Paris polyphylla var chinensis endophytic fungus strain and application thereof

Abstract

The invention discloses a paris polyphylla var chinensis endophytic fungus strain and application thereof. The strain number of the paris polyphylla var chinensis endophytic fungi is CL-26, and the paris polyphylla var chinensis endophytic fungi are classified and named as Clonostachys sp. And are preserved in Guangdong Province Microbial Culture Collection Center, the address of the preservation unit is the 5th floor of the Experimental Building, No.100 Court, Middle Road, Xianlie District, Guangzhou City, Guangdong Province, and the preservation date is March 31, 2025; the preservation number of the strain is GDMCC 66079. The paris polyphylla var chinensis endophytic fungi come from healthy rhizomes of paris polyphylla var chinensis, have the functions of dissolving inorganic phosphorus and producing IAA, and can promote paris polyphylla var chinensis rhizome buds to break dormancy, shorten the germination time and improve the emergence rate.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, in particular to an endophytic fungus strain of Paris polyphylla and an application thereof. Background Art

[0002] Paris polyphylla Smith var. chinensis, a plant of the genus Paris in the Liliaceae family, also known as Seven-Leafed Flower, is a precious traditional medicinal plant in my country. Paris polyphylla Smith has the functions of clearing heat and detoxifying, reducing swelling and relieving pain, cooling the liver and calming convulsions. It is mainly used to treat boils, carbuncles, sore throats, snake and insect bites, injuries from falls, and convulsions. Modern medical pharmacological research has shown that Paris polyphylla Smith also has anti-tumor, antibacterial, hemostatic, immune-regulating, and antioxidant effects. Paris polyphylla is the raw material for over 80 Chinese patent medicines, including Yunnan Baiyao, Ji Desheng Snake Medicine Tablets, Re Du Qing, and Gong Xuening, and has significant clinical efficacy and economic value. However, due to natural reproductive problems and overharvesting, the wild Paris polyphylla is on the verge of extinction and has been listed as a Class II endangered and rare protected plant in China. Artificial cultivation also faces problems such as low seedling propagation efficiency and a long growth cycle (5-10 years).

[0003] At present, the artificial cultivation of Paris polyphylla mainly relies on rhizome cuttings for propagation, and the dormancy of rhizome buds is the core bottleneck problem faced by its large-scale production. The rhizome buds of Paris polyphylla have a long dormancy period under natural conditions. The rhizomes that have not been released from dormancy after cutting and planting are prone to rot or rigidification, resulting in a low field emergence rate, low planting efficiency, extended growth cycle and high costs. Relieving the dormancy of rhizome buds is the key to shortening the breeding cycle of Paris polyphylla and achieving large-scale planting, but the current production mainly relies on soaking in exogenous cytokinins (6-BA), gibberellins (GA3) and the like to release dormancy, and chemical hormones can easily cause seedling deformities and pose an environmental pollution risk. Therefore, the development of green and efficient dormancy-breaking technology is an urgent need to solve the Paris polyphylla resource crisis and industrial development.

[0004] Endophytic fungi promote the growth of host plants by synthesizing substances required by the host or secreting certain inducing factors, and thus have great potential in the cultivation of medicinal plants. Previous studies have shown that symbiotic endophytes with plants can significantly affect the physiological metabolism of the host, thereby breaking dormancy and promoting germination. However, there are currently no reports on the use of endophytic fungi to promote the breaking of bud dormancy in the rhizomes of Paris polyphylla, shortening germination time, and increasing seedling emergence rate. The development of new, green, efficient, and targeted germination promotion technologies based on endophytes is of great significance to the conservation of Paris polyphylla resources and the development of its industry.

[0005] In view of this, the present invention provides an endophytic fungus strain of Paris polyphylla, which is used to promote the germination of Paris polyphylla rhizomes, break the dormancy of Paris polyphylla rhizomes in advance, shorten the germination time, and improve the emergence rate, so as to solve the drawbacks of the existing technology. Summary of the Invention

[0006] To address the issues of prolonged dormancy and uneven germination of rhizomes of the plant, the present invention provides an endophytic fungus strain for promoting rhizome germination. This strain can help rhizome buds of the plant break dormancy, shorten germination time, and increase germination rate, making it suitable for factory-scale seedling cultivation and field production applications.

