A strain G7 and its application

Abstract

The present invention belongs to the field of microbiology and specifically relates to a strain G7 and its application. The strain G7 has the taxonomic name Bacillus subtilis and the deposit number GDMCC No. 63938. The present invention identifies the selected strain through morphological observation, Gram staining, and 16S rRNA sequencing, and measures its inhibitory effect on the cane mildew pathogen Arthrotrichum spp., resulting in a strain G7 that exhibits a strong inhibitory effect against the cane mildew pathogen Arthrotrichum spp., offering broad application prospects in the field of biological control of cane mildew diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and in particular relates to a strain G7 and an application thereof. Background Art

[0002] Consuming sugarcane moldy due to Arthrinium arundinis can cause severe food poisoning. 3-Nitropropionic acid, produced by Arthrinium arundinis, is the primary toxicant in spoiled sugarcane poisoning. Using chemical preservatives with bactericidal active ingredients can, to a certain extent, prevent sugarcane mold and thereby maintain freshness. For example, the commonly used prochloraz, a highly effective, broad-spectrum imidazole fungicide and preservative, primarily inhibits ergosterol biosynthesis, causing bacterial cell membrane dysfunction and resulting in bacterial death. It is highly effective in preserving fruits and vegetables after harvest. However, long-term use of chemical preservatives can lead to drug resistance and also poses the problem of pesticide residues. With increasing attention to food safety, the development of safe preservatives is of great significance.

[0003] The inventors isolated a bacterial strain from the rhizosphere soil of sugarcane. By culturing it against Arthrospora spp. on plates, they found that this strain has a strong inhibitory effect on the cane mold pathogen Arthrospora spp., providing technical support for the development of new fruit and vegetable preservatives. Morphological observation, Gram staining, and 16S rRNA sequencing identified the strain as Bacillus subtilis, a previously unreported strain.

[0004] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0005] The purpose of the present invention is to provide a strain G7 and an application thereof.

[0006] The strain G7 provided by the present invention was obtained from the rhizospheric soil of healthy cane from the Guangxi Zhuang Autonomous Region Academy of Agricultural Sciences, and was identified as Bacillus subtilis by morphology and molecular biology. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 27, 2023, with the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No. 63938.

[0007] The second object of the present invention is to provide the use of the metabolites of the strain G7 in inhibiting the growth of Arthrospira.

[0008] The third object of the present invention is to provide a microbial metabolite, which is prepared by the strain G7.

[0009] The fourth object of the present invention is to provide a method for preparing the microbial metabolite, specifically: the strain G7 is inoculated into LB liquid culture medium and fermented at 28°C and 150r / min for 3 days; the obtained fermentation product is centrifuged at 4°C and 7000r / min for 25 minutes to remove the bacteria and collect the supernatant; the obtained supernatant is adjusted to pH 3.0 with 6.0mol / L hydrochloric acid and precipitated in a refrigerator at 4°C overnight; the supernatant is then centrifuged at 4°C and 7000r / min for 20 minutes to collect the precipitate; the obtained precipitate is extracted twice with methanol, the methanol extracts are combined, and the methanol extracts are rotary evaporated to obtain a crude methanol extract, which is the metabolite of Bacillus subtilis G7.

[0010] Preferably, the LB liquid culture contains 8.0 g of peptone, 2.0 g of casein, 5.0 g of sodium chloride, 5.0 g of yeast powder, and 0.2 g of sodium carbonate.

[0011] The fourth object of the present invention is to provide the use of the microbial metabolites in preventing and controlling the cane mildew pathogen Arthrotrichum spp.

[0012] Compared with the prior art, the present invention has the following beneficial technical effects:

[0013] The present invention identifies the screened strains through morphological observation, Gram staining, and 16S rRNA sequence sequencing, and measures their inhibitory effects on the fruit cane mildew pathogen Arthrotrichum spp., thereby obtaining a strain G7 that has a strong inhibitory effect on the fruit cane mildew pathogen Arthrotrichum spp., thus bringing broad application prospects to the field of biological control of fruit cane mildew diseases.

