Bacillus velezensis for antagonizing cryphonectria parasitica and application thereof

Abstract

The application discloses a bacillus velezensis for antagonizing chestnut blight fungus and an application thereof, the classification and naming of the bacillus velezensis is Bacillus velezensis G16, the preservation unit is China General Microbiological Culture Collection Center, the preservation number is CGMCC NO.33101, and the preservation time is December 31, 2024. Experimental results prove that the bacillus velezensis G16 has significant antagonistic activity on the chestnut blight fungus, the sterile fermentation supernatant of the bacillus velezensis G16 has certain control effect on chestnut branch infection of the chestnut blight fungus, and can obviously inhibit the expansion of a disease spot. In addition, the bacillus velezensis G16 provided by the application has good bacteriostatic activity on Aureobasidium pullulans, Colletotrichum gloeosporioides, Vitis phialangium, Lecanicillium laicium, Alternaria alternata and new shell crenospora, that is, the strain has the characteristics of inhibiting a broad spectrum, greatly increases the application range, and has a good development and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of Bacillus vesicularis antagonistic to chestnut blight and its application. Background Technology

[0002] Biological control of plant diseases refers to the use of one or more organisms other than humans to reduce the number of pathogens or weaken their pathogenicity, thereby reducing the occurrence of diseases and benefiting beneficial organisms such as crops, trees, animals, beneficial insects, and microorganisms. The types of microorganisms that can be utilized in the biological control of plant diseases include fungi, actinomycetes, bacteria, viruses, and even higher plants. With the increasing awareness of environmental protection, biological control has gradually become one of the important research directions.

[0003] Chestnut blight is caused by the chestnut blight fungus (Phytophthora infestans). Cryphonectria parasitica Chestnut blight is a serious disease that can infect chestnuts at all stages of growth. It primarily affects the branches and trunks, initially manifesting as water-soaked cankers. As the disease progresses, the symptoms worsen, eventually leading to dehydration of the branches and trunks, wilting of the leaves, and ultimately, the death of the entire tree. Because chestnut blight reduces fruit yield and quality, it negatively impacts the economic benefits of the entire industry chain.

[0004] Currently, the main control measures for chestnut blight are breeding disease-resistant varieties and chemical control. Breeding disease-resistant varieties requires significant manpower and effort, and is time-consuming. While chemical control is effective, long-term use can easily lead to increased drug resistance in pathogens, damage the ecological environment, and harm human health. Therefore, environmentally friendly biological control is gradually becoming the mainstream trend in disease control.

[0005] Currently, biological control methods for chestnut blight focus on inoculating attenuated strains to suppress highly virulent strains. However, the control efficacy of attenuated strains varies significantly depending on region and environmental conditions. In contrast, antagonistic bacteria can specifically inhibit the growth of pathogens and can colonize in plants and soil, providing long-term protection. Furthermore, antagonistic bacteria are natural microorganisms, leaving no harmful residues in the environment after application, making them more environmentally friendly. Therefore, screening for antagonistic strains for chestnut blight is necessary, as it is beneficial for the development of microbial resources and the sustainable development of agriculture and forestry. Summary of the Invention

[0006] In view of the technical problems in the background art, the purpose of the present invention is to provide a strain of Bacillus vesiculosus antagonistic to chestnut blight and its application.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a strain of Bacillus belye that antagonizes chestnut blight, said Bacillus belye being classified as... Bacillus velezensis G16, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 33101, on December 31, 2024.

[0009] A second aspect of the invention is to provide the application of Bacillus vesiculosus in the biological control of chestnut blight.

[0010] Preferably, the application includes: preparing a sterile fermentation supernatant of Bacillus belysium based on Bacillus belysium, and then spraying it onto wounded chestnut plants.

[0011] Preferably, the method for preparing sterile fermentation supernatant of Bacillus berleis based on Bacillus berleis includes the following steps: after activating Bacillus berleis, it is inoculated into LB liquid medium and placed in a constant temperature shaker at 25-28℃ and 150-180 r / min for 24-72 h to obtain the strain fermentation broth, which is then centrifuged and filtered to obtain sterile fermentation supernatant of Bacillus berleis.

[0012] A third aspect of the present invention is to provide the application of Bacillus belye in the biological control of fungal diseases of forest trees, wherein the fungi are Aureobasidium aureum, Colletotrichum gloeosporioides, Staphylococcus aureus, Verticillium dahliae, Alternaria alternata, and Clostridium neoformans.

