An endophyte strain Gliocladium roseum NG-12 and its application

Abstract

The present invention provides an endophyte strain Gliocladium roseum NG-12, named Gliocladium roseum ( Clonostachys rosea ) NG-12, with a deposit number of CGMCC No.41801. The endophyte Glechoma rosea NG-12 provided by the present invention has good root-knot nematode killing and antibacterial activity, and has a good antagonistic effect on a variety of plant pathogens such as Fusarium oxysporum, Colletotrichum gloeosporioides, and Colletotrichum gloeosporioides. It is a potential biological agent for preventing and treating root-knot nematode disease, wilt, anthracnose, etc., and the fungus also has the ability to solubilize potassium, fix nitrogen, dissolve phosphorus, produce siderophores, produce proteases, and produce cellulases. The Glechoma rosea of the present invention has broad development space and good development and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an endophyte Gliocladium roseum NG-12 and an application thereof. Background Art

[0002] The genus Russula belongs to the family Russulaceae, order Russulaes, phylum Basidiomycetes. Internationally, over 300 species of Russula have been reported, with over 90 species found in my country, including over 60 edible species, widely distributed across 30 provinces and autonomous regions. Most Russula species are edible, and many have high economic and nutritional value, serving as both edible and medicinal. Russula boasts numerous health benefits, including nourishing blood, dispelling wind and cold, enhancing beauty, and promoting blood circulation. It has long been considered a medicinal and edible fungus, making it a rare, all-natural, and health-promoting edible fungus worldwide. However, due to its wild growth and uncontrollable growth, Russula is inevitably susceptible to pests and diseases, resulting in poor quality, impacting its yield and quality, and even hindering the development of my country's edible fungus industry. Currently, physical and chemical control methods are the primary means of controlling edible fungi pests and diseases. However, due to increasing concerns about pesticide residues, environmental pollution, and the development of pathogenic bacteria caused by chemical pesticides, the effectiveness and operational limitations of physical control methods are becoming increasingly prominent. Consequently, attention is shifting towards more environmentally friendly and effective biological control methods. Biological pest control is safe and efficient, with relatively stable and long-lasting effects and high specificity. It typically targets specific pests and diseases, with minimal impact on non-target organisms. This helps protect the diverse life forms within ecosystems, maintain biodiversity, and contribute to a more stable and healthy ecosystem. Currently, biological control is considered one of the most important control methods with the greatest potential for development.

[0003] Chinese patent CN119214171A discloses a microbial agent for controlling root-knot nematodes and a preparation method thereof, wherein the microbial agent comprises Paecilomyces lilacinus ( Paecilomyces lilacinus ), Bacillus thuringiensis ( Bacillus thuringiensis )、Beauveria bassiana( Beauveria ), Bacillus subtilis ( Bacillus subtilis ) are mainly used for the prevention and treatment of root-knot nematodes in the form of fertilizers, but there is no mention of the prevention and treatment effects on other diseases.

[0004] Edible fungi pests and diseases have a serious impact on edible fungi production. Therefore, isolating and screening biocontrol bacteria with effective control effects on edible fungi is an important approach to edible fungi pest and disease control. Russula endophytes, as special beneficial microorganisms residing within Russula tissues, can prevent crop diseases by colonizing the body. They produce a wide variety of secondary metabolites that promote growth, provide antibacterial and insect resistance, and benefit plant growth. Furthermore, Russula endophytes have long coexisted with and evolved with their hosts, forming a long-term mutually beneficial symbiotic relationship. Compared to microbial agents used in edible fungi cultivation from other sources, these agents are safer, avoid food safety issues, and can replace pesticides and chemical fertilizers to achieve environmentally friendly yield increases, maintaining the stability and diversity of the ecosystem. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an endophytic pink Gliocladium NG-12 of Russula and its application.

[0006] To achieve the above objectives:

[0007] The first aspect of the present invention is to provide an endophyte Gliocladium roseum NG-12, named: Gliocladium roseum ( Clonostachys rosea ) NG-12, deposited at the General Microbiology Center of China Culture Collection Administration on January 20, 2025, with the deposit number CGMCC No.41801, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.

