminthosporium, with an inhibition rate of up to 75.69%.

[0018] Preferably, the plant pathogenic fungi also include Fusarium moniliforme, Fusarium graminearum, Rhizoctonia graminearum, Aspergillus flavus and Aspergillus niger, and the antibacterial rates thereof are 73.33%, 72.94%, 71.57%, 70.98% and 68.63% respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 hereinafter refers to the size of the inhibition zone produced by the single colony after separation and purification in Example 1 of the present invention and its inhibition rate on the lactic spore fungus; wherein Figure 1 A in the figure is the size of the inhibition zone produced by a single colony after separation and purification. Figure 1 B in the figure is the inhibition rate of three biocontrol bacteria HXYT-2, HXYT-7 and HXYT-20 on Lactobacillus spp.;

[0020] Figure 2 : is a characteristic diagram of the bacterial morphology of Bacillus velez HXYT7 in Example 1 of the present invention; wherein Figure 2 A in the figure is the colony morphology of Bacillus velez HXYT7; Figure 2 B in the figure is a scanning electron micrograph of Bacillus velez HXYT7 strain; Figure 2 C in the figure is the microscopic morphology of Bacillus velez HXYT7 strain after Gram staining; Figure 2 D in the figure is the microscopic morphology of the spores of Bacillus velez HXYT7 after Gram staining;

[0021] Figure 3 The phylogenetic tree constructed based on the 16S rDNA gene sequence in Example 1 of the present invention;

[0022] Figure 4 The phylogenetic tree constructed based on the gyrB DNA gene sequence in Example 1 of the present invention;

[0023] Figure 5 This is a graph showing the results of determining the optimum fermentation time of the fermentation liquid of Bacillus Velez HXYT7 in Example 2 of the present invention;

[0024] Figure 6 This is a comparative diagram of the inhibition of Helminthosporium by fermentation broth of Bacillus Velez subtilis HXYT7 at different concentrations in Example 2 of the present invention, wherein CFS is the cell-free supernatant of fermentation broth of Bacillus Velez subtilis HXYT7;

[0025] Figure 7The graph is a graph showing the inhibitory effect of the fermentation liquid of Bacillus Velezii HXYT7 on pathogenic fungi (Aspergillus niger, Aspergillus flavus, Fusarium moniliforme, Rhizoctonia graminearum, Fusarium graminearum, and Helminthosporium) in Example 2 of the present invention;

[0026] Figure 8 The results of the effects of different concentrations of Bacillus Velezii HXYT7 fermentation broth on the germination of Helminthosporium spores in Example 2 of the present invention are shown in FIG. Figure 8 A in the figure is the germination of Helminthosporium spores under a microscope when the concentration of the fermentation liquid of Bacillus Velezii HXYT7 is 1%, 5%, 10%, 20% and 50%; Figure 8 B in the figure is the germination rate of Helminthosporium spores when the concentration of Bacillus Velezii HXYT7 fermentation liquid is 1%, 5%, 10%, 20% and 50%; Figure 8 C in the formula is the inhibition rate of Helminthosporium spores when the concentration of Bacillus Velezii HXYT7 fermentation liquid is 1%, 5%, 10%, 20% and 50%; Figure 8 The CFS in A to C is the cell-free supernatant of the fermentation broth of Bacillus velezin HXYT7;

[0027] Fig. 9 This is a microscopic structure diagram of the mycelium of Helminthosporium in Example 2 of the present invention after being treated with the fermentation broth of Bacillus Velez subtilis HXYT7, wherein CFS is the cell-free supernatant of the fermentation broth of Bacillus Velez subtilis HXYT7, and Control is the control group;

[0028] Fig.10 This is a fluorescence image produced by the mycelium of Helminthosporium treated with the fermentation broth of Bacillus Velezii HXYT7 after being stained with propidium iodide (PI) under a fluorescence microscope in Example 2 of the present invention, wherein CFS is the cell-free supernatant of the fermentation broth of Bacillus Velezii HXYT7;

[0029] Fig.11 This is a graph showing the results of measuring the malondialdehyde (MDA) content using the thiobarbituric acid (TBA) method after treating Helminthosporium mycelium with Bacillus Velez HXYT7 fermentation broth in Example 2 of the present invention, wherein CFS in the abscissa is the cell-free supernatant of the Bacillus Velez HXYT7 fermentation broth;

[0030] Fig.12 This is a fluorescence image produced by treating Helminthosporium hyphae with Bacillus Velez HXYT7 fermentation broth under a fluorescence microscope in Example 2 of the present invention and then staining with dihydrorhodamine 123, wherein CFS is the cell-free supernatant of Bacillus Velez HXYT7 fermentation broth;

