Bacillus velezensis and application thereof in prevention and treatment of plant fungal diseases

Abstract

The invention relates to the technical field of functional microorganism screening and application, and particularly provides bacillus velezensis and application thereof in prevention and treatment of plant fungal diseases. The bacillus velezensis provided by the invention is a bacillus velezensis separated from rhizosphere of pepper, the Latin name of the bacillus velezensis is bacillus velezensis, the preservation number of the bacillus velezensis is CGMCC (China General Microbiological Culture Collection Center) No.29890, and the bacillus velezensis has a strong inhibition effect on fusarium oxysporum CMTT4-1 of pepper fusarium wilt, pathogenic bacteria SYDW1-1 of rice curvularia and colletotrichum gloeosporioides TCNKBLTJ1-1. And on the other hand, the fungicide prepared by utilizing the bacillus velezensis provided by the invention can be used for effectively preventing and treating pepper fusarium wilt and promoting pepper growth when being applied to peppers, and compared with traditional chemical pesticides and chemical fertilizers, the fungicide for biological prevention and treatment has the characteristics of being healthy to human and livestock, low in cost, environment-friendly and the like, conforms to the sustainable development principle and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of functional microorganism screening and application, and specifically provides a Bacillus Velezii and application thereof in the prevention and treatment of plant fungal diseases. Background Art

[0002] Pepper (Capsicum annuum L.) is an annual or limited perennial herb or shrub of the genus Capsicum in the Solanaceae family. It is also known as sweet pepper, big pepper, vegetable pepper, millet pepper, etc. It is native to Mexico and South America and is widely distributed worldwide. It is planted in all provinces of my country and is an important vegetable and condiment in my country. However, with the expansion of planting and cultivation area year after year, the occurrence of pepper diseases and insect pests has become more and more serious. In addition, due to unfavorable environmental conditions such as high temperature, high humidity, and high land multiple cropping index, diseases and insect pests are very likely to break out and become a disaster. Fusarium wilt is the most stubborn soil-borne disease in the production process of pepper. It is caused by Fusarium oxysporum and occurs in tropical, subtropical and temperate pepper planting areas around the world. At present, the occurrence of pepper wilt is getting worse year by year, with an incidence rate of generally 10-20%, 30-50% in severe cases, and even a total crop failure, causing serious economic losses to pepper production and becoming one of the main diseases affecting pepper production.

[0003] In order to control the diseases caused by this pathogen, plant pathologists from all over the world have conducted a lot of research on Fusarium oxysporum from different angles. However, it has been difficult to control plant diseases caused by Fusarium oxysporum for a long time because it is difficult to obtain durable resistance sources from host plants and there is a lack of effective fungicides against soil-dwelling organisms such as Fusarium oxysporum. At present, the prevention and control of this disease in production is still mainly based on the treatment of soil with chemical pesticides. However, the large-scale use of chemical pesticides not only increases the risk of drug resistance of pepper wilt, but also has an adverse effect on the ecological environment and the health of humans and animals. Therefore, it is urgent to study a new method for preventing and controlling pepper wilt that is environmentally friendly and sustainable for agricultural development.

[0004] "Using bacteria to treat bacteria" is a method that uses one or more microorganisms and their active products to inhibit the vitality and reproductive capacity of harmful organisms. It has attracted more and more attention from researchers at home and abroad. Among them, plant endophytes live in the tissues of healthy plants at a certain stage or all stages of their life history, which can cause systemic infection of plants, but will not cause obvious tissue changes or obvious damage to the host plants. Because they are directly derived from healthy plant tissues, they are non-toxic, pollution-free, easy to colonize, not easy to develop drug resistance, and environmentally friendly. They not only meet people's demand for green food, but also can overcome the drawbacks brought by chemical pesticides, provide guarantees for the sustainable development of agriculture, and will gradually replace traditional chemical control methods. They have a relatively broad research, development and application prospect.

