Endophyte gliocladium roseum NG-12 and application thereof

Abstract

The invention provides an endophyte Gliocladium roseum NG-12, which is named as Gliocladium roseum NG-12, and the preservation number of the endophyte Gliocladium roseum NG-12 is CGMCC (China General Microbiological Culture Collection Center) No. 41801. The endophyte Gliocladium roseum NG-12 provided by the invention has good root-knot nematode killing activity and antibacterial activity, has a good antagonistic effect on various plant pathogens such as fusarium oxysporum, colletotrichum gloeosporioides and colletotrichum gloeosporioides, is a potential biological preparation for preventing and treating root-knot nematode diseases, fusarium wilt, anthracnose and the like, and also has the effects of dissolving potassium, fixing nitrogen, dissolving phosphorus, preventing and treating the root-knot nematode diseases and the like. The capability of producing siderophore, protease and cellulase is realized. The gliocladium roseum provided by the invention has a wide development space and a good development and application prospect.

Description

Technical Field

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

[0002] The genus Russula belongs to the family Russula of the order Russula of the phylum Basidiomycetes. According to international reports, there are more than 300 species of Russula fungi, of which more than 90 species are found in my country, of which more than 60 are edible and widely distributed in 30 provinces and regions in my country. Most Russula are edible fungi, and many species of Russula have extremely high economic and nutritional value and can be used for food or medicine. Russula has many health benefits such as nourishing blood and removing blood stasis, dispelling wind and cold, beautifying and nourishing the skin, and promoting blood circulation. It has long been regarded as a fungus with the same origin of medicine and food, and is a rare pure natural health edible fungus in the world. However, due to its wild growth and its uncontrollable growth, it is inevitable that it will be affected by pests and diseases, resulting in poor quality of Russula, affecting the yield and quality of Russula, and even affecting the development of my country's edible fungi industry. At present, the control of edible fungi pests and diseases is mainly based on physical control and chemical control. However, due to the increasingly prominent problems of pesticide residues, environmental pollution, enhanced resistance of pathogenic bacteria caused by chemical pesticides, and limited effect and scope of operation of physical control, people gradually shift their attention to more environmentally friendly and effective biological control methods. Biological control of pests and diseases is safe and efficient, with relatively stable and lasting effects and high specificity. It usually only targets specific pests and diseases and has little impact on other non-target organisms. This helps protect various organisms in the ecosystem, maintain biodiversity, and make the ecosystem more stable and healthy. Currently, biological control is considered to be one of the most important control methods with the greatest development potential.

[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 the prevention and treatment effects on other diseases are not mentioned.

[0004] Edible fungi diseases and pests have a serious impact on the production of edible fungi. Therefore, isolating and screening out biocontrol bacteria that have a preventive effect on edible fungi is an important way to prevent and control edible fungi diseases and pests. As a special beneficial microorganism existing in the tissues of red mushrooms, endophytic bacteria of red mushrooms can prevent crop diseases by colonizing in the body. They produce a wide variety of secondary metabolites, which can promote the growth of red mushrooms, resist bacteria, and resist pests, which are beneficial to plant growth. In addition, endophytic bacteria of red mushrooms have long coexisted with the red mushroom host and evolved with it, forming a long-term mutually beneficial symbiotic relationship. Compared with other sources of microbial agents used in the edible fungi cultivation industry, they are safer and avoid food safety issues. They can also replace pesticides and fertilizers to achieve environmentally friendly and increased production effects, 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 an application thereof.

[0006] In order to achieve the above objectives: The first aspect of the present invention is to provide an endophyte, Gliocladium roseum NG-12, named: Gliocladium roseum ( Clonostachys rosea )NG-12, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 20, 2025, with the deposit number CGMCC No.41801, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Tel: 010-64807355.

[0007] The second aspect of the present invention provides the use of the endophyte Gliocladium roseum NG-12 in the preparation of an iron carrier-producing, potassium-solubilizing and nitrogen-fixing preparation.

[0008] The third aspect of the present invention provides the use of the endophyte Gliocladium roseum NG-12 in the preparation of protease and / or cellulase preparations.

[0009] The fourth aspect of the present invention provides a fermentation broth of the endophyte Gliocladium roseum NG-12.

[0010] The present invention provides a fermentation liquid, using any one of the preferred endophytic bacteria Gliocladium roseum NG-12 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 endophytic fungus Gliocladium roseum NG-12.

[0011] Preferably, in step 1, the shaking culture condition is 26°C.

