Beauveria bassiana for preventing and treating caffeinia longhorn beetles and application of Beauveria bassiana

Abstract

The invention discloses a beauveria bassiana strain for preventing and treating caffeinia and application of the beauveria bassiana strain, and belongs to the technical field of biological prevention and treatment of pests. The strain disclosed by the invention is Beauveria bassiana which is separated from a larva body of the caffeine calandria for the first time, the Beauveria bassiana is biologically preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.40829. The invention further discloses a preparation method of the Beauveria bassiana. The strain has the advantages of being simple in culture, large in sporulation quantity and high in pathogenicity to larvae of the caffeine, and is suitable for being used for developing a biological preparation for preventing and treating the pest.

Description

Technical Field

[0001] The invention relates to the technical field of biological pest control, and in particular to a Beauveria bassiana strain for controlling Xylocarpus coffee beetle. Background Art

[0002] The coffee beetle is also known as the coffee beetle ( Xylotrechus quadripes ), which is more common in the south of the Yangtze River in my country, and is mainly distributed in all planting areas in Yunnan. It is distributed in India, Myanmar, Thailand, Vietnam, Laos, Cambodia, Sri Lanka, the Philippines, Indonesia, Malaysia, and the East Indies. The tiger longhorn beetle mainly harms mature coffee trees. The larvae bore into the xylem of the trunk, and bore the xylem into horizontal, vertical or circular tortuous tunnels. Sometimes they also bore down to harm the roots, causing the leaves and branches of the affected coffee trees to wilt, or even the whole plant to die; there are as few as 1 larvae in the affected trunk, and as many as 57 larvae, and some different generations of insects exist in the same trunk at the same time; the affected coffee trees form ring-shaped lumps due to tissue stimulation, resulting in the breakage of the epidermal cork layer, obstruction of water transport, yellowing of the branches and leaves above, and lateral buds often sprouting at the base of the stem; the affected trunks are easily broken by pushing, pulling or wind. The damage rate is generally 2% to 5%, and in severe cases it reaches 10% to 25%. Therefore, in-depth research on the occurrence patterns of the black beetle and exploring simple and effective prevention and control methods are of great significance to improving coffee yield and quality, reducing insect control costs, and promoting coffee production.

[0003] Beauveria bassiana Beauveria bassiana ) is an insect pathogenic fungus widely used in agricultural and forestry pest control. Beauveria bassiana can also colonize in plants under natural conditions or through artificial inoculation. Endophytic Beauveria bassiana not only has no negative impact on natural enemy insects when used to control pests, but may also have a synergistic effect with them, thereby improving the control effect and having a positive significance for the stability of farmland ecosystems. Existing studies have shown that coffee beetles of all ages that come into contact with Beauveria bassiana, whether indoors or in the field, can be infected and killed. At the same time, the yield measurement results show that the yield of fresh coffee fruits in the biological control area is higher than that in the control area, so Beauveria bassiana is considered to be an economical and safe biological insecticide. However, Beauveria bassiana isolated from naturally infected coffee beetle beetles has not been reported in China, and there are limited Beauveria bassiana that can be used for biological control of coffee beetles. Summary of the invention

[0004] In view of the above-mentioned defects of the prior art, one of the objects of the present invention is to provide a strain of Beauveria bassiana that has strong pathogenicity to Xylodon coffee beetle, wherein the Beauveria bassiana ( Beauveriabassiana), isolated from the coffee beetle larvae infected with fungi and collected in Ruili City, Yunnan Province, is a strain with a high lethality rate to coffee beetle after separation and purification. Combined with the results of morphological and gene sequence determination, the strain was identified as Beauveria bassiana ( Beauveria bassiana ) and named it strain RZBB01.

[0005] Furthermore, the Beauveria bassiana RZBB01 strain is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, referred to as CGMCC, and the strain deposit number is CGMCC No. 40829. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is September 22, 2023.

[0006] Furthermore, the ITS sequence of the RZBB01 strain is shown in SEQ ID NO.1.

[0007] The second purpose of the present invention is to provide the use of the RZBB01 strain in preventing and controlling Xanthoceras coffee beetles.

[0008] Furthermore, the application of the RZBB01 strain in preventing and controlling coffee beetles is specifically to culture the Beauveria bassiana RZBB01 strain to prepare a spore liquid and a spore-containing bacterial agent, and the spore concentration of the strain during application is 1×10 6 Spores / mL - 1×10 8 spores / mL to achieve green control of coffee beetle.

