Helicoverpa armigera metarhizium ledebusii MRDL2024 strain and application thereof
By using the spore suspension of the MRDL2024 strain of L. L. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le. le.
Patent Information
- Application Number
- CN202510288877.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-04-11
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Cotton bollworms have developed resistance to a variety of chemical pesticides, resulting in poor control and treatment of chemical pesticides, accompanied by pesticide residues, safety hazards for human and animal life, and ecological environment impacts.
The MRDL2024 strain of L. Lesserella lepisces was used to biologically control the bollworm through its spore suspension. The strain was cultured in SMAY solid medium, with a temperature of 26±1°C, a relative humidity of 75%±5%, and a photoperiod L: D=14 h: 10 h. After a large number of conidia were produced, the spore suspension was prepared for spraying on the crop.
MRDL2024 of L. le. le. le. le. MRDL2024 is highly pathogenic to cotton bollworm larvae, is easy to cultivate indoors on a large scale, has a high spore germination rate, is safe to the environment, and is not easy to cause drug resistance to pests, providing a green, safe and efficient biological control method.
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Figure CN119799514A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a Metarhizium anisopliae MRDL2024 strain and a culture method thereof, and application of the strain in preventing and controlling cotton bollworm. Background Art
[0002] cotton bollworm ( Helicoverpa armigera ), is a worldwide pest. The insect has a wide host range and can harm more than 200 species of plants including cotton, tobacco, and corn. In recent years, the cotton bollworm has been one of the main pests in tobacco and corn producing areas in my country, causing serious damage to tobacco and corn production. The cotton bollworm mainly harms corn and tobacco leaves with its larvae during the seedling stage of corn and tobacco, and harms corn heart leaves and tobacco new buds and tender leaves during the corn seedling stage and before tobacco buds appear, biting holes or notches, and in severe cases, eating the entire leaf.
[0003] In the prior art, the prevention and control of cotton bollworms mainly relies on spraying chemical pesticides. However, cotton bollworms have developed strong resistance to a variety of chemical pesticides, which has brought new challenges to the prevention and control of cotton bollworms. In addition, the use of chemical pesticides for prevention and control will also bring risks of pesticide residues, hidden dangers to human and animal safety, and impact on the ecological environment. Therefore, it is imperative to explore green, safe, efficient, and sustainable measures to prevent and control cotton bollworms. Summary of the invention
[0004] The main purpose of the present invention is to provide a strain of Metarhizium anisopliae MRDL2024 to provide a new approach for the green control method of cotton bollworm.
[0005] To achieve the above objectives, the present invention provides the following technical solutions: A species of Metarhizium religiosa ( Metarhizium rileyi ) MRDL2024, classified as Metarhizium raderii Metarhizium rileyi , the deposit number is CGMCC No.41555.
[0006] Furthermore, the present invention provides a method for culturing Metarhizium reinhardtii MRDL2024, which comprises inoculating Metarhizium reinhardtii MRDL2024 into SMAY solid culture medium (40 g maltose, 10 g peptone, 10 g yeast extract, 18 g agar powder, fixed to 1000 mL with distilled water, natural pH) for culturing, and preparing a spore suspension after a large number of conidia are produced.
[0007] Furthermore, the culture conditions were: temperature 26±1°C, relative humidity 75%±5%, and photoperiod L:D=14 h:10 h.
[0008] Furthermore, the method for preparing the spore suspension is: adding conidia to a sterile aqueous solution of Tween-80 with a concentration of 0.05% to prepare spore suspensions with different concentrations.
[0009] The present invention also provides an insecticide, which contains the spores of Metarhizium reinhardtii MRDL2024.
[0010] Furthermore, the insecticide contains spores of Metarhizium anisopliae MRDL2024 in an amount of 1.0×10 4 –1.0×10 8 Pieces / mL.
[0011] The Metarhizium anisopliae MRDL2024 provided by the present invention, or a spore suspension obtained by any culture method, or any insecticide is used in controlling cotton bollworm.
[0012] Furthermore, the present invention provides a method for controlling cotton bollworms, wherein the insecticide is applied to crops infested with cotton bollworms, when the cotton bollworms are in the larval stage.
[0013] Furthermore, the crops include corn, tobacco, pepper or tomato.