[0012] Preferably, in any of the above items, in step 1, the shaking culture condition is 140 rpm.

[0013] Preferably, in any of the above items, in step 1, the culture is performed for 5 days.

[0014] 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%.

[0015] Preferably, in any one of the above items, in step 2, the fermentation temperature is 26°C.

[0016] Preferably, in any one of the above items, in step 2, the fermentation condition is 140 rpm.

[0017] Preferably, in any one of the above items, in step 2, the fermentation culture is carried out for 9 days.

[0018] Preferably, in any of the above items, in step 2, the formula of the fermentation medium is: sucrose 30.0 g / L, yeast extract powder 25.0 g / L, magnesium sulfate heptahydrate 2.5 g / L.

[0019] The fifth aspect of the present invention provides a sterile fermentation filtrate of the endophyte Gliocladium roseum NG-12.

[0020] 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 obtain the sterile fermentation filtrate.

[0021] Preferably, any of the above items is centrifuged at 4°C and 10,000 r / min for 10 min.

[0022] The sixth aspect of the present invention is to provide a preparation, which contains at least one of the endophytic fungus 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.

[0023] The seventh aspect of the present invention provides the use of at least one of the endophytic fungus Gliocladium roseum NG-12, the fermentation broth, and the sterile fermentation filtrate in antagonizing pathogens.

[0024] The preferred pathogens include Fusarium oxysporum Fusarium oxysporum Colletotrichum gloeosporioides Colletotrichum gloeosporioides , Colletotrichum oxysporum Colletotrichum acutatum At least one of .

[0025] The eighth aspect of the present invention provides the endophyte Gliocladium roseum NG-12, the fermentation liquid, and the fermentation filtrate in the preparation of the control of Fusarium oxysporum Fusarium oxysporum Colletotrichum gloeosporioides Colletotrichum gloeosporioides , or Colletotrichum oxysporum Colletotrichum acutatum Application in treating diseases caused by at least one pathogenic bacteria.

[0026] A ninth aspect of the present invention provides the use of at least one of the endophyte Gliocladium roseum NG-12, the fermentation broth, or the fermentation filtrate in controlling root-knot nematodes.

[0027] The tenth aspect of the present invention provides the use of the endophyte Gliocladium roseum NG-12, the fermentation liquid, and the fermentation filtrate in preventing and controlling pests and diseases caused by root-knot nematodes.

[0028] 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.

[0029] 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 good antagonistic effect on root-knot nematodes, and is a potential biological agent for preventing and controlling diseases and insect pests of red mushrooms. In addition, the pink Glioma of the invention also has good ability to produce iron carriers, potassium solution and nitrogen fixation, and produce protease and cellulase. The pink Glioma of the invention has broad development space and good development and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 These are the morphological characteristics of the endophyte Gliocladium roseum NG-12 screened in the preferred embodiment 1 of the present invention.

[0031] Figure 2 This is the phylogenetic tree of the endophyte Gliocladium roseum NG-12 in the preferred embodiment 2 of the present invention.

[0032] Figure 3 This is the result of the determination of iron carrier production by the endophyte Gliocladium roseum NG-12 in the preferred embodiment 3 of the present invention.

[0033] Figure 4 These are the results of potassium solubilization and nitrogen fixation determination of the endophytic fungus Gliocladium roseum NG-12 in the preferred embodiment 3 of the present invention.

[0034] Figure 5This is the enzyme production assay result of the endophyte Gliocladium roseum NG-12 in the preferred embodiment 3 of the present invention.

[0035] Figure 6 This is the antagonistic result of the endophyte Gliocladium roseum NG-12 in the preferred embodiment 4 of the present invention.

[0036] Figure 7 These are the microscopic examination results of the effect of the aseptic fermentation filtrate of the endophytic fungus Gliocladium roseum NG-12 in the preferred embodiment 5 of the present invention on controlling root-knot nematodes.

[0037] Figure 8 These are the statistical results of the effects of aseptic fermentation filtrates of different amounts of the endophytic fungus Gliocladium roseum NG-12 in preferred embodiment 5 of the present invention on controlling root-knot nematodes.

[0038] Fig. 9 This is the thermal stability result of the sterile fermentation filtrate of the endophytic fungus Gliocladium roseum NG-12 in the preferred embodiment 6 of the present invention. DETAILED DESCRIPTION

[0039] The present invention will be further described with reference to the accompanying drawings in conjunction with specific embodiments to better understand the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or in accordance with the product instructions are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0040] The bacterial culture medium used in the following examples is as follows: PDA solid culture medium (g / L): potato powder 10 g, glucose 20 g, agar 15 g.

