Bacillus velezensis LYG42 and application thereof

Abstract

The invention discloses bacillus velezensis LYG42 and application thereof, and belongs to the technical field of biology. On one hand, the invention provides the bacillus velezensis LYG42, and on the other hand, the invention provides application of the bacillus velezensis LYG42 in inhibition of colletotrichum gloeosporioides. The bacillus velezensis LYG42 obtained by separation can be fermented by utilizing squid processing leftovers, and the fermentation liquor obtained by fermentation can obviously inhibit colletotrichum gloeosporioides, so that a strain resource is provided for biological control of anthracnose, and a theoretical basis is provided for development of green biopesticide.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to Bacillus velez LYG42 and application thereof. Background Art

[0002] Fungi are a class of eukaryotic microorganisms that can cause diseases in plants, animals and humans. Fruits with high economic benefits, such as grapes, strawberries and blueberries, are commonly plagued by fungal diseases, such as downy mildew, anthracnose, white rot, gray mold, black pox and brown cob blight.

[0003] Commonly used control agents such as carbendazim, whose residues can cause liver disease and chromosomal aberrations, are toxic to mammals; although methyl thiophanate, isoprodinil, and polyoxin are low-toxic, they are still toxic if used improperly or exposed for a long time. In addition, long-term use of chemical pesticides will cause fungi to develop drug resistance. Therefore, it is urgent to develop efficient, safe, and reliable antifungal products. In recent years, researchers have discovered a series of highly safe antifungal substances, such as biopesticides, which can prevent and control diseases, avoid residues in plants, and ensure food safety. Therefore, the research and development of low-toxic, environmentally friendly green biopesticides has become an inevitable development trend in the field of pesticide science.

[0004] Squid is a marine economic animal of the cephalopod class of molluscs. 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 produced, 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.

[0005] 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-gloeosporioides anthracnose 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 Colletotrichum gloeosporioides. This provides strain resources for the biological control of anthrax and provides a theoretical basis for the development of green biological pesticides. Summary of the invention

[0006] 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 velezensis LYG42 and its application.

[0007] The present invention is specifically implemented by the following technical solutions: The first aspect of the present invention provides a Bacillus Velezii ( Bacillus velezensis ) LYG42, its deposit number is: CGMCC No.32059.

[0008] The strain was isolated from naturally fermented shrimp paste in Lianyungang City, Jiangsu 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 99.93%. It can be determined that the strain is Bacillus Velezii ( Bacillus velezensis ).

[0009] The Velez Bacillus LYG42 was observed on a solid plate. The colony was round or nearly round with regular edges, moist surface, pale white center, sticky texture, easy to pick up, Gram staining was positive, and rod-shaped. The Velez Bacillus LYG42 was cultured in LB solid medium at a constant temperature of 37°C for 24 hours to reach the end of the logarithmic phase.

[0010] The second aspect of the present invention provides a Bacillus Velez fermentation broth, which is obtained by fermenting the above-mentioned Bacillus Velez LYG42.

[0011] The third aspect of the present invention provides a method for preparing a Bacillus Velez fermentation broth, which comprises the following steps: The Velez Bacillus LYG42 strain liquid is inoculated into LB liquid medium at an inoculation rate of 1-3% by volume, and cultured at 37°C and 180 r / min for 24 hours, and then the strain is transferred into fermentation medium at an inoculation rate of 3-5% by volume, and cultured at 27-37°C and 180 r / min for 96-144 hours. The Velez Bacillus fermentation liquid is obtained after the fermentation is completed; 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.

[0012] As a preferred method for preparing the fermentation broth, the Bacillus Velez subtilis LYG42 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 32°C and 180 r / min for 120 h. The Bacillus Velez subtilis fermentation broth is obtained after the fermentation is completed.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] Furthermore, the biological pesticide or antifungal drug in the application is specifically used to inhibit Colletotrichum gloeosporioides.

[0017] The fifth aspect of the present invention provides a biopesticide, which contains the above-mentioned Bacillus Velez fermentation broth.

