Strobilanthes cusia endophytic fungus MLR15

Abstract

The invention belongs to the field of plant growth and resistance, and particularly relates to a strobilanthes cusia endophytic fungus MLR15. The strobilanthes cusia endophytic fungus MLR15 disclosed by the invention is a bacterial strain which is preserved in the China General Microbiological Culture Collection Center (CGMCC) and has a preservation number of CGMCC No.41250. Experiments show that MLR15 can effectively promote growth of strobilanthes cusia and has a certain broad-spectrum growth promoting effect on crops such as tomatoes. In addition, the MLR15 can also promote the increase of the content of the active ingredient indigo for the traditional Chinese medicine of the strobilanthes cusia. Therefore, the MLR15 provided by the invention can be developed into an active microbial agent for promoting plant growth and synthesis of medicinal components of strobilanthes cusia, and has a very good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of plant growth and resistance, and particularly relates to an endophytic fungus MLR15 of Indigofera lanuginosa. Background Art

[0002] Strobilanthes cusia (Nees) Kuntze is a traditional Chinese medicinal plant with the effects of detoxification, fever reduction, swelling and anti-inflammatory, and is widely used to treat various epidemics. As a natural antibacterial and antiviral Chinese herbal medicine, the whole plant of Strobilanthes contains triterpenes, indigo, indirubin and 2, 4 (1H, 3H) -quinazolidone, a total of about 36 alkaloids and 35 glycosides; the root contains indole glycosides, chrysophanol, various amino acids, indigo, indirubin, lupeol, betulin and β-sitosterol; while the stems and leaves are mainly indigo and indirubin, which are also the main medicinal ingredients of Strobilanthes, and are often used as important indicators for evaluating the quality of Strobilanthes.

[0003] The synthesis pathways of indigo and indirubin, the main active medicinal ingredients of Isatis indigo, have been basically clarified. Both are derived from the biosynthesis pathway of the alkaloid indole. Based on the understanding of the synthesis pathway, the use of molecular biological methods to regulate the expression of key enzyme genes of indigo and indirubin in Isatis indigo is currently a common strategy to increase the content of indigo and indirubin in Isatis indigo plants. For example, the literature "Yu Jian. Cloning and functional study of EPSPS and CS, key genes for the synthesis of indole alkaloids in Isatis indigo [D]. Fuzhou: Fujian Agriculture and Forestry University, 2018." By constructing an Agrobacterium gene overexpression vector and mediating the transformation of Isatis indigo leaf discs, regenerated plants and hairy roots were obtained, and it was verified that the key genes for the synthesis of indigo and indirubin in Isatis indigo, BcEPSPS and BcCS, were transferred into the hairy roots, and the highest expression levels of the genes could be increased by 37.8 times and 52.3 times, respectively. However, it is difficult to construct an overexpression vector and generate hairy roots in this method, and a mature indigo plant tissue culture system needs to be constructed. At the same time, since the genetic transformation system of indigo plant has not yet been established, existing studies have all used Isatis indigotica as a transgenic vector. Although Isatis indigotica also has an indole alkaloid biosynthesis pathway, its pathway is different from that of Indigo plant due to species differences. The results cannot accurately reflect the way indigo and indigotin are synthesized in Indigo plant, and there are certain limitations.

[0004] Endophytic fungi refer to a type of microorganisms that can colonize in the intercellular spaces or inside plant cells and establish a harmonious symbiotic relationship with the host plant. At present, research on plant endophytes mainly focuses on endophytic fungi. There are a large number of endophytic fungi in plants, which can be distributed in different tissues of various plants. Endophytic fungi may have the effects of promoting plant growth, increasing the content of plant secondary metabolites, and helping plants resist stress. In view of the various beneficial physiological activities of endophytic fungi on host plants, they can be used to create microbial agents to achieve the effects of promoting plant growth, increasing crop yields, and inducing plants to produce systemic resistance to biotic and abiotic stresses.

[0005] For Indigofera odorata, if we can screen out endophytic fungi that can increase the yield of Indigofera odorata and promote the synthesis of indigo or indigotin, it will have great application potential. However, in plants, the types of endophytic fungi in different parts vary greatly and are numerous. Screening endophytic fungi with specific effects is still a very challenging topic. Summary of the invention

[0006] In view of the defects of the prior art, the present invention provides an endophytic fungus MLR15.

[0007] The invention discloses an endophytic fungus of indigofera cyanobacteria, which is a fungus of the genus Trichoderma (Trichoderma sp.). The endophytic fungus of indigofera cyanobacteria is a strain preserved by the General Microbiological Center of China Microbiological Culture Collection Committee with a preservation number of CGMCC No.41250.

[0008] The invention also provides a fungal agent, which comprises the above-mentioned endophytic fungus of Indigofera ovata.

