Metarhizium Robertsii and its application in biocontrol and growth promotion

Abstract

The present invention relates to the field of biological control technology, in particular to Roberts green muscardine and its application in biological control and growth promotion. The present invention provides Roberts green muscardine and a method for using the same for biological control. The strain is a green muscardine with high toxicity and insecticidal function. The strain has a simple cultivation method, rapid growth, and a large spore production. The mortality rate of infected locusts can reach 100.00%, and it is non-toxic and non-polluting, will not damage the ecology, is not easy to produce drug resistance, and is harmless to humans. It can be widely used in the prevention and control of locusts.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, in particular to Metarhizium Robertsii and its application in biological control and growth promotion. Background Art

[0002] Metarhizium is an important insect-borne fungus used for biological control. There are about 200 species of pests in the world that can be infected and killed by this fungus. The spores of Metarhizium can attach to the epidermis of pests, penetrate the epidermis and invade the body of the pest, use the pest's nutrition to grow and reproduce, and secrete toxins until the pest dies. The dead body of the pest can release new spores, which are spread to infect other pests. There are currently about 30 species of Metarhizium worldwide (Kepler, RM, Humber, RA, Bischoff, JF, & Rehner, SA (2014). Clarification of generic and species boundaries for Metarhizium and related fungi through multigenephylogenetics. Mycologia, 106 (4), 811-829.). Different species of Metarhizium can infect and kill different pests in different ranges, and their toxicity also varies. Metarhizium robertsii JFBisch.,Rehner&Humber is a species of the genus Metarhizium, which was first isolated from the corpse of the weevil Curculio caryae (Bischoff JF, Rehner SA, Humber RA A multilocus phylogeny of the Metarhizium anisopliaelineage Mycologia, 101(4), 2009, pp. 512–530.).

[0003] The prior art reports (WO2019080167A1) show that by recombining broad-spectrum Metarhizium and knocking out the monoamine oxidase gene, the concentration of tryptamine in the broad-spectrum Roberts Metarhizium can be significantly increased, thereby significantly improving the insecticidal efficiency, and shortening the half-lethal time LT50 of the wild-type broad-spectrum Metarhizium to the male East Asian migratory locust from 7.33±0.445 days to 6.136±0.488 days; that is, the LT50 lethal time of the wild Roberts Metarhizium to the East Asian migratory locust is relatively long, and the genetically recombinant Roberts Metarhizium also has the problem of long lethal time, and the insecticidal efficiency needs to be improved.

[0004] In addition, the LT50 of most Roberts' Metarhizium reported in the literature is greater than 4 days, and the LT50 of some Roberts' Metarhizium is even as long as 16 days.

[0005] The prior art reports that the median lethal time LT50 of Roberts' Metarhizium cultured in SDAY medium to susceptible insects is 1.4 days longer than that of wild-type strains (Adenylate cyclase orthologues in twofilamentous entomopathogens contribute differentially to growth, conidiation, pathogenicity, and multistress responses.). Therefore, the insecticidal effect of Roberts' Metarhizium needs to be further improved, and a new strain with a short lethal time, rapid insecticidal ability, and significantly improved insecticidal efficiency compared to existing Roberts' Metarhizium needs to be developed.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a Roberts' Metarhizium and a method for using the fungus to control locusts. It is expected that the Roberts' Metarhizium has a simple cultivation method, rapid growth, and a large spore production; the mortality rate of infected locusts is high, and the fungus is non-toxic and non-polluting, will not damage the ecology, is not easy to produce drug resistance, and is harmless to humans.

[0008] In order to solve the above technical problems and achieve the above objectives, the present invention provides the following technical solutions:

[0009] In the first aspect, the present invention provides a strain of Metarhizium robertsii, the strain of which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 1, 2020, with the culture collection number GDMCC 61329, and the storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the classification name is Metarhizium robertsii.

[0010] In a second aspect, the present invention provides the use of the Roberts' Metarhizium described in the aforementioned embodiment in the preparation of a bacterial fertilizer, a bacterial agent or a pesticide having both biological control and growth promotion effects.

[0011] In a third aspect, the present invention provides a bacterial agent prepared using the Roberts' Metarhizium with a strain preservation number of GDMCC61329 as described in the aforementioned embodiment.

