Beauveria bassiana and its application in preventing and controlling scarab beetles

Abstract

The invention relates to a Beauveria bassiana and its application in preventing and controlling scarab beetles. The Beauveria bassiana is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.41111.

Description

Technical Field

[0001] The invention relates to a Beauveria bassiana, and in particular can be used for preventing and controlling scarab beetles. Background Art

[0002] Underground pests are important farmland pests, among which white grubs, cutworms, wireworms, root maggots, mole crickets and other groups are common and cause serious damage. In recent years, with climate warming, improvement of water conservancy facilities, changes in farming systems and adjustment of agricultural structure, especially the promotion of ground covering and insulation technology, a very favorable living environment has been created for underground pests, resulting in increasingly serious damage caused by underground pests of crops.

[0003] Because underground pests hide underground, their prevention and control has been difficult over the years. For a long time, they have mainly relied on chemical pesticides for control. The long-term and large-scale use of chemical pesticides has caused water and soil pollution, excessive pesticide residues in crops, and directly affected the economic benefits of agriculture. It has attracted widespread attention from all walks of life, so biological control has received more and more attention. Summary of the invention

[0004] One of the present invention provides a Beauveria brongniartii, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.41111.

[0005] The second aspect of the present invention provides an engineered bacterium obtained by genetically improving the Beauveria bassiana as described in one of the present invention. Among them, since the engineered bacterium is targeted at the Beauveria bassiana CGMCC No.41111 of the present invention, and the means generally adopted are to transfer and / or knock out specific genes and / or DNA fragments therein, the engineered bacterium is still Beauveria bassiana. In addition, the engineered bacterium can be an engineered bacterium with improved pest activity against scarabs (such as the North China black gill beetle or the copper green beetle). It can also be an engineered strain that has other pest activity and / or is endowed with other beneficial properties.

[0006] In a specific embodiment, the engineered bacteria is obtained by transforming the Beauveria bassiana with a plasmid carrying a disease-resistant gene and / or an insect-resistant gene.

[0007] The third aspect of the present invention provides a composition, which comprises the Beauveria bassiana as described in the first aspect of the present invention or the engineered bacteria as described in the second aspect of the present invention, and an acceptable carrier.

[0008] The fourth aspect of the present invention provides the use of Beauveria bassiana according to the first aspect of the present invention, the engineered bacteria according to the second aspect of the present invention, or the composition according to the third aspect of the present invention for controlling scarab beetles (Scarabaeidae).

[0009] In a specific embodiment, the scarab beetle is Holotrichia and / or Anomala.

[0010] In a specific embodiment, the Holotrichia is Holotrichia oblita; the Anomala is Anomala corpulenta.

[0011] In a specific embodiment, the scarab beetle (Scarabaeidae) is an egg and / or larval stage scarab beetle.

[0012] In a specific embodiment, the Holotrichia is an egg and / or larval stage of the Holotrichia; the Anomala is an egg and / or larval stage of the Anomala.

[0013] In a specific embodiment, the Holotrichia oblita is an egg and / or larval stage of the Holotrichia oblita; the Anomala corpulenta is an egg and / or larval stage of the Anomala corpulenta.

[0014] Beneficial effects of the present invention:

[0015] The present invention finds that there are strains in Beauveria bassiana that have high insecticidal activity against scarabs, especially against the eggs and larvae of scarabs, which not only broadens the use of Beauveria bassiana, but also broadens the means of preventing and controlling scarabs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The phylogenetic tree of the isolate numbered CZBJ-3-26 is shown.

[0017] Strain preservation

[0018] The Beauveria bassiana screened by the present invention is named CZBJ-3-26, and the strain is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, with a deposit number of CGMCC No.41111, a deposit date of March 5, 2024, and a deposit address of: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its system classification is Beauveria brongniartii. DETAILED DESCRIPTION

[0019] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0020] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0021] Example 1

[0022] Isolation and classification of strains

[0023] The hyphae on the body surface of the adult diseased beetle were picked up and inoculated on PDA medium, and purified by multiple separation and culture at 26°C to obtain the purified isolated strain. The isolated strains were numbered.

[0024] 1. Morphological identification of isolated strains

[0025] The isolated strain numbered CZBJ-3-26 showed white cotton-like hyphae in the early stage of colony on PDA plate. After 7 to 10 days of cultivation, conidia were produced. They were white powdery and turned light yellow in the later stage. The back was brown or red. Conidiophores and conidia were visible under the microscope. The conidia were oval and 2.0-3.0×3.0-5.5μm in size. Based on the above morphological observations, it was preliminarily identified as Beauveria.

[0026] 2. Molecular Identification of Isolated Strains

[0027] The universal fungal primers NS1 (SEQ ID No.1) and NS4 (SEQ ID No.2) were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. The genomic DNA extracted from the isolated strain numbered CZBJ-3-26 was used as a template, and NS1 and NS4 were used as primers to perform PCR amplification to obtain the PCR product of 18S rDNA. The PCR product was sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing, and the result of the sequence is shown in SEQ ID No.3. The sequencing results were subjected to homology BLAST comparison in the Genbank database, and the comparison results showed that the PCR amplified sequence of strain CZBJ-3-26 had a sequence homology of more than 99% with that of Beauveria bassiana. A phylogenetic tree was established using MEGA7 software, see Figure 1 .according to Figure 1 From the phylogenetic tree, we can see that strain CZBJ-3-26 is clustered with Beauveria bassiana.

[0028] In summary, the isolated strain numbered CZBJ-3-26 was systematically classified as Beauveria brongniartii.

[0029] The strain CZBJ-3-26 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee with the deposit number CGMCC No.41111 and the deposit date March 5, 2024. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its systematic classification is Beauveria brongniartii.