[0005] In addition, endophytic bacteria can improve the host's resistance to diseases by competing with pathogens for niches and nutrients, producing antibacterial and antimicrobial active substances, and inducing host system resistance. Endophytic bacteria can also promote host growth and development. Bacillus is the most studied and widely used type of bacteria in plant endophytes. It is directly derived from the inside of plant tissues, has strong colonization ability, is easy to separate and culture, and can produce a variety of antibacterial active substances. It is considered to be the most ideal source of biological control strains. Currently, commercialized Bacillus used in the field of biological control mainly includes Bacillus subtilis, Bacillus thuringiensis, etc. CN103772008B discloses that a mixed bacterial agent prepared by biocontrol Bacillus subtilis HL259, Bacillus megaterium and Bacillus colloidus can be used to prevent and control rice sheath blight. CN104195069B discloses that Bacillus subtilis 2012SYX04 can effectively inhibit the development of rice blast. CN114958647B discloses that Bacillus thuringiensis has insecticidal activity against fall armyworm and / or nymph moth. CN117757700B discloses that Bacillus Velezii BRI3 can be used for the prevention and treatment of rapeseed sclerotinia. Summary of the invention

[0006] In order to solve the above problems, the present invention isolates from the root system of healthy peppers a Bacillus that has strong inhibitory effects on pepper wilt pathogens. At the same time, it has inhibitory effects on a variety of pathogens and has broad-spectrum inhibitory effects. According to morphological characteristics, molecular biology and whole genome sequencing, it is identified as Bacillus velezensis. The bacterial agent can inhibit the growth of Fusarium oxysporum of peppers and significantly promote the growth of peppers, reduce the use of chemical pesticides in the field, provide material basis and technical support for the green development of the pepper industry, and have certain ecological, economic and social benefits. The specific technical scheme of the present invention is as follows:

[0007] The first aspect of the present invention provides a Bacillus velezensis HKSSLJEBR3, the Latin name of the Bacillus velezensis is Bacillus velezensis, and the deposit number is CGMCC No.29890;

[0008] Furthermore, the 16S rRNA sequence of the Bacillus Velez subtilis HKSSLJEBR3 is shown as SEQ ID NO: 1; and the gyrA gene sequence of the Bacillus Velez subtilis is shown as SEQ ID NO: 2.

[0009] The second aspect of the present invention provides an application of Bacillus Velezii HKSSLJEBR3 in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi are at least one of Fusarium oxysporum of pepper CMTT4-1, Curvularia oryzae SYDW1-1, and Colletotrichum arecae TCNKBLTJ1-1.

[0010] The third aspect of the present invention provides a microbial agent, the active ingredient of which is the above-mentioned Bacillus Velezii HKSSLJEBR3;

[0011] Furthermore, the amount of live bacteria of Bacillus velez HKSSLJEBR3 in the microbial preparation is at least 10 6 CFU / mL.

[0012] The fourth aspect of the present invention provides the use of the above-mentioned microbial agent in preventing and controlling pepper wilt disease and promoting pepper growth.

[0013] A fifth aspect of the present invention provides a method for isolating Bacillus velezensis HKSSLJEBR3, wherein the strain HKSSLJEBR3 is isolated, purified and screened from a healthy pepper root sample collected from a pepper field in Shishan Town, Xiuying District, Haikou City, Hainan Province. The strain is preliminarily identified as Bacillus velezensis through morphological characteristics and molecular biology.

[0014] The sixth aspect of the present invention provides a method for molecular identification of Bacillus velez HKSSLJEBR3. In the molecular identification, in order to further determine the strain type, DNA extraction was performed on the strain HKSSLJEBR3, and the 16S rRNA and gyrA genes of the strain HKSSLJEBR3 were amplified, and the amplification primer sequences were bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-TACGGCTACCTTGTTACGAGTT-3') and specific primers gyrA-F (5'-CAGTCAGGAAATGCGTACGTCCTT-3'), gyrA-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3'). The second generation + third generation sequencing method, i.e., Illumina + PacBio, was used, and after quality control and assembly, the complete genome map of HKSSLJEBR3 was obtained, with a genome size of 3947034bp and a GC content of 46.64%. Combining morphological characteristics, molecular biology and whole genome housekeeping gene analysis, the strain HKSSLJEBR3 was finally identified as Bacillus velezensis. Whole genome analysis found that strain HKSSLJEBR3 can produce 13 secondary metabolites that are antibacterial substances.

[0015] A seventh aspect of the present invention provides a method for culturing Bacillus Velez subtilis HKSSLJEBR3. The optimum temperature for the growth of Bacillus Velez subtilis HKSSLJEBR3 is 30°C, the fastest growth rate of the culture time is 24h, and the optimum pH for the growth is 7.0.

