Bacillus subtilis for antagonizing watermelon fusarium wilt and gummy stem blight and application thereof

Abstract

The invention discloses a bacillus subtilis for antagonizing watermelon fusarium wilt and gummy stem blight and application of the bacillus subtilis. The bacillus subtilis xi-103 provided by the invention has very strong inhibitory activity on Staronosporopsis citrulli and Fusarium oxysporum, a preparation method of a microbial inoculum is simple, high-pressure sterilization is not needed, and great convenience is provided for production. The number of viable bacteria is large, and the viable bacteria amount can reach 424 billion / mL after 10 days of fermentation. The bacillus subtilis xi-103 is separated from soil around watermelon diseased roots, has good field colonization, and is beneficial to prevention and treatment of watermelon diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biological pesticides and plant disease prevention and control, and particularly relates to a Bacillus subtilis strain antagonistic to watermelon wilt and vine blight and application thereof. Background Art

[0002] Stagonosporopsis citrulli and Fusarium oxysporum are the main pathogens that cause watermelon diseases. Stagonosporopsis citrulli is mainly spread through soil and irrigation, and high air humidity is more conducive to the survival and spread of pathogen spores. Hainan's high temperature and high humidity environment makes diseases caused by Stagonosporopsis citrulli frequent. Fusarium oxysporum colonizes in the vascular bundles of the plant roots, causing duct blockage, 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 pathogenic fungi seriously affect the growth, development, yield and quality of plants.

[0003] At present, the prevention and control of plant diseases caused by Fusarium oxysporum and Stagonosporopsis citrulli mainly rely on chemical agents. Studies have shown that the effects of some chemical pesticides are gradually weakened, and even pathogens develop resistance to mixed chemical pesticides. In addition, with the large-scale use of chemical pesticides, chemical preparations remain in the soil, causing an imbalance in the natural environment of the soil and affecting people's health. Therefore, biological control technology is particularly important. Therefore, it is necessary to develop safer, more efficient, and more stable biological control bacteria for the prevention and control of Stagonosporopsis citrulli and Fusarium oxysporum.

[0004] Bacillus subtilis is a type of G+ rod-shaped saprophytic bacteria that is used in the prevention and treatment of plant diseases. It is an ideal beneficial microorganism for biological control and plays an important role in the green prevention and control of crop diseases. At present, no Bacillus subtilis has been found that can effectively control Stagonosporopsis citrulli and Fusarium oxysporum at the same time. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a Bacillus subtilis strain antagonistic to watermelon wilt and vine blight and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] On the one hand, the present invention provides a Bacillus subtilis strain that is antagonistic to watermelon wilt and vine blight. The strain is deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit number of GDMCC No: 64951, a deposit date of February 5, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] The present invention further provides the fermentation liquid of the Bacillus subtilis, and the preparation method thereof is as follows: the Bacillus subtilis is inoculated into a liquid culture medium and shaken and cultured for 3 to 7 days to obtain the fermentation liquid.

[0009] Preferably, the liquid culture medium includes: YPG liquid culture medium, sugar-free YPG liquid culture medium, NB liquid culture medium or brown sugar bean powder liquid culture medium.

[0010] The formula of the YPG liquid culture medium is: 10 g / L yeast extract, 20 g / L tryptone, and 20 g / L glucose;

[0011] The formula of the sugar-free YPG liquid culture medium is: 10 g / L yeast extract and 20 g / L tryptone;

[0012] The formula of the NB liquid culture medium is: 3 g / L beef extract, 8 g / L tryptone, and 5 g / L sodium chloride;

[0013] The formula of the brown sugar bean powder liquid culture medium is: 23.3 g / L brown sugar, 2 g / L calcium carbonate, 11.1 g / L bean powder, 0.5 g / L potassium dihydrogen phosphate, 1.2 g / L dipotassium hydrogen phosphate, 5 g / L magnesium sulfate heptahydrate, and 3 g / L sodium chloride.

[0014] Preferably, the liquid culture medium is brown sugar bean powder liquid culture medium.

