CN113481109B - Beauveria bassiana and application thereof in preventing and treating scarab beetles - Google Patents
Beauveria bassiana and application thereof in preventing and treating scarab beetles Download PDFInfo
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Abstract
The invention relates to beauveria bassiana and application thereof in preventing and treating scarab beetles. The Beauveria bassiana is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23049.
Description
Technical Field
The invention relates to beauveria bassiana, and particularly relates to beauveria bassiana which can be used for preventing and treating scarab beetles.
Background
The soil insects are important farmland insects, particularly grubs, cutworms, wireworms, root maggots, mole cricket and other groups are common and seriously harmful. In recent years, with the warming of climate, the perfection of water conservancy facilities, the change of farming system and the adjustment of planting agriculture structure, especially the popularization of ground covering and heat preservation technology, a very favorable living environment is created for underground pests, so that the underground pests of crops are increasingly harmed.
Because underground pests are hidden underground, the prevention and the control of the underground pests are difficult points of prevention and control over the years, and the underground pests mainly depend on chemical pesticide control for a long time. The long-term use of chemical pesticides in large quantities causes water and soil environmental pollution, the pesticide residue of crops exceeds the standard, the economic benefit of agriculture is directly influenced, and the food with pesticide residue exceeding the standard also seriously threatens the safety of human and livestock and the social stability, has attracted the wide attention of governments and social circles, so that the biological control is more and more emphasized.
Disclosure of Invention
One of the invention provides Beauveria bassiana (Beauveria brongniartii) which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23049.
The second invention provides an engineering bacterium obtained by genetically modifying the beauveria bassiana according to the first invention. The beauveria bassiana CGMCC No.23049 is used as a target of the engineering bacteria, and the adopted means is generally to transfer and/or knock out a specific gene and/or DNA fragment and the like into the target, so the engineering bacteria are still beauveria bassiana. In addition, the engineering bacteria can be engineering bacteria with improved activity on scarab pests. Engineered strains may also be provided that have other pest activities and/or are endowed with other beneficial properties.
In a specific embodiment, the engineered bacterium is obtained by transforming the beauveria brockii with a plasmid carrying an anti-disease gene and/or an anti-insect gene.
The third invention provides a composition, which comprises the beauveria bassiana as described in the first invention or the engineering bacteria as described in the second invention, and an acceptable carrier.
The fourth invention provides the application of the beauveria bassiana of the first invention, the engineering bacteria of the second invention or the composition of the third invention in preventing and treating scarab beetle (Scarabaeidae).
In a specific embodiment, the chafer is a gill-tortoise (Holotrichia) and/or a tortoise (Anomala).
In a specific embodiment, the gill-gold (Holotrichia) is a Holotrichia parallela (Holotrichia parallela) and/or a Holotrichia magna (Holotrichia oblita); the Testudinis is Aeruginosa (Anomala corpulenta).
In a specific embodiment, the chafer (Scarabaeidae) is an egg and/or larval stage chafer.
In a specific embodiment, the gill-tortoise (Holotrichia) is an egg and/or larval stage gill-tortoise (Holotrichia); the tortoise (Anomala) is an egg and/or larva-stage tortoise (Anomala).
In a specific embodiment, the Holotrichia parallela is an egg and/or larval stage Holotrichia parallela; the Holotrichia parallela (Holotrichia oblite) is an egg and/or larval stage Holotrichia parallela (Holotrichia oblite); the Aerugo tortoise (Anamalia corpuscula) is the Onala corpuscle and/or larva stage Aerugo tortoise (Anamalia corpuscula).
The invention has the beneficial effects that:
the invention discovers that the beauveria bassiana has a strain with high insecticidal activity on chafer, particularly on eggs and larvae of the chafer. Not only the application of the beauveria bassiana is widened, but also the means for preventing and treating the chafer are widened.
Drawings
FIG. 1 shows a phylogenetic tree of the isolate numbered CZBJ-1.