[0016] Beneficial effects of the present invention:

[0017] The Bacillus Velez HKSSLJEBR3 provided by the present invention can not only inhibit the growth of Fusarium oxysporum of pepper, but also is a plant rhizosphere growth-promoting microorganism with broad-spectrum inhibitory resistance to plant pathogenic fungi. The bacterial agent prepared by the Bacillus Velez HKSSLJEBR3 provided by the present invention can be applied to pepper to effectively prevent and treat pepper wilt disease, promote pepper growth, and reduce the use of chemical pesticides, providing a new means for agricultural production, biological control and environmental management, having certain ecological benefits, economic benefits and social benefits, and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The colony morphology and 16S rRNA and gyrA gene phylogenetic tree of Bacillus velez HKSSLJEBR3 of the present invention;

[0019] Figure 2 The present invention is based on the phylogenetic tree analysis of the housekeeping gene of Bacillus velez HKSSLJEBR3;

[0020] Figure 3 The genome circle map of Bacillus velez HKSSLJEBR3 of the present invention;

[0021] Figure 4 The antiSMASH secondary metabolite analysis of Bacillus velez HKSSLJEBR3 of the present invention is based on;

[0022] Figure 5 The effects of different culture temperatures on Bacillus Velez HKSSLJEBR3 of the present invention;

[0023] Figure 6 The effects of different culture times on Bacillus Velezii HKSSLJEBR3 of the present invention;

[0024] Figure 7 The effect of different culture pH values ​​on Bacillus Velez HKSSLJEBR3 of the present invention;

[0025] Figure 8 The inhibitory effect of the Bacillus Velez HKSSLJEBR3 of the present invention on Fusarium oxysporum of pepper wilt;

[0026] Fig. 9 The inhibitory effect of the Bacillus Velezii HKSSLJEBR3 of the present invention on Curvularia oryzae;

[0027] Fig.10 The inhibitory effect of the Bacillus Velezii HKSSLJEBR3 of the present invention on the betel nut anthracnose bacteria;

[0028] Fig.11 The invention discloses the growth-promoting effect of the Bacillus Velez HKSSLJEBR3 bacterial agent on the growth of pepper. DETAILED DESCRIPTION

[0029] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0030] Example 1: Isolation and purification of Bacillus velez HKSSLJEBR3

[0031] 1.1 Pepper samples

[0032] On February 28, 2022, healthy pepper samples were collected in Shishan Town, Haikou City. The roots, stems, leaves, and fruits of the peppers were collected, and the sample information was recorded. The collection location was Shishan Town, Xiuying District, Haikou City, Hainan Province. The weather was sunny. The longitude and latitude were 110.24242679°E, 19.76643517°N, and the altitude was 47.6m. The samples were stored at 4°C and separated within 3 days.

[0033] 1.2 Culture medium

[0034] LB liquid medium: 10g sodium chloride, 10g peptone, 5g yeast powder, dilute to 1000mL with deionized water, sterilize at 121℃ for 30min. If LB solid medium is used, 15-20g agar powder should be added to every 1000mL LB liquid medium.

[0035] 1.3 Isolation of endophytic bacteria

[0036] Rinse the sample surface with tap water for 30 seconds to remove surface impurities, weigh 2g of plant tissue, rinse with sterile water 3 times, disinfect the surface with 75% ethanol for 1 minute, rinse with sterile water 3 times, disinfect the surface with 0.1% mercuric chloride for 1 minute, rinse with sterile water 5 times, add the sample to a sterilized mortar containing 2mL of sterile water and grind it, take 50μL with a pipette, and evenly spread it on a 90mm LB plate with a spreader, and use the last sterile water rinse as a control. After 48 hours, pick a single colony according to the colony morphology, purify it, and store it on a slant at 4℃ for later use.

[0037] 1.4 Purification of endogenous bacteria

[0038] Purification: A single colony was selected and passaged 5 times on LB plates to obtain a pure culture of Bacillus velezensis HKSSLJEBR3, which was stored in a -80°C refrigerator.

[0039] Example 2: Identification of Bacillus velez HKSSLJEBR3

[0040] 2.1 Morphological observation

[0041] The LB solid medium was used to culture the Bacillus velezensis HKSSLJEBR3 and its morphology was observed. The colony of HKSSLJEBR3 was white or light yellow, round or oval in shape, moist on the surface, often with convex wrinkles, neat edges, and opaque and viscous ( Figure 1 .A,B).