[0018] The sixth aspect of the present invention provides an antifungal drug, which contains the above-mentioned Bacillus Velez fermentation broth.

[0019] The seventh aspect of the present invention provides the use of the above-mentioned biological pesticide or the above-mentioned antifungal drug in inhibiting Colletotrichum gloeosporioides.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The Velez subsp. LYG42 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 Colletotrichum gloeosporioides, thereby providing strain resources for biological control of anthrax and providing a theoretical basis for the development of green biological pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the primary screening of dominant colonies; Figure 2 This is the colony morphology of Bacillus velez LYG42; Figure 3 is the Gram staining image of Bacillus velez LYG42; Figure 4 The 16S rDNA phylogenetic tree of strain Bacillus velez LYG42 was constructed for the Neighbor-Joining method; Figure 5 is the growth curve of Bacillus velez LYG42 in LB medium; Figure 6 Single factor experiment for optimization of fermentation conditions of Bacillus velez LYG42; Figure 7 Response surface experiment for optimization of fermentation conditions of Bacillus velez LYG42. DETAILED DESCRIPTION

[0022] The present invention will be further described below by means of specific examples.

[0023] Example 1: Bacillus Velez Bacillus velezensis ) Acquisition of LYG42 Sampling time: July 2023.

[0024] Sampling location: Naturally fermented shrimp paste was collected from Lianyungang City, Jiangsu Province.

[0025] Sample treatment: Sample gradient dilution: Use sterile saline to dilute the sample gradiently, select 10 -1 -10 -3 Serial dilutions were tested.

[0026] Screening medium: 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] Isolation of new microorganisms: 1) Plate initial screening 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.

[0031] The initial screening results of plate screening: 7 dominant bacteria that can use squid processing waste as the only nutrient source were isolated from the samples, such as Figure 1 shown.

[0032] 2) Rescreening and purification 2.1) Fermentation 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.

[0033] 2.2) Determination of antimicrobial activity of fermentation broth by mycelium growth inhibition method 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. Using Colletotrichum gloeosporioides as the indicator bacteria, on the Colletotrichum gloeosporioides plate that has grown for 5-6 days, place a 6 mm diameter bacterial cake on a fresh PDA plate containing fermentation liquid, culture in the dark at 25°C, and replace the fermentation liquid with sterile water as the blank control group. When the colony of the control group grows to about 2 / 3 of the culture dish, the cross method is used to determine the colony diameter of each treatment. The mycelial growth inhibition rate (%) of each PDA plate containing fermentation liquid is calculated according to the following formula. Three groups of each treatment are parallel, and the average value is taken.

[0034]

[0035] 2.3) Rescreening results The fermentation broth of the 7 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 5 dominant bacteria had antibacterial activity, as shown in Table 1, indicating that the screened bacteria can produce substances that inhibit the growth of Colletotrichum gloeosporioides.

[0036] Table 1 Rescreening results

[0037] 2.4) Purification of strains by streaking The dominant bacterium with the highest antibacterial activity was purified by streaking three times on LB medium plates and stored at -80°C. This strain was named LYG42.

[0038] 3) Identification of bacterial species 3.1) Colony morphology The selected LYG42 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.

[0039] The colonies of strain LYG42 on LB plates are round or nearly round with regular edges, moist surface, pale white protrusion in the center, sticky texture, easy to pick up, Gram-positive, rod-shaped ( Figure 2 , Figure 3).

[0040] 3.2) 16S rDNA identification and construction of phylogenetic tree Primer design: The universal primer 1492R was used: 5′-CTACGGCTACCTTGTTACGA-3′ (shown in SEQ ID NO. 1); 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (shown in SEQ ID NO. 2).

[0041] The 16S rDNA of strain LYG42 was amplified by PCR using primers 1492R and 27F.

[0042] The total length of the 16S rDNA nucleotide sequence of strain LYG42 is 1458 bp (shown in SEQ ID NO. 3). 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.

