An endophytic fungus from the root of Polygonum cuspidatum, Talaromyces lentulus PF12-1, and its application

Abstract

The present invention relates to an endophytic fungus Talaromyces lentulus PF12-1 from the roots of Polygonum cuspidatum and its applications. The present invention obtains an endophytic fungus Talaromyces lentulus PF12-1 from the roots of Polygonum cuspidatum through isolation and screening. The endophytic fungus Talaromyces lentulus PF12-1 can not only effectively promote the growth of Arabidopsis thaliana and Polygonum cuspidatum seedlings, but also promote the accumulation of active ingredients by regulating genes related to the phenylpropanoid metabolic pathway of Polygonum cuspidatum. This demonstrates the important role of endophytic fungi from the roots of Polygonum cuspidatum in the synthesis and accumulation of active ingredients in Polygonum cuspidatum, lays the foundation for in-depth analysis of the key regulatory mechanisms of active ingredients such as resveratrol and polydatin, and is of great significance for improving the quality of Polygonum cuspidatum.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an endophytic fungus Talaromyces lentulus PF12-1 from the roots of Polygonum cuspidatum and an application thereof. Background Art

[0002] Polygonum cuspidatum is a perennial herb of the Polygonaceae family. Its dried rhizome is used as medicine to treat liver and gallbladder diseases, lung heat and cough, and other diseases. The main active ingredients of Polygonum cuspidatum (resveratrol, resveratrol glycosides, emodin and emodin methyl ether, etc.) also have anti-tumor, cardiovascular protection, and anti-aging effects. Therefore, Polygonum cuspidatum has important medicinal value.

[0003] Root microbes refer to microorganisms tightly attached to soil particles around plant roots, encompassing both the rhizosphere and the endosphere. The soil layer, approximately 40 mm in diameter, surrounding plant roots is called the rhizosphere. This is the area where the most microorganisms live and move most frequently, creating a complex ecosystem characterized by plant-microbe-soil interactions. Rhizosphere microbes include bacteria, fungi, actinomycetes, and algae, which interact with plant roots, forming a mutually beneficial, symbiotic relationship. The endosphere refers to the endogenous tissues of plant roots, including the periderm, phloem, and xylem, where a variety of microorganisms, known as endophytes, live. Endophytes can colonize host plants for long periods of time and gradually form a stable symbiotic relationship. The mechanisms underlying this mutually beneficial symbiotic relationship have attracted widespread attention. Research has shown that plant health largely depends on the dynamic balance between rhizosphere and endosphere microbes.

[0004] Currently, most research on Polygonum cuspidatum fungi relies on traditional microbial isolation methods. Some researchers have screened endophytic fungi capable of converting polydatin from Polygonum cuspidatum roots, and other studies have shown that inoculation with Piriformosporaindica and the arbuscular fungus Funneliformis mosseae can affect the growth and development of Polygonum cuspidatum. However, the ecological role played by Polygonum cuspidatum root microorganisms, particularly their impact on the growth and development of the host plant, has not been studied. Research on the fungal community structure of Polygonum cuspidatum roots and its impact on Polygonum cuspidatum quality is still incomplete, and the functions of Polygonum cuspidatum root microorganisms need to be further explored. Summary of the Invention

[0005] The present invention uses Illumina high-throughput sequencing to analyze the fungal community composition of different sampling sites (non-rhizosphere soil, rhizosphere soil and roots) of one-, two- and three-year-old polygonum cuspidatum roots; and determines the physical and chemical properties of the soil and the content of the main active ingredients of polygonum cuspidatum; explores the relationship between the fungal community of the polygonum cuspidatum root system and environmental factors; analyzes the correlation between the fungal community of the polygonum cuspidatum root system and the active ingredients; screens out endophytic fungi of polygonum cuspidatum roots by microbial isolation and culture methods, and uses plate tests and pot tests to study the effects of endophytic fungi of polygonum cuspidatum roots on the growth of Arabidopsis thaliana and polygonum cuspidatum seedlings, laying a foundation for quality improvement of polygonum cuspidatum.

