Bacillus velezensis LYG42 and its applications

Abstract

Bacillus velezensis LYG42 and its application, belonging to the field of biotechnology. On the one hand, the present invention provides a Bacillus velezensis LYG42, and on the other hand, it provides the application of the Bacillus velezensis LYG42 in inhibiting Colletotrichum gloeosporioides. The Bacillus velezensis LYG42 isolated in the present invention can be fermented using squid processing waste, and the fermented liquid obtained by fermentation has a significant inhibitory effect on Colletotrichum gloeosporioides, providing strain resources for the biological control of anthracnose and a theoretical basis for the development of green biological pesticides.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to Bacillus velezensis LYG42 and its applications. Background Art

[0002] Fungi are eukaryotic microorganisms that can cause diseases in plants, animals, and humans. Economically valuable fruits such as grapes, strawberries, and blueberries generally have problems with fungal diseases, such as downy mildew, anthracnose, white rot, gray mold, black pox, and rachis blight.

[0003] Currently, commonly used control agents such as carbendazim have residues that can cause liver diseases and chromosomal aberrations and are toxic to mammals; although thiophanate-methyl, iprodione, polyoxin, etc. are of low toxicity, improper use or long-term exposure is still toxic. In addition, long-term use of chemical pesticides can prompt fungi to develop drug resistance. Therefore, there is an urgent need to develop highly efficient, safe, and reliable antifungal products. In recent years, researchers have discovered a series of highly safe antifungal substances, such as biopesticides, which can not only prevent and control diseases but also avoid residues in plants, thus ensuring the safety of food. Therefore, the research and development of low-toxic and 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 Cephalopoda class of mollusks. It has a short life cycle, strong reproductive ability, is rich in high protein and various essential amino acids, and is a low-fat aquatic product. During the squid processing process, about 15% of waste is generated, which is called squid processing by-products. Although it is a discarded part, the nutritional value it contains is extremely rich, rich in fat and protein. If it can be utilized, good economic benefits can be generated; the organic matter content in squid viscera is rich, which is an excellent culture medium for microorganisms. Using squid viscera as a culture medium to ferment and prepare a biopesticide 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 product has anti-fungal disease activity and has the potential to replace chemical pesticides.

[0005] The inventors isolated strains that can ferment squid viscera from a variety of fermented aquatic products, screened strains with high anti-Colletotrichum gloeosporioides activity in the fermentation broth by the mycelial growth inhibition method, and classified and identified the strains by morphological and molecular biological methods. A strain of Bacillus velezensis with obvious inhibitory effect on Colletotrichum gloeosporioides was found, providing strain resources for the biological control of anthracnose and a theoretical basis for the development of green biopesticides. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for Bacillus velezensis LYG42 and its applications.

[0007] The present invention is specifically implemented by the following technical solutions:

[0008] In the first aspect of the present invention, a Bacillus velezensis ( Bacillus velezensis ) LYG42 is provided, and its preservation number is: CGMCC No. 32059.

[0009] This strain was isolated from naturally fermented shrimp paste in Lianyungang City, Jiangsu Province. Through morphological analysis and sequencing analysis, it was found that this strain has the highest homologous similarity with Bacillus velezensis, and the highest similarity is 99.93%. It can be determined that this strain is Bacillus velezensis ( Bacillus velezensis ).

[0010] Observation of the colonies of the Bacillus velezensis LYG42 on a solid plate. The colonies are raised, round or nearly round with regular edges, the surface is moist, the center is protuberant and light white, the texture is viscous, easy to pick up, and the Gram stain is positive, rod-shaped. The Bacillus velezensis LYG42 reaches the late logarithmic phase after being cultured in LB solid medium for 24 h under the condition of 37 °C constant temperature.

[0011] In the second aspect of the present invention, a Bacillus velezensis fermentation broth is provided, and the fermentation broth is obtained by fermenting the above-mentioned Bacillus velezensis LYG42.

[0012] In the third aspect of the present invention, a preparation method of a Bacillus velezensis fermentation broth is provided, which includes the following steps:

[0013] Inoculate the Bacillus velezensis LYG42 strain solution into LB liquid medium at an inoculation amount of 1-3% by volume, culture at 37 °C and 180 r / min for 24 h, then transfer the strain to the fermentation medium at an inoculation amount of 3-5% by volume, and culture at 27-37 °C and 180 r / min for 96-144 h. After fermentation is completed, the Bacillus velezensis fermentation broth is obtained;

[0014] The fermentation medium is: containing 5-50 g of squid processing waste, 10-30 g of glucose per liter, pH 7.0-10.0, and the balance is water.

