Epichloë magellanica and its application in controlling Phytophthora infestans

Abstract

The present invention provides two strains of endophytic fungi Epichloe bromicola from Elymus tangutorum, with the preservation numbers being CGMCC No. 22448 and CGMCC No. 22449 respectively. The applicant for the first time used the plate mycelium inhibition method to test the inhibitory activities of crude extracts of different endophytic fungi from Elymus tangutorum against Phytophthora infestans. Through the activity inhibition experiment, the inhibition rates of the endophytic fungal strains 1 and 8 from Elymus tangutorum against Phytophthora infestans were strong; these two strains were preserved. In addition, the applicant tested the crude extracts of the endophytic fungal strains 1 and 8 from Elymus tangutorum for their control effects on potato late blight through in vitro leaf experiments. And the crude extracts of these two endophytic fungi may have low toxicity. Therefore, the endophytic fungal strains 1 and 8 from Elymus tangutorum can be used as promising plant-derived fungicides.

Description

Technical Field

[0001] The present invention relates to endophytic fungi of Elymus tangutorum and their application in controlling Phytophthora infestans of potatoes. Background Art

[0002] In 2018, the area of potatoes planted worldwide was nearly 17 million hectares, and the world's total output reached 368 million metric tons (data source: www.faostat.org). Potatoes are sustainable crops after wheat (734 million metric tons) and rice (782 million metric tons), and are the third most important crop for human consumption, facing the challenge of meeting the food needs of the growing world population by maintaining high yields. During the potato production process, various diseases such as early blight, brown spot, anthracnose, late blight, gray mold, bacterial wilt, viruses, and pests can cause a decline in its yield and quality, among which the potato late blight caused by Phytophthora infestans is the most serious. The occurrence of potato late blight can reduce potato yields by up to 30%. In the initial stage of Phytophthora infestans, fungicides, namely copper-based products, including pesticides such as zineb, mancozeb, dimethomorph, cyanuric acid, cyproconazole, and azoxystrobin, are used to control potato late blight. However, these synthetic fungicides are harmful to the environment and human health due to their mammalian toxicity and high residue levels.

[0003] In order to establish a more sustainable agricultural system, biopesticides have gradually become an ideal substitute for chemical fungicides for controlling plant diseases due to their strong biological activity, low residue toxicity to mammals, and substances that are easily biodegradable in the soil. Among them, secondary metabolites produced by plants and microorganisms have become an important source for the development of biopesticides. However, some plant resources are scarce and cannot be produced on a large scale. Therefore, there is an urgent need for new biopesticides in modern agriculture to control potato late blight in the potato production industry. In nature, in response to the invasion of foreign species, there are phenomena such as mutual promotion, mutual inhibition, and no mutual influence. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides two strains of endophytic fungi of Elymus tangutorum and their application in controlling Phytophthora infestans of potatoes. The preservation numbers of the two strains of endophytic fungi Epichloe bromicola (named Epichloe bromicola sqy-N-065 and Epichloe bromicola sqy-N-039 respectively) are: CGMCC No. 22448 and CGMCC No. 22449 respectively. The preservation unit is: China General Microbiological Culture Collection Center (CGMCC), and the preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0005] The present invention provides two strains of endophytic fungi Epichloe bromicola from Elymus tangutorum, and the preservation numbers are respectively: CGMCC No. 22448 and CGMCC No. 22449.

[0006] The present invention also provides a preparation method for the crude extract of the above-mentioned endophytic fungi of Elymus tangutorum. After fermenting and culturing the endophytic fungi Epichloe bromicola of Elymus tangutorum, the obtained fermentation broth is separated into a culture solution and mycelia. The culture solution and mycelia are respectively extracted with ethyl acetate and methanol at room temperature. The ethyl acetate part and methanol part are respectively evaporated to dryness on a rotary evaporator, and then combined to obtain the crude extract.

[0007] Preferably, the formula of the fermentation culture solution used in the fermentation culture is: per liter of fermentation culture solution contains: 100 g of sorbitol, 40 g of glucose, 3 g of yeast extract, 10 g of glutamic acid, 0.8 g of tryptophan, MgSO 4 ·7H 2 O 0.3 g, 1 g of potassium dihydrogen phosphate, and the rest is deionized water.

[0008] More preferably, the conditions for the fermentation culture are culturing at 140 - 150 rpm and 27 - 29 °C for 40 - 50 days.

