Aspergillus fungus A136 and its application

Abstract

The present invention discloses a strain of Aspergillus fungus A136, which is classified as Aspergillus Aspergillus sp., deposited in the General Microbiology Center of the China National Committee for the Collection of Microorganisms, with a deposit date of January 2, 2025, and a deposit number of CGMCC NO.41743. 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 Salvia miltiorrhiza seedlings, wherein the disease resistance is resistance to wilt caused by Fusarium oxysporum. The Aspergillus fungus A136 screened by the present invention can improve the resistance of Salvia miltiorrhiza seedlings to wilt caused by Fusarium oxysporum, provide support for the healthy and efficient factory-based breeding technology of Salvia miltiorrhiza seedlings, and have guiding significance for the production of Salvia miltiorrhiza.

Description

Technical Field

[0001] The present 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. While much research has focused on the effects of Aspergillus on crop growth in agriculture, little research has been conducted on its effects on traditional Chinese medicines.

[0003] Salvia miltiorrhiza ( Salvia miltiorrhiza Bunge (Salvia miltiorrhiza) is a perennial medicinal herb with the properties of promoting blood circulation, removing blood stasis, relieving pain, clearing the heart and relieving restlessness, and cooling blood and eliminating carbuncles. Salvia miltiorrhiza plays a significant role in the clinical treatment of cardiovascular and cerebrovascular diseases and is a commonly used medicinal herb in my country. With increasing awareness of cardiovascular and cerebrovascular diseases, the annual demand for Salvia miltiorrhiza is increasing, and the cultivated area is also expanding. Salvia miltiorrhiza is primarily cultivated in traditional Chinese medicine, with its main production areas located in Shandong, Sichuan, Henan, Shanxi, and Hebei. With increasing planting density and continuous cropping, the resulting pest and disease problems are becoming increasingly severe, severely hindering the quality improvement of Salvia milt and the development of the industry. In soils where Salvia milt is continuously cropped, common diseases that hinder continuous cropping include wilt. This disease is a common disease in Salvia miltiorrhiza production and often occurs concurrently with root rot, impacting its yield and quality, severely restricting its production. As a traditional medicinal plant with a large cultivated area in my country, Salvia miltiorrhiza is an ideal target for promoting high-efficiency agriculture and rural revitalization. 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 practical problems and needs in Salvia miltiorrhiza 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 A136, which is classified as Aspergillus Aspergillus sp., deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with a deposit date of January 2, 2025, and a deposit number of CGMCC NO.41743.

[0008] The second aspect of the present invention provides a bacterial agent prepared from the above-mentioned Aspergillus fungus A136, 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 A136 with a deposit number of CGMCC NO.41743 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, that is, the bacterial agent is obtained.

[0010] Furthermore, the bacterial agent is prepared by the following method: Aspergillus fungus A136 with a preservation number of CGMCC NO.41743 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 A136 in improving the disease resistance of Salvia miltiorrhiza seedlings, wherein the disease resistance is resistance to wilt caused by Fusarium oxysporum.

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

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

[0015] Furthermore, the period of time for colonization and growth after the Salvia miltiorrhiza seedlings are transplanted is 15-20 days after the Salvia miltiorrhiza seedlings are transplanted.

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

[0017] Beneficial effects of the present invention:

[0018] The present invention screened out an Aspergillus fungus A136 that can improve the disease resistance of Salvia miltiorrhiza seedlings. In a standoff test, A136 had a strong antagonistic effect on Fusarium oxysporum, the pathogen of Salvia miltiorrhiza wilt, with an inhibition rate of 43.65%. In a potted plant experiment, the incidence of wilt in Salvia miltiorrhiza seedlings treated with CK (inoculated with sterile water and then with the pathogen Fusarium oxysporum inoculant) was 55.56%, while the incidence of wilt in Salvia miltiorrhiza seedlings treated with CL (inoculated with A136 and then with the pathogen Fusarium oxysporum inoculant) was 11.11%. The prevention and control effect of A136 reached 80%. A136 can significantly reduce the incidence of wilt in Salvia miltiorrhiza seedlings and effectively improve the disease resistance of Salvia miltiorrhiza seedlings to wilt caused by Fusarium oxysporum.

[0019] The Aspergillus fungus A136 screened by the present invention can improve the resistance of Salvia miltiorrhiza seedlings to wilt disease caused by Fusarium oxysporum, provide support for the healthy and efficient factory-based breeding technology of Salvia miltiorrhiza seedlings, and have guiding significance for the production of Salvia miltiorrhiza. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a photo of the plate confrontation between strain A136 and the pathogen Fusarium oxysporum.

[0021] Figure 2 This is a photo of the colony plate of strain A136 (front of the colony).

[0022] Figure 3 This is a photo of the colony plate of strain A136 (back of the colony).

[0023] Figure 4 Phylogenetic tree constructed for the ITS gene sequence of strain A136.