[0042] 2.2 Molecular identification

[0043] The genome of Bacillus velez HKSSLJEBR3 was extracted according to the instructions of the bacterial genomic DNA extraction kit produced by Tiangen Biochemical Technology (Beijing) Co., Ltd. The DNA of the strain to be tested was used as a template, and the bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-TACGGCTACCTTGTTACGAGTT-3') and specific primers gyrA-F (5'-CAGTCAGGAAATGCGTACGTCCTT-3'), gyrA-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3') were selected to amplify the 16S rRNA and gyrA genes of endophytic bacteria. The PCR system (30 μL) was as follows: 2×Taq PCRMasterMix 15 μL, template 1 μL, 27F 1 μL, 1492R 1 μL, and ddH2O 12 μL. Amplification program: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, 35 cycles, 72°C extension for 5 min, and storage at 4°C. The amplified product was sent to Tianyi Huiyuan Gene Technology Co., Ltd. for sequencing.

[0044] The obtained nucleotide sequences were compared by BLAST tool of NCBI database, and its 16S rRNA sequence (SEQ ID NO: 1) and gyrA sequence (SEQ ID NO: 2) were most similar to Bacillus velezensis. The neighbor-joining method of MEGA 11.0 software was used to construct the multi-gene phylogenetic tree of HKSSLJEBR3 in combination with the 16S rRNA and gyrA gene sequences. The results showed that HKSSLJEBR3 and Bacillus velezensis were clustered into one branch ( Figure 1 .C).

[0045] 2.3 Whole genome sequencing of Bacillus velezensis HKSSLJEBR3

[0046] 2.3.1 Bacterial sample culture and collection

[0047] Bacillus velezensis HKSSLJEBR3 was inoculated into 50 mL of LB liquid medium at 30 °C and 180 rpm and cultured until OD 600 =1.0, collect the bacteria by centrifugation, and transfer the precipitate to a 1.5mL sterile cryotube after centrifugal enrichment, with 2 to 3 backups.

[0048] 2.3.2 Whole-genome sequencing

[0049] Illumina sequencing experimental process: library construction → bridge PCR → Illumina sequencing;

[0050] Single-molecule PacBio sequencing experimental process: genomic DNA → fragmentation → library construction → sequencing.

[0051] 2.3.3 Analysis of genomic results

[0052] The sequencing results of Bacillus velezensis HKSSLJEBR3 were assembled after quality control, and a high-quality genome of 3947034bp was obtained, with a GC content of 46.64%, no plasmid, 88 predicted tRNAs, 28 predicted rRNAs, 3 genomic islands, 3 prophages, and 8 CRISPR-Cas. Based on the whole genome data, an evolutionary tree analysis of HKSSLJEBR3 was performed based on housekeeping genes. The results showed that the strain was clustered with Bacillus velezensis ( Figure 2 ).

[0053] The outermost circle of the Circos genome map of Bacillus velez HKSSLJEBR3 is the genome size mark; the second and third circles are the CDS on the positive and negative chains, and different colors indicate the functional classification of different COGs of CDS; the fourth circle is rRNA and tRNA; the fifth circle is the GC content, the outward red part indicates that the GC content of this region is higher than the average GC content of the whole genome, and the higher the peak value, the greater the difference with the average GC content; the inward blue part indicates that the GC content of this region is lower than the average GC content of the whole genome, and the higher the peak value, the greater the difference with the average GC content; the innermost circle is the GC-Skew value, and the specific algorithm is GC / G+C, which can assist in judging the leading chain and the lagging chain. Generally, the leading chain GC skew>0, and the lagging chain GC skew<0. It can also assist in judging the replication start point (minimum cumulative offset) and end point (maximum cumulative offset), which is especially important for circular genomes ( Figure 3 ).

[0054] Based on antiSMASH (version 7.1.0), the whole genome data of Bacillus velezensis HKSSLJEBR3 were analyzed and predicted to have 13 secondary metabolites, of which 9 substances have been reported, namely surfactin, plantazolicin, butirosin A / B, macrolactin H, bacillaene, fengycin, difficidin, bacillibactin and bacilysin ( Figure 4 ).