[0015] On the other hand, the present invention also provides the use of the Bacillus subtilis or the fermentation product of Bacillus subtilis in inhibiting plant pathogenic fungi.

[0016] The plant pathogenic fungus is Stagonosporopsis citrulli or Fusarium oxysporum.

[0017] The fermentation product is the fermentation broth.

[0018] Beneficial effects of the present invention:

[0019] The invention provides a biocontrol bacterium which can effectively inhibit Stagonosporopsis citrulli and Fusarium oxysporum, namely Bacillus subtilis xi-103. The biocontrol strain is inoculated in the rhizosphere soil of the watermelon shed in the experimental farm of the fifth team of Baodao New Village, Nada Town, Danzhou City, Hainan Province, so that the strain can be effectively colonized and survived after being applied back to the watermelon field, laying a foundation for the practicality of developing microbial agents in the future.

[0020] The present invention uses three different culture medium formulas, namely YPG, sugar-free YPG and NB, and prepares 6 different fermentation broths according to whether the culture medium is sterilized. Activity tests show that the culture medium treated with high-pressure steam sterilization has strong overall activity, with YPG culture medium being the most prominent. Since the test sample is the filtrate after sterile filtration, it is shown that it mainly exerts its effect by secreting extracellular antibiotics.

[0021] The liquid culture medium of brown sugar bean powder was inoculated with Bacillus subtilis xi-103. The culture medium did not need to be sterilized. The fermentation liquid showed strong antibacterial activity, and the inhibition rate of Fusarium oxysporum reached 50%. The live bacteria count showed that the amount of bacteria in the 5th day of fermentation could reach 517 million CFU / mL, and 4.24 billion CFU / mL in 10 days. The process is simple and the cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the colony morphology of Bacillus subtilis xi-103 provided by the present invention.

[0023] Figure 2 This is a picture of the Bacillus subtilis strain xi-103 isolated from Fusarium oxysporum in the present invention.

[0024] Figure 3 This is a picture showing that the Bacillus subtilis xi-103 provided by the present invention inhibits the growth of the plant pathogenic fungus Fusarium oxysporum.

[0025] Figure 4 These are pictures of Stagonosporopsis citrulli and Fusarium oxysporum.

[0026] Figure 5 These are pictures showing the inhibitory effects of six different liquid culture medium fermentation broths on Fusarium oxysporum.

[0027] Figure 6This is a picture showing the inhibitory effect of the fermentation broth of the seventh culture medium, brown sugar bean powder culture medium, on Stagonosporopsis citrulli and Fusarium oxysporum.

[0028] Figure 7 This is a picture of the bacterial count in the fermentation broth of brown sugar bean powder liquid culture medium. DETAILED DESCRIPTION

[0029] In order to better understand the technical content of the present invention, the present invention is further described below through specific embodiments.

[0030] Example 1 Isolation and Identification of Bacillus subtilis xi-103

[0031] Preparation of culture medium

[0032] LB medium was selected: the components were prepared in the following mass volume ratio: yeast powder 5g / L, trypsin 10g / L, NaCl 10g / L, agar 20g / L.

[0033] Sample collection and processing

[0034] 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.

[0035] Strain isolation and purification

[0036] LB medium was selected as the separation plate to separate the soil strains. 1 g of soil sample was added to 10 ml of sterile water and mixed evenly. After constant temperature and 200 r / min shaking culture at 28°C for 30 min, a soil suspension of 10° was obtained. The suspension was diluted in multiples to obtain 10- 1 、10- 2 、10- 3 、10- 4 Soil sample suspension, 10- 3 、10- 4 Take 100 μL of the soil sample suspension and spread it evenly on LB medium, and culture it at 28℃ for 2 to 4 days. Pick out the colonies growing on LB medium and confront Fusarium oxysporum. Observe the results as follows: Figure 2 Bacillus subtilis strain xi-103 was alone against Fusarium oxysporum, and the results were as follows Figure 3As shown. The Bacillus subtilis strain xi-103 was isolated and cultured on LB medium for purification, and then stored in a glycerol tube at -80°C. An antagonistic strain Bacillus subtilis xi-103 that can effectively inhibit multiple pathogens was isolated and purified, and was named xi-103 for short. Figure 1 The morphological characteristics of the strains are shown in Table 1:

[0037] Table 1 Morphological characteristics of strains

[0038]

[0039]

[0040] Identification results of strain xi-103

[0041] After purification, the strain was inoculated on 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 subtilis.

