CN112646735B - Metarhizium anisopliae, microbial insecticide, preparation method and application - Google Patents

Abstract

The invention discloses a metarhizium anisopliae and microbial insecticide, a preparation method and application thereof. The strain comprises Metarhizium anisopliae, the preservation name is Metarhizium lepidiotae MN114915, the preservation number is GDMCC61330, the preservation time is 2020, 12 and 1 days, the preservation address is Guangdong province microbial strain preservation center, the preservation address is Guangzhou Zhonglu No. 100 college No. 59 building No.5 building, and the classification name is Metarhizium lepidiotae. The inventor separates and obtains a new metarhizium anisopliae strain from soil, the strain has the function of high-toxicity disinsection, the culture method is simple, the growth is rapid, and the spore yield is large. The method has the advantages of no damage to ecology, no easy generation of drug resistance, high biological safety and wide application in locust control.

Description

Metarhizium anisopliae, microbial insecticide, preparation method and application

Technical Field

The invention relates to the technical field of biological control, and particularly relates to a metarhizium anisopliae microbial insecticide, a preparation method and application thereof.

Background

Metarhizium anisopliae (Metarhizium) is an important entomogenous fungus, and more than 200 pests can be infected and killed by the fungus all over the world. The spores of the metarhizium anisopliae can be attached to the epidermis of the pests, penetrate through the epidermis to invade the bodies of the pests, and are propagated and secreted with the nutrition of the pests until the pests are ill and die. The dead corpses of pests can release new spores to infect other pests through transmission. In addition, metarhizium anisopliae is also a common saprophytic bacterium in soil, can be separated from rhizosphere soil, and has a certain relation with plant growth.

Currently, metarhizium has about 30 species worldwide (Kepler et al.2014), different metarhizium species can infect lethal pests in different ranges, and toxicity has certain difference. Metarhizium lepidiotae (Driver & Milner) belongs to one of the species of Metarhizium, is originally isolated from the corpse of Lepidium insect Lepidium consobrina collected in the field and is initially regarded as a variant of Metarhizium anisopliae (Driver et al 2000), and is then found to be a unique species through polygene comparison and identification (Bischoff et al 2009).

At present, the problems of high toxicity of chemical pesticides, pesticide residues in crops, drug resistance of pests and the like exist.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a metarhizium anisopliae, a microbial insecticide, a preparation method and application to solve the technical problems.

Aiming at the technical problems existing at present, the inventor separates and obtains two new strains of metarhizium anisopliae from soil, the strains have the function of high-toxicity disinsection, the culture method is simple, the growth is rapid, and the spore yield is large. Especially, the locust killing rate is high, the locust killing agent is non-toxic and pollution-free, does not destroy ecology, is not easy to generate drug resistance, has high biological safety, and can be widely used for controlling the locust.

The invention is realized by the following steps:

a metarhizium anisopliae comprising metarhizium anisopliae;

the internal number of the metarhizium anisopliae is MN114915, and the preservation number is GDMCC NO: 61330, the preservation time is 12 months and 1 day 2020, the preservation address is Guangdong province microorganism culture preservation center, the address is: building No. 59, building No.5 of the first-furious Zhonglu 100 yard in Guangzhou city; the institute of microbiology, Guangdong province, under the taxonomic name Metarhizium lepidiotae.

In a preferred embodiment of the present invention, the colony culture of metarhizium anisopliae is characterized by: the initial stage on a fungus culture medium is white and has flocculent or villiform protrusions, the diameter of a bacterial colony is 2.63-2.93cm after 5 days of culture, the diameter of the bacterial colony is 4.33-5.77 cm after 10 days of culture, and the diameter of the bacterial colony is 6.65-7.95 cm after 15 days of culture; when the spores are produced, green conidia grow from inside to outside, and the color is changed from light green to dark green; the hyphae have branch separation and diameter of 1.9-3.1 μm; conidium single cells are columnar, and the size is 5.7-9.3 mu m multiplied by 2.5-3.1 mu m; the optimal growth temperature of the metarhizium anisopliae is 25-30 ℃.

Preferably, the fungal culture is selected from the group consisting of SDAY medium, liquid Sabourne medium (SDB), modified Martin broth (martin broth modified), potato dextrose broth (potato dextrose broth PDB), malt extract (BD), corn meal agar (corn meal medium), high salt medium (salt dextrose agar medium), rose Bengal medium (rice bran medium), or Potato Dextrose Agar (PDA).

In one embodiment, the fungal culture medium may be Sabourne's agar medium (SDA), and the culture medium may be prepared autonomously or selected from commercially available products.

