Aspergillus fungus A68 and application thereof

Abstract

The invention discloses an aspergillus fungus A68, which is classified and named as Aspergillus sp. And is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 2, 2025, the preservation date is January 2025, and the preservation number is CGMCC NO.41742. The invention further discloses a preparation method of the aspergillus fungus A68. The invention also discloses a microbial inoculum prepared from the strain. The invention also discloses an application of the Salvia miltiorrhiza Bunge and the microbial inoculum prepared from the Salvia miltiorrhiza Bunge in improving the disease resistance of Salvia miltiorrhiza Bunge seedlings, wherein the disease resistance refers to resistance to fusarium wilt caused by fusarium oxysporum. The Aspergillus fungus A68 screened by the invention can improve the capability of resisting fusarium wilt caused by fusarium oxysporum of salvia miltiorrhiza seedlings, provides support for a healthy and efficient industrial breeding technology of the salvia miltiorrhiza seedlings, and has guiding significance for production of salvia miltiorrhiza.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microorganisms, and in particular to an Aspergillus fungus A68 and application thereof in improving the disease resistance of salvia miltiorrhiza seedlings. Background Art

[0002] Aspergillus is a common fungus that is widely distributed in nature, such as soil, water, food and air. At present, there are many studies on the effects of Aspergillus on crop growth in the agricultural field, but there is a lack of research on the effects of Aspergillus on traditional Chinese medicine.

[0003] Salvia miltiorrhiza Salvia miltiorrhiza Bunge is a perennial herbaceous medicinal plant with the effects of "activating blood circulation and removing blood stasis, relieving pain, clearing the heart and eliminating troubles, cooling blood and eliminating carbuncle". The efficacy of Salvia miltiorrhiza plays an important role in the clinical treatment of cardiovascular and cerebrovascular diseases, and it is one of the commonly used bulk medicinal materials in my country. With the attention paid to cardiovascular and cerebrovascular diseases, the annual demand for Salvia miltiorrhiza is increasing, and the planting area is also expanding. The Chinese medicinal Salvia miltiorrhiza is mainly cultivated products, and its main production areas are Shandong, Sichuan, Henan, Shanxi, Hebei and other places. With the increasing planting density of Salvia miltiorrhiza and repeated planting year by year, the various diseases and insect pests caused by it have become increasingly serious, which has seriously restricted the improvement of Salvia miltiorrhiza quality and industrial development. In the soil of continuous cropping of Salvia miltiorrhiza, the common diseases that cause continuous cropping obstacles of Salvia miltiorrhiza are wilt, etc. Salvia miltiorrhiza wilt is a common disease in Salvia miltiorrhiza production, and this disease is often complicated by root rot, which affects the yield and quality of Salvia miltiorrhiza, and seriously restricts the production of Salvia miltiorrhiza. Therefore, the healthy and efficient cultivation of Salvia miltiorrhiza seedlings has become one of the key issues that need to be solved in the current Chinese medicinal materials industry.

[0004] Therefore, the present invention screened Aspergillus fungus A68 from the rhizosphere soil of healthy Salvia miltiorrhiza plants, studied its effect on the disease resistance of Salvia miltiorrhiza seedlings, provided support for the healthy and efficient cultivation technology of Salvia miltiorrhiza industrialization, and has guiding significance for the production of Salvia miltiorrhiza. Summary of the invention

[0005] The purpose of the present invention is to provide an Aspergillus fungus A68 and its application in improving the disease resistance of Salvia miltiorrhiza seedlings in response to actual problems and needs in the production practice of Salvia miltiorrhiza.

[0006] The purpose of the present invention can be achieved through the following technical solutions: The first aspect of the present invention provides a strain of Aspergillus fungus A68, which is classified as Aspergillus Aspergillus sp., deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is January 2, 2025, and the deposit number is CGMCC NO.41742.

[0007] The second aspect of the present invention provides a bacterial agent prepared by the Aspergillus fungus A68, wherein the spore concentration in the bacterial agent is 1×108 more than 1 / mL.

[0008] Furthermore, the microbial agent is prepared by the following method: Aspergillus sp. A68 with the preservation number of CGMCC NO. 41742 is cultured on a solid medium to produce spores, the mycelia and spores are washed off with sterile water, the mycelia are filtered off to obtain a spore solution, and the spore concentration of the spore solution is adjusted to 1×10 8 more than 1 / mL, thus obtaining the microbial agent.