[0009] The present invention also provides a crude extract of endophytic fungi of Elymus tangutorum, which is prepared by using the above-mentioned preparation method.

[0010] The present invention also provides the application of the above-mentioned endophytic fungi Epichloe bromicola of Elymus tangutorum, or the above-mentioned crude extract of endophytic fungi of Elymus tangutorum in controlling Phytophthora infestans.

[0011] Preferably, for controlling Phytophthora infestans, the crude extract of endophytic fungi of Elymus tangutorum is dissolved in dimethyl sulfoxide to obtain a medicament solution, and then the potato leaves are sprayed with it.

[0012] Preferably, the concentration of the medicament solution is 62.5 - 1000 ppm.

[0013] Preferably, the concentration of the medicament solution is 500 - 1000 ppm.

[0014] More preferably, the concentration of the medicament solution is 1000 ppm.

[0015] The present invention also provides a plant-derived insecticide, and its active ingredient includes the above-mentioned endophytic fungi Epichloe bromicola of Elymus tangutorum, or the above-mentioned crude extract of endophytic fungi of Elymus tangutorum.

[0016] Preferably, in the plant-derived insecticide, the concentration of the crude extract of endophytic fungi of Elymus tangutorum is 62.5 - 1000 ppm;

[0017] Preferably, the concentration of the liquid medicine is 500 - 1000 ppm;

[0018] More preferably, the concentration of the liquid medicine is 1000 ppm.

[0019] The applicant first used the flat hypha inhibition method to test the inhibitory activities of crude extracts of different endophytic fungi from Elymus tangutorum against Phytophthora infestans. Through the activity inhibition experiment, the inhibitory rates of endophytic fungal strains 1 and 8 from Elymus tangutorum against Phytophthora infestans were strong; in addition, the applicant tested the crude extracts of endophytic fungal strains 1 and 8 from Elymus tangutorum for their control effects on Phytophthora infestans of potato through in vitro leaf experiments. Through the toxicity test on zebrafish yolk sacs, the results showed that the crude extracts of strains 1 and 8 had no obvious toxicity to zebrafish, indicating that the crude extracts of these two endophytic fungi may have low toxicity. Therefore, the endophytic fungi and main chemical components of Elymus tangutorum can be used as very promising plant-derived fungicides. Description of the Drawings

[0020] The drawings are used to provide a further understanding of the present invention, and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention, and do not constitute a limitation to the present invention. In the drawings:

[0021] Figure 1 are the morphological characteristics of endophytic fungal strain 8 of Elymus tangutorum.

[0022] Figure 2 are the inhibitory effects of crude extracts of different endophytic fungi from Elymus tangutorum on Phytophthora infestans (1000 ppm). In the figure, 1 represents strain 1, 8 represents strain 8, and so on.

[0023] Figure 3 are the effects of endophytic fungal strains 1 and 8 of Elymus tangutorum on the mycelia of Phytophthora infestans at 125 - 500 ppm.

[0024] Figure 4 are the control effects of endophytic fungal strains 1 and 8 of Elymus tangutorum on Phytophthora infestans of potato (A is the protective effect; B is the therapeutic effect, the disease condition of the leaves after 48 hours).

[0025] Figure 5 are the toxicological activities of the crude extracts of endophytic fungi from Elymus tangutorum on zebrafish. Detailed Embodiments

[0026] The following embodiments facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all commercially available unless otherwise specified.

[0027] Example 1

[0028] Tested endophytic fungi: All were from the State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University. The endophytic fungi were all isolated from Elymus tangutorum seeds collected from Gansu and Qinghai, China in September 2017.

[0029] The process of isolating endophytic fungi from Elymus tangutorum seeds was as follows: Remove the lemma of the seeds, disinfect the seeds in 75% ethanol and 0.1% sodium hypochlorite solution for 5 min and 10 min respectively, rinse with sterile water after each disinfection, dry the water with sterile filter paper, and place the seeds on PDA medium. The medium with the placed seeds was placed in an incubator at a temperature of (25±1) °C for cultivation, and observed every two days. After about 7 days, Epichloe endophytic fungal hyphae were observed growing at the edge of the seeds, and the endophytic fungi were purified to fresh PDA medium for continued growth.

[0030] According to morphological and molecular phylogenetic data, these fungi were named Epichloë bromicola of Elymus tangutorum.

[0031] Among them, the morphological characteristics of the endophytic fungal strain 8 of Elymus tangutorum are shown in Figure 1 .

