CN115838637A - Entomogenous fungus and fermentation method and application thereof - Google Patents
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Abstract
An entomogenous fungus and a fermentation method and application thereof relate to the field of microorganisms, in particular to an entomogenous fungus and application thereof. The method aims to solve the problems that the existing corn armyworm control means is low in efficiency and seriously affects the health of a human body. The fungus is beauveria bassiana BJ2, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the preservation date of 7 months and 15 days in 2022 years, and has the preservation number of CGMCC No.40247. The fungus fermentation method comprises the following steps: the preparation method comprises the steps of preparing a culture medium by taking wheat bran, sorghum, rice and sucrose as raw materials, and fermenting beauveria bassiana BJ2. The invention is used for biologically preventing and controlling corn armyworms, has high prevention and control efficiency, reduces the use of chemical pesticides, and has positive protection significance for black soil farmlands.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to an entomogenous fungus and application thereof.
Background
Corn is one of the main food crops in China and is also the most important feeding crop. Corn can be enriched in relevant plant diseases and insect pests in the planting process of successive years, and potential outbreak danger exists in special years.
Corn armyworm is a main pest in the corn production process and has a great harm degree to corn. Three generations generally occur in one year, and the second generation is mainly harmful to summer corns. The corn leaves are taken by the larvae destructively, and when the larvae seriously eat all the leaves, the larvae eat all the leaves in a short time, so that the serious yield reduction is caused and even the maize leaves are not harvested.
At present, the prevention and control means for corn armyworm mainly comprise physical prevention and control and chemical prevention and control. The physical prevention and control is mainly characterized in that the imagoes of armyworms have phototaxis and chemotaxis, and the imagoes are trapped and killed by nuisanceless prevention and control technologies such as sex traps and insecticidal lamps. The prevention and control mode is more environment-friendly, but the efficiency is lower, and the large-range prevention and control can not be easily carried out in a short time. The chemical prevention and control mainly adopts poison bait and foliar spray, wherein the poison bait is prepared by uniformly stirring 100 ml of 90 percent trichlorfon, a proper amount of water and 1.5 kg of fried bran per mu to prepare poison bait for trapping; the foliage spray is usually made by uniformly spraying 4.5% of high-effective cypermethrin 50 ml 1500-2000 times of solution or 40% of phoxim emulsifiable solution 1000 times of solution on the foliage. The chemical control efficiency is higher, but the chemical control method causes pollution to corns and the environment and seriously affects the health of human bodies.
Biological control of corn armyworms is a hot point of research in the year, but parasitic fungi are deficient in corn pest and disease damage research, and an effective technical system is not provided for support. The isolation and identification of microorganisms are at present urgent need in the research of the prevention and control of corn insect pests.
Disclosure of Invention
The invention provides an entomogenous fungus and a fermentation method and application thereof, aiming at solving the problems that the existing prevention and control means of corn armyworm has low efficiency and seriously affects the health of human bodies.
The invention provides a entomogenous fungus which is Beauveria bassiana (Beauveria bassiana) BJ2 and is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of Xilu No. 1 of Beijing market Kogyang North Chen, the preservation date is 7 and 15 days in 2022 years, and the preservation number is CGMCC No.40247.
Beauveria bassiana (Beauveria bassiana) BJ2 can grow in a solid potato sucrose culture medium in a hypha form. The colony color of the beauveria bassiana BJ2 is white, the hypha is white, the carpet shape is white, and the spore is conidiophore spore.
The preparation method of the potato sucrose culture medium comprises the following steps: cutting potato into pieces, adding water, cooking, filtering with gauze to obtain supernatant, adding 10g sucrose into the supernatant, and adding water to 1L.
The Beauveria bassiana (Beauveria bassiana) BJ2 is subjected to homology comparison with sequences in a gene bank through Blast analysis, the highest homology of the Beauveria bassiana (Beauveria bassiana) reaches 99%, and the strain BJ2 is determined by combining identification results of thallus morphological characteristics, growth conditions and the like, and is the Beauveria bassiana (Beauveria bassiana).
The invention also provides a fermentation method of beauveria bassiana BJ2, which comprises the following steps: uniformly mixing wheat bran, sorghum, rice and sucrose according to the mass ratio of 50; beauveria bassiana BJ2 is inoculated into a culture medium and is placed in an incubator for culture.
Further, the high-pressure steam sterilization conditions are as follows: sterilizing at 115-120 deg.C for 30-35min.
Further, the culture conditions are as follows: culturing at 24-25 deg.C for 30-35 days.
The invention also provides application of the beauveria bassiana BJ2 in prevention and treatment of corn insect pests.
Further, the insect pest is corn armyworm.
