阅读说明：本技术 一种布氏白僵菌及其在防治金龟子中的应用 (Beauveria bassiana and application thereof in preventing and treating scarab beetles ) 是由 吴娱 刘春琴 王庆雷 时荣荣 冯晓洁 刘福顺 张悦 束长龙 张�杰 于 2021-07-30 设计创作，主要内容包括：本发明涉及一种布氏白僵菌及其在防治金龟子中的应用。所述布氏白僵菌保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23049。(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 the China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No. 23049.)

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. An engineered bacterium obtained by genetically modifying the beauveria brockii of claim 1.

3. The engineered bacterium of claim 2, wherein the engineered bacterium is obtained by transforming the beauveria brockii with a plasmid carrying an antipathogenic and/or an antiparasitic gene.

4. A composition comprising the beauveria brockii of claim 1 or the engineered bacterium of claim 2 or 3, and an acceptable carrier.

5. Use of beauveria brookfield of claim 1, the engineered bacterium of claim 2 or 3, or the composition of claim 4 for the control of scarab beetle (Scarabaeidae).

6. The use according to claim 5, wherein the chafer is a gill-tortoise (Holotrichia) and/or a tortoise (Anomala).

7. The use according to claim 6, wherein the Holotrichia parallela (Holotrichia parallela) is Holotrichia parallela (Holotrichia parallela) and/or Holotrichia parallela (Holotrichia oblita); the tortoise (Anomala) is an Anomala corpulenta.

8. Use according to claim 5, wherein the scarab beetles (Scarabaeidae) are egg and/or larval stage scarabs.

9. Use according to claim 6, wherein the gill-tortoise (Holotrichia) is an egg and/or larval stage gill-tortoise (Holotrichia); the tortoise (Anomala) is an egg and/or larval stage tortoise (Anomala).

10. The use according to claim 7, wherein 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).

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 agricultural 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 seriously damaged.

Because underground pests are hidden underground, the prevention and the control of the underground pests are difficult points 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 environment pollution, the pesticide residue of crops exceeds the standard, the economic benefit of agriculture is directly influenced, and the food with the pesticide residue exceeding the standard also seriously threatens the safety of people 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 paid attention.

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 that have other pesticidal activities and/or are endowed with other beneficial properties may also be used.

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-chafer (Holotrichia) and/or a millipore chafer (Anomala).

In a specific embodiment, the gill-gold (Holotrichia) is a Holotrichia parallela (Holotrichia parallela) and/or a Holotrichia magna (Holotrichia oblita); the tortoise (Anomala) is an 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 larval 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. The application of the beauveria bassiana is widened, and the means for preventing and treating the scarab beetles is also 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 China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number of CGMCC No.23049, the preservation date of 2021, 7 and 15 days, and the preservation addresses are 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).

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 (Shanghai) Inc. PCR 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 strain CZBJ-1 is preserved in the China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.23049, the preservation date is 2021, 7 and 15 days, and the preservation addresses are 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 is propagated by PDA culture medium, i.e. cultured on PDA culture medium at 26 deg.C for 7-10 days, conidium powder is scraped, and then prepared into 1.4 × 10 with 0.1% Tween-80 sterile water⁸、2.8×10⁷、5.6×10⁶、 1.12×10⁵And 0.22X 10⁴Spore/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 × 10⁷， 5.05×10⁶，1.01×10⁶，2.02×10⁴，0.4×10³Spores/g soil. The mixed soil is evenly distributed into 6-hole bioassay plates with corresponding concentrations, 1 head of larvae of Holotrichia parallela of 1 year (6-day-old larvae hatched) are inoculated into each hole, and the Holotrichia parallela is placed in an incubator at the temperature of 26 ℃ and under the illumination of L: D ═ 16:8 for breeding. Each repeat was 30, 3 repeats. Sterile water 0.1% Tween-80 was used as a negative control and added to sterilized dry soil to give soil moisture of 18%. 15 days later, the death is investigated,Live worm number, mean and corrected mortality were 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 at 3 th (larvae of 45-day-old hatched larvae), 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, 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 example 2, subsection (1). Sterile water 0.1% Tween-80 was used as a negative control and added to sterilized dry soil to give soil moisture of 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 with a concentration gradient of 3.0X 10⁸，6.0×10⁷，1.2×10⁷，2.4×10⁶，4.8×10⁵Spore/ml. Mixing the spore suspension with sterilized dry soil to obtain soil sample with spore concentration of 5.4 × 10⁷，1.08×10⁷， 2.16×10⁶，4.32×10⁵，8.64×10⁴Spores/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. Sterile water 0.1% Tween-80 was used as a negative control and added to sterilized dry soil to give soil moisture of 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

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