[0024] Figure 5 This is a bar graph showing the effect of inoculation with A136 fungicide on the resistance of Salvia miltiorrhiza seedlings to wilt disease.

[0025] Figure 6 This is a phenotypic diagram showing the effect of inoculation with A136 fungicide on the resistance of Salvia miltiorrhiza seedlings to wilt disease.

[0026] Note: Different letters above the bar graph represent significant differences between different treatments ( p <0.05).

[0027] Biomaterial deposit information

[0028] A136, 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 January 2, 2025, and the deposit number is CGMCC NO.41743. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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.

[0032] The diameter of the culture dishes (plates) involved in the following examples is 90 mm.

[0033] Example 1 Isolation and identification of functional fungi

[0034] 1.1 Isolation of functional fungi

[0035] Rhizosphere soil from healthy Salvia miltiorrhiza plants was collected on a large scale. Specifically, rhizosphere soil from healthy Salvia miltiorrhiza plants was collected from the native Salvia miltiorrhiza production areas of Yanjiang District and Zhongjiang County, Ziyang City, Sichuan Province; Fangcheng County, Yuzhou City, and Mianchi County, Henan Province; and Ju County and Linqu County, Shandong Province. The collected rhizosphere soil from healthy Salvia miltiorrhiza plants was mixed, weighed, and placed in a flask containing six 4 mm diameter glass beads (45 mL of sterile water). The flask was shaken at 170 rpm at 30°C for 30 minutes, then removed and serially diluted. 100 µL of the soil suspension at different concentrations was spread onto PDA plates and 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 1×108 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.

[0036] The Fusarium oxysporum isolated from the roots of Salvia miltiorrhiza caused by Salvia miltiorrhiza wilt was used as the pathogen. The pathogen Fusarium oxysporum was inoculated onto a PDA culture medium plate and cultured at 28°C to produce spores. The culture was then stored in a 4°C refrigerator until use. The strain was preserved in a glycerol tube and stored in a -80°C refrigerator until use. The specific method for preserving the strain in a glycerol tube was the same as above.

[0037] The pathogen Fusarium oxysporum was inoculated onto PDA plates and incubated at 28°C until hyphae filled the entire plate. A 6 mm mycelial mass was obtained. Each isolated and purified fungal strain was inoculated onto a PDA plate and incubated at 28°C for 3 days to obtain a 6 mm mycelial mass. The 6 mm mycelial mass was inoculated onto the center of a new PDA plate. The 6 mm mycelial mass was then placed at two symmetrical points approximately 2 cm from the center of the 6 mm mycelial mass (opposing plates). The plates were incubated at 28°C. The inhibitory effect of each isolated and purified fungal strain on the pathogen Fusarium oxysporum was observed. A blank control (CK) was used as the blank control. When the blank control Fusarium oxysporum filled the entire plate, the inhibition rate of each isolated and purified fungal strain was calculated. Each treatment in this experiment was repeated 3 times. The antibacterial rate formula is as follows:

[0038] Inhibition rate (%) = (CK colony radius - opposing colony radius) / CK colony radius × 100, where the opposing colony radius is the colony radius of the pathogen Fusarium oxysporum in the opposing plate.

[0039] A large number of fungal strains were isolated and purified from the rhizosphere soil of healthy Salvia miltiorrhiza plants. The isolated and purified fungal strains were subjected to a plate confrontation test with the pathogen Fusarium oxysporum according to the above method, and 6 Aspergillus fungal strains with antagonistic effects were obtained (identified by ITS sequencing). The inhibition rates are shown in Table 1. Two strains with strong antagonistic effects, strain A136 and strain A68, were selected as functional fungi for subsequent tests. Strain A136 was used in the present invention (the plate confrontation photo of strain A136 and pathogen Fusarium oxysporum is shown in Figure 1). Figure 1 As shown), strain A68 is involved in another invention.

[0040] Table 1 Inhibitory effect of 6 isolated and purified Aspergillus fungi strains on the pathogen Fusarium oxysporum

[0041]

[0042] 1.2 Identification of functional fungi

[0043] After strain A136 was cultured on PDA medium plates at 28°C for 7 days, Figure 2 and Figure 3 As shown, the entire colony is scattered with the spores, and the single colony is close to a circle, and the overall distribution is relatively dense. The surface of the colony is velvety, with a certain three-dimensional sense. The color is mainly gray-black to black, the edge is slightly lighter in color, and the boundary with the culture medium is relatively clear. The colony produces black-brown spores, and its morphology is similar to that of Aspergillus niger. The ITS gene sequence of strain A136 (the ITS gene sequence of strain A136 is shown in SEQ ID NO: 1, which is obtained by PCR amplification and sequencing of the DNA of strain A136 using universal primers ITS1 (as shown in SEQ ID NO: 2) / ITS4 (as shown in SEQ ID NO: 3)) was compared with similar sequences, and a phylogenetic tree was constructed. The results are shown as follows Figure 4 As shown, strain A136 and Aspergillus welwitschiae The highest homology was 99.65%. Combining the colony morphology of strain A136 and the phylogenetic tree comparison analysis results constructed by the ITS series, strain A136 was identified as an Aspergillus fungus. Aspergillus sp. strain A136 was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on January 2, 2025, with the deposit number CGMCC NO.41743.

