Bacillus velezensis and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to bacillus velezensis and application thereof. The bacillus velezensis is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC 25698. The strain has a strong specific antagonistic effect on streptomyces scabies X-1 (Streptomyces scabies X-1), the prevention effect of a pot experiment reaches 70.90%, and the strain has no pathogenic risk on potato tubers. A further research shows that the bacillus velezensis XM18-5 has a broad-spectrum inhibition effect on 10 crop pathogenic fungi such as potato colletotrichum gloeosporioides, botrytis cinerea and the like, and has a very wide development and application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Bacillus velezensis and applications thereof. Background Art

[0002] Potato scab is a global soil-borne disease caused by various pathogenic Streptomyces spp., with Streptomyces scabies X-1 as the core pathogen. It infects tuber tissue by secreting cyclic dipeptide toxins called thaxtomins, causing brown, sunken spots and raised scabs on the tuber surface, severely impacting the commercial value of potatoes. With the expansion of continuous cropping, the incidence of this disease has climbed to 30%-60% in some areas, becoming a key constraint to the sustainable development of the potato industry.

[0003] Biological control has become a research hotspot for potato scab prevention and control due to its ecological safety. Bacillus species are widely used due to their strong stress resistance and excellent soil colonization ability. Studies have shown that strains such as Bacillus subtilis and Bacillus amyloliquefaciens can reduce disease incidence by secreting antimicrobial substances. However, the stability of their effectiveness is limited by environmental factors, and the inhibitory spectrum of individual strains is relatively narrow.

[0004] Bacillus velezensis has attracted much attention due to its broad-spectrum antibacterial and plant growth-promoting functions. It directly inhibits the growth of pathogens and activates plant systemic resistance by synthesizing antimicrobial peptides such as surfactin and iturin, as well as active substances such as proteases and glucanases. Summary of the Invention

[0005] The present invention provides a Bacillus velezensis strain and its applications. Bacillus velezensis XM18-5, isolated from soil infected with potato scab, exhibits strong and specific antagonism against Streptomyces scabies X-1, achieving a 70.90% efficacy in potted plant trials and exhibiting no pathogenicity risk to potato tubers. Further research has shown that Bacillus velezensis XM18-5 exhibits broad-spectrum inhibitory activity against ten crop pathogens, including potato anthracnose and gray mold, suggesting promising development and application prospects.

[0006] In one aspect, the present invention relates to a Bacillus velezensis, the deposit information of which is as follows:

[0007] Strain name: XM18-5;

[0008] Classification name: Bacillus velezensis;

[0009] Date of receipt by the depository: September 13, 2022;

[0010] Time of issuance of the preservation certificate: September 30, 2022;

[0011] Depository: General Microbiology Center, China Culture Collection Administration;

[0012] Deposit number: CGMCC 25698;

[0013] Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] On the other hand, the present invention relates to the use of Bacillus Velez or the biocontrol agent in inhibiting pathogens of plant diseases, wherein the pathogens of plant diseases are at least one of potato scab pathogen Streptomyces scabies X-1, potato anthracnose pathogen C. coccodes, grape gray mold pathogen B. cinerea, potato gray mold pathogen B. cinerea, potato wilt pathogen F. oxysporum, potato dry rot pathogen F. sambucinum, corn stalk rot pathogen F. verticillioides, potato early blight pathogen A. solani, watermelon wilt pathogen F. oxysporum f. sp. Niveum, corn ear rot pathogen F. graminearum, and wolfberry root rot pathogen F. oxysporum.

[0015] On the other hand, the present invention also relates to the use of the Bacillus Velezii in preventing and treating potato scab.

[0016] In the above application, the pathogen of potato scab is Streptomyces scabies X-1.

[0017] On the other hand, the present invention also relates to a method for preventing and controlling potato scab, comprising: allowing the Velez spore bacteria to act on the pathogenic bacteria of potato scab and / or their habitats;

[0018] The pathogenic bacteria is Streptomyces scabies X-1.

