Bacillus velezensis HMB28645 and application thereof
By using Bacillus berreatus HMB28645 and its fermentation products, the problems of poor control of gray mold in fruits and vegetables and drug resistance caused by chemical fungicides in existing technologies have been solved, achieving efficient control and safe growth promotion of a variety of fruit and vegetable diseases.
Patent Information
- Application Number
- CN202511855394.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-10
- Publication Date
- 2026-02-06
Smart Images
Figure CN121472092A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant disease control technology, specifically to a strain of Bacillus belye HMB28645 and its application in the control of fruit and vegetable diseases. Background Technology
[0002] Botrytis cinerea ( Botrytis cinerea Gray mold, caused by fungi, is a global fungal disease affecting grapes, tomatoes, cucumbers, and other fruits and vegetables. It can infect leaves, stems, flowers, and fruits. Leaf infection typically begins at the leaf tip, edge, or wound, forming V-shaped or irregular brown lesions with water-soaked edges. In high humidity, a gray mold layer grows on these lesions. Infected fruits develop brown rot, softening and becoming sticky. A dense layer of gray mold grows on the rotten areas, ultimately rendering the fruit unmarketable. Severe cases can result in yield losses of 30%-50%, or even total crop failure. It is estimated that gray mold causes global losses of $10 billion to $100 billion annually to vegetables and fruits, thus ranking it among the top ten diseases worldwide.
[0003] Besides agricultural control measures, chemical fungicides play a major role in controlling gray mold in fruits and vegetables. However, long-term, excessive, and unreasonable application of chemical fungicides not only causes pesticide residues and environmental pollution but also leads to severe drug resistance in pathogens, resulting in decreased efficacy. Research and practice have proven that microbial fungicides, due to their unique disease control mechanisms, low resistance rate, and advantages such as low toxicity and no residue, are an important way to effectively solve the damage caused by gray mold in fruits and vegetables.
[0004] Currently, there are reports on the use of microorganisms to control gray mold in fruits and vegetables. However, existing biocontrol strains generally have the following limitations: limited applicable fruit and vegetable varieties, unreliable actual field effects, or narrow application range. Therefore, developing novel biocontrol strains with highly effective antibacterial and disease-preventing effects against gray mold in grapes, tomatoes, cucumbers, and other fruits and vegetables, as well as other fruit and vegetable diseases, remains of practical significance for the green and efficient control of fruit and vegetable diseases. Summary of the Invention
[0005] In view of this, the present invention isolates a strain of Bacillus belye HMB28645 from the soil around the roots of cotton, aiming to provide a new biological resource for the prevention and control of gray mold and other diseases in fruits and vegetables.
[0006] The specific technical solution of the present invention is as follows: In a first aspect, the present invention provides a strain of Bacillus belyssus ( Bacillus velezensis HMB28645 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is November 20, 2025, and the accession number is CGMCC No. 36721.
[0007] In a second aspect, the present application provides a microbial inoculant containing at least Bacillus velezensis HMB28645. It can be understood that the microbial inoculant can also contain extracellular metabolites of the HMB28645 strain and / or other biocontrol microorganisms, and can also contain carriers or other adjuvants; the microbial inoculant can be a liquid preparation, a wettable powder, etc.
[0008] In a third aspect, the present application provides a fermentation product of Bacillus velezensis HMB28645. In practical applications, the fermentation product can be purified, diluted, and separated into a bacterial solution according to specific needs.
[0009] In an embodiment of the present application, the fermentation product is prepared by the following method: after the Bacillus velezensis HMB28645 is activated, a seed solution is prepared, and then inoculated into a fermentation medium with pH 7.0-7.5 and containing corn flour, soybean flour, glucose, sodium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, and calcium carbonate; the obtained fermentation broth can be used for plant disease control in the field after dilution.
[0010] In a fourth aspect, the present application provides the use of the above-mentioned Bacillus velezensis HMB28645, microbial inoculant, and fermentation product, at least including the following: 1) inhibiting the growth of pathogenic fungi, wherein the pathogenic fungi are selected from Botrytis cinerea, B. lycopersici, B. cucumeris, B. porri (B. allii) (B. porri) (B. allii), B. fabiae (B. fabae), B. cladosporioides (B. cladosporioides), B. viticola (B. viticola), B. vitis (B. vitis), and B. junghuhui (B. junghuhui); Coniella vitis Colletotrichum viniferum Alternaria viniferae Botryosphaeria dothidea Dematophora necatrix 2) preventing and treating fruit and vegetable gray mold, including grape gray mold, tomato gray mold, and cucumber gray mold; 3) preventing and treating grape diseases, wherein the grape diseases include grape gray mold, grape white rot, grape anthracnose, grape alternaria leaf spot, grape bacterial wilt, and grape white stripe wing rot; 4) promoting grape growth; 5) improving the defense enzyme activity of grape leaves, thereby improving the disease resistance of grapes.