[0012] In an optional embodiment, the dosage form of the bacterial agent includes granules, powder or liquid.

[0013] In an optional embodiment, the liquid agent contains spores of Metarhizium Robertsii at a concentration of 1.37×10 5 ~1×10 8 spores / mL.

[0014] In a fourth aspect, the present invention provides a method for preparing the liquid agent described in the aforementioned embodiment, comprising: using SDAY plates to activate the Roberts Metarhizium with a strain deposit number of GDMCC 61329, and then using a Tween 80 aqueous solution to elute the Roberts Metarhizium spores, and adjusting the concentration of the Roberts Metarhizium spores to 1.37×10 5 ~1×10 8 spores / mL.

[0015] In an optional embodiment, the content of Tween 80 in the Tween 80 aqueous solution is 0.05% to 0.2% (v / v), preferably 0.1% (v / v).

[0016] In a fifth aspect, the present invention provides a method for biological control using the aforementioned embodiment, comprising spraying the liquid agent described in the aforementioned embodiment for biological control.

[0017] In an optional embodiment, the pest species for biological control include at least one of Lepidoptera pests, Hemiptera pests, Diptera pests, Orthoptera pests, Coleoptera pests or Arachnida pests;

[0018] The lepidopteran pests include at least one of fall armyworm, beet armyworm, striped stem borer, yellow stem borer, diamondback moth or tea geometrid;

[0019] The hemiptera pests include at least one of planthoppers, whiteflies, aphids, leafhoppers or citrus psyllids;

[0020] The dipteran insects include at least one of Drosophila melanogaster, Housefly, Bactrocera dorsalis, or Mosquito;

[0021] The Orthoptera pests include the East Asian migratory locust;

[0022] The arachnid pests include at least one of two-spotted spider mites and citrus mites.

[0023] In a sixth aspect, the present invention provides a method for promoting crop growth by using the liquid agent described in the aforementioned embodiment, the method comprising soaking the crop seeds with the liquid agent described in the aforementioned embodiment before sowing, and / or watering the crop seeds with the liquid agent described in the aforementioned embodiment after sowing;

[0024] The crops include corn.

[0025] The invention provides a green muscardine fungus and a method for using the fungus for biological control. The fungus is a green muscardine fungus with high toxicity and insecticidal function. The culture method of the fungus is simple, the growth is rapid, and the spore production is large. The lethality rate of infected locusts can reach 100.00%, and the fungus is non-toxic and non-polluting, will not damage the ecology, is not easy to produce drug resistance, and is harmless to humans. The fungus can be widely used in the prevention and control of locusts. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 This is a front view of the colony of the Metarhizium Robertsii strain isolated in Example 1 of the present invention at 15 days;

[0028] Figure 2 This is a back morphological picture of a 15-day colony of the Metarhizium Roberti strain isolated in Example 1 of the present invention;

[0029] Figure 3 The spore-forming structure of the Metarhizium Robertii strain GDMCC 61329 isolated in Example 1 of the present invention (unit: 10 μm);

[0030] Figure 4 The conidia (unit: 10 μm) of the Metarhizium Robertsii strain GDMCC 61329 isolated in Example 1 of the present invention;

[0031] Figure 5 The phylogenetic tree typing result based on multiple genes of Metarhizium Robertii isolated in Example 1 of the present invention;

[0032] Figure 6 The effect of temperature on the growth of the Roberts' Metarhizium strain isolated in Example 1 of the present invention;

[0033] Figure 7 This is the dead insect on the culture medium of Example 3 of the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, 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 in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but only represents selected embodiments of the present invention.

[0035] In the first aspect, the present invention provides a strain of Metarhizium robertsii, the strain of which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on December 1, 2020, with the culture collection number GDMCC 61329, and the storage address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the classification name is Metarhizium robertsii.

