Bacillus velezensis P15 and application thereof

Abstract

The invention discloses bacillus velezensis P15 and application thereof. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC NO.31995. The invention further discloses a preparation method of the bacillus velezensis P15. The bacterial strain has broad-spectrum and efficient antagonistic action on 21 different phytopathogens belonging to 12 genus, can effectively prevent and treat anthracnose of tea trees, powdery mildew of various vegetables and postharvest diseases of mangoes and peaches in vivo, has the prevention and treatment effect higher than 85% or above, has the prevention and treatment effect equivalent to or even more excellent than that of chemical pesticides, and has a broad application prospect in construction of a biocontrol bacterium resource library. The development of safe and efficient biopesticide has important significance.

Description

Technical Field

[0001] The invention relates to the technical field of biological control, and in particular to Bacillus Velez P15 and application thereof. Background Art

[0002] Modern intensive monoculture, long-term and large-scale application of chemical fertilizers and pesticides, and global warming have led to the occurrence and aggravation of crop diseases. Fungal diseases are the main type of diseases in the field and in post-harvest storage of crops. Chemical pesticides are the main means of preventing and controlling fungal diseases in the field and post-harvest of crops at home and abroad. However, long-term and large-scale application of pesticides will cause a series of production, environmental and health problems, such as increased resistance of pathogens, reduced efficacy, increased pesticide residues in agricultural products, and harm to human health and environmental ecological safety.

[0003] The latest prediction results based on the global distribution map of plant pathogenic fungi show that by the end of this century, the diversity and invasion potential of plant pathogenic fungi will increase significantly in global forest and cultivated land ecosystems, with an increase of 34.93-82.51%. Among them, pathogenic fungi such as Botrytis sp., Fusarium sp. and Colletotrichum sp. have a wide geographical distribution and host range (involving hundreds of crops such as strawberries, mangoes, coffee beans, pears, tea trees, etc.), and are listed as the top ten plant pathogens that cause crop yield reduction and economic losses worldwide. Due to the long-term application of chemical pesticides, most pathogens currently have increased drug resistance. For example, the results of existing research on the drug resistance of anthracnose bacteria show that more than 200 strains of Colletotrichum gloeosporioides show a resistance frequency of 37 to 99.1% to the commonly used anthracnose pesticides, carbendazim and methyl thiophanate.

[0004] Biological control is a plant protection method that uses interactions between different species to mitigate the harm of pests. It is environmentally friendly and avoids pathogen resistance. It is a sustainable alternative to chemical pesticides. Therefore, it is necessary to develop safe, non-toxic and highly effective biological pesticides. Compared with developed countries such as Europe and the United States, the types and number of biocontrol bacteria available in my country are far lower than those in Europe and the United States; and although my country's microbial pesticides have continued to increase in recent years, there are few biological control products that can be used in the field of tea trees and post-harvest fruits. Therefore, in the biological control of crop diseases, there are still problems such as a small number of biocontrol strains, poor control effects, and poor environmental adaptability. It is urgent to increase the number of species, improve control effects, and enhance environmental adaptability.

[0005] Bacillus velezensis is a new species in the genus Bacillus. It has strong potential for plant growth promotion and biological disease control. It is a new type of Bacillus biocontrol bacteria that has attracted much attention in recent years. It is worth noting that the antibacterial spectrum and antibacterial effect of biocontrol bacteria are not predictable. Different strains of the same bacteria have very different antibacterial spectra and types and effects of controlling plant diseases. At present, there are about 500 patents related to pest control related to Bacillus velezensis, but the existing patented biocontrol bacteria only report the inhibition of a few pathogens and a single tea tree disease or the preservation effect of a certain fruit. There is no report of a new species of Bacillus velezensis with a broad-spectrum and highly efficient antagonistic effect. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a Bacillus Velez P15 and its application to solve the problems of narrow antibacterial spectrum, poor antagonistic effect and poor environmental adaptability of Bacillus Velez in the prior art.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] The invention discloses a Bacillus velezensis P15, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, and the deposit number is CGMCC NO.31995.