[0031] Fig.13This is a fluorescence image produced by treating Helminthosporium hyphae with Bacillus Velez HXYT7 fermentation broth under a fluorescence microscope in Example 2 of the present invention and then staining with JC-1 dye, wherein CFS is the cell-free supernatant of Bacillus Velez HXYT7 fermentation broth;

[0032] Fig.14 This is a graph showing the effect of the fermentation liquid of Bacillus Velezii HXYT7 on the prevention and treatment of wheat leaf blight caused by Helminthosporium in Example 2 of the present invention. Fig.14 A in the figure is the severity of wheat leaf blight after being treated with 20% fermentation liquid of Bacillus Velez HXYT7 and triadimefon; Fig.14 B in the figure is the disease index result after treatment with 20% fermentation broth of Bacillus velez HXYT7 and triadimefon; Fig.14 C in the figure is the control effect after treatment with 20% fermentation liquid of Bacillus Velez HXYT7 and triadimefon; Fig.14 D in the figure is the root length and plant height of wheat plants after being treated with 20% fermentation liquid of Bacillus Velez HXYT7 and triadimefon; Fig.14 E in the figure is the dry weight and fresh weight of wheat plants after being treated with 20% fermentation liquid of Bacillus Velez HXYT7 and triadimefon; Fig.14 The CFS in the abscissa of A to E is the cell-free supernatant of the fermentation broth of Bacillus velezensis HXYT7. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0034] Some of the biological materials, test reagents, test equipment, etc. involved in the following embodiments are briefly introduced as follows:

[0035] Biomaterials:

[0036] Tobacco leaves: from tobacco plantations in Xuchang County, Yulin City, Henan Province (longitude: 113.73, latitude: 33.91);

[0037] Test reagents:

[0038] Fast Pure Microbiome DNA Isolation Kit: Nanjing Novozyme Biotechnology Co., Ltd.

[0039] 2×Es Taq Master Mix (Dye) kit: Beijing Kangwei Century Biotechnology Co., Ltd.

[0040] The implementation process of the present invention is described in detail below in conjunction with specific embodiments.

[0041] 1. Specific embodiment of a strain of Bacillus Velezii of the present invention

[0042] Example 1

[0043] A strain of Bacillus velezensis, which is Bacillus velezensis HXYT7, with a deposit number of CGMCC No.31016; deposit date: June 19, 2024; deposit unit: General Microbiology Center of China Culture Collection Administration; deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0044] The following is a description of the isolation, screening and identification process of the strain:

[0045] 1. Isolation and screening of Bacillus velez HXYT7

[0046] This experiment is the separation and screening process of Bacillus Velez HXYT7. The specific operations are as follows:

[0047] Take 20g of tobacco leaves from a tobacco plantation in Xuchang County, Yulin City, Henan Province (longitude: 113.73, latitude: 33.91), cut them into pieces and put them into a 500mL conical flask, add 230mL of dispersant, shake for 5-6h, and elute the microorganisms to obtain a microbial stock solution. Dilute the microbial stock solution with sterile saline for 5 -1 , 5 -2 , 5 -3 times, respectively take the microbial stock solution, 5 -1 , 5 -2 , 5 -3 200 μL of gradient dilution solution was spread on PDA medium, and each sample was cultured at 37°C for 3 to 5 days. Microorganisms with the same morphology were grouped together, and one strain was selected from each of the same microorganisms for purification to obtain purified single colonies. The obtained single colonies were numbered for further study.

[0048] The plate confrontation method was used to conduct preliminary screening of the isolated and purified strains. A vertical cross was drawn on the PDA plate with the origin as the center, and each plate was divided into 4 equal areas. A 7mm diameter puncher was used to punch a bacterial cake on the pathogenic fungus plate (try to make the amount of hyphae on the bacterial cake consistent), and then a sterile toothpick was used to inoculate the bacterial cake to the center of the PDA plate. The bacterial solution was dipped into an inoculation loop, and the bacteria to be screened were inoculated equidistantly around the bacterial cake in the form of lines, 1 strain in each of the 4 directions, with a distance of 2cm, and the length of the strain line was about 1.5cm. The plate inoculated only with pathogenic fungi was used as a blank control, and 3 parallels were set up. Cultured in the dark at a constant temperature of 28℃, and bacteria that produced an inhibition zone or bacteriolysis phenomenon were selected for further rescreening.

