CN112940944B

CN112940944B - Entomopathogenic fungi and screening method and application thereof

Info

Publication number: CN112940944B
Application number: CN202010883964.0A
Authority: CN
Inventors: 徐利剑; 高思禹; 张李香; 孟威; 王庆贵
Original assignee: Heilongjiang University
Current assignee: Heilongjiang University
Priority date: 2020-08-28
Filing date: 2020-08-28
Publication date: 2022-09-23
Anticipated expiration: 2040-08-28
Also published as: CN112940944A

Abstract

The invention belongs to the field of biological control, and provides an entomopathogenic fungus, a screening method and application thereof. In order to solve the problems of pesticide residue, poor selectivity, short lasting period and the like existing in the pest control by chemical pesticide, two fungi are obtained by separation, the two fungi are proved to be the new species of entomopathogenic fungi metarhizium anisopliae through identification, and the biological characteristics and the insect-resistant activity of the two fungi are respectively measured. The invention not only provides an alternative strain for biological control of pests, but also solves the problem that the control effect is greatly influenced by the temperature rise of the sporulation yield of the existing entomopathogenic fungus metarhizium anisopliae.

Description

Entomopathogenic fungi and screening method and application thereof

Technical Field

The invention belongs to the field of biological control, and particularly relates to entomopathogenic fungi, and a screening method and application thereof.

Background

At present, chemical control methods are mainly adopted for controlling field pests, and chemical pesticides generate a large amount of waste materials in the production process and may harm the environment and human health. When in use, although the contact chemical pesticide has obvious pesticide effect, the contact chemical pesticide can be ensured to directly contact pests only by using a large amount of the contact chemical pesticide, and related problems such as waste or pesticide residue are easily caused. In addition, some chemical pesticides have the problems of poor selectivity, short duration, easy generation of drug resistance and the like. The use of biopesticides can partially alleviate these problems.

The use of entomopathogenic bacteria (e.g., beauveria bassiana, metarhizium anisopliae) to control pests is considered to be an effective green control means. However, some pests such as the diabrotica biflorosa lack a green control means, and reports on the control of the diabrotica biflorosa by using entomopathogenic bacteria have not been found yet. Metarhizium fungi, which are entomopathogenic fungi, can be classified into two types, namely obligate parasitic fungi such as Metarhizium acridimum (Metarhizium acridum) and non-obligate parasitic fungi. At present, the existing metarhizium anisopliae used as a biological pesticide for preventing and controlling pests has two defects: firstly, the green muscardine fungi which generate green spores such as Metarhizium anisopliae (Metarhizium anisopliae) and Metarhizium robustum (Metarhizium robertsi) are more applied in biological pest control, but different Metarhizium strains are difficult to distinguish in the market, and sometimes the phenomenon of sub-sufficiency occurs; secondly, for agricultural pests, temperature is a main factor influencing whether the pests break out or not, and the spore yield of the metarhizium anisopliae which is widely used at present at high temperature is reduced, so that the control effect of the metarhizium anisopliae is influenced.

Disclosure of Invention

To avoid the problems of chemical means for controlling pests and to provide alternative strains for biological control of pests, the present invention provides two entomopathogenic fungi: metarhizium longissimum (Metarhizium changbiensis) with strain number SGSF125 and Metarhizium dauricum (Metarhizium longingganensis) with strain number SGSF 355. The two strains are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation dates are 12 months and 5 days in 2019, the preservation unit addresses are No.1 Hospital, Xilu, North Chen, of Chaoyang, Beijing, wherein the preservation number of the strain SGSF125 is CGMCC No. 19143; the preservation number of the strain SGSF355 is CGMCC number 19144.

Furthermore, the Sinomenium xingan with the strain number SGSF355 produces 5X 10 spores at 30 ℃ for 14 days in PDA culture medium ⁹ ～9×10 ⁹ And (4) respectively.

The invention also provides a screening method of the entomopathogenic fungi, which comprises the following steps:

the screening method of the metarhizium anisopliae SGSF125 comprises the following steps: taking the litters in Changbai mountain forests as a separation source sample, drying and crushing the separation source sample, mixing the crushed separation source sample with water to prepare a particle suspension, and separating strains by using a plate dilution method;

the screening method of the metarhizium xingan SGSF355 comprises the following steps: taking litters of great Khingan forest as a separation source sample, drying and crushing the separation source sample, mixing the crushed separation source sample with water to prepare a particle suspension, and separating the strains by using a plate dilution method.

