Aspergillus fungus A74 and its application

Abstract

The present invention discloses a strain of Aspergillus fungus A74, which is classified as Aspergillus Aspergillus sp., deposited in the General Microbiology Center of the China National Committee for the Administration of Microbiological Culture Collection, with a deposit date of April 1, 2025, and a deposit number of CGMCC NO.41860. The present invention also discloses a microbial agent prepared thereby. The present invention also discloses the use of the microbial agent and the microbial agent prepared thereby in improving the disease resistance of tobacco seedlings, wherein the disease resistance is resistance to wilt caused by Fusarium solani. The Aspergillus fungus A74 screened by the present invention can improve the resistance of tobacco seedlings to wilt caused by the pathogenic fungus Fusarium solani, provide support for the healthy and efficient factory-based breeding technology of tobacco seedlings, and have guiding significance for tobacco production.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to an Aspergillus fungus A74 and its application in improving the disease resistance of tobacco seedlings. Background Art

[0002] Aspergillus ( Aspergillus As a fungal group widely distributed in nature, its ecological niche covers soil, water, air and various environments rich in organic matter. In the agricultural field, the research on the effect of Aspergillus on crop growth has been quite in-depth. However, in the economic crop tobacco ( Nicotiana tabacum L.) is still relatively scarce and needs further exploration.

[0003] Tobacco is an annual herb. It is the primary raw material for tobacco products such as cigarettes and cigars, and its products are in widespread demand worldwide. In China, tobacco cultivation not only provides a stable source of income for agriculture but also plays a central role in the production of tobacco products. Furthermore, tobacco's medicinal properties have recently attracted considerable attention. Research has shown that tobacco contains a variety of chemical components with pharmacological activities, including antiviral, antitumor, antibacterial, anti-inflammatory, and immunomodulatory properties. These components have potential applications in the development of pharmaceutical and pesticide lead compounds. Furthermore, tobacco exhibits significant biological control capabilities, useful for combating a variety of crop pests, including rice hoppers, rice weevils, aphids, and thrips. However, traditional tobacco cultivation relies excessively on chemical fertilizers and pesticides, which not only increases production costs but also poses potential risks to the ecological environment, hindering the sustainable development of the tobacco industry. Therefore, developing safe, environmentally friendly, and efficient tobacco cultivation methods is of great significance.

[0004] Therefore, the present invention screened out an Aspergillus fungus A74 from the rhizosphere soil of healthy tobacco plants and studied its effect on the disease resistance of tobacco seedlings, providing a theoretical basis and technical support for achieving green and efficient cultivation of tobacco production, and has important practical guiding significance for promoting the sustainable development of the tobacco industry. Summary of the Invention

[0005] The purpose of the present invention is to provide an Aspergillus fungus A74 and its application in improving the disease resistance of tobacco seedlings in response to practical problems and needs in tobacco production practice.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a strain of Aspergillus A74, which is classified as Aspergillus Aspergillussp., deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit date of April 1, 2025, and the deposit number is CGMCC NO.41860.

[0008] The second aspect of the present invention provides a bacterial agent prepared from the Aspergillus fungus A74, wherein the spore concentration in the bacterial agent is 1×10 8 More than / mL.

[0009] Furthermore, the bacterial agent is prepared by the following method: culturing Aspergillus fungus A74 with a deposit number of CGMCC NO.41860 on a solid culture medium to produce spores, washing the mycelia and spores with sterile water, filtering out the mycelia to obtain a spore liquid, and adjusting the spore concentration of the spore liquid to 1×10 8 More than / mL to obtain the bacterial agent.

[0010] Furthermore, the bacterial agent is prepared by the following method: Aspergillus fungus A74 with a preservation number of CGMCC NO.41860 is cultured on a solid culture medium at 26-30°C for 14-16 days to produce spores, the mycelium and spores are washed with sterile water, the mycelium is filtered out with multiple layers of sterile gauze to obtain a spore liquid, and the spore concentration of the spore liquid is adjusted to 1×10 8 More than / mL, that is, the bacterial agent is obtained.

