A new strain of talaromyces amestolkiae with broad-spectrum lepidoptera insecticidal activity and application thereof

Abstract

The present application belongs to the technical field of microorganisms, and discloses a new Talaromyces amestolkiae strain with broad-spectrum lepidopterous insecticidal activity and application thereof. The new strain is isolated from the pests of Spodoptera litura, and is named as Talaromyces amestolkiae GZTA01, which has been preserved in China Center for Type Culture Collection on January 6, 2026, and the strain preservation number is CCTCC NO: M2026033. The Talaromyces amestolkiae strain of the present application has a minimum lethal rate of 62.14% and a maximum lethal rate of 86.67% on Spodoptera litura, Myzus persicae, Plutella xylostella and Mythimna separata, and exhibits excellent broad-spectrum control potential on lepidopterous and hemipterous pests.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to a new strain of Talaromyces amestolkiae with broad-spectrum lepidopteran insecticidal activity and its application. Background Technology

[0002] The larvae of most Lepidoptera and Hemiptera species commonly damage various cultivated plants. Larger larvae often devour leaves or bore into branches, while smaller larvae tend to roll leaves, clump leaves together, form sheaths, spin webs, or burrow into plant tissues to feed. Among these, the peach aphid (Myzus persicae (Sulzer), the beet armyworm (Spodoptera litura (Fabricius), the armyworm (Mythimnaseparata (Walker), and the diamondback moth (Plutella xylostella (Linnaeus)) are common polyphagous pests. Long-term reliance on chemical pesticides for control has led to serious pesticide resistance and environmental health and safety issues. For example, the diamondback moth and beet armyworm have developed high levels of resistance to many conventional mainstream chemical insecticides, posing a significant problem for the safe production of many economically important vegetables. Therefore, seeking new and highly effective control products is essential. In response to the needs of sustainable agricultural development and major pest and disease control in the new era, green and efficient biological pesticides are a priority for development in field pesticide use.

[0003] Currently, the market-registered single-agent microbial pesticide products for controlling the above pests include Bacillus thuringiensis, Beauveria bassiana, and Metarhizium anisopliae, but the lethality and control efficacy of these biocontrol strains against the above pests are insufficient to meet the needs of efficient field control. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the first objective of the present invention is to provide a *Talaromyces amestolkiae* strain that exhibits significantly enhanced insecticidal effects against lepidopteran and hemiptera pests such as peach aphids, beet armyworms, armyworms, and diamondback moths, in order to solve the problem that existing biocontrol strains are not ideal in controlling lepidopteran and hemiptera pests.

[0005] To achieve the above objectives, the inventors have long been committed to biocontrol research on lepidopteran and hemiptera pests. During field pest surveys of cabbage at the Institute of Plant Protection, Guizhou Academy of Agricultural Sciences, they repeatedly found that lepidopteran pests such as the beet armyworm and cabbage caterpillar were infected and killed by an unknown fungus. Therefore, the inventors used microbiological and molecular biology techniques to isolate, purify, and identify this unknown fungus. The identification results showed that the fungus is *Talaromyces amestolkiae*, therefore the applicant named the fungal strain *Talaromyces amestolkiae* GZTA01, and deposited it on January 6, 2026, at the China Center for Type Culture Collection, Wuhan University, Hongshan District, Wuhan City, Hubei Province, China, with accession number CCTCC NO: M2026033.

[0006] It should be noted that the *Talaromyces amestolkiae* GZTA01 strain culture obtained in this invention exhibits typical sexual form characteristics of *Penicillium*. The colonies grow rapidly on potato dextrose agar (PDA), with a velvety to slightly flocculent texture; initially white, the color changes from grayish-green to yellowish-green or olive-green as conidia form, with the edges usually whiter or paler, and the reverse side often producing soluble yellow to yellowish-brown pigment. The surface of the strain is usually dry, with a slightly sunken, pale yellow center. After 3 days of dark incubation at 28 ℃ and 70±5% humidity, the initial morphology becomes apparent, spreading out in dots on the culture medium. After 5-6 days of incubation, the dots merge into patches, and the more powdery dots, the more likely they are to accumulate. Under a scanning electron microscope, the sporangiophores are clearly visible as umbrella-shaped, the hyphae are longitudinally dispersed, and the spores are numerous and resemble rice grains.