[0014] The technical effects achieved by the present invention are: The Metarhizium muscardine MRDL2024 provided by the present invention is simple to culture, grows quickly, has a large spore production amount, a high spore germination rate, and is easy to culture and reproduce in large quantities indoors. It is a biological control fungus for cotton bollworm that can be used to prevent and control pests that harm crops such as corn, tobacco or peppers. It has the characteristics of strong pathogenicity to cotton bollworm larvae, safety to the environment, and not easy to cause pests to develop drug resistance. It can be used for biological control of cotton bollworm. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a conidia morphology diagram of Metarhizium anisopliae MRDL2024 of the present invention; Figure 2 This is a morphological diagram of the sporangiophore of Metarhizium anisopliae MRDL2024 of the present invention; Figure 3 This is a colony morphology diagram of Metarhizium anisopliae MRDL2024 of the present invention; Figure 4 The symptom diagram of the natural infection of cotton bollworm larvae by Metarhizium religiosa MRDL2024 of the present invention (a is the symptom at 3 days of infection; b is the symptom at 7 days of infection). DETAILED DESCRIPTION
[0016] The following will describe the implementation methods of the present invention in detail with reference to the embodiments, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.
[0017] The Metarhizium muscardine MRDL2024 strain provided by the present invention is classified and named as Metarhizium muscardine Metarhizium rileyi ; Deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the storage address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Storage date: October 28, 2024; Storage number: CGMCC No.41555. The viability of the biological material was tested by the storage center on October 28, 2024, and the result was viable. Example 1
[0018] Isolation and identification of Metarhizium raderii MRDL2024 On August 20, 2024, strain samples were collected from diseased cotton bollworm larvae in the tobacco fields of Nandian Village, Wanqiao Town, Dali City, Yunnan Province (25°77'43"N, 100°12'88"E, 1950 meters above sea level). After isolation, purification, virulence determination and screening, it was determined to be Metarhizium muscardineum. Testing found that the fungus is highly pathogenic to cotton bollworm larvae.
[0019] 1.1 Materials 1.1.1 Test strains The strain was collected on August 20, 2024 from diseased cotton bollworm larvae in the flue-cured tobacco fields of Nandian Village, Wanqiao Town, Dali City, Yunnan Province (25°77'43"N, 100°12'88"E, 1950 meters above sea level).
[0020] 1.1.2 Isolation and culture of strains The collected diseased cotton bollworm bodies were placed in a 5 mL sterile EP tube and brought back to the laboratory. The streak method was used to culture them on SMAY solid medium (maltose 40 g, peptone 10 g, yeast extract 10 g, agar powder 18 g, distilled water to 1000 mL, pH natural) in an incubator at 26±1°C, relative humidity 75%±5%, and photoperiod L:D=14 h:10 h. After 7 days of culture, the conidia were transferred to a new medium for purification. The purified strain was stored in a 4°C refrigerator using SMAY slant medium.
[0021] 1.1.3 Colony morphology observation The isolated and purified fungi were inoculated on SMAY solid medium (maltose 40 g, peptone 10 g, yeast extract 10 g, agar powder 18 g, distilled water to 1000 mL, pH natural) and cultured in an incubator at 26±1℃, relative humidity 75%±5%, and photoperiod L:D=14 h:10 h. The colony diameters were measured and the colony morphology was observed at 3, 7 and 14 d.
[0022] 1.2 Results After obtaining the pure culture of Metarhizium anisopliae MRDL2024 strain, its morphological characteristics were identified. Figure 1 , Figure 2 and Figure 3 The strain was cultured on SMAY medium for 10 days, and the colony diameter reached 9.25~10.15 mm. The front of the colony had white villi and was slightly convex in the middle. The color of the back of the colony changed from light to dark from the edge to the center, and from yellow-green to olive green at the center 1 / 2. The hyphae were smooth and separated, with a hyphae diameter of 2~3 μm. The conidiophores were upright and grape-like. The conidia were oblong, smooth, with one end of the conidia pointed and the other end slightly blunt, with a size of 4.50μm×1.85μm. The ITS sequence analysis method was used for molecular systematic identification, and the ITS gene sequence of the Metarhizium reinhardtii MRDL2024 strain was measured as shown in SEQ ID No.1. After multiple sequence alignment, it was identified as Metarhizium reinhardtii ( Metarhizium rileyi ) and deposited with the deposit number: CGMCC No.41555.
