A strain of Aspergillus nomiae used to control fall armyworm

Abstract

This invention belongs to the field of microbiology, and specifically discloses a strain of Aspergillus for controlling fall armyworm. Aspergillus nomiae The technical problem this invention aims to solve is how to control plant damage caused by noctuid moths. This invention provides an Aspergillus fungus for controlling plant damage caused by noctuid moths, specifically Aspergillus nomi (…). Aspergillus nomiae WN_AN1, with accession number CGMCC No. 41741. Experiments have shown that this Aspergillus fungus has a high mortality rate against third instar larvae of the fall armyworm and can be used for the control of the fall armyworm.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to a strain of Aspergillus for controlling fall armyworm. Aspergillus nomia . Background Technology

[0002] fall armyworm Spodoptera frugiperda JE Smith is a major agricultural pest originally distributed in tropical and subtropical regions of the Americas, which can damage more than 350 crops, including corn, wheat, rice, soybeans, and sorghum. As an invasive alien species, it has caused significant harm to my country's food production security since its invasion of China at the end of 2018 and has been listed in the "List of Class I Crop Diseases and Pests".

[0003] Hainan Province has a hot and humid climate, which is very suitable for the growth, development, and reproduction of the fall armyworm. Based on biological and climatic predictions, it is estimated that 9-10 generations will occur in Hainan Province annually, making it a veritable "year-round breeding area." Hainan Province is also an important base for maize breeding in southern China, and the local areas of Hainan (Sanya, Ledong, Dongfang, etc.) have a long-standing tradition of growing fresh maize. The fall armyworm particularly favors maize and can damage maize at all stages, including the seedling, tasseling, and maturity stages. In just a few years since the fall armyworm invaded Hainan, it has become a major pest of maize in the fields, leading to a significant increase in the number of chemical pesticide applications during the growth period of a single maize crop, from 4.27-5.47 times in 2019 to 7.73-8.77 times. The overuse of chemical pesticides to control the fall armyworm also threatens local pollinating insects such as honeybees. Therefore, the situation for controlling the fall armyworm in Hainan Province is particularly severe, and there is an urgent need to develop greener and more sustainable control technologies to achieve long-term control of the fall armyworm.

[0004] Entomopathogenic fungi are a class of soil-derived insect pathogenic microorganisms widely used in biological control due to their diverse species, high virulence, ease of large-scale production, and environmental friendliness. Currently, the two most widely studied entomopathogenic fungi are *Metarhizium anisopliae* and *Beauveria bassiana*. These fungi have a broad host range and are mainly registered for the control of harmful arthropods, especially important agricultural pests of the Lepidoptera, including the fall armyworm, diamondback moth, and beet armyworm. Since the invasion of the fall armyworm into my country, the exploration and utilization of fungal biological control resources has become a research hotspot. Although strains of *Beauveria bassiana* and *Metarhizium anisopliae* with highly effective insecticidal and ovicidal activity against the fall armyworm have been reported in the Americas, the original habitat of the fall armyworm, research in my country shows that neither of these two fungi currently has ovicidal activity. Therefore, there is a need to develop and utilize more effective biocontrol fungal strains against the fall armyworm. Summary of the Invention

[0005] The technical problem this invention aims to solve is how to prevent and control plant damage caused by noctuid moths (fall armyworm). To this end, a strain of Aspergillus is provided, wherein the Aspergillus is *Aspergillus nomi* (…). Aspergillus nomiae WN_AN1 is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 41741.

[0006] The present invention also provides a composition containing the aforementioned Aspergillus. The active ingredient of the composition may contain the Aspergillus and / or its metabolites and / or its culture.

[0007] The culture may be a substance obtained by culturing the Aspergillus in a microbial culture medium. The culture may be obtained by culturing the Aspergillus in potato dextrose agar (PDAY) medium in a 28°C incubator.