The invention has the beneficial effects that:
the Beauveria bassiana (Beauveria bassiana) can be rapidly propagated under the condition that wheat bran, sorghum and paddy are taken as main culture conditions, which shows that the Beauveria bassiana BJ2 can be propagated by taking the wheat bran, the sorghum and the paddy as main matrixes. And the beauveria bassiana BJ2 has obvious effect on reducing the occurrence of corn armyworm diseases.
Sorghum and rice chaff are cheap and easily-obtained agricultural and sideline products, have low viscosity, leave more pores for the growth of fungi in the fermentation process, and are easy for the growth and the propagation of the fungi. Compared with the prior mode of fermenting by taking bran and fine rice bran as main raw materials, the method has certain advantages.
The invention utilizes the biological control means, namely utilizes entomogenous fungi to efficiently control corn armyworms, reduces the use of chemical pesticides and has positive protection significance for black soil farmlands.
Drawings
FIG. 1 is a morphological diagram of the strain BJ2 on a PDA plate;
FIG. 2 is a phylogenetic tree of molecular identification of strain BJ 2;
FIG. 3 is a photograph of strain BJ2 in a tissue culture bottle using sorghum and rice as main raw materials;
FIG. 4 shows the control effect of strain BJ2 on corn armyworm.
Detailed Description
The following examples are given to illustrate the present invention, and the following examples are carried out on the premise of the technical solution of the present invention, and give detailed embodiments and specific procedures, but the scope of the present invention is not limited to the following examples.
Example 1:
the entomogenous fungus of the embodiment is Beauveria bassiana (Beauveria bassiana) BJ2 which is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is No. 3 of No. 1 Siro-Lu-1 of the Chao-Yang district in Beijing, the preservation date is 7-15 days in 2022 years, and the preservation number is CGMCC No.40247.
The method for obtaining the Beauveria bassiana (Beauveria bassiana) BJ2 comprises the following steps:
primary screening: selecting white muscardine silkworm from the national institute of silkworm science in Yongji county, jilin province by adopting a plate separation method, cutting the white muscardine silkworm into small pieces of about 2cm, placing the small pieces of the white muscardine silkworm in 45ml of sterile water for washing for a plurality of times, placing the small pieces of the white muscardine silkworm on a sterilized filter paper sheet for airing, placing the small pieces of the white muscardine silkworm on a potato solid culture medium, inversely placing the small pieces of the white muscardine silkworm in an incubator at 25 ℃ for culturing for 2-3 days, and separating the bacterial strain BJ2 from the white muscardine silkworm. The growth of colonies and the generation of transparent circles were observed.
The preparation method of the potato solid culture medium comprises the following steps: peeling 200g of potato, cutting into blocks, boiling in 500ml of water for 10min, filtering with gauze to obtain filtrate, adding 10g of sucrose and 10g of agar powder, adding distilled water to reach the volume of 1000ml, and sterilizing with high-pressure steam at 115 ℃ for 30min. About 20ml of medium was poured per 9cm X9 cm plate.
Example 2: identification of Strain BJ2
And (3) morphological observation:
the strain BJ2 of the invention can grow in a solid potato sucrose medium in a hyphal form. The colony color of the strain BJ2 is white, the hyphae is white, the carpet is white, and the spores are conidiophore spores. The morphology of the strain BJ2 on PDA plates is shown in FIG. 1.
The preparation method of the potato sucrose culture medium comprises the following steps: cutting potato into pieces, adding water, cooking, filtering with gauze to obtain supernatant, adding 10g sucrose into the supernatant, and adding water to 1L.
Molecular characterization of strain BJ 2:
the strain BJ2 was subjected to molecular characterization, and genomic DNA of the strain BJ2 was extracted using a genome extraction Kit (FastDNA Spin Kit for Soil) and subjected to ITS PCR amplification. The PCR universal primer is: primer ITS1F5 '-TCCGTAGGTGAACCTGCGG-3') and primer ITS4R (5 '-TCCTCCGCTTATTGATATGC-3'), PCR reaction system 25 μ L: premix version 2.0.5. Mu.L, primer ITS1F (10 nM) 1. Mu.L, primer ITS4R (10 nM) 1. Mu.L, DNA template 1. Mu.L, sterile water to 25. Mu.L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4min; denaturation at 94 ℃ 30s, annealing at 53 ℃ 50s, extension at 72 ℃ 50s,35 cycles; final extension at 72 ℃ for 5min.
After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, and a distinct characteristic band was observed at 520 bp. The PCR amplification products were sent to Biotechnology engineering (Shanghai) Co., ltd for sequencing, and the sequences were uploaded to Genbank. Homology alignment with sequences in the gene bank is carried out by Blast analysis, and a phylogenetic evolutionary tree is constructed by Neihbor-joining in MEGA5.1 software, and is shown in figure 2. The strain BJ2 has the highest homology with Beauveria bassiana (Beauveria bassiana) and reaches 99%, and the strain BJ2 is the Beauveria bassiana (Beauveria bassiana) BJ2 determined by combining the identification results of morphological characteristics, growth conditions and the like of thalli.