[0006] The present invention provides an endophytic fungus Talaromyces lentulus PF12-1 from the roots of Polygonum cuspidatum, which was deposited in the China Center for Type Culture Collection on June 20, 2024, with a deposit number of CCTCC NO: M 20241325, and the deposit address is Wuhan University, Wuhan, China.

[0007] The present invention also provides the use of the above-mentioned Talaromyces lentulus PF12-1, a bacterial suspension thereof, or a composition containing the same, in any one of the following A1) to A6):

[0008] A1) Promote plant growth;

[0009] A2) preparing a bacterial agent for promoting plant growth;

[0010] A3) Phosphate solubilization, potassium solubilization, and / or indoleacetic acid production in vitro;

[0011] A4) preparing an in vitro bacterial agent that solubilizes phosphate, dissolves potassium, and / or produces indoleacetic acid;

[0012] A5) Promote the content of plant active ingredients;

[0013] A6) Preparation of a fungal agent that increases the content of active ingredients in plants.

[0014] The plant effective ingredients include total flavonoids, resveratrol and proanthocyanidins.

[0015] Furthermore, the plant growth promotion is embodied in whole or in part as follows:

[0016] B1) promoting the increase of above-ground biomass and / or below-ground biomass of the plant at different developmental stages of the plant;

[0017] B2) promoting an increase in the number of lateral roots of the plant at different developmental stages of the plant;

[0018] B3) promoting an increase in the number of leaves of the plant at different developmental stages of the plant.

[0019] Furthermore, the plant is any one of the following:

[0020] (C1) Arabidopsis thaliana;

[0021] (C2) Cruciferae;

[0022] (C3) Japanese knotweed;

[0023] (C4) Polygonaceae plants;

[0024] (C5) Dicotyledons.

[0025] The present invention also provides a bacterial agent containing the endophytic fungus Talaromyces lentulus PF12-1 from the root of polygonum cuspidatum or its bacterial suspension or fermentation product.

[0026] Furthermore, the concentration of the endophytic fungus Talaromyces lentulus PF12-1 from the root of Polygonum cuspidatum in the bacterial agent is not higher than 6.8×10 -5 g / mL.

[0027] The present invention also provides a method for promoting plant growth and / or increasing the content of plant active ingredients, comprising the step of treating plants with the above-mentioned Talaromyces lentulus PF12-1 endophytic fungus or bacterial agent, wherein the plant active ingredients include total flavonoids, resveratrol and proanthocyanidins.

[0028] Furthermore, the concentration of the endophytic fungus Talaromyces lentulus PF12-1 in the root of Polygonum cuspidatum is not higher than 6.8×10 -5 g / mL.

[0029] Furthermore, the plant growth promotion is embodied in whole or in part as follows:

[0030] B1) promoting the increase of above-ground biomass and / or below-ground biomass of the plant at different developmental stages of the plant;

[0031] B2) promoting an increase in the number of lateral roots of the plant at different developmental stages of the plant;

[0032] B3) promoting an increase in the number of leaves of the plant at different developmental stages of the plant.

[0033] Furthermore, the plant is any one of the following:

[0034] (C1) Arabidopsis thaliana;

[0035] (C2) Cruciferae;

[0036] (C3) Japanese knotweed;

[0037] (C4) Polygonaceae plants;

[0038] (C5) Dicotyledons.

[0039] Beneficial effects:

[0040] The present invention obtains an endophytic fungus Talaromyces lentulus PF12-1 from the roots of Polygonum cuspidatum through isolation and screening. The endophytic fungus Talaromyces lentulus PF12-1 can not only effectively promote the growth of Arabidopsis thaliana and Polygonum cuspidatum seedlings, but also promote the accumulation of effective ingredients by regulating the relevant genes of the phenylpropanoid metabolic pathway of Polygonum cuspidatum. This shows the important role of endophytic fungi from the roots of Polygonum cuspidatum in the synthesis and accumulation of effective ingredients of Polygonum cuspidatum, lays the foundation for in-depth analysis of the key regulatory mechanisms of effective ingredients such as resveratrol and polydatin, and is of great significance to the quality improvement of Polygonum cuspidatum. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 The purpose is to screen the endophytic fungi in the roots of Polygonum cuspidatum for their ability to solubilize phosphorus, potassium and produce IAA.

