Bacillus velezensis E17-23 and application thereof

Abstract

The invention relates to the technical field of microbial control, and discloses bacillus velezensis E17-23 and application thereof. The bacillus velezensis E17-23 disclosed by the invention can be used for inhibiting citrus liberobacter asiaticum, citrus canker pathogen, citrus brown spot pathogen, citrus colletotrichum gloeosporioides, citrus lipolytic yellow spot pathogen, banana fusarium wilt pathogen and banana long leaf spot pathogen, and the inhibition effects are all greater than 60%. The inhibition effect of the sterile fermentation filtrate of the bacillus velezensis E17-23 on the pathogenic bacteria is more than 20.83%. The bacillus velezensis E17-23 can also promote plant growth, the average growth amount of treated orange seedlings is increased by 177.57% compared with that of a control group, and the number of new leaves is increased by 208.5% compared with that of the control group. The application of the bacillus velezensis E17-23 in the aspects of biological control and plant growth promotion has important significance in green agriculture and sustainable agricultural production.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial control, and more specifically, to Bacillus velez E17-23 and application thereof. Background Art

[0002] Chemical pesticides are still the main means of controlling pests and diseases. However, the environmental pollution, pesticide residues and resistance caused by the large-scale use of chemical pesticides are becoming more and more serious. Therefore, more and more researchers have begun to pay attention to the biological control of plant diseases, among which the hot spots are the research on beneficial microorganisms as antagonistic bacteria.

[0003] Biocontrol bacteria can prevent or reduce the infection or damage caused by pathogens to plants by secreting antibacterial substances, competing for nutrients and space, and inducing plant-induced disease resistance. In addition, biocontrol bacteria can also promote plant growth and nutrient absorption by producing IAA, dissolving phosphorus, potassium, and fixing nitrogen, thereby increasing the accumulation of plant biomass and increasing production and income. Microbial pesticides developed with beneficial microorganisms as bacterial resources are not easy to develop drug resistance, are non-polluting to the environment, and do not harm natural enemies. When applied to agricultural production, it provides a new way to produce green or pollution-free agricultural products.

[0004] Bacillus spp. is a Gram-positive bacterium widely distributed in various habitats such as soil, plant surfaces, water bodies, agricultural waste, etc. It has broad-spectrum antibacterial activity, high spore yield and strong adaptability to adversity, and is considered to be one of the most promising beneficial microorganisms for application development. For example, Bacillus amyloliquefaciens and Bacillus subtilis are a type of biocontrol microorganism that is widely recognized by scientists and consumers as safe for the human body. At present, there are 137 registered fungicide products containing Bacillus active ingredients in my country.

[0005] However, no strain of Bacillus Velezii that has the ability to inhibit pathogens such as citrus Huanglongbing disease, citrus ulcer pathogen, citrus brown spot pathogen, citrus anthracnose pathogen, citrus fat spot yellow spot pathogen, banana wilt pathogen and banana long spot pathogen and has the ability to promote plant growth has been found so far. Summary of the invention

[0006] The present invention overcomes the above-mentioned defects of the prior art in that the Bacillus Velez strain has the ability to inhibit a broad spectrum of plant pathogenic bacteria and fungi in citrus and bananas and promote plant growth, and provides Bacillus Velez E17-23;

[0007] Another object of the present invention is to provide an application of Bacillus Velez E17-23.

[0008] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0009] Bacillus velezensis E17-23 (Bacillus velezensis), deposit number is GDMCC No. 64580. Deposit date: April 29, 2024, deposit unit: Guangdong Microbiological Culture Collection Center, deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0010] Furthermore, the Velez Bacillus E17-23 and its culture or metabolites can inhibit citrus Huanglongbing disease, citrus canker, citrus brown spot disease, citrus anthracnose, citrus fat spot yellow spot disease, banana wilt disease and banana long spot disease.

[0011] Furthermore, the Bacillus Velez E17-23 and its metabolites can promote plant growth.

[0012] Preferably, the plant is a citrus plant.

[0013] Preferably, the plant is a citrus plant in the seedling stage.

[0014] An application of the Bacillus Velez E17-23 is used for preparing a biological agent for preventing and controlling plant diseases and / or promoting plant growth.

