Beauveria bassiana TiBb-G1-2304 and application thereof

Abstract

The invention provides beauveria bassiana TiBb-G1-2304 and application thereof, and belongs to the technical field of biological control of pests. According to the invention, a strain is obtained by separating and purifying larva bombyx batryticatus of the eupatorium crassispina, is identified as Beauveria bassiana, is preserved in Guangdong Microbiological Culture Collection Center on April 27th, 2023, and has a preservation number of GDMCC No: 63414. According to the present invention, the virulence determination results of the strain on the larva of the eupatorium crassipes show that the strain has high virulence on the larva of the eupatorium crassipes, particularly has the optimal effect on the larva of the 4-year-old eupatorium crassipes, and the bombyx batryticatus rate of the 2-5-year-old larva under the concentration of 1.0 * 10 < 7 > spore / mL is more than 83%, such that the secondary infection probability of the strain is high, the lasting period is long, and the effect is significant; the strain is a biological prevention and control strain with potential significance in prevention and control of the eupatoriella crassipes.

Description

Technical Field

[0001] The invention belongs to the technical field of biological pest control, and in particular relates to a Beauveria bassiana TiBb-G1-2304 and an application thereof. Background Art

[0002] Thalassodes immissaria Walker, a member of the family Geometridae of the order Lepidoptera, is one of the main pests of litchi and longan during their tender shoots. The larvae mainly feed on the tender leaves of litchi and longan new shoots, preventing the new spikes from growing. They can also feed on the flower spikes, preventing the fruit from setting, or eat the functional leaves during the fruiting period, causing a large number of fruits to fall, which seriously threatens the production of litchi and longan (Xu Shu, Zeng Dongqiang, Quan Linfa, Yao Qiong, Chen Bingxu, Dong Yizhi. Effects of Temperature on the Growth, Development and Reproduction of Thalassodes Immissaria Walker [J]. Journal of Fruit Science, 2023, 40(10): 2241-2251.). In recent years, the geometrid has caused serious damage to litchi and longan. Among them, the thick-legged green moth is the dominant species of pests in the orchards of litchi and longan. It occurs in large quantities and causes the most serious damage. The damage rate of branches and shoots in general orchards is 30% to 50%, and the highest is more than 90%. Some branches and shoots are even completely eaten, leaving only bare branches, which seriously affects the nutrient accumulation of litchi and longan fruiting mother branches, and is not conducive to the high and stable yield of litchi and longan next year. The thick-legged green moth has 7 to 8 generations each year in Guangdong, with serious generation overlap. It can be seen from March to December every year. In view of the long occurrence and damage period of the insect and its explosive nature, chemical control has been widely promoted and applied, but the prevention effect is very small. In some areas, the pests are resistant to pesticides and difficult to control. Therefore, it is urgent to seek a green control technology.

[0003] Beauveria bassiana is an important entomogenous fungus that penetrates the body wall of host insects through hyphae and absorbs nutrients from the host, causing the host to gradually lose its ability to move and die. Beauveria bassiana has a wide range of hosts, including more than 700 species of insects from 15 orders and 149 families and more than 10 species of ticks and mites from 6 families. Among insect diseases found in nature, about 20% of insects killed by Beauveria bassiana are currently one of the most studied and applied entomogenous fungi in the world, and it plays an important regulatory role on pests. [1][2]([1] Cheng Yin, Zheng Jiyang, Wang Dun. Isolation and identification of three strains of Beauveria bassiana and their indoor toxicity to armyworms [J]. Chinese Journal of Biological Control, 2022, 38(02): 521-530. [2] Zimmermann G. Review on safety of theentomopathogenic fungi Beauveria bassiana and Beauveria brongniartii [J]. Biocontrol Science and Technology, 2007, 17(6): 553-596.). Due to the strong pathogenicity, broad insecticide spectrum, easy cultivation and spread, Beauveria bassiana is often developed into fungal insecticide biological agents and widely used in green control of agricultural and forestry pests. However, there are no reports on Beauveria bassiana strains with high pathogenicity to the larvae of the thick-legged green moth. Therefore, finding strains with high pathogenicity to the larvae of the thick-legged green moth has very important application value. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a strain of Beauveria bassiana TiBb-G1-2304 and its application. The present invention isolates and purifies a strain of Beauveria bassiana TiBb-G1-2304 from the larvae of the thick-legged green geometrid moth, and the strain can effectively kill the larvae of the thick-legged green geometrid moth, indicating that the strain can be used as a biological control strain to prevent and control diseases caused by the thick-legged green geometrid moth.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a strain of Beauveria bassiana TiBb-G1-2304, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 27, 2023, with a deposit number of GDMCC No: 63414. Address: No. 100, Xianlie Middle Road, Guangzhou