[0013] The present invention has the following beneficial effects:

[0014] (1) This invention provides a strain of Bacillus vesiculus antagonistic to chestnut blight and its application. Experimental results show that Bacillus vesiculus G16 has excellent antagonistic activity against chestnut blight. The supernatant of sterile fermentation of Bacillus vesiculus G16 has a certain control effect on chestnut branches infected with chestnut blight and can significantly inhibit the expansion of lesions.

[0015] (2) The Bacillus berreatus G16 provided by the present invention has high antibacterial activity against Chlorella vulgaris, Colletotrichum gloeosporioides, Staphylococcus aureus, Verticillium dahliae, Alternaria alternata, and Clostridium neoformans. That is, it has the characteristics of broad-spectrum antibacterial activity, which greatly increases its application range and has good development and application prospects. It promotes the research and development of biological control agents and is worth promoting and using.

[0016] Preservation Information

[0017] Preservation period: December 31, 2024;

[0018] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Culture Collections;

[0019] Accession number: CGMCC NO. 33101;

[0020] Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, Chaoyang District, Beijing;

[0021] Postal code: 100101;

[0022] Category Naming: Bacillus velezensis G16. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Plate confrontation antagonism plot of the five antagonistic bacteria screened against *Phytophthora spp.*

[0025] Figure 2 Colony morphology of strain G16 on LB solid medium;

[0026] Figure 3 Gram staining image of strain G16;

[0027] Figure 4 Phylogenetic tree of strain G16 based on 16S and gyrB sequences;

[0028] Figure 5 The control effect of aseptic fermentation supernatant of Bacillus vesiculosus G16 on detached chestnut branches is shown in the figure.

[0029] Figure 6 A quantitative graph showing the area of ​​lesions under different treatment methods;

[0030] Figure 7 The image shows the broad-spectrum antibacterial effect of Bacillus belyssus G16.

[0031] Figure 8 This is a quantitative graph showing the broad-spectrum inhibition rate of Bacillus belyssus G16. Detailed Implementation

[0032] In the following description, specific details such as particular system architectures and techniques are set forth for illustrative purposes and not for limitation, in order to provide a thorough understanding of the embodiments of the invention. However, those skilled in the art will understand that the invention may be implemented in other embodiments without these specific details.

[0033] Example 1

[0034] 1. Experimental Materials

[0035] LB solid plate medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, 15 g agar powder, adjust the total volume to 1000 mL with deionized water, pH 7.0-7.2. Sterilize, cool, and pour into plates for later use.

[0036] LB liquid medium: 10 g tryptone, 5 g yeast extract, 10 g NaCl, deionized water to adjust the total volume to 1000 mL, pH 7.0-7.2.

[0037] PDA solid culture medium: 200 g potato, 20 g glucose, 15-20 g agar, deionized water to adjust the total volume to 1000 mL, pH 5.6-6.0.

[0038] 2. Experimental Methods

[0039] 2.1 Isolation and activation of endophytic bacteria

[0040] (1) Isolation of endophytic bacteria: The specific operation is as follows: Healthy poplar leaves from a certain region are rinsed with sterile water, then soaked in 75% ethanol for 1 minute, followed by immersion in 3% sodium hypochlorite solution for 30 seconds, and finally rinsed repeatedly with sterile water until no reagent residue remains on the leaf surface. The disinfected leaves are then ground, and the suspension is serially diluted to 10. -1 Up to 10 -3 Then, suspensions of different gradients were evenly spread onto LB plates and incubated at 28°C for 7 days. Single colonies with different morphological characteristics were picked, purified, and stored in 30% glycerol at -80°C.

[0041] (2) Activate the strains preserved above. The specific operation is as follows: place the cryovial containing the bacterial culture on ice and thaw at 4°C. After thawing, use a sterile inoculation loop to take a loopful of bacterial culture and streak it on LB solid medium. After three consecutive subcultures, it can be used for subsequent screening of antagonistic strains.

[0042] 2.2 Screening for antagonistic bacterial strains against chestnut blight

[0043] Bacteria antagonistic to chestnut blight were screened using the plate confrontation method. Specifically:

[0044] 1) Chestnut blight pathogen Cryphonectria parasitica EP155 (a gift from Professor Chen Baoshan of Guangxi University) was inoculated in the center of a PDA plate. Two days later, a 5mm diameter punch was used to collect chestnut blight fungal cakes from the edge of the colony.