[0015] Furthermore, the biological preparation uses the Bacillus Velezii E17-23 and its culture or metabolites as described in claim 1 as an active ingredient.

[0016] Preferably, the culture comprises a sterile fermentation filtrate.

[0017] Preferably, the metabolites include generated volatile gases.

[0018] Furthermore, the plant diseases include citrus Huanglongbing, citrus canker, citrus brown spot, citrus anthracnose, citrus fat spot yellow spot, banana wilt and banana long spot.

[0019] Furthermore, the promoting plant growth is promoting the growth of citrus plants.

[0020] Furthermore, in the biological preparation, Bacillus Velezii E17-23 is cultured under shaking for 48 to 72 hours.

[0021] Furthermore, in the biological preparation, the concentration of Bacillus Velezii E17-23 is 10 6 ~10 9 CFU / mL.

[0022] Preferably, at 106 ~10 9 In the CFU / mL concentration, the higher the concentration of Bacillus Velezii E17-23, the stronger its ability to inhibit pathogens.

[0023] Furthermore, the dosage forms of the biological agent include aqueous suspensions, granules, powders and microcapsules.

[0024] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0025] 1. Broad-spectrum antibacterial effect. Bacillus Velez E17-23 showed the best inhibitory effect on citrus canker bacteria, with an inhibition zone diameter of 3.17±0.12 cm, which was significantly larger than the control group (LB) and other Bacillus Velez strains, indicating that this strain has a significant inhibitory effect on citrus canker bacteria. The inhibitory effect of Bacillus Velez E17-23 on citrus brown spot pathogen, citrus anthracnose pathogen, citrus fat spot yellow spot pathogen, banana wilt pathogen, and banana long spot pathogen was more than 60%, showing a strong broad-spectrum antibacterial effect. The volatile metabolites of Bacillus Velez E17-23 can also reduce the colony diameter of the above pathogens by more than 14.95%. After treatment with Bacillus Velez E17-23, the content of citrus Huanglongbing bacteria in citrus leaves dropped to (2.24±0.07)×10 8 / g, and the control effect reached 51%, indicating that the strain has a strong potential in controlling citrus Huanglongbing.

[0026] 2. Promote plant growth. In the orange seedlings treated with the Bacillus Velez E17-23 solution, the average growth was 2.97±0.74cm, an increase of 177.57% over the control group. In addition, the number of new leaves in the E17-23 treatment group was 6.17±2.04, an increase of 208.5% over the control group. These data show that Bacillus Velez E17-23 can significantly promote plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of rescreening of biocontrol bacteria against citrus canker pathogens are shown in Figure 1. A is the plate confrontation result, and B is a schematic diagram of the diameter of the inhibition zone (different lowercase letters indicate significant differences at the 0.01 level);

[0028] Figure 2 Phylogenetic tree based on 16S rRNA gene constructed for strain E17-23;

[0029] Figure 3 is the colony morphology characteristics of strain E17-23;

[0030] Figure 4The inhibitory effect of strain E17-23 on various pathogens, A is the plate confrontation result, and B is a schematic diagram of the diameter of the pathogen colony;

[0031] Figure 5 Schematic diagram of the inhibitory effect of the volatile products of strain E17-23 on various pathogens, A is the result of culture on separate plates, and B is a schematic diagram of the diameter of pathogen colonies;

[0032] Figure 6 The antibacterial effects of the aseptic fermentation filtrates of strain E17-23 cultured at different times, A is the plate confrontation result, and B is a schematic diagram of the diameter of the inhibition zone (different lowercase letters indicate significant differences at the 0.01 level);

[0033] Figure 7 The antibacterial effect of the filtrate of aseptic fermentation of strain E17-23 cultured for 48 hours, A is the plate confrontation result, and B is a schematic diagram of the diameter of the pathogen colony;

[0034] Figure 8 The control effect of strain E17-23 on potted citrus leaf canker, A is a schematic diagram of leaf lesions, and B is a schematic diagram of lesion size;

[0035] Fig. 9 The control effect of strain E17-23 on citrus leaf Huanglongbing and the copy number of 16S rDNA of citrus Huanglongbing bacteria;

[0036] Fig.10 This is the effect of E17-23 in promoting the growth of orange seedlings. A is a schematic diagram of the average growth amount, and B is a schematic diagram of the number of newly grown leaves. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0038] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0039] 1. Isolation, screening and identification of Bacillus velez E17-23

[0040] (1) Separation

[0041] Citrus leaf and soil samples grown indoors at the South China Agricultural University in Tianhe District, Guangzhou City, Guangdong Province, and tea-branch citrus leaf, fruit and soil samples were collected from the tea-branch citrus planting base of Jiangmen Guangying Chenpi Ganpu Tea Co., Ltd. and stored in a 4°C refrigerator.

