Bacillus Velez LS77 and its application

Abstract

The invention discloses Bacillus Velez LS77 and its application, belonging to the field of biotechnology. The invention provides Bacillus Velez LS77 on one hand, and provides the application of Bacillus Velez LS77 in inhibiting Botrytis cinerea on the other hand. The Bacillus Velez LS77 separated by the invention can be fermented with squid processing waste, and the fermentation liquid obtained by fermentation can significantly inhibit Botrytis cinerea, thereby providing strain resources for biological control of gray mold and providing a theoretical basis for the development of green biological pesticides.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to Bacillus velez LS77 and application thereof. Background Art

[0002] Common plant diseases caused by fungi include gray mold, anthracnose, powdery mildew, rust, and smut, accounting for 80% of all plant diseases. The use of biological pesticides has many advantages, such as: (1) almost no impact on humans, livestock, and the ecological environment; (2) no pollution to agricultural products; (3) strong targeting of target bacteria and pests; (4) protection of natural enemies of pests; (5) pests and pathogens are not easy to develop resistance. Biological pesticides are one of the most effective ways to prevent and control plant diseases and are also the most ideal choice for green agriculture. Therefore, the research and development of low-toxic, environmentally friendly green biological pesticides has become an inevitable development trend in the field of pesticide science.

[0003] Squid is a marine economic animal of the cephalopod class of mollusks. It has a short life cycle, strong reproductive capacity, is rich in high protein and multiple essential amino acids, and is a low-fat aquatic product. During the squid processing process, about 15% of waste will be generated, which is called squid processing by-products. Although it is a discarded part, the nutritional value contained in it is extremely rich, rich in fat and protein, and if it can be used, it can produce good economic benefits; the squid viscera are rich in organic matter and are an excellent culture medium for microorganisms. Using squid viscera as a culture medium, fermentation and preparation of biological pesticides with anti-plant fungal disease activity can, on the one hand, realize the high-value utilization of squid viscera, and on the other hand, the developed products have anti-fungal disease activity and have the potential to replace chemical pesticides.

[0004] The inventors isolated strains that can ferment and utilize squid viscera from a variety of fermented aquatic products, screened out strains whose fermentation broth had high anti-Botrytis cinerea activity through the mycelium growth inhibition method, classified and identified the strains using morphological and molecular biological methods, and found a strain of Bacillus Velezii that had a significant inhibitory effect on Botrytis cinerea. This provides strain resources for the biological control of gray mold and a theoretical basis for the development of green biological pesticides. Summary of the invention

[0005] In view of the problems existing in the prior art, the object of the present invention is to design and provide a technical solution for Bacillus Velezii LS77 and its application.

[0006] The present invention is specifically implemented by the following technical solutions:

[0007] The first aspect of the present invention provides a Bacillus Velezii ( Bacillus velezensis ) LS77, its deposit number is: CGMCC No.32058.

[0008] The strain was isolated from naturally fermented shrimp paste in Lanshan District, Rizhao City, Shandong Province. Through morphological analysis and sequencing analysis, it was found that the strain had the highest homology similarity with Bacillus Velezii, with the highest similarity of 99.99%. It can be determined that the strain is Bacillus Velezii ( Bacillus velezensis ).

[0009] The Velez Bacillus LS77 was observed on a solid plate. The colony was round or nearly round with regular edges, moist surface, pale yellow center, sticky texture, easy to pick up, Gram staining was positive, and rod-shaped. The Velez Bacillus LS77 was cultured in LB solid medium at a constant temperature of 37°C for 8 hours to reach the end of the logarithmic phase.

[0010] The second aspect of the present invention provides a Bacillus Velez subtilis fermentation broth, which is obtained by fermenting the above-mentioned Bacillus Velez subtilis LS77.

[0011] The third aspect of the present invention provides a method for preparing a Bacillus Velez fermentation broth, which comprises the following steps:

[0012] The LS77 bacterial liquid of Bacillus Velez is inoculated into LB liquid medium at an inoculation rate of 1-3% by volume, and cultured at 30-40°C and 160-200 r / min for 12-24 hours, and then the bacterial liquid is transferred into fermentation medium at an inoculation rate of 3-6% by volume, and cultured at 27-38°C and 180-200 r / min for 96-144 hours, and the fermentation is completed to obtain the fermentation liquid of Bacillus Velez;

[0013] The fermentation medium contains 5-50 g of squid processing waste materials, 10-30 g of glucose, pH 7.0-10.0, and the balance is water per liter.