[0009] Even further, the microbial agent is prepared by the following method: Aspergillus sp. A68 with the preservation number of CGMCC NO. 41742 is cultured on a solid medium at 26 - 30°C for 14 - 16 d to produce spores, the mycelia and spores are washed off with sterile water, the mycelia are filtered off with multiple layers of sterile gauze to obtain a spore solution, and the spore concentration of the spore solution is adjusted to 1×10 8 more than 1 / mL, thus obtaining the microbial agent.

[0010] Even further, the solid medium includes PDA medium.

[0011] The third aspect of the present invention provides the application of the above-mentioned Aspergillus sp. A68 in improving the disease resistance of Salvia miltiorrhiza seedlings, and the disease resistance is the fusarium wilt caused by Fusarium oxysporum.

[0012] The fourth aspect of the present invention provides the application of the above-mentioned microbial agent in improving the disease resistance of Salvia miltiorrhiza seedlings, and the disease resistance is the fusarium wilt caused by Fusarium oxysporum.

[0013] Furthermore, during application, after the Salvia miltiorrhiza seedlings are transplanted and grow for a period of time after transplantation, the microbial agent is inoculated into the seedling-raising substrate for breeding Salvia miltiorrhiza seedlings, and the inoculation ratio is 4 - 5 mL of the microbial agent per Salvia miltiorrhiza seedling.

[0014] Even further, the period when the Salvia miltiorrhiza seedlings grow for a period of time after transplantation is 15 - 20 d after the Salvia miltiorrhiza seedlings are transplanted and grow.

[0015] Even further, inoculating the microbial agent into the seedling-raising substrate for breeding Salvia miltiorrhiza seedlings is to directly pour the microbial agent into the seedling-raising substrate near the roots of the Salvia miltiorrhiza seedlings.

[0016] The beneficial effects of the present invention: The present invention screened a strain of Aspergillus fungus A68 that can improve the disease resistance of Salvia miltiorrhiza seedlings. In the confrontation test, A68 had a strong antagonistic effect on the pathogen Fusarium oxysporum of Salvia miltiorrhiza fusarium wilt, and the inhibition rate was 34.5%. In the pot experiment, the incidence of fusarium wilt of Salvia miltiorrhiza seedlings in the CK treatment, where sterile water was inoculated first and then the pathogen Fusarium oxysporum inoculum was inoculated, was 55.56%. The incidence of fusarium wilt of Salvia miltiorrhiza seedlings in the CL treatment, where A68 inoculum was inoculated first and then the pathogen Fusarium oxysporum inoculum was inoculated, was 22.22%. The prevention and control effect of A68 reached 60%. A68 can significantly reduce the incidence of fusarium wilt of Salvia miltiorrhiza seedlings and effectively improve the disease resistance of Salvia miltiorrhiza seedlings to fusarium wilt caused by Fusarium oxysporum.

[0017] The Aspergillus fungus A68 screened by the present invention can improve the ability of Salvia miltiorrhiza seedlings to resist fusarium wilt caused by Fusarium oxysporum, provide support for the healthy and efficient breeding technology of Salvia miltiorrhiza seedlings in factories, and has guiding significance for the production of Salvia miltiorrhiza. Description of the Drawings

[0018] Figure 1 It is a plate confrontation photo of strain A68 and the pathogen Fusarium oxysporum.

[0019] Figure 2 It is a colony plate photo of strain A68 (front of the colony).

[0020] Figure 3 It is a colony plate photo of strain A68 (back of the colony).

[0021] Figure 4 It is a phylogenetic tree constructed from the ITS gene sequence of strain A68.

[0022] Figure 5 It is a bar chart showing the effect of inoculating A68 inoculum on the anti-fusarium wilt effect of Salvia miltiorrhiza seedlings.

[0023] Figure 6 It is a phenotypic map showing the effect of inoculating A68 inoculum on the anti-fusarium wilt effect of Salvia miltiorrhiza seedlings.

[0024] Note: Different letters above the bar chart indicate significant differences between different treatments ( p <0.05).

[0025] Biological Material Preservation Information A68, classified and named as Aspergillus Aspergillus sp., was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was January 2, 2025, and the deposit number is CGMCC NO. 41742. Detailed Embodiments

[0026] The following examples and drawings facilitate a better understanding of the present invention, but do not limit the present invention. The test methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified.

[0027] The seedling-raising substrate involved in the following examples is the Xingxing Xiangnong brand special seedling-raising substrate produced by Jiangsu Xingnong Substrate Technology Co., Ltd., and the product number is: 161102G0097N.