[0015] As a preference for the preparation method of the fermentation broth, inoculate the Bacillus velezensis LYG42 strain solution into LB liquid medium at an inoculation amount of 2% by volume, culture at 37 °C and 180 r / min for 24 h, then transfer the strain to the fermentation medium at an inoculation amount of 4% by volume, and culture at 32 °C and 180 r / min for 120 h. After fermentation is completed, the Bacillus velezensis fermentation broth is obtained.

[0016] As a preferred fermentation medium, it contains 10 g of squid processing waste, 20 g of glucose, pH 9.0 per liter, and the balance is water.

[0017] As a preferred fermentation medium, mix squid viscera and water at a ratio of 1:100, homogenize using a tissue homogenizer, then add 2% glucose, adjust the pH value to 9.0 using 1 M NaOH, and sterilize at 121 °C for 15 min to prepare the fermentation medium.

[0018] The fourth aspect of the present invention provides the use of the above-mentioned Bacillus velezensis or the above-mentioned Bacillus velezensis fermentation broth in the preparation of a biological pesticide or an anti-fungal drug.

[0019] Furthermore, in this application, the biological pesticide or anti-fungal drug is specifically used to inhibit Colletotrichum gloeosporioides.

[0020] The fifth aspect of the present invention provides a biological pesticide, which contains the above-mentioned Bacillus velezensis fermentation broth.

[0021] The sixth aspect of the present invention provides an anti-fungal drug, which contains the above-mentioned Bacillus velezensis fermentation broth.

[0022] The seventh aspect of the present invention provides the use of the above-mentioned biological pesticide or the above-mentioned anti-fungal drug in inhibiting Colletotrichum gloeosporioides.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The Bacillus velezensis LYG42 isolated in the present invention can utilize squid processing waste for fermentation, and the obtained fermentation broth has a significant inhibitory effect on Colletotrichum gloeosporioides, providing strain resources for the biological control of anthracnose and a theoretical basis for the development of green biological pesticides. Description of the Drawings

[0025] Figure 1 It is the primary screening dominant colony map;

[0026] Figure 2 It is the colony morphology of Bacillus velezensis LYG42;

[0027] Figure 3 It is the Gram staining map of Bacillus velezensis LYG42;

[0028] Figure 4 It is the 16S rDNA phylogenetic tree of strain Bacillus velezensis LYG42 constructed by the Neighbor-Joining method;

[0029] Figure 5 It is the growth curve of Bacillus velezensis LYG42 in LB medium;

[0030] Figure 6 It is a single-factor experiment for optimizing the fermentation conditions of Bacillus velezensis LYG42;

[0031] Figure 7 It is a response surface experiment for optimizing the fermentation conditions of Bacillus velezensis LYG42. Specific implementation mode

[0032] The present invention will be further described below through specific embodiments.

[0033] Example 1: Obtaining of Bacillus velezensis ( Bacillus velezensis ) LYG42

[0034] Sampling time: July 2023.

[0035] Sampling location: The naturally fermented shrimp paste was collected from Lianyungang City, Jiangsu Province.

[0036] Treatment of the sample: Gradient dilution of the sample: The sample was gradient-diluted with sterile normal saline, and 10 -1 -10 -3 gradient dilution solutions were selected for the experiment.

[0037] Media used for screening:

[0038] Primary screening medium: 20 g of squid processing waste, made up to 1 L with distilled water, added with 20 g of agar, natural pH, autoclaved at 121 °C for 15 - 20 min.

[0039] LB solid medium: 1% peptone, 0.5% yeast extract powder, 1% sodium chloride, adjusted to pH 7.2 - 7.4 with sodium hydroxide. The medium was autoclaved at 121 °C for 15 min.

[0040] Potato dextrose agar (PDA) medium: 200 g of potato, 20 g of glucose, made up to 1 L with distilled water, natural pH, added with 20 g of agar, autoclaved at 121 °C for 15 min.

[0041] Fermentation medium: 10 g of squid processing waste, 20 g of glucose, made up to 1 L with distilled water, adjusted to pH 9.0, autoclaved at 121 °C for 15 min.

