CN112205422B

CN112205422B - Application of entomopathogenic fungi, method for preventing and controlling pests and insecticide

Info

Publication number: CN112205422B
Application number: CN202011096562.2A
Authority: CN
Inventors: 郑旭; 张超; 杨慧莹; 赵秀梅; 徐利剑; 高思禹; 张李香; 宋福强
Original assignee: QIQIHAR BRANCH OF HEILONGJIANG ACADEMY OF AGRICULTURAL SCIENCES
Current assignee: QIQIHAR BRANCH OF HEILONGJIANG ACADEMY OF AGRICULTURAL SCIENCES
Priority date: 2020-10-14
Filing date: 2020-10-14
Publication date: 2021-05-07
Anticipated expiration: 2040-10-14
Also published as: CN112205422A

Abstract

The invention provides an application of entomopathogenic fungi, a method for preventing and controlling pests and a pesticide, belonging to the field of microorganisms, in order to avoid the influence of a chemical pest control method on the ecological environment, the invention provides the application of the entomopathogenic fungi with the number of SGSF043, the SGSF043 strain has higher lethality rate on the diabrotica virgifera, the cumulative corrected mortality rate reaches 91.55% on the 8 th day of inoculation treatment, the stiff insect rate is more than 90%, when the SGSF043 strain infects corn aphid on the 3 rd day, the lethality reaches 100%, the final stiff worm rate is more than 70%, the corrected mortality rate of the SGSF043 strain on the 8 th day of the green bean weevil infection reaches more than 50%, which shows that the SGSF043 strain has good anti-insect activity, can infect various pests, has wide control range, belongs to non-host-specific entomopathogenic fungi, has good biological control potential of the pests, and can be used as a biocontrol strain.

Description

Application of entomopathogenic fungi, method for preventing and controlling pests and insecticide

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to application of entomopathogenic fungi, a method for preventing and controlling pests and a pesticide.

Background

Entomopathogenic fungi are widely applied to pest control as a commonly used biological control strain, and at present, a plurality of successful cases exist, such as scarab entomopathogenic fungi (Metarhizium anisoliae), yellow green entomopathogenic fungi (M.flavoviride) and Robert entomopathogenic fungi (M.robertsii), and the like, and the entomopathogenic fungi have an infection effect on coleoptera, orthoptera, lepidoptera and hemiptera insects such as grape black weevil (Tiotropichylurus), cricket (Anabrus simplex), diamond back moth (Plutella xylostella), gray plant louse (Laodelphax striatellus), and the like. The Rhagophthalmus biflorus (Monolepta hieroglyphic) is a omnivorous pest, has a wide feeding range, and has some reports on chemical control at present, but biological control has not been reported yet. The corn aphid (Rhopalosiphum maidis) can secrete a large amount of honeydew besides eating leaves and absorbing corn juice, influences photosynthesis of crops, enables harmful plants to grow weaker, reduces yield, mainly controls chemical control at present, and can influence ecological environment and kill part of beneficial microorganisms due to excessive pesticide spraying. Therefore, a fungus with the potential of preventing and controlling pests is found, and the use of chemical pesticides can be reduced.

Disclosure of Invention

The invention provides an application of entomopathogenic fungi, a method for controlling pests and a pesticide, in order to avoid the influence of a chemical pest control method on ecological environment.

The invention provides an application of entomopathogenic fungi in pest control, wherein the entomopathogenic fungi is Marquedor marquandii (Marquandomyces marquandii), the strain number is SGSF043, and the strain preservation number is CCTCC No. M2020555.

Further defined, the pests are one or more than two of Rhagophthalmus biflorus (Monolepta hieroglyphic), Vietnamese Chlorophiums (Callosobruchus chinensis) and Zea mays (Rhopalosiphum maidis).

The invention also provides an application of the entomopathogenic fungi in preparing a pesticide for killing or inhibiting pests.

The invention also provides a method for controlling pests, which comprises the step of treating the pests by using the spore suspension of the entomopathogenic fungi with the strain preservation number of CCTCC No. M2020555.

Further defined, the conditions for treating the pests are 24-26 ℃, relative humidity 65-75% and 24h total darkness.

Further limited, the preparation method of the spore suspension of the entomopathogenic fungi with the strain preservation number of CCTCC No. M2020555 comprises the following steps: inoculating the entomopathogenic fungi strain on a PDA culture medium for culturing, then adding a Tween 80 aqueous solution on the culture medium, and scraping surface spores to obtain a spore suspension.

Further defined, the spore suspension has a concentration of 1X 10⁸one/mL.

The invention also provides an insecticide, wherein the effective component of the insecticide is entomopathogenic fungi, the strain number is SGSF043, and the preservation number is CCTCC No. M2020555.