[0049] Antagonistic bacteria were screened again using Helminthosporium as the pathogen indicator bacteria. The screened bacteria were inoculated into a 50mL conical flask containing 20mL LB liquid culture medium, and cultured at 200rpm and 30℃ for 12h to obtain bacterial solution. A cross hole was punched at a distance of 2cm from the pathogenic fungus cake with a diameter of 7mm, and the culture medium cake was picked out with a sterile toothpick. Then, the bottom of the hole was sealed with a small amount of agar sterilized at high temperature. After cooling slightly, 20μL of bacterial solution was drawn into the hole with a pipette. The same bacteria were inoculated in 4 holes, and then the pathogenic fungus cake (7mm) was inoculated into the center of the PDA culture medium plate. The plate inoculated with only pathogenic fungi was used as a blank control, and 3 parallel repetitions were set. Cultured in the dark at 28℃, the colony in the control plate grew to a suitable size, the diameter of the pathogenic fungus was measured, and the inhibition rate of bacteria on pathogenic fungi was calculated.

[0050] The antibacterial rate formula is as follows:

[0051] Inhibition rate = (colony diameter of control group - colony diameter of experimental group) / colony diameter of control group) / colony diameter of control group × 100 (1)

[0052] The results are as follows Figure 1 As shown in A, compared with the control group, the colony diameter of Helminthosporium in the experimental group was significantly reduced, indicating that the three strains of bacteria had a certain antagonistic effect on Helminthosporium. The inhibition rate of the three biocontrol bacteria on Helminthosporium is shown in Figure 1 As shown in B, the inhibition rate of HXYT-2 was 72.11%, the inhibition rate of HXYT-20 was 66.79%, and the inhibition rate of HXYT-7 was the highest, which was 76.27%.

[0053] 2. Identification of Bacillus velez HXYT7

[0054] This experiment is the identification process of Bacillus Velez HXYT7. The specific operations are as follows:

[0055] Physiological and biochemical identification: Morphological observation of strain HXYT7 revealed that the colonies were milky white, round in shape, and had a wrinkled surface ( Figure 2 A in the figure), the strain cells are rod-shaped under scanning electron microscopy ( Figure 2 B); Gram staining of the strain HXYT7 and its spores revealed that the strain was purple ( Figure 2 C), the spore color is red ( Figure 2 D), thus proving that strain HXYT7 is a Gram-positive bacterium.

[0056] The strain HXYT7 was subjected to physiological and biochemical tests according to the method described in the Bergey's Manual of Bacterial Identification. As shown in Table 1, the strain HXYT7 VP reaction was positive, it could utilize citric acid, maltose, xylose, and mannitol, it could hydrolyze gelatin, it was anaerobic, and it had a certain salt tolerance, which were basically consistent with the physiological and biochemical characteristics of Bacillus.

[0057] Table 1 Physiological and biochemical characteristics of strain HXYT7

[0058]

[0059] Note: “+” indicates a positive reaction; “-” indicates a negative reaction.

[0060] Molecular biological identification: The total genomic DNA of strain HXYT7 was extracted using the Fast Pure Microbiome DNA Isolation Kit. Using this as a template, the upstream primer 27F (5'-AAGTTTTGATCCTGCTCA-3'; as shown in SEQ ID No.3) and the downstream primer 1492R (5'-GGTACCTTGTTACGACTT-3'; as shown in SEQ ID No.4) were selected to perform PCR amplification of its 16SrDNA gene, and the upstream primer up1 (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNA-3'; as shown in SEQ ID No.5) and the downstream primer up2 (5'-AGCAGGGTACGGATGTGCGAGCC-3'; as shown in SEQ ID No.6) were selected to perform PCR amplification of its gyrB gene, and the specific operations were as follows:

[0061] 16S rDNA and gyrB genes were amplified by PCR using 2×Es Taq Master Mix (Dye) kit, respectively. The amplification system was 25 μL: 1 μL template, 1 μL upstream and downstream primers, 12.5 μL 2×Es Taq Master Mix (Dye), and 9.5 μL ddH2O. The PCR reaction conditions were: 94°C pre-denaturation for 2 min, 35 cycles (94°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s), and 72°C total extension for 2 min. The amplified fragments were detected by 1.5% agarose gel electrophoresis, purified and recovered, and sent to BGI Biotechnology Co., Ltd. for sequencing (the 16S rDNA sequencing sequence is shown in SEQ ID No. 1; the gyrB gene sequencing sequence is shown in SEQ ID No. 2). The sequences were aligned by BLAST in NCBI and phylogenetic analysis was performed using MEGA11 software. A phylogenetic tree based on 16S rDNA and gyrB gene sequences was constructed using the neighbor joining method. The number of repeated samplings for testing the bootstrap support rate was 1000.

[0062] The 16S rDNA phylogenetic tree showed that strain HXYT7 and Bacillus velezensis FZB42 were in the same branch, with a similarity of up to 98% ( Figure 3 ); The phylogenetic tree of the gyrB gene sequence showed that strain HXYT7 and Bacillus velezensis 19573-3 were in the same branch, with a similarity of up to 100% ( Figure 4 ).

