CN116656507A - Metarhizium anisopliae MrS Gz1-1 and application thereof in plant disease and insect resistance - Google Patents

Abstract

The invention provides a strain of Metarhizium anisopliae MrS1Gz1‑1 and its application in plant resistance to diseases and insect pests. The present invention finds for the first time that Metarhizium anisopliae has higher lethality to aphids such as Spodoptera litura, rice stem borer, Asian corn borer 2nd instar larvae, corn aphid and pea aphid, and has broad biological control application prospects; Metarhizium anisopliae spore suspension root soaking treatment of soybean plant seedlings, construction of "Metarhizium anisopliae-soybean plant" symbiont, making it an endophytic bacterium, can reduce the occurrence of soybean sclerotinia and harm to Spodoptera litura The larvae have a repelling effect and can resist the feeding hazards of Spodoptera litura larvae, thereby improving the biological function of the host plant's resistance to biological stress, providing more lasting protection for the host plant, and better meeting the needs of pest control in agricultural production. need.

Description

A Strain of Metarhizium anisopliae MrS1Gz1-1 and Its Application in Plant Resistance to Diseases and Insects

technical field

The invention relates to the technical field of microorganisms, in particular to a strain of Metarhizium anisopliae MrS1Gz1-1 and its application in plant resistance to diseases and insect pests.

Background technique

Metarhizium rileyi belongs to Ascomycota, Sordariomycetes, Hypocreales, Clavcipitaceae, also known as Nomuraea rileyi. Entomopathogenic fungi. Currently, widely used and well-known entomopathogenic fungi include Beauveria bassiana and M. anisopliae, which have a wide range of hosts and can infect both Lepidoptera and Coleoptera. , Hemiptera, Diptera, Hemiptera, Orthoptera and other pests are the most widely used entomopathogenic fungi in commercial development and production for pest control. The host range of Metarhizium anisopliae (M.rileyi) is different from that of B. bassiana and M. anisopliae. Current studies have found that M. anisopliae can infect more Lepidoptera pests (Humber et al., Catalog of species (pp.517), 2011), such as Spodoptera frugiperda, Spodoptera litura, Spodoptera soybean, etc., do they have certain effects on other species of insects? Infection effect has not been reported yet. In recent years, studies have found that Beauveria bassiana (B. bassiana) and M. anisopliae (M. anisopliae) have plant endogenous properties, and can colonize plants under natural conditions or by artificial inoculation, and will not affect Injuries to host plants also play an important role in promoting plant growth and improving host plant resistance; both colonize in plants, can change the behavior and feeding specificity of herbivorous insects, and even cause insect death, which can be Play an indirect protective effect on plants, and thus build an entomophytic fungus-plant symbiosis.

Sclerotinia ([Sclerotinia sclerotiorum (Lib.) de Bary] belongs to the fungus phylum (eumycota) Ascomycotina (Ascomycotina) Discomycetes (Discomycetes) Molluscum order (Helotiales) Sclerotinia (Sclerotinia), sclerotinia Bacteria is a worldwide important plant pathogenic bacteria, which occurs more frequently and is more harmful in temperate regions. Sclerotinia has a wide range of hosts, and it can infect field crops, vegetables and annual weeds. Sclerotinia It can infect the host through the germination of sclerotia or the hyphae on the infected host, and it can also infect the host through the ascospores sprayed from the ascus disc. Under the condition of high humidity, the fungus infects the host relatively quickly, and at the beginning it is on the surface of the host It causes water-soaked lesions, which gradually expand and produce a white cotton-like mold layer on the surface of the host. At the same time, the host tissue disintegrates and rots due to the growth of mycelium. Finally, the mycelium gathers in the host or on the body surface, turns black, and gradually forms Sclerotia. After the sclerotium that falls into the soil passes through the dormant stage, it can germinate mycelia and ascus discs to produce ascospores. Mycelium and ascospores can infect the host again under appropriate conditions. At present, the domestic use of biological There are few studies on the prevention and control of Sclerotinia sclerotiorum. Under the ecological conditions of the main soybean producing areas in Northeast China, the diseases caused by S. sclerotiorum infecting host soybeans and sunflowers are becoming more and more serious. It is very necessary to ensure food security.

Spodoptera litura (Fabricius) belongs to the family Noctuidae of the order Lepidoptera, and has many hosts, wide distribution and strong migration. Spodoptera litura is a worldwide agricultural pest, and it is widely distributed in various agricultural production areas in my country, including food crops such as soybeans and sweet potatoes, economic crops such as eggplant and pepper, grapes, bananas, etc., and even feed on Sunflower leaves, weeds, etc., have a wide distribution of hosts. Spodoptera litura mainly damages the leaves, flower buds and fruits of plants by feeding on the larvae. When the feeding is serious, the whole crop will be "no harvest", causing serious economic losses to agricultural production. Spodoptera litura is a complete metamorphosis insect, and its individual development is divided into four stages: egg, larva, pupa, and adult. There are 6 instars of Spodoptera litura larvae, and the newly hatched larvae do damage on the back of leaves, eating the mesophyll, leaving only the epidermis; after the 3rd instar larvae, the leaves are notched, incomplete or even eaten up, and after the 4th instar, they enter the period of overeating. Eat up a large area of host plant leaves and migrate to other places to cause damage. Adults are dark brown moths with a medium size and slightly smaller body. The forewing patterns are complex, and the biggest feature of the patterns is that there are 3 obliquely extending white bands between the two wavy stripes, hence the name "Spodoptera litura". The optimum temperature for the development of each stage of Spodoptera litura is 26°C to 30°C, and the damage is particularly serious from July to September every year, causing serious losses to agricultural production. Due to its strong migratory ability and wide range of hosts, Spodoptera litura is very difficult to control. It has become one of the most difficult pests of Spodoptera litura, the main food crop in Jilin Province, and vegetables in protected areas, seriously affecting the safe production of protected areas.

