Bacillus velezensis for antagonizing watermelon pathogenic bacteria and application of bacillus velezensis
By screening and application of Berrez Bacteria XI-144 and its fermentation solution, the problem of prevention and treatment of watermelon fungal disease diseases is solved, and the effective suppression of watermelon shells and spyjate bacteria Advantage.
Patent Information
- Application Number
- CN202411472551.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-22
- Publication Date
- 2025-01-21
AI Technical Summary
The prior art is difficult to effectively prevent and control watermelon fungal diseases, and the use of chemical pesticides leads to pathogen resistance and environmental pollution.
A strain of Bacillus veleis xi-144, which is antagonistic to watermelon pathogenic bacteria, and its fermentation broth were screened and applied, and fermented through liquid fermentation medium to prepare an agent with the activity of inhibiting Bacillus solani and Fusarium oxysporum in watermelon.
This bacteria has significant inhibitory activity for the watermelon shell and spine -spyjylobacteria, and does not require high -pressure sterilization. The fermentation liquid still maintains inhibitory activity after high temperature sterilization, and has thermal stability and high temperature resistance.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biological pesticides, and particularly relates to a Velez subtilis strain antagonizing watermelon pathogens and application thereof. Background Art
[0002] Watermelon is a specialty industry in Hainan, and continuous planting has led to frequent diseases. The pathogens that cause watermelon diseases mainly include Stagonosporopsis citrulli and Fusarium oxysporum. Diseases caused by Stagonosporopsis citrulli can occur throughout the entire growth period of watermelon. Fusarium oxysporum colonizes in the vascular bundles of the plant roots, causing blockage of the vessels, withering of the aboveground parts, brown necrotic spots on the root epidermis, and later root rot and even death of the entire plant. The two fungi seriously affect the growth, development, yield and quality of plants.
[0003] The control of plant diseases caused by Fusarium oxysporum and Stagonosporopsis citrulli mainly relies on chemical agents. Studies have shown that the effects of some chemical pesticides are gradually weakened, and even the pathogens are resistant to mixed chemical pesticides. In addition, long-term use of chemical control methods will not only leave chemical agents in watermelons, but also cause pathogens to develop resistance and cause irreversible pollution to the environment. Therefore, it is necessary to develop safer, more efficient and more stable biocontrol bacteria for the control of Stagonosporopsis citrulli and Fusarium oxysporum.
[0004] Currently, there is little research on microbial agents specifically for watermelon fungal diseases. Summary of the invention
[0005] In view of the shortcomings of the prior art, the present invention proposes a strain of Bacillus Velezii antagonistic to watermelon pathogens and its application. The bacterial agent disclosed in the present invention has simple ingredients and has inhibitory activity against Stagonosporopsis citrulli and Fusarium oxysporum without high-pressure sterilization. The bacterial agent still has inhibitory activity after high-temperature sterilization of the fermentation liquid, indicating that it has certain thermal stability and high temperature resistance.
[0006] The technical solution of the present invention is as follows:
[0007] The invention screens out a Bacillus Velezii antagonistic to watermelon pathogens. The Bacillus Velezii xi-144 is preserved in Guangdong Microbial Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, the preservation number is GDMCC No: 64952, and the preservation time is February 5, 2024.
[0008] Furthermore, the present invention provides a fermentation broth of the Bacillus Velez subtilis, and the preparation method of the fermentation broth comprises the following steps: inoculating the bacterial liquid of the Bacillus Velez subtilis into a liquid fermentation medium for fermentation culture; the liquid fermentation medium comprises a YPG medium, a sugar-free YPG medium or a NB medium; the sugar-free YPG medium and the NB medium are sterilized by high pressure before use.
[0009] Preferably, the fermentation culture conditions are 28°C to 30°C, shaking constant temperature culture for 3 to 7 days.