[0014] Preservation Information

[0015] The strain Bacillus subtilis G7, the deposit number is GDMCC No. 63938, the deposit date is October 27, 2023, the depository is Guangdong Provincial Microbiological Culture Collection Center, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The antagonistic effect of the metabolites of strain G7 on Arthrospira;

[0017] Figure 2 This is the colony morphology of strain G7 on NA plates;

[0018] Figure 3 This is the bacterial morphology of strain G7;

[0019] Figure 4 Phylogenetic tree of strain G7

[0020] Figure 5 The inhibitory effect of the metabolites of strain G7 on the hyphae growth of Arthronotris spp.

[0021] Figure 6 The invention relates to the application of the metabolites of strain G7 in the prevention and treatment of the cane mildew pathogen Arthrotrichum spp.

[0022] Description of reference numerals:

[0023] Figure 5 From left to right: A - The first is the negative control; the second, third, fourth, and fifth are metabolites of strain G7, with concentrations of 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 3.0 mg / mL, respectively. B - The first is the negative control; the second, third, fourth, and fifth are prochloraz, with concentrations of 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 3.0 mg / mL, respectively.

[0024] Figure 6 A is the sugarcane treated with sterile water, B is the sugarcane treated with the metabolites of strain G7, and C is the sugarcane treated with 0.45 mg / kg prochloraz. DETAILED DESCRIPTION

[0025] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.

[0026] Example

[0027] 1. Strain Isolation and Purification

[0028] Soil was collected from the rhizosphere soil of healthy cane from Guangxi Academy of Agricultural Sciences. 10.0 g of soil was weighed and placed in a triangular flask containing glass beads and sterile water. The soil was shaken at 28 °C and 180 r / min for 30 min. The stock solution was used for gradient dilution. 10.0 g of the stock solution was taken. -3 , 10 -4 and 10 -5 The gradient was spread on NA solid plates and incubated at 37°C for 24 hours. The growth of the colonies was observed, and a plate with moderate density was selected. A single colony was streaked out for purification. Colonies with different morphologies were streaked out repeatedly for purification, and the purified strains were stored.

[0029] 2. Strain Screening

[0030] The antibacterial activity of isolated bacteria was studied using the plate standoff method, using Arthrinium arundinis as an indicator strain. Arthrinium arundinis was provided by the Institute of Agricultural Product Quality, Safety, and Testing Technology, Guangxi Academy of Agricultural Sciences, and was inoculated onto PDA plates and incubated at 28°C for 3 days before use.

[0031] The isolated strain was cultured in LB broth at 180 rpm and 30°C overnight. Under sterile conditions, a 5 mm diameter bacterial cake was punched out on the PDA plate inoculated with Arthrospora using a sterile puncher. The Arthrospora cake was transferred to the center of the culture dish containing PDA, and 2 μL of the culture solution of the primary screening strain was inoculated on each side 2 cm from the center (OD 600 The control group was set up with a treatment inoculated with Arthropoda spp. cake but not inoculated with bacteria, and each treatment was repeated 3 times.

[0032] After culturing at 28℃ for 6 days, the diameter of Arthroposophiae in each treatment was measured by the cross method, and the inhibition rate was calculated.

[0033]

[0034] The results showed that a strain of bacteria with an inhibitory effect on Arthroceros spp. was obtained and the strain was numbered G7. Its inhibition rate against Arthroceros spp. was 65.6%. Figure 1 .

[0035] 3. Strain Morphological Identification

[0036] 3.1 Inoculate the G7 strain on NA medium and culture it at 28℃ for 24h. Observe the colony morphology of the strain. Figure 2 .

[0037] from Figure 2 It can be seen that strain G7 appears as a single colony on the NA plate, which is round or oval, grayish white, opaque, and has a dry, wrinkled surface and irregular edges.

[0038] 3.2 Take the above cultured strain G7 and perform Gram staining and microscopic examination. The observed morphology is photographed microscopically. The results are shown in Figure 3 .

[0039] Depend on Figure 3 It can be seen that the morphology of bacterial body G7 is: Gram-negative, positive staining, and straight rod-shaped.

[0040] 4. Strain Sequencing and Phylogenetic Tree Construction

[0041] 4.1 Preparation of DNA template

[0042] (a) Place 1.5 mL of overnight bacterial culture into a sterile EP tube, centrifuge at 12,000 rpm for 1 min, discard the supernatant, and collect the bacteria;

[0043] (b) Add 400 μL of lysis buffer to the precipitate and mix by repeatedly pipetting. The lysis buffer consists of 40 mM Tris-Ac (pH 7.8), 20 mM NaAc, 1 mM EDTA, and 1% (w / v) SDS.

