Bacillus cereus B5 and application thereof

Abstract

The invention belongs to the technical field of microbial culture, and particularly relates to bacillus cereus B5 and application thereof. And separating from the high mountain environment of the Tibet Plateau. The preservation number of the bacillus cereus B5 is as follows: GDMCC No: 65417, and the preservation number of the bacillus cereus B5 is as follows: GDMCC No: 65417. The strain and the secreted metabolite thereof can improve the hypha formation rate of biocontrol fungi and insect resource fungi and accelerate the formation process of conidia. Influence of the B5 on growth of entomopathogenic fungi and efficiency of killing host insects is detected indoors, and results show that the B5 can improve the hypha formation rate of a tested strain and can accelerate the growth rate of conidia at the same time, and technical support is provided for fungal insecticides and artificially cultured fungal resource industries.

Description

Technical Field

[0001] The invention relates to the fields of microbiology and biotechnology, and in particular to a new strain Bacillus cereus B5 and a new use thereof. Background Art

[0002] Pathogenic fungi are the largest group of insect pathogenic microorganisms. Insect pathogenic fungi infect and kill insect hosts in nature and cause epidemics, so they are developed as fungal biopesticides as environmentally friendly alternatives to chemical pesticides and are used to control the number of various agricultural, forestry and health pests. After penetrating the insect body wall, insect pathogenic fungi produce blastospores to colonize the host's blood cavity. The blastospores differentiate and develop into pathogenic hyphae, kill the insect host, and penetrate the insect corpse to grow hyphae and conidia to start the next round of infection. Therefore, hyphae formation and the re-production of conidia are the key to the continuous control of pests by pathogenic fungi in the ecosystem.

[0003] At present, there are about 1,000 known insect pathogenic fungi, among which Hypocreales in Ascomycota and Entomophytales in Entomophyta are the most common and important insect pathogenic fungi. Beauveria and Metarhizium can parasitize a variety of insects and have been developed as fungal insecticides for wide application; Cordyceps fungi are specialized parasites and change the host's behavior. Among them, Cordyceps sinensis parasitizes bat moth insects to form Cordyceps sinensis, which is a traditional precious Chinese medicinal material. Due to the special habitat of Cordyceps sinensis (it can only reproduce naturally in high-altitude and cold areas), its growth rate is slow, the cycle is long, and its natural resources are extremely limited. Artificial cultivation of Cordyceps sinensis is also to meet the needs of humans and protect the ecological environment.

[0004] The slow insecticidal speed is the main factor limiting the widespread application of insect pathogenic fungi, and is also an important problem facing the fungal insecticide and Cordyceps sinensis artificial cultivation industry. Therefore, it is urgent to find out the factors that affect the formation of invasive hyphae by blastospores, so as to provide a scientific basis for the research and development of technologies to improve the pathogenicity of biocontrol fungi and insect resource fungi. Summary of the invention

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

[0006] Bacillus cereus B5 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on November 5, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCCNo: 65417.

[0007] The present invention also provides the use of the Bacillus cereus B5 in improving the formation speed of blastospore hyphae and conidia of insect pathogenic fungi.

[0008] Preferably, the insect pathogenic fungus is Metarhizium anisopliae.

[0009] Preferably, the invention is used to improve the efficiency of insect pathogenic fungi in killing host larvae.

[0010] The invention also provides the use of Bacillus cereus B5 in improving the rigidification of bat moth larvae infected by Cordyceps sinensis and improving the rigidification rate of the larvae.

[0011] Preferably, the blastospore liquid of Cordyceps sinensis is mixed with the supernatant of the culture liquid of Bacillus cereus B5 to infect the larvae of the bat moth.

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

[0013] The present invention provides a new strain B5 of Bacillus cereus, which is isolated from the high mountain environment of the Qinghai-Tibet Plateau, and a new use thereof. The strain and its secreted metabolites can increase the mycelium formation rate of insect resource fungi and accelerate the formation process of conidia. The effect of B5 on the growth of insect pathogenic fungi and the efficiency of killing host insects was tested indoors. The results showed that B5 can increase the mycelium formation rate of the tested strain and accelerate the growth rate of conidia, providing technical support for the fungal insecticide and artificial cultivation of fungal resource industries.

[0014] Bacillus cereus B5 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on November 5, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCCNo: 65417. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] Figure 2 This is a picture of Bacillus cereus B5 inducing spore formation of Metarhizium anisopliae. The spores of Metarhizium anisopliae were mixed with different concentrations of Bacillus cereus and inoculated into SMAY medium and then co-cultured at 25℃ for 4 days.

[0017] Figure 3This is a picture of the metabolites secreted by Bacillus cereus inducing spore formation of Metarhizium reinhardtii. The spores of Bacillus cereus (B5) and Metarhizium reinhardtii (MR) were inoculated into SMAY medium and cultured at 25℃ for 6 days.