[0036] The Roberts green muscardine described in the present invention is obtained by screening and separation. The specific separation method is: collect agricultural soil in Shaanxi, dry it, sieve the soil sample to remove impurities, weigh 10g of dry soil and put it into the filter device, wash the soil with tap water for 10min, and let the soil particles flow downward through a 1mm brass mesh and two polypropylene filters. Collect the soil particles at the bottom layer, put them into a sterile 50mL centrifuge tube, add 50mL of sterile water, and suspend them with a vortex mixer. Centrifuge at 10000rpm for 6min, pour out the supernatant, wash with sterile water, centrifuge again, and repeat centrifugation and washing 3 times. Add sterile sodium carboxymethyl cellulose solution at a ratio of 20:1 (v / v) of water / particles for suspension. Dilute the above suspension 10 times with sterile water, use a pipette to draw 100μL to evenly apply it on a PDA (containing chloramphenicol and tetracycline hydrochloride) plate, and place the plate in the dark at 28℃ for 2-7d. When a single white flocculent colony grows on the culture medium, a small amount of mycelium is picked up with a sterile toothpick and placed on a SDAY plate for further culture to obtain a purified strain.

[0037] The strain has the following characteristics: when cultured in SDAY medium at 28°C, the colony is 2.74 cm in 5 days, 5.55 cm in 10 days, and 7.58 cm in 15 days. The colony is white in the early stage and has a fluffy texture. It turns yellow in the transition period and produces dark green conidia in the later stage. The optimal growth temperature is 25-30°C.

[0038] In a second aspect, the present invention provides the use of the Roberts' Metarhizium described in the aforementioned embodiment in the preparation of a bacterial fertilizer, a bacterial agent or a pesticide having both biological control and growth promotion effects.

[0039] The application refers to the bacterial fertilizer, bacterial agent or pesticide prepared with the Roberts anhydride provided by the present invention as the insecticidal effective ingredient and other auxiliary agents into any dosage form allowed in the bacterial fertilizer, bacterial agent or pesticide.

[0040] In a third aspect, the present invention provides a bacterial agent prepared using the Roberts' Metarhizium with a strain preservation number of GDMCC 61329 as described in the aforementioned embodiment.

[0041] In an optional embodiment, the dosage form of the bacterial agent includes granules, powder or liquid.

[0042] In an optional embodiment, the liquid agent contains spores of Metarhizium Robertsii at a concentration of 1.37×10 5 ~1×10 8 spores / mL.

[0043] In a fourth aspect, the present invention provides a method for preparing the liquid agent described in the aforementioned embodiment, comprising: using SDAY plates to activate the Roberts Metarhizium with a strain deposit number of GDMCC 61329, and then using a Tween 80 aqueous solution to elute the Roberts Metarhizium spores, and adjusting the concentration of the Roberts Metarhizium spores to 1.37×10 5 ~1×10 8 spores / mL.

[0044] In an optional embodiment, the content of Tween 80 in the Tween 80 aqueous solution is 0.05% to 0.2% (v / v), preferably 0.1% (v / v).

[0045] In a fifth aspect, the present invention provides a method for biological control using the aforementioned embodiment, comprising spraying the liquid agent described in the aforementioned embodiment for biological control.

[0046] In an optional embodiment, the pest species for biological control involve agricultural pests and sanitary pests, including at least one of Lepidoptera pests, Hemiptera pests, Diptera insects, Orthoptera pests, Coleoptera pests or Arachnida pests;

[0047] The lepidopteran pests include at least one of fall armyworm, beet armyworm, striped stem borer, yellow stem borer, diamondback moth or tea geometrid;

[0048] The hemiptera pests include at least one of planthoppers, whiteflies, aphids, leafhoppers or citrus psyllids;

[0049] The dipteran insects include at least one of Drosophila melanogaster, Housefly, Bactrocera dorsalis, or Mosquito;

[0050] The Orthoptera pests include the East Asian migratory locust;

[0051] The arachnid pests include at least one of two-spotted spider mites and citrus mites.

[0052] In a sixth aspect, the present invention provides a method for promoting crop growth by using the liquid agent described in the aforementioned embodiment, the method comprising soaking the crop seeds with the liquid agent described in the aforementioned embodiment before sowing, and / or watering the crop seeds with the liquid agent described in the aforementioned embodiment after sowing;

[0053] The crops include corn.