[0010] Preferably, the method for preparing the fermentation broth comprises the following steps: inoculating a bacterial liquid of Bacillus Velezii into a liquid fermentation medium for fermentation culture to obtain a first fermentation broth; adding the first fermentation broth to a YPG medium sterilized by high temperature for fermentation culture to obtain a single culture fermentation broth.
[0011] Preferably, the bacterial liquid is a bacterial liquid obtained by inoculating the Bacillus Velez subtilis into LB medium and shaking and culturing it.
[0012] Preferably, the YPG medium contains 10 g / L yeast extract, 20 g / L trypsin, and 20 g / L glucose; the sugar-free YPG liquid medium contains 10 g / L yeast extract and 20 g / L trypsin; and the NB medium contains 3 g / L beef extract, 8 g / L trypsin, and 5 g / L sodium chloride.
[0013] On the other hand, the present invention also provides the use of the Bacillus Velez and / or its fermentation product in inhibiting the growth of watermelon pathogenic fungi.
[0014] Preferably, the pathogenic fungi include at least one of Stagonosporopsis citrulli and Fusarium oxysporum.
[0015] Preferably, the fermentation product comprises the fermentation broth.
[0016] Beneficial effects of the present invention:
[0017] The Bacillus Velezii xi-144 provided by the present invention is derived from the rhizosphere soil in the watermelon shed of the experimental farm of the fifth team of Baodao New Village, Nanda Town, Danzhou City, Hainan Province, which ensures that the strain can effectively colonize and survive after being returned to the watermelon field, laying a foundation for the practicality of developing microbial agents in the future.
[0018] Previous studies have mainly focused on the prevention and control of watermelon continuous cropping problems, but have not focused on pathogens. The present invention focuses on the microbial agents prepared for the important pathogens Stagonosporopsis citrulli and Fusarium oxysporum that cause Hainan watermelon diseases, and is more targeted.
[0019] The present invention uses 3 different liquid culture media, which are divided into two situations: the medium is sterilized under high pressure and the medium is not sterilized under high pressure, for a total of 6 liquid culture media. 6 different fermentation broths were prepared for activity comparison. The results show that the YPG liquid culture medium fermentation broth sterilized under high pressure has the best inhibitory activity against pathogens and the highest inhibition rate. The fermentation broth prepared without high pressure sterilization of the YPG culture medium also has inhibitory activity. If the culture medium does not need to be sterilized under high pressure when used to produce fermentation bacteria, it will save a lot of cost and time, and it will also be more convenient.
[0020] Based on the interaction of microorganisms, new antibiotics can be induced to produce and enhance the activity of fermentation broth. The present invention sets up YPG fermentation broth of xi144 monoculture and xi144 co-cultured with Fusarium oxysporum, calculates the inhibition rate, and selects the optimal culture method. The results show that the inhibitory effect of the fermentation broth of monoculture is better than that of the co-cultured fermentation broth. The thermal stability of the active substances in the fermentation broth was evaluated, and the results showed that the fermentation broth after high-pressure sterilization still has a strong inhibitory activity against pathogens, indicating that the active substances produced by the fermentation broth have a certain heat resistance. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 : Photos of the colony morphology of Bacillus velezii xi144.
[0022] Figure 2 :Image of Bacillus Velez strain xi144 isolated from Fusarium oxysporum.
[0023] Figure 3 : Pictures showing that Bacillus Velezii xi144 inhibits the growth of the plant pathogenic fungus Fusarium oxysporum.
[0024] Figure 4 : The inhibitory effect of six fermentation broths on Fusarium oxysporum.
[0025] Figure 5:The inhibitory effects of monoculture, co-culture and heat treatment groups on Stagonosporopsis citrulli and Fusarium oxysporum. DETAILED DESCRIPTION
[0026] In order to better understand the technical content of the present invention, the present invention is further described below through specific embodiments.