[0007] The present invention discloses an endophytic fungus strain of Paris polyphylla, with the strain number CL-26, classified as Clonostachys sp., and deposited in Guangdong Provincial Microbial Culture Collection Center. The depository address is 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province. The deposit date is March 31, 2025; the deposit number is GDMCC 66079.

[0008] Furthermore, the ITS gene sequence of the endophytic fungus strain of Paris polyphylla is the nucleotide sequence shown in SEQ ID No.1.

[0009] Furthermore, the colony of the endophytic fungus strain CL-26 of the Paris polyphylla on the PDA medium is cotton-like, with dense hyphae, disc-shaped on the front, white, slightly yellowish in the middle, and white and khaki on the back;

[0010] The hyphae are septate, with a diameter of 2.9 to 4.8 μm;

[0011] Conidiophores are erect, 76-203 μm long, 2.5-5.0 μm in diameter at the base, with broom-like branches at the top, 11.5-43.8 μm long, 1.5-3.0 μm in diameter at the base;

[0012] Spores are produced at the top of the phialide, are oval or kidney-shaped, have a smooth surface, and are (3.8-7.1) × (2.0-3.5) μm in diameter.

[0013] Furthermore, the endophytic fungus strain CL-26 of the Paris polyphylla is derived from the healthy rhizomes of the Paris polyphylla, has the functions of dissolving inorganic phosphorus and producing IAA, and can promote the rhizome buds of the Paris polyphylla to break dormancy.

[0014] The present invention also uses the Paris polyphylla endophytic fungus strain to prepare a Paris polyphylla endophytic fungus agent.

[0015] Furthermore, the active ingredient of the endophytic fungus agent of Paris polyphylla is the fermentation culture of the endophytic fungus strain of Paris polyphylla.

[0016] The method for preparing the fermentation broth of the endophytic fungus strain of Paris polyphylla of the present invention comprises the following steps:

[0017] The endophytic fungus strain CL-26 of the Paris polyphylla was inoculated into a PDA liquid culture medium, cultured at 28° C. and a rotation speed of 200 r / min for 3 to 5 days, centrifuged at a rotation speed of 10,000 r / min for 10 minutes, and the supernatant was collected to obtain the Paris polyphylla endophytic fungus fermentation liquid.

[0018] The PDA liquid culture medium uses an existing culture medium.

[0019] The invention discloses an application of the endophytic fungus strain and the microbial agent of the Paris polyphylla to promoting rhizome germination and improving the emergence rate of the Paris polyphylla.

[0020] Furthermore, the object of the study on the germination effect is the rhizome of Paris polyphylla with dormant terminal buds. Furthermore, the object of the study on the emergence effect is the rhizome of Paris polyphylla with terminal buds that have broken dormancy.

[0021] Furthermore, the application method is to soak the rhizomes of the Chinese Paris polyphylla with dormant terminal buds in the fermentation liquid of the endophytic fungus CL-26 for 0.5-2 hours, layer them with sterilized river sand at low temperature (10±2°C), and water them with the fermentation product again after 15 days, which can promote the dormancy of the rhizomes and buds of the Chinese Paris polyphylla and shorten the germination time.

[0022] Furthermore, the application method is to soak the rhizomes of Paris polyphylla with the terminal buds out of dormancy in the fermentation liquid of endophytic fungus CL-26 for 0.5-2 hours, and then plant them in a mixture of sterilized river sand and vermiculite at room temperature.

[0023] (1:1), watering the fermentation product again after 15 days can increase the seedling emergence rate of Paris polyphylla.

