CN116333892A

CN116333892A - Metarhizium anisopliae and granules thereof and application of Metarhizium anisopliae and granules thereof in preventing and controlling spodoptera frugiperda

Info

Publication number: CN116333892A
Application number: CN202310246611.3A
Authority: CN
Inventors: 张维; 顾买群
Original assignee: Guizhou Cailing Biotechnology Co ltd
Current assignee: Guizhou Cailing Biotechnology Co ltd
Priority date: 2023-03-15
Filing date: 2023-03-15
Publication date: 2023-06-27

Abstract

The invention discloses metarhizium anisopliae and granules thereof and application thereof in preventing and controlling spodoptera frugiperda. The Metarhizium anisopliae (Metarhizium rileyi) strain is GZUMr46 and the preservation number is CCTCC M2023161. The granule is fermented product of GZUMr46 Metarrhizium anisopliae and cereal. The GZUMr46 metarhizium anisopliae has good production property and strong spore production capability, and can kill insects with high efficiency; the GZUMr46 metarhizium anisopliae granule provided by the invention can have good growth, germination and adhesion capability in a field environment, has a good insecticidal effect on spodoptera frugiperda, and effectively controls the population and quantity of spodoptera frugiperda by applying the granule.

Description

Metarhizium anisopliae and granules thereof and application of Metarhizium anisopliae and granules thereof in preventing and controlling spodoptera frugiperda

Technical Field

The invention relates to the field of agricultural pest control, in particular to metarhizium anisopliae, a granule thereof and application thereof in controlling spodoptera frugiperda.

Background

Spodoptera frugiperda Spodoptera frugiperda (j.e. smith), belonging to Lepidoptera, noctida, spodoptera, also known as fall armyworm (english name fall armyworm), is a omnivorous major agricultural pest native to tropical and subtropical areas of america. In early 2019, the corn is invaded into Yunnan of China and then rapidly spread to regions such as southwest, south China, south of the river, north China and the like, and is mainly infected with crops, so that the corn production and grain safety of China are seriously threatened.

In order to ensure the corn yield and grain safety, pesticides are generally used for preventing and controlling spodoptera frugiperda, and conventional pesticides include chemical pesticides and biological pesticides. At present, the domestic spodoptera frugiperda prevention and control technology mainly uses chemical pesticides. However, the long-term use of chemical pesticides in large quantities results in the generation of drug resistance of pests, rampant pests and pesticide residues, and is harmful to human health and ecological environment. Therefore, an effective and low-risk green prevention and control method is constructed to realize sustainable control of spodoptera frugiperda and become a research hotspot. The biopesticide (Biological pesticide) is a preparation which kills or inhibits agricultural pests by using living organisms (fungi, bacteria, insect viruses, transgenic organisms, natural enemies and the like) or metabolites thereof (pheromones, auxins, naphthylacetic acid, 2,4-D and the like), and is one of the most effective control means of spodoptera frugiperda. Compared with chemical pesticides, the pesticide composition is safer and more effective, is environment-friendly and is not easy for pests to generate resistance.

Entomopathogenic fungi are the largest group of entomopathogenic microorganisms, and more than 60% of insect deaths in nature are caused by mycoses. Metarrhizium anisopliae is one of the most widely studied and used entomopathogenic fungi. The metarhizium anisopliae host has wide range, can parasitize more than 200 kinds of pests of 8 meshes and 30 families, is harmless to human and livestock, is safe to natural enemy insects, does not pollute the environment, is a good substitute for chemical pesticides, and has been widely used for pest control. The most common dosage forms of the metarhizium anisopliae pesticides at present are dispersible oil suspending agents and wettable powders. These formulations are mainly applied by spraying and powder spraying. The spodoptera frugiperda has the characteristic of hidden damage, namely, the larvae of spodoptera frugiperda above 2 years old usually dig into corn leaves or enter into a cluster area to damage corn, and the pests of the corn leaves are difficult to directly contact due to spraying and powder spraying, so that the stability and the control effect of the insecticidal effect of the fungal pesticide are seriously influenced. In addition, under general natural conditions, the field environment is mostly low-humidity and high-temperature, the population level of the insect pathogenic fungi surviving in the field is very low, most spores can be decomposed and disappear quickly in the environment, the germination and growth of the spores are restricted by environmental parameters such as the environmental temperature, the humidity, the time and space, and the like, the insecticidal activity is not high, and the infection control effect of the metarhizium anisopliae on pests is influenced. Therefore, there is an urgent need to develop a novel metarhizium anisopliae biological agent which has a good insecticidal effect on spodoptera frugiperda, can be applied to any part of plants, including the parts of the seedling core, the heart leaf, the bell mouth and the like of the hidden plant seedling, and can be applied to any other part where insect pests occur, including the soil or around the plant root system and the like due to the autonomous growth, germination, attachment and infection capability of the metarhizium anisopliae preparation in the field.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a Metarhizium anisopliae (Metarhizium rileyi) characterized in that the strain is GZUMr46 and the preservation number is CCTCC M2023161.