Drawings

FIG. 1 is a colony map of strain SGSF 043;

FIG. 2 is a characteristic diagram of hyphae formed on the surface of the SGSF043 strain after the SGSF043 strain infects pests, wherein A is the characteristic diagram of hyphae formed on the surface of the SGSF043 strain after the Skogabara maculata is infected, B is the characteristic diagram of hyphae formed on the surface of the SGSF043 strain after the corn aphid is infected, and C is the characteristic diagram of hyphae formed on the surface of the SGSF043 strain after the mung bean weevil is infected;

FIG. 3 is a graph showing the results of virulence determination of strain SGSF043, wherein A is the virulence determination of infecting Rhagophthalmus tarsa; b, determining the toxicity of the infecting corn aphids; c is the virulence assay for infecting the callosobruchus chinensis.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The invention selects an entomopathogenic fungus: the Markunmudenreri (Marquandomyces marquandii) has the strain number of SGSF043, the preservation time of 2020, 9 and 29 days, is preserved in China Center for Type Culture Collection (CCTCC), the preservation place is the eight-way Wuhan university preservation center No. 199 in Wuchang district, Wuhan city, Hubei, and the preservation number is CCTCC No. M2020555.

1. Experimental materials: the Dactylophora bipolaris pests, the corn aphids and the vigna radiata pests are all from scientific research and test bases of the Ziziqi Harer institute of agricultural academy of sciences in Heilongjiang province, the Dactylophora bipolaris and the corn aphids are collected on the corn, and the vigna radiata is artificially bred indoors.

2. Test materials: tween 80 was purchased commercially.

3. Preparing a test reagent:

potato glucose medium (PDA): 200g of potato, 20g of glucose and 20g of agar, and the volume is up to 1L.

Malt extract agar Medium (MEA): 30g of malt extract powder, 20g of agar and 1L of water.

Plate count agar medium (PCA): 5g of tryptone, 2.5g of yeast extract powder, 1g of glucose, 20g of agar and 1L of water.

Saki medium (SDAY): 10g of yeast extract powder, 40g of glucose, 10g of peptone, 20g of agar and 1L of water.

Oat medium (OA): 30g of oat, 20g of agar and 1L of water.

Potato dextrose liquid medium (PD): 200g of potato and 20g of glucose, and the volume is up to 1L.

Example 1.

Separation method of entomopathogenic fungi

An entomopathogenic fungus (SGSF043) is isolated from litters of greater Khingan mountains. Drying the under-forest litter sample collected in great khingan, crushing the dried under-forest litter sample into particles, and filtering the particles by using a filter screen to obtain the particles with the litter sample particle size of 100-200 mu m. The granular sample was mixed with sterile water at a mass ratio of 1:100 by a particle plate dilution method to prepare a particle suspension. And then sucking 50 muL, 100 muL, 150 muL, 200 muL and 250 muL by using a liquid transfer gun, respectively smearing the liquid on a PDA culture medium plate, coating the liquid on the PDA culture medium plate by using a coating rod until particles are uniformly dispersed, culturing at room temperature, observing every 12h until fungus colonies grow out, picking the fungus colonies to the PDA plate, and purifying. Observing the form by a microscope, and performing ITS and TEF sequence amplification sequencing to finally separate and identify 1 strain of entomopathogenic fungi, and storing the entomopathogenic fungi in a freezing storage tube.

II, molecular biological identification of strains

Firstly, extracting total DNA from a purified strain by using a CTAB method, and then carrying out PCR amplification on an Internal Transcribed Spacer (ITS) by using fungus universal primers ITS1 and TS4 for primary identification, wherein the ITS primers are ITS1 (the sequence is shown as SEQ ID NO.1, namely 5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (the sequence is shown as SEQ ID NO.2, namely 5'-TCCTCCGCTTATTGATATGC-3'). Since the Translation Elongation Factor 1- α (TEF) contains more information about the intervarietal differences of entomopathogenic fungi than the ITS sequence for the genus entomopathogenic fungi, a fragment of the TEF sequence of the target entomopathogenic fungi was further amplified using primers EF-983F (the sequence is shown in SEQ ID NO.3, i.e., GCYCCYGGHCAYCGTGAYTTYAT) and EF-2218R (the sequence is shown in SEQ ID NO.4, i.e., ATGACACCRACRGCRACRGTYTG). And (4) detecting the PCR product by electrophoresis, and then sending the PCR product to a sequencing company for bidirectional sequencing. The authentic sequence obtained after sequencing was analyzed by comparison using BLAST (Basic Local Alignment Search Tool, https:// BLAST. NCBI. nlm. nih. gov /) from NCBI website. The ITS fragment and TEF fragment of entomopathogenic fungi were amplified and compared by BLAST tool, and the most similar species of strain SGSF043 was Marhondride Marquardii (formerly Metarhizium marquardii), and the results are shown in Table 1, with 100% similarity.