[0008] The composition comprises spores, hyphae, plant tissue infected with the Aspergillus, and / or a culture medium containing the Aspergillus species. The spores and hyphae are mycelia of the Aspergillus species, and the plant tissue infected with the Aspergillus species also contains the corresponding mycelia.

[0009] The active ingredients of the above composition may also contain other biological or non-biological components, and those skilled in the art can determine the other active ingredients of the above composition based on the effects of the composition.

[0010] The above composition can also be used as a microbial agent.

[0011] The above-mentioned inoculants can be microbial inoculants.

[0012] The aforementioned microbial agents refer to live microbial preparations made by using a carrier as an adsorbent to adsorb the fermentation broth or solid fermentation products of the target microorganisms after propagation.

[0013] The above-mentioned microbial agents can be in various dosage forms, including but not limited to liquids, emulsions, suspensions, powders, granules, wettable powders, or water-dispersible granules.

[0014] Depending on the needs, the microbial agent may also include a carrier. The carrier may be a solid carrier or a liquid carrier.

[0015] The composition is any one of the following: A1) Compositions with biocontrol effects; A2) Compositions for controlling noctuid moths; A3) Composition for controlling fall armyworm.

[0016] The fall armyworm mentioned can be the fall armyworm larva.

[0017] The present invention also provides a method for preparing the above composition, the method comprising the step of using the Aspergillus as a component of the composition.

[0018] The present invention also provides the use of the aforementioned Aspergillus or composition in the preparation of products.

[0019] The product has at least one of the following properties: B1) It has biocontrol effects; B2) Control of noctuid moths; B3) Control of fall armyworm.

[0020] The present invention also provides a method for controlling the fall armyworm from damaging plants, the method comprising bringing a biocontrol product into contact with the plants to control the fall armyworm, wherein the biocontrol product is the Aspergillus or a combination thereof.

[0021] In the method, bringing the biocontrol product into contact with the plant can be done by spraying the biocontrol product onto the plant (e.g., onto the leaves).

[0022] The biocontrol product may be a suspension of the Aspergillus and an aqueous solution containing a suspending agent. The suspending agent is Tween-80 or other nonionic surfactants, preferably Tween-80, at a concentration of 0.05% (V / V).

[0023] Furthermore, the concentration of Aspergillus in the suspension can be 1.0 × 10⁻⁶. 4 - 1.0×10 8 per mL.

[0024] Furthermore, the concentration of the spore suspension can be 1.0 × 10⁻⁶. 8 per mL.

[0025] The plants mentioned above can be any of the following: C1) plants of the angiosperm phylum; C2) plants of the monocotyledonous class; C3) plants of the Poales order; C4) plants of the Poaceae family; C5) plants of the Maize genus; C6) maize.

[0026] The beneficial effects of this invention: This invention has screened a new Aspergillus strain. Aspergillus nomiae This strain has a high pathogenicity to fall armyworm, and this invention provides a new option for the biological control of fall armyworm.

[0027] Compared with existing technologies, this invention utilizes Aspergillus strains isolated from soil. Aspergillus nomiae This strain is effective in controlling the fall armyworm through biological means, showing significant control over its third instar larvae and promising application prospects.

[0028] Preservation Instructions Strain name: Aspergillus nomi Latin name: Aspergillus nomiae Strain number: WN_AN1 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: December 31, 2024 CGMCC Registration Number: CGMCC NO.41741 Attached Figure Description Figure 1 This is a colony diagram of Aspergillus WN_AN1 grown on PDAY medium for 5 days.

[0029] Figure 2 Survival curves of third-instar larvae of fall armyworm infected with Aspergillus WN_AN1 spore suspension after different application methods. A represents leaf immersion method, B represents insect immersion method, and C represents feed immersion method. The blue solid line represents the control group, and the red solid line represents the Aspergillus WN_AN1 infection group.

[0030] Figure 3 After fall armyworm larvae are infected and die by Aspergillus WN_AN1, they are covered with yellow mycelium within 1-3 days.