In one embodiment, the fungus culture medium is not limited to the above range, and any culture medium that can achieve culture of Metarhizium anisopliae can be used for enrichment culture of Metarhizium anisopliae.

The application of the metarhizium anisopliae in preparing microbial insecticides.

The metarhizium anisopliae can be used for preparing microbial pesticides, thereby playing the role in controlling agricultural pests and/or sanitary pests.

A microbial pesticide prepared from the above Metarrhizium anisopliae is provided.

In a preferred embodiment of the present invention, the microbial pesticide comprises the following effective components: contains Metarhizium anisopliae 1 orThe strain concentration of green muscardine fungus 2 or green muscardine fungus 1 is 10 ⁵ ～10 ¹⁰ cfu/ml。

In a preferred embodiment of the present invention, the microbial pesticide comprises the following effective components: contains powder of Metarhizium anisopliae 1 or Metarhizium anisopliae 2, and has a strain concentration of 10 ⁵ ～10 ¹⁰ cfu/ml。

In a preferred embodiment of the present invention, the microbial pesticide further comprises at least one of the following carriers: preservatives, emulsifiers or UV protectors. In other embodiments, the microbial pesticide may be formulated in any formulation that is agriculturally acceptable, and is not limited to the aqueous solution or powder.

The preservative is used for inhibiting the activity of microorganisms and may be selected from acidic preservatives, lipid-type preservatives, inorganic salt preservatives, biological preservatives, and the like.

The acidic preservative may be benzoic acid, sorbic acid, propionic acid and salts thereof. The lipid type antiseptic can be nipagin lipid, gallate etc. The inorganic salt preservative may be a sulfur-containing sulfite, pyrohydrochloric acid, or the like. The biological antiseptic may be lysozyme, etc.

The emulsifier may be one selected from albumin, soybean lecithin, sorbitan fatty acid and monoglyceride stearate.

The effective component as the microbial pesticide may be a main effective component or a minor effective component, that is, the metarhizium anisopliae may be used in combination with other microbes having an insecticidal effect to exert a synergistic insecticidal effect.

Use of a microbial pesticide for controlling agricultural and/or sanitary pests, the agricultural pest being selected from at least one of lepidopteran, hemipteran, orthopteran and coleopteran pests; the sanitary pests include at least one of diptera and arachnid pests;

preferably, the lepidopteran pest is selected from at least one of spodoptera frugiperda, spodoptera exigua, chilo suppressalis, tryporyza incertulas, plutella xylostella, and ectropis obliqua; the hemiptera pests are selected from at least one of plant hoppers, whiteflies, aphids, leafhoppers and diaphorina citri; the dipteran is selected from at least one of drosophila melanogaster, housefly, citrus fruit fly and mosquito; the orthoptera pest is at least one selected from locust, grasshopper and mole cricket; the Arachnida pest is at least one selected from Tetranychus urticae Koch and Panonychus citri;

preferably, the locust is selected from locusta migratoria manilensis.

In other embodiments, the sanitary pest may also be selected from the group consisting of gadflies, midges, gnats, sand flies, cockroaches (cockroaches), fleas, lice, bed bugs and ticks of the subclass arachnidae, spider mites, chiggers, mange mites, vermiform mites, mealworms, pyemotes, and the like.

It should be noted that the microbial pesticide can control one, two or three or more pests at the same time, and the control is not limited to directly killing the pests, but also includes reducing the activity of the pests, thereby indirectly reducing the agricultural yield reduction.

A preparation method of a microbial pesticide comprises the following steps:

carrying out strain fermentation of metarhizium anisopliae in a culture medium by using a liquid submerged fermentation method to obtain fermentation liquor; controlling the temperature at 25-30 deg.C, pH at 6, and fermenting for 5 days; then the fermentation liquor is prepared into water agent or powder.

The above-mentioned liquid submerged fermentation method is a technique well known to those skilled in the art.

Application of Metarhizium anisopliae in promoting plant growth; preferably, the plant is maize; preferably, the corn variety is zhengdan 958. In other embodiments, the plant species described above includes, but is not limited to, rice, sorghum, barley, cotton, canola, legumes.

The application refers to: plant seeds were treated in a spore suspension of metarhizium squamosum.