[0024] Beneficial effects of the present invention:

[0025] The endophytic fungus Clonostachys sp. CL-26 isolated from the healthy rhizomes of Paris polyphylla has the functions of dissolving inorganic phosphorus and producing IAA, can promote the rhizome buds of Paris polyphylla to break dormancy, shorten the germination time, and improve the emergence rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a colony morphology diagram of the endophytic fungus CL-26 grown on PDA medium for 7 days in Example 1;

[0027] Figure 1 A is the positive form, Figure 1 B is the reverse form;

[0028] Figure 2 This is a microscopic image of the hyphae of the endophytic fungus CL-26 grown on PDA medium in Example 1;

[0029] Figure 2 A is mycelium, Figure 2 B is conidiophore, Figure 2 C is spore;

[0030] Figure 3 This is a graph showing the in-dish test results of the inorganic phosphorus solubilization and IAA production activities of the endophytic fungus CL-26 in Example 2;

[0031] Figure 3 A is dissolved inorganic phosphorus, Figure 3 B is IAA production;

[0032] Figure 4 This is a graph showing the germination of Paris polyphylla rhizomes treated with the fermentation liquid of endophytic fungus CL-26 in Example 3 after being stored in sand at low temperature for 60 days;

[0033] Figure 4 A is the treatment with endophytic fungus CL-26, Figure 4 B is the control group;

[0034] Figure 5 This is a graph showing the germination of Paris polyphylla rhizomes treated with the fermentation broth of endophytic fungus CL-26 in Example 4 and planted in a mixture of river sand and vermiculite (1:1) for 30 days;

[0035] Figure 5 A is the treatment with endophytic fungus CL-26, Figure 5 B is the control group. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments, but the present invention is not limited thereto.

[0037] Example 1: Isolation and identification of endophytic fungus strain CL-26 from Paris polyphylla

[0038] 1. Isolation and purification of endophytic fungus CL-26:

[0039] Collect healthy rhizomes of Paris polyphylla, clean the surface dirt with tap water, then clean the surface with detergent, and then rinse with sterile water;

[0040] In a clean bench, soak the rhizomes in 75% alcohol for 30–45 seconds, rinse once with sterile water, soak in 0.1% mercuric chloride for 5 minutes, rinse five times with sterile water, dry on sterile filter paper, and trim the edges. Use a scalpel to cut the rhizomes into 0.5 × 0.5 × 0.5 cm pieces, then inoculate on PDA culture medium, placing three pieces per medium.

[0041] Sterile water after the last rinse was spread on PDA culture medium as a control;

[0042] The culture dish was placed in an incubator at 28°C for 5-7 days. The mycelium of the experimental group with no colonies in the control group was selected and inoculated into PDA culture medium for purification. The culture was inoculated 2-3 times until a single colony was formed.

[0043] PDA culture medium and preparation method: Peel fresh potatoes, weigh 200 g, cut into small pieces, add distilled water and boil for 30 minutes, filter through 8 layers of gauze, add 20 g glucose and 15 g agar to the filtrate, add distilled water to 1000 mL, and sterilize at 121°C for 20 minutes.

[0044] 2. Morphological identification of endophytic fungus CL-26

[0045] The purified strain was inoculated onto PDA culture medium using the spot planting method and cultured in an inverted manner at a constant temperature of 28°C for 3 to 7 days. The color, texture, and edge of the colony were observed and recorded accordingly. The insert culture method was used to observe the presence of septa, size and shape of the hyphae, spore production, and spore shape of the hyphae under a Leika upright microscope, and photographed and recorded.

[0046] The results are as follows Figure 1 、 Figure 2 As shown: After culturing endophytic fungus CL-26 on PDA medium at 28℃ for 7 days, the colony diameter is 43-47 mm, cotton-like, and the hyphae are dense; the front of the colony is disc-shaped, white, with a slightly yellowish center, the center is higher than the edge, and the hyphae density is greater than the edge, as shown in the figure. Figure 1 As shown in A;

[0047] The reverse side is white or khaki. Figure 1 As shown in B;

[0048] The hyphae have septa, with a diameter of 2.9 to 4.8 μm, e.g. Figure 2 As shown in A;

[0049] Conidiophores are erect, 76 to 203 μm long, 2.5 to 5.0 μm in diameter at the base, with broom-like branches at the top, 11.5 to 43.8 μm long, 1.5 to 3.0 μm in diameter at the base, such as Figure 2 As shown in B;

[0050] Spores are produced at the top of the phialide, are oval or kidney-shaped, have a smooth surface, and are (3.8-7.1)×(2.0-3.5)μm in diameter. Figure 2 As shown in C.