[0023] SEQ ID No.1 -ATATCATCGTTTATTTTGA Example 2
[0024] Pathogenicity determination of Metarhizium anisopliae MRDL2024 to cotton bollworm 1 Materials 1.1.1 Test insects Cotton bollworms were collected from the flue-cured tobacco fields of Nandian Village, Wanqiao Town, Dali City, Yunnan Province (25°77'43"N, 100°12'88"E, 1950m above sea level) and raised indoors for three generations in an intelligent artificial incubator (RG-300) at a temperature of 26±1℃, a relative humidity (RH) of 75±5%, and a photoperiod of L:D=14h:10h. Cotton bollworm larvae were raised on fresh flue-cured tobacco leaves. After molting five times, the larvae developed into pupae. After 7 days of pupal feeding, the adults emerged. After the adults laid eggs, the egg masses were continued to be raised on flue-cured tobacco leaves. Cotton bollworm larvae were obtained in about 3 days. For bioassay.
[0025] 1.1 Preparation of spore suspension The isolated strain was inoculated into SMAY solid medium and cultured in an incubator at 26±1℃, relative humidity 75%±5%, and photoperiod L:D=14h:10h. After a large number of conidia were produced (about 20 days), conidia on the surface of the medium were scraped with a sterile scalpel and placed in a sterile aqueous solution of 0.05% Tween-80 to prepare 1.0×10 4 –1.0×10 8 spores / mL of spore suspension.
[0026] 1.1.2 Impregnation method The third-instar larvae of cotton bollworm that were hatched on the same day, of the same size and healthy were selected and inoculated by the dipping method. The third-instar larvae of cotton bollworm were selected for the experiment. After inoculation, the cotton bollworm larvae were placed in a homemade plastic wrap insect cage (20 cm×20 cm×20 cm), and fresh flue-cured tobacco leaves were placed regularly every day. Five replicates were set for each group of experiments, and 10 replicates were set for each treatment. After inoculation, the death and infection of the larvae were observed regularly every day. When dead insects were found, the dead insects were taken out in time and placed in a sterilized culture dish with a diameter of 9 cm and a sterilized filter paper on the bottom. The culture dish was covered and placed in a light incubator at (25±1)℃, relative humidity (75%±5%), L:D=14 h:10 h for cultivation. The growth of hyphae or conidia on the surface of the insects was observed regularly every day, and the morphology of conidia and hyphae was examined under a microscope to determine that the bacteria grown on the insects were the inoculated bacteria.
[0027] 1.1.3 Data Analysis Excel 2010 was used for data collation, and the cumulative mortality rate was calculated using DPS data processing system 14.0 software.
[0028] 2. Results 2.1 Cumulative mortality of cotton bollworm The strain MRDL2024 showed significant pathogenicity to the third-instar larvae of cotton bollworm at different inoculation concentrations, and the mortality rate of the larvae gradually increased with the increase of spore concentration (Table 1). Among the third-instar larvae of cotton bollworm inoculated with spore suspensions of different concentrations, death occurred on the second day after inoculation, and the peak period of death was concentrated between 6 and 10 days.
[0029] As shown in Table 1, in the case of 1.00×10 4 / mL~1.00×10 8 The inoculation treatment with spore concentration of 1.00×10 / mL had an infection-lethal effect on the larvae of cotton bollworm. 4 , 1.00×105 , 1.00×10 6 , 1.00×10 7 and 1.00×10 8 After treatment with 1×10 / mL, the cumulative mortality rates of cotton bollworm larvae were 0, 1.11%, 1.11%, 3.33%, and 7.78% on the second day; the cumulative mortality rates of cotton bollworm larvae were 2.22%, 6.67%, 6.67%, 7.78%, and 15.56% on the third day; and the cumulative mortality rates of cotton bollworm larvae were 11.11%, 20.00%, 26.67%, 34.44%, and 48.89% on the fifth day after treatment, of which 1×10 8 The cumulative mortality rate of the treatment group reached about 50%, which was significantly higher than that of other treatment groups. On the 7th day after treatment, the mortality rates of the third-instar larvae of cotton bollworm were 16.67%, 32.22%, 43.33%, 51.11% and 66.67%, respectively. 8 and 1×10 7 The mortality rates of the third instar larvae of cotton bollworm in the spore / mL treatment groups were all above 80%, 81.11% and 83.33% respectively, which were significantly higher than those in other treatments. The above results indicate that the strain MRDL2024 of Metarhizium anisopliae has good toxicity and toxicity to cotton bollworm larvae and a strong lethal effect.