[0009] The present invention provides the use of the above-mentioned Bacillus Velez P15 in preventing and controlling plant-derived fungi, wherein the plant-derived fungi include one or more of the following strains:

[0010] Tea tree anthracnose pathogens: Colletotrichum fructicola, Colletotrichum camelliae, Colletotrichum gloeosporioides, Colletotrichum jiangxiense, Colletotrichum karsti, Colletotrichum chrysophilum, Colletotrichum perseae;

[0011] Tea tree leaf blight pathogens: Pestalotiopsis telopeae and Diaporthe sp;

[0012] Tea tree leaf spot pathogens: Alternaria sp and Fusarium sp;

[0013] Peach rot pathogens: Neofusicoccum hyperici and Diaporthe nobilis;

[0014] Peach black spot pathogen: Alternaria tenuissima;

[0015] Mango stem rot pathogens: Lasiodiplodia hormozganensis and Lasiodiplodia gilanensis;

[0016] Mango Anthracnose Pathogen: Aspergillus sp;

[0017] Melon vine blight pathogen: Didymella bryoniae;

[0018] Tomato gray mold: Botrytis cinerea;

[0019] Rhizoctonia solani: Rhizoctonia solani;

[0020] Sclerotinia sclerotiorum.

[0021] The invention provides application of the Bacillus Velezii P15 in preventing and controlling tea tree anthracnose, cucumber powdery mildew and green bean powdery mildew.

[0022] The invention provides application of the Bacillus Velez P15 in fruit preservation.

[0023] The invention also provides a biocontrol agent, the active ingredient of which is the above-mentioned Bacillus Velezii P15.

[0024] The invention also provides a biocontrol agent, the active ingredient of which is the fermentation liquid of the above-mentioned Bacillus Velezii P15.

[0025] Preferably, the fermentation broth is prepared by the following steps:

[0026] Bacillus Velez P15 was inoculated into LB liquid culture medium, cultured at 25°C for 24 hours, and then centrifuged to obtain a sterile fermentation broth.

[0027] The invention provides application of the biocontrol agent in preventing and controlling anthracnose and powdery mildew of tea trees.

[0028] The invention provides application of the biocontrol agent in fruit preservation.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The new species of Bacillus Velezii of the present invention has excellent performance and can have a broad-spectrum and efficient antagonistic effect on 21 different plant pathogens of 12 genera. It can effectively prevent and control tea anthracnose, powdery mildew of various vegetables, and post-harvest diseases of mango and peach in plants, with a prevention and control effect of more than 85%. Compared with chemical pesticides, the prevention and control effect is equivalent to or even better, which is of great significance to the construction of a biocontrol bacteria resource library and the development of safe and efficient biological pesticides.

[0031] 2. The present invention compares the antibacterial spectrum and antibacterial ability of the Velez subtilis with other strains disclosed in the prior art. Other strains in the prior art not only have a narrow antibacterial spectrum, but also have poor antibacterial effects on the plant pathogens targeted by the present invention. For example, the strain Velez subtilis LSR7 disclosed in the prior art has a good antibacterial effect on potato late blight and oak anthracnose, but has a poor antibacterial effect on Fusarium sp, and the antibacterial zone of confrontation culture is not obvious; Velez subtilis HY19 has a poor antibacterial effect on tomato gray mold, while Velez subtilis YJDG has a poor antibacterial effect on pathogenic strains other than Fusarium (for example, Rhizoctonia solani, Phytophthora, Fusarium sp), which are not as good as the Velez subtilis described in the present invention; this proves from the side that different types of Velez subtilis have different antibacterial spectra and completely different antibacterial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The colony, bacterial morphology and Gram staining of the Bacillus Velez P15 of the present invention; A is the colony of Bacillus Velez P15, and B is the bacterial morphology and Gram staining.

[0033] Figure 2 This is a multi-gene concatenation phylogenetic tree constructed based on whole genome sequencing of the Bacillus Velez P15 described in the present invention.

[0034] Figure 3 This is the antibacterial spectrum of the Bacillus Velezii P15 described in the present invention.