[0019] In another aspect, the present invention also relates to a method for preventing and controlling plant diseases, comprising: allowing the Velezella spore to act on pathogenic bacteria of plant diseases and / or their habitats;

[0020] The pathogenic bacteria is at least one of potato anthracnose fungus C. coccodes, grape gray mold fungus B. cinerea, potato gray mold fungus B. cinerea, potato wilt fungus F. oxysporum, potato dry rot fungus F. sambucinum, corn stalk rot fungus F. verticillioides, potato early blight fungus A. solani, watermelon wilt fungus F. oxysporum f. sp. Niveum, corn ear rot fungus F. graminearum, and wolfberry root rot fungus F. oxysporum.

[0021] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0022] The Bacillus Velez strain XM18-5 provided by the present invention exhibits significant antagonistic activity against Streptomyces scabies, with an inhibition zone diameter of 45.30 mm, directly inhibiting hyphal expansion and toxin secretion of the pathogen. Potted plant trials demonstrated a control efficacy of 70.90%, significantly improved compared to similar Bacillus Velez strains. It is also non-pathogenic to potato tubers, eliminating the risk of rot caused by the biocontrol agent itself and demonstrating excellent safety.

[0023] 2. The Bacillus Velezii XM18-5 provided by the present invention has broad-spectrum antibacterial activity and can achieve the excellent performance of one bacterium for multiple preventions. The strain XM18-5 has inhibitory effects on 10 crop pathogenic fungi, including potato anthracnose, gray mold, and wilt, covering key groups of fungal diseases, reducing the use of multiple chemical agents, meeting the integrated needs of green agricultural prevention and control, and significantly reducing the cost of comprehensive prevention and control.

[0024] 3. The Bacillus Velezii XM18-5 provided by the present invention has natural environmental adaptability. Strain XM18-5 was isolated from soil affected by potato scab and naturally has a strong ability to colonize the target ecological niche. Its spore structure has strong stress resistance and can survive stably and continue to function in continuously cropped soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 These are the antibacterial effect diagrams of Bacillus velezensis XM18-5; among them, a is the primary screening control diagram, b is the primary screening effect diagram, c is the rescreening control diagram, and d is the rescreening effect diagram.

[0027] Figure 2 These are morphological images of Bacillus velezensis XM18-5; a is the colony morphology, b is the Gram staining microscopic examination result, and c is the electron microscope scanning image.

[0028] Figure 3 This is the 16S rDNA phylogenetic tree of Bacillus velez XM18-5.

[0029] Figure 4 This is the gyrB phylogenetic tree of Bacillus velez XM18-5.

[0030] Figure 5 This is a scanning electron micrograph of Streptomyces scabies X-1.

[0031] Figure 6 This is a diagram showing the antagonistic effect between strain XM18-5 and Streptomyces scabies X-1.

[0032] Figure 7 Figure 1. Potato chips treated with sterile distilled water.

[0033] Figure 8 A diagram of potato chips treated with LB culture medium.

[0034] Figure 9 A picture of potato chips treated with the culture medium of Bacillus Velez XM18-5.

[0035] Figure 10 This is a diagram showing the antibacterial activity of strain XM18-5 against C. coccodes.

[0036] Figure 11 This is a diagram showing the antibacterial activity of strain XM18-5 against B. cinerea.

[0037] Figure 12 This is a diagram showing the antibacterial activity of strain XM18-5 against B. cinerea.

[0038] Figure 13 This is a diagram showing the antibacterial activity of strain XM18-5 against F.oxysporum.

[0039] Figure 14 This is a diagram showing the antibacterial activity of strain XM18-5 against F. sambucinum.

[0040] Figure 15 This is a diagram showing the antibacterial activity of strain XM18-5 against F. verticillioides.

[0041] Figure 16 This is a diagram showing the antibacterial activity of strain XM18-5 against A. solani.

[0042] Figure 17 This is a diagram showing the antibacterial activity of strain XM18-5 against F.oxysporum.