[0011] Compared with the prior art, the present application has the following beneficial effects: The *Bacillus belyceae* HMB28645 strain provided by this invention can colonize the surface of crop leaves. Through competition with pathogens for infection sites and the secretion of antibacterial active substances, it effectively controls various gray mold diseases in fruits and vegetables. Specifically, pot and field trials show that its control efficacy against grape gray mold is over 89%, against tomato gray mold over 86%, and against cucumber gray mold over 87%. Simultaneously, the HMB28645 strain has a broad antibacterial spectrum, exhibiting excellent inhibitory effects against *Alternaria alternata*, *Alternaria gracilis*, *Alternaria gracilis*, *Alternaria gracilis*, and *Alternaria gracilis* root rot pathogens, in addition to *Alternaria gracilis*. Furthermore, in vitro leaf experiments demonstrate that the HMB28645 strain can indeed reduce the pathogenicity of these grape pathogens. In addition, the HMB28645 strain can promote grape growth and increase the activity of defensive enzymes in grape leaves, thereby enhancing the grape's disease resistance.
[0012] Compared to chemical agents, the biopreventive agent developed based on the HMB28645 strain has the advantages of being safe, efficient, non-toxic, residue-free, and environmentally friendly. Moreover, the preparation method is simple, low-cost, and suitable for industrial production. Attached Figure Description
[0013] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.
[0014] Figure 1 This is the phylogenetic tree of strain HMB28645 constructed based on the 16S sequence in Example 1; Figure 2 As in Example 1, based on gyrB Phylogenetic tree of strain HMB28645 constructed from its sequences; Figure 3 As in Example 1, based on rpoB Phylogenetic tree of strain HMB28645 constructed from its sequence. Detailed Implementation
[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.
[0016] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.
[0017] The LB solid medium used in the examples was composed of 10 g of proteose peptone, 5 g of yeast powder, 5 g of sodium chloride, 12 g of agar, and 1000 mL of water, with a pH of 7.0-7.5, and sterilized at 121°C for 30 min. The LB liquid medium used in the examples did not contain agar and was the same as the LB solid medium. The grape Botrytis cinerea pathogen, tomato Botrytis cinerea pathogen, and cucumber Botrytis cinerea pathogen used in the examples were isolated from Botrytis cinerea diseased leaves of grape, tomato, and cucumber, respectively, and identified as Botrytis cinerea. The strains of grape Botrytis cinerea pathogen, grape Botrytis cinerea pathogen, grape Botrytis cinerea pathogen, grape Botrytis cinerea pathogen, and grape Botrytis cinerea pathogen used in the examples were all isolated from the corresponding grape disease tissues.
[0018] In the examples, the specific techniques or conditions not specified were carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments used, if not specified by the manufacturer, were all conventional products that can be obtained commercially.
[0019] Example 1 This example provides the isolation and identification of Bacillus velezensis HMB28645, as follows: (1) Screening and isolation process of HMB28645 strain.
[0020] In 2022, the Plant Protection Institute of Hebei Academy of Agriculture and Forestry Sciences collected soil from the rhizosphere of cotton in Jingzhou City, Hubei Province, weighed 10 g of soil sample into a triangular flask containing 100 mL of sterile water, and shook it on a 180 r / min shaking table for 30 min. After standing for 30 min, 1 mL of supernatant was added to 9 mL of sterile water to make a 10 mL 10 -2 times soil microbial suspension. Then the soil suspension was diluted to 10 -3 , 10 -4 , 10 -5 , and 10 -6 times dilutions. 100 µL of each concentration of microbial suspension was spread on LB medium plates, and each concentration was repeated 3 times. The plates were incubated at 30°C for 1-3 days for bacterial isolation and purification. Using grape Botrytis cinerea as the target, plate confrontation test, in vitro leaf test, and pot test were used to screen and evaluate biocontrol bacteria. As a result, a strain with good control effect on grape Botrytis cinerea was selected and named HMB28645.
[0021] (2) Morphological characteristics identification of HMB28645 strain.