[0035] Figure 4 The antibacterial effect of the Bacillus Velez P15 described in the present invention is compared with other common Bacillus biocontrol bacteria; wherein A is a comparison of the antibacterial effect on seven types of tea anthracnose pathogens on a plate; B is a comparison of the antibacterial effect on three other types of pathogens on a plate; and C is the antibacterial rate against seven types of tea anthracnose pathogens.

[0036] Figure 5 This is a diagram showing the antagonistic effect of the sterile fermentation liquid of Bacillus Velez P15 of the present invention on tea anthracnose, peach rot pathogen, Rhizoctonia solani and rapeseed sclerotinia.

[0037] Figure 6 This is a diagram showing the antagonistic effect of the volatile gas of the Bacillus Velez P15 described in the present invention on mango stem rot pathogen, Rhizoctonia solani, tea leaf spot pathogen and tea anthracnose pathogen.

[0038] Figure 7 This is a diagram showing the biocontrol effect of the Bacillus Velez P15 described in the present invention on tea tree anthracnose.

[0039] Figure 8 This is a diagram showing the biological control effect of the Bacillus Velez P15 described in the present invention on powdery mildew. DETAILED DESCRIPTION

[0040] The present invention will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the present invention belong to the scope of protection of the present invention.

[0041] Unless otherwise indicated in specific cases in the present invention, the numerical ranges listed herein include the upper and lower limits, and all integers and fractions within the range, and are not limited to the specific values ​​listed when defining the range.

[0042] 1. Bacillus velezensis P15

[0043] 1. Culture medium and its preparation

[0044] PDA solid culture medium (liquid culture medium without agar): 200g potato, 20g glucose, 20g agar, 1L deionized water, pH 6.50. Sterilize at 121℃, 1.5 atmospheres, and cool for 30min before pouring onto a plate (solid culture medium) or taking 20mL into a 150mL Erlenmeyer flask (liquid culture medium) for later use.

[0045] LB solid medium (liquid medium without agar): 10 g tryptone, 10 g NaCl, 10 g yeast powder, 1 L deionized water, pH 7.0.

[0046] 2. Isolation and screening of the Bacillus Velez P15 strain

[0047] Isolation method: strain P15 was isolated from the rhizosphere soil of plants that had been fertilized with the beneficial fungus Ceroplastes lacerae HG2011 for many years in Chongqing. The microorganisms in the soil were isolated using the dilution gradient method. 10.0 g of soil was weighed and placed in a 250 mL Erlenmeyer flask filled with 90 mL of sterile water. The soil suspension was prepared by shaking in a shaker at 25°C and 150 rpm for 1 hour. Then, the soil suspension was diluted to 10-2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 Different concentration gradients. Pipette 0.1mL 10 -4 , ~10 -6 The soil suspension with different dilution multiples was evenly spread on the LB plate, and each dilution gradient was repeated 5 times. After the operation was completed, the plate was incubated at 25°C for 2 days, and different bacteria were selected according to the morphology, color and size of the bacterial colonies, and purified 4 times.

[0048] Screening method: Inoculate the activated tea tree anthracnose pathogen Colletotrichum fructicola HX4-5 into 200 mL PDB, shake and culture at 150 rpm for 3-5 days, centrifuge at 1000 rpm to remove the bacteria, and collect the spore liquid. Prepare PDA medium, sterilize and cool to 40-50°C, add 10 mL of pathogen spore suspension (spore concentration 10 3 spore·mL -1 ) and poured into a 24-well microplate. After cooling and solidification, the purified bacteria were inoculated into the PDA containing the fungal spore suspension. After culturing at 25°C for 3 days, the presence of a fungus-free transparent zone around the bacterial cells was observed. The size of the transparent zone represents the antibacterial potential of the biocontrol bacteria.