[0009] Preferably, the bacterial agent is made from a spore and mycelium mixture or a fungal culture solution of the Indigofera endophytic fungus.

[0010] The present invention also provides use of the above-mentioned Indigofera endophytic fungus or bacterial agent in promoting plant growth.

[0011] Preferably, the plant is a Solanaceae plant or a Acanthaceae plant.

[0012] Preferably, the plant is Nicotiana tabacum L., Solanum lycopersicum L. or Strobilanthes cusia (Nees) Kuntze.

[0013] Preferably, the Indigofera endophytic fungus or bacterial agent is used to promote plant root growth.

[0014] The present invention also provides use of the above-mentioned indigo endophytic fungus or bacterial agent in promoting the increase of indigo content in indigo.

[0015] Preferably, the indigofera stolonifera is Strobilanthes cusia (Nees) Kuntze.

[0016] The present invention also provides a plant planting method, during which the above-mentioned Indigofera endophytic fungus or fungal agent is applied.

[0017] The present invention separates 70 endophytic fungi from Indigofera tinctoria, and screens out the strain MLR15 which has a good growth-promoting effect on crops such as Indigofera tinctoria and a certain broad-spectrum growth-promoting effect on crops such as tomatoes. In addition, MLR15 can also promote the increase of the indigo content in Indigofera tinctoria. Therefore, the MLR15 provided by the present invention can be developed into an active bacterial agent for promoting plant growth and the synthesis of Indigofera tinctoria medicinal ingredients, and has a good application prospect.

[0018] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0019] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of endophytic fungi of Indigofera punctata on the growth promotion of 12-day-old NC89 tobacco tissue culture seedlings;

[0021] Figure 2 The growth promotion of endophytic fungi of Indigofera serrata on 15-day-old NC89 tobacco tissue culture seedlings;

[0022] Figure 3 The effect of the endophytic fungus of Indigofera punctata on the growth promotion of 20-day-old NC89 tobacco tissue culture seedlings;

[0023] Figure 4 The growth promotion of endophytic fungi of Indigofera lanuginosa on 30-day-old NC89 tobacco tissue culture seedlings;

[0024] Figure 5 Effects of endophytic fungi of Indigofera salsa on the biomass of NC89 tobacco tissue culture seedlings;

[0025] Figure 6 Effects of soaking tobacco seeds with a mixture of endophytic fungi spores and hyphae on the growth of tobacco plants;

[0026] Figure 7Effect of seed soaking treatment with the spore and hypha mixture of endophytic fungi from Strobilanthes cusia on the whole-plant phenotype of tobacco plants;

[0027] Figure 8 Effect of seed soaking treatment with the spore and hypha mixture of endophytic fungi from Strobilanthes cusia on the maximum leaf area of tobacco plants;

[0028] Fig. 9 Effect of seed soaking treatment with the spore and hypha mixture of endophytic fungi from Strobilanthes cusia on the growth of tobacco plants grown in soil; where A: root biomass; B: aboveground biomass; C: maximum leaf area; D: plant height.

[0029] Fig.10 Detection of the colonization of endophytic fungi in the roots of tobacco tissue culture seedlings; Note: 10×40 Leica fluorescence microscope;

[0030] Fig.11 Growth of Strobilanthes cusia cuttings after treatment with growth-promoting strains; Note: "GPE" represents the growth-promoting effect ("+" represents different degrees of growth-promoting effects; "-" represents no growth-promoting effect);

[0031] Fig.12 Analysis of the growth indexes of Strobilanthes cusia cuttings after treatment with growth-promoting strains; where A: aboveground dry weight; B: aboveground fresh weight; C: root dry weight; D: root fresh weight; E: aboveground length; F: root length;

[0032] Fig.13 Root scanning image of Strobilanthes cusia cuttings after treatment with growth-promoting strains;

[0033] Fig.14 Detection of the colonization of growth-promoting bacteria in the roots of Strobilanthes cusia; Note: 10×40 Leica fluorescence microscope;

[0034] Fig.15 Growth of tomato plants after irrigation of the roots with the culture solution of growth-promoting bacteria;

[0035] Fig.16 Detection of the growth indexes of tomatoes after irrigation of the roots with the culture solution of growth-promoting bacteria; A: aboveground biomass; B: root biomass; C: plant height;

[0036] Fig.17 Effect of treatment with growth-promoting bacteria on the indigo content of Strobilanthes cusia potted cuttings;

[0037] Fig.18 Effect of treatment with growth-promoting bacteria on the expression of the ScCYP4501 gene related to indigo synthesis in Strobilanthes cusia.