[0022] The HMB28645 strain was cultured on LB solid medium, and the colony appearance was recorded when cultured at 30℃ for 24 h. The HMB28645 strain was inoculated into a flask containing 100 mL of LB liquid medium, and cultured at 30℃. The samples were taken at 24 h and 48 h, and were dyed and made into a film by using crystal violet staining method. The cell and spore morphology of the strain were observed under a microscope (model DM6B, Leica Microsystems CMS GmbH).
[0023] When cultured at 30℃ for 24 h, the single colony of the HMB28645 strain on the LB solid medium plate was round and raised, milky white, opaque, viscous and thick, and no pigment was secreted. The strain was long rod-shaped, and could produce spores. Through these morphological characteristics, it was preliminarily determined that the HMB28645 strain belonged to Bacillus.
[0024] (3) Molecular biology identification of the HMB28645 strain.
[0025] ① Identification and classification by using 16S rDNA sequence.
[0026] The genomic DNA of the HMB28645 strain was extracted by using a genomic DNA extraction kit of Tiangen Biosciences (Beijing) Co., Ltd. as a template, and the 16S rDNA sequence of the HMB28645 strain was amplified by using 16S rDNA universal primers.
[0027] The sequence of the primer used is as follows: 27F: 5'-AGAGTTTGATCATGGCTCAG-3' (SEQ ID NO. 1); 1492R: 5'-GGCTACCTTGTTACGACTT-3' (SEQ ID NO. 2).
[0028] The PCR reaction system (50 μL in total) was composed of 5×PCR buffer (Mg 2+ Plus) 10 μL, dNTP (2.5 mM) 4 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, DNA 1 μL, Taq DNA polymerase (0.5 U / μL) 1 μL, and the rest was sterilized ddH2O. The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles; 72℃ extension for 7 min.
[0029] The PCR amplification product was detected by 1% agarose gel electrophoresis and sent to Shanghai Biotechnology Co., Ltd. for sequencing to obtain the 16S rDNA sequence of HMB28645 strain. The obtained 16S rDNA sequence was compared for homology in GenBank, and the phylogenetic tree was constructed by the neighbor-joining method using MEGA 5.0 software (see Figure 1 ), and it was found that HMB28645 strain clustered together with Bacillus velezensis and Bacillus safensis, which initially indicated that HMB28645 strain was Bacillus.
[0030] ②Classification by using gyrB gene sequence.
[0031] The genome of HMB28645 strain was used as a template, and the primer of gyrB gene was used for PCR amplification. The primers used were: gyrB -F: 5'-CGGTTCGACAAACAGCAAAG-3' (SEQ ID NO. 3); gyrB -R: 5'-ACGTATCTCGCATTCGCTTC-3' (SEQ ID NO. 4); The PCR reaction system (total 50 μL) was composed of 5×PCR buffer (Mg 2+ Plus) 10 μL, dNTP (2.5 mM) 4 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, DNA 1 μL, Taq DNA polymerase (0.5 U / μL) 1 μL, and the rest was sterilized ddH2O. The PCR amplification program was: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 32 cycles; 72℃ extension for 7 min.
[0032] The PCR amplification product was detected by 1% agarose gel electrophoresis and sent to Shanghai Biotechnology Co., Ltd. for sequencing to obtain the gyrB sequence of HMB28645 strain. The obtained sequence was analyzed for homology in NCBI, and the phylogenetic tree was constructed by the neighbor-joining method using MEGA5 software (see Figure 2 ), and it was found that HMB28645 strain clustered into a branch with Bacillus velezensis, indicating that HMB28645 strain was Bacillus velezensis and was a new strain.
[0033] ③Classification by using rpoB gene sequence.
[0034] The genome of HMB28645 strain was used as a template, and the primer of rpoBThe primers used were: rpoB F: 5'-ACTCAGCTCCGCTAAGAGTG-3' (SEQ ID NO. 5); rpoB R: 5'-ACTACATCGCGTTCAACGTC-3' (SEQ ID NO. 6).
[0035] The PCR reaction system was composed of 5x PCR buffer (Mg 2+ Plus) 10 μL, dNTP (2.5 mM) 4 μL, upstream primer (10 μM) 1 μL, downstream primer (10 μM) 1 μL, DNA 1 μL, Taq DNA polymerase (0.5 U / μL) 1 μL, sterilized ddH2O 32 μL. The PCR amplification program was as follows: 95°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, 32 cycles; 72°C extension for 7 min.