[0018] Figure 4 This is a picture of Bacillus cereus inducing the production of conidia of Metarhizium reinhardtii. The blastospores of Metarhizium reinhardtii (MR) and Bacillus cereus (B5) at different dilution multiples were mixed and inoculated into SMAY medium and then co-cultured at 25℃ for 8 days. The green part on the edge of the hyphae is the produced conidia. DETAILED DESCRIPTION

[0019] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0020] Embodiment 1:

[0021] The Bacillus cereus B5 described in the present invention is isolated from the high mountain environment of the Qinghai-Tibet Plateau. After the collected soil samples are sieved, they are gradiently diluted to different multiples with sterile water, and 100 μL is taken to coat the LB plate, which is placed in a 37°C incubator for dark culture. The colonies obtained by culture are purified and identified as Bacillus cereus by PCR. The sequencing sequence of the 16SrRNA 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] ACCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTA

[0038] CACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAACCGTTCTCAGTTCGG

[0039] ATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCG

[0040] GGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGGGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGACAGATGATTGGGG (SEQ ID NO. 1)

[0041] The Bacillus cereus B5 of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on November 5, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and the deposit number is: GDMCC No: 65417.

[0042] A single colony of Bacillus cereus B5 was added to LB liquid culture medium and cultured at 37°C, 200 rpm to obtain a Bacillus cereus B5 bacterial solution, and the concentration was adjusted using LB liquid culture medium.

[0043] Metarhizium anisopliae ( Figure 1 A) Inoculate into autoclaved SMY medium (120 mL of water containing 4.8 g maltose, 0.6 g peptone, and 0.6 g yeast extract) and culture in the dark at 25°C for 3 days to obtain spores of Metarhizium reinhardtii. Use 1×PBS (pH 7.4) to adjust the concentration of spores to 4×10 4 / mL, respectively, with a concentration of 4×10 4 cfu / mL, 4×10 5 cfu / mL, 4×106 cfu / mL of Bacillus cereus B5 were mixed in equal volumes, and 10 μL of the mixture was inoculated into a sterile SMAY medium (120 mL of water containing 4.8 g maltose, 0.6 g peptone, 0.6 g yeast extract, and 2.16 g agar). After the mixture was absorbed, the plate was placed in a dark culture room at 25±2°C for incubation. After 4 days of incubation, the bacterial solution on the plate was taken and the formation of mycelium by blastospores was examined under a microscope ( Figure 2 ), and blastospores without Bacillus cereus B5 were used as controls. The results showed that the blastospores and Bacillus cereus mixed groups at 1:1, 1:10 and 1:100 all had three-level branched hyphae, while the blastospore control group without Bacillus cereus B5 did not form hyphae.

[0044] Embodiment 2:

[0045] Cordyceps sinensis ( Figure 1 B) Inoculate into autoclaved G5 liquid 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) at 12°C, 110 rpm, and dark culture for 50 days to obtain Cordyceps sinensis blastospores. Use 1× PBS (pH 7.4) to adjust the blastospore concentration to 4×10 4 / mL, respectively, with a concentration of 4×10 4 cfu / mL, 4×10 5 cfu / mL, 4×10 6 cfu / mL of Bacillus cereus B5 were mixed in equal volumes, 100 μL of the mixture was inoculated into 900 μL of sterile G5 liquid culture medium, and cultured in a 12±2℃ culture room at 110 rpm in the dark. After 8 days of culture, the culture solution was taken under a microscope to examine the proportion of blastospore-forming hyphae, and the control group without the addition of Bacillus cereus B5 was used as the control group. The results showed that the proportion of blastospore-forming hyphae in the control group without the addition of Bacillus cereus B5 was 11±5%, and the proportion of blastospore-forming hyphae in the mixed groups of blastospore and Bacillus cereus B5 at 1:1, 1:10 and 1:100 were 27±4%, 42±4% and 37±6%, respectively.

[0046] Embodiment 3:

[0047] M. anisopliae was inoculated into autoclaved SMY medium (120 mL of 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 spores of M. anisopliae. The spore concentration was fixed to 4 × 10 4 / mL, respectively, with a concentration of 4×10 4cfu / mL, 4×10 5 cfu / mL, 4×10 6 cfu / mL of Bacillus cereus B5 were mixed in equal volume ratios, and 10 μL of the mixture was inoculated into a sterile SMAY medium (120 mL of water containing 2.4 g maltose, 0.3 g peptone, 1.2 g yeast extract, and 2.16 g agar) plate. After the mixture was absorbed, the plate was placed in a dark culture room at 25±2°C for incubation. After 4 days of incubation, the plate bacterial solution was taken and the formation of hyphae by blastospores was examined under a microscope. The blastospores without the addition of Bacillus cereus B5 were used as controls. The results showed that the blastospores and Bacillus cereus B5 mixed groups at 1:1, 1:10, and 1:100 all had three-level branched hyphae, while the blastospore control group without the addition of Bacillus cereus had no hyphae formation.