[0041] PDA seed culture medium (g / L): potato powder 10 g, glucose 20 g.

[0042] Fermentation medium (g / L): 10 g potato extract powder, 30 g sucrose, 25 g yeast extract powder, and 2.5 g magnesium sulfate heptahydrate.

[0043] CAS detection 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.

[0044] 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.

[0045] Potassium feldspar powder treatment method: Add potassium feldspar powder to 10% HCl and soak for 8~10 hours (stir once every 1~2 hours), and soak overnight. After discarding the supernatant, rinse with deionized water, stir thoroughly, discard the supernatant after standing, and then add deionized water to rinse; repeat this several times until the pH value test does not change. Dry the treated potassium feldspar powder, grind it, pass it through a 100-mesh sieve, and put it into a bottle for later use.

[0046] Sodium carboxymethyl cellulose (CMC) medium (g / L): 5 g sodium carboxymethyl cellulose, 0.5 g (NH4)2SO4, 0.4 g Congo red, 0.1 g MgSO4·7H2O, 0.25 g K2HPO4, 16 g agar, dilute to 1000 mL.

[0047] Skim milk powder culture medium (g / L): 8g skim milk powder was dissolved in 300mL water and sterilized at 115℃ for 10min 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

[0048] Example 1 provides a method for obtaining the endophyte Gliocladium roseum NG-12.

[0049] 1. Sample source: In September 2023, the red mushroom fruiting bodies were provided by Sanming, Fujian.

[0050] 2. Isolation and screening of strains: Select healthy, undamaged caps of Russula, wash with water, clean the surface with 75% ethanol, soak in 5% NaClO solution for 5 min, rinse with sterile water 3 times, and dry with sterile filter paper. Use a sterile knife to cut the internal tissue of the cap of Russula, put it in a sterile mortar, add sterile water and grind it into a homogenate, and dilute it stepwise to get 10 -1 , 10 -2 , 10 -3 dilution. Pipette 100 μL of the original 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 and incubate for several days for observation and recording. 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 slope of a test tube and store it at 4 ℃. Figure 1 Shown are the morphological characteristics of the screened endophyte Gliocladium roseum NG-12. Example 2

[0051] Example 2 The endophyte Gliocladium roseum NG-12 obtained in Example 1 was sequenced for 18S rRNA gene, using the extracted genomic DNA as a template and the fungal 18S rDNA gene primers: The ITS1 sequence shown in SEQ ID NO: 2 is: 5'-TCCGTAGGTGAACCTGCGG-3'; The ITS4 sequence shown in SEQ ID NO: 3 is: 5′-TCCTCCGCTTATTGATATGC-3′; 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 Figure 2 As shown, it was identified as Gliocladium roseum ( Clonostachys rosea ).

[0052] Based on the above test results, combined with morphological, cultural and biological characteristics, the endophyte Glechoma rosea NG-12 was identified as a new species of Glechoma rosea and named: Glechoma rosea ( Clonostachys rosea )NG-12 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number: CGMCC No.41801. Example 3

[0053] Example 3 The biological characteristics of the endophyte Gliocladium roseum NG-12 were determined.

[0054] The plate method was used to determine the ability of the endophytic fungus Gliocladium roseum NG-12 to produce siderophore, potassium, nitrogen fixation, cellulase and protease.

[0055] (1) Determination of iron carrier production, nitrogen fixation and potassium dissolution: The endophytic fungus Glechoma rosea NG-12 was inoculated in a herringbone pattern on CAS test medium, Ashby nitrogen-free medium, and potassium-solubilizing medium by the plate method. The growth status of the strain and the presence of transparent circles were observed, and the diameter size was recorded. The ratio of the transparent circle diameter D (mm) on the CAS test medium to the colony diameter d (mm) was calculated, and the iron carrier production reached 3.10. Growing in Ashby nitrogen-free medium and potassium-solubilizing medium indicates that it has potassium-solubilizing and nitrogen-fixing functions.

[0056] (2) Enzyme production assay The endophytic fungus Glechoma rosea NG-12 was inoculated on sodium carboxymethyl cellulose (CMC) medium by the plate method. After culturing for 2 days, the colony diameter was measured and recorded, and the transparent circle was observed and the diameter was recorded. The endophytic fungus Glechoma rosea NG-12 was inoculated on skimmed milk powder medium in a square shape, and the transparent circle was observed and recorded. The ratio of the transparent circle diameter D (mm) to the colony diameter d (mm) was calculated, and the production of cellulase reached 2.56 and protease reached 2.67.