[0036] The PCR amplification product was detected by 1% agarose gel electrophoresis and then sent to Shanghai Shengong Biotechnology Co., Ltd. for sequencing, and the sequence of the HMB28645 strain was obtained. rpoB The obtained sequence was subjected to homology analysis in NCBI, and a phylogenetic tree was constructed by using the neighbor-joining method in MEGA5 software (see Figure 3 ). It was found that the strain clustered with Bacillus velezensis into a branch, indicating that the HMB28645 strain was Bacillus velezensis and was a new strain.
[0037] Example 2 The present example provides a method for preparing a fermentation broth of the HMB28645 strain, comprising the following operations: The frozen HMB28645 strain was activated on LB solid medium, cultured at 30°C for 12 h, and the activated strain was obtained; a sterile inoculation loop was used to pick a single colony and inoculated into 100 mL of LB liquid medium, which was placed in a shaking incubator at 30°C and 180 r / min for 12-16 h to obtain the seed liquid of the strain; the seed liquid was inoculated into 200 mL of fermentation medium at a ratio of 2% by volume, and cultured at 30°C with a shaking speed of 180 rpm for 48 h to obtain the fermentation broth of the HMB28645 strain.
[0038] The preparation method of the fermentation medium used in this example is as follows: 25 g of corn flour, 15 g of soybean flour, 5 g of glucose, 2 g of sodium dihydrogen phosphate, 0.5 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 2 g of calcium carbonate are added to 1000 mL of water, mixed uniformly, adjusted to pH 7.0-7.5, and sterilized at 121°C for 30 min.
[0039] Example 3 In this example, the antagonistic effect of HMB28645 strain on Botrytis cinerea was determined by the flat plate confrontation method (performed in the Plant Disease and Biological Control Laboratory of the Plant Protection Institute of Hebei Academy of Agriculture and Forestry in February 2023).
[0040] After the Botrytis cinerea was cultured on PDA plates for 4-5 days, a sterile punch with a diameter of 6 mm was used to punch a hole at the edge of the colony to make a dish and transfer it to the center of another PDA plate, and then the activated HMB28645 strain was inoculated at a distance of 2 cm on both sides of the dish. The PDA plate was placed in a constant temperature incubator at 25°C, and when the blank control was almost covered with the dish, the colony radius was measured and the inhibition rate was calculated. The Botrytis cinerea without inoculation of HMB28645 strain was used as a blank control. Each treatment was repeated 4 times.
[0041] Inhibition rate (%) = (control colony radius - treatment colony radius) / control colony radius x 100.
[0042] The results showed that the inhibition rate of HMB28645 strain on Botrytis cinerea was 74.3%, and the inhibition zone width was 7.6 mm (Table 1). This indicates that HMB28645 strain has strong antagonistic effect on Botrytis cinerea and has potential for preventing and controlling grape gray mold.
[0043] Table 1 Antagonistic effect of HMB28645 strain on Botrytis cinerea
[0044] Note: The data in the table are mean ± standard deviation. The same below.
[0045] Example 4 In this example, the control effect of HMB28645 strain on grape gray mold was evaluated by the in vitro leaf method (performed in the Plant Disease and Biological Control Laboratory of the Plant Protection Institute of Hebei Academy of Agriculture and Forestry from March to May 2023).
[0046] One-year-old grape plants (variety: Kyoho) were planted in plastic flowerpots (pot opening diameter x pot bottom diameter x height = 34.0 cm x 17.5 cm x 21.5 cm), and normal fertilization and water management were carried out in a sunlight greenhouse. After 6-7 leaves were grown, they were used. Healthy grape leaves were selected, washed with sterile water, soaked in 5-fold diluted HMB28645 strain fermentation broth for 30 min, and then placed in a glass culture dish (15 cm in diameter) covered with sterile wet filter paper. The petioles were wrapped with sterile wet absorbent cotton to keep them moist. One leaf was placed in each culture dish and incubated at 25°C. The control was treated with water, and 6 leaves were treated per treatment. After 24 h, grape Botrytis cinerea discs (5 mm in diameter) were inoculated in the middle of the leaves, sealed with parafilm, and incubated at 25°C. When the control was fully diseased, the lesion diameter was measured using the cross method, and the lesion area and control efficiency were calculated according to the following formula.
[0047] Lesion area (mm 2 ) = lesion length x lesion width x π / 4; Control efficiency (%) = (control lesion area - treatment lesion area) / control lesion area x 100.
