CN112522112B

CN112522112B - Fungus strain for preventing and treating alfalfa aphids and application thereof

Info

Publication number: CN112522112B
Application number: CN202011428714.4A
Authority: CN
Inventors: 王广君; 陈佳杰; 田野; 张泽华; 农向群; 李红梅; 涂雄兵; 李贝贝; 李霜
Original assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Plant Protection of Chinese Academy of Agricultural Sciences
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-11-05
Anticipated expiration: 2040-12-07
Also published as: CN112522112A

Abstract

The invention relates to the field of plant protection, and particularly relates to a fungal strain for preventing and controlling alfalfa aphids and application thereof. The preservation number of the strain is CGMCC No. 19034. The bacterial strain has the best effect of preventing and controlling the aphis medicaginis. The strain is simple to culture, high in growth speed, large in spore yield, high in spore germination rate, pathogenic to the aphis medicaginis at different time, low in pathogenic concentration under different concentrations, environment-friendly, not easy to generate drug resistance and capable of being used for preventing and treating the aphis medicaginis.

Description

Fungus strain for preventing and treating alfalfa aphids and application thereof

Technical Field

The invention relates to the field of plant protection, and particularly relates to a fungal strain for preventing and controlling alfalfa aphids and application thereof.

Background

Aphids are one of the omnivorous pests widely distributed around the world, and harm various crops and economic plants, the main harm to alfalfa is alfalfa aphids which are often clustered on young tips and young leaves to suck juice, so that the leaves are curled and yellow, the plants are dwarf and slow in growth, the young tips are withered when the leaves are serious, chlorophyll is damaged, honeydew is excreted to reduce photosynthesis, pathogenic bacteria are bred, and virus diseases can be spread. Due to the characteristics of short growth cycle and large breeding amount, the fertilizer quickly explodes under proper conditions, and causes unbearable loss to the farming and animal husbandry.

At present, chemical agents are mainly used for preventing and controlling the aphis medicaginis in production, and the existing indoor insecticidal activity determination is to screen some chemical agents with higher effect. However, long-term, large and unjustified use has resulted in varying degrees of resistance to aphid medicago. Research personnel are required to deeply research the drug resistance mechanism and metabolism of the lucerne aphis, so that a better and more environment-friendly prevention and treatment method is provided.

The biological assay is an important technical means for monitoring the drug resistance of pests, can reflect the resistance of the pests to certain pesticides, can also reflect the degree of the resistance, and has practical significance for adjusting resistance control strategies. The two most commonly used biocontrol microorganisms in biological control are metarhizium anisopliae and beauveria bassiana. The biological characteristics of beauveria bassiana and metarhizium anisopliae are deeply researched, the control effect of the beauveria bassiana and the metarhizium anisopliae is enhanced, and a theoretical basis is provided for future production and application and biological control of alfalfa pests.

Disclosure of Invention

The invention aims to provide a fungal strain for treating lucerne aphid.

It is a further object of the present invention to provide the use of the above mentioned strains.

It is a further object of the present invention to provide a method for controlling aphid medicago sativa.

Still another object of the present invention is to provide a biological agent for controlling aphis medicaginis.

The Beauveria bassiana (Beauveria bassiana) IPPHBP 094 strain has the preservation number of CGMCC No.19034 and the preservation date of 2019, 12 months and 11 days, and is preserved in the China general microbiological culture Collection center, the Address of Beijing City North Chen West Lu No.1 Hospital in the sunny region.

The method for controlling the aphis medicaginis comprises the step of killing the aphis medicaginis by utilizing the Beauveria bassiana (Beauveria bassiana) IPPHBB094 strain.

The biological agent for controlling the aphis medicaginis provided by the invention comprises Beauveria bassiana (Beauveria bassiana) IPPHBB094 strain.

The invention also provides application of the Beauveria bassiana (Beauveria bassiana) IPPHBP 094 strain in preventing and treating the aphis medicaginis.

The beauveria bassiana IPPHBP 094 has the best effect of preventing and treating the aphis medicaginis. The strain is simple to culture, high in growth speed, large in spore yield, high in spore germination rate, pathogenic to the aphis medicaginis at different time, low in pathogenic concentration under different concentrations, environment-friendly, not easy to generate drug resistance and capable of being used for preventing and treating the aphis medicaginis.

Detailed Description

Example 1 isolation of strains

The strain is derived from aphid stiff insects collected from suburb fields in the Beijing sea lake region. The stiff insects were transferred to a sterile petri dish with moist filter paper and incubated at a constant temperature of 30 ℃. After hyphae grow out, picking the front end mycelium by using an aseptic inoculating ring, inoculating the mycelium onto a flat plate (PDA culture medium), carrying out purification culture at constant temperature of 30 ℃ to obtain the beauveria bassiana IPPHBB094, repeatedly culturing for 2-3 times, and storing in a laboratory at 4 ℃.