[0031] Figure 4 Survival curves of 3rd instar larvae of fall armyworm infected with different concentrations of Aspergillus WN_AN1 spore suspension (leaf immersion method). Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] The following examples use the survival analysis tool in GraphPad Prism 8 statistical software to process the data and plot them. P < 0.05 (*) indicates a significant difference.

[0035] Example 1: Isolation and Identification of Strains 1.1 Soil sampling: Approximately 200g of soil samples were collected from a cornfield at a depth of 5-10cm in Wanning City, Hainan Province (N18.713598°, E110.173617°).

[0036] 1.2 Isolation and Identification of Biocontrol Fungi in Soil: 1g of soil sample that has passed through a 40-mesh sieve was weighed and added to 10mL of 0.5% sterile Tween water. The mixture was vortexed and allowed to stand for 15 minutes. The supernatant was then transferred to PDAY medium containing 50μg / mL streptomycin and spread. The medium was incubated at 28±2℃ for 3-5 days. Single colonies were selected and transferred to PDAY medium containing streptomycin for 3-5 days. Then, the colonies were transferred to PDAY medium without streptomycin for another 3-5 days. Pure single colonies were observed. The colonies were yellowish-green with good mycelial growth. After about 5 days of incubation, conidia were visible to the naked eye on the surface of the mycelium. Microscopic observation revealed that the morphology was similar to Aspergillus. Aspergillus mycelia are slender, and many round spores are distributed within and around the mycelium. (See [link to relevant documentation]). Figure 1 .

[0037] The PDAY culture medium is prepared as follows: Weigh 200 g of peeled potatoes, place them in 800 mL of distilled water, boil and filter to obtain juice in a 1 L beaker, then weigh 5 g of yeast extract, 20 g of sucrose and 15 g of agar powder, place them in the above 1 L beaker and stir well, add distilled water to make up to 1 L, and sterilize at 121℃ under high temperature and high pressure for 15 min.

[0038] 1.3 Molecular identification of strains: Genomic DNA was extracted from single strains using the CTAB method and amplified using the universal fungal primer pair ITS4 (5'-TCCTCCGCTTATTGATATGC-3') / ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG). Sequence alignment was performed using tools such as NCBI's BLAST website, and the results were compared with OP905648.1. Aspergillus nomiae With a sequence identity of 100%, the isolated strain was identified as Aspergillus. Aspergillus nomiae It was named Aspergillus WN_AN1.

[0039] The amplification product is sequence 1 in the sequence listing (SEQ ID NO: 1, 580bp), as follows: 5'--3'.

[0040] Example 2: Pathogenicity determination of Aspergillus WN_AN1 against fall armyworm 2.1 Test Materials Test strain: The test strain was Aspergillus WN_AN1, which was isolated and identified in Example 1 above.

[0041] Experimental insects: Third instar larvae of the fall armyworm were collected from the wild in Sanya City, purified and reared indoors for multiple generations, and then used for pathogenicity testing.

[0042] 2.2 Preparation of the inoculum: First, prepare a 0.05% Tween-80 solution with distilled water, sterilize it, and obtain sterile Tween water for later use. Incubate *Aspergillus* WN_AN1 on PDAY medium at 28±2℃ for 7-8 days. Scrape the conidia of *Aspergillus* WN_AN1 using a sterile spatula and place them into a glass tube containing 5 mL of the sterile Tween water and glass beads to obtain a spore suspension. Vortex for 2 minutes and count the number of spores under a microscope. Dilute the spore suspension with the sterile Tween water to adjust the spore concentration to 1.0 × 10⁻⁶. 8cells / mL (abbreviated as 1.0×10⁻⁶) 8 Inoculant or 1.0×10 8 A spore suspension of 1 spores / mL was prepared and diluted sequentially to obtain spore concentrations of 1.0 × 10⁻⁶ spores / mL. 7 cells / mL (abbreviated as 1.0×10⁻⁶) 7 Inoculant or 1.0×10 7 A fungal agent (spore suspension at 1.0 × 10⁻⁶ cells / mL), 1.0 × 10⁻⁶ 6 cells / mL (abbreviated as 1.0×10⁻⁶) 6 Inoculant or 1.0×10 6 (spore suspension of 1.0 × 10⁶ cells / mL) 5 cells / mL (abbreviated as 1.0×10⁻⁶) 5 Inoculant or 1.0×10 5 A fungal agent (spore suspension at 1.0 × 10⁻⁶ cells / mL), 1.0 × 10⁻⁶ 4 cells / mL (abbreviated as 1.0×10⁻⁶) 4 Inoculant or 1.0×10 4 (Spore suspension per mL).

