CN115838637B - Entomogenous fungi and fermentation method and application thereof - Google Patents
Entomogenous fungi and fermentation method and application thereof Download PDFInfo
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- CN115838637B CN115838637B CN202211302002.7A CN202211302002A CN115838637B CN 115838637 B CN115838637 B CN 115838637B CN 202211302002 A CN202211302002 A CN 202211302002A CN 115838637 B CN115838637 B CN 115838637B
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- 241000751139 Beauveria bassiana Species 0.000 claims abstract description 44
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- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 29
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 29
- 235000005822 corn Nutrition 0.000 claims abstract description 29
- 241001477931 Mythimna unipuncta Species 0.000 claims abstract description 17
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 12
- 229930006000 Sucrose Natural products 0.000 claims abstract description 12
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- 235000015099 wheat brans Nutrition 0.000 claims abstract description 8
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- 240000007594 Oryza sativa Species 0.000 claims abstract 2
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- 241001302652 Bassiana Species 0.000 claims 1
- 241000223679 Beauveria Species 0.000 claims 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
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 corn pest control method aims at solving the problems that the efficiency of the existing corn pest control means is low and the health of a human body is seriously affected. The fungus is beauveria bassiana BJ2, and is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation date of 2022, 7 months and 15 days, and a preservation number of CGMCC No.40247. The fungus fermentation method comprises the following steps: the method is characterized in that wheat bran, sorghum, rice and sucrose are used as raw materials to prepare a culture medium, and beauveria bassiana BJ2 is fermented. The invention is used for biologically preventing corn armyworms, has high preventing and controlling 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 a entomogenous fungus and application thereof.
Background
Corn is one of the main grain crops in China and is also the most main feed crop. The corn can be enriched with related diseases and insect pests in the successive year planting process, and potential outbreak danger exists in special years.
Corn armyworm is a main pest in the corn production process, and has quite large damage degree to corn. Three generations generally occur in one year, and summer corns are often damaged mainly in the second generation. The corn leaves are taken by the larvae destructively, and when serious, all the leaves are eaten in a short time, so that serious yield reduction and even harvest failure are caused.
At present, the prevention and treatment means for corn armyworms are mainly physical prevention and treatment and chemical prevention and treatment. Wherein the physical control mainly adopts pollution-free control technologies such as sex traps, insecticidal lamps and the like to trap and kill adults according to the characteristic that the adults of the armyworms have phototaxis and chemotaxis. The control mode is environment-friendly, but has low efficiency, and is difficult to be widely carried out in a short time. The chemical control mainly adopts poisoning baits and foliar spray, wherein the poisoning baits are prepared by uniformly stirring 100 milliliters of 90 percent trichlorfon per mu with a proper amount of water and 1.5 kilogram of stir-fried bran to prepare the poisoning baits for attracting; the foliar spray is generally to uniformly spray 50 ml 1500-2000 times of high-efficiency cypermethrin or 1000 times of 40% phoxim emulsifiable concentrate on the foliar. The chemical control efficiency is higher, but the pollution to corn and environment is caused, and the health of human body is seriously affected.
Biological control of corn armyworms is a hotspot studied in recent years, but parasitic fungi are deficient in corn pest and disease research, and no effective technical system support exists. At present, the separation and identification of microorganisms in the research of corn pest control process are urgent.
Disclosure of Invention
The invention provides a entomogenous fungi and a fermentation method and application thereof, and aims to solve the problems that the efficiency of the existing corn armyworm control means is low and the health of a human body is seriously influenced.
The invention provides a entomogenous fungus, which is beauveria bassiana (Beauveria bassiana) BJ2 and is preserved in China general microbiological culture Collection center (CGMCC), wherein the preservation address is North Chen Xili No. 1, 3 in the Korean region of Beijing, the preservation date is 2022, 7 months and 15 days, and the preservation number is CGMCC No.40247.
The beauveria bassiana (Beauveria bassiana) BJ2 of the present invention can be grown in mycelium form in solid potato sucrose medium. The colony of beauveria bassiana BJ2 is white, hypha is white, carpet is white, and spores are conidia.
The preparation method of the potato sucrose culture medium comprises the following steps: cutting potato, adding water, boiling, 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 Blast analysis and sequence homology comparison in a gene library, the homology with beauveria bassiana (Beauveria bassiana) is highest and reaches 99%, and the strain BJ2 is beauveria bassiana (Beauveria bassiana) determined by combining with identification results such as morphological characteristics and growth conditions of the fungus.
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 50:25:25:1, then adding an MS sugar-free culture solution, and sterilizing by high-pressure steam to obtain a culture medium; inoculating beauveria bassiana BJ2 into a culture medium, and culturing in an incubator.