[0051] 3. Molecular biological identification of endophytic fungus CL-26

[0052] The hyphae of endophytic fungi were selected and inoculated into PDA medium, cultured at 28℃ for 5-7 days, and the hyphae were scraped and the total DNA of the strain was extracted by CTAB method.

[0053] The total DNA of the strain was amplified by PCR using rDNA-ITS primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) / ITS4 (5′-TCCTCCGCTTATTGATATGC-3′).

[0054] The PCR reaction system was as follows: template DNA 1 μL, primer ITS1 (10 μM) 1 μL, primer ITS4 (10 μM) 1 μL, PCR Master Mix 12.5 μL, and ddH2O to a volume of 25 μL.

[0055] The PCR reaction conditions were as follows: 94°C for 3 min; 94°C for 30 s, 55°C for 30 s, 72°C for 45 min, 30 cycles; 72°C for 5 min.

[0056] The PCR products were detected by 1.0% agarose gel electrophoresis and then sent to Wuhan Qingke Biotechnology Co., Ltd. for sequencing. The rDNA-ITS sequences of the strains obtained by sequencing were compared with the GenBank database by BLAST homology analysis.

[0057] The results showed that the endophytic fungus CL-26 had the highest similarity with Clonostachys rosea (MG345099.1), which was 99.65%. The specific nucleotide sequence of the ITS sequence (5'-3') of the endophytic fungus CL-26 is shown in SEQ ID No.1.

[0058] SEQ ID No. 1:

[0059] .

[0060] After identification, the present invention isolated a strain of the genus Clonosporum from the healthy rhizome of Paris polyphylla and named it CL-26. The identified endophytic fungus of Paris polyphylla was sent to a preservation institution for preservation and classified as Clonostachys sp., with the strain number CL-26. It was deposited in the Guangdong Provincial Microbiological Culture Collection on March 31, 2025, with the deposit number GDMCC 66079. The deposit address is 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0061] Example 2: Identification of the Growth-Promoting Function of Endophytic Fungus CL-26

[0062] 1. Detection of potassium-dissolving ability

[0063] Inoculate the mycelium of endophytic fungus CL-26 into potassium-dissolving medium and culture at 28℃ for 3-5 days. Observe whether transparent circles appear around the fungal colonies. If transparent circles can be observed, it indicates that the fungus has potassium-dissolving ability. +Potassium-solubilizing ability test medium and preparation method: glucose 10.0g / L, Na2HPO4 0.2g / L, MgSO4 0.2g / L, NaCl 0.2g / L, CaCO3 5.0g / L, CaSO4 0.1g / L, potassium feldspar 2.5g / L, agar 15g / L, sterilized at 121℃ for 20min.

[0064] The results showed that no obvious transparent zone was observed in the potassium-dissolving medium for endophytic fungus CL-26, indicating that it did not have the ability to dissociate K. + ability.

[0065] 2. Detection of nitrogen-fixing ability of endophytic fungi

[0066] The mycelium of the endophytic fungus CL-26 was inoculated into nitrogen-fixing medium, cultured at 28°C for 3 to 5 days, and transferred three times continuously. If the strain can continue to grow normally, it indicates that it has the ability to fix nitrogen.

[0067] Nitrogen fixation ability test culture medium and preparation method:

[0068] KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaCO3 5.0 g / L, mannitol 10.0 g / L, CaSO4·2H2O 0.1 g / L, agar 15.0 g / L, sterilize at 121℃ for 20 min.

[0069] The results showed that the endophytic fungus CL-26 could not grow normally in nitrogen-fixing medium, indicating that it did not have the ability to fix nitrogen.