[0055] Example 3: Optimization of culture conditions of Bacillus velez HKSSLJEBR3

[0056] 3.1 Effects of different temperatures on the growth of Bacillus velezensis HKSSLJEBR3

[0057] LB liquid medium: 10 g sodium chloride, 10 g peptone, 5 g yeast powder, dilute to 1000 mL with deionized water, and sterilize at 121°C for 30 min.

[0058] Culture conditions: 100 mL of LB medium was placed in a 250 mL Erlenmeyer flask, the inoculum was 1%, the inoculum was the fresh bacterial solution from the previous day, the shaker speed was 180 rpm, and the culture time was 24 h. The culture temperature gradient was 20°C, 25°C, 30°C, 35°C, and 40°C, and each temperature was set for 3 replicates, and the OD of each replicate was measured. 600 The absorbance value was measured 3 times.

[0059] The experimental results are shown in Table 1 below, and the line graph is shown in Figure 5 The results showed that the OD of Bacillus velez HKSSLJEBR3 culture was 600 As the temperature increases, the OD 600 It reached the highest value, and then OD600 gradually decreased with the continuous increase of temperature, indicating that the optimal temperature for the growth of Bacillus velezensis HKSSLJEBR3 was 30℃.

[0060] Table 1. Effect of different temperatures on the OD of Bacillus velez HKSSLJEBR3 600 Determination

[0061]

[0062] 3.2 Effects of different culture times on the growth of Bacillus velezensis HKSSLJEBR3

[0063] LB liquid medium: 10 g sodium chloride, 10 g peptone, 5 g yeast powder, dilute to 1000 mL with deionized water, and sterilize at 121°C for 30 min.

[0064] Culture conditions: 100 mL of LB medium was placed in a 250 mL Erlenmeyer flask, the inoculum was 1%, the inoculum was the fresh bacterial solution from the previous day, the shaker speed was 180 rpm, the culture temperature was 30 °C, the shaking time was set to 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h, and 3 replicates were set for each time. The OD of each replicate was measured. 600 The absorbance value was measured 3 times.

[0065] The experimental results are shown in Table 2 below, and the line graph is shown in Figure 6The results showed that the OD of Bacillus velez HKSSLJEBR3 culture was 600 It continued to increase over time. At 48 h of culture, OD 600 Reach the highest, then high OD over time 600 also gradually decreased, indicating that the optimal growth time of Bacillus velez HKSSLJEBR3 was 48h. Figure 5 It can be clearly seen that at 24 h, Bacillus Velezii HKSSLJEBR3 was in the logarithmic phase of growth and had the fastest growth rate.

[0066] Table 2. Effect of different culture time on the OD of Bacillus velez HKSSLJEBR3 600 Determination

[0067]

[0068] 3.3 Effect of different pH values ​​on the growth of Bacillus velezensis HKSSLJEBR3

[0069] LB liquid medium: 10 g sodium chloride, 10 g peptone, 5 g yeast powder, dilute to 1000 mL with deionized water, and sterilize at 121°C for 30 min.

[0070] Culture conditions: 100 mL of liquid culture medium was placed in a 250 mL conical flask. The liquid culture medium for different treatments was adjusted to pH = 5.0, pH = 6.0, pH = 7.0, pH = 8.0, and pH = 9.0, respectively. The inoculum was 1%, and the inoculum was the fresh bacterial liquid shaken the day before. The shaker speed was 180 rpm, the culture temperature was 30 ° C, and the shaking time was 24 h. Each treatment was repeated 3 times, and the OD of each repeat was detected. 600 The absorbance value was measured 3 times.

[0071] The experimental results are shown in Table 3 below, and the line graph is shown in Figure 7 The results showed that the OD of Bacillus velez HKSSLJEBR3 culture was 600 As the pH value continued to increase, the OD 600 The OD value reaches the highest value, and then increases with the increase of pH value. 600 It also gradually decreased, indicating that the optimal pH for the growth of Bacillus Velezii HKSSLJEBR3 is 7.0.