[0049] 3. Morphological and molecular identification (16S rRNA gene and whole genome sequencing) of the Bacillus Velez P15:

[0050] (1) Morphological identification: The strain P15 was inoculated on LB solid medium and cultured at 25°C for 1-3 days. The colony morphology was observed and photographed. The smear was fixed, dried, and then stained with ammonium oxalate crystal violet for 1 minute, rinsed with pure water; then iodine solution was added to cover the surface and stained for about 1 minute, washed with water, and then the water was absorbed with absorbent paper; then a few drops of 95% alcohol were added, decolorized for 20 seconds, washed with water, and the water was absorbed; finally, it was stained with safranin staining solution for 2 minutes, rinsed, dried, and observed under a microscope. Figure 1 The P15 strain obtained by the present invention has smooth and white colonies at the early stage, and gradually wrinkles appear in the later stage of growth ( Figure 1 A). The morphology under an optical microscope is rod-shaped, usually arranged singly or in pairs, and a few are arranged in short chains; the length of the bacteria is 1.5-5.2μm, and the diameter is 0.4-0.6μm ( Figure 1 B); Gram staining result was positive ( Figure 1 B, positive purple, negative red).

[0051] (2) Genome sequencing: The strain P15 was inoculated into LB liquid medium, cultured at 25°C for 24h, and centrifuged at 10000rpm to collect the bacteria. The genomic DNA of strain P15 was extracted using a bacterial genomic DNA extraction kit, and the genomic DNA quality was tested by TBS380 or Nanodrop2500 (no degradation, OD260 / 280 = 1.8-2.0, and the total amount was not less than 10μg). It was purified, library-built, and sequenced (PacBio SMRT single-molecule sequencing and Illumina HighSeq PE300 sequencing) by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. Based on the construction of the 16Sr DNA gene phylogenetic tree of strain P15, it was preliminarily determined to be a Bacillus sp. bacterium. The strains were further compared with more than 30 house-keeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf) of the genomes of other known Bacillus and their source species reference strains on NCBI, and multi-gene concatenated phylogenetic analysis was performed (maximum likelihood method, bootstrap1000). Figures 2-3 As shown, the genome size of the strain P15 of the present invention is 3.86Mb (GC content 46.7%), and the strain P15 and the model strain Bacillus velezensis NRRL-B-41580 (NCBI RefSeq assembly number: GCF_001461825.1) are located in the same branch of the phylogenetic tree (support 100), indicating that the two have high homology and are closely related in taxonomic status. Finally, combined with the morphological and molecular identification results, the above strain was identified as Bacillus velezensis P15.

[0052] The strain B. velezensis P15 provided by the present invention was deposited in the General Microbiology Center of the China Microorganism Culture Collection Committee of the Institute of Microbiology, Chinese Academy of Sciences (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on September 18, 2024, with the deposit number CGMCC No. 31995 and the classification name Bacillus velezensis.

[0053] 2. Embodiment

[0054] Example 1: Determination of the antibacterial spectrum of B. velezensis P15

[0055] The antibacterial spectrum of biocontrol bacteria was evaluated by confrontation culture method. Specifically, 7 different species of tea anthracnose fungi (including Colletotrichum fructicola, Colletotrichum camelliae, Colletotrichum gloeosporioides, Colletotrichum jiangxiense, Colletotrichum karsti, Colletotrichum chrysophilum, Colletotrichum perseae), 2 tea leaf blight pathogens (Pestalotiopsis telopeae and Diaporthe sp), 2 tea leaf spot pathogens (Alternaria sp and Fusarium sp), Didymella bryoniae, Botrytis cinerea, Rhizoctonia solani, Sclerotiniasclerotiorum, 3 common mango postharvest pathogens (Lasiodiplodia hormozganensis, Lasiodiplodia gilanensis and anthracnose bacteria Aspergillus sp), two peach rot pathogens (Neofusicoccum hyperici, Diaporthe nobilis) and peach black spot pathogen (Alternaria tenuissima) were inoculated on PDA solid culture medium and cultured at 25°C for 5 days. After activation, a 5mm diameter bacterial block was taken along the edge of the colony and inoculated in the middle of the PDA culture medium. Then the activated bacteria were inoculated on both sides of the fungal block (3cm away from the block) for confrontation culture. The control in the middle of the PDA culture medium was not inoculated with biocontrol bacteria P15, and cultured in the dark at 25°C for 3-6 days. The appearance of an obvious antibacterial band in the radial bacterial direction of the fungus indicated that it had antibacterial potential. After the culture was completed, the colony diameter was measured and the antibacterial rate was calculated = 100×(D CK -D 生防菌 ) / D CK . Where D CK (Control) is the diameter of the fungal colony without inoculation of biocontrol bacteria, D 生防菌 It is the diameter of the fungal colony in the radial direction of the biocontrol bacteria in the biocontrol bacteria inoculation treatment. Figure 3As shown, when B. velezensis P15 was co-cultured with 21 different plant pathogenic fungi from 12 genera, obvious inhibition zones were present in the radial bacterial direction, with an inhibition rate of 63% to 77%, indicating that P15 has a broad-spectrum, stable and high resistance to pathogenic fungi.