The particle size in the above method is 100-200 μm.

The invention also provides the application of the entomopathogenic fungi in biological control.

In one embodiment of the present invention, the above biological control refers to pest control.

In one embodiment of the present invention, the pest is one or more of diabrotica sinensis (Monolepta heliotropica), Pistacia lentinus chinensis (Callosobruchus chinensis) and Aphis zeae maidis (Rhopalosiphum maidis).

The invention also provides the application of the entomopathogenic fungi as an effective component of the pesticide for biological control.

The pest killed or inhibited by the pesticide is one or more than two of Monolepta heliotropica (Monolepta heliotropica), Phaseolus aureus (Callosobruchus chinensis) and corn aphid (Rhopalosiphum maidis).

In one embodiment of the present invention, the molecular biological identification of said strains SGSF125 and SGSF355 is performed by: two fungi were preliminarily identified by extracting DNA and performing PCR amplification. In addition, the similarity of the two fungi was determined by combining the results of amplification of TEF fragments of the two fungi, and was identified as a new species of metarhizium anisopliae.

In one embodiment of the invention, the morphological characterization of said strains SGSF125 and SGSF355 comprises the following: and counting the growth speed, the colony size, the color and the colony characteristics of the two fungi on different culture media, and observing the characteristics of fungal spores, pigments, exudates, spore-forming structures, hyphae and the like formed by the two new species on different culture media and different insect surfaces by using an optical microscope.

In one embodiment of the invention, the biological properties of the SGSF125 and SGSF355 strains are measured, including the growth rate and sporulation of the strains at different temperatures and pH values.

In one embodiment of the invention, the two new species of entomopathogenic fungi are assayed for their insect-resistance activity by preparing spore suspensions of the two new species of entomopathogenic fungi separately and treating diabrotica virescens, chlorophyta elephanta, and corn aphid with the spore suspensions by different methods. After inoculation is completed, cultivation is carried out, the mortality rate and the corrected mortality rate of the pests are counted daily, and the infection of the three insects by the two new species is observed.

Advantageous effects

Experiments prove that firstly, the entomopathogenic fungi (SGSF125 or SGSF355) has the characteristics of large sporulation quantity, easy culture and wide pH tolerance range on various culture media, and has certain favorite and high temperature resistance capability, and the sporulation quantity is relatively less influenced by temperature rise. Secondly, spores of the entomopathogenic fungi are yellow and can be distinguished from common green spore-producing metarhizium anisopliae. Thirdly, the entomopathogenic fungi have good activity of resisting diabolo, callosobruchus chinensis and corn aphids, belong to non-host specialized metarhizium anisopliae, can infect various pests, and have wide control range. In conclusion, the new fungus species have good potential for pest control and are good candidate strains for pest biological control. The application of the new entomopathogenic fungus species in biological control can not only avoid adverse effects caused by chemical control of pests, but also solve the problems that the entomopathogenic fungus metarhizium anisopliae has large influence on control effect due to the fact that the sporulation quantity is greatly influenced by temperature rise, and the like.

Drawings

FIG. 1.A is the colony morphology of strain SGSF125 on PDA medium; b is the colony morphology characteristic of the bacterial strain SGSF355 on the PDA culture medium; c is the hypha characteristics formed on the surface of the bischofite infected by the SGSF125 strain; d is the hypha characteristics formed on the surface of the bischofite infected by the strain SGSF 355;

FIG. 2.A is the results of a Bispongo Blastus virulence test for strains SGSF125 and SGSF 355; b is the toxicity test result of the mung bean weevil of the bacterial strains SGSF125 and SGSF 355; c is the test result of the toxicity of corn aphids of the SGSF125 and SGSF355 strains.

Detailed Description

The present invention will be described in further detail with reference to the following embodiments and the accompanying drawings, and the present invention is not limited to the following embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Preparing a culture medium according to the following culture medium formula for later use:

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.