[0014] As a preferred method for preparing the fermentation broth, the Bacillus Velez LS77 strain liquid is inoculated into LB liquid culture medium at an inoculation rate of 2% by volume, and cultured at 37°C and 180 r / min for 24 h. The strain is then transferred to the fermentation medium at an inoculation rate of 4% by volume, and cultured at 37°C and 180 r / min for 120 h. The Bacillus Velez fermentation broth is obtained after the fermentation is completed.

[0015] The preferred fermentation medium contains 10 g of squid processing waste, 20 g of glucose, pH 9.0, and the balance is water per liter.

[0016] As a preferred fermentation medium, squid viscera and water were mixed at a ratio of 1:100, homogenized using a tissue homogenizer, and then 2% glucose was added. The pH value was adjusted to 9.0 using 1 M NaOH, and the fermentation medium was sterilized at 121°C for 15 min.

[0017] The fourth aspect of the present invention provides the use of the above-mentioned Bacillus Velez subtilis or the above-mentioned Bacillus Velez subtilis fermentation liquid in the preparation of biological pesticides or antifungal drugs.

[0018] Furthermore, the biological pesticide or antifungal drug in the application is specifically used to inhibit Botrytis cinerea.

[0019] The fifth aspect of the present invention provides a biopesticide, which contains the above-mentioned Bacillus Velez fermentation broth.

[0020] The sixth aspect of the present invention provides an antifungal drug, which contains the above-mentioned Bacillus Velez fermentation broth.

[0021] A seventh aspect of the present invention provides use of the above-mentioned biological pesticide or the above-mentioned antifungal drug in inhibiting Botrytis cinerea.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The Velez subsp. LS77 isolated by the present invention can be fermented with squid processing waste, and the fermentation liquid obtained by fermentation has the function of significantly inhibiting Botrytis cinerea, thereby providing strain resources for biological control of gray mold and providing a theoretical basis for the development of green biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the primary screening of dominant colonies;

[0025] Figure 2 This is the colony morphology of Bacillus velez LS77;

[0026] Figure 3 This is the Gram staining image of Bacillus velez LS77;

[0027] Figure 4 The 16S rDNA phylogenetic tree of strain Bacillus velez LS77 was constructed for the Neighbor-Joining method;

[0028] Figure 5 is the growth curve of Bacillus velez LS77 in LB medium;

[0029] Figure 6 Single factor experiment for optimization of fermentation conditions of Bacillus velez LS77;

[0030] Figure 7 Response surface experiment for optimization of fermentation conditions of Bacillus velez LS77. DETAILED DESCRIPTION

[0031] The present invention will be further described below by means of specific examples.

[0032] Example 1: Bacillus Velez Bacillus velezensis ) Acquisition of LS77

[0033] Sampling time: July 2023.

[0034] Sampling location: Naturally fermented shrimp paste was collected from Lanshan District, Rizhao City, Shandong Province.

[0035] Sample treatment: Sample gradient dilution: Use sterile saline to dilute the sample gradiently, select 10 -1 -10 -3 Serial dilutions were tested.

[0036] Screening medium:

[0037] Primary screening culture medium: 20 g of squid processing waste, distilled water to 1 L, add 20 g agar, natural pH, and autoclave at 121℃ for 15-20 min.

[0038] LB solid medium: 1% peptone, 0.5% yeast extract powder, 1% sodium chloride, adjust pH to 7.2-7.4 with sodium hydroxide. Sterilize the medium at 121°C for 15 min.

[0039] Potato dextrose agar (PDA) medium: 200 g potato, 20 g glucose, distilled water to 1 L, natural pH, add 20 g agar, and autoclave at 121°C for 15 min.

[0040] Fermentation medium: 10 g of squid processing waste, 20 g of glucose, distilled water to 1 L, adjust the pH to 9.0, and sterilize at 121℃ for 15 min.

[0041] Isolation of new microorganisms:

[0042] 1) Plate initial screening

[0043] The conventional dilution plate method was used to isolate the bacterial strains in the samples using LB solid medium as the primary screening medium for strain screening of squid processing waste. 0.1 mL of the sample solution of each concentration was evenly spread on the LB medium plate, cultured at 37°C for 48 h, and single colonies of different morphologies were picked and placed in LB liquid medium, cultured at 37°C and 180 r / min constant temperature shaker for 24 h, and then set aside.

[0044] The initial screening results of plate screening: 9 dominant bacteria that can use squid processing waste as the only nutrient source were isolated from the samples, such as Figure 1 shown.

[0045] 2) Rescreening and purification

[0046] 2.1) Fermentation

[0047] The selected dominant bacteria were inoculated into the fermentation medium at a 5% (V / M) inoculation rate and cultured at 180 r / min and 37°C for 72 h. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4°C, the precipitate was discarded, the supernatant was filtered through a 0.45 μm filter to remove the bacteria, and stored at 4°C for later use.