[0056] Example 2: Comparison of antibacterial effects of B. velezensis P15 and other common Bacillus

[0057] The confrontation culture method was used to compare the antibacterial ability of B. velezensis P15 and other common Bacillus biocontrol bacteria. The operating steps of the test method are the same as those in "Example 1", and the tested Bacillus biocontrol strains are Bacillus velezensis B2, Bacillus mojavensis 21, Bacillus amyloliquefaciens 11, Bacillus subtilis 09 and Bacillus tequilensis 13. The control (CK) was not inoculated with biocontrol bacteria, and each treatment was repeated 3 times. After 6-8 days of culture, the colony diameter was measured and the antibacterial rate was calculated. The calculation method was the same as in "Example 1". Figure 4 As shown in the figure, the antibacterial rate of B. velezensis P15 ranged from 64.6% to 76.7% for the 9 pathogens tested, which was significantly higher than that of other Bacillus velezensis strains B2 and other common Bacillus biocontrol bacteria tested at the same time ( Figure 4 AC), which is also higher than the Bacillus velezensis F85 (the antibacterial rate against C. fructicola and C. karsti is 42.5-57.1%) and B. velezensis SF327 (the antibacterial rate against C. gloeosporioides and C. acutatum is 50.7-67.4%) disclosed in the prior art.

[0058] Example 3: Application of secondary metabolites of Bacillus velezensis P15

[0059] Soluble secondary metabolites: B. velezensis P15 was activated in LB liquid medium and inoculated into new LB liquid medium (inoculation volume 10 4cfu / mL culture fluid), 150rpm, 25℃ constant temperature culture for 24h, centrifuge at 10000rpm to remove most of the bacteria, and further filter the filtrate with a 0.22μm filter membrane to obtain a sterile fermentation fluid (mainly containing soluble secondary metabolites). The fermentation fluid was added to the PDA solid culture medium sterilized and cooled to 50℃ to form four concentrations of 0% (no fermentation fluid, control), 5%, 10% and 15%, respectively, and poured into a 9cm diameter culture dish to make a poisonous plate.

[0060] After activating each pathogen according to Example 1, take a bacterial block with a diameter of 5 mm along the edge of the colony and inoculate it in the center of the above-mentioned virus-carrying plate. Invert and culture at 25°C for 5 to 7 days, and calculate the antibacterial rate. Antibacterial rate = 100 × (D CK -D 生防菌 ) / D CK . Where D CK (Control) is the diameter of the fungal colony without inoculation of biocontrol bacteria, D 生防菌 is the diameter of the fungal colony in the biocontrol bacteria treatment. Figure 5 As shown in the figure, compared with the blank control (CK), on the plate with a fermentation broth concentration of 5%, the colony diameters of the above-mentioned pathogens have begun to decrease significantly, and the specific inhibition rate ranges from 57% to 82% (Table 1); on the plate with a fermentation broth concentration of 15%, the above-mentioned pathogens almost do not grow at all ( Figure 5 ). The antibacterial rates of the aseptic fermentation broths of the patented strains of Bacillus velezensis disclosed in the prior art are generally not high, specifically: 20.84% ​​to 56.41% (5% B. velezensis HY19 fermentation broth), 54.44% (5% B. velezensis SB023 fermentation broth against mango gloeosporium anthracnose) and 56.39% to 58.36% (10% B. velezensis ZJ fermentation broth), indicating that the soluble secondary metabolites of B. velezensis P15 have a strong inhibitory effect.