The invention has the following beneficial effects:

the invention separates two strains of green muscardine fungus with high toxicity and pesticidal function, and the culture method is simple, fast in growth and great in spore yield. Especially, the locust killing rate is high, and the locust killing agent is non-toxic and pollution-free, does not destroy ecology, is not easy to generate drug resistance, has high biological safety, and can be widely used for preventing and controlling agricultural pests. In addition, the metarhizium anisopliae provided by the invention has the potential of being developed into a microbial insecticide. The metarhizium anisopliae also has a certain effect of promoting plant growth.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a micrograph of white hyphae picked and filmed after 4-5 days of inoculation;

FIG. 2 is a micrograph of conidia selected after 10-12 days of inoculation and subjected to flaking;

FIG. 3 is a front view of the colony morphology of MN113832 strain 15 days after inoculation;

FIG. 4 is a back image of colony morphology of MN113832 strain 15 days after inoculation;

FIG. 5 is a front view of the colony morphology of MN114915 strain 15 days after inoculation;

FIG. 6 is a back view of the colony morphology of the MN114915 strain 15 days after inoculation;

FIG. 7 is stiff insects on culture medium;

FIG. 8 is a phylogenetic tree that is built based on sequencing results;

FIG. 9 is a graph showing the effect of temperature on strain growth.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The Metarhizium anisopliae in this example is stored in the Guangdong province culture collection center and is named as Metarhizium lepididae (Metarhizium lepididae) MN114915, and the storage number is GDMCC NO: 61330, the preservation time is 12 months and 1 day 2020, the preservation address is Guangdong province microorganism culture preservation center, the address is: building No. 59, building No.5 of the first-furious Zhonglu 100 yard in Guangzhou city; the institute of microbiology, Guangdong province, is classified under the name Metarhizium lepidiotae.

The separation and purification method of the strain comprises the following steps in sequence:

weighing soil: collecting agricultural soil in Guangxi, airing, sieving a soil sample to remove impurities, weighing 10g of dry soil, and filling into a filtering device.

Soil washing: the soil was washed with tap water for 10min, allowing soil particles to flow down through a 1mm brass mesh and two polypropylene filters. The bottom most soil particles were collected, placed into a sterile 50mL centrifuge tube, 50mL sterile water was added, and the mixer was vortexed to suspend them. Then, the mixture was centrifuged at 10000rpm for 6min, the supernatant was decanted, washed with sterile water, centrifuged again, and the centrifugal washing was repeated 3 times.

Then diluting the precipitate: sterile sodium carboxymethylcellulose solution was added at a ratio of 20:1(v/v) water/granulate.

Coating: diluting the precipitation suspension by 10 times with sterile water, sucking 100 mu L of the dilution by a pipette to a PDA (containing chloramphenicol and tetracycline hydrochloride) plate, uniformly coating the PDA plate, and then culturing the PDA plate in the dark at 28 ℃ for 2-7 days. In other embodiments, other antibiotic plates may be coated as desired.

And (3) fungus purification: when a single white flocculent colony grows on the culture medium, picking a small amount of hyphae with a sterile toothpick to an SDAY plate for continuous culture to obtain a purified strain.

Example 2

The strain isolated in example 1 was subjected to morphological identification and molecular biological identification, respectively.

Morphological identification:

the isolated metarhizium anisopliae is inoculated on an SDAY medium plate and cultured for 15 days at 28 ℃ to observe the morphological characteristics, sporulation structure and spore morphology size of the colony. Picking white hyphae after 4-5 days to prepare slices, and observing the hyphae and conidiophore forms under a microscope; selecting conidia for 10-12 days to prepare a slice, and observing the size and the shape of the conidia. The colony morphology characteristics were recorded at 15 days of observation.

FIG. 1 is a micrograph of white hyphae taken after 4-5 days of inoculation and pelleted, at 400-fold magnification. FIG. 2 is a micrograph of conidia picked and produced into a slide 10-12 days after inoculation, and the magnification is 400 times. FIG. 3 is a front view of the colony morphology of the MN113832 strain 15 days after inoculation, and FIG. 4 is a back view of the colony morphology of the MN113832 strain 15 days after inoculation. FIG. 5 is a front view of the colony morphology of the MN114915 strain 15 days after inoculation, and FIG. 6 is a back view of the colony morphology of the MN114915 strain 15 days after inoculation.

The strain is white in the initial stage on the SDAY culture medium, has flocculent or villous protrusions, produces spores from a central position, and makes conidia bright green to dark green. The hypha has a septum with a diameter of 1.9-3.1 μm; conidiophore single cell is columnar, and has size of (5.7-9.3) μm x (2.5-3.1) μm.

Molecular biological identification:

the green muscardine fungus growing for 15 days is taken to extract genome DNA which is used as a template for gene amplification, and the following primers are used for polygene amplification and sequencing.