The second object of the invention is to provide a green muscardine fungus granule, wherein the green muscardine fungus used in the granule is a deposited strain of GZUMr46, and the granule is a fermentation product of GZUMr46 and grains. The cereal can be wheat, corn, sorghum, rice, etc. Any method for fermenting the metarhizium anisopliae GZUMr46 and the grains is feasible, the metarhizium anisopliae is adhered to the grains after the fermentation is finished, namely the grains are granules, the metarhizium anisopliae can be directly applied without crushing, spodoptera frugiperda can be prevented and treated by placing any part of the plant, and particularly, the metarhizium anisopliae can be conveniently placed in a hidden position, such as a seedling core, a heart leaf, a horn mouth and the like of the plant seedling, so that the metarhizium anisopliae has good comprehensive insecticidal and plant protection effects. Of course, to facilitate storage of the granule, the fermented granule can be dried in a forced air drying oven at 30-35deg.C for 24 hr, reduced in water, vacuum-bag sealed and stored at 4-25deg.C

Further, the preparation method of the ferment comprises the following steps: the GZUMr46 metarhizium anisopliae is subjected to expanded culture by an SMAY liquid culture medium, and the culture product is liquid seeds; soaking grains in clear water for 24-48h, taking the soaked sterilized grains as a solid culture medium; inoculating the liquid seed to a solid culture medium according to the volume-mass ratio of 10-20mL/100g, and then carrying out solid fermentation.

Further, the method comprises the steps of,

the preparation method of the liquid seed comprises the following steps:

(1) Preparing an SMAY solid culture medium, sterilizing by high-pressure steam, and pouring the SMAY solid culture medium into a sterile culture dish to obtain an SMAY flat plate;

(2) Preparing an SMAY liquid culture medium, and sterilizing by high-pressure steam for later use;

(3) Inoculating the GZUMr46 metarhizium anisopliae on the SMAY plate in the step (1), and carrying out light cycle 14L at 25-30 ℃: culturing for 7-25 days in 10D;

(4) Scraping spores on the plate after culturing in the step (3) to obtain 1×10 ⁸ Inoculating spore/mL spore suspension into the SMAY liquid culture medium in the step (2) according to the volume ratio of the inoculum size of (1000-2000), and shaking and expanding culture for 4-6 days at 25-30 ℃ at 180-250r/min, wherein the culture product is liquid seeds;

the solid culture medium is subpackaged into inoculation bags, and then is subjected to high-pressure sterilization and cooling, and then liquid seeds are inoculated, wherein the specification of each inoculation bag is (20-50 cm) x (30-100 cm), and the content of each inoculation bag is 20-70% of the volume of each inoculation bag;

the fermentation condition of the solid fermentation is 28 ℃, the humidity (70+/-5)%, and the photoperiod L: d=16: 8 for 15-30 days.

The invention further aims to provide an application of the Metarhizium anisopliae GZUMr46 in preventing and controlling spodoptera frugiperda.

Further, the Metarhizium anisopliae GZUMr46 is used in combination with emamectin benzoate or Bt formulation to control spodoptera frugiperda. The GZUMr46 and emamectin benzoate or Bt preparation have a synergistic effect, and the toxicity to spodoptera frugiperda is improved.

The invention aims at providing an application of the GZUMr46 metarhizium anisopliae granules in preventing and controlling spodoptera frugiperda.

Further, the metarhizium anisopliae granule is used for preventing and controlling spodoptera frugiperda by being placed in any part of a plant or being directly applied to the root of the plant or surrounding soil, wherein any part comprises the plant horn mouth, the plant seedling core or the plant heart leaf. The spodoptera frugiperda has the characteristic of hidden harm, and can be drilled into heart leaves, seedling cores and bell mouths to cause harm.

Further, the GZUMr46 Metarrhizium anisopliae granule is used in combination with one or more of sex attractant, emamectin benzoate, bt preparation and black egg bees. The GZUMr46 Metarrhizium anisopliae granule is used in combination with one or more of sex attractant, emamectin benzoate, bt preparation and black egg bee, and has synergistic effect and improved control effect.

The invention directly separates and screens a high-efficiency insecticidal metarhizium anisopliae strain GZUMr46 with good production property and strong spore production capability from naturally infected spodoptera littoralis stiff worms, and the strain is subjected to grain solid fermentation to obtain metarhizium anisopliae fungus granules. The strain GZUMr46 has good insecticidal effect on spodoptera frugiperda, and the metarhizium anisopliae fungus granule has good growth germination attachment capability in a field environment, can be applied to any part of the plant, including hidden positions in the plant, and has good insecticidal effect on spodoptera frugiperda. The green muscardine fungus granule is applied to effectively control the population and the quantity of spodoptera frugiperda.