[0032] Figure 1 are the morphological characteristics of the endophytic fungal strain 8 of Elymus tangutorum. Among them, Figure A is the front morphology of strain 8 on the medium, and Figure B is the back morphology of strain 8 on the medium.

[0033] Table 1 The tef and tub sequence accession numbers of the endophytic fungal strain 8 isolated from Elymus tangutorum and the blast comparison results

[0034]

[0035] Tested pathogenic fungi: Phytophthora infestans, the pathogen of potato late blight, was provided by the Gansu Academy of Agricultural Sciences.

[0036] Table 2 The geographical coordinates, altitude and location of the host plants of endophytic fungi

[0037]

[0038]

[0039] Preparation method of crude extract of endophytic fungi of Elymus tangutorum: 20 strains of endophytic fungi Epichloe bromicola of Elymus tangutorum at different altitude gradients were cultured on potato dextrose agar (PDA) medium at 28 °C for 15 days. Then the thalli were inoculated into 1000 mL shake flasks, each flask containing 400 mL of M104T (per liter of M104T contains: 100 g of sorbitol, 40 g of glucose, 3 g of yeast extract, 10 g of glutamic acid, 0.8 g of tryptophan, MgSO 4 ·7H 2 O 0.3 g, 1 g of potassium dihydrogen phosphate, and made up to volume with deionized water), and then cultured with shaking at 145 rpm and 28 °C for 45 days. The fermentation broth of Epichloe bromicola was placed in a gauze folded 8 layers. After the gauze was rolled up, it was tightened in the reverse direction to separate the culture solution and the mycelium. High-speed centrifugation or vacuum filtration can also be used for separation. The culture solution was extracted 3 times with 1.2 L of ethyl acetate at room temperature, and the mycelium was extracted 3 times with 300 mL of methanol at room temperature. The culture solution was weakly acidic, and ethyl acetate had a good extraction effect; methanol could dissolve the lipids contained in the mycelium and improve the purity of the crude extract. After extraction, the 3 ethyl acetate extracts were combined, and the 3 methanol extracts were combined. Then the ethyl acetate extract and the methanol extract were evaporated to dryness on a rotary evaporator respectively, and the crude extract was obtained by combining them.

[0040] Example 2 Inhibitory effect of crude extract of endophytic fungi of Elymus tangutorum on Phytophthora infestans

[0041] Experimental method:

[0042] The growth rate method with toxic medium was used to determine the inhibitory effect of crude extracts of endophytic fungi of Elymus tangutorum at different altitudes on the mycelial growth of Phytophthora infestans.

[0043] Dissolve 20 mg of the crude extract prepared in Example 1 in 100 μL of dimethyl sulfoxide (DMSO) to obtain a test concentration of the medicinal solution. This medicinal solution (0.1 mL) is thoroughly mixed with 20 mL of sterilized and warm-to-the-touch rye medium (55 g / L rye, 20 g / L sucrose, 18 g / L agar), and then the mixture is evenly poured into three petri dishes with a diameter of 6 cm to prepare a medium with the medicinal solution. Add dimethyl sulfoxide (DMSO) to the rye medium as a blank control. Use a 0.25 cm puncher to evenly punch out fungal disks from the edge of the Phytophthora infestans colony. The fungal disks with the mycelium facing down are placed on the central surface of the prepared medium. After culturing for 7 days under dark conditions, calculate the inhibition rate according to the following formula: Inhibition rate (%) = (C - T) / (C - 0.25 cm). T is the average diameter on the rye medium treated with the medicinal solution, and C is the average diameter on the rye medium treated with DMSO. All inoculations are carried out by the same person, and the inoculated medium is placed in an incubator at a constant temperature of 18°C for culturing. Measure the colony diameter using the cross method and correct it to calculate the percentage of antibacterial activity. Conduct parallel experiments 3 times and take the average of the results.

[0044] Experimental results:

[0045] Through the activity inhibition experiment, the antibacterial activities of the crude extracts of 20 endophytic fungal strains against Phytophthora infestans were tested. Compared with the blank control, the inhibitory effects of these endophytic fungal crude extracts (1000 ppm, the calculation process is 20 mg / 20 ml = 1 mg / ml = 1000 μg / ml = 1000 ppm) on Phytophthora infestans were observed on the 7th day (Table 3). The crude extract of strain 1 had the strongest antibacterial activity against Phytophthora infestans, with an inhibition rate as high as 88%. The crude extracts of strains 8, 10, and 20 had obvious inhibitory activities against Phytophthora infestans, and their inhibition rates were 81%, 72%, and 76% respectively (Table 3). The crude extracts of strains 11 and 19 had moderate inhibitory activities against Phytophthora infestans, with inhibition rates of 50% and 53% respectively. The remaining crude extracts (strains 2 - 7, 9, 12 - 18) had weak antibacterial activities, with inhibition rates ranging from 9% to 40%. The results of this experiment indicate that strains 1 and 8 can be used as candidate strains for the biological control of Phytophthora infestans and have the potential for application and promotion in the pesticide industry.