[0061] Table 1

[0062]

[0063] Volatile gas: The antibacterial activity of volatile gases of biocontrol bacteria was evaluated by the plate-stitching method. After activating each pathogen according to Example 1, take a bacterial block with a diameter of 6 mm along the edge of the colony and inoculate it in the center of the culture dish containing PDA. At the same time, inoculate the activated B. velezensis P15 on the surface of another PDA plate. Stitch the two plates together and seal them with sealing film. The control is a PDA culture medium without biocontrol bacteria inoculation, and the plate is cultured in the dark at 25°C for 5 to 7 days. The method for calculating the antibacterial rate is the same as that for the fermentation broth treatment. Figure 6As shown in Table 2, the volatile gas of B. velezensis P15 exhibited different degrees of antibacterial effects on several tested pathogens, with the antibacterial rate ranging from 18% to 56%.

[0064] Table 2

[0065] Pathogens Antibacterial rate / % Rhizoctonia solani 24.90±5.32 Mango stem rot 56.38±7.72 Tea tree anthracnose 18.11±7.67 Tea leaf spot pathogen 17.90±20.91

[0066] Example 4: Application of Bacillus velezensis P15 in preventing and controlling fungal diseases of crops

[0067] Example of tea tree anthracnose control: The in vitro leaf method was used to evaluate the effect of B. velezensis P15 in controlling tea tree anthracnose disease. The experiment set up two treatments, including single anthracnose pathogen inoculation (CK) and co-inoculation of the pathogen and B. velezensis P15. Take tea leaves with consistent growth, disinfect the surface and inoculate the pathogen (4 places / leaf) using the conventional wound inoculation method, and each treatment has 8 biological replicates. In the biocontrol bacteria treatment, 2 to 4 hours after the pathogen inoculation, the bacterial suspension of strain P15 is sprayed on the leaves. The preparation method of the bacterial suspension of P15 is to collect the bacterial liquid before filtration in Example 3, centrifuge at 12000rpm, remove the supernatant, and collect the bacteria. The bacteria are resuspended in a phosphate buffer (pH7.0) containing 0.5% Tween 80, and the final bacterial concentration is 10 7 cfu / mL. All treatments were sealed in large dishes (13×13 cm) and cultured at 25°C. During the culture period, the development of lesions was observed regularly. When the area of ​​a single lesion treated with PI was 1 to 2 cm 2 The leaves of each treatment were photographed, and the lesion area on the leaves was measured using ImageJ to calculate the control effect. Control effect = (lesion area of ​​control treatment - lesion area of ​​treatment group) / lesion area of ​​control treatment. Figure 7 As shown, when treated with the biocontrol bacteria B. velezensis P15, the incidence and morbidity of tea anthracnose were significantly reduced, and the control effect reached 92.93%.

[0068] Example of powdery mildew control: In July 2024, a farm in Guizhou Province used B. velezensis P15 fermentation liquid (containing bacteria, bacterial concentration> 10 7 cfu·mL -1 ), diluted 4 times with water and added 1% Tween 80, sprayed on vegetable crops such as zucchini and green beans with obvious powdery mildew symptoms (30 plants of each crop), and the powdery mildew spots on the leaves disappeared after 1 day ( Figure 8 ).