[0007] Preferably, the ITS sequence of Beauveria bassiana TiBb-G1-2304 is as shown in SEQ ID NO.1.

[0008] The invention provides application of Beauveria bassiana TiBb-G1-2304 in preventing and controlling larvae of the thick-legged green geometrid moth.

[0009] Preferably, the Beauveria bassiana TiBb-G1-2304 is the Beauveria bassiana TiBb-G1-2304 and / or a spore liquid containing the Beauveria bassiana TiBb-G1-2304.

[0010] Preferably, the spore liquid of Beauveria bassiana TiBb-G1-2304 is prepared by adding the Beauveria bassiana TiBb-G1-2304 into water containing a surfactant and shaking the mixture.

[0011] Preferably, the surfactant is Tween-80.

[0012] Preferably, the concentration of the surfactant in water is 0.01% to 0.10%.

[0013] Preferably, the concentration of the spore solution of Beauveria bassiana TiBb-G1-2304 is 1.0×10 4 ~1.0×10 8 Spores / mL.

[0014] Preferably, the larvae of the thick-legged green geometrid moth are 2nd to 5th instar larvae of the thick-legged green geometrid moth.

[0015] The present invention also provides a method for preventing and controlling the larvae of the broad-legged green geometrid moth, which comprises spraying and / or pouring a solution containing the Beauveria bassiana TiBb-G1-2304 into crops damaged by the larvae of the broad-legged green geometrid moth.

[0016] Compared with the prior art, the present invention has the following beneficial effects: the present invention obtains a strain of Beauveria bassiana TiBb-G1-2304 by isolating and purifying the dead insect body of the larvae of the thick-legged green geometrid moth, and the toxicity of the strain to the larvae of the thick-legged green geometrid moth is determined by the insect immersion method. The results show that Beauveria bassiana TiBb-G1-2304 has a high pathogenicity to the larvae of the thick-legged green geometrid moth, especially the best effect on the 4th instar larvae of the thick-legged green geometrid moth, with a toxicity of 1.0×10 7 After 7 days of inoculation at a concentration of spores / mL, the corrected mortality rate of 2nd to 4th instar larvae reached more than 78%; 1.0×10 8 After 7 days of treatment at the concentration of spores / mL, the corrected mortality of 2nd to 5th instar larvae reached 100%; at 1.0×10 7 The dead insect rate of 2nd to 5th instar larvae at the concentration of spores / mL reached more than 83%, indicating that the strain had a high probability of secondary infection and a long lasting effect. It is a biological control strain with potential significance in the prevention and control of the thick-legged green moth. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The indoor culture and conidia images of Beauveria bassiana strain TiBb-G1-2304 are shown;

[0018] Figure 2 It is an agarose gel electrophoresis diagram of the specific ribosomal DNA-ITS sequence of Beauveria bassiana strain TiBb-G1-2304;

[0019] Figure 3 This is the phylogenetic diagram of the TiBb-G1-2304 strain based on rDNA-ITS sequence analysis, where the numbers in brackets are the accession numbers of known strains in GenBank, the numbers on each branch indicate the confidence level, and the evolutionary tree scale is 0.005;

[0020] Figure 4 These are pictures of the advanced larvae and prepupae of the thick-legged green moth infected with the Beauveria bassiana strain TiBb-G1-2304.

[0021] Biological Deposit Description

[0022] Beauveria bassiana TiBb-G1-2304, Latin name Beauveria bassiana, is deposited in Guangdong Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, preservation date: April 27, 2023, preservation number: GDMCC No: 63414. DETAILED DESCRIPTION

[0023] The present invention provides a strain of Beauveria bassiana TiBb-G1-2304, wherein the Beauveria bassiana TiBb-G1-2304 was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 27, 2023, with a deposit number of GDMCC No: 63414.