[0044] Example 2 Preparation of bacterial agent

[0045] 2.1 Preparation of A136 inoculant

[0046] Strain A136 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 (counted on a hemocytometer), the A136 bacterial agent was obtained.

[0047] 2.2 Preparation of pathogenic Fusarium oxysporum inoculant

[0048] The pathogenic fungus 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 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 oxysporum agent is obtained.

[0049] Example 3 Potted experiment on the effect of inoculation with A136 bacterial agent on the disease resistance of Salvia miltiorrhiza seedlings

[0050] 150 g of dry weight seedling medium was added to a plastic flower pot 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. Healthy Salvia miltiorrhiza seedlings (one-year-old purple Salvia miltiorrhiza seedlings, purchased from a planting base of a Shandong farmer) with uniform growth and size were selected as test plants and transplanted into the flower pots. One Salvia miltiorrhiza seedling was planted in each flower pot. The flower pots were placed in a glass greenhouse at the Binjiang Campus of Nanjing Agricultural University and grown for 15-20 days (15 days in this embodiment). During the planting and growth period, the plant fill light in the greenhouse was automatically turned off from 22:00 at night to 6:00 in the morning of the next day. The greenhouse temperature was 30°C and the greenhouse humidity was 80%. Water was poured every 2 days to keep the seedling medium basically moist (the greenhouse lighting, temperature, humidity and watering frequency of subsequent experiments were the same). After planting, each pot in the experimental group (CL) was inoculated with 5 mL of the A136 inoculant (directly poured into the seedling medium near the roots of the Salvia miltiorrhiza seedlings). Each pot in the control group (CK) was inoculated with an equal volume of sterile water (directly poured into the seedling medium near the roots of the Salvia miltiorrhiza seedlings). Seven days later, each pot in both the experimental group (CL) and the control group (CK) was inoculated with 5 mL of the pathogen Fusarium oxysporum (directly poured into the seedling medium near the roots of the Salvia miltiorrhiza seedlings). This experiment was divided into two treatments: the CK treatment (inoculated with sterile water followed by the pathogen Fusarium oxysporum) and the CL treatment (inoculated with A136 followed by the pathogen Fusarium oxysporum). A completely randomized block design was used, with three independent biological replicates per treatment and six plant pots per replicate. Seedlings were harvested 30 days after inoculation with the pathogen Fusarium oxysporum, and the incidence of Salvia miltiorrhiza seedling wilt disease was determined. The wilt incidence rate (%) of replicates = the number of diseased pots (with wilt symptoms) in 6 pots / 6×100, and the wilt incidence rate (%) of a treatment = the sum of the wilt incidence rate (%) of replicates of the same treatment / 3.

[0051] because Figure 5 and Figure 6 The results showed that the incidence of wilt in Salvia miltiorrhiza seedlings treated with CK (inoculated with sterile water and then with the pathogen Fusarium oxysporum) was 55.56%, while the incidence of wilt in Salvia miltiorrhiza seedlings treated with CL (inoculated with A136 and then with the pathogen Fusarium oxysporum) was 11.11%, with A136 achieving an 80% control efficacy. These results indicate that Aspergillus A136 can significantly reduce the incidence of wilt in Salvia miltiorrhiza seedlings and effectively improve their resistance to Fusarium oxysporum.

Claims

1. A strain of Aspergillus A136, classified as Aspergillus Aspergillus sp., deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with a deposit date of January 2, 2025, and a deposit number of CGMCC NO.41743.

2. The bacterial agent prepared by the Aspergillus fungus A136 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 A136 with a preservation number of CGMCC NO.41743 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, that is, the bacterial agent is obtained.

4. The microbial agent according to claim 3, characterized in that The bacterial agent is prepared by the following method: Aspergillus fungus A136 with a preservation number of CGMCC NO.41743 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 A136 according to claim 1 in improving the disease resistance of Salvia miltiorrhiza seedlings, wherein the disease resistance is resistance to wilt caused by Fusarium oxysporum.

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

8. The use according to claim 7, characterized in that When used, after the Salvia miltiorrhiza seedlings are transplanted and planted for a period of time, the bacterial agent is inoculated into the seedling medium for breeding the Salvia miltiorrhiza seedlings, and the inoculation ratio is 4-5 mL of the bacterial agent per Salvia miltiorrhiza seedling.

9. The use according to claim 8, characterized in that The period of time for the Salvia miltiorrhiza 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 medium for breeding Salvia miltiorrhiza seedlings by directly pouring the bacterial agent into the seedling medium near the roots of the Salvia miltiorrhiza seedlings.

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