TABLE 1 molecular identification of the strain SGSF043

Thirdly, morphological identification of the strains

Transferring the entomopathogenic fungi strain obtained by separation and purification to PDA, MEA, SDAY, OA and PCA culture medium, and recording the growth rate, colony size, color and colony characteristics of the entomopathogenic fungi after culturing for 14 days. Observing characteristics of fungal spores, pigments and exudates, spore-forming structures and hyphae and the like formed by the fungi on different culture media and different insect surfaces by using an optical microscope, and comparing related documents to perform morphological identification, wherein the results are shown in figures 1 and 2, the front surface of a colony is white cotton-shaped, no folds are formed, and no obvious pigments and exudates are generated; the back of the colony is light yellow, the conidium is oval, and the size of the conidium is (2.11-4.31) mum multiplied by (1.85-3.04) mum; round chlamydospores can be seen in a PD (potato glucose liquid culture medium) liquid culture medium, under a body type microscope, conidiophores are branched in multiple stages, white hyphae grow from the abdomen after the insects are infected, and then white spore-forming structures and conidia are generated and spread all over the body. In combination with morphological characteristics and molecular characterization, strain SGSF043 was identified as Marquadamycin (Marquanomyces marqualdii) (formerly Metarrhizium marqualdii).

Thirdly, biological characteristics of entomopathogenic fungi

1. Determination of growth rate and spore yield of strain at different temperatures

The purified SGSF043 strain was punched with a punch and transferred to PDA medium, and placed in incubators at 20 deg.C, 25 deg.C and 30 deg.C, respectively, three times per treatment, and the colony growth diameter was measured periodically every day for a total of 14 d. At the end of the measurement experiment, 5mL of sterilized 1% Tween 80 aqueous solution was added to the plate, surface spores were scraped off, excess mycelia and agar were filtered off with sterile cotton, and the amount of spores produced was measured using a hemocytometer.

Tables 2 and 3 show the results of measuring the growth diameter and the spore yield of the strains at different temperatures, and it can be seen from table 2 that the optimum growth temperature of the strain SGSF043 is 25 ℃, the diameter of the colony after 14d culture reaches 44mm, the growth diameter is slightly less than 25 ℃ at 20 ℃, but the growth of the strain is slightly slowed down at 30 ℃, and it can be seen from the result of the spore yield of table 3 that the influence of the temperature on the strain is small and the spore yield is maximum at 25 ℃.

TABLE 2 measurement results of growth diameter of strains at different temperatures

Note: in mm, the diameter of the colony has been removed the cake diameter is 6.5 mm.

TABLE 3 determination of sporulation of the strains at different temperatures

Note: the unit is "number", and the data in the table are the total spore yield after 14 days of PDA culture medium culture

2. Determination of growth rate and spore yield of strain under different pH values

The purified SGSF043 strain was punched on colonies with a punch, transferred to

PDA medium pH

4, 5, 6, 7, 8, 9, each treatment was repeated three times, cultured in 25 ℃ incubator for 14d, and the colony growth diameter was measured periodically every day. At the end of the measurement experiment, 5mL of sterilized 1% Tween 80 aqueous solution was added to the plate, surface spores were scraped off, excess mycelia and agar were filtered off with sterile cotton, and the amount of spores produced was measured using a hemocytometer.

Tables 4 and 5 show the results of measuring the growth rate and spore yield of the SGSF043 strain at different pH values, and according to the results in table 4, the SGSF043 strain is greatly influenced by pH, the growth state of the strain under alkaline conditions is obviously better than that under acidic culture conditions, the growth diameter is increased at pH8 and 9, and according to the results in table 5, the spore yield is also significantly increased at pH8 and 9, so that the SGSF043 strain is suitable for growth under alkaline conditions.

TABLE 4 measurement results of growth diameter of strains at different pH

TABLE 5 determination of sporulation of the strains at different pH

Note: the units are "ones", and the data in the table are the total sporulation yield after 14 days of culture per PDA medium.

Example 2 method for determining Pest resistance Activity

Method for preparing spore suspension of entomopathogenic fungi

Firstly, spore suspension is prepared, strains are inoculated on a PDA culture medium for 14 days, 5mL of sterilized 0.05% Tween 80 aqueous solution is added to a plate, and surface spores are scraped. Filtering off excess mycelia and agar with sterilized cotton, counting with a hemocytometer, and adjusting the spore suspension to a final concentration of 1 × 10⁸one/mL.