Strain preservation
The Beauveria bassiana screened by the invention is named as CZBJ-1, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.23049, the preservation date is 2021 year, 7 months and 15 days, and the preservation address is as follows: the institute of microbiology, national academy of sciences, no.3, west Lu No.1, beijing, chaoyang, beicheng, area, beichen. Its system is classified as Beauveria brangniartii (Beauveria brongniartii).
Detailed Description
The above-described aspects of the invention are explained in more detail below by means of preferred embodiments, but they are not intended to limit the invention.
The reagents in the examples of the present invention were all commercially available unless otherwise specified.
Example 1
Isolation and Classification of strains
Selecting and inoculating the epihyphae of the adult Holotrichia parallela on a PDA culture medium, and performing multiple separation culture and purification at 26 ℃ to obtain a purified isolated strain. The isolated strains were numbered.
1. Morphological characterization of isolated strains
The isolated strain named CZBJ-1 appears white flocculent hypha in the early stage of colony on PDA plate, and can produce conidium in white powder form after culturing for 7-10 days, and the back of the strain is red or purple red. Conidiophores peduncles and conidia can be seen under a microscope, and the conidia are oval and have the size of 2.0-3.0 multiplied by 3.0-6.0 mu m. Based on the above morphological observations, beauveria was preliminarily identified as Beauveria (Beauveria).
2. Molecular characterization of isolated strains
Fungal universal primers ITS1 (SEQ ID No. 1) and ITS4 (SEQ ID No. 2) were synthesized by Biotechnology engineering (Shanghai) Ltd. PCR amplification was carried out using the extracted genomic DNA of the isolate numbered CZBJ-1 as a template and ITS1 and ITS4 as primers to obtain a PCR product. And (3) sending the PCR product to Shanghai biological engineering Co., ltd for sequencing, wherein the result of the sequence is shown as SEQ ID No. 3. And (3) carrying out homology BLASTE comparison on the sequencing result in a Genbank database, wherein the comparison result shows that the homology of the PCR amplification sequence of the strain CZBJ-1 and the sequence of beauveria bassiana is more than 98%. Phylogenetic trees were created using MEGA7 software, see FIG. 1. As can be seen from the phylogenetic tree of FIG. 1, the strain CZBJ-1 was aggregated with Beauveria bassiana.
In conclusion, the isolated strain with the number CZBJ-1 was systematically classified as Beauveria brangniartii (Beauveria brongniartii).
The CZBJ-1 strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.23049, the preservation date is 2021 years, 7 months and 15 days, and the preservation address is as follows: the institute of microbiology, national academy of sciences No.3, xilu No.1, beijing, chaoyang, beijing. Its system is classified as Beauveria brangniartii (Beauveria brongniartii).
Example 2
Biological activity assay
(1) Biological activity determination of 1-instar larvae of Holotrichia parallela
The purified isolated strain was propagated using PDA medium by culturing at 26 ℃ for 7 to 10 days on PDA medium, scraping conidium powder, and formulating into 1.4X 10% with 0.1% Tween-80 sterile water 8 、2.8×10 7 、5.6×10 6 、1.12×10 5 And 0.22X 10 4 Spores/ml spore suspension.
Preparing shredded potatoes, washing the shredded potatoes with clear water, and airing until no moisture exists on the surface. Soaking shredded potato in spore suspension for about 20min, taking out, uniformly placing in 6-hole bioassay plate with 4-5 pieces per hole, mixing the rest spore suspension with 100g sterilized dry soil, and mixing to make soil humidity 18% and final lifetime concentration of 2.52 × 10 7 ，5.05×10 6 ，1.01×10 6 ，2.02×10 4 ，0.4×10 3 Spores/g soil. And (3) uniformly subpackaging the mixed soil into 6-hole bioassay plates with corresponding concentrations, inoculating 1 head of larvae of Holotrichia parallela of 1 year (6-day-old larvae hatching) into each hole, and feeding in an incubator at the temperature of 26 ℃ and under the illumination L: D = 16. Each repeat was 30, 3 repeats. Tween-80 (0.1%) as a negative control was added to sterilized dry soil so that the soil humidity was 18%. Dead and live insect counts were investigated after 15 days, mean and corrected mortality was calculated using Excel2007 and anova was performed using SPSS 17.0.