[0009] The application of the Beauveria bassiana RZBB01 strain provided by the present invention in preventing and controlling Xylotreya coffee beetle has the following beneficial effects: The invention provides a Beauveria bassiana RZBB01 strain. The spore liquid prepared from the RZBB01 strain after cultivation has strong pathogenicity to both adults and larvae of Xylocarpus coffeei, can be used for preparing Xylocarpus coffeei insecticide, and has the characteristics of low production cost, short cycle, environmental friendliness, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 : These are the morphological characteristics of the Beauveria bassiana RZBB01 strain in the examples, where a is the colony morphology, b is the conidia structure, and c is the hyphae morphology.

[0011] Figure 2 The molecular identification results of Beauveria bassiana RZBB01 strain in the examples are shown.

[0012] Figure 3 The figure shows the infection of the Beauveria bassiana RZBB01 strain on the coffee beetle larvae, wherein a is the state three days after infection, and b is the state six days after infection. DETAILED DESCRIPTION

[0013] The above contents of the present invention are further described in detail below in the form of preferred implementation cases. The examples are only used to explain the present invention and do not constitute a limitation of the present invention.

[0014] The reagents in the embodiments of the present invention can all be purchased through commercial channels.

[0015] Example 1

[0016] Isolation and Purification of Beauveria bassiana RZBB01

[0017] The dead insects of the naturally infected coffee X. longipes larvae were obtained in the coffee garden of Dehong Institute of Tropical Agricultural Sciences in Ruili, Yunnan Province. The dead insects of the infected coffee X. longipes larvae were brought back to the laboratory. In the clean bench, the dead insects of the coffee X. longipes larvae were soaked in 75% alcohol for 30 seconds, and then rinsed with sterile water for several times. After rinsing, the treated coffee X. longipes larvae tissues were taken with an inoculation needle and inoculated on PDA medium (the formula of PDA medium is: 200g peeled potato, 20g glucose, 20g agar, 1000ml distilled water, pH 7.0). The inoculated PDA medium was placed in a constant temperature incubator at 28℃ for lightless culture. After 5 to 7 days, the mycelium in the center of the colony was picked and inoculated on a new PDA plate. This was repeated for more than 3 times to obtain a pure strain with strong spore production ability. Then the isolated strain is cultured on a PDA medium, and after a large number of conidia are produced, the spores are collected and a spore suspension is prepared using 1% Tween-80 sterile water. Then the spore suspension is evenly sprayed on the body surface of the coffee beetle larvae, and the insect bodies are cultured under a humidity condition of more than 80%. After the larvae are infected and die, a strain with stronger pathogenicity is isolated according to the above method.

[0018] Example 2

[0019] Morphological and Molecular Identification of Beauveria bassiana RZBB01

[0020] 1. Morphological identification

[0021] The obtained highly virulent strain was transferred to PDA medium. Colonies began to appear on the third day after culture. The edges of the colonies were neat and white and fluffy. One week after inoculation, the colonies began to produce spores. When observed from the back, the center of the colony slightly changed to a light sandy yellow. The middle of the colony was slightly convex, and the spore layer was powdery and grew evenly. After two weeks of culture on PDA medium, the colony diameter was 60.54 mm. Under an optical microscope, the vegetative hyphae were slender and transparent, and conidiophores were formed on the elongated mycelium, on which conidia were produced. The conidia were round or oval, colorless, and smooth in surface, with a diameter of 1.22 to 2.83 μm. It was preliminarily identified as Beauveria. Beauveria .

[0022] 2. Molecular identification

[0023] (1) DNA extraction

[0024] The genomic DNA of RZBB01 strain was extracted using TSINGKE Plant DNA Extraction Kit (Universal).

[0025] (2) PCR amplification and molecular identification

[0026] The extracted DNA samples were diluted appropriately and used as PCR templates for amplification with 1×TSE101 Gold Mix (Qingke Biotechnology Co., Ltd.). The universal primer ITS for fungi was used for PCR amplification. The upstream primer ITS1 is shown in SEQ ID NO.2, 5´-TCCGTAGGTGAACCTGCGG-3´, and the downstream primer ITS4 is shown in SEQ ID NO.3, 5´-TCCTCCGCTTATTGATATGC-3´. The amplification system was: 1×TSE101 Gold Mix 45 μL, ITS1 (10P) 2 μL, ITS4 (10P) 2 μL, and template DNA 1 μL. PCR amplification program: 98 ℃ pre-denaturation for 2 min; 98 ℃ denaturation for 10 s, 56 ℃ annealing for 10 s, 72 ℃ extension for 10 s, 35 cycles; 72 ℃ extension for 5 min, and storage at 4 ℃. After PCR amplification, 2 μL of PCR product was taken for agarose gel electrophoresis to detect the PCR amplified fragment. After confirmation, it was sequenced by Qingke Biotechnology Co., Ltd. to obtain a 538 bp sequence, as shown in SEQ ID NO. 3. The sequencing results were spliced ​​with Contig Express, and the inaccurate parts at both ends were removed. The spliced ​​sequences were subjected to Blast comparison analysis in the NCBI database (blast.ncbi.nlm.nih.gov). The results showed that the RZBB01 strain was similar to Beauveria bassiana ( Beauveria bassianaThe similarity between strain RZBB01 and Beauveria bassiana was as high as 100%. The phylogenetic tree was constructed using MEGA 11.0 software, and the results showed that strain RZBB01 had a high homology with Beauveria bassiana. At the same time, the sequence of strain RZBB01 was submitted to Genbank with the accession number PP023877.