Second, method for treating pest by spore suspension of entomopathogenic fungi

Soaking Dastarcus tarsal (adult) in water at a temperature of 1 × 10⁸Spore suspension was added at a concentration of 5 sec per mL and then transferred to a flask containing corn kernels for observation, and 35 adults were inoculated per treatment. Soaking Phaseolus vulgaris (imago) in 1 × 10⁸spores/mL (spores spore count) concentration of spore suspension for 5s, then transferred with a brush pen into a flask containing mung beans for observation, and 20 adults were inoculated per treatment. Transferring wingless corn aphids into a culture dish by using a writing brush, and spraying 1 multiplied by 10⁸Uniformly spraying each/mL spore suspension on the surface of the insect body, spraying 1mL spore suspension each time, transferring the treated corn aphids to cabbage leaves, and inoculating 4 corn aphids for each treatment. Each treatment was repeated three times using sterile 0.05% aqueous tween 80 as a solvent control. After inoculation, all flasks were transferred to 25. + -.1 ℃ in an incubator with a relative humidity of 70. + -. 5% for 24h and incubated in a total dark condition, the mortality and corrected mortality were counted daily and the dead insects were transferred to a laboratoryThe entomopathogenic fungi infection condition is observed in a culture dish paved with sterile moist filter paper.

Virulence determination was performed on diabrotica diplodialis and adult zea mays, and the results are shown in a in fig. 3 and table 6, SGSF043 strain has a high mortality rate on diabrotica diplodialis, the mortality rate gradually increases with the inoculation time, the cumulative corrected mortality rate on the 8 th day of inoculation treatment reaches 91.55%, the median lethal time is about 3 days, and the stiff worm rate is above 90% after 8 days of inoculation.

According to the results of the corn aphids on the 3 rd day of the inoculation treatment, as shown in B in figure 3 and table 6, the lethality reaches 100%, and the final stiff insect rate is more than 70%.

According to the results of the bruchid vaccination experiments, as shown in C in FIG. 3 and Table 6, the SGSF043 strain corrected mortality rate was more than 50% at day 8 of bruchid infestation. The results prove that the SGSF043 strain has good insect-resistant activity, can infect various pests, has wide control range, belongs to non-host-specific entomopathogenic fungi, and shows that the strain has good biological control potential of the pests and can be used as a biocontrol strain.

TABLE 6 determination of insect-resistant Activity of SGSF043 Strain

SEQUENCE LISTING

<110> ziqi hall division of agriculture academy of sciences of Heilongjiang province

<120> application of entomopathogenic fungi, method for controlling pests and insecticide

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tcctccgctt attgatatgc 20

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Claims

1. The application of entomopathogenic fungi in pest control is characterized in that the entomopathogenic fungi is Marquedor marquandii (Marquandomyces marquandii), the strain number is SGSF043, and the strain preservation number is CCTCC No. M2020555.

2. The use according to claim 1, wherein the pest is one or more of Rhynchosia bimaculata (Monolepta hieroglyphica), Phaeophora lunata (Callosobruchus chinensis) and Myzus zeae (Rhopalosiphum maidis).

3. The use according to claim 1, wherein said use is the use of said entomopathogenic fungus for the preparation of a pesticide for killing or inhibiting pests.

4. The use according to claim 3, wherein the pests are one or more of Rhynchosia bimaculata (Monolepta hieroglyphica), Phaeophora lunata (Callosobruchus chinensis) and Myzus zeae (Rhopalosiphum maidis).

5. A method for controlling pests, which is characterized by comprising the step of treating the pests by using spore suspension of entomopathogenic fungi with the strain preservation number of CCTCC No. M2020555.

6. The method according to claim 5, wherein the pest is one or more of Rhynchosia bimaculata (Monolepta hieroglyphica), Phaeophora lunata (Callosobruchus chinensis) and Myzus zeae (Rhopalosiphum maidis).

7. The method of claim 5, wherein the conditions for treating pests are 24 ℃ to 26 ℃, relative humidity 65% to 75% and 24h total darkness.

8. The method according to any one of claims 5 to 7, wherein the spore suspension of the entomopathogenic fungus with the strain accession number CCTCC No. M2020555 is prepared by: inoculating the entomopathogenic fungi strain on a PDA culture medium for culturing, then adding a Tween 80 aqueous solution on the culture medium, and scraping surface spores to obtain a spore suspension.

9. The method of claim 8, wherein the spore suspension is at a concentration of 1 x 10⁸one/mL.

10. The pesticide is characterized in that the effective component of the pesticide is entomopathogenic fungi, the strain number is SGSF043, and the preservation number is CCTCC No. M2020555.