[0043] 2.3 Determination of pathogenicity of strains: 2.3.1 Leaf dipping method: The experiment included 6 treatments, with 1.0 × 10⁻⁶ treatments. 4 Inoculum group, 1.0×10 5 Inoculum group, 1.0×10 6 Inoculum group, 1.0×10 7 Inoculum group, 1.0×10 8 Microbial agent group. Each treatment was set up with 3 replicates, and each replicate contained 20 test insects.

[0044] 2.3.1.11.0×10 4 Inoculum group: The experiment was repeated 3 times, and the experimental method for each repetition was as follows: Fresh corn leaves with a diameter of 1.5 cm were cut and soaked in 1.0 × 10⁻⁶ microbial solution obtained in 2.2. 4 The bacteria were incubated in the inoculum for 1 minute, then allowed to dry. The bacteria were then fed to selected third-instar fall armyworm larvae of uniform size in a 12-well culture plate. Each larva was fed two corn leaves. After 24 hours, the feed was replaced with artificial feed. The number of dead larvae in each treatment group was counted every 24 hours for survival analysis.

[0045] 1.0×10 5 Inoculum group: The experiment was repeated 3 times, and the experimental method for each repetition was as follows: Fresh corn leaves with a diameter of 1.5 cm were cut and soaked in 1.0 × 10⁻⁶ microbial solution obtained in 2.2. 5After 1 minute in the inoculum and drying, the larvae were selected as 3rd instar fall armyworms of uniform size and fed to them in 12-well culture plates. Each larva was fed two corn leaves, and after 24 hours, the feed was replaced with artificial feed. The number of dead larvae in each treatment group was counted every 24 hours for survival analysis.

[0046] 1.0×10 6 Inoculum group: The experiment was repeated 3 times, and the experimental method for each repetition was as follows: Fresh corn leaves with a diameter of 1.5 cm were cut and soaked in 1.0 × 10⁻⁶ microbial solution obtained in 2.2. 6 After 1 minute in the inoculum and drying, the larvae were selected as 3rd instar fall armyworms of uniform size and fed to them in 12-well culture plates. Each larva was fed two corn leaves, and after 24 hours, the feed was replaced with artificial feed. The number of dead larvae in each treatment group was counted every 24 hours for survival analysis.

[0047] 1.0×10 7 Inoculum group: The experiment was repeated 3 times, and the experimental method for each repetition was as follows: Fresh corn leaves with a diameter of 1.5 cm were cut and soaked in 1.0 × 10⁻⁶ microbial solution obtained in 2.2. 7 After 1 minute in the inoculum and drying, the larvae were selected as 3rd instar fall armyworms of uniform size and fed to them in 12-well culture plates. Each larva was fed two corn leaves, and after 24 hours, the feed was replaced with artificial feed. The number of dead larvae in each treatment group was counted every 24 hours for survival analysis.

[0048] 1.0×10 8 Inoculum group: The experiment was repeated 3 times, and the experimental method for each repetition was as follows: Fresh corn leaves with a diameter of 1.5 cm were cut and soaked in 1.0 × 10⁻⁶ microbial solution obtained in 2.2. 8 After 1 minute in the inoculum and drying, the larvae were selected as 3rd instar fall armyworms of uniform size and fed to them in 12-well culture plates. Each larva was fed two corn leaves, and after 24 hours, the feed was replaced with artificial feed. The number of dead larvae in each treatment group was counted every 24 hours for survival analysis.

