Bacillus cereus b5 and use thereof

Abstract

The application belongs to the technical field of microbial culture, and particularly relates to a bacillus cereus B5 and application thereof. The bacillus cereus B5 is isolated from an alpine environment of Qinghai-Tibet Plateau, and the preservation number is GDMCC No: 65417. The strain and secreted metabolites thereof can improve the mycelium formation rate of biocontrol fungi and insect resource fungi, and accelerate the formation process of conidia. Indoor detection is conducted on the influence of B5 on the growth of insect pathogenic fungi and the efficiency of killing host insects, and the results show that B5 can improve the mycelium formation rate of the tested strains, and simultaneously accelerate the growth rate of conidia, thereby providing technical support for fungal insecticides and artificial cultivation of fungal resource industry.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, specifically to a novel strain Bacillus cereus, strain B5, and its novel uses. Background technology:

[0002] Pathogenic fungi constitute the largest group of entomopathogenic microorganisms. In nature, entomopathogenic fungi infect and kill their insect hosts, causing epidemics, and have thus been developed into fungal biopesticides as environmentally friendly alternatives to chemical pesticides, used to control various agricultural, forestry, and sanitary pests. After penetrating the insect's body wall, entomopathogenic fungi produce budding spores to colonize the host's hemocoel. These budding spores differentiate and develop into pathogenic hyphae, killing the insect host, and then emerge from the dead insect to grow hyphae and conidia, initiating the next round of infection. Therefore, hyphal formation and conidia regeneration are crucial for the sustained control of pests by pathogenic fungi in the ecosystem.

[0003] Currently, approximately 1000 species of entomopathogenic fungi are known, among which the Hypocreales order within the Ascomycota and the Entomotheria order within the Entomophthea are the most common and important groups. Fungi of the genera *Beauveria* and *Metarhizium* can parasitize various insects and have been developed into widely used fungal insecticides. Fungi of the genus *Cordyceps* are obligate parasites that alter host behavior; *Cordyceps sinensis*, formed by parasitizing insects of the family Hepialidae, is a traditional and precious Chinese medicinal herb. Due to the special habitat of *Cordyceps sinensis* (naturally reproducing only in high-altitude and cold regions), its slow growth rate, and long growth cycle, its natural resources are extremely limited. Artificial cultivation of *Cordyceps sinensis* is necessary to meet human needs and protect the ecological environment.

[0004] Slow insecticidal speed is a major factor limiting the widespread application of entomopathogenic fungi, and a significant problem facing the fungal insecticide and Cordyceps sinensis artificial cultivation industries. Therefore, it is urgent to identify the factors affecting the formation of invasive hyphae from budding spores, providing a scientific basis for developing technologies to improve the pathogenicity of biocontrol fungi and entomopathogenic fungi. Summary of the Invention:

[0005] Based on the above problems, the purpose of this invention is to overcome the difficulties of the prior art and to provide a strain of Bacillus cereus B5 and its new uses, which can improve the mycelial formation rate of biocontrol fungi and insect resource fungi and accelerate the formation process of conidia.

[0006] Bacillus cereus B5 was deposited on November 5, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCCNo: 65417.

[0007] The present invention also provides the application of the above-mentioned Bacillus cereus B5 in improving the rate of mycelial and conidial formation of blastomonas fungi.

[0008] Preferably, the entomopathogenic fungus is *Metarhizium anisopliae*.

[0009] Preferred application is in improving the efficiency of insect pathogenic fungi in killing host larvae.

[0010] This invention also provides the application of Bacillus cereus B5 in improving the silencing of Cordyceps sinensis-infected ghost moth larvae and increasing the silencing rate.

[0011] Preferably, the budding spore liquid of Cordyceps sinensis is mixed with the supernatant of Bacillus cereus B5 culture medium to infect the larvae of the ghost moth.

[0012] The present invention also provides a method for improving the efficiency of insect pathogenic fungi in killing host larvae, which involves mixing a bacterial solution of Bacillus cereus B5 with Metarhizium anisopliae or its budding spores to control host larvae.

[0013] This invention provides a novel strain of Bacillus cereus, B5, and its new applications. This strain was isolated from the high-altitude environment of the Qinghai-Tibet Plateau. This strain and its secreted metabolites can increase the mycelial formation rate of insect-derived fungi and accelerate the conidia formation process. The effects of B5 on the growth of insect pathogenic fungi and their lethality to host insects were investigated in the laboratory. The results showed that B5 can increase the mycelial formation rate of the tested strain and accelerate the conidia growth rate, providing technical support for the development of fungal insecticides and the artificial cultivation of fungal resources.