Description of biological preservation

Metarhizium anisopliae GZUMr46, latin Metarhizium rileyi, deposited in China center for type culture Collection, accession number: cctccc M2023161, the preservation date 2023, 2 months and 20 days, eight paths 299 of the armed forces district of armed chinese, hubei province.

Drawings

FIG. 1 is a diagram showing the isolation and identification of the strain GZUMr46 and a phylogenetic tree of the present invention;

FIG. 2 is a schematic view ofThe GZUMr46 of the invention is 1 multiplied by 10 ⁸ Pathogenicity result diagram of spore/mL on spodoptera frugiperda 2, 4-instar larvae;

FIG. 3 shows a GZUMr46 of the present invention of 1X 10 ⁸ The pathogenicity and semi-lethal time results of the combined use of spores/mL and emamectin benzoate on spodoptera frugiperda 2, 4-year larvae are shown;

FIG. 4 shows a GZUMr46 of the present invention of 1X 10 ⁸ Pathogenicity and semi-lethal time results of spodoptera frugiperda 2, 4-instar larvae in combination with 6 g/LBt;

FIG. 5 is a graph of field test results of single doses and their synergistic effect with the Metarhizium anisopliae granules of the present invention.

Detailed Description

The present invention will be described in detail below with reference to examples and drawings, which are for illustrative purposes only and are not limiting to the scope of application of the present invention. The present invention is not limited to the following embodiments or examples, and modifications and variations made without departing from the spirit of the present invention are intended to be included in the scope of the present invention.

Experimental example 1: identification of strains

(1) Morphological identification

Picking a trace of fresh spores of spodoptera frugiperda stiff worm collected naturally under a sterile condition by using an inoculating loop, streaking on an SMAY plate, and placing the plate at 28 ℃ with a photoperiod of 14L: culturing under 10D conditions. Observing the growth of the strain for 3-6 days, purifying and separating, inoculating the separated strain on an SMAY culture medium, wherein the temperature is 28 ℃ and the light cycle is 14L: culturing for 15 days under 10D conditions. The microstructure such as colony morphology, hypha and conidium morphology of the strain was observed by using an optical microscope. The results show that: the colonies on the medium were white short villiated, and after about 5-7 days, the colonies began to produce spores, and the colonies turned light green, and the spores turned dark green or yellow green at the later stage, and were powdery (FIGS. 1C, D, E). Conidiophores are oval, slightly pointed at one end and slightly rounded at the other end (fig. 1F). From the above features, the isolated metarhizium anisopliae Metarhizium rileyi was designated as strain number: GZUMr46.

(2) Molecular characterization

According to fungal genomic DNA extraction testKit instructions for isolating genomic DNA from mycelium. The isolated genomic DNA of the strain is used as a template, and the fungus universal primers ITS1,5'-CCGTAGGTGAACCTGCGG-3', ITS4 and 5' TCCTCCGCTTATTGATATGATGC are used for carrying out PCR amplification of the strain ITS-rDNA sequence to identify the fungus strain. PCR reaction system: templite (genomic DNA20-50 ng/. Mu.l) 0.5. Mu.l, 10 XBuffer (with Mg) ²⁺ ) 2.5. Mu.L of dNTPs (2.5 mM each) 1. Mu.L, enzyme 0.2. Mu.L, F (10. Mu.M) 0.5. Mu.L, R (10. Mu.M) 0.5. Mu.L, double distilled H was added ₂ O to 25. Mu.L; amplification procedure: pre-denaturation at 94℃for 4min, denaturation at 94℃for 45s, annealing at 55℃for 45s, extension at 72℃for 1min for 35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃. The PCR products were detected by 1% agarose gel electrophoresis and the gel imaging results were recorded. The ITS-PCR amplification product was sequenced, and the sequencing result was shown in SEQ ID NO.1, by the company of Shanghai, inc. The strain sequencing results were submitted to NCBI database for BLAST alignment, the sequences with highest similarity were selected, MEGA software (ver 7.0) (Kumar, 2016) was used, clustalW multiple alignment was used and the phylogenetic tree was constructed using the maximum likelihood method (maximum likelihood, ML).

Several entomogenous fungi Metarhizium cylindrosporae (HQ 165693.1, GU 980047.1), metarhizium flavoviride (AY 646390.1), pochonia rubescens (AB 709860.1), pochonia suchlasporia (FJ 439582.2), nomuraea riley i (AF 368501.1), metarhizium rileyi (MH 856926, MZ 151847.1), metarhizium anisopliae (FJ 609314.1, KY 437680.1), metarhizium acridum (HQ 331457.1), metarhizium guizhouense (JF 827150.1), metarhizium pinghaense (MH 249930.1, HM 055447.1), beauveria supgli (HQ 880812.1) ITS sequence was selected and a phylogenetic tree was constructed using MEGA7.0 as an outer group with Beauveria supgli (fig. 1G). As seen in FIG. 1G, the highest ITS sequence homology to strain GZUMr46 is Metarhizium anisopliae Metarhizium rileyi (MZ 151847.1), with a similarity of 98%. The strain GZUMr46 is identified as Metarhizium anisopliae Metarhizium rileyi by combining morphological characteristics, culture characteristics and molecular biological characteristic analysis.