[0042] The strain xi-103 was deposited in Guangdong Provincial Microbiological Culture Collection Center with a preservation date of February 5, 2024 and a preservation number of GDMCC No: 64951.

[0043] The gene sequence of strain xi-103 is as follows (SEQ ID NO.1):

[0044]

[0045] Example 2 Antibacterial activity test of Bacillus subtilis xi-103 strain against plant pathogenic fungi

[0046] Plant pathogenic fungi tested

[0047] Stagonosporopsis citrulli, Fusarium oxysporum Figure 4 shown.

[0048] Culture medium

[0049] PDA medium

[0050] Test Method

[0051] Plate confrontation method: On the activated test 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 subtilis xi-103 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 test results are as follows: Figure 3 shown.

[0052] Example 3 Preparation of seven fermentation broths of strain xi-103 and testing of resistance to plant pathogens

[0053] The preparation method of Bacillus subtilis xi-103 bacterial liquid is as follows: the colonies of Bacillus subtilis xi-103 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 an 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 200 r / min for 2 days, and cultured to obtain a bacterial liquid for use.

[0054] Fermentation broth medium

[0055] YPG liquid medium, sugar-free YPG liquid medium, NB liquid medium and brown sugar bean powder liquid medium.

[0056] YPG liquid medium: yeast extract 10 g / L, tryptone 20 g / L, glucose 20 g / L;

[0057] Sugar-free YPG liquid medium: yeast extract 10 g / L, tryptone 20 g / L;

[0058] NB liquid culture medium: beef extract 3g / L, tryptone 8g / L, sodium chloride 5g / L;

[0059] Brown sugar bean powder liquid culture medium: brown sugar 23.3g / L, calcium carbonate 2g / L, bean powder 11.1g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1.2g / L, magnesium sulfate heptahydrate 5g / L, sodium chloride 3g / L.

[0060] YPG liquid culture medium, sugar-free YPG liquid culture medium and NB liquid culture medium, divided into two conditions: medium autoclaved and medium not autoclaved, and red sugar bean powder liquid culture medium not subjected to high temperature sterilization, a total of 7 liquid culture media. LB bacterial solution of xi-103 was added to the 7 liquid culture media, the volume ratio of LB bacterial solution: liquid culture medium = 1:50, and 7 different fermentation broths were obtained by constant temperature culture at 28℃~30℃ and 200r / min shaking for 3~7d.

[0061] Test Method

[0062] Plate confrontation method: On the activated Fusarium oxysporum plant pathogenic fungus plate, 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 to filter 6 kinds of fermentation broth to obtain sterile fermentation broth (YPG liquid culture medium with high pressure sterilization and medium without high pressure sterilization, sugar-free YPG liquid culture medium and NB liquid culture medium), and add 110μL of the same sterile fermentation broth to the 4 wells. Incubate at a constant temperature of 28℃ for 4 to 7 days, and record the growth status of the pathogen strain. 7d results: The fermentation broths of the three different liquid culture media with sterilized culture media all have inhibitory effects on Fusarium oxysporum ( Figure 5 ).

[0063] The seventh fermentation liquid, brown sugar bean powder fermentation liquid, was directly added with 110 μL into three wells, and the pathogenic bacteria cake was pasted in the center of the plate. The fermentation liquid-fungus co-culture (Bacillus subtilis xi-103 brown sugar bean powder fermentation liquid and two pathogenic fungi) was selected and cultured under the same conditions for 4 to 7 days. The observation results at 7 days showed that the fermentation liquid had strong inhibitory activity against Stagonosporopsis citrulli and Fusarium oxysporum ( Figure 6 ).