[0048] Embodiment 4:

[0049] M. anisopliae was inoculated into autoclaved SMY medium (120 mL of 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 spores of M. anisopliae. 50 μL of a 1×10 6 A blastospore solution of 10 × 10 / mL was inoculated vertically on one side of a sterile SMAY medium (120 mL of water containing 4.8 g of maltose, 0.3 g of peptone, 0.6 g of yeast extract, and 2.16 g of agar) plate, and 50 μL of a blastospore solution of 4 × 10 / mL was inoculated on the left side in the same manner. 6 cfu / mL of Bacillus cereus B5, after the bacterial solution was absorbed by the plate, the plate was cultured in a confrontation manner without Bacillus cereus B5 as the control group, and the plate was cultured in the dark at 25±2℃. After 4 days of culture, the bacterial solution on the plate edge of the colony close to the Bacillus cereus side was taken, and the formation of hyphae by blastospores was examined under a microscope. The results showed that the confrontation culture group of blastospores and Bacillus cereus B5 had obvious hyphae, while the blastospore control group without Bacillus cereus B5 had no obvious hyphae, and there was no significant difference in the colony morphology after 12 days of culture ( Figure 3 ).

[0050] Embodiment 5:

[0051] M. reinhardtii was inoculated into autoclaved SMY medium (120 mL of 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 spores of M. reinhardtii. The spore concentration was fixed to 1×10 using 1×PBS (pH 7.4). 6 / mL, respectively with Bacillus cereus B5 (OD 600=1.5) 10×, 100× and 1000× diluted bacterial solutions were mixed in equal volume ratios, and 10 μL of the mixed solution was inoculated onto a sterile SMAY medium (120 mL of water containing 4.8 g maltose, 0.3 g peptone, 0.6 g yeast extract, and 2.16 g agar) plate. After the mixed solution was absorbed, the plate was placed in a dark culture room at 25±2°C for incubation. After 8 days of co-culture, the colony morphology was checked, and the blastospores without the addition of Bacillus cereus B5 were used as the control group. The results showed that green conidia were formed at the edges of the colonies in the group where blastospores and Bacillus cereus B5 were mixed in proportion, while no green conidia were formed in the blastospore control group without the addition of Bacillus cereus B5 ( Figure 4 ).

[0052] Embodiment 6:

[0053] M. anisopliae was inoculated into autoclaved SMY medium (120 mL of 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 spores of M. anisopliae. The spore concentration was fixed to 2 × 10 6 / mL, Bacillus cereus B5 culture solution (OD 600 =1.5) at room temperature, centrifuged at 4000 rpm, filtered through a 0.22 μm sterile filter membrane to obtain the supernatant, the blastospore solution and the sterile supernatant were mixed in equal volume ratios, and microinjected into 5-instar wax moth larvae at an injection concentration of 2×10 6 The control group was treated with a mixture of blastospores and 1×PBS in equal volume ratio, and each larva was injected with 4μL. After injection, the larvae were cultured in a 28℃ culture room. Three replicates were set up in each group, and the mortality rate of the larvae was checked every day. The results showed that the cumulative dead insect rate of the control group without the addition of Bacillus cereus B5 was 71±5% on the 5th day, and the cumulative mortality rate of the group injected with blastospores and Bacillus cereus mixture was 100% on the 7th day.

[0054] Embodiment 7:

[0055] Cordyceps sinensis was inoculated into autoclaved G5 liquid 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) and cultured in the dark at 12°C, 110 rpm for 50 days to obtain Cordyceps sinensis blastospores. The blastospore concentration was fixed to 2 × 10 using 1 × PBS (pH 7.4). 6 / mL, Bacillus cereus B5 culture solution (OD 600=1.5) at room temperature, centrifuged at 4000 rpm, filtered through a 0.22 μm sterile filter membrane to obtain the supernatant, the blastospore solution and the sterile supernatant were mixed in equal volume ratios, and microinjected into 5-year-old golden bat moths at an injection concentration of 2×10 6 The control group was treated with a mixture of blastospores and 1×PBS in equal volume ratio, and each larva was injected with 4μL. After injection, the larva was cultured in a dark room at 8±2℃, with 3 replicates per group. The dead insect rate was counted after 120 days. The results showed that the dead insect rate of the control group without Bacillus cereus was 31±5%, and the dead insect rate of the group injected with blastospores and Bacillus cereus was 42±6%.

Claims

1. Bacillus cereus B5, deposit number: GDMCC No: 65417.

2. Use of the Bacillus cereus B5 according to claim 1 in increasing the speed of blastospore hyphae formation and conidia formation of insect pathogenic fungi.

3. The use according to claim 2, characterized in that: The insect pathogenic fungus is Metarhizium anisopliae.

4. The use according to claim 2, characterized in that: It is an application in improving the efficiency of insect pathogenic fungi in killing host larvae.

5. Use of the Bacillus cereus B5 according to claim 1 in increasing the rigidification rate of bat moth larvae infected by Cordyceps sinensis.

6. The use according to claim 5, characterized in that: The blastospores of Cordyceps sinensis are mixed with the supernatant of Bacillus cereus B5 culture medium to infect the larvae of bat moths.

7. A method for improving the efficiency of insect pathogenic fungi in killing host larvae, characterized in that: The bacterial liquid of Bacillus cereus B5 described in claim 1 is mixed with insect pathogenic fungi or blastospores thereof to control host larvae.

8. The method according to claim 7, characterized in that The insect pathogenic fungus is Metarhizium anisopliae.

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