[0027] Example 1 Isolation and Identification of Bacillus velezensis xi144
[0028] (1) Preparation of culture medium
[0029] LB medium was prepared according to the following components in mass volume ratio: 5 g / L yeast powder, 10 g / L tryptone, 10 g / L NaCl, and 20 g / L agar.
[0030] (2) Sample collection and processing
[0031] By collecting soil samples from the watermelon shed of the Experimental Farm of the Fifth Team of Baodao New Village, Danzhou City, Hainan Province, China, an antagonistic strain that can effectively inhibit a variety of pathogens was isolated and purified.
[0032] (3) Strain isolation and purification
[0033] LB culture medium was selected as the separation plate to separate the soil strains. Take 1g of soil sample and add 10ml of sterile water to mix. After constant temperature and 200r / min shaking culture at 28℃ for 30min, a soil sample suspension of 10° was obtained. The soil sample suspensions of 10-1, 10-2, 10-3, and 10-4 were obtained by multiple dilution. 100μL of the 10-3 and 10-4 soil sample suspensions were evenly spread on the LB culture medium and cultured at 28℃ for 2-4 days. The colonies grown on the LB culture medium were selected to confront Fusarium oxysporum. The results were observed as follows: Figure 2 The results are shown in Figure 2. The Velez Bacillus strain xi144 was tested against Fusarium oxysporum alone. Figure 3 As shown. The Velez Bacillus strain xi144 was isolated and cultured on LB medium for purification, and then stored in a glycerol tube at -80°C. An antagonistic strain Bacillus velezensis xi144 that can effectively inhibit a variety of pathogens was isolated and purified. It was abbreviated as xi144. Its colony morphology is shown in Figure 1 As shown, its gene sequence is shown in SEQ ID NO.1.
[0034] The morphological characteristics of the strains are shown in Table 1:
[0035] Table 1 Morphological characteristics of strains
[0036] index Colony characteristics Colony morphology Round Colony color milky pigment none Colony surface Rough Degree of bulge Slightly raised Edge growth conditions irregular transparency opaque
[0037] (4) Identification results of strain xi144
[0038] After the strain xi144 was purified, it was inoculated on an LB plate and cultured at 28°C for 5 days. The plate with bacteria was directly handed over to Hainan Lifefit Biotechnology Co., Ltd. for sequencing and strain identification. The results showed that the strain was Bacillus Velezii.
[0039] The strain xi144 was deposited in Guangdong Microbiological Culture Collection Center with the deposit number: GDMCC No: 64952.
[0040] Example 2 Antibacterial Test of Bacillus velezensis xi144 Strain against Plant Pathogenic Fungi
[0041] The plant pathogenic fungi tested: Stagonosporopsis citrulli and Fusarium oxysporum were donated by the Winter Vegetable Research Center of the Institute of Tropical Crops Variety Resources, Chinese Academy of Tropical Agricultural Sciences.
[0042] Culture medium: PDA medium
[0043] Test method:
[0044] Plate confrontation method: On the activated plant pathogenic fungus plate, take the pathogen and inoculate it in the center of the PDA medium plate, then use a sterile inoculation loop to inoculate the bacteria at the four corners (2×4cm range) with a spacing of 2cm, select bacteria-fungus co-culture (Bacillus Velez xi144 and pathogenic fungi), and use the plate with only pathogens on the left side of the plate as the control group, keep it at a constant temperature of 28℃, and record the growth status of the strains at each period. The results show that Bacillus Velez can significantly inhibit the growth of pathogenic fungi.
[0045] Example 3 Preparation of six fermentation broths of strain xi144 and testing of resistance to plant pathogens
[0046] The preparation method of the bacterial liquid of Bacillus Velez xi144 is as follows: the colonies of the Bacillus Velez xi144 are collected and inoculated on a sterile LB medium, cultured at a constant temperature of 28°C for 2 to 4 days, purified twice on the LB medium, cultured under the same conditions for 2 to 4 days, inoculated the bacterial blocks into an LB liquid culture medium, placed in a shaker at 28°C to 30°C and 180 to 200 r / min for 2 days, and cultured to obtain a bacterial liquid for use.