[0048] 2.2) Determination of antimicrobial activity of fermentation broth by mycelium growth inhibition method

[0049] On the clean bench, transfer 3 mL of the above fermentation liquid into 22 mL of PDA medium that has been sterilized and cooled to 50-60°C, shake well and pour the plate to prepare a PDA plate containing fermentation liquid. Take Botrytis cinerea as the indicator bacteria, and on the Botrytis cinerea plate that has grown for 5-6 days, place a 6 mm diameter bacterial cake on a fresh PDA plate containing fermentation liquid, and culture it in the dark at 25°C. Use sterile water instead of fermentation liquid as the blank control group. When the colony of the control group grows to about 2 / 3 of the culture dish, use the cross method to determine the colony diameter of each treatment. Calculate the mycelial growth inhibition rate (%) of each PDA plate containing fermentation liquid according to the following formula. Three groups of each treatment were parallel, and the average value was taken.

[0050]

[0051] 2.3) Rescreening results

[0052] The fermentation broth of the five dominant bacteria obtained in the initial screening was subjected to antibacterial test analysis, and it was found that after culturing at 25°C for 5 days, the fermentation broth of one of the dominant bacteria had obvious antibacterial activity, as shown in Table 1, indicating that the screened bacteria can produce substances that inhibit the growth of Botrytis cinerea.

[0053] Table 1 Rescreening results

[0054]

[0055] 2.4) Purification of strains by streaking

[0056] The bacterial strain obtained by streaking and rescreening on LB medium plates for three times was stored at -80°C and named LS77.

[0057] 3) Identification of bacterial species

[0058] 3.1) Colony morphology

[0059] The selected LS77 was inoculated onto LB medium plates and cultured at 37°C for 24 h. The morphological characteristics of the colonies on the solid medium, including their size, color, and ridge shape, were observed, and Gram staining was performed.

[0060] The colonies of strain LS77 on LB plates are round or nearly round with regular edges, moist surface, light yellow center protrusion, sticky texture, easy to pick up, Gram-positive, rod-shaped ( Figure 2 , Figure 3 ).

[0061] 3.2) 16S rDNA identification and construction of phylogenetic tree

[0062] Primer design:

[0063] The universal primer 1492R was used: 5′-CTACGGCTACCTTGTTACGA-3′ (shown in SEQ ID NO. 1);

[0064] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (shown in SEQ ID NO. 2).

[0065] The 16S rDNA of strain LS77 was amplified by PCR using primers 1492R and 27F.

[0066] The total length of the 16S rDNA nucleotide sequence of strain LS77 is 1458 bp, and the sequence is shown in SEQ ID NO.3:

[0067]

[0068] The sequencing results were submitted to the NCBI database for comparison, and sequences similar to the sequencing results were searched, and the phylogenetic tree was constructed using MEGA 11 software.

[0069] Comparison with the GenBank database showed that strain LS77 was similar to strain Bacillus velezensis The CCUG similarity reached 98.40%, and combined with the phylogenetic tree (e.g. Figure 4 LS77 was identified as Bacillus velezensis . The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 25, 2024, with the deposit number CGMCC No.32058. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0070] 3.3) Physiological and biochemical test results

[0071] The results of physiological and biochemical identification of strain LS77 are shown in Table 2. As shown in Table 2, the VP test, propionate, D-xylose, and L-arabinose utilization tests were negative; the citrate, gelatin liquefaction, nitrate reduction, and starch hydrolysis tests were positive; the NaCl and pH tolerance tests showed that strain LS77 could not grow under 7% NaCl conditions, but could grow under pH=5.7 growth conditions; it is a facultative anaerobe and can grow in both aerobic and anaerobic environments. Combined with the morphological characteristics, strain LS77 was preliminarily judged to be Bacillus.

[0072] Table 2 Physiological and biochemical identification results of strain LS77

[0073]

[0074] 4) Growth curve of strain LS77

[0075] The strain LS77 was inoculated into LB liquid medium at a 1% (V / V) inoculum and cultured at 37°C and 180 rpm to obtain the following Figure 5 The growth curve shown in Figure 1 is that strain LS77 experienced a short delay period in the culture medium, entered a logarithmic growth period at about 2 h, and entered a stable period at about 8 h, during which the total number of cells reached a maximum.

[0076] Example 2: Preparation of Bacillus Velez LS77 Seed Solution

[0077] The seed liquid of Bacillus Velez LS77 was inoculated into LB liquid culture medium at a volume ratio of 2%, and cultured at 37°C and 180 r / min for 24 hours to obtain the seed liquid of Bacillus Velez LS77.