[0043] Figure 2 This is a phylogenetic tree of Talaromyces species constructed based on multiple gene sequences (ITS, BenA, CaM).

[0044] Figure 3 Morphological characteristics of the endophytic fungus F12 (Talaromyces lentulus) from Polygonum cuspidatum roots. Figure a: Colony morphology (MEA) after 7-day culture at 28°C; b-d: Conidiophores, phialides, and conidia; e: Conidia; Scale bars: b-e = 10 μm.

[0045] Figure 4 Morphological images of endophytic fungi from Polygonum cuspidatum roots co-cultured with Arabidopsis thaliana for 14 days. A: CK; B: F12 co-cultured with Arabidopsis thaliana.

[0046] Figure 5 Figure 14 shows the morphology of endophytic fungi from Polygonum cuspidatum roots cultured separately from Arabidopsis thaliana. A: CK; B: F12 cultured separately from Arabidopsis thaliana.

[0047] Figure 6 The morphology of Polygonum cuspidatum seedlings treated with F12 suspension. From left to right, the treatments are the initial concentration, 10-fold dilution, 100-fold dilution, and 1000-fold dilution of the fungal suspension.

[0048] Figure 7 These are the results of gene detection of the phenylpropanoid metabolic pathway in Polygonum cuspidatum root treated with endophytic fungus F12. DETAILED DESCRIPTION

[0049] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention with this. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0050] Example 1 Isolation of endophytic fungus F12 from the rhizosphere of Polygonum cuspidatum root

[0051] (1) Isolation, purification and identification of endophytic fungi from the roots of Polygonum cuspidatum

[0052] Plant material: Knotweed was planted in the nursery of Yangtze University. Soil material: Yangtze University nursery.

[0053] Rinse the roots of Polygonum cuspidatum with tap water for 2 hours, absorb the moisture with filter paper, and treat the sample with a three-step surface disinfection method. After disinfection with 75% alcohol for 3 minutes, 5% sodium hypochlorite for 3 minutes, and 75% alcohol for 1 minute, rinse with sterile water 5 times, absorb the moisture with sterile filter paper, and use a sterile knife to cut the surface-sterile Polygonum cuspidatum roots into small pieces of 0.5 cm long, place them on the corresponding culture medium, and culture them at 28°C for 2 to 3 days. Take 100 μL of sterile water from the last wash and apply it to the surface of the corresponding culture medium as a control to test whether the surface of the Polygonum cuspidatum root is thoroughly disinfected. Weigh 5g of air-dried rhizosphere soil sample, dissolve it in 45mL of sterile water, and incubate at 28°C, 250rpm / min, for 30 minutes to fully disperse the microorganisms in the soil sample, and then carry out 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 Perform gradient dilutions and evenly spread 100 μL of the dilutions on the corresponding culture medium. Culture at 28°C for 2-3 days. Finally, based on differences in colony morphology, size, color, etc., isolate hyphae from the edges of different colonies and transfer them to purification medium for isolation and purification.

[0054] The isolated root fungus from Polygonum cuspidatum was purified using the tip hyphae purification method until a single strain was obtained. Preliminary morphological observation and PCR molecular identification were then performed. 30 μL of lysate was transferred to a 1.5 mL centrifuge tube. Purified hyphae were picked with a toothpick and placed in the lysate. The mixture was incubated at 80°C for 15 minutes to serve as a PCR template. PCR amplification was performed using universal primers for the fungal ITS region, ITS1 (5′-TCCGTAGGTGAACCTG CGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The PCR reaction system (50 μL) consisted of 25 μL 2× Mix, 1 μL Primer-F, 1 μL Primer-R, 5 μL DNA, and ddH2O to make up the remaining volume to 50 μL. PCR reaction conditions were: 95°C denaturation for 10 minutes, 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, followed by extension at 72°C for 5 minutes. The PCR products were detected by gel electrophoresis and then sent to Shanghai Bioengineering Co., Ltd. for sequencing.