[0043] The complete nucleotide sequence of 16S rDNA sequence of strain LYG42:

[0044] Comparison with the GenBank database showed that strain LYG42 was similar to strain Bacillus velezensis The CCUG similarity reached 99.86%, and combined with the phylogenetic tree (e.g. Figure 4 LYG42 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.32059. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0045] 3.3) Physiological and biochemical test results The results of physiological and biochemical identification of strain LYG42 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 LYG42 could not grow under 7% NaCl conditions, but could grow under pH=5.7 growth conditions; it was a facultative anaerobe and could grow in both aerobic and anaerobic environments. Combined with the morphological characteristics, strain LYG42 was preliminarily judged to be Bacillus.

[0046] Table 2 Physiological and biochemical identification results of strain LYG42

[0047] 4) Growth curve of strain LYG42 The strain LYG42 was inoculated into LB liquid medium at a 2% (V / V) inoculum and cultured at 37°C and 180 rpm to obtain the following Figure 5 The growth curve shown in Figure 2 shows that strain LYG42 experienced a short delay period in the culture medium, entered a logarithmic growth phase at about 5 h, and entered a stable phase at about 24 h, during which the total number of cells reached a maximum.

[0048] Example 2: Preparation of seed solution of Bacillus velez LYG42 The seed liquid of Bacillus Velez LYG42 was inoculated into LB liquid culture medium at an inoculum volume ratio of 2%, and cultured at 37°C and 180 r / min for 24 hours to obtain the seed liquid of Bacillus Velez LYG42.

[0049] Example 3: Preparation of Bacillus Velez LYG42 fermentation broth 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.

[0050] A single factor experiment was carried out on the fermentation conditions of strain LYG42. According to the single factor experimental design and response surface experiment, the optimal fermentation conditions were obtained as follows: fermentation temperature 32℃, inoculation size 4%, fermentation time 5 d. The inhibition rate of the fermentation broth of strain LYG42 against Colletotrichum gloeosporioides could reach 58.50%.

[0051] 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 LYG42 were investigated. Results are shown in Figure 6 : 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 .

[0052] Table 3 Factors and levels of response surface experiment

[0053] Table 4

[0054] Example 4: Preparation of Bacillus Velez LYG42 fermentation broth 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 6 days. 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 LYG42 had an antibacterial rate of 52.70% against Colletotrichum gloeosporioides.

[0055] Example 5: Preparation of Bacillus Velez LYG42 fermentation broth The seed solution obtained in Example 2 was inoculated into the fermentation medium at a 5% inoculum, and cultured at 180 r / min and 30°C for 5 days. 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 antibacterial rate of the fermentation broth of strain LYG42 against Colletotrichum gloeosporioides can reach 55.11%.

[0056] Experiments show that the antibacterial effects of Examples 4 and 5 are similar to that of Example 3.

Claims

1. A Bacillus velezensis LYG42, whose deposit number is: CGMCC No.32059.

2. A Bacillus Velez fermentation broth, characterized in that: The fermentation broth is obtained by fermenting the Bacillus Velez subtilis LYG42 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 Velez Bacillus LYG42 strain liquid is inoculated into LB liquid medium at an inoculation rate of 1-3% by volume, and cultured at 37°C and 180 r / min for 24 h, and then the strain is transferred into fermentation medium at an inoculation rate of 3-5% by volume, and cultured at 27-37°C and 180 r / min for 96-144 h. The Velez Bacillus fermentation liquid is obtained after the fermentation is completed; 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 biological pesticides or antifungal drugs.

5. The use according to claim 4, characterized in that The biological pesticide or antifungal drug is specifically used to inhibit Colletotrichum gloeosporioides.

6. A biological pesticide, characterized in that: The biological pesticide contains the Bacillus Velez fermentation liquid described in claim 2.

7. An antifungal drug, characterized in that: The medicine contains the Bacillus Velez fermentation liquid as claimed in claim 2.

8. Use of the biological pesticide according to claim 6 or the antifungal drug according to claim 7 in inhibiting Colletotrichum gloeosporioides.

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