[0078] Example 3: Preparation of Bacillus Velez LS77 fermentation broth

[0079] The seed solution obtained in Example 2 was inoculated into the fermentation medium at a solid-liquid ratio of 1:100, 180 r / min, and cultured for a certain period of time. The fermentation liquid was centrifuged at 8000 rpm for 20 min at 4°C, the precipitate was discarded, the supernatant was filtered through a 0.45 μm filter to remove the bacteria, and stored at 4°C for later use.

[0080] A single factor experiment was carried out on the fermentation conditions of strain LS77. According to the single factor experimental design and response surface experiment, the optimal fermentation conditions were obtained as follows: fermentation temperature 37℃, inoculation size 4%, and fermentation time 5 d. The inhibition rate of the fermentation broth of strain LS77 against Botrytis cinerea could reach 76.6%.

[0081] Single factor experiment: The effects of fermentation temperature (fixed initial pH of fermentation broth = 7.0, fermentation time 72 h, 3% inoculation amount, set temperature range 27-47°C), fermentation medium pH (fixed fermentation temperature 37°C, fermentation time 72 h, 3% inoculation amount, solid-liquid ratio 1:100, set initial pH of fermentation broth 5.0-11.0), inoculation amount (fixed initial pH of fermentation broth = 7.0, fermentation temperature 37°C, fermentation time 72 h, solid-liquid ratio 1:100, set inoculation amount 1-5%), and fermentation time (fixed fermentation temperature 37°C, initial pH of fermentation broth = 7.0, 3% inoculation amount, solid-liquid ratio 1:100, set fermentation time 24 h-216 h) on the fermentation effect of LS77 were investigated. Results are shown in Figure 6 :

[0082] Based on the results of the single-factor experiment, three factors, namely fermentation temperature (A, °C), inoculation amount (B,%) and fermentation time (C, d), were selected, and the supernatant inhibition rate was used as the response value to design a response surface experiment (Table 3). The results are shown in Tables 4 and Figure 7 .

[0083] Table 3 Factors and levels of response surface experiment

[0084]

[0085] Table 4

[0086]

[0087] Example 4: Preparation of Bacillus Velez LS77 fermentation broth

[0088] The seed solution obtained in Example 2 was inoculated into the fermentation medium at a 4% inoculum, and cultured at 180 r / min and 32°C for 120 h. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4°C, the precipitate was discarded, the supernatant was filtered through a 0.45 μm filter to remove the bacteria, and stored at 4°C for later use. Through the above experiments, the antibacterial rate of the fermentation broth of strain LS77 against Botrytis cinerea can reach 71.5%.

[0089] Example 5: Preparation of Bacillus Velez LS77 fermentation broth

[0090] The seed solution obtained in Example 2 was inoculated into the fermentation medium at a 5% inoculum, and cultured at 180 r / min and 38°C for 144 h. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4°C, the precipitate was discarded, the supernatant was filtered through a 0.45 μm filter membrane to remove the bacteria, and stored at 4°C for later use. Through the above experiments, the fermentation broth of strain LS77 had an antibacterial rate of 72.1% against Botrytis cinerea.

[0091] Experiments show that the antibacterial effects of Examples 4 and 5 are similar to that of Example 3.

Claims

1. A Bacillus Velezii ( Bacillus velezensis ) LS77, its deposit number is: CGMCCNo.32058.

2. A Bacillus Velez fermentation broth, characterized in that: The fermentation broth is obtained by fermenting the Bacillus Velez LS77 according to claim 1.

3. The method for preparing a Bacillus Velez fermentation broth according to claim 2, wherein: The following steps are involved: The LS77 bacterial liquid of Bacillus Velez is inoculated into LB liquid medium at an inoculation rate of 1-3% by volume, and cultured at 30-40°C and 160-200 r / min for 12-24 hours, and then the bacterial liquid is transferred into fermentation medium at an inoculation rate of 3-6% by volume, and cultured at 27-38°C and 180-200 r / min for 96-144 hours, and the fermentation is completed to obtain the fermentation liquid of Bacillus Velez; The fermentation medium contains 5-50 g of squid processing waste materials, 10-30 g of glucose, pH 7.0-10.0, and the balance is water per liter.

4. Use of the Bacillus Velez subtilis according to claim 1 or the fermentation liquid of the Bacillus Velez subtilis according to claim 2 in the preparation of a biological pesticide or an antifungal drug, wherein the biological pesticide or the antifungal drug is specifically used to inhibit Botrytis cinerea.

5. A biological pesticide, characterized in that: The biological pesticide contains the Bacillus Velez fermentation liquid described in claim 2, and the biological pesticide is used in inhibiting Botrytis cinerea.

6. An antifungal drug, characterized in that: The medicine contains the fermentation liquid of Bacillus Velezii according to claim 2, and the antifungal medicine is used in inhibiting Botrytis cinerea.

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