[0042] Treatment of plant samples: Cut leaves into 0.5×0.5 mm pieces, cut roots and tubers into 1 cm pieces, treat with 75% ethanol for 30 seconds, soak in 5% sodium hypochlorite for 10 minutes, rinse with sterile water for 3 to 5 times, dry, pick 3 tissue blocks, grind in a sterile mortar with 3 to 5 mL of sterile water, draw 1 mL of grinding solution into a 10 mL centrifuge tube with 9 mL of sterile water, and mix well to obtain 10 -1 Then dilute to 10 with sterile water. -2 , 10 -3 and 10 -4 Take 10 times of each sample solution. -3 and 10 -4 0.1 mL of the sample solution was evenly spread on LB, PDA, and NA culture medium plates, and the treatment was repeated three times. After inverted culture at 37°C for 48 hours, the growth of microorganisms on the plates was observed, and single colonies of different bacteria were picked and purified 2 to 3 times and stored for later use.

[0043] Soil sample treatment: Weigh 1g of soil and add it to a 10mL centrifuge tube containing 9mL of sterile water to prepare a soil suspension. After each sample suspension was shaken and cultured at 200rpm for 30min, the supernatant was diluted to 10% with sterile water. -1 Up to 10 -6 Concentration. Take 0.1 mL of diluted bacterial solution of each concentration and evenly spread it on LB, PDA, and NA medium plates, and repeat the treatment three times. After inverted culture at 37℃ for 48 hours, observe the growth of microorganisms on the plate, pick out single colonies of different bacteria for purification 2 to 3 times, and save them for later use.

[0044] (2) Biocontrol bacteria screening

[0045] The plate confrontation method was used for preliminary screening. The strains isolated above were inoculated on LB liquid culture medium and cultured at 37°C for 24 hours. The test bacterial liquid was adjusted to OD600 = 1.0 with LB for standby use. Heat the NA solid culture medium. When the NA culture medium cools to about 40°C, add the pre-cultured citrus canker bacteria liquid at a ratio of 1:100, pour the mixed liquid into the culture dish, and prepare the bacterial plate. Use a 5mm puncher to punch holes, pick out the culture medium with a toothpick, add 20μL of the test bacterial liquid to each hole, add 20μL of LB culture medium to the control, blow dry, and seal. Put it in a 28°C incubator for constant temperature culture. After 24 hours, observe whether a transparent antibacterial zone appears and record the results.

[0046] According to the results of the initial screening, the 10 strains with the best antibacterial effect were selected for rescreening. Prepare the bacterial plate according to the initial screening test method, punch holes with a 5mm puncher, remove the culture medium with a toothpick, add 20μL of the test solution to each hole, repeat each treatment three times, and the test control is 20μL LB culture medium and 20μL E3-01 bacterial solution of the strain with no antibacterial effect, blow dry, and seal. Put it in a 28℃ incubator for constant temperature culture, and observe whether a transparent antibacterial circle appears after 24 hours.

[0047] according to Figure 1 The results showed that compared with the control group LB and E3-01, the 10 strains including E17-23 showed obvious transparent inhibition zones after treatment, indicating that the above 10 strains had a significant inhibitory effect on citrus canker pathogens. Figure 1 As shown in Figure B, E17-23 had the best antibacterial effect, and the diameter of the inhibition zone was significantly different from that of E3-02 and E17-20.

[0048] (3) Molecular biological identification of strains

[0049] The total DNA of the above 10 strains was extracted using a DNA extraction kit (Nanjing Novozyme Biotechnology Co., Ltd.).