[0044] (c) Add 200 μl of 5 M NaCl, mix thoroughly, and centrifuge at 12,000 rpm for 10 min.

[0045] (d) Take 600 μL of the supernatant from step (c), add an equal volume of phenol / chloroform (1:1), gently invert several times to mix, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to another clean EP tube;

[0046] (e) Add an equal volume of chloroform to the supernatant, mix thoroughly, centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to another clean EP tube.

[0047] (f) Add an equal volume of isopropanol to the supernatant, mix well, incubate at room temperature for 10 min, centrifuge at 12,000 rpm for 15 min, discard the supernatant, and retain the precipitate;

[0048] (g) washing the resulting precipitate with 70% ethanol and drying it in air to obtain DNA;

[0049] (h) Dissolve the dried DNA in 30 μL of double-distilled water (ddH2O) and store at -20°C.

[0050] 4.2 PCR amplification

[0051] Amplification was performed using the 16S rDNA sequence PCR reaction system shown in Table 2 below. The sequence of 16S rDNA is shown in Table 3 below.

[0052] Table 2 16S rDNA sequence PCR reaction system

[0053]

[0054]

[0055] Table 3 16S rDNA sequences

[0056]

[0057] PCR reaction program: denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, for a total of 35 cycles; and extension at 72°C for 5 min.

[0058] 4.3 The PCR amplification product was sequenced by Guangzhou Qingke Biotechnology Co., Ltd., and its nucleotide sequence is shown in SEQ ID No. 3.

[0059] 4.4 The measured 16S rDNA sequences were compared with the bacterial 16S rDNA sequences in the GenBank gene library, and the phylogenetic tree was constructed by the neighbor-joining algorithm of Mega6.0. Figure 4 .

[0060] Through comparison with the Genebank database and Mega6.0 analysis, the strain G7 was determined to be Bacillus subtilis, the strain code was G7, and it was named Bacillus subtilis G7.

[0061] 5. Inhibitory effect of strain G7 metabolites on hyphal growth of Arthropoda spp.

[0062] 5.1 Fermentation of strain G7 and extraction of metabolites of strain G7

[0063] A single colony of G7 was picked and inoculated into 50 mL of LB liquid medium, and cultured on a shaker (180 r / min, 37°C) for 24 h to prepare a seed solution, which was then inoculated at a 2% inoculum into a 2L conical flask containing 1000 mL of LB medium (8.0 g of peptone, 2.0 g of casein, 5.0 g of sodium chloride, 5.0 g of yeast powder, and 0.2 g of sodium carbonate). The solution was placed in a shaker at 28°C and 150 r / min for fermentation for 3 days. The resulting fermentation product was centrifuged at 7000 r / min and 4°C for 25 min to remove the bacteria and collect the supernatant; the supernatant was adjusted to pH 3.0 with 6.0 mol / L hydrochloric acid and precipitated in a refrigerator at 4°C overnight. The mixture was then centrifuged at 4°C and 7000 rpm for 20 minutes to collect the precipitate. At room temperature, the precipitate was extracted twice with 10 mL of methanol (chromatographic grade) for 10 minutes each time. The methanol extracts were combined and rotary evaporated to dryness to obtain a crude methanol extract, which was the metabolite of Bacillus subtilis G7.

[0064] 5.2. Inhibitory effect of strain G7 metabolites on hyphal growth of Arthropoda spp.

[0065] The mycelial growth method was used to determine the inhibitory activity of the G7 metabolite of the strain on the mycelial growth of Nysspora spp. The crude extract of the G7 metabolite of the strain was dissolved in chromatographic grade methanol to prepare drug-containing plates containing 0.6 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 3.0 mg / mL of G7 metabolite. The same concentration of myclobutanil was used as a positive control, and 3.0 mg / mL of methanol was added as a negative control. Under sterile conditions, the pathogenic fungus was made into a 5 mm cake using a puncher, and the Nysspora spp. cake was transferred to the center of each drug-containing plate as a treatment. All treatments and controls were repeated three times, and all plates were cultured at 28°C. When the negative control filled the culture dish, the cross-cross method was used to control the growth diameter of the colony and each treated Nysspora spp., and the inhibition rate was calculated according to the following formula. The results are shown in Table 4. Among them, the inhibitory effect of the metabolite of strain G7 on Nysspora spp. is shown in Table 4. Figure 5 .