[0011] Furthermore, the solid culture medium includes PDA culture medium.

[0012] A third aspect of the present invention provides the use of the Aspergillus fungus A74 in improving the disease resistance of tobacco seedlings, wherein the disease resistance is resistance to wilt caused by Fusarium solani.

[0013] A fourth aspect of the present invention provides the use of the above-mentioned bacterial agent in improving the disease resistance of tobacco seedlings, wherein the disease resistance is resistance to wilt disease caused by Fusarium solani.

[0014] Furthermore, during application, after the tobacco seedlings are transplanted and planted for a period of time, the bacterial agent is inoculated into the seedling medium for breeding the tobacco seedlings, and the inoculation ratio is 4-5 mL of the bacterial agent per tobacco seedling.

[0015] Furthermore, the period of time for the tobacco seedlings to grow after transplanting is 15-20 days after transplanting.

[0016] Furthermore, the bacterial agent is introduced into the seedling medium for growing tobacco seedlings by directly pouring the bacterial agent into the seedling medium near the roots of the tobacco seedlings.

[0017] Beneficial effects of the present invention:

[0018] The present invention screened out a strain of Aspergillus fungus A74 that can improve the disease resistance of tobacco seedlings. In the confrontation test, A74 had a strong antagonistic effect on the tobacco wilt pathogen Fusarium solani, with an inhibition rate of 69.1%. In the pot test, sterile water was first added and then the pathogen Fusarium solani was added. F.solani The incidence of wilt disease in tobacco seedlings treated with A74 was 41.67%, and the incidence of wilt disease in tobacco seedlings treated with A74 was 41.67%. F.solani+ The incidence of tobacco seedling wilt disease treated with A74 was 8.33%, and the prevention and control effect of A74 reached 80%. F.solani+ The growth indicators of tobacco seedlings treated with A74 (plant height, stem diameter, leaf chlorophyll content) were significantly higher than those of F. solani Treated tobacco seedlings. Aspergillus fungus A74 can significantly reduce the incidence of tobacco seedling wilt disease, promote the growth of tobacco seedlings, improve the quality of tobacco seedlings, and effectively improve the disease resistance of tobacco seedlings.

[0019] The Aspergillus fungus A74 screened by the present invention can improve the resistance of tobacco seedlings to wilt disease caused by the pathogenic fungus Fusarium solani, provide support for the healthy and efficient factory breeding technology of tobacco seedlings, and have guiding significance for tobacco production. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a photo of the plate confrontation between strain A74 and the pathogenic bacterium Fusarium solani.

[0021] Figure 2 This is a photo of the colony plate of strain A74.

[0022] Figure 3 Phylogenetic tree constructed for the ITS gene sequence of strain A74.

[0023] Figure 4 This is a bar graph showing the effect of inoculation with A74 fungicide on the incidence of tobacco seedling wilt.

[0024] Figure 5 This is a bar graph showing the effect of inoculation with A74 fungicide on the plant height of tobacco seedlings resistant to wilt disease.

[0025] Figure 6 This is a bar graph showing the effect of inoculation with A74 fungicide on the stem diameter of tobacco seedlings resistant to wilt disease.

[0026] Figure 7 This is a bar graph showing the effect of inoculation with A74 fungicide on the chlorophyll content of tobacco seedlings resistant to wilt disease.

[0027] Figure 8 This is a photo of potted tobacco seedlings inoculated with A74 fungicide to show their resistance to wilt disease.

[0028] Note: * and different letters above the bar graph indicate significant differences between different treatments ( p <0.05).

[0029] Biomaterial deposit information

[0030] A74, classified as Aspergillus Aspergillus sp., deposited in the General Microbiology Center of China Culture Collection Administration, the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is April 1, 2025, and the deposit number is CGMCC NO.41860. DETAILED DESCRIPTION

[0031] The following examples and accompanying drawings are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0032] The seedling culture medium involved in the following examples is the Xingxing Xiangnong brand seedling culture medium produced by Jiangsu Xingnong Matrix Technology Co., Ltd., with the product number: 161102G0097N.