[0008] The second aspect of the present invention provides the use of the endophyte Gliocladium roseum NG-12 in the preparation of a siderophore-producing, potassium-solubilizing, and nitrogen-fixing preparation.

[0009] The third aspect of the present invention provides the use of the endophyte Gliocladium roseum NG-12 in the preparation of a protease and / or cellulase preparation.

[0010] The fourth aspect of the present invention provides a fermentation broth of the endophyte Gliocladium roseum NG-12.

[0011] The present invention provides a fermentation broth, using any one of the preferred endophytes Gliocladium roseum NG-12 as a fermentation bacterium, and a method for preparing the fermentation broth comprises the following steps:

[0012] Step 1: shaking culture the endophyte Gliocladium roseum NG-12 in a PDA seed culture medium to obtain a fermentation seed liquid of the endophyte Gliocladium roseum NG-12;

[0013] Step 2: The fermentation seed liquid prepared in step 1 is inoculated into a fermentation medium to obtain a fermentation liquid of the endophyte Gliocladium roseum NG-12.

[0014] Preferably, in step 1, the shaking culture condition is 26°C.

[0015] Preferably, in any of the above items, in step 1, the shaking culture condition is 140 rpm.

[0016] Preferably, in any of the above items, in step 1, the culture is performed for 5 days.

[0017] Preferably, in any of the above items, in step 2, the fermentation seed liquid is inoculated into the fermentation medium at a volume percentage of 5%.

[0018] Preferably, in any of the above items, in step 2, the fermentation temperature is 26°C.

[0019] Any of the above is preferably that in step 2, the fermentation condition is 140 rpm.

[0020] Preferably, in any of the above items, in step 2, the fermentation culture is carried out for 9 days.

[0021] Preferably, in any of the above items, in step 2, the fermentation medium has a formula of: 30.0 g / L sucrose, 25.0 g / L yeast extract powder, and 2.5 g / L magnesium sulfate heptahydrate.

[0022] The fifth aspect of the present invention provides a sterile fermentation filtrate of the endophyte Gliocladium roseum NG-12.

[0023] The present invention provides a sterile fermentation filtrate, which is prepared from any of the fermentation broths described above. After centrifuging any of the fermentation broths described above, the supernatant is filtered through a 0.22 μm filter to prepare the sterile fermentation filtrate.

[0024] Preferably, any of the above items is centrifuged at 4° C. and 10,000 rpm for 10 min.

[0025] The sixth aspect of the present invention is to provide a preparation containing at least one of the endophyte Gliocladium roseum NG-12 described in any one of the above items, the fermentation broth described in any one of the above items, and the sterile fermentation filtrate described in any one of the above items.

[0026] A seventh aspect of the present invention provides the use of at least one of the endophyte Gliocladium roseum NG-12, the fermentation broth, and the sterile fermentation filtrate in antagonizing pathogens.

[0027] The preferred pathogens include Fusarium oxysporum Fusarium oxysporum Colletotrichum gloeosporioides Colletotrichum gloeosporioides , Colletotrichum oxysporum Colletotrichum acutatum At least one of .

[0028] The eighth aspect of the present invention is to provide the endophyte Gliocladium roseum NG-12, the fermentation liquid, and the fermentation filtrate in the preparation of the method for preventing and treating Fusarium oxysporum. Fusarium oxysporum Colletotrichum gloeosporioides Colletotrichum gloeosporioides , or Colletotrichum oxysporum Colletotrichum acutatum Application in treating diseases caused by at least one pathogenic bacteria.

[0029] A ninth aspect of the present invention provides a use of at least one of the endophyte Gliocladium roseum NG-12, the fermentation broth, or the fermentation filtrate in controlling root-knot nematodes.

[0030] The tenth aspect of the present invention provides the use of the endophyte Gliocladium roseum NG-12, the fermentation liquid, and the fermentation filtrate for preventing and controlling pests and diseases caused by root-knot nematodes.

[0031] The eleventh aspect of the present invention is the 18S rRNA gene sequence of the endophyte Gliocladium roseum NG-12, as shown in SEQ ID NO: 1.