ITS: ITS4 (5'-TCCTCCGCTTATTGATATGC-3') and ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3')

BenA Bt1F (5'-GGTCCCTTCGGTCAGCTCTTCC-3') and Bt1R (5'-CAGCCATCATGTTCTTAGGGTC-3')

EF 983F (5 '-GCYCCYGGHCAYCGTGAYTTYAT-3') and 2218R (5 '-ATGACACCRACRGCRACRGTYTG-3')

RPB1 Mz1F1(5 '-CGRACMYTRCCYCATTTCACAA-3') and Mz1R1(5 '-TTGAGCGGAAGYTGCATCATCTCC-3')

RPB2:5F (5 '-GAYGAYMGWGATCAYTTYGG-3') and 7CR (5 '-CCCATRGCTTGYTTRCCCAT-3')

The above primers were synthesized by Biotechnology engineering (Shanghai) GmbH.

Extracting template DNA: genomic DNA Extraction was performed according to the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, Bioer Technology Co., Ltd.).

Gene amplification reaction system: preparing a reaction system in a PCR tube:

2 XTaq PCR Master Mix 12. mu.L, DMSO 1. mu.L, upstream and downstream primers (10. mu. mol/L) each 0.6. mu.L, ddH ₂ O10.8. mu.L, template DNA 2. mu.L.

The PCR amplification conditions were as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 40s, annealing at 55 ℃ for 40s, extension at 72 ℃ for 1min, and 35 cycles from denaturation to extension; extending at 72 ℃ for 10min, and keeping the temperature at 10 ℃ for 5 min. Samples after PCR were sent for mental only sequencing.

Wherein, sequences of ITS, BenA, EF, RPB1 and RPB2 of the strain MN113832 are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and sequences of ITS, BenA, EF, RPB1 and RPB2 of the strain MN114915 are respectively shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10.

Phylogenetic trees based on the sequencing sequences of the polygenes (ITS, BenA, EF, RPB1, RPB2) are shown in fig. 8, and it is known from the evolutionary tree that the isolated strains MN113832, MN114915 belong to Metarhizium lepidiotae (Metarhizium lepidiotae).

Experimental example 1

In the experimental example, biological measurements are respectively carried out on the strains MN113832 and MN114915 separated in the example 2, the influence of temperature on the strains MN113832 and MN114915 is researched, and meanwhile, the colony growth rate, the spore yield and the spore germination rate are measured.

(1) Effect of temperature on growth of the strain.

The cultured metarhizium squamosum spores are washed down by 0.1% Tween80, 10 mul of the spores are absorbed and inoculated to a new SDAY culture medium, the spores are cultured at constant temperature at five different temperatures of 20 ℃, 25 ℃, 28 ℃, 30 ℃ and 37 ℃ respectively, the diameters of bacterial colonies are measured every day, and each temperature treatment group is set to be 3 times.

(2) And (4) determining the growth rate of the colonies.

The cultured metarhizium squamosum spores are washed down by 0.1% Tween80, 10. mu.l of the spores are sucked and inoculated to a new SDAY culture medium for 3 times, the diameter of the spores is measured at regular time every day, and data are recorded until the colonies grow over the culture medium.

(3) And (4) measuring the sporulation quantity.

After 100. mu.l of spore suspension of Metarhizium anisopliae was applied to SDAY and cultured at 28 ℃ for 12 days, 8 mm-diameter pellet was cut out from the plate with a punch, conidia on the pellet were washed with 10ml of 0.1% Tween80 by shaking on an ultrasonic disruptor, and the spore concentration was measured by counting with a hemocytometer and converted to the amount of spores produced per cm of 2 plate. Spore content was determined by punching 3 wells per plate and averaged to give the number of spores for 1 replicate. Each strain was assayed in duplicate 3 times.

(4) And (4) measuring the germination rate of the spores.

The method comprises the steps of taking a 4 mm-diameter fungus sheet by using a puncher on a scaly green muscardine fungus flat plate cultured for 10-15 days, washing conidia on the fungus sheet into 2ml of 0.1% Tween80 on an ultrasonic crusher in a vibration mode, adding 20 mu l of spore suspension to a glass slide with a groove, putting the glass slide into a flat dish containing wet filter paper, culturing for 24 hours in an incubator under the same condition, randomly observing 3 visual fields of the glass slide by using a 400-time inverted biomicroscope, and counting the spore germination rate by taking the length of a germ tube of spores larger than or equal to the minor axis diameter of the spores as a standard.

As shown in FIG. 9, it can be seen from FIG. 9 that the growth rates of MN113832 and MN114915 are fastest at 25-30 ℃, and the growth of the strain is effectively inhibited at both low temperature (20 ℃) and high temperature (37 ℃). From the experimental results, the optimal growth temperature of the metarhizium anisopliae is 25-30 ℃.