[0007] Further biocontrol experiments revealed that the Talamoyces amestolkiae GZTA01 strain obtained in this invention has significant insecticidal effects against beet armyworm, peach aphid, armyworm and diamondback moth. Therefore, the second objective of this invention is to provide the following new uses for the Talamoyces amestolkiae GZTA01 strain: (1) its application in the control of lepidopteran or hemiptera pests; (2) its application in the preparation of insecticides for the control of lepidopteran or hemiptera pests.

[0008] More preferably, the lepidopteran pests include noctuid moths, armyworms, and diamondback moths; the noctuid moths are beet armyworms, fall armyworms, or cabbage armyworms; the armyworms are corn armyworms; and the diamondback moths are diamondback moths.

[0009] More preferably, the hemiptera pests include aphids; even more preferably, the aphids are peach aphids or tobacco aphids.

[0010] Compared with the prior art, the Talaromyces amestolkiae GZTA01 strain and its application provided by the present invention have the following significant advantages and advancements:

[0011] (1) Novel strain: Talamoyces amestolkiae is a fungus found in some marine or symbiotic systems. As a newly emerging member of the Talamoyces genus, this type of fungus has been reported in studies on industrial enzymes, metabolites, and certain fungal-insect interactions, but there are no literature reports on its use in controlling lepidopteran pests such as the beet armyworm. The GZTA01 strain isolated and identified from the beet armyworm pest for the first time in this invention may be a mutant strain, and experiments have shown that it can control a variety of common economic pests.

[0012] (2) Significantly improved insecticidal efficacy: The Talamoyces amestolkiae strain isolated from the beet armyworm in this invention has a mortality rate of up to 62.14% and up to 86.67% against beet armyworm, peach aphid, diamondback moth and armyworm, showing excellent potential for controlling beet armyworm, peach aphid, diamondback moth and armyworm.

[0013] (3) Eco-friendly and long-term sustainability: It can suppress pathogens and pests in a natural way, reduce dependence on chemical pesticides, and reduce environmental burden; it has little impact on non-target organisms and can protect the diversity of beneficial insects and soil microbial communities; through breeding and screening, it can be produced and applied under specified conditions. Attached Figure Description

[0014] Figure 1 Symptoms of an unknown fungal infection in the beet armyworm.

[0015] Figure 2 :Talaromyces amestolkiae GZTA01 Fungal morphology.

[0016] Figure 3 :Talaromyces amestolkiae GZTA01 Fungal sporophyte morphology.

[0017] Figure 4 Symptoms of infection with Spodoptera litura: Talaromyces amestolkiae GZTA01.

[0018] Figure 5 Symptoms of peach aphid infection in *Talaromyces amestolkiae* GZTA01.

[0019] Figure 6Symptoms of diamondback moth infection: Talaromyces amestolkiae GZTA01.

[0020] Figure 7 Symptoms of infection with Talamoyces amestolkiae GZTA01. Detailed Implementation

[0021] The following are specific embodiments of the present invention, which describe the technical solutions of the present invention in detail. Unless otherwise specified, the technical operations or conditions in the embodiments are performed using conventional techniques or conditions described in the literature in this field. Unless otherwise specified, the reagents or instruments used are all conventional products that can be purchased normally.

[0022] Example 1: Isolation and Identification of Talaromyces amestolkiae GZTA01 strain

[0023] 1. Sample collection

[0024] On October 11, 2025, the Institute of Plant Protection, Guizhou Academy of Agricultural Sciences, collected samples of beet armyworms infected with an unknown fungus from cabbage fields for the isolation and identification of the unknown fungus. Fifty samples of beet armyworms infected with the unknown fungus were collected at one time. Figure 1 After numbering, aliquot the samples into 1.5mL EP tubes and bring them back to the laboratory for storage at -80℃ until testing.

[0025] 2. Main reagents and instruments

[0026] Culture media: Potato dextrose agar (PDA, potato extract, glucose, agar, pH: 5.6±0.2) and potato dextrose liquid medium (PDB, potato extract, glucose, pH: 5.6±0.2). Reagents and instruments: Premix Taq (EX Taq version 2.0 Plus Dye), DL2000 DNA Marker, and DNA lysis buffer were all purchased from Takara Bio Inc. Universal primers for sequence amplification were synthesized by Shanghai Jierui Biotechnology Co., Ltd. CTAB reagents were purchased from Beijing Noribod Technology Co., Ltd., DNA purification and recovery kit (DP214) was purchased from Beijing Tiangen Biotech Co., Ltd., NEB Next® Ultra™ II FS DNA PCR-free Library Prep Kit (NEB / E7430L) library preparation kit was purchased from Beijing New England Biotechnology Co., Ltd., PCR instrument (Alpha-SE) was purchased from ABI Corporation, USA, and universal electrophoresis system (PowerPac Uni-versal 1645070) was purchased from Bio-Rad Corporation, USA.