Further, the autoclaving conditions are: sterilizing at 115-120deg.C for 30-35min.
Further, the culture conditions are as follows: culturing at 24-25deg.C for 30-35 days.
The invention also provides application of beauveria bassiana BJ2 in preventing and controlling 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 of taking wheat bran, sorghum and paddy as main culture conditions, which indicates that beauveria bassiana BJ2 can be propagated by taking wheat bran, sorghum and paddy as main matrixes. Moreover, beauveria bassiana BJ2 has remarkable effect on reducing the occurrence of corn myxomycete diseases.
Sorghum and rice chaff are cheap and easily available agricultural and sideline products, have low viscosity, leave more pores for fungus growth in the fermentation process, and are easy for fungus growth and propagation. Compared with the traditional fermentation mode which takes bran and fine rice bran as main raw materials, the method has certain advantages.
The invention utilizes biological control means, namely utilizes the entomogenous fungi to control corn armyworms with high efficiency, reduces the use of chemical pesticides, and has positive protection significance on black soil farmlands.
Drawings
FIG. 1 is a morphology of strain BJ2 on a PDA plate;
FIG. 2 is a phylogenetic tree of molecular characterization of strain BJ 2;
FIG. 3 is a photograph of strain BJ2 in a tissue culture flask with sorghum and rice as the major raw materials;
FIG. 4 shows the control effect of strain BJ2 on corn armyworm.
Detailed Description
The following examples of the present invention are described in detail, and are provided by taking the technical scheme of the present invention as a premise, and the detailed embodiments and specific operation procedures are given, but the scope of the present invention is not limited to the following examples.
Example 1:
the entomogenous fungi of the embodiment is beauveria bassiana (Beauveria bassiana) BJ2 which is preserved in China general microbiological culture Collection center (CGMCC), the preservation address is 1 # 3 of North Chen Xili in the Korean area of Beijing, the preservation date is 2022, 7 months and 15 days, and the preservation number is CGMCC No.40247.
The method for obtaining beauveria bassiana (Beauveria bassiana) BJ2 in the embodiment comprises the following steps:
and (3) primary screening: selecting Bombyx Batryticatus from Yongji county silkworm industry science research institute in Jilin, cutting Bombyx Batryticatus into small pieces of about 2cm, washing with 45ml sterile water for several times, air drying on sterilized filter paper sheet, placing the small pieces on potato solid culture medium, culturing in incubator at 25deg.C for 2-3 days, and separating to obtain strain BJ2. The colony growth and the transparent ring generation were observed.
The preparation method of the potato solid culture medium comprises the following steps: peeling 200g of potato, cutting, 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 volume of 1000ml, and sterilizing with high pressure steam at 115 ℃ for 30min. Each 9cm by 9cm plate was poured into about 20ml of medium.
Example 2: identification of Strain BJ2
Morphological observation:
the strain BJ2 of the present invention can be grown in a hyphal form in a solid potato sucrose medium. The bacterial colony of the strain BJ2 is white, hypha is white, carpet is white, and spores are conidium. A morphology of strain BJ2 on a PDA plate is shown in FIG. 1.
The preparation method of the potato sucrose culture medium comprises the following steps: cutting potato, adding water, boiling, filtering with gauze to obtain supernatant, adding 10g sucrose into the supernatant, and adding water to 1L.
Molecular characterization of strain BJ 2:
molecular identification is carried out on the strain BJ2, and genome DNA of the strain BJ2 is extracted by utilizing a genome extraction kit (FastDNA Spin Kit for Soil) to carry out PCR amplification of ITS. The PCR universal primers are as follows: primer ITS1F5 '-TCCGTAGGTGAACCTGCGG-3') and primer ITS4R (5 '-TCCTCCGCTTATTGATATGC-3'), 25 μl of PCR reaction system: premix version 2.0.12.5. 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 were: pre-denaturation at 94℃for 4min; denaturation at 94℃for 30s, annealing at 53℃for 50s, extension at 72℃for 50s,35 cycles; finally, the extension is carried out for 5min at 72 ℃.
After PCR amplification, the product was checked by 1.5% agarose gel electrophoresis, resulting in a distinct characteristic band at 520 bp. The PCR amplified product was sent to the biological engineering (Shanghai) Co., ltd for sequencing and the resulting sequence was uploaded to Genbank. Homology alignment is performed with sequences in the gene library by Blast analysis, and a phylogenetic tree is constructed by Neihbor-Joing in MEGA5.1 software, and is shown in FIG. 2. The homology of the strain BJ2 with beauveria bassiana (Beauveria bassiana) is highest and reaches 99%, and the strain BJ2 is beauveria bassiana (Beauveria bassiana) BJ2 according to the embodiment determined by combining the identification results of the morphological characteristics, growth conditions and the like of the strain.