The result of measuring the growth rate of the bacterial colonies shows that the bacterial strains MN113832 and MN114915 grow rapidly on the SDAY plate from the second day, and reach the fastest growth on the fourth day, and after the 5 th day, the growth rate of the bacterial colonies which start sporulation also decreases correspondingly, but still grows 0.40-0.61 cm per day, and the surface bacterial strains grow rapidly.

The determination results of sporulation yield and spore germination rate show that: the spore yield can reach 0.8-1.1 multiplied by 10 after the culture on the SDAY culture medium to day 15 ⁸ Spore/cm ² . The spore yield is higher at this time. The germination rate of the spores can reach over 90 percent in 24 hours. The results show that the strain has simple nutritional requirements, fast growth and propagation, fast spore production and good biological characteristics.

Experimental example 2

In this experimental example, the pathogenicity of the strain MN113832 and the strain MN114915 isolated in example 2 to locusta migratoria scoparia was measured.

The experimental method is as follows:

taking out the strain MN113832 and the strain MN114915 from minus 80 ℃, activating on an SDAY plate, and culturing for 7-15 days at 28 ℃ until sporulation. Washing Metarrhizium anisopliae spore with 0.1% Tween80, counting with blood counting plate to obtain spore suspension with specific concentration, diluting or concentrating to obtain 1 × 10 spore suspension ⁵ cfu/ml、1×10 ⁶ cfu/ml、1×10 ⁷ cfu/ml、1×10 ⁸ cfu/ml、1×10 ⁹ cfu/ml suspension.

Selecting locusta nymphs with consistent size and activity at 3 ages. And (3) taking 10 mu l of spore suspension by using a liquid transfer gun, dropping the spore suspension on the anterior dorsal shield of the locust, continuously feeding the locust and feeding fresh wheat seedlings every day. Each treatment was repeated 3 times, with 15 locust nymphs each. Sterile 0.1% Tween80 water was used as a blank. Culturing at 28-30 deg.C with RH 55% +/-5% and 16L 8D, continuously observing for 10 days, and recording death condition. And (4) judging the death of the locust, and if the locust is lightly touched by tweezers, the locust is dead when the locust is not moved.

And (3) inoculating dead locusts: this step verifies whether the locust is infected and killed by metarhizium anisopliae. The dead locusts are subjected to surface disinfection, and the specific method is that the dead locusts are washed twice with 75% alcohol for 15s, 0.05% sodium hypochlorite for 1min and sterile water for 1min, then the dead locusts are aired on sterilizing filter paper, finally the locusts are placed on an SDAY culture medium (containing chloramphenicol and tetracycline hydrochloride), and whether the metarhizium anisopliae grows on the surfaces of the locusts or not is observed. The stiff insects on the medium are shown in FIG. 7.

The calculation formula of the data is as follows:

the results of the experiments are shown in tables 1 to 8, where table 1 is the corrected mortality (%) of MN113832, table 2 is the corrected mortality (%) of MN114915, tables 3 and 4 are the LC50 regression equation and LC50 of MN113832 and MN114915, respectively, tables 5 and 6 are the LT50 regression equation and LT50 of MN113832 and MN114915, respectively, and tables 7 and 8 are the statistical tables (28 ℃) of the growth rates of the colony diameters of MN113832 and MN114915, respectively.

The experimental result shows that the metarhizium anisopliae MN113832 and MN114915 have obvious pathogenic effects on the locust. The lethal middle concentration (LC50) on day 10 under the treatment of the metarhizium anisopliae spore suspension with different concentrations is 0.783X 10 ⁶ ～1.94×10 ⁶ cfu/ml; at 1X 10 ⁸ Under the cfu/ml concentration, the cumulative corrected lethality rate of the metarhizium anisopliae to the locust on the 10 th day is 100%, the cumulative corrected lethality rate (LT50) during the middle death is 1.41-2.55 days, the lethality rate of the infected locust is 95.24% -100.00%, and the rate of the metarhizium anisopliae of the dead locust is 100%. The experimental results show that the metarhizium anisopliae strain has the potential of being developed into a microbial pesticide for preventing and controlling locusts.

Table 1 corrected mortality (%) for MN 113832.

Table 2 corrected mortality (%) for MN 114915.

Table 3 LC50 regression equation for MN113832 and LC 50.

Table 4 LC50 regression equation and LC50 for MN 114915.

Table 5 MN113832 for LT50 regression equation and LT 50.

Table 6 LT50 regression equation for MN114915 and LT 50.

Table 7 growth rate statistics (28 ℃) for colony diameters of MN 113832.

TABLE 8 growth Rate statistics (28 ℃) for colony diameters of MN 114915.

Experimental example 3

In this example, the growth promoting effect of Metarhizium anisopliae on plants was measured.