1. Test strains: IPPHBP 094, IPPBSX-1, IPPB34-3, IPPB18514, IPPB2010-15, IPPB2010-16, IPPB2010-17, IPPBFrM2019-137, IPPM189 and IPPM202 are all provided by plant protection research institute of Chinese academy of agricultural sciences, and are separated and purified by adopting a PDA culture medium and then stored in a laboratory at 4 ℃.

2. The method comprises the following steps: and (4) carrying out a germination rate test by adopting a culture dish germination method.

3. And (4) checking the result: and (3) washing conidia on the PDA with sterile water to prepare a suspension with 20-30 spores per visual field under a 10X microscope, and performing a germination test in the sterile water by a culture dish germination method. And (3) randomly observing for 3 times by each repetition, counting 100 spores, and calculating the germination rate.

TABLE 1 comparison of germination rates of different strains

Whether spores of a strain germinate is an indicator of the viability or virulence of the strain and is also a prerequisite for infection. As can be seen from Table 1, in this experiment, the germination rate of spores of 10 strains was measured, and the germination rate of spores was measured at 10h and 22h, respectively, by culturing in PDA medium. As can be seen in Table 1, the difference in spore germination was significant for both times. The germination rate of each strain reaches over 90% in 22h, which shows that the test strains have excellent properties and can be used for indoor insecticidal activity tests. Wherein the strain with the highest germination rate of 22 hours is IPPHBP 094, and the germination rate is 97.33%.

Example 2 virulence assay for A. lucerne Aphis species

1. The method comprises the following steps: and (4) inoculating by adopting a dipping method, and carrying out indoor toxicity measurement. Culturing each strain at 30 deg.C with PDA, scraping spore powder with a sporulator after the spore powder grows over the whole culture dish, transferring into sterile water containing 0.01% Tween-80 to prepare spore suspension, placing on an oscillator, oscillating sufficiently, measuring spore concentration with a blood counting plate, and diluting with sterile water to desired concentration (1x 10)⁸spores/mL).

2. Test strains: IPPHBP 094, IPPBSX-1, IPPB34-3, IPPB18514, IPPB2010-15, IPPB2010-16, IPPB2010-17, IPPBFrM2019-137, IPPM189 and IPPM202 are all provided by plant protection research institute of Chinese academy of agricultural sciences, and are separated and purified by adopting a PDA culture medium and then stored in a laboratory at 4 ℃.

3. And (3) selecting the tested lucerne aphids: the alfalfa leaf aphids are collected from a Korean village and a Richeng modern agricultural alfalfa garden in Cangzhou city and county of Hebei province, collected insect states are adults, and the collected adults are placed in an incubator with the temperature of 25 +/-1 ℃ and the relative humidity of 65 +/-5 percent and the light period (L/D) of 16/8 hours to be fed with fresh alfalfa leaves. Selecting active alfalfa leaf aphids with consistent sizes, inoculating by adopting a dipping method, repeatedly selecting 20 active alfalfa leaf aphids with consistent sizes in a culture dish, uniformly spraying on the body of the aphid by using a spraying pot, ensuring that the body surface of each aphid contains spore suspension, repeatedly treating for 5 times, and using sterile water of 0.01 percent Tween-80 as a blank control.

4. Determining the mortality rate of different strains to the lucerne aphids: is placed at 25Continuously observing for 6 days in an artificial climate box with the relative humidity of 75% and the photoperiod of 16h/8h at L/D, picking out the newly born aphids and the dead aphids every day, putting the newly born aphids and the dead aphids into a culture dish with filter paper for moisturizing so as to observe the growth condition of hyphae, recording the death number of the lucerne aphis, and counting the death rate. Data analysis IBMSPSSSstatins 21 software is used for analysis to obtain virulence regression equation and correlation coefficient R²、LT₅₀。

Mortality (%). ratio (number of dead insects/number of test insects). times.100

Corrected mortality (%) - (treatment mortality-control mortality)/(1-control mortality) ] × 100

5. Results and analysis

As can be seen from Table 2, 1-7 days after treatment of different fungal strains has a certain control effect on the aphis medicaginis. The difference of mortality rate after treatment 1 and 2d is not significant, and after treatment 3d, the lethality rates of IPPM189, IPPHBP 094, IPPBSX-1, IPPB34-3, IPPB2010-16 and IPPB2010-17 all reach more than 50%. As can be seen from Table 3, the regression equation and the correlation coefficient of the indoor toxicity measurement of the 10 strains of fungal strains on the aphis medicaginis are better when the R values of the correlation coefficients are all above 0.84. Spore concentration of 1 × 10 was used for each strain⁸Half-lethal time statistics is carried out on spore/mL bacterial suspension, and the results show that the pathogenicity is IPPHBP 094, the half-lethal time is 66.26h, the half-lethal time is 67.37h for IPPB34-3, the worst is IPPBFrM2019-137, and the half-lethal time is 84.22 h.