[0030] Table 1 Cumulative mortality of cotton bollworm larvae at different time periods after inoculation with different concentrations of Metarhizium raderii MRDL2024 spore suspension
[0031] 2.2 Effect of lethal time of Metarhizium religiosa MRDL2024 on cotton bollworm The infection and pathogenicity time of strain MRDL2024 to cotton bollworm larvae was shortened with the increase of inoculation concentration (Table 2). 8 , 1.0×10 7 , 1.0×10 6 , 1.0×10 5 and 1.0×10 4 spores / mL, half of the larvae die at LT 50 The length of time of infection was 5.9d, 6.9d, 8.0d, 9.7d and 12.7d respectively. It can be seen that the strain MRDL2024 has a strong ability to infect and cause disease to the third instar larvae of cotton bollworm.
[0032] Table 2
[0033] 2.3 Lethal dose effect of Metarhizium religiosa MRDL2024 on cotton bollworm The strain MRDL2024 showed high pathogenicity to the third instar larvae of cotton bollworm. 50 From the values of LC, it can be found that with the gradual extension of the post-inoculation treatment time, the lethal dose showed a decreasing trend, indicating that the virulence was continuously increasing. 50 The value is from 5.64×10 7 The spores / mL was significantly reduced to 2.53×10 6 spores / mL (Table 3).
[0034] Table 3 The lethal dose of different spore concentrations of Metarhizium raderii MRDL2024 suspension to the third instar larvae of cotton bollworm
[0035] The research group of the present invention found in August 2023 that the Metarhizium muscardine MRDL2024 strain in the flue-cured tobacco field of Nandian Village, Wanqiao Town, Dali Prefecture, Yunnan Province (25°77'43"N, 100°12'88"E, 1950 meters above sea level) has a strong pathogenicity to the larvae of cotton bollworm ( Figure 4 ).
[0036] In summary, the Metarhizium muscardine MRDL2024 provided by the present invention has a strong lethal effect on cotton bollworm larvae. Metarhizium muscardine MRDL2024 is a biocontrol fungus that can be used for green control of cotton bollworm. It has the characteristics of strong pathogenicity to cotton bollworm, no pollution to the environment, and pests are not easy to develop drug resistance. It can be widely used in biological control of cotton bollworm.
[0037] The above description shows and describes several preferred embodiments of the present invention, but as mentioned above, it should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications, and can be modified within the scope of the present invention through the above teachings or the technology or knowledge of the relevant field. And the changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.
Claims
1. A Metarhizium anisopliae MRDL2024, characterized in that Metarhizium anisopliae Metarhizium rileyi , the deposit number is CGMCC No.41555.
2. The method for culturing Metarhizium anisopliae MRDL2024 according to claim 1, characterized in that: The method comprises the following steps: inoculating Metarhizium anisopliae MRDL2024 into a SMAY solid culture medium for culturing, and preparing a spore suspension after a large number of conidia are produced.
3. The culture method according to claim 2, characterized in that The culture conditions were as follows: temperature 26±1°C, relative humidity 75%±5%, and photoperiod L:D=14 h:10 h.
4. The culture method according to claim 2, characterized in that The method for preparing the spore suspension is as follows: conidia are added into a sterile aqueous solution of Tween-80 with a concentration of 0.05% to prepare spore suspensions with different concentrations.
5. An insecticide, characterized in that: The insecticide contains spores of Metarhizium anisopliae MRDL2024 described in claim 1.
6. The insecticide according to claim 5, characterized in that The insecticide contains 1.0×10 4 –1.0×10 8 spores / mL.
7. Use of the Metarhizium anisopliae MRDL2024 described in claim 1, or the spore suspension prepared by the culturing method described in any one of claims 2 to 4, or the insecticide described in any one of claims 5 to 6 in controlling cotton bollworm.
8. A method for controlling cotton bollworm, characterized in that: The insecticide according to any one of claims 5 to 6 is applied to crops infested with cotton bollworms, when the cotton bollworms are in the larval stage.
9. The control method according to claim 8, characterized in that: The crops include corn, tobacco, pepper or tomato.
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