[0064] The inhibition rate of the fermentation liquid with antibacterial activity was calculated, inhibition rate = (control colony growth diameter - treatment colony growth diameter) / control colony growth diameter × 100%. The results are shown in Table 2 below:

[0065] Table 2 Results of the inhibition of pathogens by fermentation broth

[0066]

[0067] Finally, the brown sugar bean powder medium with the best comprehensive conditions such as good activity, low cost, simple preparation method and no need for sterilization was selected as the bacterial fertilizer fermentation medium.

[0068] Example 4 Preparation technology of bacterial fertilizer of strain xi-103 brown sugar bean powder culture medium

[0069] Add LB culture of xi-103 to the liquid culture medium of brown sugar bean powder, LB culture medium: liquid culture medium = 1:50, and culture in a fermenter at 28℃~30℃ and 200r / min for 7~10 days. During this period, count the bacteria in the fermentation liquid in the fermenter. The count was 517 million / mL at 5 days of fermentation, and 4.24 billion / mL at 10 days of fermentation. The microscopic screenshot is as follows Figure 7 shown.

[0070] In summary, the Bacillus subtilis xi-103 provided by the present invention uses brown sugar bean powder culture medium as bacterial fertilizer fermentation liquid, the number of live bacteria in the fermentation liquid is large, and it can reach 4.24 billion in 10 days, and it has strong activity and low cost. Brown sugar bean powder liquid culture medium: brown sugar 23.3g / L, calcium carbonate 2g / L, bean powder 11.1g / L, potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 1.2g / L, magnesium sulfate heptahydrate 5g / L, sodium chloride 3g / L, the required raw material price is low; the preparation method is simple, and the culture medium does not need to be sterilized, which provides great convenience for the production process. And it has a significant antibacterial effect on Stagonosporopsis citrulli and Fusarium oxysporum, providing new strain resources and bacterial fertilizer production process for biological control of watermelon diseases.

[0071] 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 Bacillus subtilis strain antagonistic to watermelon wilt and vine blight, characterized in that: The strain is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 64951.

2. The fermentation broth of Bacillus subtilis according to claim 1, characterized in that The preparation method comprises the following steps: inoculating the bacillus subtilis into a liquid culture medium and carrying out shaking culture for 3 to 7 days to obtain a fermentation liquid.

3. The fermentation broth according to claim 2, characterized in that The liquid culture medium includes: YPG liquid culture medium, sugar-free YPG liquid culture medium, NB liquid culture medium or brown sugar bean powder liquid culture medium.

4. The fermentation broth according to claim 3, characterized in that The formula of the YPG liquid culture medium is: 10 g / L yeast extract, 20 g / L tryptone, and 20 g / L glucose; The formula of the sugar-free YPG liquid culture medium is: 10 g / L yeast extract and 20 g / L tryptone; The formula of the NB liquid culture medium is: 3 g / L beef extract, 8 g / L tryptone, and 5 g / L sodium chloride; The formula of the brown sugar bean powder liquid culture medium is: 23.3 g / L brown sugar, 2 g / L calcium carbonate, 11.1 g / L bean powder, 0.5 g / L potassium dihydrogen phosphate, 1.2 g / L dipotassium hydrogen phosphate, 5 g / L magnesium sulfate heptahydrate, and 3 g / L sodium chloride.

5. The fermentation broth according to claim 2, characterized in that The liquid culture medium is brown sugar bean powder liquid culture medium.

6. Use of the Bacillus subtilis according to claim 1 or the fermentation product of Bacillus subtilis according to claim 1 in inhibiting plant pathogenic fungi.

7. The use according to claim 6, characterized in that: The plant pathogenic fungus is Stagonosporopsis citrulli or Fusarium oxysporum.

8. The use according to claim 6, characterized in that: The fermentation product is the fermentation broth according to any one of claims 2 to 5.

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