[0043] Figure 18 This is a diagram showing the antibacterial activity of strain XM18-5 against sp. Niveum.

[0044] Figure 19 This is a diagram showing the antibacterial activity of strain XM18-5 against F. graminearum.

[0045] Figure 20 This is a diagram showing the antibacterial activity of strain XM18-5 against F.oxysporum. DETAILED DESCRIPTION

[0046] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental and detection methods described in each example are conventional methods unless otherwise specified. The reagents and materials described are commercially available unless otherwise specified. The percentages in the following examples are percentages by mass unless otherwise specified. The ratios in the following examples are ratios by mass unless otherwise specified.

[0047] Example 1

[0048] This example provides the isolation and identification results of Bacillus velezensis XM18-5 (antagonistic strain), demonstrating that strain XM18-5 has significant antibacterial activity against Streptomyces scabies X-1 (strain X-1).

[0049] 1. Isolation and screening of antagonistic strains:

[0050] Isolation of strains: Soil dilution separation method was used for separation. After drying the soil sample, weigh 1g and add 9mL sterile water to make 10 -4 , 10 -5 , 10 -6 and 10 -7 Take 100 μL of the dilution solution and spread it on the OMA solid culture medium plate. Repeat each gradient 3 times. After culturing in the dark at 30℃ for 18-24h, pick out single colonies with different morphologies and streak to separate them to obtain pure culture. Transfer them into the slant of freezing tubes for later use.

[0051] Screening of strains: Adjust the bacterial suspension of strain X-1 to 1×10 8cfu / mL, 100 μL of the bacterial suspension was spread on a Gao's medium plate. The isolated strain was then activated and a 5 mm diameter cake was made. This cake was then inoculated in the center of the plate. Uninoculated strains served as controls. Each treatment was repeated three times, incubated at 30°C for 5–7 days, and the size of the inhibition zone was compared. For rescreening, strains with antibacterial activity were placed in LB liquid medium and cultured at 30°C with constant shaking at 180 rpm for 24 hours. The resulting culture was centrifuged at 10,000 rpm for 1–2 minutes, and the supernatant was collected. A sterile Oxford cup was placed on the Gao's medium plate coated with strain X-1. 200 μL of the supernatant was pipetted into the cup using a pipette. A medium without supernatant served as a control. Each treatment was repeated three times, incubated at 30°C for 5–7 days, and the appearance of an inhibition zone around the cup was observed and its diameter was measured.

[0052] like Figure 1 As shown in the study, using strain X-1 as the target strain, 53 potato rhizosphere soil samples were collected from potato-growing areas in Yuanzhou District, Pengyang County, Xiji County, and Longde County. Soil microorganisms were isolated and purified, yielding 174 bacterial strains. Initial screening of these strains using the plate standoff growth method yielded 34 strains with significant antagonistic effects against strain X-1. Further screening using the Oxford cup test revealed that strain XM18-5, isolated from a soil sample from Malian Township, Xiji County, exhibited significant antibacterial activity against strain X-1, with an inhibitory diameter of 45.30 mm and an inhibition rate of 60.72%.

[0053] 2. Identification of morphological and physiological and biochemical characteristics of antagonistic strains:

[0054] The selected antagonistic strains were streaked onto LB solid medium, and the colony morphology was observed and identified. Physiological and biochemical indicators such as VP, glucose glycolysis, starch hydrolysis, gelatin liquefaction, and nitrate reduction of the antagonistic strains were identified using HBI Bacillus bioassay strips.

[0055] The morphological identification results are as follows Figure 2 As shown in a, the antagonistic strain grows well on LB solid medium. The single colony is milky white, spherical, opaque, slightly convex, with slightly wrinkled surface, irregular edges, and no pigmentation around it. Figure 2 As shown in Figure b, the strain was found to be Gram-positive under microscopic examination. Figure 2 As shown in Figure c, scanning electron microscopy results show that the antagonistic strain has a rod-shaped body with a smooth surface and no wrinkles. Based on morphological identification, it can be preliminarily determined that the antagonistic strain belongs to the genus Bacillus.