[0032] The endophyte Gliocladium roseum NG-12 of the present invention has good antibacterial activity against Fusarium oxysporum Fusarium oxysporum , and / or Colletotrichum gloeosporioides Colletotrichum gloeosporioides , and / or Colletotrichum oxysporum Colletotrichum acutatum The invention has a good antagonistic effect on the root-knot nematode and has a good lethal effect on root-knot nematodes, making it a potential biological agent for controlling red mushroom diseases and insect pests. In addition, the pink Glycoscouril of the present invention has good ability to produce siderophores, potassium and nitrogen fixation, and protease and cellulase. The pink Glycoscouril of the present invention has broad development space and good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are the morphological characteristics of the endophyte Gliocladium roseum NG-12 screened in the preferred embodiment 1 of the present invention.

[0034] Figure 2 This is the phylogenetic tree of the endophyte Gliocladium roseum NG-12 in the preferred embodiment 2 of the present invention.

[0035] Figure 3 This is the determination result of siderophore production by the endophyte Gliocladium roseum NG-12 in preferred embodiment 3 of the present invention.

[0036] Figure 4 These are the results of potassium solubilization and nitrogen fixation assays of the endophyte Gliocladium roseum NG-12 in preferred embodiment 3 of the present invention.

[0037] Figure 5This is the enzyme production assay result of the endophyte Gliocladium roseum NG-12 in preferred embodiment 3 of the present invention.

[0038] Figure 6 This is the antagonistic result of the endophyte Gliocladium roseum NG-12 in the preferred embodiment 4 of the present invention.

[0039] Figure 7 These are the microscopic examination results of the effect of the sterile fermentation filtrate of the endophyte Gliocladium roseum NG-12 in preferred embodiment 5 of the present invention on controlling root-knot nematodes.

[0040] Figure 8 These are the statistical results of the effects of sterile fermentation filtrates of different amounts of the endophyte Gliocladium roseum NG-12 in preferred embodiment 5 of the present invention on controlling root-knot nematodes.

[0041] Figure 9 This is the thermal stability result of the sterile fermentation filtrate of the endophyte Gliocladium roseum NG-12 in the preferred embodiment 6 of the present invention. DETAILED DESCRIPTION

[0042] The present invention will be further described in conjunction with specific examples with reference to the accompanying drawings for a better understanding of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in accordance with the product instructions were used. Where the manufacturer of the reagents or instruments used is not specified, they are all commercially available conventional products.

[0043] The bacterial culture medium used in the following examples is as follows:

[0044] PDA solid medium (g / L): potato powder 10 g, glucose 20 g, agar 15 g.

[0045] PDA seed culture medium (g / L): potato extract powder 10 g, glucose 20 g.

[0046] Fermentation medium (g / L): potato extract powder 10 g, sucrose 30 g, yeast extract powder 25 g, magnesium sulfate heptahydrate 2.5 g.

[0047] CAS assay medium was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.; Ashby nitrogen-free medium was purchased from Ruichu Biotechnology (Jiangsu) Co., Ltd.

[0048] Potassium-dissolving medium (g / L): sucrose 1.5 g, MgSO4·7H2O 0.02 g, CaSO4·7H2O 0.01 g, NaCl 0.02 g, potassium feldspar powder 0.5 g, agar 15 g, pH 7.0~7.5.

[0049] Treatment of potassium feldspar powder: Soak the powder in 10% HCl for 8–10 hours (stirring thoroughly every 1–2 hours), then soak overnight. Discard the supernatant and rinse with deionized water. Stir thoroughly, let it sit, discard the supernatant, and rinse again with deionized water. Repeat this process several times until the pH value remains unchanged. Dry and grind the treated powder, pass it through a 100-mesh sieve, and bottle it for later use.

[0050] Sodium carboxymethylcellulose (CMC) medium (g / L): sodium carboxymethylcellulose 5 g, (NH4)2SO4 0.5 g, Congo red 0.4 g, MgSO4·7H2O 0.1 g, K2HPO4 0.25 g, agar 16 g, dilute to 1000 mL.