[0063] Combined with the results of physiological and biochemical identification and molecular biological identification, the strain was identified as Bacillus Velez subtilis and named Bacillus Velez subtilis HXYT7. It was deposited in the China Microorganism Culture Collection on June 19, 2024, with the deposit number CGMCC No.31016.

[0064] 2. Specific application of the present invention's Bacillus Velezii HXYT7 in preventing and controlling wheat leaf blight and inhibiting pathogenic fungi

[0065] Example

[0066] Example 2

[0067] In this example, the optimal fermentation time and the optimal fermentation liquid concentration of Bacillus Velez subtilis HXYT7 are determined to obtain the Bacillus Velez subtilis HXYT7 fermentation liquid with the best inhibitory effect, and the inhibitory effect of the fermentation liquid on Helminthosporium, Fusarium moniliforme, Fusarium graminearum, Rhizoctonia graminearum, Aspergillus flavus and Aspergillus niger is identified by a plate confrontation method; further verification shows that the fermentation liquid of Bacillus Velez subtilis HXYT7 can destroy the hyphae structure of Helminthosporium, inhibit the germination of Helminthosporium spores, and thus kill Helminthosporium; the control effect of the fermentation liquid on wheat infected with Helminthosporium is observed by root irrigation or foliar spraying on wheat, which proves that the fermentation liquid of Bacillus Velez subtilis HXYT7 has a high control effect on leaf blight caused by Helminthosporium infection in wheat.

[0068] The following is a detailed description of its operation method.

[0069] 1. Optimal fermentation time of Bacillus Velez HXYT7

[0070] In order to explore the optimal fermentation time of Bacillus velez HXYT7, the specific operation is as follows:

[0071] Pick a single colony of Bacillus Velez HXYT7 on the LB plate, inoculate it in 10mL LB liquid medium, and culture it at 37°C, 200rpm for 18h, then inoculate it in 100mL LB liquid medium at a 1% inoculation ratio, ferment and culture it at 37°C, 200rpm, collect the cell-free supernatant (Cell-Free Supernatant, CFS) of the fermentation broth at 1d, 2d, 3d, 4d and 5d, prepare the fermentation broth CFS with PDB medium to a final concentration of 10% Bacillus Velez HXYT7 fermentation broth, place 7mm off-worm spore cake (to ensure that the amount of hyphae carried by the cake is consistent) in 100mL 10% fermentation broth, and culture it at 28°C, 180rpm for 72h. Then filter the hyphae with a single layer of gauze, rinse the residual impurities with sterile water, bake them in a 55°C oven for 12h to constant weight, weigh them on an electronic balance, and calculate the inhibition rate by 3 parallels for each treatment.

[0072] The mycelium growth inhibition rate was calculated as follows:

[0073] Inhibition rate (%) = (mycelium weight of control group - mycelium weight of experimental group) / mycelium weight of control group × 100. (2)

[0074] The results showed that the inhibition rates of the fermentation broth of Bacillus Velezii HXYT7 on Helminthosporium were 76.66%, 79.72%, 79.21%, 46.75%, 38.46% and 5 days respectively. Figure 5), it can be seen that the fermentation broth of Bacillus Velez subtilis HXYT7 fermented for 2 days has the highest inhibition rate against Helminthosporium, and the fermentation broth of Bacillus Velez subtilis HXYT7 fermented for 2 days was selected in the subsequent operation.

[0075] 2. Optimal fermentation broth concentration of Bacillus velez HXYT7

[0076] In order to explore the optimal fermentation broth concentration of Bacillus velez HXYT7, the specific operation is as follows:

[0077] Pick a single colony of Bacillus Velez HXYT7 on the LB plate, inoculate it in 10 mL LB liquid culture medium, and culture it at 37° C., 200 rpm for 18 h. Then, inoculate it in 100 mL LB liquid culture medium at a 1% inoculation ratio, and ferment it at 37° C., 200 rpm for 2 days. The fermentation broth CFS and PDB culture medium are prepared into Bacillus Velez HXYT7 fermentation broth with final concentrations of 1%, 5%, 10%, 20%, and 50%, respectively (final volume of 100 mL). The control group is an equal volume of PDB culture medium without adding fermentation broth, and a 7 mm Helicospora cake (ensuring that the amount of mycelium carried by the cake is consistent) is placed therein, and cultured at 28° C., 180 rpm for 72 h. The mycelium was then filtered through a single layer of gauze, and the remaining impurities were rinsed with sterile water. The mycelium was dried in an oven at 55°C for 12 h until constant weight was reached. The mycelium growth inhibition rate was calculated using the same formula as formula (2).