[0027] The SEQ ID NO. 1 is the ITS sequence of the Beauveria bassiana RZBB01 strain, specifically:.

[0028] Example 3

[0029] Determination of the toxicity of Beauveria bassiana RZBB01 strain to Xylophora coffeae larvae in laboratory

[0030] (1) Preparation of spore suspension of RZBB01 strain

[0031] The RZBB01 strain of Beauveria bassiana was inoculated into PDA medium and cultured in a constant temperature incubator at 28°C without light for 2 weeks. Then, conidia were scraped from the colonies and prepared with sterile water to a spore concentration of 1×10 8 / mL, 1×10 7 / mL, 1×10 6Spore suspension with a concentration of 100 / mL.

[0032] (2) Indoor toxicity determination.

[0033] The toxicity of X. longipes larvae was determined by immersion method. Healthy and active X. longipes larvae with uniform body size were obtained from coffee tree trunks for the test. Ten larvae were treated in each experiment with three replicates, and sterile water was used as a control. The experimental larvae were immersed in the corresponding suspension concentration for 5 seconds, and then placed in a culture dish with sterile filter paper, and an appropriate amount of sterile water was added to the culture dish to keep it moist. The larvae were cultured at 28 ℃ and 85% relative humidity for 7 days. The mortality was observed and recorded every day.

[0034] (3) Results

[0035] The Beauveria bassiana RZBB01 strain (CGMCC No. 40829) discovered in the present invention is the first to be isolated and purified from the larvae of the coffee beetle. It has a strong infection and pathogenic effect on the larvae of the coffee beetle, and the results are shown in Table 1. The variance analysis results show that there is no significant difference in the corrected mortality rate between the concentrations on the same observation day.

[0036] The Beauveria bassiana RZBB01 strain discovered by the present invention is the Beauveria bassiana RZBB01 separated and purified from the coffee beetle larvae for the first time in my country, CGMCC No. 40829, and its spore suspension has high pathogenicity to the coffee beetle larvae, and can be widely used in the prevention and treatment of the coffee beetle larvae. At the same time, the strain can grow on a PDA culture medium, has a wide source of raw materials, is low in price, has a simple culture method, is suitable for large-scale production, has great development and application potential, and uses the strain to carry out biological control of the coffee beetle larvae, which can avoid the problems of drug resistance and environmental pollution caused by chemical pesticides, which will provide a basis for the green prevention and control of the coffee beetle.

[0037] Table 1 Pathogenicity of strain RZBB01 to Xylophora coffeae larvae (mean ± SE)

[0038] The above-mentioned embodiments are only examples and are not the only way to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made on the basis of understanding the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A strain of Beauveria bassiana RZBB01 for controlling coffee beetle, characterized by: Beauveria bassiana Beauveria baasiana Beauveria bassiana Beauveria baasiana ; Deposited in the General Microbiology Center of China Microorganism Culture Collection, referred to as CGMCC, the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit date is September 22, 2023; the deposit number is CGMCC No. 40829.

2. The RZBB01 strain according to claim 1, characterized in that The ITS sequence of the RZBB01 strain is specifically shown in SEQ ID NO.

1.

3. Use of the Beauveria bassiana RZBB01 strain according to claim 1 in preventing and treating Xylotreya coffee beetle.

4. The use according to claim 3, characterized in that: The conidia of the RZBB01 strain are effective ingredients.

5. The RZBB01 strain conidia according to claim 4, characterized in that The conidia of the RZBB01 strain are conidia grown on sporangia of the Beauveria bassiana RZBB01 strain under specific culture conditions, and can be used to prepare spore liquid or a bacterial agent containing conidia.

6. The RZBB01 strain conidia agent according to claim 5, characterized in that: The inoculant is made from conidia of the RZBB01 strain, with a spore concentration range of 1×10 6 Pieces / mL - 1×10 8 Pieces / mL.

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