[0045] 2) Transfer the collected chestnut blight fungal cake to the center of a new PDA plate (9cm), and inoculate the activated bacteria at four equidistant points 2.5cm away from the fungal cake, i.e., 4 points on each plate;

[0046] 3) Place in a 28℃ incubator for constant temperature incubation for 6 days.

[0047] A control group was set up: *Phytophthora chinensis*, the pathogen inoculated solely in the center of the PDA plate, was selected. Once the mycelium of *Phytophthora chinensis* from the control group had completely covered the entire plate, the inhibition rate was calculated. The inhibition rate was calculated using the formula: I = (D...) C -D T ) / (D C -0.5)×100, where I is the inhibition rate (%), and D C The control group colony diameter (cm), D T The colony diameter (cm) is for the treatment group. Inhibition rate results are shown in Table 1, and plate confrontation screening results are shown below. Figure 1 A total of 5 bacteria with antagonistic activity against chestnut blight pathogens were obtained.

[0048] Table 1

[0049]

[0050] From Table 1 and Figure 1 The results showed that five bacterial strains exhibited good antagonistic activity against *Phytophthora chinensis*, all exceeding 60%. Among them, strain G16 showed the highest antagonistic activity, with a mycelial inhibition rate of up to 80.73%, demonstrating considerable biocontrol potential, and was therefore selected as the target strain.

[0051] 2.3 Identification of strain G16

[0052] The selected strain G16 was identified using a combination of colony morphology, Gram staining, and multigene phylogenetic tree analysis.

[0053] 2.3.1 Morphological analysis and Gram staining identification

[0054] Streak strain G16 on LB solid medium and incubate at 28°C for 1-2 days. Observe the colony color and morphology, and pick an appropriate amount of cells for Gram staining. Observe the cell morphology and staining color under a microscope.

[0055] 2.3.2 Genetic Identification

[0056] Genomic DNA was extracted from bacterial strain G16 using a bacterial genomic DNA extraction kit. Amplification was performed using universal primers for the 16S rRNA gene and universal primers UP-1 and UP-2r for the gyrB gene. The universal primers for the 16S rRNA gene are as follows:

[0057] 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′;

[0058] 1492R: 5′-TACGGYTACCTT GTT ACGACTT -3′;

[0059] The DNA sequences of the universal primers UP-1 and UP-2r for the gyrB gene are as follows:

[0060] UP-1:5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′;

[0061] UP-2:5′-AGCAGGATACGGATGTGCGAGCCRTCACRTCNGCRTCNGTCAT-3′;

[0062] PCR amplification was performed on 16S rRNA and gyrB gene, respectively.

[0063] The PCR reaction system consisted of 25 μL of 2×Taq PCR Mix, 1 μL each of forward and reverse primers (10 μM), 1.0 μL of DNA template, and ddH2O to a final volume of 50 μL.

[0064] The PCR reaction conditions were: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 1 min, 55℃ annealing for 1 min, 72℃ extension for 1.5 min, 30 cycles, and a final extension at 72℃ for 5 min.

[0065] After the PCR products were detected by 1.5% agarose gel electrophoresis, DNA sequencing was performed. The sequencing results showed that the 16S sequence of strain G16 was as shown in SEQ ID NO.1, with a total length of 1526 bp; and the gyrB sequence of strain G16 was as shown in SEQ ID NO.2, with a total length of 1275 bp.

[0066] 2.3.3 Morphological and Molecular Biological Identification Results

[0067] (1) such as Figure 2 As shown, on LB solid medium, the colonies of strain G16 are milky white, with a smooth surface, a raised center, and irregular edges.

[0068] (2) such as Figure 3 As shown, the Gram staining result is purple, indicating that strain G16 is a Gram-positive bacterium, and the bacterial cells are elliptical at both ends and short rod-shaped overall, which is consistent with the bacterial cell morphology of Bacillus.

[0069] (3) The 16S rRNA sequence of strain G16 was compared with that of the type strain on the Ezbiocloud website. Bacillus velezensis CR-502 showed the highest similarity, reaching 99.93%. A phylogenetic tree of the 16S and gyrB gene sequences of strain G16 was constructed as follows: Figure 4 As shown, based on morphology, Gram staining, and a polygenetic phylogenetic tree, strain G16 was identified as *Bacillus belyesense*. Bacillus velezensis ), categorized and named Bacillus velezensis G16, designated as Bacillus bereaves G16, was preserved.