Selecting healthy and consistent corn seeds (Zhengdan 958), surface sterilizing, and placing the seeds at 1 × 10 ⁷ Soaking the cfu/mL of the zoophobous squamosa spore suspension for half an hour, then sowing, sowing 5 seeds in each pot, sowing 3 pots in total, and irrigating 1mL of each seed after sowingAnd (4) treating the liquid. Soaking in sterile water for 0.5h as control group, placing in a greenhouse at 25 deg.C, culturing with 12L:12D photoperiod, and periodically supplementing water to the nursery pot and the chassis during experiment to ensure sufficient water content. After potted plant cultivation for 20 days, the harvested plants were cleaned and then placed in a 65 ℃ oven to be dried to constant weight, and then the dry weight of the plants was measured. (experimental-control)/control 100%.

Table 9 dry weight growth index.

The results of table 9 above show that: compared with a control group (CK), the dry weight of the maize plant in the metarhizium anisopliae treatment group is respectively increased by 25.00% and 62.50%, which shows that the strain has obvious growth promoting effect on plant growth.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> Muen (Guangzhou) Biotechnology Ltd

<120> Metarhizium anisopliae, microbial insecticide, preparation method and application

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caggtcatcg ttctcaacca ccccggccag gtcggtgctg gttacgctcc cgtcctcgat 600

