Aspergillus fungus A136 and application thereof

Abstract

The invention discloses an aspergillus fungus A136, 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.41743. The invention further discloses a preparation method of the aspergillus fungus A136. 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 A136 screened by the invention can improve the capability of resisting fusarium wilt caused by fusarium oxysporum of the salvia miltiorrhiza seedlings, provides support for the industrialized healthy and efficient breeding technology of the salvia miltiorrhiza seedlings, and has guiding significance for the 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 A136 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 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 traditional Chinese medicine 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 with root rot, which affects the yield and quality of Salvia miltiorrhiza, and seriously restricts the production of Salvia miltiorrhiza. As a traditional medicinal plant, Salvia miltiorrhiza has a wide planting area in my country. It is an ideal promotion project for the development of high-efficiency agriculture and the realization of rural revitalization today. Therefore, the healthy and efficient cultivation of Salvia miltiorrhiza seedlings has become one of the key issues that need to be urgently addressed in the current Chinese medicinal materials industry.

[0004] Therefore, the present invention screened Aspergillus fungus A136 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 A136 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 A136, 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.41743.

[0007] In the second aspect of the present invention, a microbial agent prepared from the above-mentioned Aspergillus sp. A136 is provided, and the spore concentration in the microbial agent is 1×10 8 or more per mL.

[0008] Furthermore, the microbial agent is prepared by the following method: The Aspergillus sp. A136 with the preservation number of CGMCC NO.41743 is cultured on a solid medium to produce spores, the mycelia and spores are washed off with sterile water, the mycelia are filtered off, a spore solution is obtained, and the spore concentration of the spore solution is adjusted to 1×10 8 or more per mL with sterile water, thus obtaining the microbial agent.

[0009] Even further, the microbial agent is prepared by the following method: The Aspergillus sp. A136 with the preservation number of CGMCC NO.41743 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, a spore solution is obtained, and the spore concentration of the spore solution is adjusted to 1×10 8 or more per mL with sterile water, thus obtaining the microbial agent.

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

[0011] In the third aspect of the present invention, the application of the above-mentioned Aspergillus sp. A136 in improving the disease resistance of Salvia miltiorrhiza seedlings is provided, and the disease resistance is resistance to Fusarium oxysporum wilt.

[0012] In the fourth aspect of the present invention, the application of the above-mentioned microbial agent in improving the disease resistance of Salvia miltiorrhiza seedlings is provided, and the disease resistance is resistance to Fusarium oxysporum wilt.

[0013] Furthermore, during application, after the Salvia miltiorrhiza seedlings are transplanted and grow for a certain 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 time when the Salvia miltiorrhiza seedlings grow for a certain 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 means directly pouring the microbial agent onto the seedling-raising substrate near the roots of the Salvia miltiorrhiza seedlings.

[0016] Advantages of the present invention: The present invention screened a strain of Aspergillus fungus A136 that can improve the disease resistance of Salvia miltiorrhiza seedlings. In the confrontation test, A136 had a strong antagonistic effect against Fusarium oxysporum, the pathogen causing Salvia miltiorrhiza wilt, with an inhibition rate of 43.65%. In the pot experiment, the incidence of wilt disease in Salvia miltiorrhiza seedlings in the CK treatment, where sterile water was inoculated first and then the pathogen Fusarium oxysporum inoculum was added, was 55.56%. The incidence of wilt disease in Salvia miltiorrhiza seedlings in the CL treatment, where A136 inoculum was added first and then the pathogen Fusarium oxysporum inoculum was added, was 11.11%. The prevention and control effect of A136 reached 80%. A136 can significantly reduce the incidence of wilt disease in Salvia miltiorrhiza seedlings and effectively improve the disease resistance of Salvia miltiorrhiza seedlings to wilt disease caused by Fusarium oxysporum.

[0017] The Aspergillus fungus A136 screened by the present invention can improve the ability of Salvia miltiorrhiza seedlings to resist wilt disease caused by Fusarium oxysporum, providing support for the healthy and efficient breeding technology of Salvia miltiorrhiza seedlings in factories and having guiding significance for the production of Salvia miltiorrhiza. Description of the Drawings

[0018] Figure 1 It is a plate confrontation photo of strain A136 and the pathogen Fusarium oxysporum.

[0019] Figure 2 It is a colony plate photo of strain A136 (front of the colony).

[0020] Figure 3 It is a colony plate photo of strain A136 (back of the colony).

[0021] Figure 4 It is a phylogenetic tree constructed from the ITS gene sequence of strain A136.

[0022] Figure 5 It is a bar chart showing the effect of inoculating A136 inoculum on the anti-wilt disease effect of Salvia miltiorrhiza seedlings.

[0023] Figure 6 It is a phenotypic map showing the effect of inoculating A136 inoculum on the anti-wilt disease effect of Salvia miltiorrhiza seedlings.