[0048] The results are shown in Table 2. The lesion area after treatment with HMB28645 strain fermentation dilution was 74.8 mm 2 , which was significantly lower than that of the blank control (377.2 mm 2 ). The control efficiency of HMB28645 strain fermentation dilution on grape Botrytis cinerea was 80.2%. This indicates that HMB28645 strain and its liquid preparation have good control effect on grape Botrytis cinerea.
[0049] Table 2 Control effect of HMB28645 strain on grape Botrytis cinerea (in vitro leaf)
[0050] Example 5 In this example, the potting method was used to evaluate the control effect of HMB28645 strain on grape Botrytis cinerea (2023 May-June in the Plant Disease and Biological Control Laboratory of Hebei Academy of Agriculture and Forestry Sciences).
[0051] One-year-old grape plants (variety: Kyoho) were planted in plastic flowerpots (pot opening diameter 20 cm, pot bottom diameter 13 cm, pot height 17 cm), and normal fertilization and water management were carried out in a sunlight greenhouse. After 8-10 leaves were grown, the grape seedlings with uniform growth were selected, and the HMB28645 strain fermentation liquid was 5 times diluted and uniformly sprayed on the grape plants. After 24 h of culture in the sunlight greenhouse, the grape gray mold pathogen discs were inoculated on the leaves, and the self-sealing bag was sealed and kept moist for further culture. The blank control was sprayed with water, and the chemical agent control was sprayed with Luna Sensation (43% fluoro fungicide·triflumizole, PD20152429, produced by Bayer AG) at 1000 times dilution. Each treatment was repeated 3 times, and 6 leaves were inoculated in each repeat. After the blank control was fully diseased, the lesion diameter was measured by the cross method, and the lesion area and control effect were calculated.
[0052] The results showed that the lesion area of grape leaves treated with the HMB28645 strain fermentation dilution liquid was 56.7 mm 2 , which was not significantly different from the lesion area of the chemical agent (64.1 mm 2 ), and was significantly lower than the lesion area of the blank control (283.7 mm 2 ). The control effect of the HMB28645 strain fermentation dilution liquid on grape gray mold was 80.0% (Table 3). It was shown that the HMB28645 strain and its liquid preparation had good control effect on grape gray mold.
[0053] Table 3 Control effect of HMB28645 strain on grape gray mold (pot experiment)
[0054] Example 6 This example used a field plot test to evaluate the control effect of the HMB28645 strain on grape gray mold (carried out in a facility grape shed in Ligang Village, Hengshui City, Hebei Province from April to May 2024).
[0055] The grape variety was Tungren, 2 years old, and the field was normally managed. The grape plants with uniform growth and health were selected, and the same position of grape leaves was selected. The sterile inoculation needle was used to make holes in the middle of the grape leaves, and then the HMB28645 strain fermentation 5 times dilution liquid was uniformly sprayed on the grape leaves. After 24 h, the grape gray mold pathogen discs were inoculated at the hole position and sealed with a self-sealing bag. The blank control was sprayed with water, and the chemical agent control was sprayed with Luna Sensation at 1000 times dilution. Each treatment was repeated 3 times, and 6 leaves were inoculated in each repeat. After the blank control was fully diseased, the lesion diameter was measured by the cross method, and the lesion area and control effect were calculated.
[0056] The results showed that the lesion area of grape leaves treated with the HMB28645 strain fermentation dilution liquid was 28.6 mm2 The disease spot area of the HMB28645 strain fermentation liquid treatment was 21.8 mm 2 , which was not significantly different from the chemical agent disease spot area (24.1 mm 2 ), and was significantly lower than the blank control disease spot area (269.5 mm 2 ). The prevention and control effect of the HMB28645 strain fermentation liquid on grape gray mold was 89.4% (Table 4). It shows that the HMB28645 strain and its liquid preparation have good prevention and control effect on grape gray mold.
[0057] Table 4 Prevention and control effect of HMB28645 strain on grape gray mold (field test)
[0058] Example 7 This example uses potting method to evaluate the prevention and control effect of HMB28645 strain on tomato gray mold (carried out in September 2024 in the plant disease and biological control laboratory of Hebei Academy of Agriculture and Forestry Sciences).