[0050] PCR amplification was performed using the universal primers 27F and 1492R of bacterial 16S rRNA and the total DNA of the above 10 strains as templates. The reaction system was 10 μL of 2×TaqMasterMix (DyePlus) (Shanghai Sangon Biotech Co., Ltd.), 1 μL of DNA template, 1 μL of each of the front and back primers, and ddH2O was added to 10 μL.

[0051] The PCR reaction program was: pre-denaturation at 94°C for 1 min; 25 cycles: denaturation at 94°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1.5 min; after the cycle was completed, standby at 4°C.

[0052] Among them, the 27F sequence is: 5'-AGAGTTTGATCCTGGCTCAG-3'; the 1492R sequence is: 5'-TACGGCTACCTTGTTACGACTT-3'.

[0053] The PCR product was detected by electrophoresis gel and a band of about 1400 bp was obtained. The PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for purification and sequencing.

[0054] The sequencing results were subjected to BLAST analysis on the NCBI website, and the results are shown in Table 1. Among them, the strain E17-23 with the best antibacterial effect and the strains E3-02 and E17-20 with the worst antibacterial effects had the highest similarity with Bacillus Velezii, indicating that not all strains of Bacillus Velezii have the ability to inhibit citrus canker bacteria.

[0055] Table 1 Comparison results of strain 16S rRNA

[0056]

[0057] The 16S rRNA reference sequences of different Bacillus species were downloaded from NCBI and the phylogenetic tree was constructed using the Neighbor-Joining method (NJ) of MEGA11.0. The reliability of the constructed phylogenetic tree was estimated using the bootstrap test. The number of repetitions was 1000. The results are shown in the figure. Figure 2 Finally, the strain E17-23 was identified as Bacillus velezensis and named Bacillus velezensis E17-23. Its colony morphology is shown in Figure 3 As shown, the colonies on the LB medium appear as white opaque colonies with irregular raised edges, a rough surface and a crater-like protrusion in the center. The strain Bacillus velezensis E17-23 is deposited in the Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No. 64580, the deposit date: April 29, 2024, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0058] (4) Physiological and biochemical identification of strain E17-23

[0059] The test solution was prepared with reference to the "Common Bacteria System Identification Manual" and "Berger's Bacteria Identification Manual" to conduct physiological and biochemical tests on strain E17-23. The results of the physiological and biochemical characteristics test are shown in Table 2. Strain E17-23 is a Gram-positive bacterium; the VP test is positive; the MR test is negative; it cannot produce indole; it can produce protease; it can produce amylase; it can produce catalase; it has nitrogen fixation function; it can produce siderophore; it can liquefy gelatin.

[0060] Table 2 Physiological and biochemical characteristics test results

[0061]

[0062] 2. Application of Bacillus Velez E17-23

[0063] Example 1

[0064] Take out the stored Bacillus Velezii E17-23 from -80℃, streak it on LB plate, and culture it at 37℃ for 48h. Pick a single colony and culture it in LB liquid medium at 37℃ and 200r / min. -1 Incubate in a shaker for 24 hours. Pipette 1 mL of bacterial solution into a flask containing 40 mL of LB liquid medium and culture at 37°C, 200 r·min -1 The culture was shaken in a shaker for 24 h, and the obtained bacterial solution was adjusted by spectrophotometer to OD 600 to about 1.0 (concentration of 8×10 8 ~10 9 CFU / mL) for later use.

[0065] Example 2

[0066] Velezella E17-23 were inoculated into LB liquid medium and incubated at 37°C and 200 r·min -1 The culture was shaken in a shaker for 48 h and 72 h respectively. The bacterial solution was centrifuged at 6000 rpm for 10 min and filtered through a 0.22 μm filter to obtain a sterile fermentation filtrate.

[0067] Example 3

[0068] Take out the stored ulcer bacteria from -80℃, streak them on NA plates, and culture them at 28℃ for 72h. Pick a single colony and culture it in NA liquid medium at 28℃ and 200r·min. -1 Incubate in a shaker for 48 hours. Pipette 1 mL of bacterial solution into a flask containing 40 mL of NA liquid medium and culture at 28°C, 200 r·min -1 The culture was shaken in a shaker for 24 h, and the obtained bacterial solution was adjusted to an OD600 of about 0.6 (concentration of 10 6 ~10 8 CFU / mL) for later use.