[0055] A total of 56 fungal strains were isolated and identified from the roots and rhizosphere soil of Polygonum cuspidatum of different ages. Of these, 42 endophytic fungi (F1-F42) were isolated from the roots, and 14 (F43-F56) were isolated from the rhizosphere soil. At the phylum level, these strains belonged to the Basidiomycota, Mucoromycota, and Ascomycota phyla. One strain belonged to the Basidiomycota, accounting for 2%; one to the Mucoromycota, accounting for 2%; and 55 strains belonged to 13 genera within the Ascomycota, accounting for 96%. At the genus level, these strains belonged to 15 genera, with the dominant genera being Aspergillus, Penicillium, and Talaromyces, accounting for 30%, 23%, and 11%, respectively.

[0056] (2) Determination of the strain's ability to solubilize phosphate, dissolve potassium, and produce IAA

[0057] Fifty-six isolated fungal strains were screened for their phosphate-solubilizing and potassium-solubilizing abilities. The ability to solubilize and dissolve phosphates was determined by observing the formation of halos in the corresponding culture media. The isolated strains were inoculated onto solid plates containing organic phosphate, inorganic phosphate, and potassium-solubilizing media, respectively, in triplicate. The plates were incubated in an inverted culture at 28°C for 7 days and observed for the formation of clearing zones. If clearing zones were observed, the strain possessed phosphate-solubilizing and potassium-dissolving abilities. The isolated strains were inoculated into 5 mL of PDB medium containing 1 mg / mL L-tryptophan, in triplicate, with uninoculated PDB as a control. The culture was shaken at 28°C and 180 rpm / min for 2 days. The culture was then centrifuged at 12,000 rpm / min for 5 minutes, and 1 mL of the supernatant was pipetted into a test tube. An equal amount of Salkowski's reaction solution (10.8 mol / L H₂SO₄ containing 4.5 g of FeCl₃ per liter) was added and mixed. Uninoculated PDB medium served as a blank control. Each strain was tested in triplicate. The mixture was allowed to react in the dark for 30 min. When the color turned pink, it indicated that the product had the ability to secrete IAA. The color depth was positively correlated with the secretion ability. The OD was measured. 530 The curve was drawn using IAA standard, with OD 530 The absorbance value (Y) under the condition of α-D was used as the ordinate and the IAA concentration (X) was used as the abscissa for linear regression. The linear regression equation was: y = 0.0246x + 0.1127 (R 2 =0.9929).

[0058] The results are as follows Figure 1 As shown, 19 fungal strains, including F12, could grow on organic phosphorus media and form clear zones, accounting for 33.93% of the total strains. Thirteen fungal strains, including F12, could grow on inorganic phosphorus media and form clear zones, accounting for 23.21% of the total strains. Seven fungal strains, including F12, could grow on potassium feldspar media and form clear zones, accounting for 10.71% of the total strains. The Salkowski colorimetric method was used to determine the IAA production capacity of the strains. Seven of the 56 fungal strains, representing 12.50% of the total isolates, were capable of producing IAA. F12 had a particularly strong IAA production capacity, synthesizing 13.43±0.05 mg / L.

[0059] In summary, F12 has the ability to dissolve organic phosphorus, inorganic phosphorus, potassium and produce IAA, and has strong growth-promoting potential.

[0060] (3) Identification of F12 based on multi-gene phylogenetic tree

[0061] The total length of the concatenated polygenic sequence used to construct the phylogenetic tree is 1606 bp, including 500 bp of ITS, 534 bp of BenA, and 572 bp of CaM. The phylogenetic tree was calculated using the GTR+G model and the maximum likelihood algorithm using the Find Model software. The results are shown in Figure 2. Figure 2 As shown in the figure, F12 and the type strain T.lentulusAS3.15689T of the Talaromyces group clustered together. Based on the phylogenetic tree identification of multiple genes, F12 was determined to belong to Talaromyceslentulus, and it was named: Talaromyces lentulus PF12-1 (hereinafter referred to as F12). The strain was deposited in the China Center for Type Culture Collection on June 20, 2024, with the deposit number CCTCC NO: M 20241325. The deposit address is: Wuhan University, Wuhan, China.