The Asian corn borer [Ostrinia furnacalis (Guenée)] is an insect belonging to the family Pyralidae of the order Lepidoptera. It has a wide range of hosts, is the most serious harm to corn crops, and has a wide geographical distribution. It is harmful to all corn production areas in my country, especially in continuous cropping areas. It is listed as a "provincial insect" by the agricultural department of Jilin Province.

Chilo suppressalis (Walker) belongs to Lepidoptera: Pyrlidae. The insect has the characteristics of fast reproduction, large food intake, borer, cold resistance and strong drug resistance. pose a serious threat.

Aphids, also known as greasy insects and honey worms, are a class of herbivorous insects, including all members of the superfamily Aphidoidea. There are about 4,400 species of aphids in 10 families, most of which belong to Aphididae. Aphids are also one of the most destructive pests on earth. About 250 of them are serious pests for agriculture, forestry and horticulture. Aphids vary in size, ranging from 1 mm to 10 mm in length.

Corn aphid [Rhopalosiphum maidis (Fitch)], commonly known as putty insect, belongs to Hemoptera (Homoptera) Aphidoidea (Aphidoidea), is an important piercing-sucking pest that damages corn, and occurs all over my country. In recent years, the occurrence and damage of corn aphids in Jilin Province have been increasing year by year, and it has become an important pest in corn production in our province.

The pea aphid (Acyrthosiphon pisum) has a short development period and a large reproduction rate, so it is known as the most destructive pest on the earth. It has a wide range of hosts and is extremely harmful to most leguminous crops and pastures. At the same time, it is also a model species for studying the relationship between host plants and pests, the symbiotic relationship between parasites and hosts, and the interaction between plant viruses and insect phenotypic plasticity.

At present, the main means of controlling agricultural pests is to adopt chemical control methods, but they often treat the symptoms rather than the root cause, which will not only cause insecticide resistance, but also bring about a series of worrying problems such as environmental pollution, ecological crisis, food safety and health hazards. . Therefore, it is particularly important to develop "green" biological control methods.

Contents of the invention

Aiming at the defects of the prior art, the present invention provides a strain of Metarhizium anisopliae MrS1Gz1-1 and its application in plant resistance to diseases and insect pests. Metarhizium anisopliae provided by the invention is effective against Lepidoptera litura litura (Fabricius) larvae, rice stem borer (Chilo suppressalis) larvae, corn aphid [Rhopalosiphummaidis (Fitch)] and pea aphid (Acyrthosiphon pisum) All aphidoids (Aphidoidea) are highly virulent and can infect the larvae of the Asian corn borer [Ostrinia furnacalis (Guenée)], and their virulence is lower than that of the above-mentioned pests. In addition, Metarhizium anisopliae can inhibit the growth of Sclerotinia sclerotiorum in vitro, and treating soybean plant seedlings with conidia suspension of Metarhizium anisopliae can make the bacteria grow in soybean plants. The colonization rate reaches 66.7%, which can reduce the incidence of Sclerotinia sclerotiorum and the size of lesions, and has a repelling effect on Spodoptera litura larvae, reducing pest damage.

One of the objects of the present invention is to provide a strain of Metarhizium anisopliae MrS1Gz1-1, which is preserved in the China General Microorganism Culture Collection and Management Center, with a preservation number of CGMCC No.40481 and a preservation date of January 6, 2023. Address of Preservation Unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing. It is classified as Metarhizium.rileyi.

Preferably, the 16S rDNA of the strain is shown in SEQ ID NO:1.

Another object of the present invention is to provide an application of Metarhizium anisopliae MrS1Gz1-1 in plant resistance to pests and diseases.

Preferably, the disease is selected from plant diseases caused by Sclerotinia sclerotiorum (Lib.) de Bary].

Preferably, the pests are selected from plants caused by Lepidoptera litura litura (Fabricius), rice stem borer (Chilo suppressalis), aphids (Aphidoidea) and/or Asian corn borer [Ostrinia furnacalis (Guenée)] pests.

Preferably, the application is to suppress plant diseases and insect pests by constructing a fungus-plant symbiosis.

Further preferably, the application is to treat plant seedlings with the bacterial agent containing the conidia of Metarhizium anisopliae MrS1Gz1-1 through root filling/root soaking, so as to construct Metarhizium anisopliae-plant symbiosis.

Further preferably, the Metarhizium anisopliae conidia root irrigation/root soaking treatment of plant seedlings for 24 hours, so that the Metarhizium anisopliae colonizes in the plant, thereby constructing Metarhizium anisopliae-plant symbiosis .

Preferably, the bacterial agent of the Metarhizium anisopliae conidia is a suspension prepared from the Metarhizium anisopliae conidia and an aqueous solution containing a suspending agent.

Preferably, the concentration of conidia of Metarhizium anisopliae in the suspension is 1× ¹⁰⁸ spores/mL, and the suspending agent is Tween-80 or other nonionic surfactants, preferably Tween-80, The concentration is 0.1% (v/v).

One of the purposes of the present invention is also to provide a method for cultivating Metarhizium anisopliae, comprising the following steps:

a) Inoculate Metarhizium anisopliae into SMAY solid medium for cultivation, and collect conidia;

b) Soak the conidia suspension in the body surface of the tested insects for 0.2min to 1min to measure the pathogenicity. The tested insects were reared in a 5mL sterile centrifuge tube, and the spores were collected after the corpses of the insects were covered with spore powder.