[0069] Comparative Example 1

[0070] The preparation scheme of Comparative Example 1 is similar to that of Example 2, except that the shaking culture time in the shaker is 24 h and 96 h, respectively.

[0071] Verification Method

[0072] (1) Antibacterial effect of strain E17-23

[0073] Prepare a PDA medium plate, and inoculate the bacterial cakes (5 mm in diameter) of citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen, and banana long spot pathogen in the center of the plate. With this as the center, 5 μL of the bacterial solution of Example 1 was dropped at the midpoints of the four sides of the square 2.5 cm away from the center using a pipette. LB was used as a control, and the plate was cultured at a constant temperature of 28°C. After 5 days, the colony diameter was measured by the cross method, and the distance between the edge of the endophytic fungus colony and the edge of the pathogen colony was measured to observe whether the pathogen colony formed an isolation zone. Each treatment was repeated 3 times.

[0074] (2) Verification of the antibacterial effect of strain E17-23 by split plate method

[0075] PDA culture medium was poured on both sides of the partition plate, 50 μL of the bacterial solution of Example 1 was dripped on one side for coating, and the other side was inoculated with bacterial cakes of citrus brown spot pathogen, citrus anthracnose pathogen, citrus lipid spot yellow spot pathogen, banana wilt pathogen and banana long spot pathogen, with LB as the control. The plates were cultured at a constant temperature of 28°C, and the diameter of the pathogen colonies was observed and measured after 5 days.

[0076] (3) Verification of the antibacterial effect of the sterile filtrate of strain E17-23 at different shaking times

[0077] Prepare a perforated plate with citrus canker bacteria by referring to the method in "(2) Biocontrol bacteria screening". Add 20 μL of sterile filtrate of Example 2 and Comparative Example 1 with different shaking times to each well. Repeat each treatment three times. The experimental control is 20 μL LB medium and 20 μL of strain E3-01 bacterial solution with no antibacterial effect. Blow dry and seal. Put it in a 28°C incubator for constant temperature culture. After 24 hours, observe whether a transparent antibacterial zone appears.

[0078] (3) Antibacterial effect of aseptic fermentation filtrate of strain E17-23 on different pathogens

[0079] The sterile fermentation filtrate obtained by shaking for 48 hours in Example 2 was mixed with PDA medium at a ratio of 1:9 and poured into plates, and the middle of the medium was inoculated with the bacterial cakes of citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen and banana long spot pathogen. The control group added LB medium in the same proportion and inoculated the same pathogenic bacteria cake in the middle of the medium. The culture was kept at 28°C, and the diameter of the pathogen colony was observed and measured after 5 days.

[0080] (4) Control effect of strain E17-23 on citrus leaf canker

[0081] On the well-growing grafted plants, select new leaves that have reached the maximum leaf area but have not yet completely turned green. Wipe the front and back of the leaves with alcohol. After the alcohol evaporates, gently turn the leaves over so that the backs of the leaves are facing up. Use a 1mL syringe needle to lightly puncture two small holes in the veins on both sides, being careful not to pierce the leaves. Then use a syringe to inject the citrus canker pathogen solution into the mesophyll tissue. The injection volume at each point is about 10 μL, and the mesophyll tissue between the veins on both sides is completely infiltrated. Subsequently, the same method is used to inject about 10 μL of the E17-23 bacterial solution obtained in Example 3 into the two holes on the right side of the leaf, and LB is injected on the left side as a control. After each injection, wipe off the residual liquid on the leaf surface with absorbent paper, and then place the plants in a greenhouse for unified fertilizer and water management. After 7 days, collect the leaves, take pictures, and measure the size of the lesions. (5) Control effect of strain E17-23 on citrus leaf Huanglongbing

[0082] The citrus half-leaf method was used to determine the control effect of biocontrol bacteria on citrus Huanglongbing. Citrus leaves with symptoms and similar morphological characteristics such as size were collected and brought back to the laboratory and stored at 4℃ for use.

[0083] The leaves were cut into two halves perpendicular to the veins. One half was placed in LB, while the other half was placed in the bacterial solution of Example 3. Each treatment included 6 leaves, and the experiment was repeated 3 times. After standing at room temperature for 24 hours, the plant genomic DNA extraction kit (axygen) was used to extract the leaf midrib DNA and perform RT-PCR.