[0054] Some embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

[0055] Example 1 Isolation, purification and identification of Metarhizium Robertsii

[0056] 1.1 Separation and purification

[0057] Weighing the soil: Agricultural soil was collected from Shaanxi Province. After drying, the soil samples were sieved to remove impurities. 10 g of dry soil was weighed and loaded into a filtering device.

[0058] Soil washing: Wash the soil with tap water for 10 minutes, allowing the soil particles to flow downward through a 1mm brass mesh and two polypropylene filters. Collect the soil particles at the bottom layer, place them in a sterile 50mL centrifuge tube, add 50mL of sterile water, and vortex mixer to suspend them. Centrifuge at 10,000rpm for 6 minutes, pour out the supernatant, wash with sterile water, centrifuge again, and repeat the centrifugation and washing 3 times.

[0059] Dilute the pellet: Add sterile sodium carboxymethylcellulose solution at a water / pellet ratio of 20:1 (v / v).

[0060] Spreading: dilute the above precipitation suspension 10 times with sterile water, pipette 100 μL onto a PDA plate (containing chloramphenicol and tetracycline hydrochloride) and spread evenly, and incubate the plate at 28°C in the dark for 2 to 7 days.

[0061] Fungal purification: When a single white flocculent colony grows on the culture medium, use a sterile toothpick to pick up a small amount of mycelium and continue to culture it on a SDAY plate to obtain a purified strain.

[0062] 1.2 Morphological identification

[0063] The isolated green anisopliae were inoculated on SDAY medium plates and cultured at 28°C for 15 days to observe the morphological characteristics of the colonies, the spore production structure, and the morphological size of the spores. After 4 to 5 days, white hyphae were picked up to make slides, and the morphology of hyphae and conidiophores was observed under a microscope; after 10 to 12 days, conidia were picked up to make slides to observe the size and morphology of conidia. After 15 days, the morphological characteristics of the colonies were observed and recorded, and the front and back sides were as follows: Figure 1 and Figure 2As shown in the figure, the strain is white at the beginning on SDAY medium, and then changes to light yellow or yellow, with flocculent or velvety protrusions in the shape, and green spores appear in the later stage. Figure 3 As shown, conidia are single-celled and columnar. Conidia are oblong and arranged in chains, with a size of (5.17-6.55) μm × (2.56-3.43) μm. Figure 4 shown.

[0064] 1.3 Molecular Biological Identification

[0065] The genomic DNA was extracted from the mycelium of the strain and used as a template for gene amplification to perform multi-gene amplification and sequencing on the strain. The genes and primers to be amplified are:

[0066] ITS: ITS4 (5′-TCCTCCGCTTATTGATATGC-3′SEQ ID No: 1) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′SEQ ID No: 2)

[0067] BenA: Bt1F (5'-GGTCCCTTCGGTCAGCTCTTCC-3'SEQ ID No: 3) and Bt1R (5'-CAGCCATCATGTTCTTAGGGTC-3'SEQ ID No: 4)

[0068] EF:983F(5'-GCYCCYGGHCAYCGTGAYTTYAT-3'SEQ ID No:5) and 2218R(5'-ATGACACCRACRGCRACRGTYTG-3'SEQ ID No:6)

[0069] RPB1:Mz1F1(5'-CGRACMYTRCCYCATTTCACAA-3'SEQ ID No:7) and Mz1R1(5'-TTGAGCGGAAGYTGCATCATCTCC-3'SEQ ID No:8)

[0070] RPB2: 5F (5'-GAYGAYMGWGATCAYTTYGG-3'SEQ ID No: 9) and 7CR (5'-CCCATRGCTTGYTTRCCCAT-3'SEQ ID No: 10)

[0071] The above primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0072] Template DNA extraction: Genomic DNA was extracted according to the Biospin Fungus Genomic DNA Extraction Kit (BioFlux, Bioer Technology Co., Ltd.).

[0073] For each gene amplification reaction system: prepare the reaction system 2×Taq PCR Master Mix 12 μL, DMSO 1 μL, primer 1 and primer 2 (10 μmol / L) 0.6 μL each, ddH2O 10.8 μL, and template DNA 2 μL in a PCR tube.