[0070] 3. Detection of phosphate solubilizing ability of endophytic fungi, such as Figure 3 As shown:

[0071] The hyphae of endophytic fungus CL-26 were inoculated into organic and inorganic phosphorus culture medium and cultured at 28℃ for 3-5 days. If a transparent circle was formed around the colony, it indicated that it had the ability to solubilize phosphorus.

[0072] Organophosphorus culture medium and preparation method:

[0073] Glucose 10.0 g / L, (NH4)2SO4 0.5 g / L, NaCl 0.3 g / L, KCl 0.3 g / L, FeSO4·7H2O 0.03 g / L, MnSO4·4H2O 0.03 g / L, CaCO3 5.0 g / L, yeast extract powder 0.4 g / L, agar 15 g / L, pH 7.0-7.5, sterilize at 121°C for 20 min, wait until the temperature drops to 50-60°C, and add 0.2 g / L sterile lecithin.

[0074] Inorganic phosphorus culture medium and preparation method:

[0075] Glucose 10.0 g / L, (NH4)2SO4 0.5 g / L, NaCl 0.3 g / L, KCl 0.3 g / L, MgSO4·7H2O0.3 g / L, FeSO4·7H2O 0.03 g / L, MnSO4·4H2O 0.03 g / L, Ca3(PO4)2 5.0 g / L, agar 15 g / L, pH 7.0-7.5, sterilization at 121℃ for 20 min.

[0076] The results are as follows Figure 3 As shown in A: The endophytic fungus CL-26 only has a transparent zone in the inorganic phosphorus culture medium, and the diameter of the transparent zone is 0.5 cm, indicating that the endophytic fungus CL-26 has the ability to dissolve inorganic phosphorus but not organic phosphorus.

[0077] 4. Detection of IAA production capacity of endophytic fungi, such as Figure 3 As shown:

[0078] The mycelium of the endophytic fungus CL-26 was inoculated into the IAA production detection medium and cultured at 28°C and 200 rpm for 3-5 days. The mycelium was removed after filtration, and the filtrate was centrifuged in a sterile centrifuge tube at 4°C and 10,000 rpm for 15 minutes to collect the fermentation liquid. The fermentation liquid was mixed with Salkowski colorimetric solution in a ratio of 1:2 and allowed to stand in the dark for 30 minutes. If the mixed liquid is pink or purple-red, it indicates that IAA is produced.

[0079] IAA production capacity detection culture medium and preparation method:

[0080] 0.5 g / L KH2PO4, 1 g / L yeast extract, 10 g / L mannitol, 0.2 g / L MgSO4, 0.1 g / L NaCl, pH 6.8-7.2, sterilize at 121°C for 20 min. Once the temperature drops to 50-60°C, add 0.1 g / L sterile tryptophan. Salkowski colorimetric solution: 250 mL distilled water, 150 mL concentrated sulfuric acid, 7.5 mL 0.5 mol / L FeCl3.

[0081] The results are as follows Figure 3 As shown in B: The mixture of the fermentation broth of endophytic fungus CL-26 and Salkowski colorimetric solution (1:2) is pink, indicating that the endophytic fungus CL-26 has the ability to produce IAA.

[0082] Example 3: Effect of endophytic fungus CL-26 on the germination of Paris polyphylla

[0083] 1. Preparation of endophytic fungus CL-26 fermentation broth

[0084] The hyphae of the endophytic fungus CL-26 were selected and inoculated into PDA liquid culture medium, cultured at 28°C and 200 r / min for 3-5 days, and centrifuged at 10000 r / min for 5 minutes to collect the supernatant, which was the fermentation broth.

[0085] 2. Test to promote root and stem bud germination, such as Figure 4 As shown:

[0086] Three- to four-year-old rhizomes of Paris polyphylla with dormant terminal buds were immersed in a fermentation broth of the endophytic fungus CL-26 for 1 hour. Uninoculated PDA liquid medium served as a control. Sterilized river sand was then placed at low temperature (10 ± 2°C). Fifteen rhizomes were placed in each treatment, with three replicates. After 15 days, the fermentation product was watered. The effects of the fermentation broth of the endophytic fungus CL-26 on the dormancy release of rhizomes and buds of Paris polyphylla were regularly observed.