[0028] The PDA medium involved in the following examples, that is, the potato dextrose agar medium formula is (1L): 200 g of peeled potatoes cut into pieces about 1 cm 3 small pieces, add 800 mL of deionized water to the pot, pour the potatoes into the pot after the water boils, cook for 15 - 20 min until the water becomes viscous, add 20 g of glucose to the beaker, place two layers of gauze on the cup mouth, pour the viscous potato liquid from the gauze, then add deionized water to make up to 1 L, add 20 g of agar powder, and sterilize at 121 °C for 20 min.

[0029] The diameter of the culture dishes (plates) involved in the following examples is 90 mm.

[0030] Example 1 Isolation and Identification of Functional Fungi 1.1 Isolation of Functional Fungi Collect the rhizosphere soil of healthy-growing Salvia miltiorrhiza plants on a large scale. Specifically: collect the rhizosphere soil of healthy-growing Salvia miltiorrhiza plants in the Salvia miltiorrhiza genuine producing areas of Yanjiang District and Zhongjiang County in Ziyang City, Sichuan Province, Fangcheng County, Yuzhou City, and Mianchi County in Henan Province, and Juxian County and Linqu County in Shandong Province. Mix the collected rhizosphere soil of healthy-growing Salvia miltiorrhiza plants, weigh 5 g, place it in a triangular flask containing 45 mL of sterile water (there are 6 glass beads with a diameter of 4 mm in the triangular flask), take it out after shaking at 30 °C and 170 rpm for 30 min, and perform gradient dilution. Pipette 100 μL of soil suspension with different concentration gradients onto the PDA medium plate for spreading, and incubate the spread plates at 28 °C for 7 d. After the colonies grow, select the different colonies and purify them on the PDA medium plate at 28 °C. Inoculate each of the isolated and purified fungal strains onto the PDA medium plate and incubate at 28 °C to produce spores, then store them in a 4 °C refrigerator for later use, and store each fungal strain in a -80 °C refrigerator using a glycerol tube. The specific method for storing strains in a glycerol tube is: wash the mycelium and spores on the PDA medium plate with sterile water, and filter out the mycelium with four layers of sterile gauze to obtain a spore solution. Mix the spore solution with a sterile 50 v / v% glycerol solution evenly at a volume ratio of 1:1 to obtain a glycerol spore solution. Count the spore concentration of the glycerol spore solution with a hemocytometer at 1×10 8When the number is above / mL, transfer the glycerol spore solution to a sterile centrifuge tube. When preserving, first pre-freeze it at -20°C for 12 - 15 h under low-temperature conditions, and then transfer it to -80°C for ultra-low temperature preservation.

[0031] Take Fusarium oxysporum isolated from the weak-growing Salvia miltiorrhiza roots caused by Salvia miltiorrhiza wilt as the pathogen. Inoculate the pathogen Fusarium oxysporum onto a PDA medium plate and culture it at 28°C to produce spores. Then place it in a 4°C refrigerator for storage and use. And preserve the strain in a glycerol tube in an -80°C refrigerator for use. The specific method of preserving the strain in a glycerol tube is the same as above.

[0032] Inoculate the pathogen Fusarium oxysporum onto a PDA medium plate and culture it at 28°C until the mycelium covers the PDA medium plate to obtain mycelium blocks (6 mm) of the pathogen Fusarium oxysporum. Inoculate each of the isolated and purified fungal strains onto a PDA medium plate and culture it at 28°C for 3 d to obtain mycelium blocks (6 mm) of each of the isolated and purified fungal strains. Inoculate the mycelium block (6 mm) of the pathogen Fusarium oxysporum in the center of a new PDA medium plate, and place the mycelium blocks (6 mm) of each of the isolated and purified fungal strains at two symmetric points about 2 cm away from the center of the mycelium block (6 mm) of the pathogen Fusarium oxysporum (confrontation plate). Culture it confrontationally at 28°C and observe the antibacterial (inhibiting the pathogen Fusarium oxysporum) situation of each of the isolated and purified fungal strains. At the same time, use the plate inoculated only with the mycelium block (6 mm) of the pathogen Fusarium oxysporum as a blank control (CK). When the blank control pathogen Fusarium oxysporum covers the entire plate, calculate the antibacterial rate of each of the isolated and purified fungal strains. This experiment is repeated 3 times for each treatment. The antibacterial rate formula is as follows: Antibacterial rate (%) = (CK colony radius - confrontation colony radius) / CK colony radius × 100, where the confrontation colony radius is the colony radius of the pathogen Fusarium oxysporum in the confrontation plate.