[0047] Fermentation broth culture medium: YPG liquid culture medium, sugar-free YPG liquid culture medium, NB liquid culture medium.
[0048] YPG liquid culture medium: yeast extract 10g / L, trypsin 20g / L, glucose 20g / L; sugar-free YPG liquid culture medium: yeast extract 10g / L, trypsin 20g / L; NB liquid culture medium: beef extract 3g / L, trypsin 8g / L, sodium chloride 5g / L.
[0049] The three liquid culture media are divided into two conditions: medium autoclaved and medium not autoclaved, for a total of 6 liquid culture media. LB bacterial solution of xi144 was added to the 6 liquid culture media, LB bacterial solution: liquid culture medium = 1:50, and cultured at 28°C to 30°C, 200r / min shaking constant temperature for 3 to 7 days to obtain 6 different fermentation broths.
[0050] Test method:
[0051] Plate confrontation method: On the activated Fusarium oxysporum plant pathogenic fungus plate, use a sterile puncher to make a bacterial cake for use, close to the center of the PDA culture medium plate, and then use a sterile puncher to punch holes in the four corners with a spacing of 2cm (2×4cm range). Use a 0.22μm sterile filter to filter 6 kinds of fermentation broth to obtain sterile fermentation broth, and add 110μL of the same sterile fermentation broth to 4 holes. Select fermentation broth-fungus co-culture (six kinds of sterile fermentation broth of Bacillus Velez xi144 and Fusarium oxysporum), with the pathogenic fungus cake attached to the center of the plate and 110μL of sterile water added to the 4 holes as the blank control group, and culture at a constant temperature of 28℃ for 4-7 days, and record the growth status of the pathogen strain. Results of 7 days of culture: The fermentation broths of the three different liquid culture media sterilized by the medium all had an inhibitory effect on Fusarium oxysporum. In the group of medium not sterilized by high pressure, only YPG had an inhibitory effect on the pathogen. Figure 4 shown.
[0052] The inhibition rate of the fermentation broth was calculated, inhibition rate = (control colony growth diameter - treated colony growth diameter) / control colony growth diameter × 100%. The results are shown in Table 2 below.
[0053] Table 2 Results of the inhibition of pathogens by fermentation broth
[0054]
[0055] Example 4 Single co-culture activity test and thermal stability test of strain xi144
[0056] Fermentation medium: YPG liquid medium, PDA liquid medium. PDA liquid medium: potato 200g / L, glucose 20g / L.
[0057] On the activated Fusarium oxysporum PDA plate, the bacteria were inoculated into the PDA liquid culture medium, and placed in a shaker at 28°C and 180-200rpm for 3-7 days to obtain the PDA fermentation liquid. LB fermentation liquid was added to the YPG liquid culture medium sterilized at a ratio of 2% to obtain a single culture group; LB fermentation liquid and PDA fermentation liquid were added to the YPG liquid culture medium sterilized at a ratio of 2% to obtain a co-culture group. Two different fermentation liquids were obtained by constant temperature culture at 28°C and 200r / min for 3-7 days. Appropriate amounts of bacterial liquid were taken from the single culture group and the co-culture group and placed in an autoclave for high-pressure sterilization to obtain heat-treated single culture group and co-culture group, which were used to test the thermal stability of the fermentation liquid, with a total of 4 fermentation liquids to be tested.