[0030] Example 2

[0031] Biological activity analysis

[0032] (1) Determination of the biological activity of the first-instar larvae of the North China black beetle

[0033] The purified isolated strain CZBJ-3-26 was propagated using PDA medium, that is, cultured on PDA medium at 26°C for 7 to 10 days, conidia powder was scraped, and then prepared with a sterile aqueous solution containing 0.1wt% Tween-80 to a concentration of 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 spores / mL of spore suspension.

[0034] Prepare potato shreds, rinse them with clean water, and dry them until there is no moisture on the surface. Soak the potato shreds in the spore suspension, take them out after about 20 minutes, and evenly place them in a 6-well bioassay plate, with about 4 to 5 strips per well. Mix the remaining spore suspension in 100g of sterilized dry soil and mix well to make the soil humidity 18%. The final bioassay concentration is about 2×10 3 , 2×10 4 , 2×10 5 , 2×10 6 , 2×10 7 Spores / g soil. The mixed soil was evenly distributed into 6-well bioassay plates at the corresponding concentrations, and one 1-year-old (6-day-old larvae) larva of the North China black gill beetle was placed in each well and raised in an incubator with a temperature of 26°C and a light intensity of L:D=16:8. There were 36 larvae in each replicate, with 3 replicates. A sterile aqueous solution containing 0.1% Tween-80 was used as a negative control and added to the sterilized dry soil to make the soil humidity 18%. After 15 days, the number of dead and live insects was investigated, and the average mortality rate and adjusted mortality rate were calculated using Excel2010, variance analysis was performed using SPSS17.0, and the lethal median concentration LC was calculated using PoloPlus software. 50 value.

[0035] in,

[0036] The corrected mortality results at each concentration for this age are shown in Table 1.

[0037] (2) Determination of the biological activity of the second-instar larvae of the North China black beetle

[0038] The test insects used were the 2nd instar larvae of Holotrichia nigromaculata (15-day-old larvae after hatching), and the rest was the same as that in section (1) of Example 2.

[0039] The corrected mortality results at each concentration for this age are shown in Table 1.

[0040] LC at this age 50 4.76×10 5 spores / g soil, with a confidence interval of 2.78×10 5 to 8.15×10 5 spores / g soil, toxicity correlation equation Y=0.6876X+6.8039, correlation coefficient is 0.9876.

[0041] (3) Determination of the biological activity of the third-instar larvae of the North China black beetle

[0042] The test insects used were the third-instar larvae of Holotrichia nigromaculata (larvae 45 days old after hatching), and the rest was the same as that in section (1) of Example 2.

[0043] The corrected mortality results at each concentration for this age are shown in Table 1.

[0044] (4) Determination of the biological activity of eggs of Holothuria chinensis

[0045] Add the spore suspension to 100 g of sterilized dry soil, mix well, and obtain a soil sample to be tested. Place the soil sample to be tested in a 16.5 cm × 17 cm × 12 cm (length × width × height) food plastic box, pick out the eggs newly laid by the North China black gill beetle for 5 days and evenly place them in the soil sample to be tested, 50 eggs per box, and repeat 3 times. The preparation and concentration gradient of the spore suspension are the same as those in subsection (1) of Example 2. A sterile aqueous solution containing 0.1% Tween-80 is used as a negative control and is added to the sterilized dry soil so that the humidity of the soil is 18%. Investigate the infection of the eggs after 15 days.

[0046] The corrected mortality results at each concentration for this age are shown in Table 1.

[0047] Table 1

[0048]

[0049] Note: P<0.05

[0050] (5) Determination of biological activity of first-instar larvae of A. cooperi

[0051] The test insects used were 1st instar larvae of A. corydalis (6-day-old larvae after hatching), and the rest was the same as that in section (1) of Example 2.

[0052] The corrected mortality results at each concentration for this age are shown in Table 2.

[0053] (6) Determination of biological activity of the second-instar larvae of the copper green beetle

[0054] The test insects used were the second-instar larvae of A. corydalis (15-day-old larvae after hatching), and the rest was the same as that in section (1) of Example 2.

[0055] The corrected mortality results at each concentration for this age are shown in Table 2.

[0056] LC at this age 50 2.33×10 5 spores / g soil, with a confidence interval of 1.37×10 5 to 3.96×10 5 spores / g soil, the toxicity correlation equation is Y=0.6485X+6.9026, and the correlation coefficient is 0.9978.

[0057] (7) Determination of biological activity of the third-instar larvae of the copper green beetle

[0058] The test insects used were the third-instar larvae of A. corydalis (larvae 45 days old after hatching), and the rest was the same as that in section (1) of Example 2.

[0059] The corrected mortality results at each concentration for this period are shown in Table 2.

[0060] (8) Determination of the biological activity of the eggs of the copper green beetle

[0061] The test objects used were eggs laid 5 days ago by A. cooperi. The rest was the same as that in section (4) of Example 2.

[0062] The corrected mortality results at each concentration for this age are shown in Table 2.

[0063] Table 2

[0064]

[0065] Note: P<0.05.

Claims

1. A Beauveria brongniartii, which is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration with a deposit number of CGMCC No. 41111.

2. A composition comprising the Beauveria bassiana according to claim 1, and an acceptable carrier.

3. Use of the Beauveria bassiana according to claim 1 or the composition according to claim 2 for controlling scarab beetles (Scarabaeidae); The scarab beetle is Holotrichia oblita and / or Anomalacorpulenta.

4. The use according to claim 3, characterized in that: The scarab beetles (Scarabaeidae) are scarab beetles in the larval stage.

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