[0042] Isolation of new microorganisms:

[0043] 1) Primary screening on plates

[0044] Using the conventional dilution plate method, LB solid medium was used as the primary screening medium for screening strains that can utilize squid processing waste, and bacterial strains in the sample were isolated. Take 0.1 mL of the sample solution at each concentration and evenly spread it on the LB medium plate. Incubate at 37 °C for 48 h. Pick single colonies with different morphologies into LB liquid medium and incubate in a constant temperature shaker at 37 °C and 180 r / min for 24 h for standby.

[0045] Primary screening results on the plate: Seven dominant bacteria that can use squid processing waste as the sole nutrient source were isolated from the sample, as Figure 1 shown.

[0046] 2) Re-screening and purification

[0047] 2.1) Fermentation

[0048] Inoculate the screened dominant bacteria into the fermentation medium at an inoculation amount of 5% (V / M), and culture at 180 r / min and 37 °C for 72 h. Centrifuge the fermentation broth at 8000 rpm for 20 min at 4 °C, discard the precipitate, and remove the bacteria from the supernatant with a 0.45 μm filter membrane. Store at 4 °C for standby.

[0049] 2.2) Determination of the antibacterial activity of the fermentation broth by the mycelial growth inhibition method

[0050] In a laminar flow hood, transfer 3 mL of the above-mentioned fermentation broth into 22 mL of sterilized PDA medium cooled to 50 - 60 °C, shake well and pour plates to prepare PDA plates containing the fermentation broth. Using Colletotrichum gloeosporioides as the indicator bacterium, on the Colletotrichum gloeosporioides plate that has grown for 5 - 6 days, punch out a 6 mm diameter bacterial cake and place it on the fresh PDA plate containing the fermentation broth. Incubate in the dark at 25 °C. Use sterile water instead of the fermentation broth as the blank control group. When the colonies in the control group grow to about 2 / 3 of the culture dish, use the cross method to measure the colony diameter of each treatment. Calculate the mycelial growth inhibition rate (%) of each PDA plate containing the fermentation broth according to the following formula. Each treatment has three parallels, and the average value is taken.

[0051]

[0052] 2.3) Re-screening results

[0053] The fermentation broths of the seven dominant bacteria obtained from the primary screening were analyzed for antibacterial activity. It was found that after culturing at 25 °C for 5 days, the fermentation broths of 5 of the 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.

[0054] Table 1 Re-screening results

[0055]

[0056] 2.4) Purification of strains by streaking on a plate

[0057] The dominant bacterium with the highest antibacterial activity was purified by streaking on an LB medium plate three times and stored at -80 °C. This strain was named LYG42.

[0058] 3) Strain identification

[0059] 3.1) Colony morphological characteristics

[0060] The selected LYG42 was inoculated onto an LB medium plate and incubated statically at 37 °C for 24 h. The morphological characteristics of the colonies on the solid medium, including their size, color, and raised shape, were observed, and Gram staining was performed.

[0061] The colonies of strain LYG42 on the LB plate were raised, round or nearly round with regular edges, the surface was moist, the center was protuberant and light white, the texture was viscous, and it was easy to pick up. Gram staining was positive, and it was rod-shaped ( Figure 2 、 Figure 3 ).

[0062] 3.2) 16S rDNA identification and construction of phylogenetic tree

[0063] Design of primers:

[0064] The universal primer 1492R: 5’-CTACGGCTACCTTGTTACGA-3’ (shown in SEQ ID NO.1) was used;

[0065] 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ (shown in SEQ ID NO.2).

[0066] Using 1492R and 27F as primers, the 16S rDNA of strain LYG42 was amplified by PCR.

[0067] The total length of the nucleotide sequence of the 16S rDNA of strain LYG42 was 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. The MEGA 11 software was used to construct a phylogenetic tree.

[0068] The complete nucleotide sequence of the 16S rDNA of strain LYG42:

[0069]

[0070] After comparison with the GenBank database, the results showed that the similarity between strain LYG42 and strain Bacillus velezensis CCUG reached 99.86%. Combining with the phylogenetic tree (as Figure 4 shown), strain LYG42 was identified as Bacillus velezensis . This strain was deposited in the China General Microbiological Culture Collection Center on September 25, 2024, with the deposit number CGMCC No. 32059. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0071] 3.3) Results of physiological and biochemical tests

[0072] The physiological and biochemical identification results of strain LYG42 are shown in Table 2. As can be seen from Table 2, the V-P 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 and could grow under pH = 5.7 growth conditions; it was a facultative anaerobe and could grow in both aerobic and anaerobic environments. Combining with the morphological characteristics, strain LYG42 was preliminarily judged to be of the genus Bacillus.