tgccacaccg cccacattgc ctgcaagttc tctgagatca aagagaagat tgaccgacgt 660

accggtaagg ctgttgagtc tgcccccaag ttcatcaagt ctggtgactc tgccatcgtc 720

aagatggttc cctccaagcc tatgtgcgtt gaggcttcac cgactacc 768

<210> 4

<211> 511

<212> DNA

<213> Artificial sequence

<400> 4

catttcacaa aggatgacta ctctcccgaa gcacgtggct ttgtcgagaa ctcttacctc 60

cgtggtctca caccctctga attcttcttc cacgccatgg ctggtagaga aggtctcatt 120

gatactgctg tcaagactgc agagacaggt tatattcagc gacgacttgt gaaggctctt 180

gaagacttga gcgctcggta cgatggcact gtacgaaact cgttgggtga catcgtccag 240

tttctctatg gagaagatgg tctcgatgcc atgtgcattg agaagcagaa gctcggtatc 300

ctcaaaatgt cagatcctgc gttcgagaaa aagtatcgtc tggacttggc caacccacct 360

gaatggttca agaaggatta cgagtatggc aacgagttgg ctggtgacaa ggaatcgatg 420

gatctgctcg attctgaatg ggaaacgctg ctgtctgacc gacagactgt gcggctcatc 480

aataagtcta agatgggaga ggagatgatg c 511

<210> 5

<211> 1054

<212> DNA

<213> Artificial sequence

<400> 5

aatgaatacg gaattgtcca attatttgcg aagatgcgtt gaaggtaacc ggcatttcaa 60

cttggcagtt ggtatcaagc ccggaacact ttccaacggt ttgaaatatt cccttgccac 120

tggcaactgg ggagaccaga agaaggccat gagttcgact gccggcgtgt cccaagtgtt 180

gaataggtat acttttgctt cgacactctc tcacttgcga cgaaccaata caccgattgg 240

tagagatggt aagctcgcta aaccgcgtca gctgcacaac acgcactggg gcttggtctg 300

tcctgccgag acgccagaag gtcaggcttg cggtctggtc aagaacctgt cattgatgtg 360

ttatgtcagt gtgggttcac cggccgagcc attgattgaa ttcatgatca accgaggcat 420

ggaagtggta gaagagtacg agccgctgag atatccccat gccaccaaga tctttgtcaa 480

tggtgtttgg gttggtgtac accaagatcc taagcacctg gtcagtcaag tcttggatac 540

tagacgaaag tcgtatctgc agtacgaggt gtctctcgtc cgagaaatca gggatcaaga 600

gttcaagatt ttctccgacg ctggccgggt tatgagacca gtgtttactg ttcagcaaga 660

agatgatccc gagactggca ttgaaaaagg ccatctcgtt ttgaccaaag agttggttaa 720

caagcttgct aaagaacaag ctgaaccacc tgaagaccca agcgagaaaa ttggctggga 780

aggattgatt cgtgccggcg ccgtcgagta cctcgatgcc gaagaagaag agacatcaat 840

gatctgcatg acgccagaag atcttgagct gtatcgtctg cagaaagccg gtgttgctct 900

tgatgacgac attggagatg acctgaataa gcgtctcaag accaagacca accccacaac 960

gcacatgtat acgcattgtg aaattcaccc cagtatgatt cttggtattt gcgctagtat 1020

tattccgttt cccgatcaca atcaggtaag cagc 1054

<210> 6

<211> 545

<212> DNA

<213> Artificial sequence

<400> 6

ggaggacatt accgagtttt caactcccaa acccactgtg aacatatacc tttgttttcg 60

ttgcctcggc ggttcacgcc gccgggtgac acctaaaccc tgattttaat tacagaagtc 120

tttctgagta aaacattcta aatgaatcaa aactttcaac aacggatctc ttggttctgg 180

catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat tcagtgaatc 240

atcgaatctt tgaacgcaca ttgcgcccgc cagtattctg gcgggcatgc ctgttcgagc 300

gtcatttcaa ccctcaagac cccttcgggg gacttggtgt tggggaccgg cataggggcc 360

ttgcgcccct cgccgccccc gaaatgaatt ggcggcctcg tcgcggcctc ccctgcgtag 420

tagcacaacc tcgcaacggg aacgtgacgg cggccactgc cgtaaaacaa cccaatttta 480

ttagagttga cctcgaatca ggtaggaata cccgctgaac ttaagcatat caataggccg 540

gagga 545

<210> 7

<211> 649

<212> DNA

<213> Artificial sequence

<400> 7

cgtcagctct tccgtcccga caacttcgtc tttggtcagt ctggtgctgg caacaactgg 60

gccaagggtc actacactga gggtgctgag cttgtcgaca atgtccttga tgttgtccgt 120

cgcgaggcgg aaggttgtga ctgcctccag ggcttccaga tcacccactc tctcggtggt 180

ggtaccggtg ccggtatggg tactctgttg atctccaaga ttcgtgaaga gtttcccgac 240

cgaatgatgg ccactttctc cgtcgttccc tctcccaagg tttccgacac cgttgtcgag 300

ccctacaacg ccactctctc cgtccatcag ctcgttgaga actctgacga gactttctgc 360

atcgacaacg aggctctgta cgacatctgc atgcgcactc tcaagctgtc taacccttcg 420

tacggtgacc tgaactatct cgtctctgcc gtcatgtctg gcgtcaccac atgcttgcgt 480

ttccccggtc agttgaactc tgatctgcgt aagctggctg tcaacatggt ccccttccct 540

cgtctgcact ttttcatggt cggcttcgcc cccctgacca gccgtggtgc tcactctttc 600

cgcgctgtca gcgtacctga gctcacccag cagatgttcg accctaaga 649

<210> 8

<211> 812

<212> DNA

<213> Artificial sequence

<400> 8

caagcagctc attgtcgcca tcaacaagat ggacaccacc aagtggtccg aggcccgtta 60

ccaggaaatc atcaaggaga cttccaactt catcaagaag gtcggctaca accccaagac 120

cgtcgccttc gtccccatct ccggtttcca cggtgacaac atgcttcagg cctctaccaa 180

ctgcccctgg tacaagggtt gggagaagga gaccaaggct ggcaagtcca ccggcaagac 240

cctccttgag gccattgacg ccattgagcc ccccaagcgt cccaccgaca agcccctccg 300

tcttcccctc caggatgtgt acaagatcgg cggtattgga actgtccctg tcggccgtat 360

cgagactggt gtcctcaagc ccggtatggt cgttaccttc gctccctcca acgtcaccac 420

tgaagtcaag tccgtggaaa tgcaccacga gcagcttact gagggtgtcc ccggcgacaa 480

cgttggtttc aacgtgaaga acgtttccgt caaggaaatc cgccgtggta acgttgctgg 540

tgactccaag aacgaccccc cctctggtgc cgcttccttc gatgcccagg tcatcgttct 600

caaccacccc ggccaggtcg gtgctggtta cgctcccgtc ctcgattgcc acaccgccca 660