Experimental example 2: strain indoor virulence determination

Laboratory bioassays and field applications were performed by purchasing the chemical pesticide emamectin benzoate microemulsion (Dongguan Rui national defense biotechnology Co., ltd.), bt formulation (32000 IU/mg Bacillus thuringiensis wettable powder, wuhan Keno biotechnology Co., ltd.) and sex attractant (Nanjing south China green biotechnology Co., ltd.).

1. Virulence of GZUMr46 against spodoptera frugiperda 2, 4-instar larvae

For laboratory bioassays of fungal pathogens, 1X 10 formulations with paraffinic oil solutions ⁸ spore/mL spore suspension treated with paraffin oil solution served as control. Dropping 1×10 of healthy 2-and 4-instar larvae on the back of larvae ⁸ spore/mL spore suspension (0.2 μl/head drop of 2-instar larvae, 5 μl/4-instar) the virulence of the fungus on spodoptera frugiperda larvae was determined.

2. Toxicity to spodoptera frugiperda 2, 4-year larvae by combined use of GZUMr46 and emamectin benzoate

(1) Single-dose treatment of emamectin benzoate: the larvae of 2 years and 4 years are treated by using 5 percent and 10 percent emamectin benzoate as single agents respectively, and are taken out after being respectively soaked for 15 seconds, and are fed by artificial feed.

(2) GZUMr46 single dose treatment: soaking 2-and 4-instar larvae in 1mL of 1X 10 ⁸ Soaking in spore/mL Tween spore suspension for 15s, removing excessive bacterial liquid on the surface of the larva body with filter paper, and feeding with artificial feed.

(3) To analyze the synergy between emamectin benzoate and fungi, GZUMr46 and emamectin benzoate were treated in combination: methyl benzoate at a concentration of 5% and 10% and 1X 10, respectively ⁸ The spore/mL spore suspension is mixed to form a mixed solution, 2-age and 4-age larvae are soaked for 15s, the excessive bacterial liquid on the surfaces of the larvae is removed by filter paper, and the larvae are fed with artificial feed.

3. Toxicity to spodoptera frugiperda 2, 4-instar larvae by GZUMr46 in combination with Bt

(1) GZUMr46 single dose treatment: soaking 2-and 4-instar larvae in 1mL of 1X 10 ⁸ Soaking in spore/mL Tween spore suspension for 15s, filtering to remove larvaThe superfluous bacterial liquid is removed and the record is observed.

(2) Single-dose Bt treatment: preparing Bt liquid medicine with the concentration of 6g/L, cutting fresh corn leaves into small pieces, immersing the corn leaves into the Bt liquid medicine with the concentration of 6g/L, and taking out the corn leaves after 15s of immersion; and 3, on the leaf transferring water absorbing paper, in a light-shading area with good ventilation condition, naturally airing the leaf, and feeding 2-year and 4-year larvae, and observing and recording.

(3) To analyze synergy between Bt and fungi, GZUMr46 was combined with Bt: soaking 2-and 4-instar larvae in 1mL of 1X 10 ⁸ The spores/mL Tween spore suspension is soaked for 15 seconds, and then 6g/L Bt liquid medicine soaked corn leaves are used for feeding, and observation and record are carried out.

4. Experimental results

(1) Virulence effects of GZUMr46 on spodoptera frugiperda 2, 4-instar larvae:

indoor biological activity measurement shows that the metarhizium anisopliae strain GZUMr46 has stronger virulence to 2 and 4-year larvae. Metarhizium anisopliae strain GZUMr46×10X ⁸ Mortality of spodoptera frugiperda larvae at 2 and 4 ages at 9 days after spore/mL concentration treatment was 68% (fig. 2A) and 72% (fig. 2C), respectively, whereas mortality of larvae at 2 and 4 ages was only 19% and 18% for the control group; the half-life time of the metarhizium anisopliae strain GZUMr46 for infecting 2-age and 4-age larvae is 3.843 days (figure 2B) and 4.38 days (figure 2D) respectively, and the half-life time of the control group 2-age and 4-age larvae is 18D and 21D respectively, and the mortality of the control group and the treatment group are obviously different; after the metarhizium anisopliae GZUMr46 is infected and killed, the insects become stiff, white hypha grows on the surfaces of the insects after the insects are placed in an incubator for one day of moisture culture, green conidia grow after 3-4 days of moisture culture, and the spore color becomes scattered after 7 days (fig. 1A and B). The metarhizium anisopliae GZUMr46 has remarkable insecticidal effect on spodoptera frugiperda larvae.