[0072] Table 3. Effect of different pH values ​​on the OD of Bacillus velez HKSSLJEBR3 growth 600 Determination

[0073]

[0074] Example 4: Inhibitory effect of Bacillus velez HKSSLJEBR3 on various pathogens

[0075] 4.1 Test strains

[0076] Pathogens: The CMTT4-1 strain of Fusarium oxysporum of pepper was isolated and preserved by the Biological Control Research Laboratory of the Institute of Plant Protection, Hainan Academy of Agricultural Sciences. The strain was isolated from diseased pepper plants in Chengmai County, Hainan Province, and was identified as Fusarium oxysporum by morphology and molecular biology; the SYDW1-1 strain of Curvularia lunata of rice was isolated and preserved by the Biological Control Research Laboratory of the Institute of Plant Protection, Hainan Academy of Agricultural Sciences. The strain was isolated from diseased rice leaves in Sanya City, Hainan Province, and was identified as Curvularia lunata by morphology and molecular biology; the TCNKBLTJ1-1 strain of Colletotrichum gloeosporiodes of areca nut was isolated and preserved by the Biological Control Research Laboratory of the Institute of Plant Protection, Hainan Academy of Agricultural Sciences. The strain was isolated from diseased areca nut leaves in Nankun Town, Tunchang County, Hainan Province, and was identified as Colletotrichum gloeosporiodes by morphology and molecular biology.

[0077] Endophytic bacteria: Bacillus velezensis HKSSLJEBR3, which was deposited on February 26, 2024 in the General Microbiology Center of China Culture Collection (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), with the deposit number CGMCC No.29890.

[0078] 4.2 Culture medium

[0079] PDA medium: 200 g of potato, boiled for 15 min, filtered through double-layer gauze and collected the filtrate, 20 g of glucose, 15-20 g of agar powder, dilute to 1000 mL with deionized water, sterilized at 121°C for 30 min.

[0080] LB liquid medium: 10g sodium chloride, 10g peptone, 5g yeast powder, dilute to 1000mL with deionized water, sterilize at 121℃ for 30min. If LB solid medium is used, 15-20g agar powder should be added to every 1000mL LB liquid medium.

[0081] 4.3 Strain activation

[0082] Activation of pathogenic bacteria: culture on PDA plates for 7 days, use a hole puncher to make a 6mm diameter bacterial cake, and place it in the center of a new PDA plate; activation of endogenous bacteria on LB plates: use a sterilized toothpick to select a single colony, and use the gradient dilution method to streak on the LB plate to obtain a single colony.

[0083] 4.4 Flat Plate Confrontation Method

[0084] The activated Bacillus Velezii HKSSLJEBR3 was inoculated in LB liquid culture medium and cultured at 30°C, 180rpm for 24h. Sterilized filter paper with a diameter of 5mm was placed at 4 diagonals 3cm away from the pathogen cake, and 5μL of the endophytic bacterial solution to be tested was inoculated. At the same time, 5μL of LB liquid culture was inoculated on the filter paper as a control. Each treatment was repeated 3 times and placed in a 26°C incubator for culture. The diameter of the pathogen was recorded from the third day and recorded every 24h until the control covered the entire plate. The antibacterial rate was calculated as follows: Antibacterial rate = [(untreated pathogen diameter - treated pathogen diameter) / untreated pathogen diameter] × 100%.

[0085] 4.5 Inhibitory effect of Bacillus velezensis HKSSLJEBR3 on three pathogens

[0086] As shown in Tables 4 and 5 below, the inhibition rates of Bacillus velezensis HKSSLJEBR3 against pepper wilt pathogen, rice curvature spore pathogen, and betel nut anthracnose pathogen were 48.99%, 63.00%, and 49.49%, respectively, with a broad antibacterial spectrum ( Figure 8 , 9 , 10).

[0087] Table 4. Inhibitory effect of Bacillus velez HKSSLJEBR3 on three pathogens (unit: mm)

[0088]

[0089] Table 5. Inhibition rate of Bacillus velezensis HKSSLJEBR3 against three pathogens

[0090]

[0091] Example 5: HKSSLJEBR3 bacterial agent promotes the growth of pepper

[0092] 5.1 Preparation of pepper seedlings

[0093] Potted pepper seedlings were planted in an artificial climate box at 25°C, 70% humidity, and 12h light / 12h dark photoperiod. Thirty plants were planted in each treatment, repeated three times. When the peppers had 4 leaves and 1 core, the HKSSLJEBR3 inoculant was inoculated by root irrigation.