[0073] Table 2 Physiological and biochemical identification results of strain LYG42

[0074]

[0075] 4) Growth curve of strain LYG42

[0076] Strain LYG42 was inoculated into LB liquid medium at an inoculum size of 2% (V / V) and cultured at 37 °C with constant shaking at 180 rpm, obtaining the growth curve as Figure 5 shown. Strain LYG42 experienced a short lag phase in the medium, entered the logarithmic growth phase at about 5 h, and entered the stationary phase at about 24 h, at which time the total cell number reached the maximum.

[0077] Example 2: Preparation of Bacillus velezensis LYG42 seed liquid

[0078] The Bacillus velezensis LYG42 strain liquid was inoculated into LB liquid medium at an inoculum size of 2% (V / V) and cultured at 37 °C and 180 r / min for 24 h to obtain the Bacillus velezensis LYG42 seed liquid.

[0079] Example 3: Preparation of Bacillus velezensis LYG42 fermentation broth

[0080] The seed liquid obtained in Example 2 was inoculated into the fermentation medium at a material-liquid ratio of 1:100 and cultured at 180 r / min for a certain period of time. The fermentation broth was centrifuged at 8000 rpm for 20 min at 4°C, the precipitate was discarded, and the supernatant was filtered through a 0.45 μm filter membrane to remove the bacteria and stored at 4°C for later use.

[0081] Single-factor experiments were carried out on the fermentation conditions of strain LYG42. According to the single-factor experiments, response surface experiments were designed, and the optimal fermentation conditions were obtained as follows: fermentation temperature 32°C, inoculation amount 4%, fermentation time 5 d. The antibacterial rate of the fermentation broth of strain LYG42 against Colletotrichum gloeosporioides could reach 58.50%.

[0082] Single-factor experiments: The effects of fermentation temperature (fixing the initial pH of the fermentation broth at 7.0, fermentation time at 72 h, inoculation amount at 3%, and setting the temperature range at 27 - 47°C), pH of the fermentation medium (fixing the fermentation temperature at 37°C, fermentation time at 72 h, inoculation amount at 3%, material-liquid ratio at 1:100, and setting the initial pH value of the fermentation broth at 5.0 - 11.0), inoculation amount (fixing the initial pH of the fermentation broth at 7.0, fermentation temperature at 37°C, fermentation time at 72 h, material-liquid ratio at 1:100, and setting the inoculation amount at 1 - 5%), and fermentation time (fixing the fermentation temperature at 37°C, initial pH of the fermentation broth at 7.0, inoculation amount at 3%, material-liquid ratio at 1:100, and setting the fermentation time at 24 h - 216 h) on the fermentation effect of LYG42 were investigated respectively. The results are shown in Figure 6 :

[0083] Based on the results of the single-factor experiments, three factors, namely fermentation temperature (A, °C), inoculation amount (B, %), and fermentation time (C, d), were selected, and the inhibition rate of the supernatant was used as the response value to design the response surface experiment (Table 3). The results are shown in Table 4 and Figure 7 .

[0084] Table 3 Factors and levels of the response surface experiment

[0085]

[0086] Table 4

[0087]

[0088] Example 4: Preparation of the fermentation broth of Bacillus velezensis LYG42

[0089] The seed liquid obtained in Example 2 was inoculated into the fermentation medium at an inoculation amount of 4%, 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, and 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 52.70%.

[0090] Example 5: Preparation of the fermentation broth of Bacillus velezensis LYG42

[0091] The seed liquid obtained in Example 2 was inoculated into the fermentation medium at an inoculation amount of 5%, 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, and 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%.

[0092] Experiments have proved that the antibacterial effects of Examples 4 and 5 are similar to that of Example 3.

Claims

1. A Bacillus Velezii ( Bacillus velezensis ) LYG42, its 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 a biological pesticide or an antifungal drug, wherein the biological pesticide or the antifungal drug is specifically used to inhibit Colletotrichum gloeosporioides.

5. A biological pesticide for inhibiting Colletotrichum gloeosporioides, characterized in that: The biological pesticide contains the Bacillus Velez fermentation liquid described in claim 2.

6. An antifungal drug for inhibiting Colletotrichum gloeosporioides, characterized in that: The antifungal drug contains the Bacillus Velez fermentation liquid according to claim 2.

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