[0033] A large number of fungal strains were isolated and purified from the rhizosphere soil of healthy-growing Salvia miltiorrhiza plants. Each of the isolated and purified fungal strains was subjected to a plate confrontation test with the pathogen Fusarium oxysporum according to the above method, and 6 Aspergillus fungal strains with antagonistic effects (identified by ITS sequencing) were obtained. The antibacterial rates are shown in Table 1. Select two strains with stronger antagonistic effects, strain A136 and strain A68, as functional fungi for subsequent experiments. Strain A68 is involved in the present invention (the plate confrontation photo of strain A68 and the pathogen Fusarium oxysporum is as Figure 1 shown), and strain A136 is involved in another invention.

[0034] Table 1 Antibacterial rates of 6 Aspergillus fungal strains isolated and purified against the pathogen Fusarium oxysporum

[0035] 1.2 Identification of Functional Fungi After strain A68 was cultured on a PDA medium plate at 28 °C for 7 days, as Figure 2 and Figure 3 shown, the whole colony dispersed with the spores. The single colony was nearly circular and was relatively densely distributed as a whole. The texture of the colony was generally villous or flocculent, the surface of the colony was relatively fluffy, and it had a certain three-dimensional sense. The color of the central part of the colony was darker, being black, while the color of the edge part was lighter, being white or off-white. This was usually due to the different degrees of spore maturity. The spores in the central part had a high maturity and a darker color. The colony produced black-brown spores, and their morphology was relatively close to that of Aspergillus niger. By comparing the ITS gene sequence of strain A68 (the ITS gene sequence of strain A68 is shown as SEQ ID NO: 1, which was obtained by PCR amplification and sequencing of the DNA of strain A68 using the universal primers ITS1 (shown as SEQ ID NO: 2) / ITS4 (shown as SEQ ID NO: 3)) with similar sequences and constructing a phylogenetic tree, the result was as Figure 4 shown. Strain A68 had the highest homology with Aspergillus tubingensis , reaching 98.57%. Combining the colony morphological characteristics of strain A68 and the comparative analysis results of the phylogenetic tree constructed by the ITS series, strain A68 was identified as a fungus of the genus Aspergillus Aspergillus sp. Strain A68 was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 2, 2025, and the deposit number was CGMCC NO. 41742.

[0036] Example 2 Preparation of Bacterial Agent 2.1 Preparation of A68 Bacterial Agent Strain A68 was cultured on a PDA medium plate at 28 °C for 14 days to produce spores. The mycelium and spores on the PDA medium plate were washed off with sterile water, and the mycelium was filtered off with four layers of sterile gauze to obtain a spore suspension. The spore suspension was adjusted with sterile water to a spore concentration of 1×10 8 cells / mL (counted by a hemocytometer), and thus the A68 bacterial agent was obtained.

[0037] 2.2 Preparation of the Bacterial Agent of the Pathogenic Fusarium oxysporum The pathogenic Fusarium oxysporum was cultured on a PDA medium plate at 28 °C for 14 days to produce spores. The mycelium and spores on the PDA medium plate were washed off with sterile water, and the mycelium was filtered off with four layers of sterile gauze to obtain a spore suspension. The spore suspension was adjusted with sterile water to a spore concentration of 1×10 8 cells / mL (counted by a hemocytometer), and thus the bacterial agent of the pathogenic Fusarium oxysporum was obtained.