[0059] Tomato seedlings with good growth, about 10 cm in height, and 3-4 true leaves (variety is Rui Li Hong Sheng, produced by Qingxian Qingfeng Seed Industry Co., Ltd.) were selected from the seedling tray and transplanted into flowerpots containing seedling substrate (pot opening diameter 11.8 cm, pot bottom diameter 8.5 cm, pot height 10 cm), and placed in a 25°C artificial climate chamber for culture. When the seedlings grew to 7-8 true leaves, the test was started. First, the HMB28645 strain fermentation liquid 5 times dilution was uniformly sprayed on the tomato plants, and after 24 h of culture, the tomato gray mold pathogen disc was inoculated on the leaves, and the humidity was maintained in the artificial climate chamber (humidity > 90%) for further culture. Spraying water was used as blank control, and spraying Luna 1000 times dilution was used as chemical agent control. Each treatment was repeated 3 times, each repetition inoculated 3 compound leaves, and each compound leaf inoculated 3 small leaves at the top. After the blank control was fully diseased, the disease spot diameter was measured by cross method, and the disease spot area and prevention effect were calculated.
[0060] The results show that the disease spot area of tomato leaves treated by HMB28645 strain fermentation dilution liquid was 21.8 mm 2 , which was not significantly different from the chemical agent disease spot area (24.5 mm 2 ), and was significantly lower than the blank control disease spot area (134.8 mm 2 ). The prevention and control effect of the HMB28645 strain fermentation liquid on tomato gray mold was 83.8% (Table 5). It shows that the HMB28645 strain and its liquid preparation have good prevention and control effect on tomato gray mold.
[0061] Table 5 Prevention and control effect of HMB28645 strain on tomato gray mold (potting test)
[0062] Example 8 This example uses field plot test to evaluate the control effect of HMB28645 strain on tomato gray mold (December 2024-March 2025 in Longhua Village, Dingxing County, Hebei Province).
[0063] The tomato variety is Provence, with uniform growth. The HMB28645 fermentation liquid 5 times water dilution liquid is uniformly sprayed on the surface of the tomato plant leaves using a backpack electric sprayer, and the spraying of clean water is used as a blank control, and the spraying of Luna 1000 times dilution liquid is used as a chemical pesticide control. Each treatment is repeated 3 times, with 2 rows per repeat (1 row of double rows), and is randomly arranged in groups. The first application is at the flowering stage of the third inflorescence, and there is no gray mold. Then spray every 7 days, a total of 4 times. After the blank control is fully diseased, the number of diseased leaves is investigated, and the control effect is calculated.
[0064] The results show that after the HMB28645 strain fermentation dilution liquid treatment, the tomato gray mold leaf rate is 0.5%, which is not significantly different from the chemical pesticide disease leaf rate (0.4%), and is significantly lower than the blank control disease leaf rate (3.4%). The control effect of the fermentation dilution liquid of the strain on tomato gray mold is 86.1% (Table 6). It shows that the HMB28645 strain and its liquid preparation have good control effect on tomato gray mold.
[0065] Table 6 Control effect of HMB28645 strain on tomato gray mold (field test)
[0066] Example 9 This example uses potting method to evaluate the control effect of HMB28645 strain on cucumber gray mold (October 2024 in the Plant Disease and Biological Control Laboratory of Hebei Academy of Agriculture and Forestry Institute).
[0067] Select cucumber (variety is Jinyou No. 1, produced by Tianjin Kelun Agricultural Science and Technology Co., Ltd.) seedlings with good growth, uniform size, and 2-3 true leaves from the seedling tray, transplant them into plastic pots filled with seedling substrate (pot opening diameter 11.8 cm, pot bottom diameter 8.5 cm, pot height 10 cm), and cultivate them in a 25°C artificial climate chamber. When 4-5 true leaves grow, start the test. First, uniformly spray the HMB28645 strain fermentation liquid 5 times dilution liquid on the cucumber plants, and after 24 hours of cultivation, inoculate the cucumber gray mold pathogen disc on the leaves. Continue to cultivate in the artificial climate chamber (humidity > 90%). Spray clean water as a blank control, and spray Luna 1000 times dilution liquid as a chemical pesticide control. Each treatment is repeated 5 times, with 1 cucumber seedling per repeat. After the blank control is fully diseased, measure the lesion diameter using the cross method, calculate the lesion area, and calculate the control effect.
[0068] The results showed that the disease spot area of cucumber leaves treated with HMB28645 strain fermentation diluent was 51.4 mm 2 , which had no significant difference with that of chemical agent (49.0 mm 2 ), and was significantly lower than that of the blank control (424.3 mm 2 ). The prevention and control effect of the fermentation diluent of the strain on cucumber gray mold was 87.9% (Table 7). It was shown that the HMB28645 strain and its liquid preparation had good prevention and control effect on cucumber gray mold.