The ITS sequence of the strain BJ2 is as follows:
ACCGGGAGTTTTTCACTCCCTAACCCTTCTGTGAACCTACCTATCGTTGCTTCGGCGGACTCGCCCCAGCCCGGACGCGGACTGGACCAGCGGCCCGCCGGGGACCTCAAACTCTTGTATTCCAGCATCTTCTGAATACGCCGCAAGGCAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATCCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCGACCTCCCCTTGGGGAGGTCGGCGTTGGGGACCGGCAGCACACCGCCGGCCCTGAAATGGAGTGGCGGCCCGTCCGCGGCGACCTCTGCGTAGTAATACAGCTCGCACCGGAACCCCGACGCGGCCACGCCGTAAAACACCCAACTTCTGAACGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTT。
example 3: growth of Beauveria bassiana (Beauveria bassiana) BJ2
Indoor propagation experiments were performed on Beauveria bassiana (Beauveria bassiana) BJ2. Uniformly mixing wheat bran, sorghum, rice and sucrose according to the proportion of 50. Then, the PDA bacterial block of the 1cm directly punched bacterial strain BJ2 is inoculated under the aseptic condition. Culturing in 25 deg.C incubator for 30 days.
Beauveria bassiana BJ2 was able to grow well in a matrix of wheat bran: sorghum: rice: sucrose = 50. It is demonstrated that Beauveria bassiana (Beauveria basssana) BJ2 can grow well in culture medium using sorghum and rice (coarse rice chaff) as main raw materials.
Example 4: effect of Beauveria bassiana (Beauveria bassiana) BJ2 on corn mythimna
Corn armyworm was placed in petri dishes with 10 larvae of 2 instar per dish. Feeding armyworm feed (wheat bran as main material). Beauveria bassiana BJ2 is treated according to the spore concentration of 10 5 Spore/ml and 10 6 Preparing each spore/ml, and then uniformly spraying the spore/ml on the surface of the corn armyworm. The control group was not inoculated. Each treatment was 3 replicates. Culturing in 25 deg.C incubator for 5-10 days. And (5) observing the disease condition of corn armyworms. And calculating the survival rate of the insects. The calculation formula of the insecticidal rate is as follows: insecticidal rate = 1-survival rate.
The effect of beauveria bassiana BJ2 on corn armyworm is shown in fig. 4. In FIG. 4, a is a control group, and b is a spore concentration of 10 5 Spore/ml treatment group, c is spore concentration 10 6 Spores/ml treatment group. The measurement result shows that the beauveria bassiana BJ2 can obviously infect corn armyworm, 10 6 The insecticidal rate of the bacterial liquid of each spore/ml can reach 73.33%.
And (4) conclusion: beauveria bassiana BJ2 can obviously kill corn armyworms.
Claims (6)
1. An entomogenous fungus is characterized in that the fungus is Beauveria bassiana (Beauveria bassiana) BJ2, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation address is No. 3 of Xilu No. 1 North Chen of Chaoyang district, beijing, the preservation date is 2022 years, 7 months and 15 days, and the preservation number is CGMCC No.40247.
2. The method of claim 1, which is a fermentation process of beauveria bassiana BJ2, characterized in that the method comprises the steps of: uniformly mixing wheat bran, sorghum, rice and sucrose according to the mass ratio of 50; beauveria bassiana BJ2 is inoculated into a culture medium and is placed in an incubator for culture.
3. Fermentation process according to claim 2, characterized in that the autoclaving conditions are: sterilizing at 115-120 deg.C for 30-35min.
4. Fermentation process according to claim 2, characterized in that the culture conditions are: culturing at 24-25 deg.C for 30-35 days.
5. The use of beauveria bassiana BJ2 as defined in claim 1 for controlling corn pest.
6. The use of claim 5, wherein the insect pest is corn armyworm.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004069A (en) * | 2019-05-14 | 2019-07-12 | 西南林业大学 | A kind of Strain of Beauveria bassiana and its application |
| CN114317289A (en) * | 2022-01-11 | 2022-04-12 | 云南农业大学 | Beauveria bassiana Bbsfa202007 strain and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110004069A (en) * | 2019-05-14 | 2019-07-12 | 西南林业大学 | A kind of Strain of Beauveria bassiana and its application |
| CN114317289A (en) * | 2022-01-11 | 2022-04-12 | 云南农业大学 | Beauveria bassiana Bbsfa202007 strain and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| MUHAMMAD NAEEM MALIK 等: "Microbial Control of Maize Army Worm, Mythimna separata (Lepidoptera: Noctuidae) by Entomopathogenic Fungi", 《PAKISTAN J. ZOOL.》, pages 1607 - 1614 * |
| 程茵 等: "三株球孢白僵菌的分离鉴定与其对粘虫的室内毒力", 《中国生物防治学报》, pages 521 - 530 * |
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