[0033] The PDA medium involved in the following examples, that is, the potato dextrose agar medium, is formulated as follows (1 L): 200 g peeled potatoes cut into approximately 1 cm 3 Add 800mL of deionized water to a pot, pour in potatoes after the water boils, and cook for 15-20 minutes until the water becomes viscous. Add 20g of glucose to a beaker, place two layers of gauze on the mouth of the cup, pour the viscous potato liquid from the gauze, then add deionized water to make the volume 1L, add 20g of agar powder, and sterilize at 121℃ for 20 minutes.

[0034] The diameter of the culture dishes (plates) involved in the following examples is 90 mm.

[0035] Example 1 Isolation and identification of functional fungi

[0036] 1.1 Isolation of functional fungi

[0037] Rhizosphere soil from healthy tobacco plants was collected in tobacco-growing areas in Yibin City, Sichuan Province. 5 g of the collected rhizosphere soil was mixed and placed in a flask containing 45 mL of sterile water (containing six 4 mm diameter glass beads). The flask was shaken at 30°C and 170 rpm for 30 minutes, then removed and serially diluted. 100 µL of the soil suspension at different concentrations was spread onto polydimethylsiloxane (PDA) plates. The plates were incubated at 28°C for 7 days. Once colonies emerged, differentially expressed colonies were selected and purified on PDA plates at 28°C. The fungal strains isolated and purified were inoculated onto PDA culture plates and cultured at 28°C for spore production, and then stored in a 4°C refrigerator for use. Glycerol tubes were used to preserve the fungal strains in a -80°C refrigerator for use. The specific method for preserving the strains in glycerol tubes was as follows: the hyphae and spores on the PDA culture plates were washed with sterile water, and the hyphae were filtered out with four layers of sterile gauze to obtain a spore solution. The spore solution was mixed evenly with a sterile 50 v / v% glycerol solution in a volume ratio of 1:1 to obtain a glycerol spore solution. The spore concentration of the glycerol spore solution was counted with a hemocytometer to determine whether it was 1×10 8 The glycerol spore solution was transferred to a sterile centrifuge tube; when stored, it was first pre-frozen at -20°C for 12-15 hours, and then transferred to -80°C ultra-low temperature for storage.

[0038] Fusarium solani (Fusarium solani ) were inoculated onto PDA culture plates and cultured at 28°C until the mycelium filled the PDA culture plates. Mycelial blocks (6 mm) of the pathogenic Fusarium solani were obtained. The isolated and purified fungal strains were inoculated onto PDA culture plates and cultured at 28°C for 3 days to obtain mycelial blocks (6 mm) of the isolated and purified fungal strains. The mycelial blocks (6 mm) of the pathogenic Fusarium solani were inoculated onto the center of a new PDA culture plate. The mycelial blocks (6 mm) of the isolated and purified fungal strains were placed at four points approximately 2 cm away from the mycelial blocks (6 mm) of the pathogenic Fusarium solani (opposite plates). The plates were cultured at 28°C in opposite plates to observe the antibacterial activity of the isolated and purified fungal strains (inhibition of the pathogenic Fusarium solani). A plate inoculated only with mycelial blocks (6 mm) of the pathogenic Fusarium solani was used as a blank control (CK). When the blank control pathogenic Fusarium solani filled the entire plate, the inhibition rate of the isolated and purified fungal strains was calculated. Each treatment in this experiment was repeated three times. The inhibition rate formula is as follows:

[0039] Inhibition rate (%) = (CK colony radius - opposing colony radius) / CK colony radius × 100, where the opposing colony radius is the colony radius of the pathogenic fungus Fusarium solani in the opposing plate.

[0040] By analyzing the inhibition rate of each fungal strain isolated and purified against the pathogenic fungus Fusarium solani, we finally obtained 5 functional bacteria with an inhibition rate of more than 40%, numbered A90, A20, A25, A44, and A74, with inhibition rates of 44.9%, 56.0%, 57.4%, 61.5%, and 69.1%, respectively. The strain A74 with the highest inhibition rate was subsequently selected for testing. The plate confrontation photo of strain A74 and the pathogenic fungus Fusarium solani is shown in the figure below. Figure 1 shown.