Preferably, the culture conditions of step a) are: temperature 25-26°C, culture time 10d-14d;

Preferably, the feeding conditions of step b) are: photoperiod 14L:10D, temperature 26±1°C, relative humidity 70±10%;

Preferably, the insect soaking method of the step b) is as follows: immerse the tested insect body in the spore suspension of Metarhizium anisopliae for 0.2 min to 1 min, and then use sterile filter paper to absorb excess water.

Further preferably, the tested insects are all in the 2nd instar stage, and both corn aphids and pea aphids are of the aphidless type.

Preferably, the plants of the present invention are selected from leguminous plants;

Further preferably, the leguminous plant is soybean;

Further preferably, the soybean plant seedlings are in the three compound leaf stage.

Beneficial effects of the present invention: the present invention finds for the first time that a strain of Metarhizium anisopliae MrS1Gz1-1 has a higher lethality to the 2nd instar larvae of Spodoptera litura and Chilo borer and aphids such as corn aphid and pea aphid, and has a higher lethality to Asian aphids. The 2nd instar larvae of the corn borer also have a good lethal effect, and have broad application prospects in biological control; use the Metarhizium anisopliae spore suspension to treat the soybean plant seedlings, and construct the symbiont of "Metarhizium anisopliae-soybean plants" , making it an endophyte in plants, which can reduce the occurrence and harm of soybean sclerotinia, avoid the larvae of Spodoptera litura, and resist the feeding hazards of Spodoptera litura larvae, thereby improving the life of the host plant's resistance to biological stress It can provide more durable protection for host plants, and can better meet the needs of pest control in agricultural production.

Description of drawings

Fig. 1 is the bacterium colony figure of Metarhizium anisopliae provided by the present invention on the SMAY medium;

Fig. 2 is the molecular morphology diagram of the hyphae and spore morphology of Metarhizium anisopliae provided by the present invention;

Fig. 3 is the phylogenetic tree of Metarhizium anisopliae provided by the present invention;

Fig. 4 is the morphological diagram of the dead insects formed by the conidia of Metarhizium anisopliae provided by the present invention through the dipping method to treat 5 kinds of important agricultural pests; wherein, a is the 2nd instar larvae of Spodoptera litura, and b is the rice stem borer 2 instar larvae, c is the 2nd instar larvae of O. corn borer, d is the corn aphid, e is the pea aphid;

Figure 5 The cumulative corrected mortality of the tested insects on the 1st day, the 2nd day, and the 3rd day after being infected by Metarhizium anisopliae strain MrS1Gz1-1; There was a significant difference in difference method test (P<0.05);

Figure 6 is the colonization detection of Metarhizium anisopliae SMAY plate; A blank control; B Metarhizium anisopliae MrS1Gz1-1;

Fig. 7 is the detection of colonization rate of Metarhizium anisopliae SMAY plate;

Figure 8 is the confrontation culture result of Metarhizium anisopliae and Sclerotinia provided by the present invention in a petri dish;

Figure 9 is the infection symptoms of Sclerotinia sclerotiorum on soybean leaves after 4 days of inoculation; Control: blank control; Ss: treatment group inoculated with Sclerotinia sclerotiorum; Mr: treatment group with Metarhizium anisopliae MrS1Gz1-1; Metarhizium anisopliae MrS1Gz1-1 and Sclerotinia treatment group;

Fig. 10 is different treatment soybean leaves inoculated with Sclerotinia sclerotiorum infection symptom on the 3rd day in embodiment 1, Ss: soybean leaves are inoculated with Sclerotinia sclerotiorum; Control: soybean leaves are not inoculated with Metarhizium anisopliae and Sclerotinia sclerotiorum; Mr: soybean Leaves were inoculated with Metarhizium anisopliae MrS1Gz1-1; Mr+Ss: soybean leaves were inoculated with Metarhizium anisopliae MrS1Gz1-1 and Sclerotinia;

Figure 11 is the incidence rate of the 4d, 5d, 6d, and 7d after Metarhizium anisopliae MrS1Gz1-1 was inoculated with Sclerotinia bacterium cake;

Figure 12 is the lesion size of the 4th day after Metarhizium anisopliae MrS1Gz1-1 was inoculated with Sclerotinia bacterium cake;

Figure 13 is a non-selective experimental figure;

Fig. 14 is a selectivity experiment figure;

Figure 15 is the feeding rate of the 2nd instar larvae of Spodoptera litura at different times on the Metarhizium anisopliae-soybean symbiont and control soybean leaves in the non-selective test;

Figure 16 is the feeding rate of the 2nd instar larvae of Spodoptera litura on the Metarhizium anisopliae-soybean symbiont and control soybean leaves at different times in the selection test.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1 Isolation and cultivation of Metarhizium anisopliae strain MrS1Gz1-1

1 Test materials and methods

1.1 Samples of Spodoptera litura zombies

Samples of Spodoptera litura worms were collected in the autumn of 2021 in the soybean experimental field of Jilin Academy of Agricultural Sciences in Gongzhuling City (N43°50′42″, E124°82′58″).

1.2 Test materials

The strains were isolated using potato dextrose agar medium (Potato Dextrose Agar, PDA): 200g fresh potatoes, 20g glucose, 18g agar powder, 1000mL deionized water, appropriate amount of antibiotics. Strains were cultured using Sabouraud maltose agar with 1% yeast extract (SMAY): 40 g of maltose, 10 g of tryptone, 10 g of yeast powder, 18 g of agar powder, 0.6 g of chitin, 1000 mL of deionized water, Appropriate amount of antibiotics. The suspending agent is Tween-80 solution. 0.1% (v/v) Tween-80, 75% (v/v) ethanol, 1% (v/v) sodium hypochlorite, self-prepared.