[0014] Bacillus cereus B5 was deposited on November 5, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCCNo: 65417. Attached image description:

[0015] Figure 1 The colony morphology (A) of the biocontrol fungus *Metarhizium anisopliae* inoculated on SMAY medium and producing green conidia, and the colony morphology (B) of *Cordyceps sinensis* inoculated on G5 medium.

[0016] Figure 2 This is a hyphal diagram of Metarhizium anisopliae bud spore formation induced by Bacillus cereus B5. The Metarhizium anisopliae bud spores were mixed with different concentrations of Bacillus cereus, inoculated into SMAY medium, and co-cultured at 25°C for 4 days.

[0017] Figure 3This is a hyphal diagram of metabolites secreted by Bacillus cereus inducing the formation of budspores of Metarhizium repens. Bacillus cereus (B5) and Metarhizium repens (MR) budspores were inoculated into SMAY culture medium and cultured in opposition at 25°C for 6 days.

[0018] Figure 4 This image shows the conidia of Metarhizium anisopliae induced by Bacillus cereus. The blastospores of Metarhizium anisopliae (MR) were mixed with Bacillus cereus (B5) at different dilutions, inoculated into SMAY medium, and co-cultured at 25°C for 8 days. The green part at the edge of the hyphae is the conidia produced. Detailed implementation method:

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Example 1:

[0021] The *Bacillus cereus* B5 described in this invention was isolated from the high-altitude environment of the Qinghai-Tibet Plateau. Soil samples were sieved and serially diluted with sterile water to different concentrations. 100 μL of each diluted sample was plated onto LB agar plates and incubated in the dark at 37°C. The resulting colonies were purified and identified by PCR as *Bacillus cereus*. The sequencing sequence of the 16S rRNA gene PCR product is as follows:

[0022] >Bacillus cereus B5 16S ribosomal RNA,partial sequence

[0023] GCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGA

[0024] GTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGAACCGC

[0025] ATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGG

[0026] CTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACG

[0027] GGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGT

[0028] CGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACG

[0029] GCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGG

[0030] TGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGA

[0031] GGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCT

[0032] GTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTG

[0033] CTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGC

[0034] TGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTAC

[0035] CAGGTCTTGACATCCTCTGAAAACCCTAGAGATAGGGCTTCTCCTTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCG

[0036] TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGC

[0037] ACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTA

[0038] CACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGG

[0039] ATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCG

[0040] GGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGG (SEQ ID NO. 1)

[0041] The Bacillus cereus B5 of this invention was deposited on November 5, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China, with accession number GDMCC No: 65417.

[0042] Single colonies of Bacillus cereus B5 were cultured in LB liquid culture medium at 37°C and 200 rpm to obtain Bacillus cereus B5 bacterial suspension, and the concentration was adjusted with LB liquid culture medium.

[0043] Metarhizium anisopliae ( Figure 1 A) Beauveria bassiana budspores were obtained by inoculating the culture medium (120 mL of water containing 4.8 g maltose, 0.6 g peptone, and 0.6 g yeast extract) into autoclaved SMY medium and incubating in the dark at 25°C for 3 days. The budspore concentration was then adjusted to 4 × 10⁻⁶ using 1×PBS (pH 7.4). 4 Cells / mL, respectively compared with a concentration of 4×10 4 cfu / mL, 4×10 5 cfu / mL, 4×106 A mixture of CFU / mL Bacillus cereus B5 was prepared in equal volume ratios. 10 μL of this mixture was inoculated onto sterile SMAY agar plates (120 mL water containing 4.8 g maltose, 0.6 g peptone, 0.6 g yeast extract, and 2.16 g agar). After absorption, the plates were incubated in the dark at 25 ± 2 °C for 4 days. The bacterial culture was then examined under a microscope to observe the formation of hyphae from budding spores. Figure 2 The study used budding spores without Bacillus cereus B5 as a control. The results showed that the mixtures of budding spores and Bacillus cereus at ratios of 1:1, 1:10, and 1:100 all produced long hyphae with tertiary branching, while the control group of budding spores without Bacillus cereus B5 did not produce long hyphae.