[0069] Example 4: Application of Bacillus velezensis P15 fermentation liquid in fruit preservation

[0070] The experiment set up three treatments: control, common fruit preservative prochloraz, and strain P15 fermentation broth. 7 mL -1 ), diluted 4 times with water and added 1% Tween 80, soaked the fruits in the diluted P15 fermentation liquid for 8-10 minutes, fished out and dried and placed in commercial fruit boxes. The control was clean water, and prochloraz was purchased from a local pesticide company (active ingredient content 450g / L). The dosage and usage were referred to the instructions for use of the pesticide. Other operations were the same as the P15 fermentation liquid treatment. Each treatment contained 8-16 fruits, and each treatment was repeated 3 times. According to the rottenness of the control treated fruits (the morbidity rate exceeded 50%), they were stored for 5-20 days. The number of rotten fruits and the disease level of each treatment were counted, and the rotten fruit rate, disease index and control effect were calculated according to the following formula:

[0071] Rotten fruit rate (%) = 100 × rotten fruit number / total number of tested fruits

[0072] Disease index = [Σ(disease level × number of fruits at that level) / (highest disease level × total number of fruits)] × 100

[0073] Control effect (%) = 100 × (control disease index - treatment disease index) / control disease index

[0074] The disease level standard for peaches (determined by the area of ​​disease and rot): Level 0: no disease and rot; Level 1: the diseased area is less than 1 / 4 of the fruit surface; Level 2: the diseased fruit surface is between 1 / 4 and 1 / 2; Level 3: the diseased fruit surface is between 1 / 2 and 3 / 4; Level 4: the diseased fruit surface is between 3 / 4 and completely rotten.

[0075] The disease grade standards for mango stem rot are as follows: Grade 0: no disease or rot; Grade 1: diseased area ≤ 20% of the fruit surface; Grade 2: diseased area 21-40% of the fruit surface; Grade 3: diseased area 41-60% of the fruit surface; Grade 4: diseased area 61-80% of the fruit surface; Grade 5: diseased area > 80% of the fruit surface.

[0076] As shown in Table 3, the fermentation liquid of biocontrol bacteria P15 can effectively reduce the fruit rot rate and disease index of fruits such as peaches and mangoes during storage, with a control effect of 55.56-86.21%. The preservation effect is equivalent to or even slightly better than that of the commonly used fruit chemical preservative prochloraz.

[0077] Table 3

[0078]

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the technical solution. Those skilled in the art should understand that those modifications or equivalent substitutions of the technical solution of the present invention that do not depart from the purpose and scope of the technical solution should be included in the scope of the claims of the present invention.

Claims

1. A Bacillus Velezii P15, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC NO.31995.

2. The use of Bacillus Velez P15 as claimed in claim 1 in preventing and controlling plant-derived fungi, wherein the plant-derived fungi are one or more combinations of Colletotrichum fructicola, Colletotrichum camelliae, Colletotrichum gloeosporioides, Colletotrichum jiangxiense, Colletotrichum Karsti, Colletotrichum chrysophilum, Colletotrichum perseae, Pestalotiopsis telopeae, Diaporthe sp, Alternaria sp, Fusarium sp, Neofusicoccum hyperici, Diaporthenobilis, Alternaria tenuissima, Lasiodiplodia hormozganensis, Lasiodiplodiagilanensis, Aspergillus sp, Didymella bryoniae, Botrytis cinerea, Rhizoctoniasolani, and Sclerotinia sclerotiorum.

3. The use of Bacillus Velez P15 as claimed in claim 1 in preventing and treating anthracnose of tea trees, powdery mildew of cucumbers and powdery mildew of kidney beans.

4. Application of Bacillus Velez P15 in fruit preservation as claimed in claim 1.

5. A biocontrol agent, It is characterized in that The active ingredient in the biocontrol agent is the Bacillus Velez P15 bacteria of claim 1, and the number of viable bacteria is not less than 1×10 7 cfu·mL -1 .

6. A biocontrol agent, It is characterized in that The active ingredient in the biocontrol agent is the fermentation broth of Bacillus Velez P15 according to claim 1.

7. The biocontrol agent according to claim 6, It is characterized in that The fermentation broth is prepared by the following steps: Bacillus Velez P15 was inoculated into LB liquid culture medium, cultured at 25°C for 24 hours, and then centrifuged to obtain a sterile fermentation broth.

8. Use of the biocontrol agent as claimed in claim 5 or 6 in preventing and controlling anthracnose and powdery mildew of tea trees.

9. Use of the biocontrol agent as claimed in claim 5 or 6 in fruit preservation.

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