[0056] The results of physiological and biochemical characterization are shown in Table 1. The antagonistic strain exhibited catalase and oxidase activity, could utilize citrate, propionate, D-xylose, L-arabinose, glucose, and sucrose, tested positive for VP and nitrate reduction, and grew normally in media with a pH of 5.7 and a 7% NaCl concentration, respectively. It was capable of hydrolyzing starch and gelatin, but was unable to utilize D-mannitol, maltose, and lactose. It produced urease but not esterase. Based on the physiological and biochemical test results, combined with morphological characteristics, the strain was preliminarily identified as Bacillus velezensis.

[0057] Table 1: Physiological and biochemical characteristics of strain XM18-5

[0058]

[0059] Note: +: positive; -: negative.

[0060] 3. Molecular biological identification of antagonistic strains:

[0061] The genomic DNA of the antagonistic strain was extracted according to the instructions of the Solarbio Bacterial Genomic Kit (Cat#D1600), and the 16S rDNA sequence was amplified using bacterial universal primers using this as a template;

[0062] The bacterial universal primers are specifically:

[0063] 27F(5′-AGTTTGATCMTGGCTCAG-3′);

[0064] 1492R(5′-GGTTACCTTGTTACGACTT-3′).

[0065] In this example, the sequence of the gyrase β subunit gene (gyrB) was amplified using bacterial housekeeping gene primers.

[0066] The bacterial housekeeping gene primers are specifically:

[0067] gyrB-F(5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYG A-3′);

[0068] gyrB-R(5′-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCN GTCAT-3′).

[0069] The above primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the PCR amplification products were sequenced by Shanghai Sangon Biotechnology Co., Ltd. The sequencing results were uploaded to the NCBI website and compared with the 16S rDNA and gyrB gene sequences of other strains by BLAST. The strain sequences with high similarity in GenBank were selected, and a phylogenetic tree was constructed using the neighbor-joining method using MEGA.11 software.

[0070] Based on 16S rDNA gene sequencing, a 1446 bp sequence was obtained and registered at NCBI with the accession number OK560566. BLAST comparison revealed that the antagonistic strain had a sequence similarity of greater than 98% with multiple Bacillus strains, confirming that the antagonistic strain belonged to the genus Bacillus. The 16S rDNA sequence of the strain with high sequence homology to the target strain was downloaded and constructed as follows: Figure 3 The phylogenetic tree of the antagonistic strain shown in the figure shows that the antagonistic strain is clustered on the same root branch with Bacillus velezensis BCRC 17467, with a similarity of 100%, indicating that the antagonistic strain has a high homology with it and can be preliminarily determined to be Bacillus velezensis.

[0071] Based on the sequencing of gyrB gene, a sequence of 1137 bp was obtained, which was registered at NCBI and obtained the sequence accession number OK557802. Figure 4 The phylogenetic tree of the strains shown in the figure shows that strain XM18-5 and Bacillus velezensis BCRC 17467 clustered into a branch with a sequence similarity of 100%. Combined with the 16S rDNA gene sequencing results, the antagonistic strain was identified as Bacillus velezensis and named Bacillus velezensis XM18-5.

[0072] Example 2

[0073] This example is intended to illustrate the effect of Bacillus velezensis XM18-5 on the morphology of Streptomyces scabies X-1.

[0074] Samples were collected at a position of about 5 mm from the interface between strain XM18-5 and Streptomyces scabies X-1, placed on a cover slip to prepare a slide, and cultured in the dark at 30°C for 12 h. The cover slips were then removed and placed in a 4°C refrigerator, fixed with 2.5% glutaraldehyde for 1 day, and sent to Sewell Biotech for electron microscopy scanning.