[0027] 3. Isolation and purification of unknown fungi

[0028] In a clean bench, the preserved samples were treated with 70% ethanol for 30-60 seconds, followed by sodium hypochlorite (0.5-1%) for 2-5 minutes, and finally rinsed with sterile water at least three times to remove surface contaminants as much as possible. The samples were cut into sterile pieces (preferably from the internal tissue of lesions) and inoculated onto PDA, AEA, and OA media containing 1% kanamycin. This process was repeated three times, and the samples were incubated at 30°C in the dark for 7 days, with daily observation until distinguishable colony characteristics appeared. The culture was gradually purified to a single-spore pure strain. Each pure strain was assigned a unique number, and the incubation temperature, time, colony morphology, and growth rate were recorded. The color, surface texture, and edge characteristics of the pure strains were also recorded. All purified strains were stored in 50% glycerol at 4°C.

[0029] 4. Molecular biological identification

[0030] The isolated fungal strain was inoculated onto PDA medium and cultured for 7 days. Hyphae were picked with a sterile toothpick and transferred to a centrifuge tube containing 30 μL of lysis buffer. The tube was then incubated at 80 °C for 15 min to serve as a DNA template. Primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used. The PCR reaction mixture consisted of 15 μL PremixTaq (EX Taq version 2.0 plus dye), 2 μL DNA template, 1.5 μL each of forward and reverse primers (20 μmol / L), and ddH2O to a final volume of 30 μL. The PCR amplification program was as follows: 94 °C pre-denaturation for 3 min; 94 °C denaturation for 30 s, 55 °C annealing for 30 s, 72 °C extension for 45 s, for 30 cycles, followed by a final extension at 72 °C for 5 min. After PCR products were detected by 1% agarose gel electrophoresis, they were sent to Shanghai Sangon Biotech Co., Ltd. for fungal ITS gene sequencing. The sequencing results were then compared with BLAST homology in the NCBI database (https: / / www.ncbi.nlm.nih.gov) to obtain information on the isolation and identification of the strain.

[0031] 5. Appraisal Results

[0032] Figure 2 Under a scanning electron microscope, the sporangiophores of this fungus are clearly visible as umbrella-shaped, the hyphae are scattered longitudinally, and the spores are numerous and resemble rice grains. Figure 3 ).

[0033] Example 2: Application of Talaromyces amestolkiae GZTA01 in the control of Lepidoptera and Hemiptera pests

[0034] 1. Insecticidal activity of Talaromyces amestolkiae GZTA01 against second instar larvae of the beet armyworm.

[0035] Select cabbage leaves of similar age and size, gently rinse them with tap water, then rinse with distilled or deionized water. Pat dry with absorbent paper, and cut leaves of uniform size (approximately 5 cm long and 4 cm wide). Use tweezers to pick up the leaves and immerse them completely in a solution of 0.6 × 10⁻⁶ water. 6 Immerse the leaves in a T. amestolkiae GZTA01 spore suspension (number of insects / ml) for 30-40 seconds. After the time is up, immediately remove the leaves and hold them vertically above the plastic bottle for a few seconds to allow excess spore suspension to drip off. Lay the treated leaves flat on clean newspaper with the underside facing up and allow them to air dry naturally at room temperature. Simultaneously, select second-instar Spodoptera litura (treated with 24-hour starvation) of the same instar, size, and health status. Place a suitably sized circular filter paper at the bottom of a 90 mm petri dish to absorb excess spore suspension. Use a soft brush to transfer the test insects to the center of the petri dish with the filter paper. Place the petri dish at a fixed distance and use a fine mist sprayer (30 mL) to spray the spore suspension evenly onto the test insects until the filter paper is completely wet but without any liquid accumulation. Use 500 μL each time, spraying 2-3 times, for a total of 3 replicates, with 10 test insects per replicate. After standing for 2 hours, place one treated leaf in the dish and seal the petri dish with sealing film to prevent the test insects from escaping and to prevent excessive evaporation of moisture. A 0.1% Triton aqueous solution was used as a control. All inoculated petri dishes were placed on an insect rearing rack for observation at a temperature of (24±1)℃, a relative humidity of (65±5)%, and a photoperiod of L / / D = 16h / / 8h. The treated leaves were replaced regularly with fresh leaves until the end of the experiment. The number of dead beet armyworms was monitored daily or intermittently for 8 days after treatment. Insects were considered dead if they remained completely still within 10 seconds after being gently touched with a brush. Infection photographs were taken using a super-depth-of-field microscope (Nikon, Research Stereo Microscope, SMZ25 / SMZ18) and a desktop scanning electron microscope (JEOL, JCM-700).