[0084] Control effect = (Huanglongbing bacteria content in the control group - Huanglongbing bacteria content in the treatment group) / Huanglongbing bacteria content in the control group (6) Strain E17-23 can promote the growth of orange seedlings

[0085] Prepare test plants, take orange seedlings of similar seedling age and growth conditions, rinse with clean water, plant them in a seedling cup filled with sterilized nutrient soil, and place them in an artificial climate incubator with a light intensity of 16h and 25°C. After 7 days of routine management, treat. Dilute the E17-23 bacterial solution obtained in Example 1 10 times with clean water, and irrigate the roots. Each orange seedling is irrigated with about 10mL of bacterial solution. Control: Each orange seedling is irrigated with about 10mL of LB diluted 10 times. Regularly measure the growth indicators of citrus seedlings.

[0086] Results Analysis

[0087] (1) Inhibitory effects of different Bacillus spores on citrus canker pathogen

[0088] according to Figure 1 A and Figure 1B The results showed that compared with the control group LB and strain E3-01, the 10 strains including strain E17-23 all showed obvious transparent inhibition zones after treatment, and the diameters of the inhibition zones were all >2.60 cm, indicating that the above 10 strains had a significant inhibitory effect on citrus canker bacteria, and strain E17-23 had the best inhibitory effect ( Figure 1 B). Combined with the results in Table 1, the strain E17-23 with an average inhibition zone diameter of 3.17±0.12cm is Bacillus Velez, and the other two strains E3-02 and E17-20 with average inhibition zone diameters of 2.75±0.05cm and 2.60±0.10cm are also Bacillus Velez. Different strains have different abilities to secrete antibiotics, produce volatile organic compounds, activate plant immunity, have different adaptability to culture conditions, and have genetic differences between strains. Therefore, not all strains of Bacillus Velez have excellent inhibitory effects on citrus canker, and the antibacterial effect of Bacillus Velez E17-23 is better than that of other Bacillus biocontrol strains.

[0089] (2) Antibacterial effect of Bacillus velezensis E17-23 on various pathogens

[0090] like Figure 4 As shown in A, in the control group, all pathogens grew normally, covering the surface of the culture medium and showing typical colony morphology. In the culture dishes treated with Bacillus Velez E17-23, the growth of pathogens was significantly inhibited, and obvious inhibition zones were visible on some culture dishes. Figure 4 The inhibitory effect of Bacillus Velez E17-23 on citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen and banana long spot pathogen is greater than 60%.

[0091] (3) The volatile products of Bacillus velezensis E17-23 have antibacterial effects

[0092] like Figure 5 As shown in A, in the control group where LB was applied on one side of the separation plate, all pathogens grew normally. However, after applying the E17-23 bacterial solution, the colony diameters of all pathogens were significantly reduced. Figure 5 As shown in B, after applying the bacterial solution of Bacillus Velez E17-23, the colony diameters of citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen and banana long spot pathogen were all reduced by more than 14.95%, indicating that the volatile products of strain E17-23 can significantly inhibit the growth of pathogens.

[0093] (4) Antibacterial effect of sterile fermentation filtrates of Bacillus velezensis E17-23 cultured at different times

[0094] like Figure 6As shown in Figure 6A, the plate confrontation results show that compared with the LB control, the sterile fermentation filtrates of strain E17-23 with different shaking times in Example 2 and Comparative Example 1 can produce obvious inhibition zones. As shown in Figure 6B, the best antibacterial effects were obtained after culturing for 48h and 72h, with the diameters of the inhibition zones being 1.62±0.03cm and 1.63±0.03cm, respectively. This indicates that the biocontrol bacteria E17-23 has the most effective antibacterial substances accumulated in its fermentation broth when the culturing time is between 48h and 72h.

[0095] (5) Antibacterial effect of 48h sterile fermentation filtrate of Bacillus velezensis E17-23

[0096] like Figure 7 As shown in A, all pathogens on the PDA plate with LB liquid medium grew normally. However, the colony diameters of all pathogens on the PDA plate with the sterile fermentation filtrate of Bacillus Velezii E17-23 cultured for 48 hours were significantly reduced. Figure 7 B shows that the inhibitory effect of the sterile fermentation filtrate of Bacillus Velez E17-23 cultured for 48 hours on citrus brown spot pathogen, citrus anthracnose pathogen, citrus yellow spot pathogen, banana wilt pathogen and banana long spot pathogen is greater than 20.83%, indicating that the sterile fermentation filtrate of Bacillus Velez E17-23 cultured for 48 hours can significantly inhibit the growth of pathogens.