[0024] In the present invention, the ITS sequence of Beauveria bassiana TiBb-G1-2304 is shown in SEQ ID NO.1, which is as follows:

[0025] .

[0026] The invention provides application of Beauveria bassiana TiBb-G1-2304 in preventing and controlling larvae of the thick-legged green geometrid moth.

[0027] In the present invention, the Beauveria bassiana TiBb-G1-2304 is the Beauveria bassiana TiBb-G1-2304 and / or a spore solution containing the Beauveria bassiana TiBb-G1-2304. The preparation method of the spore solution of the Beauveria bassiana TiBb-G1-2304 is as follows: adding the Beauveria bassiana TiBb-G1-2304 to water containing 0.01% to 0.10% Tween-80, and shaking well to obtain the obtained product. The concentration of the Tween-80 is preferably 0.03% to 0.07%, and more preferably 0.05%. The concentration of the spore solution of the Beauveria bassiana TiBb-G1-2304 is 1.0×10 4 ~1.0×10 8 Spores / mL, preferably 1.0×10 7 Spores / mL; the larvae of the thick-legged green geometrid moth are 2nd to 5th instar larvae of the thick-legged green geometrid moth.

[0028] The present invention also provides a method for preventing and controlling the larvae of the broad-legged green geometrid moth, which comprises spraying and / or pouring a solution containing the Beauveria bassiana TiBb-G1-2304 into crops damaged by the larvae of the broad-legged green geometrid moth.

[0029] In the present invention, the solution of Beauveria bassiana TiBb-G1-2304 has a concentration of 1.0×10 4 ~1.0×10 8 Spores / mL of Beauveria bassiana TiBb-G1-2304 spore solution, preferably 1.0×10 7 Spores / mL.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Preparation of PDA plate culture medium: weigh 200 g of potato, 20 g of glucose, and 20 g of agar, place in 1 L of distilled water, and sterilize in an autoclave at 121°C for 20 min.

[0032] The PDA plate culture medium used in the subsequent examples was prepared according to the above method.

[0033] Example 1 Isolation and purification of Beauveria bassiana TiBb-G1-2304 strain

[0034] The infected and lethal larvae of the green geometrid moth collected from the litchi garden in Baiyun District, Guangzhou City, Guangdong Province were cut into 1-2 mm small segments, and the surface was disinfected with 75% alcohol for 2-3 minutes under sterile conditions, washed with sterile water for 3-5 times, and then inoculated with PDA flat culture medium (with 0.1% penicillin added). Three pieces were placed in each culture dish in a triangle shape, and cultured in a constant temperature incubator at 26°C for 7 days. The white and thick hyphae on the edge were picked with an inoculation loop and inoculated into an inclined test tube as a strain. After culturing for 12 days, the test tube was full of white spore layers and no foreign bacteria, and the TiBb-G1-2304 strain was obtained.

[0035] Example 2 Identification of Beauveria bassiana TiBb-G1-2304 strain

[0036] The TiBb-G1-2304 strain obtained in Example 1 was morphologically identified: the pure strain was inoculated on a PDA plate medium for culture, the colony growth and morphological characteristics were observed and recorded, and the colony front and back morphology and color were recorded after culturing in a constant temperature incubator at 26°C for 7 days. An optical microscope (OLYMPUS CX31RTSF) and its matching TUCSEN microscopic imaging system were used to observe the colony hyphae morphology, spore production structure and conidia morphology, etc., and photographed and recorded. According to the morphological characteristics of the strain, the classification status of the entomogenous fungi was preliminarily identified with reference to the method of Pu Zhelong and Li Zengzhi (Pu Zhelong, Li Zengzhi. Insect Mycology [M]. Hefei: Anhui Science and Technology Press, 1996.).

[0037] Depend on Figure 1 It can be seen that when the TiBb-G1-2304 strain was inoculated on a PDA plate medium and cultured at 26°C for 7 days, the colony grew to a diameter of 22-26 mm, with an average daily growth rate of 3.1-3.7 mm / d. The colony was white on the front and light yellow on the back. It was a round flat colony with a velvety initial appearance and a white powdery spore layer in the later stage. Microscopic examination showed that the vegetative hyphae was colorless, the conidiophores were smooth and spike-shaped, and multiple cylindrical spore-producing cells were produced at the top; the conidia were single spores, transparent spherical or nearly spherical.