[0051] Skim milk powder culture medium (g / L): 8 g of skim milk powder was dissolved in 300 mL of water and sterilized at 115°C for 10 min.

[0052] Add water to 9 g of agar to make up to 300 mL, sterilize at 121°C for 20 min, and sterilize A and B separately before mixing. Example 1

[0053] Example 1 provides a method for obtaining the endophyte Gliocladium roseum NG-12.

[0054] 1. Sample source:

[0055] In September 2023, the red mushroom fruiting bodies were provided by Sanming, Fujian.

[0056] 2. Isolation and screening of strains:

[0057] Select healthy, undamaged caps of red mushrooms, wash them with clean water, clean the surface with 75% ethanol, soak them in 5% NaClO solution for 5 minutes, rinse them with sterile water three times, and dry them with sterile filter paper. Use a sterile knife to cut the inner tissue of the cap of red mushrooms into a sterile mortar, add sterile water and grind it into a homogenate, and then dilute it stepwise to obtain 10 -1 , 10 -2 , 10 -3 dilution. Pipette 100 μL of the original liquid homogenate and dilution onto the PDA solid culture medium and spread it evenly on the surface of the PDA solid culture medium; take another 100 μL of sterile water from the last wash to spread on the plate as a control group. If no bacteria grow, it means that the surface of the sample is sterile. Repeat the experiment 3 times. Place the culture medium upside down in a 28 ℃ incubator for several days and observe and record. The selected colonies are round, white or milky white in color, with neat edges, clear outlines, creeping hyphae, and are numbered. After picking a single colony, scratch the inclined surface of the test tube and store it at 4 ℃. Figure 1Shown are the morphological characteristics of the screened endophyte Gliocladium roseum NG-12. Example 2

[0058] Example 2 The endophyte Gliocladium roseum NG-12 obtained in Example 1 was subjected to 18S rRNA gene sequencing, using the extracted genomic DNA as a template and the fungal 18S rDNA gene primers:

[0059] The ITS1 sequence shown in SEQ ID NO: 2 is: 5′-TCCGTAGGTGAACCTGCGG-3′;

[0060] The ITS4 sequence shown in SEQ ID NO: 3 is: 5′-TCCTCCGCTTATTGATATGC-3′;

[0061] PCR amplification and sequencing were performed. The sequencing results were compared by BLAST on the NCBI website to determine the species of closely related fungi. The phylogenetic tree constructed based on the 18S rRNA gene sequence is shown in the figure. Figure 2 As shown, it was identified as Gliocladium roseum ( Clonostachys rosea ).

[0062] Based on the above test results, combined with morphological, cultural and biological characteristics, the endophyte G. rosea NG-12 was identified as a new species of G. rosea and named: G. rosea ( Clonostachys rosea ) NG-12 has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit number: CGMCC No.41801. Example 3

[0063] Example 3 The biological characteristics of the endophyte Gliocladium roseum NG-12 were determined.

[0064] The plate method was used to determine the ability of the endophyte Gliocladium roseum NG-12 to produce siderophores, potassium, nitrogen, cellulase and protease.

[0065] (1) Determination of siderophore production, nitrogen fixation, and potassium dissolution:

[0066] Using the plate method, the endophyte Gliocladium roseum NG-12 was inoculated in a square pattern onto CAS test medium, Ashby nitrogen-free medium, and potassium-solubilizing medium. The strain's growth and the presence of clearing zones were observed, and their diameters were recorded. The ratio of the clearing zone diameter D (mm) on CAS test medium to the colony diameter d (mm) was calculated. Siderophore production reached 3.10. Growth on Ashby nitrogen-free medium and potassium-solubilizing medium indicates potassium-solubilizing and nitrogen-fixing capabilities.

[0067] (2) Enzyme production assay

[0068] The endophyte G. rosea NG-12 was spotted onto sodium carboxymethyl cellulose (CMC) medium using the plate method. After two days of incubation, colony diameters were measured and recorded, and the clearing zone was observed and recorded. The endophyte G. rosea NG-12 was spotted onto skim milk powder medium in a square pattern, and the clearing zone was observed and recorded. The ratio of the clearing zone diameter (D (mm)) to the colony diameter (d (mm)) was calculated. Cellulase production reached 2.56, and protease production reached 2.67.