Example 1 isolation and identification of entomopathogenic fungi

(1) Isolation of Strain SGSF125

And (4) separating strains by taking litters in Changbai mountain forests as a separation source. Firstly, crushing the collected air-dried litter sample into particles, and filtering the particles by using a filter screen to obtain particles with the particle size of 100-. Then, the granular sample was mixed with sterile water at a mass ratio of 1:100 by a plate dilution method to prepare a granular suspension. And (3) sucking 50 mu L, 100 mu L, 150 mu L, 200 mu L and 250 mu L of the mixture by using a pipette, respectively smearing the mixture on a PDA culture medium plate, coating the mixture on a coating rod until particles are uniformly dispersed, culturing at room temperature, observing every 12h until fungus colonies grow out, picking the colonies on the PDA plate, and purifying. Purifying and culturing to obtain a fungus strain, and numbering as SGSF 125.

(2) Isolation of Strain SGSF355

The strain is separated by taking litters in the great Khingan forest as a separation source. Firstly, crushing the collected air-dried litter sample into particles, and filtering the particles by using a filter screen to obtain the particles with the particle size of 100-200 mu m of the litter sample. Then, the granular sample was mixed with sterile water at a mass ratio of 1:100 by a plate dilution method to prepare a granular 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. A strain of fungus was obtained by purification and cultivation and was designated SGSF 355.

(3) Molecular biological identification and morphological identification of entomopathogenic fungi

The purified strain was first subjected to extraction of total DNA by CTAB method, and then Internal Transcribed Spacer (ITS) was subjected to PCR amplification using fungal universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') for preliminary identification. Since the Translation elongation factor 1-alpha (TEF) contains more information on the interspecies difference of Metarrhizium than ITS sequence for Metarrhizium, the TEF sequence fragment of the target Metarrhizium was further amplified using primers EF-983F (GCYCCYGGHCYCGTGAYTATAT) and EF-2218R (ATGACACCCRACCRACGCRACTYTTG). And (4) detecting the PCR product by electrophoresis, and 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.

By amplifying ITS fragments and TEF fragments of fungi and comparing with a BLAST tool, the strain closest to the SGSF125 strain is Metarhizium anisopliae (Metarhizium aciculum), and the similarity is 91.67%; the most similar strain to strain SGSF355 is Metarhizium anisopliae (Metarhizium carneum), the similarity is 92.66%, and the specific molecular identification results are shown in Table 1. These two strains have low ITS and TEF similarity to known species and appear as highly supportive independent branches on the phylogenetic tree and thus belong to new species of fungi that have not been described.

TABLE 1 molecular identification results of strain SGSF125 and strain SGSF355

Transferring the metarhizium anisopliae strain obtained by separation and purification to PDA, MEA, SDAY, OA and PCA culture medium, culturing for 14 days, and recording the growth rate, colony size, color and colony characteristics of the metarhizium anisopliae. In addition, characteristics of fungal spores, pigments, exudates, spore-forming structures, hyphae and the like formed on different culture media and different insect surfaces of the fungi are observed by using an optical microscope, and morphological identification is carried out by comparing related documents.

The results show that the bacterial colony diameter of the bacterial strain SGSF125 after being cultured in the PDA culture medium for 14d is 21mm (including the bacterial cake diameter is 6.5mm) and the front surface of the bacterial colony is golden yellow and has wrinkles as shown in A and C in figure 1; the strain appears as a conidiophore oval to spherical under an optical microscope and has a size of (1.56-2.83) × (1.33-2.17) μm; the conidiophores are transparent, and have three branches, each branch has 2-4 phialides, the phialides are cylindrical, and the tip of part of the phialides is narrowed to have the size of (2.74-19.85) × (1.41-2.47) μm. The morphological characteristics are distinguished from the description of other known species of fungi. As shown in B and D in FIG. 1, after the bacterial strain SGSF355 is cultured in the PDA culture medium for 14D, the diameter of a colony is 27mm (including the diameter of a bacterial cake is 6.5mm), the front surface of the colony is yellowish and has folds, and no obvious exudate is generated; the strain shows that the shape of a conidium oval is from round to round under an optical microscope, and the size is (1.08-3.31) × (1.0-2.6) mu m; conidiophores have more branches with 2-3 grades, each branch has 2-4 phialides, the phialides are in ampoule shape, the tops of the phialides gradually narrow to form obvious bottle necks, and the size of the phialides is (5.23-25.56) × (1.27-2.36) mu m. Morphological features are distinguished from descriptions of other known species of fungi.