[0057] The results are as follows Figures 3-5 As shown: Figure 3 The results of the determination of siderophore production by the endophytic fungus Gliocladium roseum NG-12; Figure 4 The results of potassium solubilization and nitrogen fixation of endophytic fungus Gliocladium roseum NG-12 are shown in the figure. Figure 4 The left picture in the middle shows the results of potassium dissolution, and the right picture shows the results of nitrogen fixation; Figure 5 The results of enzyme production assay of endophytic fungus 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

[0058] Example 4 provides an antagonism assay for the endophyte Gliocladium roseum NG-12.

[0059] The isolated and purified endophyte Gliocladium roseum NG-12 was used as the antagonist, and Fusarium oxysporum ( Fusarium oxysporum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Colletotrichum oxysporum ( Colletotrichum acutatum ) was used as the indicator bacteria for pathogens, and the plate confrontation method was used to determine the antagonistic effect of the endophytic fungus Glechoma rosea NG-12 on pathogens. A 5 mm diameter puncher was used to punch holes in the bacterial cake, and the mycelium was placed in the center of the PDA culture dish. Pathogens were inoculated around the four sides. The bacterial cake containing only the tested pathogens was used as the control (CK). Each group was repeated 3 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%.

[0060] Corrected inhibition rate = (colony diameter of control group - colony diameter of experimental group) / (colony diameter of control group - 5 mm) × 100%. Example 5

[0061] Example 5 provides the effect of aseptic fermentation filtrate of strain NG-12 on controlling root-knot nematodes After strain NG-12 was cultured at 26°C and 140 rpm for 9 days, the fermentation liquid was centrifuged at 4°C and 10,000 r / min for 10 min, and the supernatant after centrifugation was filtered with a 0.22 μm syringe filter to obtain a sterile fermentation filtrate. 1 mL of the incubated second-instar larvae of root-knot nematodes (about 100) 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 3 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 The following are the results of microscopic examination of nematode mortality. Figure 7 The left picture in the middle 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 on the effects of 0.5mL, 1mL, 1.5mL and 2mL fermentation filtrate on controlling root-knot nematodes. Example 6

[0062] Example 6 provides the thermal stability of the sterile fermentation filtrate of the endophyte Gliocladium roseum NG-12.

[0063] 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 for use.

[0064] The effect of temperature on the antibacterial active substances of the endophytic fungus Gliocladium roseum NG-12 was detected. The sterile filtrate of the endophytic fungus Gliocladium roseum NG-12 was treated at 7 different temperatures: (a) treated in a water bath at 30℃ for 1h; (b) treated in a water bath at 40℃ for 1h; (c) treated in a water bath at 50℃ for 1h; (d) treated in a water bath at 60℃ for 1h; (e) treated in a water bath at 70℃ for 1h; (f) treated in a water bath at 80℃ for 1h; (g) treated in a water bath at 90℃ for 1h. After the treatment, an experiment on the prevention and control of 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.

[0065] The results are as follows Fig. 9As shown in the data, within the range of 30℃~70℃, the corrected mortality rate of root-knot nematodes was relatively stable and the effect of temperature was small. When the treatment temperature increased to above 70℃, the effect decreased significantly. After treatment at 80℃ for 1h, 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 1h was 41.83%.

[0066] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An endophyte Gliocladium roseum NG-12, characterized in that: The deposit number is CGMCC No.41801.

2. A fermentation broth, characterized in that: The endophyte Gliocladium roseum NG-12 described in 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: Take the fermentation seed liquid prepared in step 1 and inoculate it into the fermentation medium to obtain the fermentation liquid of the endophytic fungus Gliocladium roseum NG-12.

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

4. The fermentation broth according to claim 2, characterized in that 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 is prepared from the fermentation broth according to any one of claims 2 to 4. After the fermentation broth according to any one of claims 2 to 4 is centrifuged, the supernatant is filtered 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 iron carriers, potassium dissolving, and nitrogen fixing.

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 protease and / or cellulase preparations.

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. The use according to claim 8, characterized in that: The pathogens include at least one of Fusarium oxysporum, Colletotrichum gloeosporioides, and Colletotrichum oxysporum.

10. A preparation, characterized in that The method comprises at least one of the endophytic fungus 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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