[0069] The results are as follows Figures 3-5 As shown:

[0070] Figure 3 The results of the determination of siderophore production by the endophyte Gliocladium roseum NG-12;

[0071] Figure 4 The results of potassium solubilization and nitrogen fixation of endophyte Gliocladium roseum NG-12 are shown. Figure 4 The middle left picture shows the results of potassium dissolution assay, and the right picture shows the results of nitrogen fixation assay;

[0072] Figure 5 This is the result of enzyme production assay of endophyte Gliocladium roseum NG-12. Figure 5 In the figure, the upper figure shows the results of cellulase production, and the lower figure shows the results of protease production. Example 4

[0073] Example 4 provides an antagonism assay for the endophyte Gliocladium roseum NG-12.

[0074] The isolated and purified endophyte Gliocladium roseum NG-12 was used as the antagonistic bacterium, and Fusarium oxysporum ( Fusarium oxysporum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Colletotrichum oxysporum ( Colletotrichum acutatum ) was used as the pathogen indicator bacteria, and the plate confrontation method was used to determine the antagonistic effect of the endophyte Gliocladium roseum NG-12 on pathogens. A 5 mm diameter punch was used to punch a bacterial cake, and the mycelium was placed in the center of a PDA culture dish. Pathogens were inoculated around the four sides. A bacterial cake containing only the test pathogen was used as the control (CK). Each group was repeated three times and cultured at 28°C for 3 days. The inhibition zones were observed and recorded. The inhibition rates were 51.64%, 49.09%, and 47.30%, respectively. Figure 6 As shown, Figure 6 From left to right in the middle, the antagonistic inhibition rate against Fusarium oxysporum is 51.64%, the antagonistic inhibition rate against Colletotrichum gloeosporioides is 49.09%, and the antagonistic inhibition rate against Colletotrichum oxysporum is 47.30%.

[0075] Corrected inhibition rate = (colony diameter of control group - colony diameter of experimental group) / (colony diameter of control group - 5 mm) × 100%. Example 5

[0076] Example 5 provides the effect of the sterile fermentation filtrate of strain NG-12 on controlling root-knot nematodes

[0077] After culturing strain NG-12 at 26°C and 140 rpm for 9 days, the fermentation broth was centrifuged at 4°C and 10,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 μm syringe filter to obtain a sterile fermentation filtrate. One mL of the incubated second-instar root-knot nematode larvae (approximately 100 larvae) was placed in a culture dish, and 0.5 mL, 1 mL, 1.5 mL, and 2 mL of the fermentation filtrate were added, respectively. Each group was repeated three times. After 24 hours at 28°C, the number of dead root-knot nematodes was counted and the corrected mortality rate was calculated. The results were 39.69%, 67.44%, 67.41%, and 69.61%, respectively. Figure 7 Shown are the results of microscopic examination of nematode mortality. Figure 7 The middle left picture shows the survival of root-knot nematodes without adding fermentation liquid, and the right picture shows the survival of root-knot nematodes after adding 1 mL of fermentation liquid. Figure 8 The results are statistical results of the control effect of root-knot nematodes using 0.5mL, 1mL, 1.5mL, and 2mL fermentation filtrate. Example 6

[0078] Example 6 provides the thermal stability of the sterile fermentation filtrate of the endophyte Gliocladium roseum NG-12.

[0079] Preparation of fermentation broth of endophytic fungus Gliocladium roseum NG-12: A single colony cake of the strain was inoculated into PDA seed medium and cultured in a shaking incubator at 26°C and 140 rpm for 5 days. After obtaining the seed liquid, it was inoculated into fermentation medium and cultured at 26°C and 140 rpm for 9 days.