[0070] 2.4 Prevention and Control Trials

[0071] (1) Preparation of sterile fermentation supernatant of Bacillus belyssus G16: One loop of activated G16 cells was picked up with a sterile inoculation loop and inoculated into 5 ml of LB liquid medium. The culture was placed in a shaker at 28℃ and 180 r / min for 1 day to obtain the seed culture of strain G16. The seed culture was added to 200 ml of LB liquid medium at an inoculation rate of 1%, and the culture was placed in a shaker at 28℃ and 180 r / min for 3 days. The fermentation broth after 3 days of shaker culture was centrifuged at 4000 r / min for 10 min to obtain the supernatant. Finally, the supernatant was filtered through a 0.22 μm filter membrane to remove bacteria and obtain sterile fermentation supernatant of Bacillus belyssus G16.

[0072] (2) Chestnut disease control experiment

[0073] A control experiment was conducted using scalded inoculation of detached chestnut branches to evaluate the control effect of Bacillus berberis G16 aseptic fermentation supernatant on chestnut blight. Three treatment methods were used: 1) After scalding chestnut branches, PDA agar blocks were inoculated at the scalded sites as a negative control; 2) After scalding chestnut branches, chestnut blight fungal cakes were inoculated at the scalded sites as a positive control; 3) After scalding chestnut branches, Bacillus berberis G16 aseptic fermentation supernatant was sprayed onto the scalded sites, air-dried at room temperature, and then inoculated with chestnut blight fungus. The lesion area was counted after 14 days of incubation. Results are shown below. Figure 5 and Figure 6 .

[0074] Depend on Figure 5 and Figure 6 The results showed that the lesion area in the PDA agar block treatment group (negative control) was 180.40 ± 4.14 mm. 2 The lesion area in the group treated with the aseptic fermentation supernatant of Bacillus belyss G16 was 226.90 ± 7.14 mm. 2 The lesion area in the chestnut blight pathogen treatment group (positive control) was 325.26 ± 9.65 mm. 2Compared to the group treated with chestnut blight pathogens, the area of ​​lesions on chestnut branches treated with the supernatant of Bacillus vesiculosus G16 aseptic fermentation was reduced by 30.24%. Therefore, it can be concluded that the supernatant of Bacillus vesiculosus G16 aseptic fermentation can significantly inhibit the further expansion of lesions and achieve a certain control effect.

[0075] (3) Study on the broad-spectrum antibacterial activity of Bacillus belyssus G16

[0076] Select forest tree pathogenic fungus Aureobasidium aureum ( Cytospora chrysosperma ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), Staphylococcus aureus ( Botryosphaeria dothidea Verticillium dahliae Verticillium dahliae Alternaria ( Alternaria alternata ) and Clostridium neoformans ( Neofusicoccum laricinum The broad-spectrum antibacterial activity of *Bacillus belyssus* G16 was determined using the plate confrontation method, targeting the pathogenic fungus. Results are shown in [Figure number missing]. Figure 7 and Figure 8 .

[0077] Depend on Figure 7 and Figure 8 The results showed that Bacillus belye G16 not only had a good antagonistic effect on Pythium spp., but also showed a significant inhibitory effect on the above six target pathogenic fungi, with inhibition rates all exceeding 75%.

[0078] This invention is not limited to the specific embodiments described above. Any modifications made by those skilled in the art based on the above concept without creative effort are within the scope of protection of this invention.

Claims

1. The application of aseptic fermentation supernatant of a strain of Bacillus belye in the control of chestnut blight on chestnut branches, characterized in that, The classification of Bacillus belysium is named Bacillus velezensis G16, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO. 33101, and deposited on December 31, 2024; Its application method includes: spraying the sterile fermentation supernatant of Bacillus vesiculosus onto the wounded chestnut branches; The specific preparation steps of the sterile fermentation supernatant of Bacillus belysae are as follows: after activating Bacillus belysae, it is inoculated into LB liquid medium and placed in a constant temperature shaker at 25-28℃ and 150-180r / min for 24-72h to obtain the strain fermentation broth. After centrifugation and filtration, the sterile fermentation supernatant of Bacillus belysae is obtained.

2. The application of a strain of Bacillus belye in the biological control of fungal diseases of forest trees, characterized in that, The classification of Bacillus belysium is named Bacillus velezensis G16, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO. 33101, deposited on December 31, 2024; the fungus described is *Cyclosporium chrysogenum*, *Verticillium dahliae*, *Alternaria alternata*, or *Clostridium neoformans*.

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