[0062] (4) Morphological characteristics of endophytic fungus F12 from Polygonum cuspidatum roots

[0063] After 7 days of culture on MEA medium at 28°C, F12 colonies were 49-50 mm in diameter, with a raised center. The texture was slightly velvety and granular. The conidia were abundant and gray-green in color. The mycelium was light yellow. The soluble pigment was red. The reverse side was yellow. Conidiophores were formed on the stroma or aerial hyphae. The conidiophores had smooth walls and bi-whorls of broom-like branches. The conidia were spherical to nearly spherical, such as Figure 3 shown.

[0064] Example 2 Verification of the Growth-Promoting Effect of Endophytic Fungus F12 from Polygonum cuspidatum Roots

[0065] (1) Cultivation of sterile seedlings of Arabidopsis thaliana

[0066] Wild-type Arabidopsis seeds were shaken with 75% ethanol and 1.5% sodium hypochlorite for 1 minute, then rinsed with sterile water 5 to 6 times. Finally, the sterilized seeds were spotted on 1 / 2MS solid culture medium, with about 30 seeds in each culture dish. After vernalization in a 4°C refrigerator for 2 days, they were taken out and placed in an incubator with a culture temperature of 23°C, a light duration of 16h / d, and a light intensity of 66%.

[0067] (2) Co-culture of endophytic fungi from Polygonum cuspidatum roots with Arabidopsis thaliana and separate culture on plates

[0068] 2.1 Co-cultivation of endophytic fungi from Polygonum cuspidatum roots with Arabidopsis thaliana

[0069] 7-day-old Arabidopsis seedlings (two cotyledons) with basically the same growth were selected as the test subjects. A horizontal line of about 8 cm was drawn at the bottom of each 1 / 2 MS culture dish. Sterile seedlings were placed on the horizontal line, with 5 seedlings per culture dish. A 0.5 cm hole was punched with a hole punch.2 The bacterial cake was placed on the lower side of the culture medium, 1 cm away from the bottom of the culture dish, and a PDA block of the same size was used as a blank control. Each group was repeated 3 times and cultured in the light culture medium.

[0070] After all plant materials were cultured in a light incubator for 7 days, the number of Arabidopsis lateral roots was counted. The plants were harvested on the 14th day, the residual culture medium was removed, and growth indicators such as fresh weight and main root length were measured. The fresh plants were placed in an oven to dry at 50°C for 8 hours and then weighed. The lateral root density was calculated based on the fixed root length.

[0071] Lateral root density = number of lateral roots / main root length

[0072] The results are as follows Figure 4 As shown in Table 1, the growth of sterile Arabidopsis seedlings in the experimental group was better than that in the CK group when endophytic fungi from Polygonum cuspidatum roots were co-cultured with Arabidopsis thaliana. Statistical analysis of the experimental data using SPSS 22.0 software showed that the total fresh weight of F12 seedlings in the treated group increased by 619.49% compared to the CK group; the total dry weight increased by 2188.84%; the taproot length decreased by 23.30%; the number of lateral roots increased by 65.62%; the lateral root density increased by 115.55%; and the number of leaves increased by 41.34% (p < 0.05).

[0073] Table 1 Effects of co-cultivation of endophytic fungi from Polygonum cuspidatum roots with Arabidopsis thaliana on plant growth

[0074]

[0075] The results showed that co-cultivation of the endophytic fungus F12 from the roots of Polygonum cuspidatum with Arabidopsis thaliana could significantly promote the number of lateral roots and leaves and increase the biomass of Arabidopsis thaliana.

[0076] 2.2 Culture of endophytic fungi from Polygonum cuspidatum roots and Arabidopsis thaliana on separate plates

[0077] Use a two-divided culture dish for isolation culture. Pour 1 / 2 MS culture medium on one side of the culture dish and PDA culture medium on the other side. Then place the Arabidopsis seedlings in the 1 / 2 MS culture medium. Place 3 seedlings in each culture dish. Use a hole puncher to punch a 0.5 cm 2 The bacterial cake was placed in the middle of the PDA culture medium, and a PDA block of the same size was used as a blank control. Each group was replicated 3 times and cultured in a light culture medium.