1.3 Test method

1.3.1 Metarhizium anisopliae isolation and colony morphology observation

Spodoptera litura critters were sterilized with 75% (v/v) ethanol for 0.5 min, 1% (v/v) sodium hypochlorite for 2 min, and finally rinsed with sterile water for 3 times, and cut them with a sterile scalpel. Divided into five segments, placed on a PDA plate in a five-point distribution, and cultivated in a biochemical incubator at 26°C for 2d to 3d. Observe whether there are hyphae growing on the surface of the insect body, pick a small amount of mycelium with a sterile needle, place it on a PDA plate, and observe and cultivate it in a biochemical incubator at 26°C for 2d to 3d.

Pick a small amount of newly germinated mycelium from the edge of the colony, place it in a sterile 0.1% (v/v) Tween-80 solution, make a bacterial suspension, and draw 100 μL ~ 150 μL of the bacterial suspension with a sterile glass applicator Spread it on a new PDA plate, then culture it in a biochemical incubator at 26°C for 1d to 2d, and observe the growth of single colonies on the PDA plate. Pick 10 to 15 single colonies from a well-growing PDA plate on a new PDA plate, and culture them in a 26°C biochemical incubator for 2d to 3d to ensure that there is only one single colony on each PDA plate. Pick a small amount of fresh mycelia from the edge and place them in sterile water, and use an inoculation loop to streak and culture them on a plate until a purified strain is obtained.

Select a well-growing strain from the purified strains, pick a small amount of hyphae with a sterile syringe needle in the ultra-clean workbench, put it on a glass slide dripped with sterile water, cover it with a cover glass and put it under a microscope Observe the mycelium and spore morphology of pathogenic bacteria and take pictures for records. Refer to the "Handbook of Fungal Identification" and other materials for systematic classification.

1.3.2 Molecular identification of Metarhizium anisopliae

Genomic DNA was extracted by SDS method. The fungal transcriptional spacer specific primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used as primers to amplify the fungal ITS region by PCR. It was sent to Jilin Province Kumei Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared and analyzed by BLAST.

1.3.3 Preparation of conidia of Metarhizium anisopliae

The spore-forming medium of Metarhizium anisopliae was selected from SMAY medium containing chitin. Cultivate in a biochemical incubator at 26°C for 14 days, scrape conidia and place them in a sterile 0.1% (v/v) Tween-80 solution, make a spore suspension, count with a hemocytometer, and dilute to 1×10 ⁸ individual/mL.

2. Test results

2.1 Strain morphology

Spot the isolated and purified strains on the center of SMAY medium and PDA medium plates, cultivate them in a biochemical incubator at 26°C, and observe their growth. After culturing for 10 days, the diameter of the colony on the PDA plate is 0.4-0.6 cm, and the diameter of the colony on the SMAY plate is 0.5 cm-0.7 cm; the colony of this strain on the PDA plate is white and has no depression in the center, and no spores are produced. Culture in SMAY The edge of the colony on the base is white and fluffy, and the center of the colony is slightly depressed and green spores are produced in the center of the colony (Figure 1). Overall, it showed that the strain grew poorly on PDA medium. Its morphology is similar to that of Metarhizium anisopliae under microscope observation, the hyphae are slender and have a septum, and the conidia are born on both ends of the extension axis of the hyphae, which are oval (Figure 2).

2.2 Molecular biological identification of strains

The endogenous transcriptional spacer universal primer ITS1/ITS4 was used to amplify the bacterial transcriptional spacer, and the ITS sequence (SEQ ID NO: 1) with a length of 588 bp was obtained by sequencing. Through BLAST comparison analysis, it was found that the sequence identity with M. rileyiMN602591 was as high as 100%. The results of comprehensive morphological observation and molecular biological identification showed that the pathogen isolated from Spodoptera litura worms in the soybean experimental field in Gongzhuling City was M. rileyi, so the pathogenic strain was named MrS1Gz1-1, see Figure 3.

Example 2 Study on the host range of Metarhizium anisopliae strain MrS1Gz1-1

1. Test materials and methods

1.1 Test material

The 2nd instar larvae of Spodoptera litura were provided by the Biocontrol Microbe Research and Utilization Team of the Institute of Plant Protection, Jilin Academy of Agricultural Sciences. The 2nd instar larvae of rice stem borer were provided by Beijing Channostar Biotechnology Co., Ltd. The 2nd instar larvae of the corn borer were provided by Beijing Channostar Biotechnology Co., Ltd. Corn aphids were provided by Beijing Zannosite Biotechnology Co., Ltd. Pea aphids were provided by Beijing Zannosite Biotechnology Co., Ltd. Metarhizium anisopliae strain MrS1Gz1-1 was isolated from Spodoptera litura larvae.

1.2 Test reagents

SMAY solid medium was selected as the test medium, the same as in Example 1. 0.1% (v/v) Tween-80 was prepared by itself, and a sterile spoon was used.

1.3 Test method

Select the bacterial strain MrS1Gz1-1 that was cultured on the SMAY plate for 15 days, scrape the spores on the plate into 20mL sterile 0.1% (v/v) Tween-80 with a sterile spoon, mix well, and use 4 layers without Filter the spore suspension with sterile lens tissue and a sterile funnel, count the filtrate with a hemocytometer, and dilute to a spore suspension of 1.0×10 ⁸ spores/mL. Then use the dipping method to inoculate the 2nd instar larvae of the Lepidoptera pest Spodoptera litura, the 2nd instar larvae of the rice stem borer, and the 2nd instar larvae of the Asian corn borer. , set 3 replicates for each insect, and each replicate had 20 insect populations. The control group chose to immerse in 0.1% (v/v) Tween-80 solution. Then they were cultivated in an insect culture room at 25±1°C, L:D=14h:10h, relative humidity 70±10%, fed with corresponding food, observed regularly and counted their mortality.

