Beauveria bassiana Bb1Bm for preventing and treating tomato leaf miner larvae and application thereof
By screening and identifying the strain Bb1Bm of the highly virulent coccidioidae, the problem of insufficient pathogenicity of tomato leaf moth larvae in the existing technology was solved, and efficient biotechnology prevention and control effect was achieved, with a mortality rate of 85.3%. Green prevention and control provides a reliable basis.
Patent Information
- Application Number
- CN202411231529.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-04
- Publication Date
- 2024-12-13
AI Technical Summary
The existing strains of Cyperus leukobassium are insufficient to cause pathogenicity to tomato leaf moth larvae, making it difficult to achieve efficient and green prevention and control effects.
Screen and provide a strain of Bb1Bm, a highly virulent coccidioidae strain Bb1Bm, which is used to prevent and control the larvae of tomato leaf moth by isolation, identification and preservation, combined with biotechnology, and is cultured using PDA and PDB culture medium, and is rDNA- ITS sequence analysis and phylogenetic tree construction confirm its identity.
The 14-day correction lethality rate of this strain on tomato leaf moth larvae can reach 85.3%, providing a reliable basis for biotechnology prevention and control. The lethality effect increases significantly with the increase of spore suspension concentration, providing an efficient green prevention and control approach.
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Figure CN119120207A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of microbial control, and in particular to the control of Beauveria bassiana Bb1Bm of tomato leafminer larvae and applications thereof. Background Art
[0002] The tomato leafminer (Tuta absoluta), also known as the South American tomato leafminer, belongs to the family Gelechiidae of the order Lepidoptera. It is native to Peru in South America. Its larvae feed on the leaves of tomatoes, potatoes, eggplants, ginseng fruit, tobacco and other Solanaceae plants, or bore into their fruits, especially tomatoes.
[0003] The use of microorganisms (such as Beauveria bassiana) to control agricultural and forestry pests is environmentally friendly and an important part of reducing the use of chemical pesticides. Beauveria bassiana is an entomogenous fungus with a certain host specificity, and different strains have significant differences in pathogenicity to target pests. Therefore, screening for highly toxic Beauveria bassiana strain resources can provide an effective way to achieve efficient, green, and sustainable prevention and control of tomato leafminers. Summary of the invention
[0004] The invention aims to provide a Beauveria bassiana strain for preventing and controlling the larvae of tomato leafminer.
[0005] Another object of the present invention is to provide the application of the above strain.
[0006] The Beauveria bassiana strain according to the present invention is preserved in the China General Microbiological Culture Collection Center, with a preservation date of June 14, 2024 and a preservation number of CGMCC No.41260.
[0007] The invention provides the Beauveria bassiana and application thereof in preventing and controlling larvae of tomato leafminer.
[0008] Compared with the existing Beauveria bassiana strains, the strain provided by the present invention has a 14-day corrected lethality rate of up to 85.3% for tomato leafminer larvae, providing a reliable basis for controlling tomato leafminer by biotechnology means. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Figure 1 The colony morphology of Beauveria bassiana Bb1Bm cultured on PDA medium for 3 days is shown;
[0010] Figure 2 The phylogenetic tree of Beauveria bassiana Bb1Bm is shown;
[0011] Figure 3 The corrected mortality of the second instar larvae of the tomato leafminer is shown in Figure 2 for spore suspensions of the Bb1Bm strain at different concentrations.
[0012] The Beauveria bassiana strain Bb1Bm of the present invention was deposited on June 14, 2024 in the General Microbiology Center of the China Microorganism Culture Collection Committee (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and was classified and named Beauveria bassiana, with a preservation number of CGMCC No.41260. DETAILED DESCRIPTION
[0013] Example 1 Isolation of Beauveria bassiana
[0014] The isolation and culture method of Beauveria bassiana comprises the following steps:
[0015] Dead insects were found in silkworms purchased from Jingzhou City, Hubei Province. The mycelium and spore powder grown on the insects infected with Beauveria bassiana were taken and spread on PDA medium with antibiotics for culture. PDA medium was selected: potato powder 6g / L, glucose 20g / L, agar 20g / L, chloramphenicol 0.02g / L, dodine 0.01g / L, distilled water 1L, sterilized at 121℃ for 20min. The screened and activated strains were inoculated into PDB liquid medium and cultured on a shaker at 27℃ for 3d to obtain the culture solution of the test strain Bb1Bm. PDB liquid medium: potato powder 5g / L, peptone 10g / L, glucose 15g / L, sodium chloride 5g / L, distilled water 1L, sterilized at 121℃ for 20min. Take 200μL of the culture solution of the test strain and inoculate it into solid PDA medium using a plate spreader. The inoculated culture dish was placed in a sterile incubator at 27°C for 14 days for the preparation of spore suspension.