[0041] 1.2 Identification of functional fungi

[0042] After strain A74 was cultured on PDA medium plates at 28°C for 7 days, Figure 2 As shown, the colony is dark brown-gray in color, and the colony shows a clear radial growth pattern, expanding from the center to the surrounding areas. The edges of the colony are relatively uniform, no obvious edge features are observed, and the overall morphology is relatively regular. By comparing the ITS gene sequence of strain A74 (the ITS gene sequence of strain A74 is shown in SEQ ID NO: 1, which is obtained by PCR amplification and sequencing of the DNA of strain A74 using universal primers ITS1 (as shown in SEQ ID NO: 2) / ITS4 (as shown in SEQ ID NO: 3)) with similar sequences, and constructing a phylogenetic tree, the results are shown in the figure below. Figure 3 As shown, strain A74 and Aspergillus assiutensis The highest homology was 99.81%. Combining the colony morphology of strain A74 and the phylogenetic tree analysis results constructed by ITS sequence, strain A74 was identified as Aspergillus fungus. Aspergillus sp. strain A74 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on April 1, 2025, with the deposit number CGMCC NO.41860.

[0043] Example 2 Preparation of bacterial agent

[0044] 2.1 Preparation of A74 inoculant

[0045] Strain A74 was cultured on PDA medium plates at 28°C for 14 days to produce spores. Mycelia and spores on the PDA medium plates were washed with sterile water, and the mycelia were filtered through four layers of sterile gauze to obtain spore liquid. The spore liquid was adjusted to a spore concentration of 1×10 8 / mL (hemocytometer count), and the A74 bacterial agent was obtained.

[0046] 2.2 Preparation of pathogenic fungus Fusarium solani inoculum

[0047] The pathogenic fungus Fusarium solani was cultured on a PDA medium plate at 28°C for 14 days to produce spores. The mycelia and spores on the PDA medium plate were washed with sterile water, and the mycelia were filtered through four layers of sterile gauze to obtain a spore solution. The spore solution was adjusted with sterile water to a spore concentration of 1×10 8 / mL (hemocytometer count), the pathogenic fungus Fusarium solani agent is obtained.

[0048] Example 3 Potted experiment on the effect of inoculation with A74 bacterial agent on disease resistance of tobacco seedlings

[0049] The seedling medium was distributed into 6-well seedling trays (each well was approximately 4.5 cm × 4.5 cm × 5 cm, with approximately 26 g of dry medium per well) and watered thoroughly with sterile water. Healthy tobacco seedlings (grown from Nicotiana benthamiana seeds to the 4-leaf stage) of uniform growth and size were transplanted into the 6-well seedling trays, with one seedling per well. The 6-well seedling trays were placed in a glass greenhouse at the Binjiang Campus of Nanjing Agricultural University and grown for 15-20 days (15 days in this example). During this period, the greenhouse lights automatically turned off from 10:00 PM to 6:00 AM the following morning. The greenhouse temperature was maintained at 30°C and the humidity at 80%. Water was applied every two days to keep the seedling medium substantially moist. (Subsequent experiments maintained the same greenhouse lighting, temperature, humidity, and watering frequency.) After planting, each tobacco seedling was inoculated with 5 mL of A74 inoculant and poured directly into the seedling medium near the roots of the tobacco seedlings. Seven days after inoculation with A74 inoculant, each tobacco seedling was inoculated with 5 mL of the pathogenic fungus Fusarium solani inoculant and poured directly into the seedling medium near the roots of the tobacco seedlings. The experiment was divided into three treatments: CK treatment (each tobacco seedling was not inoculated with A74 inoculant or the pathogenic fungus Fusarium solani inoculant, and was inoculated with 5 mL of sterile water twice), F.solani Treatment (each tobacco seedling was not inoculated with A74 inoculant, but was first inoculated with 5 mL of sterile water and then with 5 mL of pathogenic fungus Fusarium solani inoculant) and F. solani + The A74 treatment (each tobacco seedling was first inoculated with 5 mL of the A74 inoculant, followed by 5 mL of the pathogenic Fusarium solani) was performed. Three replicates were set up for each treatment, with six tobacco seedlings per replicate. The tobacco seedlings were grown for 30 days after inoculation. The incidence of wilt disease was determined. The aboveground plant height of the tobacco seedlings was measured with a ruler, the stem diameter at the junction of the aboveground and underground parts was measured with a vernier caliper, and the chlorophyll content of the mid-leaf of the tobacco seedlings was measured with a chlorophyll meter.