[0046] Table 3 Inhibition rates (%) of crude extracts of Elymus tangutorum endophytic fungi at different altitudes against Phytophthora infestans

[0047]

[0048]

[0049] To further confirm the sensitivity of the crude extracts of two strains of endophytic fungi, strain 1 and strain 8 from Elymus tangutorum, to Phytophthora infestans, and to confirm whether the crude extracts of endophytic fungi strain 1 and strain 8 from Elymus tangutorum have an inhibitory effect on Phytophthora infestans at low concentrations, the applicant further tested their antibacterial activities at 500 ppm, 200 ppm, and 125 ppm.

[0050] It was found that when the concentrations of the crude extracts of strain 1 and strain 8 were 500 ppm, 250 ppm, and 125 ppm, the inhibition rates against Phytophthora infestans gradually decreased. The specific results are as Figure 2 shown.

[0051] Figure 2 Inhibitory effects of crude extracts of different endophytic fungi from Elymus tangutorum on Phytophthora infestans (1000 ppm). In the figure, 1 represents strain 1, 8 represents strain 8, and so on.

[0052] Figure 3 Effects of endophytic fungi strain 1 and strain 8 from Elymus tangutorum on the mycelia of Phytophthora infestans at 125 - 500 ppm.

[0053] On June 8, 2021, the applicant sent two strains of endophytic fungi Epichloe bromicola, strain 1 and strain 8 from Elymus tangutorum, to the China General Microbiological Culture Collection Center (CGMCC) for preservation. The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation number of endophytic fungi strain 1 from Elymus tangutorum (named Epichloe bromicola sqy - N - 065) is CGMCC No. 22448, and the preservation number of endophytic fungi strain 8 from Elymus tangutorum (named Epichloe bromicola sqy - N - 039) is: CGMCC No. 22449.

[0054] Example 3 Prevention and treatment effects of endophytic fungi from Elymus tangutorum on Phytophthora infestans

[0055] Experimental method:

[0056] Preparation of Phytophthora infestans spore suspension: Inoculate Phytophthora infestans on rye medium and culture it in the dark at 18 °C for 11 days, and then prepare a spore suspension (Yao Yanpo, 2015; Zeng Yan, 2010). Wash the mycelia of Phytophthora infestans with 10 mL of sterile deionized water, place it in a refrigerator at 4 °C in the dark and let it stand for 4 h to release zoospores, and then filter it through a 100 μm nylon mesh to remove the mycelia. Adjust the spore suspension solution to 1×10 5spores / mL. Potatoes (Longshu No. 6) were cultivated in the greenhouse of Lanzhou University in Yuzhong. Fresh potato leaves of uniform size were collected, washed with distilled water, wiped with 75% ethanol, and then sterile wet cotton was wrapped around the petioles.

[0057] This part involves two experiments: one is to test the preventive effect of the crude extract on Phytophthora infestans: 2 hours before inoculating the spore suspension of Phytophthora infestans (100 ppm / leaf, 1×10 5 spores / mL), the crude extracts of Epichloë gansuensis strains 1 and 8 at different concentrations (0, 62.5, 125, 250, 500, and 1000 ppm) were sprayed on the leaves; the other is to test the therapeutic effect of the crude extract on Phytophthora infestans: the spore suspension was sprayed on the potato leaves, and 2 hours later, the crude extracts of Epichloë gansuensis strains 1 and 8 at different concentrations (0, 62.5, 125, 250, 500, and 1000 ppm) were sprayed again.

[0058] Experimental results:

[0059] Regardless of the therapeutic or protective effect, compared with the crude extract of Epichloë gansuensis strain 1, the latter showed obvious effects. For the crude extract of strain 1, the protective effect was stronger compared with the therapeutic effect; for the crude extract of strain 8, the therapeutic effect was significantly better than the protective effect. Therefore, the crude extracts of strains 1 and 8 can be used to control potato late blight to reduce economic losses in agricultural production.