[0075] The test results showed that strain XM18-5 could inhibit the mycelial growth of Streptomyces scabies X-1. Figure 5As shown in the figure, the mycelium surface is smooth, straight, without shriveling or wrinkles, etc., while after being treated with the biocontrol bacteria XM18-5, the mycelium of Streptomyces scabies X-1 is as shown in the figure. Figure 6 As shown, deformation and wrinkling occurred, and part of the hyphae wall collapsed and broke.

[0076] Example 3

[0077] This example provides a safety test for potato tubers using Bacillus velezensis XM18-5.

[0078] The surface of potato tubers was disinfected with 75% ethanol, washed with sterile water and dried, and the potato tubers were cut into chips with a thickness of about 0.5 cm using a sterile knife. The chips were mixed with water at a concentration of 1×10 8 After being infiltrated in the culture medium of the antagonistic strain with a concentration of 1.5 cfu / mL, the chips were placed in a culture dish covered with sterile filter paper, with sterile distilled water and LB culture medium as controls. Each treatment was repeated 3 times, and the degree of potato chip decay was observed after being placed at 28°C for 3 to 5 days.

[0079] The results are as follows Figures 7-9 As shown, the concentration was 1×10 8 cfu / mL of the antagonistic strain culture medium was used to treat potato chips. After being placed in the culture dish for 5 days, it was found that the potato chips did not rot, and there was no significant difference from the control group, indicating that the strain XM18-5 was safe for potato tubers and would not cause the rot of potato tubers.

[0080] Example 4

[0081] This example demonstrates that Bacillus velezensis XM18-5 has a broad-spectrum antibacterial effect on crop pathogens.

[0082] Ten common crop pathogens were selected and the antibacterial spectrum was determined using the plate standoff method. A 5 mm cake was made on the edge of the activated pathogen plate and placed in the center of a PDA plate. A 5 mm sterile filter paper was placed 2.5 cm from the pathogen. 3 μL of the antagonistic strain culture solution was dripped onto the filter paper and incubated in a dark incubator at 28°C for 5-7 days. The width of the inhibition zone was observed and measured, and the antibacterial effect was calculated. Analysis of variance was performed using DPS17.0. Each treatment was replicated three times. The antibacterial effect (%) = (width of the inhibition zone / distance between the biocontrol agent and the cake) × 100.

[0083] The 10 common crop pathogens are detailed in Table 2.

[0084] Table 2: Tested pathogens

[0085]

[0086] The results of the inhibition spectrum determination of the antagonistic strain XM18-5 are as follows Figures 10-20As shown in Table 3, XM18-5 has different degrees of inhibitory effects on 10 plant pathogens.

[0087] Table 3: Inhibition rate of strain XM18-5 against 10 pathogens

[0088]

[0089]

[0090] Note: The data are the average of three replicates. The same lowercase letters after the data indicate no significant difference at the 0.05 level.

[0091] Among them, XM18-5 had the best antibacterial activity against potato anthracnose pathogen C. coccodes, with an inhibition rate of 88.23%, which was significantly higher than that of other treatments (P<0.05); it also had no effect on grape gray mold B. cinerea, potato gray mold B. cinerea, potato wilt F. oxysporum, potato dry rot F. sambucinum, corn stalk rot F. verticillioides and potato early blight. A. solani was the second most effective, with inhibition rates of 71.67%, 71.07%, 69.79%, 67.27%, 65.42%, and 63.44%, respectively. The inhibition rates against watermelon wilt pathogen F. oxysporum f. sp. niveum, corn ear rot pathogen F. graminearum, and wolfberry root rot pathogen F. oxysporum were all lower than 60%, at 57.50%, 56.25%, and 54.25%, respectively. This suggests that strain XM18-5 has broad-spectrum antipathogenic activity and has great potential in biocontrol applications.

[0092] Example 5

[0093] This example demonstrates that Bacillus Velezii XM18-5 has excellent effects on potato scab.

