Aspergillus ochraceus strain and application thereof

Abstract

The invention discloses an Aspergillus ochraceus Slt L1 strain and application thereof. The Aspergillus ochraceus Slt L1 strain has the morphological characteristics that after the Aspergillus ochraceus Slt L1 strain is cultured on a PDA culture medium for 7 days, typical Aspergillus ochraceus colony characteristics are formed, hyphae are separated and branched, conidiophore is upright, and conidia are spherical to nearly spherical. The prodenia litura strain has obvious pathogenicity to prodenia litura eggs, can lead to 100% of egg death rate, and has wide metabolic adaptability and environmental tolerance. Through the determination of a Biolog phenotype chip system, the bacterium can efficiently utilize a carbon source, a nitrogen source, a phosphorus source, a sulfur source and a plurality of biosynthetic pathways, and can keep the growth activity under the extreme conditions of osmotic pressure and pH. The microbial inoculum can be used for biological control of prodenia litura, and has the characteristics of greenness, high efficiency, environmental friendliness and the like.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, specifically to an ochratoxin strain, and also to the use of the ochratoxin strain in controlling the beet armyworm. Background Technology

[0002] Spodoptera litura ( Spodoptera litura Entomopathogenic fungi are a globally distributed, omnivorous lepidopteran pest that severely damages various crops, including those in the Solanaceae and Brassicaceae families, causing enormous economic losses annually. Currently, control still primarily relies on chemical pesticides, but problems such as pesticide resistance and environmental pollution are becoming increasingly prominent. Entomopathogenic fungi, as biological control agents, have advantages such as high host specificity and good environmental compatibility; however, most commercially available products target larvae, and biocontrol agents targeting the egg stage are extremely scarce.

[0003] Therefore, developing a fungal strain that can effectively control the egg stage of the beet armyworm is of great significance for achieving green and sustainable agriculture. Summary of the Invention

[0004] The purpose of this invention is to provide an Ochratus strain that is significantly pathogenic to the beet armyworm.

[0005] Another object of the present invention is to provide the use of the Aspergillus ochraceus strain in the control of crop pests caused by the beet armyworm.

[0006] An Ochratus Splt L1 of the present invention ( Aspergillus ochraceus The strain Splt L1, with the following morphological characteristics, formed typical Aspergillus ochraceus colonies after 7 days of culture on PDA medium. The hyphae were septate and branched, the conidiophores were erect, and the conidia were spherical to nearly spherical. This strain was deposited on September 17, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China (accession number: CCTCC No.: M 20252053), and its taxonomic name is [not specified]. Aspergillus ochraceus Splt L1.

[0007] An Ochratus Splt L1 of the present invention ( Aspergillus ochraceus The method for isolating Splt L1 strain includes the following steps: (1) Aspergillus ochraceus Splt L1 ( Aspergillus ochraceus Isolation of Splt L1 strain The egg masses of the infected Spodoptera litura were collected using the "tissue isolation method" and placed on sterile filter paper. The eggs were rinsed three times with sterile water, and the cleaned egg masses were then placed on sterile filter paper and thoroughly dried. Under sterile conditions, the tissue was placed on a PDA plate and incubated in the dark at 28°C. After fungal growth, single spores were picked and purified. (2) Culture medium Potato glucose agar (PDA): 200 g / L potato (peeled), 20 g / L glucose, 20 g / L agar. The prepared culture medium is sterilized at 121°C for 20 minutes and then set aside for use.

[0008] (3) Preservation and subculturing of strains Use an 18×180 mm test tube, fill it with 9 ml of PDA medium, make a slant, inoculate with fungi, incubate at 28℃ for 7-10 days, store at 4℃, and subculture once every 3 months; The purified fungi were cultured on PDA medium. After the fungi sporulated, the fungal spores were collected using a moistened sterile cotton swab. The swab was then washed in a storage tube containing 700 µl of 30% sterile glycerol to collect the fungal spores. The storage tube was then stored in a freezer at -20°C for long-term preservation. (4) Morphological characteristics of the strain The colonies formed by this bacterium were cultured on PDA plates at 28°C for 7 days. After 7 days of cultivation on PDA medium, the strain exhibited typical colony characteristics of *Aspergillus ochraceus*, with septate and branched hyphae, erect conidiophores, and spherical to nearly spherical conidia. ITS and β-tubulin gene sequence analysis (GenBank accession numbers: PQ21937, PV874200) identified it as... Aspergillus ochraceus This strain was named Aspergillus ochraceus Splt L1 ( Aspergillus ochraceus Splt L1 strain.

[0009] (5) Culture characteristics of the strain This bacterium can grow in a pH range of 3.5 to 10.0, with an optimal pH of around 6.0. It can tolerate osmotic pressure conditions including up to 6% potassium chloride, 20% ethylene glycol, 5% urea, and 12% sodium lactate. It can efficiently utilize carbon sources including L-arabinose, D-xylose, D-galactose, D-mannose, D-trehalose, and sucrose, among which L-arabinose is one of the most suitable carbon sources. It can utilize a wide range of nitrogen sources, including various amino acids and inorganic nitrogen sources. Among them, there are 92 nitrogen metabolism pathways represented by various amino acids, showing a significant metabolic advantage for amino acid nitrogen sources.

[0010] An Ochratus Splt L1 of the present invention ( Aspergillus ochraceus The use of Splt L1 strain in controlling crop pests caused by beet armyworm.