[0038] In the above figures, CK1, CK2, and CK3 are the controls for each batch of experiments; One-way ANOVA was used, and * was marked for 0.01 < p < 0.05, ** for 0.001 < p < 0.01, *** for 0.0001 < p < 0.001, and **** for p < 0.0001. DETAILED DESCRIPTION

[0039] Unless otherwise specified, the reagents and materials used in the following examples and experimental examples are commercially available.

[0040] Example 1 Strain screening and identification

[0041] 1. Strain Isolation

[0042] Endophytic fungi were isolated from the roots, stems and leaves of Indigofera serrata. A total of 70 strains with different morphologies were isolated. According to the source number, the endophytic fungi from the roots were numbered MLR1-32, the endophytic fungi from the stems were numbered MLS1-16, and the endophytic fungi from the leaves were numbered MLL1-22. After ITS sequence determination, Blast homologous sequence alignment and phylogenetic evolution analysis, they can be classified into 16 species-level taxa under 7 genera.

[0043] After physical and chemical analysis, category identification, screening of growth-promoting effects and screening of effects on increasing indigo content, the endophytic fungus MLR15, which has the ability to promote the growth of Indigo and increase the indigo content, was obtained.

[0044] 2. Identification and preservation

[0045] After identification, MLR15 was found to be a Trichoderma sp. fungus and was deposited in the General Microbiology Center of China Culture Collection Administration on July 1, 2024; the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing; the deposit number is: CGMCC No.41250.

[0046] Its specific identification characteristics are as follows:

[0047] (1) Strain morphology and characteristics

[0048] Mycelium morphology: underdeveloped, sparse in the middle and dense at the edges;

[0049] Spore morphology: strip-shaped;

[0050] Texture and protrusion of colony: smooth surface, flower-shaped;

[0051] Color: white and light yellow;

[0052] Edge characteristics: smooth;

[0053] Aerial hyphae: None.

[0054] (3) rDNA-ITS sequence analysis

[0055] The sequence (SEQ ID NO.1) is as follows:

[0056] >MLR15

[0057] .

[0058] Homologous sequences were compared using the Blast function of the online analysis database NCBI (URL: https: / / www.ncbi.nlm.nih.gov / ) to find related homologous sequences. The results showed that the ITS sequence of MLR15 was 99.82% similar to that of Trichoderma sp. Combined with the morphological and physiological and biochemical characteristics, strain MLR15 was identified as a Trichoderma sp. fungus.

[0059] The beneficial effects of the present invention are further illustrated by experiments below.

[0060] Experimental Example 1 Study on the Growth-Promoting Effect of MLR15

[0061] 1. Experimental Methods

[0062] 1. Experimental Materials

[0063] 1.1 Plant materials

[0064] The indigofera plant is Yunnan indigofera, biologically classified as indigofera of the Acanthaceae family, collected in the indigofera industrial park in Lushan County, Ya'an City, Sichuan Province. The collection site is located in the western edge of the mountainous area of ​​the Sichuan Basin, northwest of Ya'an City, 102.52′-103.11′ east longitude, 30.01′-30.49′ north latitude, covering an area of ​​1,364.42 square kilometers. It belongs to the mid-latitude inland subtropical humid climate, with mild climate, abundant rainfall, distinct four seasons, long frost-free period, less sunshine, wet summer and dry winter. This variety likes warm and humid weather, less sunshine, and cutting seedlings are easy to survive.

[0065] 1.2 Test strains

[0066] 70 strains of endophytic fungi isolated from the roots, stems and leaves of Indigofera lanuginosus were stored in a -4°C refrigerator using the slant preservation method. All of them were preserved by our laboratory and needed to be activated on PDA plates for 5-7 days before use.

[0067] 1.3 Main Reagents

[0068] The drugs and reagents used in this study are listed in Table 1.

[0069] Table 1 Main experimental reagents and their uses

[0070]

[0071]

[0072] 1.4 Main culture matrix

[0073] (1) PDA medium: Take 200 g of peeled potatoes, add 900 mL of distilled water, boil for 30 min, filter through 8 layers of gauze, take the filtrate, add 20 g of anhydrous glucose and 18-20 g of microbial agar, make up to 1000 mL, set the pH to natural, sterilize with high pressure steam at 121°C for 20 min.

[0074] (2) MS medium: Murashige and Skoog 4.64 g, sucrose 30 g, plant agar 9 g, pH 6.2, distilled water 1000 mL, 121°C, high pressure steam sterilization, 20 min.

[0075] (3) 1 / 2MS medium: Murashige and Skoog 2.32 g, sucrose 30 g, plant agar 9 g, pH 6.2, distilled water 1000 mL, 121°C, high pressure steam sterilization, 20 min.

[0076] (4) Composition of potting soil: seedling soil: vermiculite: perlite = 4:2:1.