[0094] Potato seed potatoes were cut into triangular pieces of similar size, weighing 20-25 g, and planted in pots with a diameter of 25 cm. Three potato pieces were planted in each pot. When the plants grew to the 4-leaf stage, different treatment liquids were irrigated. There were three treatments in total, with three replicates for each treatment. Treatment 1: irrigated with 200 mL of water; Treatment 2: irrigated with 100 mL of 1×10 9 cfu / mL of Streptomyces scabies X-1 suspension and then irrigate with 100 mL of clean water; Treatment 3: irrigate with 100 mL of 1×10 9 cfu / mL of Streptomyces scabies X-1 suspension and then poured into 100mL of 1×10 6cfu / mL of XM18-5 culture medium. Disease status of potato plants was observed in real time during the growth period, and scab on tubers was investigated during the harvest period. The incidence rate, disease index, and disease control efficacy were calculated.

[0095] As shown in Table 4, the potatoes in the blank control group (Treatment 1) showed no disease and were growing healthily. The potatoes treated with the pathogen suspension (Treatment 2) showed more severe disease, with a 53.56% incidence rate and a disease index of 49.50. The potato tubers treated with the culture medium of the antagonistic strain XM18-5 (Treatment 3) showed a 25.00% disease rate and a disease index of 14.40. While some cases did develop, the overall rate was significantly lower than that of Treatment 2, demonstrating a 70.90% efficacy against potato scab. This demonstrates that strain XM18-5 is effective in preventing and controlling potato scab caused by Streptomyces scab.

[0096] Table 4: Disease prevention effect of strain XM18-5 on potato scab

[0097] deal with Incidence / % Disease index / % Prevention effect / % 1 0 0 / 2 53.56±1.20a 49.50±2.30a / 3 25.00±1.40a 14.40±1.45b 70.90±0.40

[0098] Note: The same lowercase letters after the data indicate no significant difference at the 0.05 level.

[0099] In summary, strain XM18-5, a strain of Bacillus velezensis, significantly inhibited the growth of Streptomyces scab, with an inhibition zone diameter of 45.30 mm. It exhibited broad-spectrum antibacterial activity against 10 crop pathogens and was highly safe for potato tubers, not causing rot. It achieved a 70.90% efficacy against potted potato scab. This suggests that Bacillus velezensis XM18-5 is an excellent biocontrol strain that can expand strain resources for the biological control of potato scab and has broad prospects for development and application.

[0100] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

Claims

1. A Bacillus Velezii, characterized in that The Bacillus velezensis is named Bacillus velezensis XM18-5 and is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCC No. 25698.

2. The use of the Bacillus Velezii according to claim 1 in inhibiting pathogens of plant diseases, characterized in that: The pathogen of the plant disease is at least one of potato anthracnose C. coccodes, grape gray mold B. cinerea, potato gray mold B. cinerea, potato wilt F. oxysporum, potato dry rot F. sambucinum, corn stalk rot F. verticillioides, potato early blight A. solani, watermelon wilt F. oxysporum f. sp. Niveum, corn ear rot F. graminearum, and wolfberry root rot F. oxysporum.

3. Use of the Bacillus Velezii of claim 1 in preventing and treating potato scab.

4. The use according to claim 3, characterized in that The pathogen of potato scab is potato scab pathogen Streptomyces scabies X-1.

5. A method for preventing and treating potato scab, characterized in that: include: The Velez spore bacteria according to claim 1 act on the pathogenic bacteria of potato scab and / or their habitats; The pathogenic bacteria is Streptomyces scabies X-1.

6. A method for preventing and controlling plant diseases, characterized in that: include: The Velez spore bacillus according to claim 1 acts on pathogenic bacteria of plant diseases and / or their habitats; The pathogenic bacteria is at least one of potato anthracnose fungus C. coccodes, grape gray mold fungus B. cinerea, potato gray mold fungus B. cinerea, potato wilt fungus F. oxysporum, potato dry rot fungus F. sambucinum, corn stalk rot fungus F. verticillioides, potato early blight fungus A. solani, watermelon wilt fungus F. oxysporum f. sp. Niveum, corn ear rot fungus F. graminearum, and wolfberry root rot fungus F. oxysporum.

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