A kind of Beauveria bassiana and its application in preventing and controlling leaf beetles

Abstract

The invention relates to a Beauveria bassiana and its application in preventing and controlling leaf beetles. The Beauveria bassiana is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with a preservation number of CGMCC No.41112.

Description

Technical Field

[0001] The invention relates to the field of biocontrol microorganisms, in particular to Beauveria bassiana which can be used for controlling leaf beetles. Background Art

[0002] Monolepta hieroglyphica belongs to the order Coleoptera, family Chrysomelidae, subfamily Galerucinae, and is referred to as the monolepta hieroglyphica. The insect is polyphagous and has many hosts, and occurs on beans, corn, potatoes, cotton, and vegetables. In my country's corn-producing areas, the monolepta hieroglyphica has risen from a minor pest to a major pest, and its damage has become more severe year by year, causing crop yield reduction or even crop failure.

[0003] Traditional prevention and control mainly relies on chemical pesticides, but chemical pesticides can easily cause water and soil pollution, excessive pesticide residues on crops, and threaten human and animal safety and social stability. This has attracted widespread attention from the government and all sectors of society, so biological control has received increasing attention. Summary of the invention

[0004] One of the present invention provides a Beauveria bassiana, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC No.41112.

[0005] The second aspect of the present invention provides an engineered bacterium obtained by genetically improving the Beauveria bassiana as described in the first aspect of the present invention. Among them, since the engineered bacterium is targeted at the Beauveria bassiana CGMCC No.41112 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 leaf beetles (e.g., two-spotted long-tarsed leaf beetles). 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 the 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 Galerucinae.

[0009] In a specific embodiment, the leaf beetle is Monoleptahieroglyphica.

[0010] In a specific embodiment, the leaf beetle is a larva and / or adult of Monoleptahieroglyphica.

[0011] Beneficial effects of the present invention:

[0012] The present invention finds that there are strains in Beauveria bassiana that have high insecticidal activity against leaf beetles, especially against two-spotted long-tarsed leaf beetles, which not only broadens the use of Beauveria bassiana, but also broadens the means of preventing and controlling leaf beetles. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The phylogenetic tree of the isolated strain numbered CZBJ-3 is shown.

[0014] Strain preservation

[0015] The Beauveria bassiana screened by the present invention is named CZBJ-3, 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.41112, 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 bassiana. DETAILED DESCRIPTION

[0016] 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.

[0017] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0018] Example 1

[0019] Isolation and classification of strains

[0020] The hyphae on the body surface of the adult diseased insect of the two-spotted firefly leaf beetle were picked up and inoculated on the PDA medium, and purified by multiple separation and culture at 26°C to obtain the purified isolated strain. The isolated strains were numbered.

[0021] 1. Morphological identification of isolated strains

[0022] The isolated strain numbered CZBJ-3 was cultured on a PDA medium plate. The colony was white and hairy at the beginning, the hyphae were transparent, and had branched septa. Conidia were produced after about one week of culture. The spore layer was thick and uniform, and later turned light yellow, with yellow or brown on the back. Conidiophores and conidia were visible under a microscope. The conidia were round or oval, and the spore size was 2.0-3.0×2.0-2.5μm. Based on the above morphological observations, it was preliminarily identified as belonging to the genus Beauveria.

[0023] 2. Molecular Identification of Isolated Strains

[0024] 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 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 Biopharmaceutical Technology Co., Ltd., and the sequence result 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 amplification sequence of strain CZBJ-3 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 is clustered with Beauveria bassiana.

[0025] In summary, the isolated strain numbered CZBJ-3 was systematically classified as Beauveria bassiana.

[0026] The CZBJ-3 strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration Committee with the deposit number CGMCC No.41112 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 bassiana.

[0027] Example 2

[0028] Analysis of biological activity against the two-spotted leaf beetle

[0029] The purified isolated strain CZBJ-3 was propagated using PDA medium, that is, cultured on PDA medium at 26°C for 7 to 10 days, conidia powder was scraped and prepared with a sterile aqueous solution containing 0.05% 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.