[0044] Example 2:

[0045] Cordyceps sinensis fungus ( Figure 1 B) Cordyceps militaris buds were obtained by inoculating the culture medium (150 mL of water containing 30 g of cooked potato filtrate, 1.5 g of peptone, 3 g of maltose, 0.08 g of magnesium sulfate, 0.25 g of potassium dihydrogen phosphate, and 3 mg of vitamin B1) into autoclaved G5 liquid medium (150 mL of water) and incubating in the dark at 12°C and 110 rpm for 50 days. The buds were then adjusted to a concentration of 4 × 10⁻⁶ using 1×PBS (pH 7.4). 4 Cells / mL, respectively compared with a concentration of 4×10 4 cfu / mL, 4×10 5 cfu / mL, 4×10 6 A mixture of CFU / mL Bacillus cereus B5 was prepared in equal volumes. 100 μL of this mixture was inoculated into 900 μL of sterile G5 liquid medium and incubated in the dark at 110 rpm for 8 days. The proportion of budding spores forming hyphae was then examined under a microscope. The control group was prepared without Bacillus cereus B5. The results showed that the proportion of budding spores forming hyphae in the control group without Bacillus cereus B5 was 11±5%. The proportions of budding spores forming hyphae in the groups with Bacillus cereus B5 mixtures at ratios of 1:1, 1:10, and 1:100 were 27±4%, 42±4%, and 37±6%, respectively.

[0046] Example 3:

[0047] Metarhizium anisopliae was inoculated into autoclaved SMY medium (120 mL water containing 2.4 g maltose, 0.3 g peptone, and 1.2 g yeast extract) and cultured in the dark at 25°C for 3 days to obtain Metarhizium anisopliae budspores. The budspore concentration was adjusted to 4 × 10⁻⁶ using 1×PBS (pH 7.4). 4 Cells / mL, respectively compared with a concentration of 4×10 4cfu / mL, 4×10 5 cfu / mL, 4×10 6 A mixture of CFU / mL *Bacillus cereus* B5 was prepared in equal volume ratios. 10 μL of this mixture was inoculated onto sterile SMAY agar plates (120 mL water containing 2.4 g maltose, 0.3 g peptone, 1.2 g yeast extract, and 2.16 g agar). After absorption, the plates were incubated in the dark at 25 ± 2 °C for 4 days. The bacterial culture was then examined under a microscope to observe the formation of hyphae in the budding spores. Budding spores without *Bacillus cereus* B5 served as a control. The results showed that the budding spores mixed with *Bacillus cereus* B5 at ratios of 1:1, 1:10, and 1:100 all exhibited long hyphae with tertiary branching, while the budding spore control group without *Bacillus cereus* showed no long hyphae formation.

[0048] Example 4:

[0049] Metarhizium anisopliae was inoculated into autoclaved SMY medium (120 mL water containing 2.4 g maltose, 0.3 g peptone, and 1.2 g yeast extract) and incubated in the dark at 25°C for 3 days to obtain Metarhizium anisopliae budding spores. 50 μL of a 1×10⁻⁶ concentration was then used. 6 Inoculate a solution of budding spores per mL in a vertical line onto one side of a sterile SMAY medium plate (120 mL water containing 4.8 g maltose, 0.3 g peptone, 0.6 g yeast extract, and 2.16 g agar). Inoculate the same way on the left side with 50 μL of a solution containing 4 × 10⁶ spores per mL. 6 Bacillus cereus B5 at cfu / mL was added to the plates. After the bacterial suspension was absorbed by the plates, a control group without Bacillus cereus B5 was used. The plates were incubated in the dark at 25±2℃ for 4 days. After incubation, the bacterial suspension from the edge of the colony near the Bacillus cereus was taken for microscopic examination of the mycelial formation of budding spores. The results showed that the budding spores in the Bacillus cereus B5 control group had obvious mycelial clusters, while the budding spores control group without Bacillus cereus B5 did not yet have obvious mycelial clusters. After 12 days of incubation, there was no significant difference in colony morphology. Figure 3 ).