[0078] The results showed that the inhibition rates of the fermentation broth of Bacillus Velezii HXYT7 at the final concentrations of 1%, 5%, 10%, 20%, and 50% against Helminthosporium were 62.54%, 78.66%, 84.2%, 95.06%, and 96.27%, respectively. Figure 6 ), because the inhibition rates of 20% and 50% of the fermentation broth of Bacillus Velez subtilis HXYT7 on Helminthosporium were basically the same, based on cost considerations, the fermentation broth of Bacillus Velez subtilis HXYT7 with a concentration of 20% was selected in the subsequent operations.

[0079] 3. Analysis of antibacterial activity of Bacillus velez HXYT7

[0080] In this experiment, the plate confrontation method was used to analyze the antibacterial activity of Bacillus Velezii HXYT7. The specific operation is as follows:

[0081] Draw a vertical cross with the origin of the PDA medium as the center, divide the plate into 4 equal areas, punch a hole with a hole diameter of 7 mm in the middle of each equally divided area and 2 cm away from the origin, pick it with a sterile toothpick, and seal the bottom of the hole with a small amount of agar sterilized at high temperature. After solidification, use a pipette to draw 20 μL of the fermentation liquid CFS of the strain HXYT7 with a concentration of 20% and cultured for 2d (culture conditions are 37°C, 200rpm) in the hole, and use 6 pathogenic fungi, including Bacillus cereus, Aspergillus flavus, Aspergillus niger, Fusarium moniliforme, Fusarium graminearum, and Rhizoctonia graminearum, as indicator bacteria, and inoculate them in the center of the PDA medium respectively. The blank control is the PDA medium inoculated with only the indicator bacteria. Set 3 parallels and culture them in the dark at 28°C until the colonies in the control plate grow to a suitable size. Measure the diameter of the indicator bacteria and calculate the inhibition rate of Bacillus Velezii HXYT7 on each indicator bacteria.

[0082] The antibacterial rate calculation formula is as follows:

[0083] Inhibition rate (%) = (colony diameter of control group - colony diameter of experimental group) / colony diameter of control group × 100.

[0084] The results show that ( Figure 7 ), Bacillus Velezii HXYT7 had a strong inhibitory effect on Tremella thunbergii, Fusarium moniliforme, Fusarium graminearum, Rhizoctonia graminearum, Aspergillus flavus and Aspergillus niger, with the inhibition rates being 75.69%, 73.33%, 72.94%, 71.57%, 70.98% and 68.63%, respectively.

[0085] 4. Effect of Bacillus Velezii HXYT7 fermentation broth on spore germination of Helminthosporium

[0086] This experiment verifies the effect of the fermentation broth of Bacillus Velezii HXYT7 on the germination of Helminthosporium spores. The specific operation is as follows:

[0087] Add 1 mL of conidia suspension of Helminthosporium (10 6 CFU / mL), then 1 mL of PDB medium containing different concentrations of Bacillus Velezii HXYT7 fermentation broth CFS (cultured at 37°C, 200 rpm for 2 days) was added to make the final concentration of the fermentation broth 1%, 5%, 10%, 20%, 50% (v / v), and the control group was added with 1 mL of sterilized PBD liquid medium, cultured at 28°C for 24 h, and the germination of spores was observed under a microscope ( Figure 8 A) in the figure, and count them to calculate the spore germination rate and inhibition rate.

[0088] The calculation formulas for spore germination rate and inhibition rate are as follows:

[0089] Spore germination rate (%) = number of germinated spores / total number of spores × 100;

[0090] Inhibition rate (%) = (spore germination rate of control group - spore germination rate of experimental group) / spore germination rate of control group × 100.

[0091] The results showed that compared with the control group, the number of spores of the treated group was significantly reduced, and the germination rates were 54%, 35%, 14%, 12%, and 11% respectively under the treatment of 1%, 5%, 10%, 20%, and 50% of the fermentation broth of Bacillus Velezii HXYT7 (the germination rate of the control group was 73%). Figure 8 B), the inhibition rates were 26%, 52%, 81%, 84.1% and 84.5% respectively ( Figure 8 C), it can be concluded that the fermentation broth of Bacillus Velezii HXYT7 has a significant inhibitory effect on the germination of Helminthosporium spores.

[0092] 5. Effect of Bacillus Velezii HXYT7 fermentation broth on the microstructure of helminth mycelium

[0093] This experiment verifies the effect of the fermentation broth of Bacillus Velezii HXYT7 on the microstructure of Helminthosporium hyphae. The specific operation is as follows:

[0094] The fermentation broth CFS of Bacillus Velezii HXYT7 (cultured for 2 days at 37°C and 200rpm) was added to the sterile PDB medium to make the final concentrations 5%, 10% and 20% (total volume 50mL), respectively, and the control group was an equal volume of sterile PDB medium, and then 0.5g of worm spore hyphae was added, the sealing film was sealed, and the shaking table was cultured at 28°C and 180rpm for 48h, and the hyphae was collected by single-layer gauze filtration, and impurities were washed with sterile water, and the hyphae was fixed with 2.5% glutaraldehyde at 4°C overnight, and the supernatant was removed by centrifugation. Wash three times with PBS buffer, each time for 15min, and then wash twice with pure water, and the supernatant was removed by centrifugation. Then, the samples were dehydrated with 30%, 50%, 70%, and 90% ethanol in turn, with a dehydration time of 10 to 15 minutes, and then dehydrated twice with 100% ethanol, each time for 15 minutes, and then replaced with a mixture of ethanol and tert-butanol in a ratio of 2:1 and 1:1 with 100% tert-butanol, each time for 10 minutes, and the supernatant was removed by centrifugation, and finally the mycelium sample was made into a bacterial suspension containing tert-butanol. The tert-butanol bacterial suspension of appropriate concentration was dripped onto aluminum foil, precooled to -80°C, and then dried in a freeze dryer for about 4 hours. After drying, the sample was taken out and fixed on the sample table, sprayed with gold, and finally tested and analyzed.

[0095] Scanning electron microscopy results show that ( Fig. 9), after 24 hours of action of the fermentation broth of Bacillus Velez subtilis HXYT7 on Helminthosporium, the mycelium became dehydrated and deformed, with irregular deformation, wrinkled and shriveled surface, and wrinkles and depressions. In the control group, the mycelium was uniform in thickness, dense in structure, smooth and full, revealing that the fermentation broth of Bacillus Velez subtilis HXYT7 could destroy the mycelium structure of Helminthosporium, affect the normal physiological function of Helminthosporium, cause cell membrane damage and lead to cytoplasm loss.

[0096] 6. Effect of Bacillus Velezii HXYT7 fermentation broth on the integrity of cell membrane of Helminthospore hyphae

[0097] This experiment verifies the effect of the fermentation broth of Bacillus Velezii HXYT7 on the integrity of the mycelial cell membrane of Helminthosporium. The specific operation is as follows:

[0098] The fermentation broth CFS of Bacillus Velezii HXYT7 (cultured for 2 days at 37°C and 200rpm) was added to the sterile PDB medium to make the final concentrations 5%, 10% and 20% (total volume 50mL), respectively. The control group was an equal volume of sterile PDB medium, and then 0.5g of worm spore hyphae was added, the sealing film was sealed, and the culture was shaken at 28°C and 180rpm for 48h. The hyphae was collected by filtering with a single layer of gauze, and the impurities were washed with sterile water. The hyphae were stained with 5mM propidium iodide (PI) at a constant temperature of 37°C for 20min in the dark. The stained hyphae were then washed several times with PBS buffer until the supernatant was completely colorless to thoroughly remove the residual propidium iodide staining solution. A small amount of stained hyphae was placed on a slide, picked up and spread flat, and covered with a coverslip. Three parallels were made for each treatment.

[0099] Observation under a microscope revealed that mycelia treated with fermentation broth of Bacillus Velez HXYT7 at different concentrations had extremely strong red fluorescence, which increased with increasing fermentation broth concentrations. The mycelia treated with fermentation broth of Bacillus Velez HXYT7 at a final concentration of 20% had the strongest red fluorescence intensity, while mycelia in the control group had normal morphology and weak red fluorescence intensity ( Fig.10 ). This indicates that propidium iodide dye entered the cells and the fermentation liquid of Bacillus velezensis HXYT7 seriously destroyed the cell membrane structure of Helminthosporium.

[0100] 7. Effect of Bacillus subtilis HXYT7 fermentation broth on the MDA content of Helminthospore hyphae

[0101] In this experiment, the thiobarbituric acid (TBA) method was used to detect the malondialdehyde (MDA) content. The specific operation is as follows:

[0102] The fermentation broth CFS of Bacillus Velez HXYT7 (cultured for 2 days at 37°C and 200rpm) was added to the sterile PDB medium to make the final concentrations 5%, 10% and 20% (total volume 50mL), respectively. The control group was an equal volume of sterile PDB medium, and then 0.5g of worm spore hyphae was added, the sealing film was sealed, and the culture was shaken at 28°C and 180rpm for 48h. The hyphae treated with different concentrations of Bacillus Velez HXYT7 fermentation broth were collected by centrifugation. Then the malondialdehyde (MDA) content was quantified by the thiobarbituric acid (TBA) method, the method was as follows: 1g of hyphae treated with Bacillus Velez HXYT7 fermentation broth was mixed with 2mL of pre-cooled phosphate buffered saline solution and a small amount of quartz sand, and ground thoroughly in ice water. The mixture was transferred to a 5 mL centrifuge tube, and the mortar was rinsed with 0.2 mmol / L PBS buffer (pH 7.0), and added to the centrifuge tube until the total volume was 5 mL. The mixture was centrifuged at 4500 rpm for 10 min. 2 mL of the supernatant and 3 mL of 5% (V / V) thiobarbituric acid (TBA) solution were added to the test tube, and the tube was boiled in a water bath for 10 min. The tube was cooled to room temperature in ice water, and centrifuged at 4500 rpm for 10 min. The absorbance was measured at wavelengths of 532 nm and 600 nm, and three parallel results were set for each result.