[0016] The identification method of Beauveria bassiana comprises the following steps:
[0017] Morphological identification: A test strain was isolated from Bombyx mori by tissue separation method. The purified strain was inoculated on PDA plate and cultured in a 27℃ incubator for 3 days. Figure 1 As shown, the colony diameter is 5.2mm, the colony is white, the matrix is yellow, the colony is generally thick, powdery, protruding in the middle, without radial grooves, concentric rings, and wrinkles, and the spores of the strain are spherical, transparent, and smooth. The collected strains were identified by morphological methods, and the strains were identified as Beauveria bassiana through Olympus microscope examination and retrieval table.
[0018] rDNA-ITS sequence analysis: The obtained strains were isolated and cultured using an inoculation loop under sterile conditions, and genomic DNA was extracted from the obtained single colonies using a fungal genome extraction kit, followed by specific PCR amplification of the TS1-5.8S-ITS2 rDNA region:
[0019] Primers ITS1F (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4
[0020] (5'-TCCTCCGCTTATTGATATGC-3').
[0021] The strain DNA was used as a template for PCR amplification, and the resulting PCR product was sequenced, resulting in a sequence of 598 bp. The sequence was submitted to the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) for homology comparison, and the comparison results showed that Bb1Bm had a similarity of 99% with Beauveria bassiana LHYF (Beauveria bassiana) ON796013.1 (published gene fragment length of 573 bp). A phylogenetic tree was constructed based on the ITS sequence using MEGA7.0 ( Figure 2 ). According to the phylogenetic tree, it is most closely related to the Beauveria bassiana strain. Therefore, the Bb1Bm strain is systematically classified as Beauveria bassiana.
[0022] Analysis of the insecticidal activity of the strain against the tomato leafminer:
[0023] The tomato leafminers for the test were collected from Datong City, Shanxi Province. The 2nd-3rd instar larvae of tomato leafminers collected from the greenhouse were raised in insect cages in an artificial insect room (temperature 25°C, relative humidity 65%, light-dark ratio 14:10h), and laboratory-grown tomato seedlings were provided for feeding every day. When the larvae of tomato leafminers reached the 2nd instar, they were used for bioassays. Before the experiment, a small hole with a diameter of 4mm was drilled and burned on the lid of a 1.5mL centrifuge tube using an electric soldering iron. The quickly cut tomato side branches were passed through the small hole and placed at the bottom of a centrifuge tube filled with water. The tube was then placed on a 150mm culture dish with filter paper on the bottom for the tomato leafminers to feed on. During the experiment, 2nd instar larvae of basically the same size were taken and placed in a 1×10 8 , 1×10 7 , 1×10 6 , 1×10 5 , 1×10 4spores / mL of Bb1Bm spore suspension for 10s, use filter paper to absorb excess water and place on tomato leaves for feeding, and use sterile water containing 0.05% Tween-80 as a control. Each treatment contains 8 replicates, and each replicate contains 5 test larvae. After the treatment, the culture dish was placed in an incubator with a temperature of 27°C, a relative humidity of 70%, and a light-dark ratio of 14:10h. Thereafter, water was added to the centrifuge tube every day, and the death of the larvae was observed. The experiment was investigated and recorded for a total of 14 days, and the corrected mortality rate of each treatment was calculated. The experimental results show that the strain (Bb1Bm) of the present invention has a good lethal effect on tomato leafminers, and the concentration of 1×10 8 The corrected lethality of strain Bb1Bm with spores / mL against tomato leafminer larvae was 85.3% after 14 days. 50 1.4×10 5 spores / mL. Figure 3 The results show that the mortality rate of Bb1Bm strains against the second-instar larvae of tomato leafminers increased significantly with the increase of spore suspension concentration. 8 When the concentration was 100 μg / mL, the corrected mortality rate against the second-instar larvae of tomato leafminer could reach 85.3%.
[0024] Table 1
[0025]
[0026] Table 1 shows the toxicity regression equations of the Bb1Bm strain at 7, 10, and 14 days. The results showed that the LC50 of the Bb1Bm strain at 7, 10, and 14 days were 7.94×10 6 , 3.65×10 5 , 1.40×10 5 spores / mL.
[0027] The above embodiments are only used to understand the technical solution of the present application and do not limit the protection scope of the present application.
Claims
1. A Beauveria bassiana strain, characterized in that The preservation number of the Beauveria bassiana strain is CGMCC No.41260.
2. The Beauveria bassiana strain according to claim 1 is used for controlling tomato leafminer.
3. The use according to claim 2, characterized in that: The tomato leafminer is a larva of the tomato leafminer.
4. A fungal agent for controlling tomato leafminer, characterized in that: The bacterial agent comprises spores of the Beauveria bassiana strain according to claim 1.
5. The fungal agent for controlling tomato leafminer according to claim 4, characterized in that: The bacterial agent is in liquid dosage form or solid dosage form.