[0074] Gene amplification conditions: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at 55°C for 40 s, extension at 72°C for 1 min, 35 cycles of denaturation to extension; extension at 72°C for 10 min, and insulation at 10°C for 5 min. The samples after PCR were sent to Genewise for sequencing.

[0075] The sequence obtained by sequencing was compared with the sequence of Metarhizium strains in the NCBI gene database. Figure 5 As shown, the strain is Metarhizium robertsii.

[0076] Example 2 Biological Assay of Metarhizium anisopliae

[0077] 1.1 Effect of temperature on strain growth

[0078] The cultured M. anisopliae spores were washed with 0.1% Tween80, and 10 μl was inoculated on new SDAY medium. The culture was kept constant at five different temperatures: 20°C, 25°C, 28°C, 30°C, and 35°C. The colony diameter was measured every day, with 3 replicates in each group. The results are shown in the figure. Figure 6 As shown in the figure, it can be seen that the strain can grow at a temperature of 20-30°C, and the growth rate at 28°C is higher than that at other temperatures; the growth rate at low temperature (20°C) is lower than the growth rate at 25°C and 30°C. The optimum temperature for strain growth is 25-30°C.

[0079] 1.2 Colony growth rate determination

[0080] Wash the cultured Metarhizium spores with 0.1% Tween80, take 10 μl and inoculate it on new SDAY medium, repeat 3 times, measure the diameter regularly every day and record the data until the colony fills the medium. Colony growth rate: As can be seen from the table below

[0081] Table 1 Colony growth rate

[0082] Days 0d 1d 2d 3d 4d 5d 6d Colony diameter (cm) 0.93 1.14 1.55 2.00 2.44 2.88 3.26

[0083] On the first day, the strain grew slowly and failed to form a shape visible to the naked eye. From the second day, the strain began to grow rapidly and reached the fastest on the fourth day. On the fourth day, as the strain began to produce spores, the growth rate of the colony also decreased accordingly.

[0084] 1.3 Determination of spore production

[0085] 100 μl of spore suspension was spread on SDAY and cultured at 28°C for 10-15 days. Then, a bacterial sheet with a diameter of 8 mm was cut from the plate with a hole puncher. The conidia on the bacterial sheet were vibrated and washed into 10 ml of 0.1% Tween80 on an ultrasonic crusher. The spore concentration was determined by counting with a hemocytometer and then converted to per cm 2 The spore production on the plate. Each plate was punched with 3 holes for spore content determination and the average value was taken as the spore number of 1 replicate. Each strain was measured 3 times. The results showed that the spore production on SDAY medium could reach 0.53×10 on the 15th day. 8 Spores / cm 2 . The spore production is relatively high.

[0086] 1.4 Determination of spore germination rate

[0087] Wash the spores from the green anisopliae plate cultured for 10-15 days with 0.1% Tween80, take 10μl and inoculate on 1 / 4SDAY medium, inoculate 3 places per dish, place incubator under the same conditions for 24 hours, observe randomly under a 400x microscope, and count the number of spore germination and calculate the germination rate based on the spore germ tube length greater than or equal to the spore short axis diameter. The results showed that the germination rate of spores in 24 hours can reach more than 80%.

[0088] The above experimental results all indicate that the strain has simple nutritional requirements, grows and reproduces quickly, produces spores quickly, and has good biological characteristics.

[0089] Example 3 Determination of pathogenicity to pests

[0090] The M. anisopliae strain isolated in Example 1 was taken out from -80°C, activated on a SDAY plate, and cultured at 28°C for 7-15 days until spores were produced. The M. anisopliae spores were washed with 0.1% Tween80, counted with a hemocytometer, and a spore suspension of a specific concentration was obtained, which was then diluted or concentrated to prepare 1×10 5 Spores / ml, 1×10 6 Spores / ml, 1×10 7 Spores / ml, 1×10 8 Spores / ml, 1×10 9 Spores / ml suspension.

[0091] Select good 3rd-instar locust nymphs with consistent size and activity. Use a pipette to take 10 μl of spore suspension and spot it on the pronotum of the locust. Continue to feed it and feed it with fresh wheat seedlings every day. Each treatment is repeated 3 times, with 15 locust nymphs in each repeat. Use sterile 0.1% Tween80 water as a blank control. Cultivate under conditions of 28℃~30℃, RH 55%±5%, 16L∶8D, observe for 10 consecutive days, and record the death. To judge the death of the locust, gently touch the locust with tweezers. If it does not move, it is dead.