[0030] (1) Determination of the biological activity of the first-instar larvae of the two-spotted leaf beetle

[0031] Pick out the first-instar larvae of the two-spotted leaf beetle (1-day-old larvae after hatching) of uniform size and health, spray the above-mentioned spore suspensions of different concentrations on the body surface of the test insects, and use filter paper to absorb excess water. Place them under the conditions of 30±1℃ and 14h:10h (L:D) photoperiod and feed them with fresh corn leaves, which are replaced in time. A sterile aqueous solution containing 0.05% Tween-80 was used as a blank control. For each treatment of 30 test insects, 3 replicates were set. Starting from the second day, the number of dead insects was investigated and counted daily, and the number of dead insects was recorded. The investigation lasted for 7 days in total, and finally the mortality rate was counted, the corrected mortality rate was calculated, and variance analysis was performed using SPSS17.0. The lethal median concentration LC was calculated using PoloPlus software. 50 value.

[0032] The results of the corrected mortality at each concentration are shown in Table 1. 50 The results are shown in Table 2.

[0033] (2) Determination of the biological activity of the second-instar larvae of the two-spotted leaf beetle

[0034] The test insects used were the second-instar larvae of the two-spotted leaf beetle (larvae 7 days old after hatching), and the rest was the same as section (1) in Example 2.

[0035] The results of the corrected mortality at each concentration are shown in Table 1. 50 The results are shown in Table 2.

[0036] (3) Determination of the biological activity of the third-instar larvae of the two-spotted leaf beetle

[0037] The test insects used were the third-instar larvae of the two-spotted leaf beetle (larvae 15 days old after hatching), and the rest was the same as section (1) in Example 2.

[0038] The results of the corrected mortality at each concentration are shown in Table 1. 50 The results are shown in Table 2.

[0039] (4) Determination of the biological activity of adult two-spotted leaf beetles

[0040] 30 healthy and active adults of the two-spotted leaf beetle were inoculated on two healthy corn plants of the same size, and the prepared spore suspensions of different concentrations were sprayed on the corn leaves with the two-spotted leaf beetle to form water droplets, which were placed in a 90-mesh net insect cage (30×30×30cm) to ensure that the two-spotted leaf beetle could move in the cage. The corn leaves with the two-spotted leaf beetle were sprayed with a 0.05% Tween-80 sterile aqueous solution as a control, and repeated 3 times.

[0041] The results of the corrected mortality at each concentration are shown in Table 1. 50 The results are shown in Table 2.

[0042] Table 1

[0043]

[0044] Note: P<0.05

[0045] Table 2

[0046] Insect stage <![CDATA[LC 50 (Spores / mL)]]> 95% confidence interval (spores / mL) Virulence related equation Correlation coefficient 1 year old <![CDATA[2.98×10 5 ]]> <![CDATA[1.58×10 5 to 5.62×10 5 ]]> Y=0.6516X+7.2971 0.9947 2 years old <![CDATA[5.48×10 5 ]]> <![CDATA[2.82×10 5 to 1.06×10 6 ]]> Y=0.5638X+6.8387 0.9857 3 years old <![CDATA[1.47×10 6 ]]> <![CDATA[7.78×10 5 to 2.78×10 6 ]]> Y=0.5828X+6.6506 0.9943 adult <![CDATA[3.03×10 6 ]]> <![CDATA[1.57×10 6 to 5.84×10 6 ]]> Y=0.6051X+6.5243 0.9978

Claims

1. A Beauveria bassiana ( Beauveria bassiana ), which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 41112.

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

3. The Beauveria bassiana according to claim 1 or the composition according to claim 2 is used for controlling the two-spotted long-tarsed leaf beetle ( Monolepta hieroglyphica ) in applications.

4. The use according to claim 3, characterized in that: The two-spotted long-tarsed leaf beetle ( Monolepta Hieroglyphics ) is the two-spotted long-tarsed leaf beetle ( Monolepta hieroglyphica ) larvae and / or adults.

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