[0087] The results are as follows Figure 4 As shown in the results, only the morphological changes of rhizome buds of Paris polyphylla treated with the fermentation liquid of endophytic fungus CL-26 were obvious. The proportion of rhizome buds breaking through the leaf sheath was 42.22% at 60 days, indicating that the fermentation liquid of endophytic fungus CL-26 can promote the dormancy of rhizome buds of Paris polyphylla and shorten the germination time.

[0088] Example 4: Effect of endophytic fungus CL-26 on the emergence of Paris polyphylla

[0089] 1. Preparation of endophytic fungus CL-26 fermentation broth was the same as step 1 in Example 3.

[0090] 2. Test to promote root and stem bud germination, such as Figure 5 As shown:

[0091] Two- to three-year-old rhizomes of Paris polyphylla, whose terminal buds had emerged from dormancy, were immersed in a fermentation broth of the endophytic fungus CL-26 for one hour. Uninoculated PDA liquid medium served as a control. Thirty rhizomes were planted in a sterilized river sand and vermiculite mixture (1:1) in a greenhouse at room temperature, with three replicates. After 15 days, the fermentation product was irrigated. Rhizome germination was regularly observed, and the emergence rate was calculated after 30 days.

[0092] The results are as follows Figure 5 As shown in the results, the germination rate of the treatment with the fermentation liquid of endophytic fungus CL-26 was 61.11%, which was 57.14% higher than that of the control group, indicating that the fermentation liquid of endophytic fungus CL-26 can increase the germination rate of Paris polyphylla.

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An endophytic fungus strain of Paris polyphylla, characterized by: The endophytic fungus strain number of the Paris polyphylla is CL-26, and its classification is named Clonostachys sp., deposited in Guangdong Provincial Microbial Culture Collection Center, the deposit address of which is 5th Floor, Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, deposit date: March 31, 2025; deposit number: GDMCC 66079.

2. The endophytic fungus strain of Paris polyphylla according to claim 1, characterized in that: The ITS gene sequence of the endophytic fungus strain of Paris polyphylla is the nucleotide sequence shown in SEQ ID No.

1.

3. The endophytic fungus strain of Paris polyphylla according to claim 1, characterized in that: The colony of the endophytic fungus strain CL-26 of the Paris polyphylla on the PDA medium is cotton-like, with dense hyphae, disc-shaped on the front, white, slightly yellowish in the middle, and white and khaki on the back; The hyphae are septate, with a diameter of 2.9–4.8 μm; Conidiophores are erect, 76-203 μm long, 2.5-5.0 μm in diameter at the base, with broom-like branches at the top, 11.5-43.8 μm long, 1.5-3.0 μm in diameter at the base; Spores are produced at the top of the phialide, are oval or kidney-shaped, have a smooth surface, and are (3.8~7.1)×(2.0~3.5)μm in diameter.

4. The endophytic fungus agent of Paris polyphylla prepared from the endophytic fungus strain of Paris polyphylla according to claim 1. The endophytic fungus agent of Paris polyphylla according to claim 4 , wherein the active ingredient is the fermentation broth of the endophytic fungus strain of Paris polyphylla according to claim 1 .

6. The method for preparing the fermentation liquid of the endophytic fungus strain of Paris polyphylla according to claim 1, characterized in that: The steps include: The endophytic fungus strain CL-26 of the Paris polyphylla was inoculated into a PDA liquid culture medium, cultured at 28° C. and a rotation speed of 200 r / min for 3 to 5 days, centrifuged at a rotation speed of 10,000 r / min for 10 minutes, and the supernatant was collected to obtain the Paris polyphylla endophytic fungus fermentation liquid.

7. Use of the endophytic fungus strain of Paris polyphylla according to any one of claims 1 to 3 in promoting rhizome germination and increasing seedling emergence rate.

8. Use of the endophytic fungal agent of Paris polyphylla according to claim 4 or 5 in promoting rhizome germination and increasing the emergence rate of Paris polyphylla.

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