[0078] The results are as follows Figure 5As shown in Table 2, observation of endophytic fungi in the roots of Polygonum cuspidatum and cultured separately from Arabidopsis thaliana revealed that the growth of sterile Arabidopsis seedlings in the experimental group was better than that in the CK group. Statistical analysis of the experimental data using SPSS 22.0 software showed that the total fresh weight of F12 in the treatment group increased by 252.04%; the total dry weight increased by 877.44%; the taproot length decreased by 4.31%; the number of lateral roots increased by 121.47%; the lateral root density increased by 129.80%; and the number of leaves increased by 26.30% compared to the CK group (p < 0.05).

[0079] Table 2 Effects of endophytic fungi in Polygonum cuspidatum roots and Arabidopsis thaliana cultured separately on plant growth

[0080]

[0081] The results showed that the endophytic fungus F12 from the roots of Polygonum cuspidatum significantly increased the number of lateral roots and leaves, and increased the biomass of Arabidopsis thaliana when cultured separately from Arabidopsis thaliana. It is speculated that the volatile organic compounds (MVCs) produced by the endophytic fungus F12 from the roots of Polygonum cuspidatum promote the growth of Arabidopsis thaliana plants.

[0082] Example 3 Effect of Polygonum cuspidatum root endophytic fungus F12 on the growth of Polygonum cuspidatum seedlings

[0083] Soak knotweed seeds in warm water at around 35°C for 4 hours, then soak in 3% hydrogen peroxide for 10 minutes, disinfect with 75% ethanol for 1 minute, and then 5% sodium hypochlorite for 10 minutes. Rinse with sterile water 5-6 times before germinating in a petri dish lined with moistened filter paper. Incubate at 23°C, with a light intensity of 16 hours per day and 66% illumination. After 14 days of incubation, transplant the germinated seeds into pots for later use.

[0084] F12 was inoculated into PDB medium at 25°C and 180 rpm / min. After 10 days, the fungal suspension was collected and the mixture was stirred at 12000 rpm / min for 10 min to obtain a precipitate. Approximately 6.8 g of mycelium was homogenized in a tissue homogenizer using 100 mL of sterile water to obtain an initial suspension (6.8 × 10 -2 Then, the prepared fungal suspension was serially diluted with sterile water to 10 times (6.8×10 -3 g / mL), 100 times (6.8×10 -4 g / mL) and 1000 times (6.8×10 -5 The undiluted, 10-fold, 100-fold, and 1000-fold diluted fungal suspensions were numbered F12-1, F12-10, F12-100, and F12-1000, respectively.

[0085] Using the greenhouse potting method, 14-day-old Polygonum cuspidatum seedlings with consistent growth were selected for transplanting. After the seedlings had established, the roots were irrigated with 20 mL of different concentrations of F12 bacterial suspension. Six plants were treated per treatment, with the roots irrigated twice, 7 days apart. Irrigation with an equal volume of water served as a blank control. Temperature and water and fertilizer management remained consistent during this period. After 70 days of incubation in a light incubator, the Polygonum cuspidatum plants were harvested, the soil on the root surface of the seedlings was cleaned, the taproot length was measured, and the root system was scanned. Aboveground and underground fresh weights were weighed, and the roots were then oven-dried at 50°C for 24 hours before determination of active ingredient content.

[0086] Measuring growth indicators can most intuitively understand the growth of knotweed seedlings. Figure 6 As shown in Table 3-4, after 70 days of pot culture, the F12-1000 treatment (6.8×10 -5 g / mL) grew fastest compared with the control group ( Figure 6 and Tables 4-5), and the root system was more developed (Table 4-6). Specifically, plant height, root length, aboveground fresh weight, belowground fresh weight, root volume, and number of root branches increased by 32.15%, 22.41%, 24.30%, 42.61%, 199.81%, and 75.95% (p < 0.05) compared to the control group. The number of root tips, projected area, surface area, and average diameter of the roots were also higher than those in the control group. There were no significant differences between the treatments with other F12 concentrations and the control group.