[0080] The effect of temperature on the antifungal activity of G. rosea NG-12 was examined. Sterile filtrates of G. rosea NG-12 were treated at seven different temperatures: (a) 30°C in a water bath for 1 hour; (b) 40°C in a water bath for 1 hour; (c) 50°C in a water bath for 1 hour; (d) 60°C in a water bath for 1 hour; (e) 70°C in a water bath for 1 hour; (f) 80°C in a water bath for 1 hour; and (g) 90°C in a water bath for 1 hour. After the treatment, an experiment on controlling root-knot nematodes was carried out. 1 mL of root-knot nematode liquid (about 100 nematodes) was placed in a culture dish, and 1 mL of fermentation filtrate was added. Each group was repeated 3 times and placed at 28°C for 24 hours. The number of dead root-knot nematodes was counted, and the corrected mortality rates were calculated to be 63.72%, 64.11%, 61.71%, 61.70%, 59.60%, 46.11%, and 41.83%, respectively.

[0081] The results are as follows Figure 9 As shown in the data, within the range of 30℃~70℃, the corrected mortality rate of root-knot nematodes was relatively stable, and the influence of temperature was small. When the treatment temperature increased to above 70℃, the effect showed a significant downward trend. After treatment at 80℃ for 1 hour, the corrected mortality rate of root-knot nematodes decreased by 17.61%, and the mortality rate of root-knot nematodes after treatment at 90℃ for 1 hour was 41.83%.

[0082] The above embodiments are provided for illustrative purposes only and are not intended to limit the scope of implementation. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to provide an exhaustive list of all implementations. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. An endophyte strain of Gliocladium roseum NG-12, characterized by: The deposit number is CGMCC No. 41801. The endophyte Gliocladium roseum NG-12 has the functions of producing siderogen, dissolving potassium, and fixing nitrogen; at the same time, it produces protease and cellulase; at the same time, it inhibits Fusarium oxysporum, Colletotrichum gloeosporioides, and Colletotrichum oxysporum; at the same time, it has the function of preventing and controlling nematodes.

2. A fermentation broth, characterized in that: The endophyte Gliocladium roseum NG-12 according to claim 1 is used as a fermentation bacterium, and the method for preparing the fermentation liquid comprises the following steps: Step 1: shaking culture the endophyte Gliocladium roseum NG-12 in a PDA seed culture medium to obtain a fermentation seed liquid of the endophyte Gliocladium roseum NG-12; Step 2: The fermentation seed liquid prepared in step 1 is inoculated into a fermentation medium to obtain a fermentation liquid of the endophyte Gliocladium roseum NG-12.

3. The fermentation liquid according to claim 2, wherein In step 1, the shaking culture conditions are 26° C., 140 rpm, and culture for 5 days.

4. The fermentation liquid according to claim 2, wherein In step 2, the fermentation seed liquid is inoculated into the fermentation medium at a volume percentage of 5%, and the fermentation conditions are: 26° C., 140 rpm, and culture for 9 days; the formula of the fermentation medium is: 30.0 g / L sucrose, 25.0 g / L yeast extract powder, and 2.5 g / L magnesium sulfate heptahydrate.

5. A sterile fermentation filtrate, characterized in that The fermentation broth according to any one of claims 2 to 4 is prepared by centrifuging the fermentation broth according to any one of claims 2 to 4, and filtering the supernatant with a 0.22 μm filter to obtain a sterile fermentation filtrate.

6. Use of the endophyte Gliocladium roseum NG-12 according to claim 1, the fermentation broth according to any one of claims 2 to 4, or the sterile fermentation filtrate according to claim 5 in the preparation of at least one preparation for producing siderophores, potassium solubilizers, and nitrogen fixers.

7. Use of the endophyte Gliocladium roseum NG-12 according to claim 1, the fermentation broth according to any one of claims 2 to 4, or the sterile fermentation filtrate according to claim 5 in the preparation of a protease and / or cellulase preparation.

8. Use of the endophyte Gliocladium roseum NG-12 according to claim 1, the fermentation broth according to any one of claims 2 to 4, or the sterile fermentation filtrate according to claim 5 in controlling root-knot nematodes and preparing an agent for controlling diseases caused by root-knot nematodes.

9. A preparation, characterized in that The method comprises at least one of the endophyte Gliocladium roseum NG-12 according to claim 1, the fermentation broth according to any one of claims 2 to 4, and the sterile fermentation filtrate according to claim 5.

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