The results are shown in Table 1.
(2) Biological activity assay for Holotrichia parallela 2-instar larvae
The adopted test insects are larvae of Holotrichia parallela of 2 th age (15-day-old larvae hatched), and the other test insects are same as those in the section (1) in the example 2.
The results are shown in Table 1.
(3) Biological activity determination of 3-instar larvae of Holotrichia parallela
The adopted test insects are larvae of Holotrichia parallela of 3 th age (larvae of 45 th day), and the other test insects are same as those in the section (1) in the example 2.
(4) Biological activity assay of Holotrichia parallela eggs
Adding the spore suspension into 100g of sterilized dry soil, mixing uniformly to obtain a soil sample to be tested, putting the soil sample to be tested into a food plastic box with the length being multiplied by 17cm multiplied by 12cm (length being multiplied by width being multiplied by height), picking eggs newly produced by Holotrichia parallela for 5 days, uniformly placing the eggs into the soil sample to be tested, and repeating the steps of 50 eggs in each box for 3 times. Spore suspensions were prepared and concentration gradients as in example 2, subsection (1). Tween-80 (0.1%) as a negative control was added to sterilized dry soil so that the soil humidity was 18%. After 15 days, the eggs were investigated for infestation.
The results are shown in Table 1.
TABLE 1
Note: p <0.05
(5) Biological activity determination of 1-instar larvae of Holotrichia parallela
The spore suspension was prepared in a concentration gradient of 3.0X 10 8 ，6.0×10 7 ，1.2×10 7 ，2.4×10 6 ，4.8×10 5 Spores/ml. Mixing the spore suspension with sterilized dry soil to obtain soil sample with spore concentration of 5.4 × 10 7 ，1.08×10 7 ，2.16×10 6 ，4.32×10 5 ，8.64×10 4 Spores/g soil.
The adopted test insects are 1 st larvae (6 th day larvae) of Holotrichia parallela.
The rest is the same as the section (1) in example 2.
The results are shown in Table 2.
(6) Biological activity determination of 2-instar larvae of Holotrichia parallela
The adopted test insects are 2-year larvae of Holotrichia parallela (15-day-old larvae hatched), and the other test insects are the same as those in the section (5) in the example 2.
The results are shown in Table 2.
(7) Biological activity determination of Holotrichia parallela 3-instar larvae
The adopted test insects are larvae of Holotrichia parallela at 3 rd age (larvae of 45 days old), and the other test insects are same as those in the section (5) in the example 2.
(8) Biological activity determination of Holotrichia parallela eggs
Adding the spore suspension into 100g of sterilized dry soil, uniformly mixing to obtain a soil sample to be detected, putting the soil sample to be detected into a food plastic box with the length being multiplied by 17cm multiplied by 12cm (length being multiplied by width being multiplied by height), picking eggs newly produced by Holotrichia parallela for 5 days, uniformly placing the eggs into the soil sample to be detected, and repeating the steps for 3 times, wherein 50 eggs are placed in each box. Spore suspensions were prepared and concentration gradients as in subsection (5) of example 2. Tween-80 (0.1%) as a negative control was added to sterilized dry soil so that the soil humidity was 18%. After 15 days, the eggs were investigated for infestation.
The results are shown in Table 2.
TABLE 2
Note: p <0.05
(9) Biological activity determination of 1-instar larva of Aerugo chafer
The adopted test insects are 1 st larva (6 th larva after hatching) of the anomala aeruginosa, and the other test insects are the same as the section (5) in the example 2.
The results are shown in Table 3.
(10) Biological activity determination of 2-instar larva of Aerugo chafer
The adopted test insects are 2-instar larvae of the Aerugo chafer (15-day-old larvae), and the rest is the same as the section (5) in the example 2.
The results are shown in Table 3.
(11) Bioactivity determination of 3-instar larva of Aerugo chafer
The adopted test insects are larvae of the 3 rd instar of the Aerugo chafer (larvae of 45 days old), and the rest is the same as the subsection (5) in the example 2.