[0011] Compared with existing technologies, this invention has significant beneficial effects. As shown in the above technical solution, indoor spraying experiments on egg masses demonstrate that this strain can significantly inhibit the growth and development of the beet armyworm. Egg masses die 7-10 days after infection, effectively controlling beet armyworm egg masses. It has a control effect comparable to chemical insecticides such as cypermethrin and deltamethrin. This bacterial agent is prepared using wet bacteria and a bacterial culture preservation solution. The culture preservation solution consists of 0.01 mol / L phosphate buffer and 0.001 mol / L Tween 40 nonionic surfactant. This bacterial agent can be sprayed directly onto crop leaves or mixed with insect bait and placed in the field. This strain has a 100% lethality rate against beet armyworm eggs and can efficiently utilize multiple carbon, nitrogen, phosphorus, and sulfur sources, possessing broad biosynthetic capabilities and tolerance to extreme environments (osmotic pressure, pH). Because it is a microbial agent, it is environmentally friendly and does not have any of the problems caused by the use of chemical pesticides, which is conducive to pollution-free and organic production. Detailed Implementation

[0012] Example 1: An Ochratus Splt L1 of the present invention ( Aspergillus ochraceus The method for isolating Splt L1 strain includes the following steps: (1) Aspergillus ochraceus Splt L1 ( Aspergillus ochraceus Isolation of Splt L1 strain The "egg mass tissue isolation method" was used. Moldy beet armyworm egg mass samples were taken and placed on sterile filter paper. After rinsing the surface with sterile water, they were placed on PDA plates and incubated in the dark at 28°C for 5-7 days. After the colonies grew, single spores were picked and purified. Preliminary identification was performed based on colony morphology, sporulation structure and microscopic characteristics. Further molecular verification was performed by ITS and β-tubulin sequences. (2) Culture medium Potato glucose medium (PDA) was used: the composition was 200 g / L potato (peeled), 20 g / L glucose, and 20 g / L agar, and it was autoclaved at 121℃ for 20 minutes before use. (3) Preservation and fermentation of microbial strains Use an 18×180 mm test tube, fill it with 9 ml of PDA medium, make a slant, inoculate with fungi, incubate at 28℃ for 7-10 days, store at 4℃, and subculture once every 3 months; The purified fungi were cultured on PDA medium. After the fungi sporulated, the fungal spores were collected using a moistened sterile cotton swab. The swab was then washed in a storage tube containing 700 µl of 30% sterile glycerol to collect the fungal spores. The storage tube was then stored in a freezer at -20°C for long-term preservation. The purified strain was inoculated onto PDA slant, cultured at 28℃ for 7 days, stored at 4℃ and subcultured every 3 months; for large-scale sporulation, PD liquid medium was used for shaking culture (28℃, 150 rpm, 5-7 days), conidia were collected and stored long-term at -20℃ or -80℃ with 30% glycerol. (4) Preparation of microbial agents Mix the suspension of Aspergillus ochraceus L1 spores with the cell preservation solution at a ratio of 1:50 (w / v), resulting in a final spore concentration of 1×10⁻⁶. 6 –1×10 8 CFU / mL, the cell preservation solution is 0.01 mol / L phosphate buffer (pH 6.5–7.0) containing 0.001 mol / L Tween 40; The strain L1, which controls the beet armyworm, was cultured on PDA medium for 10 days. 5 ml of sterile water was added to the petri dish to wash off the spores, and the spore solution was collected.

[0013] Mix L1 spore suspension and mycelial culture solution at a mass-to-volume ratio of 1:50 to prepare a microbial inoculum. The viable cell concentration of the finished inoculum is 1×10⁻⁶. 3 -1×10 6 The bacterial cell density was [number] cells / ml, and the cell preservation solution was 0.01 mol / L phosphate buffer, also containing 0.001 mol / L Tween 40 nonionic surfactant. The PDA culture medium was prepared as follows: 200 g / L potato (peeled), 20 g / L glucose, and 20 g / L agar. The prepared culture medium was sterilized at 121℃ for 20 minutes and then set aside for use.

[0014] Experimental Example 1: Control Experiment of Ochratoxin L1 on Spodoptera litura egg masses Collect healthy Spodoptera litura egg masses, with 30 eggs per treatment group, and set up the following treatment groups: Ochratus L1 treatment group: The inoculum was sprayed evenly onto the surface of the egg masses; Blank control group: only sterile water (containing 0.001 mol / L Tween 40) was sprayed; Deltamethrin treatment group: spray with 50 mg / L deltamethrin solution.

[0015] After treatment, the eggs were placed in an incubator at 25±1℃, RH 75%, and a photoperiod of 14L:10D. Egg mass development and mold growth were observed daily, and hatching mortality was recorded on day 10. Results showed that the ochratoxin L1 treatment group achieved 100% control efficacy against *Spodoptera litura* egg masses, significantly higher than the chemical treatment group and the blank control group (Table 1). The control efficacy was statistically analyzed based on mortality rate using the following method: …………………………………………(2) In the formula: P2 – efficacy, expressed as a percentage (%); P1—Treatment mortality rate, in percentage (%). P0 – Mortality rate in the blank control group, expressed as a percentage (%).

[0016] The efficacy test results of the cabbage leaf fungicide treatment showed that the L1 fungicide treatment group was able to effectively control the beet armyworm, with a control effect of 100%.

[0017] Table 1. Control efficacy of L1 fungicide against beet armyworm 12 days after application. deal with Preventive efficacy (%) Ochratoxin L1 treatment group 100 deltamethrin 100 Blank control group / The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments without departing from the technical essence of the present invention shall still fall within the scope of the present invention.

Claims

1. An Aspergillus ochraceus Splt L1 (A. ochraceus Splt L1) strain, characterized by: Aspergillus ochraceus A. ochraceus Splt L1) strain, characterized by: The deposit number of the strain is CCTCC No: M 20252053.

2. Use of Aspergillus ochraceus Splt L1 strain in the prevention and treatment of crop insect pests caused by Spodoptera litura.