TABLE 2 indoor bioassay of lucerne aphids by different strains

Note: the data in the table are mean values, and different letters in the same column of data respectively show significant difference at the 0.05 level.

TABLE 3 determination of LT for toxicity of strains on lucerne aphid with the same spore concentration₅₀Regression equation of virulence (1X 10)⁸spore/mL)

Example 3 virulence strategy for the concentration of different strains on the E.lucerne population

2. Test strains: IPPHBP 094, IPPBSX-1, IPPB34-3, IPPM189 and IPPM202 are all provided by plant protection research institute of Chinese academy of agricultural sciences, and are stored in a laboratory at 4 ℃ after being separated and purified by adopting a PDA culture medium.

3. And (3) selecting the tested lucerne aphids: the alfalfa leaf aphids are collected from a Korean village and a Richeng modern agricultural alfalfa garden in Cangzhou city and county of Hebei province, collected insect states are adults, and the collected adults are placed in an incubator with the temperature of 25 +/-1 ℃ and the relative humidity of 65 +/-5 percent and the light period (L/D) of 16/8 hours to be fed with fresh alfalfa leaves.

Selecting active alfalfa leaf aphids with consistent sizes, inoculating by adopting a dipping method, repeatedly selecting 20 active alfalfa leaf aphids with consistent sizes in a culture dish, uniformly spraying on the body of the aphid by using a spraying pot, ensuring that the body surface of each aphid contains spore suspension, repeatedly treating for 5 times, and using sterile water of 0.01 percent Tween-80 as a blank control.

4. The mortality of the lucerne aphid was determined with different concentrations of the strains, which were designed as given in table 4 below. Each treatment was repeated 5 times.

TABLE 4 concentration of different strains

And (3) placing the aphids in an artificial climate box with the temperature of 25 ℃, the relative humidity of 75% and the light period of 16h/8h, continuously observing for 6D, picking out the new aphids and dead aphids every day, placing the new aphids and dead aphids in a culture dish with filter paper for moisturizing so as to observe the growth condition of hypha, recording the death number of the lucerne aphis, and counting the death rate. Data analysis IBMSPSSSstatins 21 software is used for analysis to obtain virulence regression equation and correlation coefficient R²、LT₅₀。

Mortality (%). ratio (number of dead insects/number of test insects). times.100

5. Results and analysis

As can be seen from Table 5, the difference in the semi-lethal concentration of each strain was significant at the third day (72h) of the bioassay, with the semi-lethal concentration being lowest for the strain IPPHBP 094 and LC₅₀Is 0.5X 10⁴spore/mL, strain IPPB34-3, strain IPPM189, strain IPPM202, and half-lethal concentration of strain IPPBSX-1, LC₅₀Is 1.1X 10⁶spore/mL shows that the strain IPPHBB094 has the strongest toxicity to the aphis medicaginis, and the strain IPPBSX-1 has the weakest toxicity to the aphis medicaginis.

TABLE 5 virulence of different strains against lucerne aphid (72h)

In conclusion, the beauveria bassiana IPPHBP 094 has the best effect of preventing and treating the aphis medicaginis. The strain is simple to culture, high in growth speed, large in spore yield, high in spore germination rate, pathogenic to the aphis medicaginis at different time, low in pathogenic concentration under different concentrations, environment-friendly, not easy to generate drug resistance and capable of being used for preventing and treating the aphis medicaginis.

Claims

1. Beauveria bassiana (Beauveria bassiana) IPPHBH 094 strain, and is characterized in that the preservation number of the strain is CGMCC No. 19034.

2. A method for controlling aphid medicago, said method comprising the step of killing aphid medicago using beauveria bassiana (beauveria bassiana) IPPHBB094 strain of claim 1.

3. A biological agent for controlling aphis medicaginis, comprising beauveria bassiana (beauveria bassiana) IPPHBB094 strain of claim 1.

4. Use of the beauveria bassiana (beauveria bassiana) IPPHBB094 strain of claim 1 to control aphis medicaginis.