[0038] Molecular identification of the TiBb-G1-2304 strain obtained in Example 1:

[0039] The total DNA of Beauveria bassiana TiBb-G1-2304 strain was obtained using a fungal genomic DNA extraction kit (purchased from Beijing Liuhe BGI Gene Technology Co., Ltd.), and its ITS segment was amplified with the help of primers ITS1-TCCGTAGGTGAACCTGCGG (SEQ ID No. 2) and ITS4-TCCTCCGCTTATTGATATGC (SEQ ID No. 3). The amplification system was: 1 μL each of ITS1 primer and ITS4 primer, 5 μL of Beauveria bassiana TiBb-G1-2304 strain DNA template, 21 μL of PCR mix, and then supplemented to 25 μL with ddH2O; the amplification program was: 96℃ pre-denaturation for 5 min, 96℃ denaturation for 20 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, a total of 35 cycles, and finally extension at 72℃ for 10 min. The amplified product was detected by agarose gel electrophoresis ( Figure 2 ), after confirming the concentration and band size, the amplified product was sent to BGI for sequencing. The sequencing results were compared with the known sequences in NCBI by BLAST, and the relevant sequences were selected and downloaded using MEGA 11.0. The neighbor-joining method was used to construct a phylogenetic tree. The phylogenetic tree is shown in Figure 3 .

[0040] Depend on Figure 2 It can be seen that the amplified product was detected by agarose gel electrophoresis, and the base number of the amplified product obtained was 540bp. After sequencing, BLAST comparison was performed, and the ITS sequence of the strain was shown in SEQ ID No.1, specifically:

[0041] .

[0042] BLAST comparison with known sequences in NCBI showed that the strain was most similar to Beauveria bassiana, with an ITS sequence homology of 100%. Combined with morphological identification, the strain was identified as Beauveria bassiana, that is, the TiBb-G1-2304 strain obtained in Example 1 was Beauveria bassiana.

[0043] Example 3 Determination of the toxicity of Beauveria bassiana strain TiBb-G1-2304 to the larvae of the thick-legged green geometrid moth The test insect source was collected from the litchi garden of the Baiyun Experimental Base of Guangdong Academy of Agricultural Sciences, and was stably subcultured with young litchi leaves in the artificial climate room of the Institute of Plant Protection of Guangdong Academy of Agricultural Sciences for more than 7 generations. The larvae of the thick-legged green geometrid moth for the test were 2-5 years old, and the test insects were placed in a plastic insect box (length = 19.0 cm, width = 12.5 cm, height = 7.5 cm) disinfected with 75% alcohol. There were 30 larvae in each box, and clean young leaves of Feizixiao litchi were replaced once a day as food. Rearing conditions: temperature 26±1°C, relative humidity 75±5%, light L14:D10.

[0044] In a sterile operating table, use an inoculating loop to scrape the conidia of the Beauveria bassiana strain TiBb-G1-2304 obtained in a single test tube in Example 1 into a 50 mL sterile centrifuge tube, add 30 mL of distilled water containing 0.05% Tween-80, shake well, count under a microscope using a hemocytometer to obtain the initial concentration, and then dilute it to the required concentration with distilled water containing 0.05% Tween-80. The spore solution was prepared into 1.0×10 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 , 1.0×10 4 There are 5 concentrations of spores / mL. Each concentration is a treatment, with 0.05% Tween-80 solution as the control group, and each group is repeated 3 times, with 15 heads in each repeat. Inoculation was carried out by immersion method, and the larvae were immersed in the spore solution for 5s and then taken out, dried at room temperature and placed in an insect box. The insect bodies were placed in an insect box disinfected with 75% alcohol for breeding. The death of the larvae of the thick-legged green geometrid moth was investigated at regular intervals every day (9:00 a.m.), and observed for 10 consecutive days. If dead insects were found, they were removed from the culture dish and placed in another disinfected culture dish for moisturizing culture to observe whether the dead insects grew hyphae. Dead insect culture conditions: temperature 26±1℃, relative humidity 75±5%, light L14:D10. The effects of different concentrations of Beauveria bassiana TiBb-G1-2304 on the corrected mortality and dead insect rate of 2-5 instar larvae of the thick-legged green geometrid moth were determined, and the results are shown in Tables 1 and 2.