(2) Toxicity effects of GZUMr46 in combination with emamectin benzoate on spodoptera frugiperda 2, 4-instar larvae:

1) Toxicity effects of GZUMr46 in combination with 5% emamectin benzoate on spodoptera frugiperda 2-year larvae

The result shows that 5d after treatment, through GZUMr46, 5% emamectin benzoateThe cumulative survival rates of 2-year-old larvae after single-dose treatment of the mycotinyl benzoate and combined treatment of the GZUMr46 and 5% emamectin benzoate are respectively as follows: 73.3%, 50%, 13.3% (fig. 3A); 5% methyl methylamino avermectin benzoate and 1X 10 ⁸ Half-life time of spores/mL GZUMr46 single dose to 2-instar larvae was 4.2d and 7.65d (FIG. 3B), respectively, and 1X 10 ⁸ The half-life time of the spore/mL GZUMr46 and 5% emamectin benzoate combined treatment group is 3.16d (figure 3B), and the remarkable difference shows that after the 5% emamectin benzoate and the destruxer GZUMr46 are mixed, the mortality rate is increased, the destruxer action is slow, the emamectin benzoate action is fast, and the insecticidal toxicity is increased by the mixed use of the reduced emamectin benzoate and the destruxer GZUMr46.

2) Virulence effects of GZUMr46 and 10% emamectin benzoate in combination treatment on spodoptera frugiperda 4-instar larvae

The result shows that the mixed solution of 10% emamectin benzoate and metarhizium anisopliae GZUMr46 has remarkable control effect on the 4-instar larvae of spodoptera frugiperda, and the cumulative survival rates of the treated 6d, the combined treatment of the metarhizium anisopliae GZUMr46, 10% emamectin benzoate and metarhizium anisopliae on the 4-instar larvae are 86.67%, 80% and 43.3% respectively (figure 3C); 10% methyl methylamino avermectin benzoate and 1X 10 ⁸ The half-lethal time of the combined treatment of spores/mL of Metarrhizium anisopliae GZUMr46 on 4-instar larvae was 5.68D (FIG. 3D), which is significantly shorter than 8.67D and 1×10 of 10% emamectin benzoate ⁸ spores/mL GZUMr46 alone treated 10.37D (fig. 3D). The combined use of 10% emamectin benzoate and metarhizium anisopliae GZUMr46 is shown to accelerate the insecticidal effect.

(3) Toxicity effects of GZUMr46 in combination with Bt on spodoptera frugiperda 2, 4-instar larvae:

1) Experimental results show that the GZUMr46 and Bt combined use has a certain difference in pathogenicity of spodoptera frugiperda 2 and 4-year larvae.

2) Virulence effects of GZUMr46 in combination with 6g/L Bt on spodoptera frugiperda 2-year larvae

On the 4.5 th day after treatment, the cumulative survival rates of 2-year-old larvae subjected to combined treatment of the metarhizium anisopliae GZUMr46, bt, and GZUMr46 and Bt are respectively 83.3%, 36.7% and 3% (figure 4A); LT50 is respectively: 7.64d, 3.66d and 2.58d (figure 4B), which have obvious differences, show that the insecticidal effect is quickened after the metarhizium anisopliae GZUMr46 is mixed with Bt;

3) Virulence effects of GZUMr46 in combination with 6g/L Bt on spodoptera frugiperda 4-instar larvae

The cumulative mortality rate of the combined use of the metarhizium anisopliae GZUMr46 and Bt on 4-year-old larvae is higher than that of the single-dose treatment of Bt and GZUMr46, and the cumulative survival rates of the 4-year-old larvae after the combined treatment of the metarhizium anisopliae GZUMr46, bt and GZUMr46 and Bt are 86.67%, 53.3% and 16.67% respectively (figure 4C); 1X 10 ⁸ The half-life time of spores/mL of Metarrhizium anisopliae GZUMr46 and 6g/L Bt mixed with 4-instar larvae is 3.81D (FIG. 4D), which is shorter than 1×10 ⁸ spore/mL GZUMr46 (LT ₅₀ =10.37d) and 6g/L Bt alone (LT ₅₀ =5.18d) (fig. 4D).

Experimental example 3: metarrhizium anisopliae fungus granule production

1. Method for producing metarhizium anisopliae fungus granule

(1) Preparing an SMAY solid culture medium: weighing 40g of maltose, 10g of peptone, 10g of yeast extract and 20g of agar, and fixing the volume to 1L by pure water; steam sterilization under high pressure for 20min, cooling to about 50deg.C, and introducing the culture medium into a sterile culture dish in a sterile operation table to obtain an SMAY plate.

(2) Preparing an SMAY liquid culture medium: weighing 40g of maltose, 10g of peptone and 10g of yeast extract, and sterilizing with steam under high pressure for 20min with pure water to 1L.