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

[0025] A136, taxonomically named Aspergillus Aspergillus sp., is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is January 2, 2025, and the preservation number is CGMCC NO. 41743. 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 mouth of the beaker, pour the viscous potato liquid from the gauze, 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 authentic Salvia miltiorrhiza production 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 (the triangular flask contains 6 glass beads with a diameter of 4 mm), 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, incubate the spread plate 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 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 glycerol tube in an -80 °C refrigerator for later use. The specific method for storing the strains in glycerol tubes is: wash the mycelia and spores on the PDA medium plate with sterile water, filter out the mycelia 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, and count the spore concentration of the glycerol spore solution with a hemocytometer at 1×10 8When the concentration of glycerol spore solution is more than 1×10⁶ / mL, transfer the glycerol spore solution to a sterile centrifuge tube. When preserving, first pre-freeze it at -20°C for 12 - 15 hours 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 fusarium wilt as the pathogen. Inoculate the pathogen Fusarium oxysporum on 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 on a PDA medium plate and culture it at 28°C until the mycelium covers the PDA medium plate to obtain mycelial blocks (6 mm) of the pathogen Fusarium oxysporum. Inoculate each of the isolated and purified fungal strains on a PDA medium plate and culture it at 28°C for 3 days to obtain mycelial blocks (6 mm) of each of the isolated and purified fungal strains. Inoculate the mycelial block (6 mm) of the pathogen Fusarium oxysporum in the center of a new PDA medium plate, and place the mycelial 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 mycelial block (6 mm) of the pathogen Fusarium oxysporum (confrontation plate). Culture it at 28°C for confrontation, 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 mycelial 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 A136 is involved in the present invention (the plate confrontation photo of strain A136 and the pathogen Fusarium oxysporum is as Figure 1 shown), and strain A68 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 A136 was cultured on a PDA medium plate at 28 °C for 7 days, as Figure 2 and Figure 3 shown, the whole colony drifted with the spores. The single colony was nearly circular and was relatively densely distributed as a whole. The surface of the colony was villous, with a certain three-dimensional sense. The color was mainly grayish-black to black, and the color at the edge was slightly lighter, and the boundary with the medium was relatively clear. The colony produced dark brown spores, and their morphology was relatively close to that of Aspergillus niger. By comparing the ITS gene sequence of strain A136 (the ITS gene sequence of strain A136 is shown in SEQ ID NO: 1, which was obtained by PCR amplification and sequencing of the DNA of strain A136 using universal primers ITS1 (shown in SEQ ID NO: 2) / ITS4 (shown in SEQ ID NO: 3)) with similar sequences and constructing a phylogenetic tree, the results were as Figure 4 shown. Strain A136 had the highest homology with Aspergillus welwitschiae , reaching 99.65%. Combining the colony morphological characteristics of strain A136 and the comparison and analysis results of the phylogenetic tree constructed by the ITS series, strain A136 was identified as a fungus of the genus Aspergillus Aspergillus sp. Strain A136 was deposited in the China General Microbiological Culture Collection Center on January 2, 2025, and the deposit number was CGMCC NO. 41743.

[0036] Example 2 Preparation of microbial inoculum 2.1 Preparation of A136 microbial inoculum Strain A136 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 off with sterile water, and the mycelia were 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 with a hemocytometer), and the A136 microbial inoculum was thus obtained.

[0037] 2.2 Preparation of the pathogen Fusarium oxysporum microbial inoculum The pathogen Fusarium oxysporum 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 off with sterile water, and the mycelia were 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 with a hemocytometer), and the pathogen Fusarium oxysporum microbial inoculum was thus obtained.

[0038] Example 3 Pot experiment on the disease resistance effect of inoculating A136 microbial inoculum 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 of Nanjing Agricultural University and let them be planted and grow for 15 - 20 days (in this example, they are planted and grow for 15 days). During the planting and growth period, the plant supplementary lights in the greenhouse are automatically turned 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 A136 bacterial agent (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 the pathogen Fusarium oxysporum bacterial agent (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, the CK treatment (adding sterile water and then adding the Fusarium oxysporum bacterial agent) and the CL treatment (adding the A136 bacterial agent and then adding the Fusarium oxysporum bacterial agent). The experiment is a completely randomized block design, with 3 independent biological replicates for each treatment, and each replicate contains 6 pots of plants. The seedlings are harvested 30 days after adding the Fusarium oxysporum bacterial agent, and the incidence of Fusarium wilt of Salvia miltiorrhiza seedlings is measured. The incidence of Fusarium wilt of the replicate (%) = the number of pots with disease (showing symptoms of Fusarium wilt) among 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] Since Figure 5 and Figure 6 it can be seen that the incidence of Fusarium wilt of Salvia miltiorrhiza seedlings in the CK treatment (adding sterile water and then adding the Fusarium oxysporum bacterial agent) is 55.56%, and the incidence of Fusarium wilt of Salvia miltiorrhiza seedlings in the CL treatment (adding the A136 bacterial agent and then adding the Fusarium oxysporum bacterial agent) is 11.11%, and the prevention and control effect of A136 reaches 80%. The results show that the Aspergillus sp. A136 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 A136, taxonomically named Aspergillus Aspergillus sp., deposited in the China General Microbiological Culture Collection Center on January 2, 2025, with the deposit number CGMCC NO. 41743.

2. The microbial agent prepared from Aspergillus sp. A136 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, wherein The microbial agent is prepared by the following method: Aspergillus sp. A136 with the preservation number of CGMCC NO. 41743 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 or more per mL to obtain the microbial agent.

4. The microbial agent according to claim 3, wherein The microbial agent is prepared by the following method: Aspergillus sp. A136 with the preservation number of CGMCC NO.41743 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 to obtain the microbial agent.

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

6. Use of the Aspergillus fungus A136 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 introduced into the seedling-raising substrate for breeding Salvia miltiorrhiza seedlings, and the introduction ratio is 4-5 mL of the bacterial agent per Salvia miltiorrhiza seedling.

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

10. The application according to claim 8, wherein Introducing 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.

Images