Pick out the dead insects in time, put the dead insects in a sterilized glass petri dish with a sterile filter paper at the bottom, cover the dish, and place it in a light incubator at 25°C for moisturizing cultivation. Observe the surface of the dead insects every day Whether there is mycelial growth, regularly take pictures to record the growth of mycelium or conidia, observe and record the appearance of the colony, and check the conidia and mycelium morphology under an electron microscope. Observe whether the colony shape and conidia shape of the re-isolated strain are consistent with the original inoculated strain.

1.4 Data Analysis

Input the recorded raw data into an Excel table, and statistically calculate the initial death time of worms and the cumulative death rate of worms in each treatment. All experimental data were analyzed using IBM SPSS Statistics 25 software, and GraphPadPrism 8.0.2 was used for drawing.

Mortality (%) = number of dead insects/number of tested insects × 100%; (2.1)

Corrected mortality rate (%) = (mortality rate in treatment group - mortality rate in control group) / (1 - mortality rate in control group) × 100% (2.2)

2. Test results

2.1 Host range of Metarhizium anisopliae strain MrS1Gz1-1

The strain MrS1Gz1-1 was used to inoculate five important agricultural pests, and the results showed that the five pests of Lepidoptera litura, rice stem borer, Asian corn borer, hemiptera corn aphid and pea aphid were all affected. Infection death situation (Fig. 4), and the pathogenic bacteria consistent with bacterial strain MrS1Gz1-1 colony morphology and conidia morphology can be isolated from zombies.

2.2 Toxicity test results

Table 1 Toxicity of Metarhizium anisopliae strain MrS1Gz1-1 to various pests

It can be seen from Table 1 that the strain MrS1Gz1-1 has high pathogenicity to 2nd instar Spodoptera litura, 2nd instar rice stem borer, 2nd instar Asian corn borer, corn aphid, and pea aphid. After being infected by Metarhizium anisopliae strain MrS1Gz1-1, the corrected mortality of Spodoptera litura, rice stem borer, Asian corn borer, hemiptera corn aphid and pea aphid gradually increased with the treatment time , and the corrected mortality of Spodoptera litura was significantly higher than that of other tested insects (Figure 5).

Example 3 Colonization of Metarhizium anisopliae strain MrS1Gz1-1 in soybean

1. Test materials and methods

1.1 Plant material

Soybean seeds were provided by the utilization team of biocontrol microbiology researchers at the Institute of Plant Protection, Jilin Academy of Agricultural Sciences.

1.2 Test reagents

0.1% (v/v) Tween-80, 75% (v/v) ethanol, 1% (v/v) sodium hypochlorite, self-prepared.

1.3 Test method

1.3.1 Preparation of conidia suspension of Metarhizium anisopliae strain MrS1Gz1-1

First prepare a sterile 0.1% (v/v) Tween-80 solution, add 200 μL of Tween-80 stock solution to 200 mL of deionized water, sterilize at 115° C. for 30 minutes, and cool to room temperature for later use. Immediately scrape an appropriate amount of conidia of the strain MrS1Gz1-1 into a 50mL centrifuge tube, pour an appropriate amount of sterile 0.1% (v/v) Tween-80 solution, and shake in a vortex oscillator for 3 minutes. Use 4 layers of sterile gauze and sterile Filter the spore suspension with a bacterial funnel, transfer the filtrate to a new sterile 50mL centrifuge tube, adjust the concentration of the spore suspension to 1× ¹⁰⁸ spores/mL, and store at 4°C for later use.

1.3.2 Experimental design

This experiment adopts randomized block design, setting two treatments of control group and Metarhizium anisopliae strain MrS1Gz1-1. When the soybean grows to the stage of three compound leaves, 25 days, and the plant height is about 15cm, the plants treated with 0.1% (v/v) Tween-80 are used as controls, and the Metarhizium anisopliae strain MrS1Gz1-1 treatment group is treated with 10 mL concentration of 1 The spore suspension of ×10 ⁸ spores/mL was inoculated by root irrigation, 10 soybean plants in each treatment group, and repeated three times.

1.3.3 Planting of soybeans

Select healthy and plump soybean seeds and place them in a petri dish filled with sterile water, and accelerate germination at 26°C. During this period, replace the sterile water in the petri dish every day, and they will germinate after 2 to 3 days. After they germinate, they can be transplanted and planted .

Before transplanting and planting, you should choose a flower pot with good drainage. The soil should be loose and moist flower soil as the soybean growth substrate. Wet, cultivate in the greenhouse until the three compound leaf stage, pay attention to water supply during this period.

1.3.4 Soybean root irrigation and inoculation treatment

When the soybean plants grow to the stage of three compound leaves, carry out the root irrigation treatment, shake the conidia suspension of Metarhizium anisopliae strain MrS1Gz1-1 prepared in advance, and use a 10mL sterile syringe to inject 10mL of Metarhizium anisopliae The conidia suspension of the bacterial strain MrS1Gz1-1 was injected into the surface soil of the root system, avoiding contact with the leaves of the plants. The control group was injected with 10 mL of sterile 0.1% (v/v) Tween-80 solution. Three replicate groups were set up for each treatment group, and each replicate treated 10 soybean plants.