[0058] Test method:
[0059] Plate confrontation method: On the activated Stagonosporopsis citrulli and Fusarium oxysporum plant pathogenic fungi plates, use a sterile hole puncher to make a bacterial cake for later use, close to the center of the PDA culture medium plate, and then use a sterile hole puncher to punch holes at the four corners with an interval of 2 cm (2×4 cm range). Use a 0.22μm sterile filter head to filter the four fermentation broths to be tested to obtain four sterile fermentation broths. Add 110μL of the same sterile fermentation broth to the four wells. Select the fermentation broth-fungus co-culture (Bacillus Velez xi144 fermentation broth and two pathogenic fungi) and culture under the same conditions for 4 to 7 days. The results of 7-day observations showed that the fermentation broth had inhibitory activity against Stagonosporopsis citrulli and Fusarium oxysporum, such as Figure 5 shown.
[0060] The inhibition rate of the fermentation liquid to be tested was calculated, inhibition rate = (control colony growth diameter - treatment colony growth diameter) / control colony growth diameter × 100%. The results are shown in Table 3 below:
[0061] Table 3 Results of the inhibition of pathogens by fermentation broth
[0062]
[0063] The final results showed that the monoculture effect of the fermentation broth of Bacillus Velezii xi144 YPG liquid medium was better than that of the co-culture, and the monoculture effect after heat treatment was also better than that of the co-culture. The monoculture and co-culture fermentation broths still had certain inhibitory activity after high-pressure sterilization, indicating that the active substances produced by the fermentation broth had certain thermal stability and high temperature resistance.
[0064] In summary, the fermentation liquid prepared by the Bacillus velezensis xi144 provided by the present invention using YPG medium has significant antibacterial activity against Stagonosporopsis citrulli and Fusarium oxysporum, and has antibacterial activity without high-temperature sterilization liquid, which provides great convenience for future production processes. The fermentation liquid still has a certain antibacterial effect after high-pressure sterilization, and the active substances produced by the fermentation liquid have certain thermal stability and high temperature resistance, providing new strain resources for biological control of plant diseases.
[0065] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.
Claims
1. A strain of Bacillus Velezii antagonistic to watermelon pathogens, characterized in that: The Bacillus Velez is deposited in Guangdong Microbiological Culture Collection Center with the deposit number of GDMCC No: 64952.
2. The fermentation liquid of Bacillus Velezii according to claim 1, characterized in that The method for preparing the fermentation liquid comprises the following steps: inoculating the bacterial liquid of Bacillus Velez into a liquid fermentation medium for fermentation culture; the liquid fermentation medium comprises a YPG medium, a sugar-free YPG medium or a NB medium; the sugar-free YPG medium and the NB medium are sterilized by high pressure before use.
3. The fermentation broth according to claim 2, characterized in that The fermentation culture conditions are 28°C to 30°C and shaking constant temperature culture for 3 to 7 days.
4. The fermentation broth according to claim 2, characterized in that The method for preparing the fermentation liquid comprises the following steps: inoculating the bacterial liquid of Bacillus Velezii into a liquid fermentation medium for fermentation culture to obtain a first fermentation liquid; adding the first fermentation liquid into a YPG medium that has been sterilized at high temperature for fermentation culture to obtain a single culture fermentation liquid.
5. The fermentation broth according to claim 2, characterized in that The bacterial solution is obtained by inoculating the Bacillus Velez subtilis into LB medium and shaking and culturing it.
6. The fermentation broth according to claim 2, characterized in that The YPG culture medium contains 10 g / L yeast extract, 20 g / L tryptone, and 20 g / L glucose; the sugar-free YPG liquid culture medium contains 10 g / L yeast extract and 20 g / L tryptone; and the NB culture medium contains 3 g / L beef extract, 8 g / L tryptone, and 5 g / L sodium chloride.
7. Use of the Bacillus Velez and / or its fermentation product according to claim 1 in inhibiting the growth of watermelon pathogenic fungi.
8. The use according to claim 7, characterized in that: The pathogenic fungi include at least one of Stagonosporopsis citrulli and Fusarium oxysporum.
9. The use according to claim 7, characterized in that: The fermentation product comprises the fermentation broth according to any one of claims 2 to 6.