According to the polygene sequence similarity analysis, phylogenetic tree analysis and morphological characteristics, the strain SGSF125 and the strain SGSF355 are new fungal species, the most similar genus is Metarrhizium anisopliae, but the genetic sequence, colony morphology, spore cell morphology, conidiophore shape and size, spore shape and size and other aspects of the known Metarrhizium anisopliae are obviously different. These two strains are hereby assumed to be new species of fungi, the taxonomically most recent genus being Metarhizium sp. The strain SGSF125 is named as Metarhizium changbiensis, the strain SGSF355 is named as Metarhizium xingan (Metarhizium changganensis), and the two strains of fungi are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, wherein the preservation number of the strain SGSF125 is CGMCC No. 19143; the preservation number of the strain SGSF355 is CGMCC No. 19144. The specific strain information is shown in Table 2.

TABLE 2 Strain information of Strain SGSF125 and Strain SGSF355

Example 2 biological Properties of novel Metarrhizium anisopliae

1. Test method

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

The bacterial colonies of the bacterial strain SGSF125 and the bacterial strain SGSF355 are respectively punched by a puncher, then are respectively transferred to a PDA culture medium and are respectively cultured in incubators with the temperature of 20 ℃, 25 ℃ and 30 ℃, each treatment is repeated three times, the growth diameter of the bacterial colonies is regularly measured every day, and the total measurement is 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.

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

The colonies of the strain SGSF125 and the strain SGSF355 are respectively punched by a puncher, then are respectively transferred to PDA culture media with pH values of 4, 5, 6, 7, 8 and 9, each treatment is repeated three times, the PDA culture media are placed in an incubator at 25 ℃ for 14d, and the growth diameter of the colonies is measured regularly 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.

2 results

(1) Results of measuring growth rate and spore yield of strains at different temperatures

Table 3 shows the growth diameter measurement results of the strains at different temperatures, and from Table 3, the optimum growth temperature of the strain SGSF125 is 25 ℃, the diameter of the bacterial colony reaches 14.5mm after the bacterial colony is cultured for 14 days, and the growth speed is obviously slowed down at 30 ℃; the optimum growth temperature of the strain SGSF355 is 30 ℃ or above, and the growth speed is reduced at 20 ℃. Table 4 shows the results of measuring the spore yields of the strains at different temperatures, and it can be seen from Table 4 that the spore yields of the 2 strains are correlated with the temperature, and the spore yield is the greatest at the optimum growth temperature. And (3) sporulation yield: the sporulation yield of the strain SGSF125 at 20 ℃ and 25 ℃ is not obviously different, but the sporulation yield at 30 ℃ is obviously reduced. The strain SGSF355 produces the largest spore amount at 30 ℃, the temperature for the optimum growth and the maximum spore amount of the strain SGSF355 can be seen to be 30 ℃ or above, and the growth rate and the spore amount at the normal temperature of 25 ℃ are higher and are obviously better than those of the strain SGSF 125. Therefore, the strain SGSF355 is characterized by preference and resistance to high temperature of 30 ℃ or more.

TABLE 3 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 4 determination of sporulation of the strains at different temperatures

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

(2) Results of measuring growth rate and spore yield of strains under different pH values

Table 5 shows the growth diameter measurements of the strains at different pH values, and it can be seen from Table 5 that 2 strains grow at the fastest growth rate at 5 and 6 and at the significantly slower growth rate at 4. The growth rate of the strain SGSF355 is reduced at the pH of 7, 8 and 9. Table 6 shows the spore yields of the strains at different pH values, and it can be seen from table 6 that there is no significant difference between the spore yields of the strain SGSF125 at pH5, 6 and 7, and the spore yield of the strain SGSF355 at pH7, 8 and 9 gradually decreases, from which it can be seen that 2 strains are suitable for growth under acidic conditions at pH5 and 6; the strain SGSF355 produced the largest amount of spores at pH 5.

TABLE 5 measurement results of growth diameter of strains at different pH

Note: the unit is mm, and the diameter of the colony is 6.5mm after the fungus cake is removed.