[0050] Example 5:

[0051] Metarhizium anisopliae was inoculated into autoclaved SMY medium (120 mL water containing 2.4 g maltose, 0.3 g peptone, and 1.2 g yeast extract) and cultured in the dark at 25°C for 3 days to obtain Metarhizium anisopliae budspores. The budspore concentration was adjusted to 1×10⁻⁶ using 1×PBS (pH 7.4). 6 CFU / mL, respectively with Bacillus cereus B5 (OD 600=1.5) Equal volumes of bacterial solutions diluted 10×, 100×, and 1000× were mixed. 10 μL of this mixture was inoculated onto sterile SMAY agar plates (120 mL water containing 4.8 g maltose, 0.3 g peptone, 0.6 g yeast extract, and 2.16 g agar). After absorption, the plates were incubated in the dark at 25±2℃ for 8 days. Colony morphology was then examined, with budding spores without Bacillus cereus B5 serving as the control group. Results showed that green conidia formed at the edges of colonies in the mixture of budding spores and Bacillus cereus B5, while no green conidia formed in the budding spore control group without Bacillus cereus B5. Figure 4 ).

[0052] Example 6:

[0053] Metarhizium anisopliae was inoculated into autoclaved SMY medium (120 mL water containing 2.4 g maltose, 0.3 g peptone, and 1.2 g yeast extract) and cultured in the dark at 25°C for 3 days to obtain Metarhizium anisopliae budspores. The budspore concentration was adjusted to 2 × 10⁻⁶ using 1×PBS (pH 7.4). 6 Bacillus cereus B5 culture medium (OD) / mL 600 =1.5) Centrifuged at room temperature and 4000 rpm, the supernatant was obtained by filtration through a 0.22 μm sterile filter membrane. The budding spore solution was mixed with the sterile supernatant in an equal volume ratio and microinjected into 5th instar larvae of the large wax moth at an injection concentration of 2 × 10⁻⁵. 6 A mixture of blastomonas / mL and 1×PBS in an equal volume ratio served as a control. Each larva was injected with 4 μL of the mixture, and the larvae were incubated at 28°C. Each group had three replicates, and larval mortality was checked daily. Results showed that the cumulative larval stunting rate in the control group without Bacillus cereus B5 was 71±5% on day 5, while the cumulative mortality rate in the blastomonas / Bacillus cereus mixed group was 100% on day 7.

[0054] Example 7:

[0055] Cordyceps sinensis was inoculated into autoclaved G5 liquid medium (150 mL water containing 30 g cooked potato filtrate, 1.5 g peptone, 3 g maltose, 0.08 g magnesium sulfate, 0.25 g potassium dihydrogen phosphate, and 3 mg vitamin B1) and cultured in the dark at 12°C and 110 rpm for 50 days to obtain Cordyceps sinensis budspores. The budspore concentration was adjusted to 2 × 10⁻⁶ using 1×PBS (pH 7.4). 6 Bacillus cereus B5 culture medium (OD) / mL 600=1.5) Centrifuged at room temperature and 4000 rpm, the supernatant was obtained by filtration through a 0.22 μm sterile filter membrane. The budding spore solution was mixed with the sterile supernatant in an equal volume ratio and microinjected into 5th instar *Hepialus chinensis* at an injection concentration of 2 × 10⁻⁵. 6 A mixture of blastomonas / mL and 1×PBS in an equal volume ratio served as a control. Each larva was injected with 4 μL of the mixture, and the larvae were incubated in the dark at 8±2℃. Each group had three replicates, and the worm mummification rate was recorded after 120 days. The results showed that the worm mummification rate in the control group without Bacillus cereus was 31±5%, while the rate in the mixed blastomonas / Bacillus cereus group was 42±6%.

Claims

1. Bacillus cereus Bacillus cereus B5, with accession number GDMCC No: 65417.

2. The application of Bacillus cereus B5 as described in claim 1 in improving the rate of mycelial and conidial formation of Metarhizium repens budspores.

3. The application according to claim 2, characterized in that, This is an application in improving the efficiency of Metarhizium anisopliae in killing larvae of the large wax moth.

4. The application of Bacillus cereus B5 as described in claim 1 in improving the mummification rate of small golden worm larvae infected with Cordyceps sinensis.

5. The application according to claim 4, characterized in that, The method involves mixing the budding spores of Cordyceps sinensis with the supernatant of Bacillus cereus B5 culture medium to infect the larvae of the small golden pheasant.

6. A method for improving the efficiency of insect pathogenic fungi in killing host larvae, characterized in that, The bacterial solution of Bacillus cereus B5 as described in claim 1 is mixed with Metarhizium anisopliae or its budding spores to control the larvae of the large wax moth.

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