[0094] 5.2 Preparation of HKSSLJEBR3 inoculant

[0095] HKSSLJEBR3 strain was activated on LB plate: single colonies were selected with sterile toothpicks and streaked on LB plate using gradient dilution method to obtain single colonies. HKSSLJEBR3 strain was inoculated into a 1000mL Erlenmeyer flask containing 500mL LB liquid medium and cultured at 30℃ and 180rpm for 24h. The culture solution was then prepared to a concentration of 10 6 CFU / mL of HKSSLJEBR3 bacterial agent is kept for later use.

[0096] 5.3 Experimental treatment

[0097] Take 4-leaf 1-core pepper seedlings, treat with HKSSLJEBR3 fungicide and plant 30 plants, repeat 3 times, 20 mL per pepper plant. Use sterile water as control, inoculate once every 10 days, inoculate twice in succession, and investigate pepper plant height, root length, leaf number and fresh weight 20 days after the last inoculation.

[0098] 5.4HKSSLJEBR3 bacterial agent has a promoting effect on pepper growth

[0099] 20 days after the last inoculation of HKSSLJEBR3, the plant height, leaf number, fresh weight and root length of peppers under different treatments were counted. The results are shown in Table 6. The average plant height of the three different replicates of the control pepper treatment was (23.49+23.27+24.04) / 3=23.6 cm, the average leaf number was (23.20+23.20+24.00) / 3=23.54 pieces, the average fresh weight was (11.78+11.60+10.28) / 3=11.22 g, and the average root length was (8.52 +8.38+8.51) / 3=8.47cm; the average plant height of three different replicates of pepper treated with HKSSLJEBR3 fungus was (28.37+29.08+29.01) / 3=28.82cm, the average number of leaves was (33.83+34.53+35.13) / 3=34.50 pieces, the average fresh weight was (16.70+17.70+18.22) / 3=17.54g, and the average root length was (10.43+10.00+9.94) / 3=10.12cm.

[0100] Compared with the control group, the pepper plant height was 5.22 cm higher, the number of pepper leaves was 10.96 more, the fresh weight of pepper was 6.32 g, and the length of pepper root was 1.65 cm longer after the treatment with HKSSLJEBR3 fungus, indicating that the growth of pepper can be promoted after the treatment with HKSSLJEBR3 fungus ( Fig.11 ).

[0101] Table 6. HKSSLJEBR3 bacterial agent to promote pepper growth index determination

[0102]

[0103] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Bacillus Velez, characterized in that The Latin name of the Bacillus velezensis is Bacillus velezensis, and the deposit number is CGMCC No.29890.

2. The 16S rRNA sequence of the Bacillus Velez subtilis according to claim 1 is shown as SEQ ID NO: 1; the gyrA gene sequence of the Bacillus Velez subtilis according to claim 1 is shown as SEQ ID NO:

2.

3. The use of the Bacillus Velezii according to claim 1 in inhibiting plant pathogenic fungi, characterized in that: The plant pathogenic fungus is at least one of Fusarium oxysporum of pepper CMTT4-1, Curvularia oxysporum of rice SYDW1-1, and Colletotrichum sphaerocephalae TCNKBLTJ1-1.

4. A microbial agent, characterized in that: The active ingredient is the Bacillus Velezii described in claim 1.

5. The microbial agent according to claim 4, characterized in that The amount of live bacteria of Bacillus Velez in the microbial preparation is at least 10 6 CFU / mL.

6. Use of the microbial agent as claimed in claim 4 or 5 in preventing and controlling pepper wilt and promoting pepper growth.

7. A method for isolating the Bacillus Velezii according to claim 1, characterized in that: The Bacillus Velez is isolated from the rhizosphere of pepper.

8. A method for molecular identification of the Bacillus Velezii of claim 1, characterized in that: The primer sequences for amplifying the 16S rRNA and gyrA genes of the Bacillus velez were bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-TACGGCTACCTTGTTACGAGTT-3') and specific primers gyrA-F (5'-CAGTCAGGAAATGCGTACGTCCTT-3'), gyrA-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3').

9. A method for culturing the Bacillus Velezii according to claim 1, characterized in that: The optimum temperature for the growth of the Velez subtilis is 30° C., the fastest growth rate in culture is 24 hours, and the optimum pH for the growth is 7.0.

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