[0097] (5) Control effect of Bacillus Velezii E17-23 on leaf canker of potted citrus

[0098] like Figure 8 As shown in A, leaves inoculated with Xac alone show larger lesion areas, indicating that Xac successfully infected and spread. Leaves inoculated with Xac and Bacillus Velez E17-23 show significantly smaller lesions, indicating that Bacillus Velez E17-23 slowed down the spread of Xac on leaves. Figure 8 B shows the quantitative comparison of the lesion area. The lesion area of ​​the Xac+E17-23 treatment group was smaller, at 0.47±0.13cm 2 The lesion area in the Xac group was larger (0.93±0.04cm 2 ), indicating that Bacillus Velezii E17-23 has a significant inhibitory effect on citrus canker.

[0099] (6) Control effect of Bacillus Velezii E17-23 on citrus leaf Huanglongbing

[0100] like Fig. 9 As shown in the figure, the 16S rDNA copy number of citrus Huanglongbing disease fungus treated with LB medium was (4.54±0.24)×10 8 / g, but it was significantly reduced to (2.24±0.07)×10 8 / g, the control effect reaches 51%.

[0101] (7) Bacillus Velez E17-23 can promote the growth of orange seedlings

[0102] Fig.10 A shows the comparison of plant growth after treatment with Bacillus Velez E17-23 and LB. The average growth of the E17-23 treatment group was 2.97±0.74 cm, and the average growth of the LB treatment group was 1.07±0.48 cm. Fig.10 B shows a comparison of the number of newly grown leaves of plants after treatment with Bacillus Velez E17-23 and LB. The strain E17-23 treatment group grew an average of 6.17±2.04 new leaves, and the LB treatment group grew an average of 2.00±0.63 new leaves. The plant growth and number of newly grown leaves of the plant treated with Bacillus Velez E17-23 were higher than those of the LB treatment, indicating that the Bacillus Velez E17-23 of the present invention can significantly promote plant growth.

[0103] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Bacillus Velezii E17-23, characterized in that The deposit number is GDMCC No.64580, the deposit date is April 29, 2024, the deposit unit is Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The Bacillus Velezii E17-23 according to claim 1, characterized in that The Velez Bacillus E17-23 and its culture or metabolites can inhibit citrus Huanglongbing fungus, citrus canker fungus, citrus brown spot fungus, citrus anthracnose fungus, citrus fat spot yellow spot fungus, banana wilt fungus and banana long spot fungus.

3. The Bacillus Velez E17-23 according to claim 1, characterized in that The Velez Bacillus E17-23 and its metabolites can promote plant growth.

4. A use of the Bacillus Velezii E17-23 according to any one of claims 1 to 3, characterized in that: Used to prepare biological preparations for preventing and controlling plant diseases and / or promoting plant growth.

5. The use of Bacillus Velez E17-23 according to claim 4, characterized in that: The biological preparation uses Bacillus Velezii E17-23 and its culture or metabolites as active ingredients.

6. The use of Bacillus Velez E17-23 according to claim 4, characterized in that: The plant diseases include citrus Huanglongbing, citrus canker, citrus brown spot, citrus anthracnose, citrus fat spot yellow spot, banana wilt and banana long spot pathogen.

7. The use of Bacillus Velez E17-23 according to claim 4, characterized in that: The promoting plant growth is promoting the growth of citrus plants.

8. The use of Bacillus Velez E17-23 according to claim 4, characterized in that: In the biological preparation, Bacillus Velez E17-23 is cultured with shaking for 48 to 72 hours.

9. The use of Bacillus Velez E17-23 according to claim 4, characterized in that: In the biological preparation, the concentration of Bacillus Velez E17-23 is 10 6 ~109CFU / mL.

10. The use of Bacillus Velez E17-23 according to claim 4, characterized in that: The dosage forms of the biological preparation include water suspension, granules, powders and microcapsules.

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