[0069] Table 7 Prevention and control effect of HMB28645 strain on cucumber gray mold (pot experiment)
[0070] Example 10 In this example, the inhibition effect of HMB28645 strain on other grape important disease pathogens was determined by plate confrontation method (in November 2024 at the Plant Disease and Biological Control Laboratory of Hebei Academy of Agriculture and Forestry Sciences). The grape disease pathogens included grape white rot pathogen, grape anthracnose pathogen, grape alternaria leaf spot pathogen, grape bacterial wilt pathogen and grape white stripe wing root rot pathogen.
[0071] After the above 5 kinds of pathogens were cultured on PDA plates for 4-5 days, sterile punchers with a diameter of 6 mm were used to punch the colony edge to make a fungus tray and transfer it to the center of another PDA plate, and then HMB28645 strain was inoculated at 2 cm on both sides of the fungus tray. The PDA plates were placed in a constant temperature incubator at 25°C, and when the blank control was about to be full, the colony radius was measured and the inhibition rate was calculated. The pathogen without inoculation of HMB28645 strain was used as blank control. Each treatment was repeated 4 times.
[0072] The results showed that HMB28645 strain had different degrees of inhibition on the 5 kinds of grape disease pathogens, with inhibition rate of 63.2%-71.9%. The highest inhibition rate was 71.9% on grape anthracnose pathogen, indicating that HMB28645 strain had a wide inhibition spectrum.
[0073] Table 8 Antagonistic effect of HMB28645 strain on 5 kinds of grape disease pathogens
[0074] Example 11 In this example, the prevention and control effect of HMB28645 strain on grape alternaria leaf spot was determined by in vitro leaf method (from April to May 2025 at the Plant Disease and Biological Control Laboratory of Hebei Academy of Agriculture and Forestry Sciences).
[0075] One-year-old grape plants (variety: Kyoho) were planted in plastic flowerpots (pot opening diameter x pot bottom diameter x height = 34.0 cm x 17.5 cm x 21.5 cm), and normal fertilization and water management were performed in a sunlight greenhouse. After 6-7 leaves were grown, healthy grape leaves were selected, washed with sterile water, soaked in 5-fold diluted HMB28645 strain fermentation liquid for 30 min, and then placed in a glass culture dish (diameter 15 cm) covered with sterile wet filter paper. The petioles were wrapped with sterile wet absorbent cotton to keep them moist. One leaf was placed in each culture dish, and the culture dishes were placed in a 25°C incubator. The control was treated with clean water, and 6 leaves were treated per treatment. After 24 h, grape Alternaria alternata spore suspension (1 x 10 5 After the control was fully diseased, the disease condition of the leaves was observed and recorded, and the leaf disease rate was calculated.
[0076] Leaf disease rate = number of diseased leaves / total number of inoculated leaves x 100%.
[0077] The results showed that the leaf spot disease rate of grape Alternaria alternata was 20.8% after treatment with the HMB28645 strain, which was significantly lower than that of the blank control, and the control effect reached 77.3% (Table 9). This indicated that the HMB28645 strain could effectively control grape Alternaria alternata leaf spot disease.
[0078] Table 9 Control effect of HMB28645 strain on grape Alternaria alternata leaf spot disease
[0079] Example 12 In this example, the potting method was used to determine the growth-promoting effect of the HMB28645 strain on grape (April-May 2025 in the Plant Disease and Biological Control Laboratory of the Hebei Academy of Agricultural and Forestry Sciences).
[0080] One-year-old grape plants (variety: Kyoho) were planted in plastic flowerpots (pot opening diameter x pot bottom diameter x height = 34.0 cm x 17.5 cm x 21.5 cm), and normal fertilization and water management were performed in a sunlight greenhouse. After 6-7 leaves were grown, healthy grape leaves were selected, washed with sterile water, soaked in 5-fold diluted HMB28645 strain fermentation liquid for 30 min, and then placed in a glass culture dish (diameter 15 cm) covered with sterile wet filter paper. The petioles were wrapped with sterile wet absorbent cotton to keep them moist. One leaf was placed in each culture dish, and the culture dishes were placed in a 25°C incubator. The control was treated with clean water, and 6 leaves were treated per treatment. After 24 h, grape Alternaria alternata spore suspension (1 x 10
[0081] Wherein, the relative growth amplitude of new shoots (%) = (the final new shoot length - the initial new shoot length) / the initial new shoot length * 100. The leaf greenness is determined by using a SPAD portable chlorophyll meter (SY-S02, Shijiazhuang Shiyate Science and Technology Co., Ltd.), 3 leaves at the same position are determined in each repetition, 3 points are taken from each leaf, and a total of 5 repetitions are performed.