[0045] Table 1 Corrected mortality of different concentrations of Beauveria bassiana TiBb-G1-2304 against 2nd-5th instar larvae of the thick-legged moth

[0046]

[0047]

[0048] Table 2 Dead insect rate of 2-5th instar larvae of the thick-legged green moth after treatment with different concentrations of Beauveria bassiana TiBb-G1-2304

[0049]

[0050] The results in Tables 1 and 2 show that (1) at the same spore concentration, the pathogenicity of Beauveria bassiana strain TiBb-G1-2304 to 2-5 instar larvae varies, with the best effect on 4-instar larvae. 6 spores / mL, the corrected mortality rate reached 100% after 7 days of inoculation; (2) Under the insect immersion method, the mortality rate of the larvae of the thick-legged green geometrid moth inoculated with different spore concentrations increased with the extension of time within 5-10 days after treatment, and the cumulative corrected mortality rate of the larvae of the thick-legged green geometrid moth reached the maximum 10 days after treatment; (3) The pathogenicity to the larvae of the thick-legged green geometrid moth was high, at 1.0×10 7 After 7 days of inoculation at a concentration of spores / mL, most of the larvae died, especially the 4th-5th instar larvae, with a corrected mortality rate of more than 80%; 1.0×10 8 After 7 days of treatment with spores / mL, the corrected mortality of 2-5 instar larvae reached 100%; (4) The larval dead insect rate of Beauveria bassiana strain TiBb-G1-2304 was higher, at 1.0×10 7 The rate of dead insects in 2-5 instar larvae at the concentration of spores / mL reached more than 83%, indicating that the strain has a high probability of secondary infection and a long duration of efficacy. Based on the above experimental results, the Beauveria bassiana strain TiBb-G1-2304 has high pathogenicity to the larvae of the thick-legged green moth, with a concentration of 1.0×10 7 The spore / mL concentration can effectively kill the larvae and has a high insect deadening rate. It is a biological control strain with potential significance in the prevention and control of the thick-legged green moth.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Beauveria bassiana TiBb-G1-2304, characterized in that: The Beauveria bassiana TiBb-G1-2304 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on April 27, 2023, with the collection number GDMCCNo: 63414.

2. The Beauveria bassiana TiBb-G1-2304 according to claim 1, characterized in that The ITS sequence of Beauveria bassiana TiBb-G1-2304 is shown in SEQ ID NO.

1.

3. Use of Beauveria bassiana TiBb-G1-2304 according to claim 1 or 2 in controlling larvae of the thick-legged green moth.

4. The use according to claim 3, characterized in that: The Beauveria bassiana TiBb-G1-2304 is the Beauveria bassiana TiBb-G1-2304 described in claim 1 and / or a spore liquid containing the Beauveria bassiana TiBb-G1-2304 described in claim 1.

5. The use according to claim 4, characterized in that: The preparation method of the spore liquid of Beauveria bassiana TiBb-G1-2304 is as follows: adding the Beauveria bassiana TiBb-G1-2304 described in claim 1 into water containing a surfactant and shaking the mixture to obtain the spore liquid.

6. The use according to claim 5, characterized in that: The surfactant is Tween-80.

7. The use according to claim 5 or 6, characterized in that: The concentration of the surfactant in water is 0.01% to 0.10%.

8. The use according to claim 4, characterized in that: The concentration of the spore liquid of Beauveria bassiana TiBb-G1-2304 is 1.0×10 4 ~1.0×10 8 Spores / mL.

9. The use according to claim 4, characterized in that: The larvae of the thick-legged green geometrid moth are 2nd to 5th instar larvae of the thick-legged green geometrid moth.

10. A method for controlling the larvae of the broad-legged moth, characterized in that: The solution containing the Beauveria bassiana TiBb-G1-2304 according to claim 1 is sprayed and / or poured into crops damaged by the larvae of the broad-legged moth.

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