[0038] Example 3 Pot Experiment on the Disease Resistance Effect of Inoculating A68 Bacterial Agent on Salvia miltiorrhiza Seedlings Add 150 g of dry weight seedling-raising substrate into a plastic flowerpot with an upper inner diameter of about 8.8 cm, a bottom inner diameter of about 6.4 cm, and a depth of about 12 cm. Select healthy Salvia miltiorrhiza seedlings with the same growth vigor and size (one-year-old purple-flowered Salvia miltiorrhiza seedlings, purchased from the planting base of Shandong farmers) as test plants, transplant them into the flowerpots, and plant one Salvia miltiorrhiza seedling in each flowerpot. Place the flowerpots in the glass greenhouse of Binjiang Campus, Nanjing Agricultural University, and let them be planted and grow for 15 - 20 days (planted and grown for 15 days in this example). During the planting and growth period, the plant supplementary light in the greenhouse automatically turns off from 22:00 at night to 6:00 in the early morning of the next day, the greenhouse temperature is 30 °C, the greenhouse humidity is 80%, and water is poured once every 2 days to keep the seedling-raising substrate basically moist (the greenhouse light, temperature, humidity, and watering frequency in subsequent tests are the same). After the planting is completed, 5 mL of A68 inoculant (poured directly into the seedling-raising substrate near the roots of Salvia miltiorrhiza seedlings) is added to each flowerpot in the experimental group (CL), and 5 mL of sterile water of the same volume (poured directly into the seedling-raising substrate near the roots of Salvia miltiorrhiza seedlings) is added to each flowerpot in the control group (CK). After 7 days, 5 mL of pathogen Fusarium oxysporum inoculant (poured directly into the seedling-raising substrate near the roots of Salvia miltiorrhiza seedlings) is added to each flowerpot in the experimental group (CL) and the control group (CK). That is, this experiment is divided into two treatments, CK treatment (adding sterile water and then adding Fusarium oxysporum inoculant) and CL treatment (adding A68 inoculant and then adding Fusarium oxysporum inoculant). The experiment is a completely randomized block design, with 3 independent biological replicates for each treatment, and each replicate contains 6 flowerpots. The seedlings are harvested 30 days after adding the Fusarium oxysporum inoculant, and the incidence of Fusarium wilt of Salvia miltiorrhiza seedlings is measured. The incidence of Fusarium wilt of the replicates (%) = the number of diseased pots (showing symptoms of Fusarium wilt) in 6 pots / 6 × 100, and the incidence of Fusarium wilt of the treatment (%) = the sum of the incidence of Fusarium wilt of the replicates of the same treatment (%) / 3.

[0039] It can be seen from Figure 5 and Figure 6 that the incidence of Fusarium wilt of Salvia miltiorrhiza seedlings in the CK treatment of adding sterile water and then adding Fusarium oxysporum inoculant is 55.56%, and the incidence of Fusarium wilt of Salvia miltiorrhiza seedlings in the CL treatment of adding A68 inoculant and then adding Fusarium oxysporum inoculant is 22.22%, and the prevention and control effect of A68 reaches 60%. The results show that the Aspergillus sp. A68 can significantly reduce the incidence of Fusarium wilt of Salvia miltiorrhiza seedlings and effectively improve the disease resistance of Salvia miltiorrhiza seedlings to Fusarium wilt caused by Fusarium oxysporum.

Claims

1. A strain of Aspergillus fungus A68, taxonomically named Aspergillus Aspergillus sp., deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 2, 2025, with the deposit number CGMCC NO. 41742.

2. The microbial agent prepared from Aspergillus sp. A68 according to claim 1, characterized in that, The spore concentration in the microbial agent is 1×10 8 or more per mL.

3. The microbial agent according to claim 2, characterized in that, The microbial agent is prepared by the following method: Aspergillus sp. A68 with the preservation number of CGMCC NO. 41742 is cultured on a solid medium to produce spores. The mycelia and spores are washed off with sterile water, and the mycelia are filtered off to obtain a spore solution. The spore concentration of the spore solution is adjusted to 1×10 8 or more per mL, thus obtaining the microbial agent.

4. The microbial agent according to claim 3, wherein The microbial agent is prepared by the following method: Aspergillus sp. A68 with the preservation number of CGMCC NO.41742 is cultured on a solid medium at 26-30 °C for 14-16 d to produce spores. The mycelia and spores are washed off with sterile water, and the mycelia are filtered off with multiple layers of sterile gauze to obtain a spore solution. The spore concentration of the spore solution is adjusted to 1×10 8 or more / mL with sterile water, thus obtaining the microbial agent.

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

6. Use of the Aspergillus fungus A68 according to claim 1 in improving the disease resistance of Salvia miltiorrhiza seedlings, wherein the disease resistance is resistance to Fusarium oxysporum wilt.

7. Use of the bacterial agent according to any one of claims 2-5 in improving the disease resistance of Salvia miltiorrhiza seedlings, wherein the disease resistance is resistance to Fusarium oxysporum wilt.

8. The application according to claim 7, characterized in that, During application, after the Salvia miltiorrhiza seedlings are transplanted and grow for a period of time after transplantation, the bacterial agent is inoculated into the seedling-raising substrate for breeding Salvia miltiorrhiza seedlings, and the inoculation ratio is 4-5 mL of the bacterial agent per Salvia miltiorrhiza seedling.

9. The application according to claim 8, wherein Growing for a period of time after the Salvia miltiorrhiza seedlings are transplanted and grow is 15-20 days after the Salvia miltiorrhiza seedlings are transplanted and grow.

10. The application according to claim 8, characterized in that Inoculating the bacterial agent into the seedling-raising substrate for breeding Salvia miltiorrhiza seedlings is to directly pour the bacterial agent into the seedling-raising substrate near the roots of the Salvia miltiorrhiza seedlings.

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