1.3.5 Colonization detection of Metarhizium anisopliae strain MrS1Gz1-1 in soybean

48 hours after soybean inoculation, the colonization of Metarhizium anisopliae in soybean plants was detected by plate colonization assay. Soybean samples were collected using alcohol-sterilized scissors, and a leaf was collected from each soybean plant, and then the plant leaf specimens were surface-sterilized, and these samples were sequentially soaked in 75% (v/v) ethanol solution, 1% ( v/v) Sodium hypochlorite solution, 75% (v/v) ethanol solution and sterile water, each soaked for 30s; blot excess water on the surface of the tissue with sterile filter paper, cut off the edge of the soybean leaf with sterile scissors, and left Cut 9 small squares of the same size from the leaf tissue, place the leaf tissue evenly on the SMAY plate with sterile tweezers, and culture it in a light incubator at 26°C. After 4 days, observe whether Metarhizium anisopliae grows around the leaf tissue in the plate, and record the colonization situation of Metarhizium anisopliae in the plant leaves. Specific determination method: If there is Metarhizium anisopliae growing around a leaf tissue in the plate, it indicates that Metarhizium anisopliae has successfully colonized the soybean plant. The formula for calculating the colonization rate of Metarhizium anisopliae in soybean leaves is as follows:

Colonization rate=number of soybean strains colonized by Metarhizium anisopliae/total number of strains in the treatment group×100% (3.1)

1.3.6 Data processing and statistical analysis

All experimental data were carried out using IBM SPSS Statistics 25 software for one-way analysis of variance (p<0.05), and GraphPad Prism 8.0.2 was used for drawing.

2 test results

2.1 Colonization rate of Metarhizium anisopliae strain MrS1Gz1-1 in soybean

The colonization results of Metarhizium anisopliae strain MrS1Gz1-1 in soybean leaves are shown in Figure 6: on the 4th day of culture on SMAY medium, the edge of the soybean leaf tissue that was rooted by Metarhizium anisopliae strain MrS1Gz1-1 was from the inner On the outside, white visible Metarhizium anisopliae hyphae began to grow. The results showed that Metarhizium anisopliae strain MrS1Gz1-1 could be colonized in soybean plants by root irrigation inoculation. As shown in Fig. 7, the colonization rate of the treatment group of Metarhizium anisopliae MrS1Gz1-1 was as high as 66.67%.

Example 4 Effect of Metarhizium anisopliae strain MrS1Gz1-1 on host soybean disease resistance

1. Test materials and methods

1.1 Experimental materials

Sclerotinia sclerotiorum, provided by the Biocontrol Microbe Research and Utilization Team of the Institute of Plant Protection, Jilin Academy of Agricultural Sciences. Soybean plants and experimental reagents are the same as in Example 3.

1.2 Test method

1.2.1 Experimental design

Control group and only accept sclerotinia treatment group and process with 0.1% (v/v) Tween-80 soaking root, experimental design is as shown in table 2:

Table 2 Experimental treatment groups

1.2.2 Culture of Sclerotinia

Before the experiment, take out the strains cultivated in the biochemical incubator at 26°C to produce sclerotia, use a sterile inoculation needle to pick a single sclerotium and inoculate it on a PDA plate, place it in a constant temperature incubator at 26°C, and cultivate it for 5-7 days for the experiment .

1.2.3 Anti-Sclerotinia activity of Metarhizium anisopliae MrS1Gz1-1 in vitro

Use a 5mm hole punch to take out the cultured Sclerotinia sclerotiorum cake and place it at a distance of 1.5cm from the edge of the petri dish. The same 5mm M. anisopliae MrS1Gz1-1 bacteria cake is placed on the opposite side of the Sclerotinia sclerotiorum and kept at a constant temperature of 26°C Cultured in an incubator, 6 replicates were set; at the same time, a control petri dish containing only Sclerotinia sclerotiorum was prepared. During the culture, it was observed whether there was a zone of inhibition between Metarhizium anisopliae and Sclerotinia.

1.2.4 Inoculation of soybean with Metarhizium anisopliae MrS1Gz1-1

The spores of Metarhizium anisopliae MrS1Gz1-1 inoculated in this experiment are conidia, the preparation method of the spore suspension is the same as in Example 1, the inoculation method is the root soaking method, and the healthy soybean plants with good growth during the three compound leaf stage are selected , take out the whole soybean seedling completely, wash the soybean plant root with tap water, and then use 0.1 (v/v) Tweeen-80 solution, 1×10 ⁸ spores/mL of Metarhizium anisopliae strain MrS1Gz1-1 spore suspension The roots were soaked for 12 hours, and each treatment was repeated 3 times, with 10 plant seedlings in each repetition, and the volume of the root soaking solution was 20 mL.

1.2.5 Sclerotinia inoculated soybeans

After soybean plants were inoculated with Metarhizium anisopliae MrS1Gz1-1 for 12 hours, three compound leaves of soybean leaves with consistent and healthy growth were selected as the control effect test objects of isolated leaves. Soybean leaves were cut with sterile scissors, then sterilized with 75% (v/v) ethanol for 30 seconds, 1% (v/v) sodium hypochlorite for 2 minutes, and finally washed twice with sterile water. On a Petri dish with moistened filter paper, wet cotton balls were placed on the petioles to keep them moist. Select the leaves used for the colonization test to artificially inoculate the sclerotinia cake, use a 5mm puncher to punch out the cultured sclerotinia cake with the same area, try to select the sclerotinia cake cultured at 26°C for 4 to 5 days for the inoculation , the colonies at a distance of 2cm from the edge of the petri dish and with the same density have the best activity, and then inoculated on the leaf surface (avoiding the veins), placed in a light incubator at 25±1°C, and the humidity was kept above 70%, L:D=16h : Under the environment of 8 hours, observe the disease state of soybean leaves every 24 hours, count the disease rate, and measure the diameter of the lesion.

1.2.6 Plant incidence statistics and disease spot observation

After the onset of soybean leaves, observe and record the incidence of soybean leaves in each treatment group every day, record the total number of diseased plants and the total number of observed plants, and the ratio of the two is the incidence of soybean sclerotinia.

Soybean sclerotinia incidence rate (%)=the total number of soybean plants with each treatment/investigation total number of soybean plants × 100% (4.1)

After the onset of soybean leaves, use a ruler to measure and record the lesion on the leaf, and take the mean value of the horizontal and vertical diameter lengths as the length of the lesion diameter, and observe for 4 to 5 days.