[0092] Dead locusts re-inoculation: This step verifies whether the locusts are killed by Metarhizium infection. The dead locusts are surface disinfected by washing twice with 75% alcohol for 15 seconds, 0.05% sodium hypochlorite for 1 minute, and sterile water for 1 minute, then dried on sterile filter paper. Finally, the locusts are placed on SDAY medium (containing chloramphenicol and tetracycline hydrochloride) to observe whether Metarhizium grows on the surface of the locusts. The results are as follows: Figure 7 shown.

[0093] Comparison of toxicity with commercial products: Commonly used insecticides, deltamethrin (Bayer) and Metarhizium anisopliae CQMa421 (Polyxin) were selected for comparison. Deltamethrin was diluted 1000 times, and Metarhizium anisopliae CQMa421 and our Metarhizium anisopliae GDMCC 61331 were diluted 1×10 8 Spores / mL, 10 μl of spore suspension was taken with a pipette and spotted on the pronotum of the locust. Each treatment was repeated 3 times, with 15 locust nymphs in each repeat.

[0094] Data calculation:

[0095]

[0096]

[0097] Control effect (%) = [1-(the number of live insects in the treated area after treatment × the number of live insects in the control area before treatment) / (the number of live insects in the treated area before treatment × the number of live insects in the control area after treatment)] × 100%.

[0098] The experimental results showed that M. anisopliae had a significant pathogenic effect on locusts. Under treatment with different concentrations of M. anisopliae spore suspension, the median lethal concentration (LC50) was 1.37×10 5 Spores / ml; at 1×10 8 At the concentration of spores / ml, the cumulative corrected lethality of Metarhizium anisopliae to locusts was 100% at 10 days, the median lethal time (LT50) was 1 day, and the rate of dead locusts being re-grafted was 100%. The above results show that Metarhizium Robertsii has the potential to be developed as a microbial pesticide for controlling locusts.

[0099] Table 2 Corrected mortality of Metarhizium Robertii GDMCC 61329 against locusts (%)

[0100]

[0101] Note: Numbers in the same column with the same letter after them indicate no significant difference at the 0.05 level (DMRT method), the same as in the following table.

[0102] Table 3 LC50 regression equation and LC50 of Metarhizium anisopliae GDMCC 61329 against locusts

[0103]

[0104] Table 4 LT50 regression equation and LT50 of Metarhizium anisopliae GDMCC 61329 against locusts

[0105]

[0106]

[0107] Table 5 Results of indoor toxicity test of Metarhizium Robertsii GDMCC 61329 against locusts (1×10 8 Spores / ml)

[0108]

[0109] Table 6 Indoor toxicity test results of Metarhizium Robertsii GDMCC 61329 and commercial products (1×10 8 Spores / ml)

[0110]

[0111] In addition, the present invention also investigates the toxicity of Roberts' Metarhizium GDMCC 61329 powder to various pests such as fall armyworm, striped stem borer and white grubs. The specific experiments are as follows:

[0112] The powder of Metarhizium anisopliae GDMCC 61329 was prepared into a wettable powder and diluted with water to 1×10 8 spores / ml of bacterial solution. Use a sprayer to spray 5ml of bacterial solution on the pests shown in Table 7 (about 30 of each pest), and continue to feed. Each treatment is repeated 3 times. Sterile 0.1% (v / v) Tween80 water is used as a blank control. Observe for 10 consecutive days, record the mortality, and select the best result to calculate the control effect in Table 7 below.