[0103] The MDA content was calculated using the following formula:

[0104] MDA(nmol / g)=(A532-A600)×V1×V) / (0.155×W×V2)

[0105] V1 = total volume of reaction solution, 5 mL;

[0106] V = total volume of the extract, 5 mL;

[0107] V2 = volume of extract in reaction solution, 2 mL;

[0108] W = weight of mycelium sample, 1 g;

[0109] 0.155 is the micromolar absorption coefficient of MDA (absorbance when 1 μmol MDA is contained in 1 L solution).

[0110] The MDA concentrations of 5%, 10% and 20% Velez subtilis HXYT7 fermentation broth were 3.790 nmol / g, 4.273 nmol / g and 6.67 nmol / g, respectively, while that of the control group was 3.36 nmol / g ( Fig.11). The data showed that the fermentation broth of Bacillus Velezii HXYT7 caused the MDA content of Helminthosporium to increase, and the higher the concentration of the fermentation broth, the greater the MDA value. Because malondialdehyde can interact with cell membrane proteins to destroy the cell membrane structure, the higher the MDA content, the more serious the damage to the cell membrane. Therefore, the fermentation broth of Bacillus Velezii HXYT7 can damage the cell membrane structure of Helminthosporium.

[0111] 8. Effect of Bacillus Velezii HXYT7 fermentation broth on ROS content in helminth mycelium

[0112] Excessive accumulation of reactive oxygen species (ROS) can cause mycelial damage. This experiment observed the changes in the mycelium of Helminthosporium to verify the effect of the fermentation broth of Bacillus Velezii HXYT7 on the ROS content of its mycelium. The specific operation is as follows:

[0113] The fermentation broth CFS of Bacillus Velezii HXYT7 (cultured for 2 days at 37°C and 200rpm) was added to sterile PDB medium to make the final concentrations of 5%, 10% and 20% (total volume 50mL), respectively. The control group was an equal volume of sterile PDB medium. Then 0.5g of Helminthosporium hyphae was added, the medium was sealed with a sealing film, and the medium was cultured in a shaking incubator at 28°C and 180rpm for 12h. The hyphae were centrifuged at 8000rpm for 5min, and the hyphae were collected and washed twice with PBS buffer. The hyphae were collected by centrifugation, stained with dihydrorhodamine 123 (DHR123) dye for 1h in the dark, the dye was washed off with PBS buffer, and the hyphae were observed under a fluorescence microscope. Three parallels were set for each treatment.

[0114] The hyphae of Helminthosporium treated with the fermentation broth supernatant were stained with DHR123 dye. The results showed that the number and fluorescence intensity of green fluorescent hyphae in the treatment group increased significantly compared with the control group, and increased with the increase of fermentation broth concentration. The fluorescence intensity of the group treated with 20% Velez subtilis HXYT7 fermentation broth reached the highest ( Fig.12 ), it can be seen that the fermentation broth of Bacillus Velezii HXYT7 can cause reactive oxygen species (ROS) to accumulate in the hyphae of Helminthosporium.

[0115] 9. Effect of Bacillus Velezii HXYT7 fermentation broth on mitochondrial membrane potential of Helminthospore hyphae

[0116] Mitochondrial damage will cause a decrease in mitochondrial membrane potential, resulting in the JC-1 dye in the cell existing in monomeric form and emitting green fluorescence. This experiment verifies the effect of Bacillus Velez HXYT7 fermentation broth on its mitochondrial potential by observing the changes in fluorescence emitted by Helminthosporium hyphae under a fluorescence microscope. The specific operation is as follows:

[0117] The fermentation broth CFS of Bacillus Velezii HXYT7 (cultured at 37°C, 200 rpm for 2 days) was added to the sterile PDB medium to make the final concentrations of 5%, 10% and 20% (total volume 50 mL), respectively. The control group was an equal volume of sterile PDB medium. Then, 0.5 g of Helminthosporium hyphae was added, the medium was sealed with a sealing film, and the medium was cultured in a shaking incubator at 28°C and 180 rpm for 12 h. The hyphae were collected by centrifugation at 8000 rpm for 5 min, washed twice with PBS, collected by centrifugation, stained with JC-1 dye for 30 min in the dark, and then the dye was washed off with PBS buffer. The hyphae were observed under a fluorescence microscope ( Fig.13 ), and 3 parallels were set for each treatment group.