The ITS sequence of strain BJ2 is:
ACCGGGAGTTTTTCACTCCCTAACCCTTCTGTGAACCTACCTATCGTTGCTTCGGCGGACTCGCCCCAGCCCGGACGCGGACTGGACCAGCGGCCCGCCGGGGACCTCAAACTCTTGTATTCCAGCATCTTCTGAATACGCCGCAAGGCAAAACAAATGAATCAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATCCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCGACCTCCCCTTGGGGAGGTCGGCGTTGGGGACCGGCAGCACACCGCCGGCCCTGAAATGGAGTGGCGGCCCGTCCGCGGCGACCTCTGCGTAGTAATACAGCTCGCACCGGAACCCCGACGCGGCCACGCCGTAAAACACCCAACTTCTGAACGTTGACCTCGAATCAGGTAGGACTACCCGCTGAACTT。
example 3: growth status of beauveria bassiana (Beauveria bassiana) BJ2
Indoor propagation experiments are carried out on beauveria bassiana (Beauveria bassiana) BJ2. Mixing wheat bran, sorghum, rice and sucrose at a ratio of 50:25:25:1, adding appropriate amount of MS sugar-free culture solution to make the mixture fully absorb water, and sterilizing with 115 deg.C high pressure steam for 30min. Then, 1cm of PDA pellet of directly-perforated strain BJ2 was inoculated under aseptic conditions. Culturing in an incubator at 25 ℃ for 30 days.
Beauveria bassiana BJ2 grew well in a wheat bran to sorghum to rice to sucrose=50:25:25:1 matrix, and a rapid propagation picture in the matrix is shown in fig. 3. It was demonstrated that beauveria bassiana (Beauveria bassiana) BJ2 was able to grow well in a culture medium with sorghum and rice (crude rice chaff) as main raw materials.
Example 4: influence of beauveria bassiana (Beauveria bassiana) BJ2 on corn armyworm
Corn armyworms were placed in petri dishes, each with 10 2-year-old larvae. Feeding the mythic fungus feed (using wheat bran as main raw material). Beauveria bassiana BJ2 with spore concentration of 10 5 Individual spores/ml and 10 6 The spores/ml are prepared and then evenly sprayed on the surface of corn armyworm. The control group is a sterile treatment. Each treatment was repeated 3 times. Culturing in incubator at 25deg.C for 5-10 days. Observing the disease condition of corn armyworm. The survival rate of the worms was calculated. 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 figure 4. In FIG. 4, a is a control group and b is spore concentration 10 5 Individual spores/ml treatment group, c is spore concentration 10 6 Individual spores/ml treatment group. Measurement results show that beauveria bassiana BJ2 can obviously infect corn armyworms, 10 6 The insecticidal rate of bacterial liquid of each spore/ml can reach 73.33 percent.
Conclusion: beauveria bassiana BJ2 can be used for remarkably killing corn armyworms.
Claims (5)
1. An entomogenous fungus is characterized in that the fungus is beauveria bassianaBeauveria bassiana) BJ2 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation address of 1 # 3 of North West Lu of the Korean area of Beijing, a preservation date of 2022, 7 months and 15 days, and a preservation number of 40247.
2. A fermentation process for beauveria bassiana BJ2 as claimed in claim 1, characterized in that the process comprises the steps of: uniformly mixing wheat bran, sorghum, rice and sucrose according to the mass ratio of 50:25:25:1, then adding an MS sugar-free culture solution, and sterilizing by high-pressure steam to obtain a culture medium; inoculating beauveria bassiana BJ2 into a culture medium, and culturing in an incubator.
3. Fermentation process according to claim 2, characterized in that the conditions of autoclaving are: sterilizing at 115-120deg.C for 30-35min.
4. Fermentation process according to claim 2, characterized in that the conditions of the culture are: culturing at 24-25deg.C for 30-35 days.
5. The use of beauveria bassiana BJ2 as claimed in claim 1 for controlling corn armyworms.
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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 |
|---|
| Microbial Control of Maize Army Worm, Mythimna separata (Lepidoptera: Noctuidae) by Entomopathogenic Fungi;Muhammad Naeem Malik 等;《Pakistan J. Zool.》;第1607-1614页 * |
| 三株球孢白僵菌的分离鉴定与其对粘虫的室内毒力;程茵 等;《中国生物防治学报》;第521-530页 * |
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