1.2.7 Data processing and statistical analysis

All experimental data were carried out using IBM SPSS Statistics 25 software for one-way analysis of variance (p<0.05), and GraphPad Prism 8.0.2 was used for drawing.

2 test results

2.1 In vitro inhibitory effect of Metarhizium anisopliae MrS1Gz1-1 on Sclerotinia

After facing each other on the plate, the relationship between S. sclerotiorum and Mr. anisopliae MrS1Gz1-1 gradually fit over time, but the two ends expanded as shown in Figure 8, which shows that Mr. Definitely antagonistic.

2.2 Morphological observation of soybean leaves infected by Sclerotinia sclerotiorum

On the 4th day after the infection of soybean leaves by Sclerotinia sclerotiorum, the disease symptoms of isolated soybean leaves were shown in Figure 9, and neither the blank control group nor the soybean leaves treated with Metarhizium anisopliae MrS1Gz1-1 had disease. The symptoms of the soybean leaves in the group treated with Sclerotinia sclerotiorum were that the leaves had obvious lesion spots, and the lesion spots were partially festered, and the yellowing degree of the leaves spread from the veins until the whole leaf turned yellow. After treatment with Metarhizium anisopliae MrS1Gz1-1, the symptoms of soybean leaves inoculated with S. sclerotiorum group were that the overall yellowing degree of the leaves was not obvious, and only part of the leaves were yellowed at the part of the cake inoculated with Sclerotinia sclerotiorum, and the size of the lesion Significantly smaller than the non-inoculated treatment group with Metarhizium anisopliae MrS1Gz1-1. The overall yellowing degree of leaves only inoculated with S. sclerotiorum was more obvious, and the degree of leaf rot at the position where the cake was inoculated with S. sclerotiorum was also more obvious, while soybean leaves treated with M. The incidence of infection was significantly reduced after infection, indicating that the inoculation of Metarhizium anisopliae MrS1Gz1-1 had a more obvious inhibitory effect on the infection of Sclerotinia sclerotiorum.

2.3 Statistics of disease incidence of soybean leaves

On the 3rd day after S. sclerotiorum infects soybean leaves, the leaves begin to become ill. From the day of onset, the observation begins. With the development of time, after 5 days of inoculation, neither the blank control nor the M. anisopliae MrS1Gz1-1 treatment group has disease. The incidence of soybean leaves in the treatment group of S. sclerotiorum reached 83.33%. The symptoms of soybean leaves inoculated with Metarhizium anisopliae MrS1Gz1-1 and then inoculated with S. sclerotiorum were mild ( FIG. 10 ). It shows that Metarhizium anisopliae MrS1Gz1-1 has an excellent inhibitory effect on the infection of Sclerotinia sclerotiorum.

2.4 Statistics of Soybean Leaf Disease Rate

Soybean leaves started to get sick on the 3rd day after being inoculated with Sclerotinia sclerotiorum, but the disease degree was not obvious, so the incidence data on the 4th, 5th, 6th, and 7th days of the onset were collected for analysis (Figure 11).

2.5 Measurement of soybean leaf lesion size

The soybean leaves were inoculated with S. sclerotiorum on the 3rd day. Except for the blank control group and the Mr. control group, the lesions on the soybean leaves inoculated with S. On the 4th day and 5th day of the disease, some diseased leaves were withered and could not be measured. The lesion diameter data on the 4th day of the disease were collected and analyzed as shown in Figure 12. The results showed that soybean leaves inoculated with S. sclerotiorum The lesion size was significantly lower than that of the treatment group only inoculated with Sclerotinia sclerotiorum, while the disease index of the blank control group and the treatment group only inoculated with Metarhizium anisopliae MrS1Gz1-1 were 0.

Example 5 Effect of Metarhizium anisopliae strain MrS1Gz1-1 on insect resistance of host soybean

1. Materials and methods

1. Test materials and methods

1.1 Experimental materials

Metarhizium anisopliae MrS1Gz1-1 was isolated from the worms of Spodoptera litura collected from the soybean experimental field of Jilin Academy of Agricultural Sciences in 2021 by our laboratory. The strain is preserved in China Agricultural Microorganism Culture Collection Center (CGMCC No. 40481). Soybean plants are the same as in Example 3. The 2nd instar larvae of Spodoptera litura (S.litura) are the same as in Example 1.

1.2 Symbiont construction method

In this experiment, the Metarhizium anisopliae-soybean symbiont was constructed by a seed soaking method, which was the same as in Example 4.

1.3 Experimental design

Experimental design: There are 2 treatments in the experiment, which are the blank control group and the root soaking treatment group of Metarhizium anisopliae MrS1Gz1-1. The effects of symbionts on the feeding behavior of the 2nd instar larvae of Spodoptera litura were studied by means of indoor control experiment and petri dish leaf disc method through selective and non-selective experiments.

1.3.1 Non-selective test

Take a plastic petri dish with a diameter of 14 cm, place a piece of filter paper with a diameter of 13 cm in the center of the bottom of the petri dish, add an appropriate amount of distilled water to keep the filter paper moist, place 10 soybean leaves of the same size along the periphery, and connect 10 larvae in the center of the filter paper (Figure 13) , after inoculation, move into (26±1)°C, RH (60±10)%, photoperiod 14L:10D insect culture room. The experiment was repeated 5 times. 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 24h, 48h and 72h after inoculation, check the location of the larvae (leaves or Petri dish wall or filter paper) ) and the number of larvae, calculate the percentage of larvae that choose to feed on soybean leaves, expressed as feeding rate.