[0050] The calculation formula for the incidence of tobacco seedling wilt disease is as follows:

[0051] The incidence of tobacco seedling wilt disease (%) = number of diseased plants (with wilt symptoms) / total number of plants × 100.

[0052] like Figure 4 As shown, F.solani The incidence of wilt disease in treated tobacco seedlings was 41.67%. F.solani+ The incidence of tobacco seedling wilt disease treated with A74 was 8.33%, and the control effect of A74 was 80%. Figure 5-8 As shown, F.solani+ The growth indicators of tobacco seedlings treated with A74 (plant height, stem diameter, leaf chlorophyll content) were significantly higher than those of F. solani Treated tobacco seedlings. F.solani+ The stem diameter and leaf chlorophyll content of tobacco seedlings treated with A74 were also significantly higher than those treated with CK. In summary, the Aspergillus fungus A74 can significantly reduce the incidence of tobacco seedling wilt, promote the growth of tobacco seedlings, improve the quality of tobacco seedlings, and effectively enhance the disease resistance of tobacco seedlings.

Claims

1. A strain of Aspergillus A74, classified as Aspergillus Aspergillus sp., deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit date of April 1, 2025, and the deposit number is CGMCC NO.41860.

2. The bacterial agent prepared by the Aspergillus fungus A74 according to claim 1, characterized in that The spore concentration in the bacterial agent is 1×10 8 More than / mL.

3. The microbial agent according to claim 2, characterized in that The bacterial agent is prepared by the following method: Aspergillus fungus A74 with a preservation number of CGMCC NO.41860 is cultured on a solid culture medium to produce spores, the mycelia and spores are washed with sterile water, the mycelia are filtered out to obtain a spore liquid, and the spore concentration of the spore liquid is adjusted to 1×10 8 More than / mL to obtain the bacterial agent.

4. The microbial agent according to claim 3, characterized in that The bacterial agent is prepared by the following method: Aspergillus fungus A74 with a preservation number of CGMCC NO.41860 is cultured on a solid culture medium at 26-30°C for 14-16 days to produce spores, the mycelia and spores are washed with sterile water, the mycelia are filtered out with multiple layers of sterile gauze to obtain a spore liquid, and the spore concentration of the spore liquid is adjusted to 1×10 8 More than / mL, that is, the bacterial agent is obtained.

5. The bacterial agent according to claim 3 or 4, characterized in that The solid culture medium includes PDA culture medium.

6. Use of the Aspergillus fungus A74 according to claim 1 in improving disease resistance of tobacco seedlings, wherein the disease resistance is resistance to wilt caused by Fusarium solani.

7. Use of the microbial agent according to any one of claims 2 to 5 for improving disease resistance of tobacco seedlings, wherein the disease resistance is resistance to wilt caused by Fusarium solani.

8. The use according to claim 7, characterized in that During application, after the tobacco seedlings are transplanted and grown for a period of time, the bacterial agent is inoculated into the seedling culture medium for breeding the tobacco seedlings, and the inoculation ratio is 4-5 mL of the bacterial agent per tobacco seedling.

9. The use according to claim 8, characterized in that The period of time for tobacco seedlings to grow after transplanting is 15-20 days.

10. The use according to claim 8, characterized in that The bacterial agent is introduced into the seedling culture medium for growing tobacco seedlings by directly pouring the bacterial agent into the seedling culture medium near the roots of the tobacco seedlings.

Images