[0087] Table 3 Effects of endophytic fungus F12 from Polygonum cuspidatum roots on the growth of Polygonum cuspidatum seedlings

[0088]

[0089]

[0090] Table 4 Effects of endophytic fungus F12 from Polygonum cuspidatum roots on the root system of Polygonum cuspidatum seedlings

[0091]

[0092] The results showed that for strain F12, the concentration was 6.8×10 -5 g / mL bacterial solution treatment can effectively promote the growth of Polygonum cuspidatum seedlings, especially has a significant promoting effect on the root growth of seedlings.

[0093] Example 4 Effect of endophytic fungus F12 from the roots of Polygonum cuspidatum on the effective components of Polygonum cuspidatum

[0094] Accurately weigh 0.1 g of dried sample into a 1.5 mL centrifuge tube, add 1 mL of 80% chromatography-grade methanol, sonicate for 5 minutes, and extract overnight. The next day, centrifuge the supernatant, filter through a 0.22 μm organic filter, and inject into a sample vial to serve as the loading sample. The sample was analyzed by high-performance liquid chromatography (HPLC) using a Spherisorb C18 column with a mobile phase of acetonitrile (A) and purified water (B). The column temperature was 30°C, the flow rate was 1 mL / min, and the injection volume was 10 μL. Polydatin, resveratrol, emodin, and emodin methyl ether were detected at 290 nm. The linear gradient elution program was: 25-50% A for 0-10 minutes; 50-90% A for 10-30 minutes. Accurately weigh 1 mg of polydatin, resveratrol, emodin and physcion (content 99%) standard products. Polydatin and resveratrol were prepared with chromatographic grade methanol to prepare standard products with concentrations of 5, 10, 50, 100, 500, and 1000 μg / mL. Emodin and physcion were prepared with chromatographic grade methanol to prepare standard products with concentrations of 0.5, 1, 5, 10, 50, and 100 μg / mL. Standard curves were prepared.

[0095] Total flavonoids were determined using a sodium nitrite-aluminum nitrate spectrophotometric method: 1 mg of Polygonum cuspidatum root was dissolved in 1 ml of 60% ethanol. To 0.02 ml of sample, 0.1 ml of 5% sodium nitrite solution was added. After 6 minutes of simmering, 0.1 ml of 10% aluminum nitrate solution was added. After 6 minutes of simmering, 1 ml of 4% sodium hydroxide solution was added. The volume was filled to 2.5 ml with purified water, mixed, and allowed to stand for 15 minutes. The absorbance of each sample was measured at 510 nm using a UV spectrophotometer, using purified water as a blank control. Rutin was used as the standard.

[0096] Polydatin, resveratrol, emodin and proanthocyanidins are the main active ingredients of Polygonum cuspidatum. Inoculation with the endophytic fungus F12 from the roots of Polygonum cuspidatum seedlings had a certain effect on the active ingredients in the roots of Polygonum cuspidatum seedlings. The results are shown in Table 5. The F12-1000 treatment group (6.8×10 -5 g / mL) can promote the synthesis of resveratrol and proanthocyanidins in the roots of Polygonum cuspidatum seedlings, which increased by 20.10% and 47.15% respectively compared with the control group (p < 0.05), and the content of total flavonoids was also higher than that of the control group, while there was no significant change in the other F12 treatment groups compared with the control.

[0097] Table 5 Effects of endophytic fungus F12 from the roots of Polygonum cuspidatum on the effective components in the roots of Polygonum cuspidatum seedlings

[0098]

[0099] In summary, the concentration is 6.8×10 -5g / mL F12 bacterial solution treatment of Polygonum cuspidatum seedlings can significantly promote the accumulation of effective ingredients such as total flavonoids, resveratrol and proanthocyanidins in the roots of Polygonum cuspidatum seedlings.

[0100] Example 5 Quantitative analysis of genes related to the synthesis of effective components of Polygonum cuspidatum by endophytic fungus F12 from the roots of Polygonum cuspidatum

[0101] The total flavonoids, resveratrol and proanthocyanidins of Polygonum cuspidatum are all products of the phenylpropanoid metabolic pathway. In order to explore the effect of inoculation of endophytic fungi on the genes related to the phenylpropanoid metabolic pathway of Polygonum cuspidatum, fluorescence quantitative PCR was used to detect the expression of 1.5-2.5 mmol / l of the F12-1000 treatment group (6.8×10 -5 g / mL) seedling roots.