(12) Bioactivity determination of eggs of Aerugo-ligonum-testudinis
The test object adopted is the eggs of the newly born 5-day tortoise plastron aeruginosa, and the rest is the same as the section (8) in the example 2. The results are shown in Table 3.
TABLE 3
Note: p <0.05
Sequence listing
<110> Cangzhou city academy of sciences for agriculture and forestry
<120> beauveria bassiana and application thereof in preventing and treating scarab beetles
<130> LHA2160430
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
tccgtaggtg aacctgcgg 19
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 545
<212> DNA
<213> Beauveria bassiana (Beauveria brongniartii)
<400> 3
ggacttcgag ttttactccc taacccttat gtgaacctac ctattgttgc ttcggcggac 60
tcgccccagc cggacgcgga ctggaccagc ggccgccggg gaccctcaaa ctcttgtatt 120
atcagcatct tctgaatacg ccgcaaggca aaacaaataa atcaaaactt tcaacaacgg 180
atctcttggc tctggcatcg atgaagaacg cagcgaaacg cgataagtaa tgtgaattgc 240
agaatccagt gaatcatcga atctttgaac gcacattgcg cccgccagca ttctggcggg 300
catgcctgtt cgagcgtcat ttcaaccctc gacctccctt tggggaagtc ggcgttgggg 360
accggcagca caccgccggc cctgaaatgg agtggcggcc cgtccgcggc gacctctgcg 420
tagtaatcca actcgcaccg gaaccccgac gtggccacgc cgtaaaacac ccaacttctg 480
aacgttgacc tcgaatcagg taggactacc cgctgaactt aagcatatca ataaggccgg 540
aggaa 545
Claims (4)
1. Beauveria bracteata (Beauveria brongniartii) which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23049.
2. A composition comprising the beauveria brookfield of claim 1, and an acceptable carrier.
3. Use of beauveria brookfield of claim 1 or the composition of claim 2 for the control of scarab beetle (Scarabaeidae);
the chafer is a gill-plate tortoise (Holotrichia) and/or a lima-plate tortoise (Anomala);
the golden cuora (Holotrichia) is an egg and/or larval stage golden cuora (Holotrichia), and the golden cuora (Anomala) is an egg and/or larval stage Aerugo's larva (Anomala corpulenta).
4. Use according to claim 3, wherein the Holotrichia parallela (Holotrichia) is Holotrichia parallela (Holotrichia parallela) and/or Holotrichia diomphalia (Holotrichia oblia).
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| CN107779408A (en) * | 2017-11-02 | 2018-03-09 | 河北省农林科学院植物保护研究所 | A kind of beauveria bassiana and its microbial bacterial agent for being used to prevent and treat chafer |
| CN112280691A (en) * | 2020-11-05 | 2021-01-29 | 河北省农林科学院植物保护研究所 | Beauveria bassiana for preventing and treating Taurus mahogany and application thereof |
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| JP2002338419A (en) * | 2001-05-14 | 2002-11-27 | Nitto Denko Corp | Pest control preparation and method for pest control using the same |
| CN108378063A (en) * | 2018-05-18 | 2018-08-10 | 中国农业科学院农业环境与可持续发展研究所 | A kind of preparation method of muscardine granule |
| CN109337822B (en) * | 2018-10-10 | 2020-05-22 | 中国农业科学院植物保护研究所 | Beauveria bassiana BRNS50206 and application thereof |
| CN109757521B (en) * | 2019-02-10 | 2021-02-23 | 中国农业科学院植物保护研究所 | Beauveria bassiana microbial inoculum and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107779408A (en) * | 2017-11-02 | 2018-03-09 | 河北省农林科学院植物保护研究所 | A kind of beauveria bassiana and its microbial bacterial agent for being used to prevent and treat chafer |
| CN112280691A (en) * | 2020-11-05 | 2021-01-29 | 河北省农林科学院植物保护研究所 | Beauveria bassiana for preventing and treating Taurus mahogany and application thereof |
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