TABLE 6 determination of sporulation of the strains at different pH

Example 3 determination of Metarrhizium anisopliae anti-insect Activity

(1) Test method

First of all makePreparing spore suspension, inoculating the strain on PDA culture medium, culturing for 14 days, adding 5mL sterilized 0.05% Tween 80 aqueous solution, and scraping surface spores. Filtering off excess mycelia and agar with sterilized cotton, counting with a hemocytometer, and adjusting the spore suspension to a final concentration of 1 × 10 ⁸ spores/mL。

Soaking diabrotica (adult) in 1 × 10 ⁸ spores/mL concentration of spore suspension for 5s, then transferred to a triangular flask containing corn kernels for observation, and 35 adults were inoculated per treatment. Soaking Phaseolus vulgaris (imago) in 1 × 10 ⁸ spores/mL spore suspension for 5s, then transferred with a brush 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 10 ⁸ spores/mL spore suspension is uniformly sprayed on the surface of the insect body, 1mL spore suspension is sprayed each time, the treated corn aphids are transferred to cabbage leaves, and 40 corn aphids are inoculated in each treatment. Each treatment was repeated three times using sterile 0.05% aqueous tween 80 as a solvent control. After inoculation is completed, all the triangular flasks are transferred to an incubator with 25 +/-1 ℃, relative humidity of 70 +/-5% and 24h of total darkness for culture, the mortality rate and the corrected mortality rate are counted daily, and the dead insects are transferred to a culture dish paved with sterile moist filter paper for observing the infection condition of the metarhizium anisopliae.

(2) Results

As shown in figure 2, the bacterial strain SGSF125 and the bacterial strain SGSF355 have higher mortality rate to the Diabrotica, the mortality rate is gradually increased along with the inoculation time, the median lethal time of the two bacterial strains is about 3d, the corrected mortality rate is about 90% when the two bacterial strains are inoculated for 8d, and the experiment proves that the two green muscardine fungi have better control capability to the Diabrotica. When the green bean weevil is inoculated with the metarhizium anisopliae for 5 days, the lethality rates of the bacterial strain SGSF125 and the bacterial strain SGSF355 are gradually increased, wherein the lethality rate of the bacterial strain SGSF355 is obviously increased, the corrected mortality rate reaches 96.97% when the green bean weevil is inoculated for 9 days, and the experiment proves that the SGSF355 has stronger infection capacity on the green bean weevil. The two metarhizium anisopliae strains have strong lethal effect on corn aphids, the mortality rate reaches 100% on the third day of inoculation, and the lethal time is within 1 d. As can be seen from Table 7, the two novel metarhizium anisopliae strains have the most significant activity effect on Diabrotica, the rate of the muscardine pests reaches more than 95%, and the activity effect on the Vibrio intervarioides and the corn aphids is the second, the rate of the muscardine pests is about 80%. The results prove that the two strains (SGSF125 and SGSF355) have good insect-resistant activity, can infect various pests, have wide control range, belong to non-host specialized metarhizium anisopliae, show that the new species of the two strains have good biological control potential of the pests, and can be used as a biocontrol strain.

TABLE 7 determination results of insect-resistant Activity of SGSF125 Strain and SGSF355 Strain

SEQUENCE LISTING

<110> university of Heilongjiang

<120> entomopathogenic fungi, and screening method and application thereof

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Claims

1. An entomopathogenic fungus, wherein said entomopathogenic fungus is:

metarhizium anisopliae with strain number SGSF125 ((S-SGSF-125))Metarhizium sp.), the preservation number is CGMCC No. 19143; or Metarhizium anisopliae with strain number SGSF355 (II)Metarhizium sp.), the preservation number is CGMCC No. 19144.

2. Use of the entomopathogenic fungus according to claim 1 for controlling pests, characterized in that said pests are coleoptera-family diabrotica bifida(s) (Monolepta hieroglyphica) Coleoptera family Viridae, Viburnum viridis (L.) Var. (Callosobruchus chinensis) And corn aphid of hemiptera Aphididae (Rhopalosiphum maidis) One or more than two of them.

3. The use according to claim 2, wherein said use is of said entomopathogenic fungi as an active ingredient of insecticides for controlling pests.