[0118] It can be seen that the intensity of green fluorescence gradually increases with the increase of the concentration of Velez Bacillus HXYT7 fermentation liquid, while the intensity of green fluorescence in the control group decreases and more red fluorescence appears. From these data, it can be concluded that Velez Bacillus HXYT7 fermentation liquid can cause the mitochondrial membrane potential of Helminthosporium hyphae to decrease.

[0119] 10. Application of Bacillus Velezii HXYT7 in the prevention and control of wheat leaf blight

[0120] This experiment verifies the control effect of Bacillus Velez HXYT7 on wheat leaf blight. The specific operation is as follows:

[0121] The seeds of Zhengmai 366 were sown in plastic pots (110×80 mm) filled with horticultural soil (10% zeolite, 20% perlite, 20% vermiculite, 50% peat moss, uniform particle size, 60% humidity). About 30 wheat plants were planted in each pot, and the pots were placed in a light incubator at a temperature of 25℃±2℃ and a relative humidity of 50±5% for 14h / d. A suspension of conidia of Helminthosporium prepared with sterile water (10 6 CFU / mL) were sprayed on the leaves of wheat at the two-leaf and one-heart stage, and then sterile water, 20% fermentation broth of Bacillus Velez HXYT7 (prepared with sterile water) and triadimefon (1%, v / v, prepared with sterile water) were used to irrigate the potted wheat. The results were observed on the 7th day ( Fig.14 ) and assess growth parameters and disease progression:

[0122] The growth parameters are as follows:

[0123] (1) Plant height: Use a ruler to measure the length from the base of the plant (i.e. the tillering node) to the top of the top leaf.

[0124] (2) Root length: Use a ruler to measure the length from the base of the root system to the longest root tip.

[0125] (3) Plant fresh weight: The whole plant was weighed to determine its fresh weight.

[0126] (4) Plant dry weight: After drying at 55°C, the dry weight of the plant was measured when the weight of the plant remained unchanged.

[0127] The condition is assessed as follows:

[0128] (5) Disease index and prevention and treatment effect:

[0129] Disease index = Σ(number of leaves with different disease levels × corresponding disease level value) / (total number of leaves surveyed × highest disease level value) × 100

[0130] Control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100

[0131] The disease grade of each replicate was counted, recorded and calculated using a 5-grade classification system, and the disease grade was assessed on a scale of 0-5, where 0 = no infection; 1 = 1%-15%; 2 = 6%-15%; 3 = 16%-25%; 4 = 26%-50%; and 5 = 51%-100% of the plant leaves were covered with yellow-brown spots.

[0132] It was found that the wheat lesion area treated with 20% of the fermentation liquid of Bacillus Velezii HXYT7 and triadimefon was significantly reduced ( Fig.14 The disease index of the control group was 63, while the disease index of 20% Velez subtilis HXYT7 fermentation broth and triadimefon were 22 and 21, respectively, which were 41 and 42 lower than those of the control group ( Fig.14 B in the above). The control effects of 20% Bacillus velez HXYT7 fermentation broth and triadimefon were 65% and 67% respectively ( Fig.14 C).

[0133] After treatment with 20% Bacillus Velez HXYT7 fermentation liquid and triadimefon, the plant heights were 23.32±2.76cm and 22.42±3.95cm, respectively, significantly higher than the control group (15.53±2.2cm); the root lengths were 7.72±1.34cm and 7.68±1.76cm, respectively, significantly longer than the control group (5.8±1.06cm) ( Fig.14 D); the fresh weights were 0.1679±0.06g and 0.1815±0.06g, which were significantly heavier than those in the control group (0.1361±0.05g); the dry weights were 0.0518±0.02g and 0.054±0.01g, which were significantly heavier than those in the control group (0.046±0.01g) ( Fig.14 E); It can be seen that 20% of the fermentation liquid of Bacillus Velez HXYT7 has a good biocontrol effect on wheat leaf blight.

Claims

1. A strain of Bacillus Velez, characterized in that: The strain is Bacillus Velez Bacillus velezensis )HXYT7, the deposit number is CGMCC No.31016.

2. Use of the Bacillus Velezii as claimed in claim 1 in preventing and treating wheat leaf blight caused by Helminthosporium.

3. The use according to claim 2, characterized in that: The control comprises using the cell-free supernatant (Cell-Free Supernatant, CFS) of the fermentation liquid of Bacillus Velez HXYT7 for root irrigation or foliar spraying.

4. A use of the Bacillus Velezii as claimed in claim 1 in inhibiting plant pathogenic fungi, characterized in that: The plant pathogenic fungi include Helminthosporium.

5. The use according to claim 4, characterized in that: The plant pathogenic fungi also include Fusarium moniliforme, Fusarium graminearum, Rhizoctonia graminearum, Aspergillus flavus and Aspergillus niger.

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