[0082] The defense enzyme activity determination method is as follows: the grape leaves at the last investigation are collected and collected, and the activities of superoxide dismutase (Superoxide Dismutase, SOD), phenylalanine ammonia-lyase (Phenylalanine Ammonia-Lyase, PAL) and polyphenol oxidase (Polyphenol Oxidase, PPO) are determined. The SOD activity is determined by NBT reduction method, the POD is determined by guaiacol colorimetry, the PAL activity is determined by phenylalanine colorimetry, and the PPO is determined by catechol method.
[0083] The results are shown in Tables 10-11, and the relative growth amplitude of new shoots, leaf greenness and leaf area of grape plants treated by HMB38645 strain are significantly higher than those of the blank control, which are increased by 36.1%, 24.2% and 25.9% respectively; at the same time, after being treated by HMB28645 strain, the defense enzyme activity in grape leaves is significantly higher than that of the control, which is increased by 38.9%, 26.0% and 18.9% respectively. It shows that HMB28645 strain can promote the growth of grape and improve the disease resistance of grape.
[0084] Table 10 Effect of HMB28645 strain on the agronomic characters of grape plants
[0085] Table 11 Effect of HMB28645 strain on the defense enzymes of grape plants
[0086] In summary, the HMB28645 strain isolated from the soil around the cotton roots is a new strain of Bacillus velezensis, and the pot experiment and field experiment show that it has excellent control effect on grape, tomato and cucumber gray mold, and also has good inhibition effect on other important pathogenic bacteria on grape. At the same time, the HMB28645 strain can also promote the growth of grape, and can enhance the disease resistance of grape plants by increasing the defense enzyme activity. Therefore, the HMB28645 strain has important significance in the prevention and control of fruit and vegetable gray mold and the promotion of grape disease resistance.
[0087] Note that the present application is not limited to the above-described embodiments. The above-described embodiments are merely examples, and embodiments having substantially the same configuration as the technical idea and achieving the same effects within the scope of the technical idea of the present application are included in the technical scope of the present application. Furthermore, other modes constructed by applying various modifications that can be thought of by those skilled in the art to the embodiments or by combining part of the constituent elements of the embodiments are also included in the scope of the present application without departing from the spirit of the present application.
Claims
1. A strain of Bacillus belye ( Bacillus velezensis HMB28645 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36721.
2. A microbial inoculant, characterized in that, It includes Bacillus belyssus HMB28645 as described in claim 1.
3. A fermentation product, characterized in that, It was obtained by culturing Bacillus belye HMB28645 as described in claim 1.
4. The fermentation product according to claim 3, characterized in that, The seed culture was prepared by activating Bacillus belye HMB28645 and then inoculating it into a fermentation medium containing corn flour, soybean flour, glucose, sodium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, and calcium carbonate at pH 7.0-7.
5.
5. The application of Bacillus berberis as described in claim 1, or the microbial agent as described in claim 2, or the fermentation product as described in claim 3 in inhibiting the growth of pathogens, wherein the pathogens are selected from the following pathogens: Grape gray mold pathogen, Tomato gray mold pathogen, Cucumber gray mold pathogen, Grape white rot pathogen, Grape anthracnose pathogen, Grape Alternaria leaf spot pathogen, Grape canker pathogen, and Grape white root rot pathogen.
6. The application of Bacillus berberis as described in claim 1, or the microbial agent as described in claim 2, or the fermentation product as described in claim 3 in the prevention and control of gray mold in fruits and vegetables.
7. The application according to claim 4, characterized in that, The fruits and vegetables mentioned include grapes, tomatoes, and cucumbers.
8. The application of Bacillus berreatus as described in claim 1, or the microbial agent as described in claim 2, or the fermentation product as described in claim 3, in the prevention and control of grape diseases, wherein the grape diseases include grape gray mold, grape white rot, grape anthracnose, grape Alternaria leaf spot, grape canker, and grape white root rot.
9. The application of Bacillus berberis as described in claim 1, or the microbial agent as described in claim 2, or the fermentation product as described in claim 3, in promoting grape growth.
10. The application of Bacillus berberis as described in claim 1, or the microbial agent as described in claim 2, or the fermentation product as described in claim 3 in improving the disease resistance of grapes.