(3) GZUMr46 metarhizium was inoculated on SMAY plates and photoperiod 14L at 28 ℃): 10D for 15 days.

(4) Scraping spores on a plate to obtain 1×10 ⁸ spore/mL spore suspension 100. Mu.L spore suspension was inoculated into 300mL Erlenmeyer flask containing 100mL of SMAY liquid medium, and cultured with shaking at 28℃for 5 days at 220r/min to obtain a culture product as liquid seed for the next solid phase fermentation.

(5) The grains soaked in clean water for 24 hours were weighed and placed in 20cm×50cm inoculation bags (purchased from plastics limited, upper jun, wenzhou) with a weight of 100g per bag, and then autoclaved at 121 ℃ for 30 minutes for use.

(6) Inoculating the liquid seeds of the step (4) into the inoculation bags filled with grains of the step (5), wherein the inoculation amount of each bag of the liquid seeds is 10mL, and then the liquid seeds are subjected to light cycle L at 28 ℃ and humidity (70+/-5 percent): d=16: 8, fermenting and culturing for 15-30 days to obtain the fermentation product of the GZUMr46 metarhizium anisopliae and the grains, wherein the fermentation product is the GZUMr46 metarhizium anisopliae granule.

2. Measuring spore yield, germination rate, pollution rate and water content of GZUMR46 Metarrhizium anisopliae granules

And after the solid fermentation is finished, the solid fermentation is the granule, and the spore yield of the granule (wheat, corn and rice) after the fermentation is finished is measured. The results show that the wheat has the highest spore yield, and the spore yields of 15d, 20d and 25d after inoculation are 1.8125 multiplied by 10 respectively ⁹ Spores/g, 3.8125X 10 ⁹ Spores/g and 8.2825 ×10 ⁹ Spores/g (Table 1). The spore yield of rice and corn is similar at 15 days, 20 days and 25 days after inoculation, and is 4.375-5.875×10 respectively ⁸ Spores/g, 1.1-1.19X10 ⁹ Spores/g and 1.2-1.3X10 ⁹ Spores/g (Table 1).

TABLE 1 conidium amount of GZUMr46 granules on different media

The moisture content, spore germination rate and pollution rate of spores produced by wheat were further analyzed. The moisture content of wheat germinated spores is 7.07%, and the moisture content of the wheat culture medium after spore removal is 7.13%. The spore germination rate is 95.81%, and the pollution rate is 1.3%.

Drying the fermented green muscardine fungus granule in a blast drying oven at 30-35 deg.c for 24 hr, lowering water content, vacuum sealing, and storing at 4-25 deg.c. Wherein the green muscardine fungus granules obtained by wheat fermentation are used in the following field experiments.

Experimental example 4: field application

The field test was performed in the county of the Pingzhou town of the Miao nationality of the southern Brix of Guizhou province for 5 to 10 months in 2022. The test field was designed for a total of 7 pairs of plots, including control and treatment two groups, as shown in table 2. Each plot comprises 7 ridge columns of a comparison and treatment group, two side ridges are isolation ridge columns which do not carry out any treatment, and 5 inner ridge columns are respectively arranged as the comparison and treatment. Each row consists of two rows, each row having about 120 seedlings of corn, initially having 3-6 leaves.

Carrying out field experiments for two times, and carrying out monitoring on the adult spodoptera littoralis in the field and investigation on the effect of the spodoptera littoralis eggs in the black egg bees of the spodoptera littoralis in the field in the first experiment; the second test applied the green muscardine fungus granule and investigated the number of spodoptera frugiperda larvae and the maize plant integrity rate.

In the first field test, the prevention and treatment effects of sex attractant and black egg bees of spodoptera frugiperda on adults and eggs of spodoptera frugiperda are analyzed respectively. The field test was performed on the CK0/T0, CK1/T1 and CK2/T2 cells on day 12 of 7 months, when the maize seedlings had 3-5 leaves. CK0 and T0 plots are adult detection groups, and two character attractants are respectively arranged for adult detection. Two character attractants are respectively placed in five inner ridges of the CK1 land block and the T1 land block, and adults are monitored; and 5 inner ridges of the CK2 and T2 plots, a black egg bee egg of the noctuid is placed on two sides of each ridge, and the egg blocks are fixed on the back surfaces of the corn leaves through double faced adhesive tape. The number of spodoptera frugiperda larvae and the number of adults monitored were investigated on day 22 of 7 months.