[0036] The test results showed that the *T. amestolkiae* GZTA01 strain had a 0.6 × 10⁻⁶ mmol / L saturation. 6The mortality rate of *Spodoptera litura* was 62.14% after 8 days of treatment with a spore suspension of 1 / ml. The dead insects showed obvious symptoms of infection with this fungus. Figure 4 This indicates that *T. amestolkiae* GZTA01 shows great potential for application in the control of the beet armyworm.

[0037] Table 1. Mortality rate of *Talaromyces amestolkiae* GZTA01 infected with *Spodoptera litura*.

[0038]

[0039] 2. Insecticidal activity of *Talaromyces amestolkiae* GZTA01 against second-instar peach aphid nymphs.

[0040] The measurement methods and environmental conditions are the same as those in Part 1, the experiment on the beet armyworm.

[0041] The test results showed that the *T. amestolkiae* GZTA01 strain had a 0.6 × 10⁻⁶ mmol / L saturation. 6 The mortality rate of peach aphids was 83.33% after 8 days of treatment with a spore suspension of 1 / ml. The dead aphids showed obvious symptoms of infection with this fungus. Figure 5 This indicates that *T. amestolkiae* GZTA01 shows great potential for application in the control of peach aphids.

[0042] Table 2 Mortality rate of *Talaromyces amestolkiae* GZTA01 infected with peach aphids

[0043]

[0044] 3. Insecticidal activity of Talaromyces amestolkiae GZTA01 against second instar diamondback moth larvae

[0045] The measurement methods and environmental conditions are the same as those in Part 1, the experiment on the beet armyworm.

[0046] The test results showed that the *T. amestolkiae* GZTA01 strain had a 0.6 × 10⁻⁶ mmol / L saturation. 6 The mortality rate of diamondback moths treated with a spore suspension of 1 / ml for 8 days was 86.67%, and the dead moths showed obvious symptoms of infection with this fungus. Figure 6 This indicates that *T. amestolkiae* GZTA01 shows great potential for application in the control of diamondback moth.

[0047] Table 3 Mortality rate of *Talaromyces amestolkiae* GZTA01 infected with diamondback moth

[0048]

[0049] 4. Insecticidal activity of Talaromyces amestolkiae GZTA01 against second instar armyworm larvae

[0050] The measurement methods and environmental conditions are the same as those in Part 1, the experiment on the beet armyworm.

[0051] The test results showed that the *T. amestolkiae* GZTA01 strain had a 0.6 × 10⁻⁶ mmol / L saturation. 6 After treatment with a spore suspension of 1 / ml for 8 days, the mortality rate of armyworms was 80.00%, and the dead armyworms showed obvious symptoms of infection with this fungus. Figure 7 This indicates that *T. amestolkiae* GZTA01 shows great potential for application in the control of armyworms.

[0052] Table 4 Mortality rate of *Talaromyces amestolkiae* GZTA01 infected with armyworms

[0053]

Claims

1. A strain of Talaromyces amestolkiae GZTA01, with the strain preservation number CCTCC NO: M2026033.

2. An insecticide composition comprising the *Talaromyces amestolkiae* GZTA01 strain as described in claim 1 and an acceptable vector.

3. Any of the following applications of the Talaromyces amestolkiae GZTA01 strain according to claim 1: (1) in the control of lepidopteran or hemiptera pests; (2) in the preparation of insecticides for the control of lepidopteran or hemiptera pests.

4. The application according to claim 3, characterized in that, The lepidopteran pests mentioned include noctuid moths, armyworms, and diamondback moths.

5. The application according to claim 4, characterized in that, The noctuid moths mentioned are the beet armyworm, the fall armyworm, or the beet armyworm.

6. The application according to claim 4, characterized in that, The aforementioned armyworm is the corn armyworm.

7. The application according to claim 4, characterized in that, The diamondback moth mentioned is the small diamondback moth.

8. The application according to claim 3, characterized in that, The hemiptera pests mentioned include aphids.

9. The application according to claim 8, characterized in that, The aphids mentioned are peach aphids or tobacco aphids.

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