[0060] Figure 4 Control effects of Epichloë gansuensis strains 1 and 8 on potato late blight (A is the protective effect; B is the therapeutic effect, the disease condition of the leaves after 48 hours).

[0061] Example 4 Toxicity test of Epichloë gansuensis

[0062] In order to further confirm the toxicity of the crude extracts of Epichloë gansuensis strains 1 and 8, the applicant further conducted a toxicological activity experiment.

[0063] Using zebrafish yolk sacs as the measurement object, when the concentration of the crude extracts of endophytic fungi strains 1 and 8 was 10 ppm, their effects on zebrafish yolk sac cells were measured. Compared with the blank control, the zebrafish vesicles showed slightly different morphological characteristics under the treatment of the crude extracts of strains 1 and 8. The crude extract of strain 1 showed slight edema on the zebrafish yolk sac; while at the same concentration, the crude extract of strain 8 showed no obvious phenomenon on the zebrafish yolk sac. The above research results indicate that the crude extracts of strains 1 and 8 have no obvious toxicity to zebrafish, suggesting that the crude extracts of these two endophytic fungi may have low toxicity.

[0064] Figure 5It is the toxicological activity of the crude extract of endophytic fungi in Elymus tangutorum on zebrafish.

[0065] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments or perform equivalent replacements on some of the technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Two strains of endophytic fungi Epichloe bromicola from Elymus tangutorum, with the preservation numbers being CGMCC No. 22448 and CGMCC No. 22449 respectively.

2. A method for preparing a crude extract of the endophytic fungi of Elymus tangutorum described in claim 1, which is characterized in that: After fermenting and culturing the endophytic fungi Epichloe bromicola of Elymus tangutorum, the obtained fermentation broth is separated into a culture solution and mycelium. The culture solution and mycelium are respectively extracted with ethyl acetate and methanol at room temperature. The ethyl acetate part and the methanol part are respectively evaporated to dryness on a rotary evaporator, and then combined to obtain a crude extract.

3. According to the preparation method described in claim 2, which is characterized in that: The formula of the fermentation culture medium used in the fermentation culture is as follows: per liter of the fermentation culture medium, it contains: 100 g of sorbitol, 40 g of glucose, 3 g of yeast extract, 10 g of glutamic acid, 0.8 g of tryptophan, 0.3 g of MgSO 4 ·7H 2 O, 1 g of potassium dihydrogen phosphate, and the rest is deionized water.

4. According to the preparation method described in claim 2 or 3, which is characterized in that: The conditions for the fermentation culture are culturing at 140 - 150 rpm and 27 - 29 °C for 40 - 50 days.

5. A crude extract of endophytic fungi of Elymus tangutorum, which is prepared by using the preparation method described in any one of claims 2 - 4.

6. The application of the endophytic fungi Epichloe bromicola of Elymus tangutorum described in claim 1, or the crude extract of endophytic fungi of Elymus tangutorum described in claim 5 in controlling Phytophthora infestans.

7. According to the application described in claim 6, which is characterized in that: For controlling Phytophthora infestans, the crude extract of endophytic fungi of Elymus tangutorum is dissolved in dimethyl sulfoxide to obtain a medicament solution, and then the potato leaves are sprayed with it.

8. According to the application described in claim 7, which is characterized in that: The concentration of the medicament solution is 62.5 - 1000 ppm.

9. According to the application described in claim 8, which is characterized in that: The concentration of the medicament solution is 500 - 1000 ppm.

10. According to the application described in claim 9, which is characterized in that: The concentration of the medicament solution is 1000 ppm.

11. A plant-derived insecticide, which is characterized in that: Its active ingredient includes the endophytic fungi Epichloe bromicola of Elymus tangutorum described in claim 1, or the crude extract of endophytic fungi of Elymus tangutorum described in claim 5.

12. According to the plant-derived insecticide described in claim 11, which is characterized in that: In the plant-derived insecticide, the concentration of the crude extract of endophytic fungi of Elymus tangutorum is 62.5 - 1000 ppm.

13. According to the plant-derived insecticide described in claim 12, which is characterized in that: The concentration of the crude extract of endophytic fungi of Elymus tangutorum is 500 - 1000 ppm.

14. According to the plant-derived insecticide described in claim 13, which is characterized in that: The concentration of the crude extract of endophytic fungi of Elymus tangutorum is 1000 ppm.

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