The second field experiment was performed on CK1-6 and T1-6 6 plots on day 31 of 7 months. A spodoptera frugiperda egg is applied to two sides of five inner ridges of the land block, which are respectively formed by CK1-6 and T1-6 6, and the spodoptera frugiperda egg blocks are fixed on the back surfaces of the corn leaves by double-sided adhesive tapes. For CK1 and T1, both control and treatment groups were provided with two character information traps, but the treatment group was supplemented with metarhizium anisopliae granules, which were applied to each corn cob, bell mouth or leaf to analyze the synergy of the leys anisopliae GZUMr46 and sex pheromone. CK2 and T2 are the noctuid black egg bees and fungus treatment group, after the noctuid black egg bees are released in the first field test, the noctuid black egg bees are not released in the CK2 and T2 groups, and the metarhizium anisopliae granule is applied to the T2 cell to analyze the synergistic effect of the Metarrhizium anisopliae GZUMr46 and the noctuid black egg bees. CK3 and T3 are single fungus treatment groups, and one seed grain (wheat seed grain is soaked in clear water for 24 hours and then sterilized by high-pressure steam for 30 minutes and cooled) or one metarhizium anisopliae granule (oval, length: 0.6mm-1 cm) is respectively put into each corn seedling core or core leaf in all corn seedlings of CK3 and T3 plots. CK4 and T4 are methyl emamectin benzoate and fungus treatment groups, methyl emamectin benzoate emulsifiable concentrate is taken from a control group and mixed with unvaccinated grains or metarhizium anisopliae granules according to the recommended field quantity, the emulsifiable concentrate is adhered to the grains or granules, and the emulsifiable concentrate is applied to corncob or heart leaves at the ridges of CK4 and T4. CK5 and T5 are groups treated by Bt and fungi, bt preparations are respectively taken and mixed with unvaccinated grains or green muscardine fungus granules, so that Bt powder is adhered to the grains or the granules and respectively applied to corncob or heart leaves or bellmouth at the ridge columns of CK5 and T5. CK6 and T6 are comprehensive prevention and control treatment groups, two character attractants and noctuid black egg bees (released by first field test) are respectively arranged on CK6 and T6 plots, and methyl methylamino avermectin benzoate and Bt are respectively mixed with sterile seeds or fungi particles and then applied to corncob, heart leaves or horn mouth.

TABLE 2 test reagents and test design and results of the 2 nd field experiment

The results show that:

(1) The field trials were conducted on days 7, 12 to 23. The first field test was performed on day 7 months 12, when the maize seedlings had 3-6 leaves and no spodoptera frugiperda eggs or larvae were present in the maize. In the field trials, two sex attractant were placed in each of the two cells of CK0 and T0 for adult monitoring in the first field trial, the average spodoptera frugiperda adult numbers of CK0 and T0 sex attractant were 7.5 and 6.5, respectively, and then in the second investigation, CK0 and T0 sex attractant further lured 1.5 and 2 spodoptera frugiperda adults (fig. 5A) and reduced the spodoptera frugiperda larva numbers from 100% (CK 3) to 30% (CK 0), 29% (T0), respectively, with plant integrity rates increased from 0% to 52.8% (CK 0), 31% (T0) (fig. 5B).

(2) In the first field test, the control of sex attractant on spodoptera frugiperda adults in CK1 and T1 cells and spodoptera frugiperda black bees in CK2 and T2 cells was mainly investigated, and 1 egg mass was released on each inner ridge of all cells of CK1-6 and T1-6 prior to application to maintain consistency. The result shows that the average number of trapped adults of each sex attractant in the T1 region is 6 (the average number of larvae of corn per hundred plants in the T1 region is 20.1 percent and is obviously lower than that in the control region (CK 1, 50.8 percent) (figure 5C). In the first field test, the control effect of the noctuid black egg bees on the corn per hundred plants can reach 17.6 percent (figure 5E) and is obviously lower than that in the control region (CK 2, 45.5 percent) (figure 5E). The result shows that both the sex attractant and the noctuid black egg bees can effectively reduce the number of the adult spodoptera frugiperda and eggs in the field test, thereby further reducing the number of the larvae.

(3) In the second field trial, we collected the egg masses from all cells and released 1 egg mass per row on average prior to application. The administration of the green muscardine fungus GZUMr46 granule alone reduced the larval count from 100% (CK 3) to 40.0% (T3), and the whole plant rate increased from 0% (CK 3) to 40.0% (T3) (FIG. 5G). In the second field trial, the single Shi Xing attractant only slightly reduced the larval load from 100.0% (CK 3) to 78.0% (CK 1), and the undamaged plant rate was slightly increased from 0.0% (CK 3) to 12.2% (CK 1) (fig. 5D). The combination of sex attractant and fungal particle (T3) did not significantly reduce the number of larvae (52.2%) (fig. 5D), but the combination of both significantly improved plant integrity from 0 (CK 3) to 71.0% (T1) (fig. 5D). The synergistic effect of the Metarhizium anisopliae particles and the sex attractant is revealed in terms of plant protection effect.