Feeding rate (%)=the number of insects that eat soybean leaves per treatment/the total number of insects tested in each treatment×100% (5.1)

1.3.2 Selective test

Fold a piece of filter paper with a diameter of 13cm into 10 equal parts, spread it in the center of the bottom of the petri dish and infiltrate it, place the symbiont material and the soybean material of the control group alternately, and the rest of the treatment is the same as 1.3.1, and the experiment is repeated 10 times (Figure 14)

1.4 Data statistics and analysis

All experimental data were carried out using IBM SPSS Statistics 25 software for one-way analysis of variance (p<0.05), and GraphPad Prism 8.0.2 was used for drawing.

2 Test results

2.1 Non-selective experiment

As shown in Figure 15, in the 72h non-selective experiment, the feeding rate of the 2nd instar larvae of Spodoptera litura on the leaves of Metarhizium anisopliae-soybean symbiont was lower than that of the control group as a whole. The feeding rate of the 2nd instar larvae of Spodoptera litura on the soybean compound leaves of the control group showed a general decreasing trend, while the feeding rate on the leaves of the fungus-soybean symbiont first decreased and then increased. Among them, the feeding rate of the 2nd instar larvae of Spodoptera litura on the leaves of the symbiont was basically unchanged from 2h to 2.5h after inoculation, and was basically the same as that of the control group at 2.5h. The feeding rate of the 2nd instar larvae of Spodoptera litura on the leaves of the symbiont fluctuated greatly at the 3rd and 24th hours, but the overall feeding rate did increase gradually after 3 hours of inoculation.

2.2 Selective experiment

As shown in Figure 16, in the 72h selective experiment, the feeding rate on the leaves of the control group was always higher than the feeding rate on the leaves of the fungus-soybean symbiont, and the feeding rate of the 2nd instar larvae of Spodoptera litura on the leaves of the control group The overall feeding rate showed a downward trend, and the feeding rate on the leaves of Metarhizium anisopliae-soybean symbiont fluctuated and increased. One hour after inoculation, the feeding rate of the 2nd instar larvae of Spodoptera litura on the leaves of the control group gradually showed a downward trend, and the feeding rate on the leaves of Metarhizium anisopliae-soybean symbionts was relatively flat, but the overall It is an upward trend, and the two finally converge at the 24th hour of inoculation.

Compared with the prior art, the present invention utilizes this strain of Metarhizium anisopliae to carry out biological control of Lepidoptera pest Spodoptera litura, rice stem borer, Asian corn borer, Hemiptera pest aphids and Sclerotinia sclerotiorum. It is an efficient and broad-spectrum biological control method with good application prospects. It utilizes the characteristics that the Metarhizium anisopliae is easy to colonize at the seedling stage and works together with soybeans to improve the resistance of soybean plants to Spodoptera litura and Sclerotinia sclerotiorum, and can better meet the requirements of agricultural production for preventing and controlling Spodoptera litura. and Sclerotinia requirements.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (18)

1. The Metarhizium anisopliae MrS Gz1-1 strain is characterized in that the strain is preserved in China general microbiological culture collection center (CGMCC) No.40481.

2. The strain of claim 1, wherein the strain has a 16S rDNA as shown in SEQ ID NO. 1.

3. Use of the metarhizium anisopliae MrS Gz1-1 according to any one of claims 1 or 2 in plant disease and pest resistance.

4. The use according to claim 3, wherein the plant disease is selected from the group consisting of a plant disease caused by sclerotinia.

5. The use according to claim 3, wherein the plant pest is selected from the group consisting of lepidoptera prodenia litura, rice stem borer, aphid and/or asian corn borer.

6. The use according to any one of claims 4 or 5, wherein the use is for inhibiting plant diseases and insect pests by constructing a fungal-plant symbiont.

7. The use according to claim 6, wherein the use is by treating young plants with a microbial inoculum containing conidia of said green muscardine fungus by root irrigation/root dipping to construct a green muscardine fungus-plant symbiont.

8. The use according to claim 7, wherein the use is to establish a green muscardine-plant symbiont by rooting/rooting the green muscardine conidia for 24 hours to colonize the plant body.

9. The use according to claim 7 or 8, wherein the bacterial agent of the metarhizium anisopliae conidium is a suspension of the metarhizium anisopliae conidium with an aqueous solution containing a suspending agent.

10. The use according to claim 9, wherein the concentration of conidia of metarhizium anisopliae in said suspension is 1 x 10 ⁸ And spores/mL, wherein the suspending agent is Tween-80 or other nonionic surfactants.

11. The method for culturing metarhizium anisopliae MrS1Gz1-1 according to any one of claims 1 or 2, wherein the method comprises the steps of:

a) Inoculating the destruxin to an SMAY solid culture medium for culturing, and collecting conidia;

b) Immersing the conidium suspension on the surface of the tested insect for 0.2-1 min for pathogenicity determination, feeding the tested insect in a 5mL sterile centrifuge tube in a single-head manner, and collecting spores after the insect corpses grow to be full of spore powder.

12. The method of claim 11, wherein the culturing conditions of step a) are: the temperature is 25-26 ℃ and the culture time is 10-14 d.

13. The method of claim 11, wherein the feeding conditions of step b) are: photoperiod 14L 10D, temperature 26+ -1deg.C, relative humidity 70+ -10%.

14. The method of claim 11, wherein the insect dipping method of step b) is as follows: immersing the tested insect body into the destruxin of the metarhizium anisopliae spore suspension for 0.2 min-1 min, and then sucking out excessive water by using sterile filter paper.

15. A method according to claim 11, wherein the test insects are all at age 2 and the corn aphid and the pea aphid are both selected from the wingless aphid class.

16. The use according to any one of claims 3 to 10, wherein the plant is selected from the group consisting of leguminous plants.

17. The use according to claim 16, wherein the leguminous plant is soybean.

18. The use of claim 17, wherein the soybean plant seedlings are in the three-out-of-multiple stage.