[0066]

[0067] Table 4 Inhibition rate of different treatments on the mycelial growth of Arthroceros spp.

[0068]

[0069] Note: The positive control in Table 4 is prochloraz emulsion in water 400 g / L (Suzhou FMC Plant Protection Chemicals Co., Ltd.).

[0070] As shown in Table 4, the inhibitory rate of strain G7 metabolites on the mycelial growth of Arthroceros spp. ranged from 57.5% to 95.0%. Compared with the positive control, prochloraz emulsion in water, when the strain G7 metabolite content in PDA was 3.0 mg / kg, the inhibitory rate on the mycelial growth of Arthroceros spp. was close to that of the positive control. This indicates that strain G7 of the present invention has the potential to be developed into a biological preservative.

[0071] 5.3 Application of Strain G7 Metabolites in Controlling Cane Mold Pathogen Arthrozoites

[0072] Fresh sugarcane of uniform size, consistent color, and no damage or pests was collected as test material. After removing the tops and roots, the sugarcane was rinsed with sterile water. The center of each cane was cut into 30cm segments. Each segment was sterilized by wiping with 75% alcohol and wrapping the ends with alcohol-disinfected film.

[0073] The sugarcane surface was stabbed (5 mm in diameter, 3 mm in depth), inoculated with 100 μL of strain G7 metabolites, dried, and then inoculated with 100 μL of 10 5 A suspension of spores of Arthrocosporium spores of 0.5 mg / mL was used as negative and positive controls, respectively, with sterile water and prochloraz (0.45 mg / mL). The disease on the cane was observed after culturing at 25°C for 10 days. Figure 6 .

[0074] Depend on Figure 6 It can be seen that in the test of strain G7 metabolites on the infection of Nyctospora cane, compared with the negative control, the effects of strain G7 metabolites treatment and prochloraz treatment were significantly better than the negative control, and the effect of strain G7 metabolites treatment was equivalent to that of prochloraz treatment.

[0075] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A strain G7, characterized in that The taxonomic name of the strain G7 is Bacillus subtilis ( Bacillus subtilis ), the deposit number is GDMCC No.63938.

2. The use of the metabolites of strain G7 according to claim 1 in inhibiting the growth of Arthroceros spp., characterized in that: The preparation method of the metabolite of the strain G7 is specifically as follows: the strain G7 is inoculated into LB liquid culture medium, and fermented at 28°C and 150 rpm for 3 days; the resulting fermentation product is centrifuged at 4°C and 7000 rpm for 25 minutes to remove the bacterial cells and collect the supernatant; the resulting supernatant is adjusted to pH 3.0 with 6.0 mol / L hydrochloric acid, and precipitated in a refrigerator at 4°C overnight; The mixture was then centrifuged at 4°C and 7000 r / min for 20 min to collect the precipitate. The precipitate was extracted twice with methanol, the methanol extracts were combined, and the methanol extracts were rotary evaporated to dryness to obtain a crude methanol extract, which was the metabolite of Bacillus subtilis G7.

3. A microbial metabolite, characterized in that: The microbial metabolite is prepared by the strain G7 according to claim 1, and is characterized in that the preparation method of the microbial metabolite is specifically as follows: the strain G7 is inoculated into LB liquid culture medium and fermented and cultured at 28°C and 150r / min for 3 days; the obtained fermentation product is centrifuged at 4°C and 7000r / min for 25 minutes to remove the bacteria and collect the supernatant; the obtained supernatant is adjusted to pH 3.0 with 6.0mol / L hydrochloric acid and precipitated in a refrigerator at 4°C overnight; the supernatant is then centrifuged at 4°C and 7000r / min for 20 minutes to collect the precipitate; the obtained precipitate is extracted twice with methanol, the methanol extracts are combined, and the methanol extracts are rotary evaporated to obtain a crude methanol extract, which is the microbial metabolite.

4. Use of the microbial metabolite according to claim 3 in preventing and treating the cane mildew pathogen Arthrotrichum spp.

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