[0049] 2.3.1.2 Control group (CK): 1.0 × 10⁻⁶ mg / L of the control group (CK) from 2.3.1.1 was used. 4 The bacterial agent is replaced with sterilized Tween water from 2.2, and all other operations are the same as in 2.3.1.1.

[0050] 2.3.2 Immersion method: Immerse 3rd instar fall armyworm larvae in 1.0 × 10⁻⁶ solution. 8 The spores were added to a spore suspension of 1 spore / mL for 1 minute (the control group was 0.5% sterile Tween water without fungal spores), and then transferred to 12-well culture plates containing artificial feed for feeding. The number of dead animals in the treatment group was counted every 24 hours for survival analysis.

[0051] 2.3.3 Feed Soaking Method: Soak 0.5cm × 0.5cm artificial feed blocks in 1.0 × 10... 8 The spores were added to a spore suspension of 1 spore / mL for 1 minute (the control group was 0.5% sterile Tween water without fungal spores), and then allowed to dry. The spores were then fed to selected 3rd instar fall armyworm larvae of uniform size. After 24 hours, the larvae were replaced with artificial feed and fed again. The number of dead larvae in the treatment group was counted every 24 hours for survival analysis.

[0052] Using leaf soaking, insect soaking, and feed soaking methods, at a concentration of 1.0 × 10⁻⁶... 8 The survival rate of fall armyworm larvae was determined using a spore suspension at a concentration of 1 spores / mL. The results showed that only the leaf immersion method (…) Figure 2 In the case of (A) immersion method, the survival rate of 3rd instar larvae of the fall armyworm was significantly lower than that of the control. Figure 2 (B) and soaking feed method ( Figure 2 There was no significant difference between the control group (C) and the treatment group. Furthermore, using the leaf-immersion method, the phenotypic changes of fall armyworm larvae infected and killed by Aspergillus WN_AN1 were observed 1-3 days later, showing a change from sparse mycelia on day 1 (…). Figure 3 (A), by the 3rd day it was completely covered by yellow mycelium ( Figure 3 (B) The leaf soaking method is the most effective method.

[0053] The leaf dipping method was used to determine the concentration of spore suspensions at different concentrations (1.0 × 10⁻⁶). 4 1.0×10 5 1.0×10 6 1.0×10 7 1.0×10 8 The survival rate of 3rd instar larvae of the fall armyworm (number per mL) was found to be: at 1.0 × 10⁻⁶ larvae / mL. 7 cells / mL and 1.0×10 8 At a concentration of 1.0 × 10⁶ cells / mL, the survival rate was significantly different from that of the control group, and this difference was even more pronounced at a concentration of 1.0 × 10⁶ cells / mL. 8 When the number of larvae is 1 / mL, the survival rate of 3rd instar larvae of the fall armyworm drops to below 15%. Figure 4 ).

[0054] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Aspergillus, characterized in that, The Aspergillus species is Aspergillus nomi ( Aspergillus nomiae WN_AN1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41741.

2. A composition, characterized in that, The composition contains the Aspergillus as described in claim 1.

3. The composition according to claim 2, characterized in that: The composition is a culture, which is a substance obtained by culturing the Aspergillus in a microbial culture medium.

4. The composition according to claim 2, characterized in that: The composition is a microbial agent.

5. The composition according to any one of claims 2-4, characterized in that, The composition is a composition for controlling fall armyworm.

6. A method for preparing the composition, characterized in that, The composition is the composition according to any one of claims 2-5, and the method includes the step of using the Aspergillus of claim 1 as a component of the composition.

7. The use of the Aspergillus of claim 1 or the composition of claims 2-5 in the preparation of products for controlling fall armyworm.

8. A method for controlling the fall armyworm's damage to plants, characterized by: The method includes contacting a biocontrol product with plants to control fall armyworm, wherein the biocontrol product is the Aspergillus of claim 1 or the composition of claims 2-5; and the plant is corn.

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