(5) After the noctuid black wasps are released from the first field test, the CK2 and T2 cells do not release additional parasitic wasps, and the parasitic wasps can be considered to continue fertilization and breeding in the field. The second test result shows that the larva quantity of spodoptera frugiperda is reduced to 68.9% (CK 2) after the spodoptera frugiperda black wasp is singly applied for about 1 month (figure 5F), but is significantly lower than the larva quantity of the control (CK 3), and the corn plant integrity rate can reach 37.2% (figure 5F). The combined treatment of the black bees and the green muscardine fungus granules can further reduce the number of larvae to 32.5% and improve the plant integrity to 78.7% compared with the black bees or green muscardine fungus granules alone (fig. 5F). This reveals the synergy of the Metarhizium anisopliae granules and the Spodoptera exigua black wasps from both the control of larva numbers and plant protection effects.

(6) Compared to the control, single administration of emamectin benzoate only reduced the larval numbers from 100.0% to 80.3%, but significantly increased the undamaged plant rate from 0.0% to 80.3% (fig. 5H). The co-treatment of emamectin benzoate with the Metarrhizium anisopliae granule (T4) reduced the larval count (40.0%) to the level of the Metarrhizium anisopliae granule (40%) of Shan Shilai and maintained the non-insect pest-receiving rate (79.1%) to the level of emamectin benzoate alone (80.3%) (FIG. 5H). Treatment with Bt formulations alone (CK 5) did not significantly reduce the number of larvae (89.1%), but significantly increased the rate of non-infested plants from 0.0% to 23.7% compared to control (CK 3) (fig. 5I). Co-treatment of bt+metarhizium anisopliae granules reduced the number of larvae to 43.0% and increased the whole plant rate to 48.0% (fig. 5I), comparable to the level of metarhizium anisopliae granules used alone. CK6 and T6 are comprehensive treatment areas and consist of sex attractant, black egg bees of noctuid (released in the first test field), methyl emamectin benzoate, bt preparation, and sterile seeds (CK 6) or Metarhizium anisopliae granules (T6). CK6 cells reduced the number of larvae to 47.2% and the whole plant rate to 76.2% compared to control CK3 (fig. 5J). The number of larvae in the T6 region was further reduced to 32.7%, and the non-insect plant rate was similar to that in the CK6 region (75.1%) (FIG. 5J).

In conclusion, the GZUMr46 metarhizium anisopliae granules are used together with one or more of sex attractant, emamectin benzoate, bt preparation and black egg bees, so that the control effect can be improved, and the method is an effective method for improving the control of spodoptera frugiperda larvae and adults and protecting the integrity rate of plants.

It should be noted that, the experimental operations related to the above experimental examples have a certain universality, so that the detailed description is not given, and the detailed description is omitted for the content of the related operations in other experimental examples or for the reference to the prior art.

Claims

1. The Metarhizium anisopliae (Metarhizium rileyi) is characterized in that the strain number is GZUMr46 and the preservation number is CCTCC M2023161.

2. The metarhizium anisopliae granule is characterized in that metarhizium anisopliae in the granule is a GZUMr46 strain, and the granule is a fermentation product of GZUMr46 and grains.

3. The metarhizium anisopliae granule according to claim 2, wherein the cereal comprises wheat, corn, sorghum or rice.

4. The metarhizium anisopliae granule as claimed in claim 3, wherein the preparation method of the ferment comprises the following steps: the GZUMr46 metarhizium anisopliae is subjected to expanded culture by an SMAY liquid culture medium, and the culture product is liquid seeds; soaking grains in clear water for 24-48h, and taking the soaked grains as a solid culture medium; the liquid seeds are inoculated to a solid culture medium according to the volume-mass ratio of 10-20mL/100g, and then solid fermentation is carried out.

5. The metarhizium anisopliae granule according to claim 4,

the preparation method of the liquid seed comprises the following steps:

the solid culture medium is sub-packaged into inoculation bags, and then is subjected to high-pressure sterilization and then is inoculated with liquid seeds, the specification of each inoculation bag is (20-50) cm multiplied by (30-100) cm, and the containing amount of each inoculation bag is 20-70% of the volume of each inoculation bag;

6. Use of metarhizium anisopliae gzur 46 as claimed in claim 1 for controlling spodoptera frugiperda.

7. The use according to claim 6, wherein the metarhizium anisopliae gzur 46 is used in combination with emamectin benzoate or Bt formulations.

8. Use of the metarhizium anisopliae granules according to any one of claims 2 to 5 for controlling spodoptera frugiperda.

9. The use according to claim 8, wherein the metarhizium anisopliae granules are used for controlling spodoptera frugiperda by being placed in any part of the plant, including in the plant's trumpet, in the seedling core or in the heart leaf, or by being directly applied to the plant's roots or surrounding soil.

10. The use according to claim 8 or 9, wherein the GZUMr46 metarhizium anisopliae granule is used in combination with one or more of sex attractant, emamectin benzoate, bt preparation and black wasp.