[0102] The results are as follows Figure 7 As shown in the results, in the synthesis pathway, the endophytic fungus F12 from the root of Polygonum cuspidatum significantly increased the expression of three common genes in the upstream of the phenylpropanoid metabolic pathway, namely the phenylalanine ammonia lyase gene (PcPAL), the cinnamate-4-hydroxylase gene (PcC4H), and the 4-coumarate-CoA ligase gene (Pc4CL) (p<0.05), and significantly increased the expression of the key gene for flavonoid synthesis, the chalcone synthase gene (PcCHS) (p<0.05), and the resveratrol synthase gene (PcRS) (p<0.05), and also significantly increased the expression of the key gene for proanthocyanidin synthesis, the dihydroflavonol 4-reductase gene (PcDF R), colorless anthocyanidin reductase gene (PcLAR), anthocyanidin synthase gene (PcANS), anthocyanidin reductase gene (PcANR) and anthocyanidin glucosyltransferase gene (PcUFGT) (p < 0.05), thereby promoting the accumulation of total flavonoids, resveratrol and proanthocyanidins in the roots of Polygonum cuspidatum seedlings.

[0103] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. An endophytic fungus from the root of Polygonum cuspidatum Talaromyces lentulus PF12-1 was deposited in the China Center for Type Culture Collection on June 20, 2024, with the deposit number CCTCC NO:M 20241325.

2. The endophytic fungus from the root of Polygonum cuspidatum according to claim 1 Talaromyces lentulus Use of PF12-1 or its bacterial suspension or a composition containing the same in any of the following A1) to A6): A1) Promote plant growth; A2) Preparation of microbial agents for promoting plant growth; A3) In vitro phosphate solubilization, potassium solubilization and / or indoleacetic acid production; A4) Preparation of in vitro bacterial agents for solubilizing phosphate, dissolving potassium and / or producing indoleacetic acid; A5) Promote the content of plant active ingredients; A6) Preparation of microbial agents for increasing the content of active ingredients in plants; The active ingredients of the plant are total flavonoids, resveratrol and proanthocyanidins; the plant is Polygonum cuspidatum or Arabidopsis thaliana; the endophytic fungus in the root of Polygonum cuspidatum Talaromy ces lentulus The concentration of PF12-1 is not higher than 6.8×10 -5 g / mL.

3. The use according to claim 2, characterized in that: The plant growth promotion is embodied in whole or in part as follows: B1) promoting the increase of aboveground biomass and / or belowground biomass of the plant at different developmental stages of the plant; B2) promoting an increase in the number of lateral roots of the plant at different developmental stages of the plant; B3) promoting an increase in the number of leaves of the plant at different developmental stages of the plant.

4. Containing the endophytic fungus of the root of Polygonum cuspidatum according to claim 1 Talaromyces lentulus A bacterial agent of PF12-1 or its bacterial suspension or its fermentation product.

5. The microbial agent according to claim 4, characterized in that: Endophytic fungi in the roots of Polygonum cuspidatum Talaromyces lentulus The concentration of PF12-1 is not higher than 6.8×10 -5 g / mL.

6. A method for promoting plant growth and / or increasing the content of active ingredients in plants, comprising using the endophytic fungus from the root of Polygonum cuspidatum according to claim 1 Talaromyces lentulus PF12-1 or the step of treating plants with a fungal agent according to any one of claims 4-5, wherein the active ingredients of the plant are total flavonoids, resveratrol and proanthocyanidins, the plant is Polygonum cuspidatum, and the endophytic fungus of the root of Polygonum cuspidatum Talaromyces lentulus The concentration of PF12-1 is not higher than 6.8×10 -5 g / mL.

7. The method according to claim 6, characterized in that: The plant growth promotion is embodied in whole or in part as follows: B1) promoting the increase of aboveground biomass and / or belowground biomass of the plant at different developmental stages of the plant; B2) promoting an increase in the number of lateral roots of the plant at different developmental stages of the plant; B3) promoting an increase in the number of leaves of the plant at different developmental stages of the plant.

Images