cattgcctgc aagttctctg agatcaagga gaagattgac cgacgtaccg gtaaggctgt 720

tgagtctgcc cccaagttca tcaagtctgg tgactctgcc atcgtcaaga tggttccctc 780

caagcccatg tgcgttgagg cttcaccgac ta 812

<210> 9

<211> 511

<212> DNA

<213> Artificial sequence

<400> 9

atttcacaaa ggatgactac tctcccgaag cacgtggctt tgtcgagaac tcttacctcc 60

gtggtctcac accctctgaa ttcttcttcc acgccatggc tggtagagaa ggtctcattg 120

atactgctgt caagactgca gagacaggtt atattcagcg acgacttgtg aaggctcttg 180

aagacttgag cgctcggtac gatggcactg tacgaaactc gttgggtgac atcgtccagt 240

ttctctatgg agaagatggt ctcgatgcca tgtgcattga gaagcagaag ctcggtatcc 300

tcaaaatgtc agatcctgcg ttcgagaaaa agtatcgtct ggacttggcc aacccacctg 360

aatggttcaa gaaggattac gagtatggca acgagttggc tggtgacaag gaatcgatgg 420

atctgctcga ttctgaatgg gaaacgctgc tgtctgaccg acagactgtg cggctcatca 480

ataagtctaa gatgggagag gagatgatgc a 511

<210> 10

<211> 1059

<212> DNA

<213> Artificial sequence

<400> 10

cgcagaatga atacggaatt gtccaattat ttgcgaagat gcgttgaagg taacaggcat 60

ttcaacttgg cagttggtat caagcccgga acactttcca acggtttgaa atattccctt 120

gccactggca actggggaga ccagaagaag gccatgagtt cgactgccgg cgtgtcccaa 180

gtgttgaata ggtatacttt tgcttcgaca ctctctcact tgcgacgaac caatacaccg 240

attggtagag atggtaagct cgctaaaccg cgtcagctgc acaacacgca ctggggcttg 300

gtctgtcctg ccgagacgcc agaaggtcag gcttgcggtc tggtcaagaa cctgtcattg 360

atgtgttatg tcagtgtggg ttcaccggcc gagccattga ttgaattcat gatcaaccga 420

ggcatggaag tggtagaaga gtacgagccg ctgagatatc cccatgccac caagatcttt 480

gtcaatggtg tttgggttgg tgtacaccaa gatcctaagc acctggtcag tcaagtcttg 540

gatactagac gaaagtcgta tctgcagtac gaggtgtctc tcgtccgaga aatcagggat 600

caagagttca agattttctc cgacgctggc cgggttatga gaccagtgtt tactgttcag 660

caagaagatg atcccgagac tggcattgaa aaaggccatc tcgttttgac caaagagttg 720

gttaacaagc ttgctaaaga acaagctgaa ccacctgaag acccaagcga gaaaattggc 780

tgggaaggat tgattcgtgc cggcgccgtc gagtacctcg atgccgaaga agaagagaca 840

tcaatgatct gcatgacgcc agaagatctt gagctgtatc gtctgcagaa agccggtgtt 900

gctcttgatg acgacattgg agatgacctg aataagcgtc tcaagaccaa gaccaacccc 960

acaacgcaca tgtatacgca ttgtgaaatt caccccagta tgattcttgg tatttgcgct 1020

agtattattc cgtttcccga tcacaatcag gtaagcagc 1059

Claims (12)

1. Metarhizium lepidiotae (Metarhizium lepidiotae) is characterized in that,

the internal number of the metarhizium anisopliae is MN114915, and the preservation number is GDMCC NO: 61330, the preservation time is 12 months and 1 day in 2020, the preservation address is Guangdong province microorganism strain preservation center, the address is No. 59 building 5 of Michelia Tokyo No. 100 of Guangzhou city, the institute of microorganisms in Guangdong province, and the taxonomic name is Metarhizium lepidiotae.

2. Use of metarhizium anisopliae according to claim 1 for the preparation of a microbial insecticide.

3. A microbial insecticide prepared from Metarrhizium anisopliae of claim 1.

4. The microbial insecticide according to claim 3, wherein said microbial insecticide comprises as active ingredients: aqueous preparation containing Metarhizium anisopliae with strain concentration of 10 ⁵ ～10 ¹⁰ Spore cfu/ml.

5. The microbial insecticide according to claim 3, wherein the effective components of said microbial insecticide are: powder containing Metarhizium anisopliae with strain concentration of 10 ⁵ ～10 ¹⁰ cfu/ml。

6. The microbial insecticide of claim 3, wherein said microbial insecticide further comprises at least one of the following carriers: preservatives, emulsifiers or UV protectors.

7. Use of a microbial insecticide as claimed in any one of claims 3 to 6 for controlling agricultural pests, wherein said agricultural pests are selected from locusts.

8. Use according to claim 7, characterized in that said locust is selected from locusta migratoria manilensis.

9. A process for the preparation of a microbial insecticide as claimed in any one of claims 3 to 6, comprising the steps of:

carrying out strain fermentation of metarhizium anisopliae in a culture medium by using a liquid submerged fermentation method to obtain fermentation liquor; controlling the temperature to be 25-30 ℃, the pH value to be 6 and the fermentation time to be 5 days during fermentation; then the fermentation liquor is prepared into water agent or powder.

10. Use of metarhizium anisopliae of claim 1 in promoting corn growth.

11. The use of claim 10, wherein said corn variety is zhengdan 958.

12. The use according to claim 10 or 11, characterized in that it is: plant seeds were treated in a spore suspension of metarhizium squamosum.

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