[0113] Table 7 The control effect of Metarhizium Robertii GDMCC 61329 on pests

[0114] Pest types Prevention effect (%) Pest types Prevention effect (%) Fall Armyworm 28.67 Beet Armyworm 85.67 Borer borer 80.74 Yellow stem borer 88.39 Plutella xylostella 75.37 Tea geometrid 86.67 Grub 91.33 Corn borer 89.93 Brown Planthopper 79.36 Laodelphax striatellus 82.57 White-backed Planthopper 78.33 Bemisia tabaci 91 aphid 88.67 Leafhopper 80.23 Citrus psyllid 90.57 Drosophila melanogaster 72.67 Bactrocera dorsalis 92.32 East Asian migratory locust 100 Two-spotted spider mite 72.33 Panonychus citri 80.26

[0115] Example 4 Determination of the plant growth-promoting effect of Metarhizium Robertsii

[0116] Healthy and uniform-sized corn seeds (Zhengdan 958) were selected for surface disinfection and then the seeds were placed in a 1×10 7 Soak in a spore suspension of Roberts's green anisopliae for half an hour and then sow. Sow 5 seeds in each pot, for a total of 3 pots. After sowing, 1 mL of treatment solution is poured on each seed. Sterile water is used as the control group. It is placed in a 25°C greenhouse and cultivated with a photoperiod of 12L:12D. During the experiment, water is regularly added to the seedling pot and the bottom plate to ensure sufficient water. After 20 days of pot cultivation, the plants are harvested, washed, and placed in a 70°C oven to a constant weight, and then the dry weight of the plants is measured.

[0117] Table 8 Dry weight growth index

[0118]

[0119]

[0120] The results showed that compared with the control group, the dry weight of corn plants in the group treated with Metarhizium Robertsii increased by 2.33%, indicating that this strain has a certain growth-promoting effect on plant growth.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Metarhizium robertsii, characterized in that The strain of Metarhizium Roberti is preserved in Guangdong Microbiological Culture Collection Center, and the culture collection number is GDMCC 61329.

2. Use of the Metarhizium Robertii described in claim 1 in the preparation of a bacterial fertilizer, a bacterial agent or a pesticide having both biological control and crop growth promoting effects; The pests for biological control are at least one of Lepidoptera pests, Hemiptera pests, Diptera insects, Orthoptera pests, Coleoptera pests or Arachnida pests; and the crop is corn.

3. Use of the Metarhizium anisopliae described in claim 1 in the preparation of bacterial fertilizers, bacterial agents or pesticides with biological control effects; The pests for biological control are at least one of Lepidoptera pests, Hemiptera pests, Diptera insects, Orthoptera pests, Coleoptera pests or Arachnida pests.

4. Use of the Metarhizium Robertsii described in claim 1 in the preparation of bacterial fertilizers, bacterial agents or pesticides that promote crop growth; the crop is corn.

5. A bacterial agent prepared using the Roberts' Metarhizium with a strain collection number of GDMCC 61329 as described in claim 1.

6. The bacterial agent according to claim 5, characterized in that The dosage form of the bacterial agent includes granules, powders or liquids.

7. The bacterial agent according to claim 5, characterized in that The liquid agent contains spores of Metarhizium Roberts at a concentration of 1.37×10 5 ~1×10 8 spores / mL.

8. A method for biological control using the microbial agent according to any one of claims 5 to 7, characterized in that: The method comprises spraying the bacterial agent according to any one of claims 5 to 7 for biological control; the pest species for biological control are at least one of lepidopteran pests, hemipteran pests, dipteran insects, orthoptera pests, coleopteran pests or arachnid pests.

9. The method according to claim 8, wherein the lepidopteran pest is at least one of Ostrinia furnacalis, Spodoptera frugiperda, Spodoptera exigua, Chilo suppressalis, Striped stem borer, Plutella xylostella or Tea geometrid; The hemiptera pest is at least one of brown planthopper, gray planthopper, whiteback planthopper, whitefly, aphid, leafhopper or citrus psyllid; The dipteran insect is at least one of Drosophila melanogaster and Bactrocera dorsalis; The Orthoptera pest is at least one of Locust migratoria or Locust nymph; The arachnid pest is at least one of two-spotted spider mite and citrus mites; The coleopteran pests are white grubs.

10. A method for promoting crop growth by using the microbial agent according to any one of claims 5 to 7, characterized in that: The method comprises soaking crop seeds with the microbial agent described in any one of claims 5 to 7 before sowing, and / or watering crop seeds with the microbial agent described in any one of claims 5 to 7 after sowing; the crop is corn.

Images