[0077] 2. Experimental operation

[0078] 2.1 Preliminary screening of endophytic fungi with growth-promoting effects on Indigofera ovata

[0079] Flue-cured tobacco NC89 was used as plant material to preliminarily screen the growth-promoting strains of endophytic fungi indigofera.

[0080] 2.1.1 Detection of the growth-promoting effect of endophytic fungi of Indigofera serrata on tobacco tissue culture seedlings

[0081] (1) Strain activation

[0082] 70 strains of endophytic fungi were picked up with the tip of a high-pressure steam-sterilized pipette tip and a small amount of mycelium preserved in the PDA slant, inoculated into the center of the PDA plate, sealed with a single-layer sealing film, and then inverted in a constant temperature incubator at 28°C for activation culture for 5-7 days.

[0083] (2) Pretreatment of tobacco seeds

[0084] ① Tobacco seed screening: Select an appropriate amount of flue-cured tobacco NC89 seeds that were harvested in the current year, are similar in size, and have no obvious diseases or insect pests, and place them in a 2 mL sterile EP tube for later use.

[0085] ② Tobacco seed disinfection: Use 70% ethanol to disinfect the surface of tobacco seeds. The disinfection steps are as follows: take an appropriate amount of NC89 seeds, put them into a sterile 2mL EP tube, add 70% ethanol, place them in your hand and shake them continuously for 2.5-3min, pour out the ethanol, rinse them with sterile water 4-5 times, add an appropriate amount of 0.01% plant agar (121℃, high pressure steam sterilization, 20min) to the disinfected seeds, suspend the tobacco seeds, and then place them in a clean bench for use.

[0086] ③ Tobacco seed germination: Pour the prepared sterilized MS culture medium into a plate (10×10cm disposable plastic square culture dish), 25mL per plate, wait for the plate to cool and solidify, suck the sterilized seeds with a 10μL pipette tip (cut off the tip), and evenly dot them on the MS plate, stagger the seeds in two adjacent rows, dot 50 seeds per plate, dot 5 rows, 10 seeds per row, seal with double-layer sealing film, and place vertically in a light incubator at 25℃, 16h light, 10000Lx, 22℃, 8h dark, for NC89 tobacco seed germination culture. When NC89 tobacco grows four true leaves (about 12 days), it can be used to establish the co-culture system of endophytic fungi-NC89 tissue culture seedlings.

[0087] (3) Establishment of co-culture system

[0088] The co-cultivation system uses a 10×10 cm disposable plastic square culture dish. The culture medium uses 1 / 2MS solid culture medium. After sterilization, pour the medium into the plate, 25 mL per plate. Before transplanting the tobacco seedlings, use sterilized tweezers to scrape off 1 / 3 of the culture medium on the upper part of the 1 / 2MS plate to leave space for the growth of NC89 tobacco leaves. Then, use sterile tweezers to gently transfer the tobacco seedlings with three true leaves, the same growth and the same size to the 1 / 2MS plate. Use tweezers to gently stretch the roots of the tobacco seedlings on the culture medium, and place the leaf part in the empty part of the previous 1 / 2MS plate. Put 3 NC89 tobacco seedlings in each 1 / 2MS plate. After transplanting the NC89 tobacco seedlings, use the tip of a sterilized pipette to pick up a small amount of endophytic fungal hyphae and inoculate it 1 cm below the root knot of the tobacco seedlings. The blank control tobacco seedlings are not inoculated with endophytic fungi. The plates are sealed with a double-layer sealing film and placed in a light incubator at a 45° angle at 25°C, 16 h of light, 10,000 Lx, 22°C, 8 h of darkness, and cultured for 20 days. By observing the growth of NC89 tobacco seedlings, the treatment groups with significantly better growth than the control were selected, and the relevant growth indicators were recorded and statistically analyzed.

[0089] (4) Growth index determination

[0090] Different seedling raising cycles (12d, 15d, 20d, 30d) were set. After 20d of co-cultivation, the maximum leaf area, aboveground and underground plant height, dry weight and fresh weight were measured to evaluate the growth-promoting effect of endophytic fungi of Indigofera umbellata on NC89 tobacco seedlings of different ages.

[0091] 2.1.2 Detection of the growth-promoting effect of endophytic fungi of Indigofera serrata on tobacco seedlings grown in soil

[0092] (1) Strain activation

[0093] The initially screened growth-promoting endophytic fungi were picked up with the tip of a gun sterilized with high-temperature and high-pressure steam, and a small amount of mycelium preserved in the PDA slant was inoculated into the center of the PDA plate. After sealing with a single layer of sealing film, the plate was placed in a constant temperature incubator at 28°C for activation culture for 5-7 days.

[0094] (2) Pretreatment of tobacco seeds

[0095] ① Tobacco seed screening: Select an appropriate amount of flue-cured tobacco NC89 seeds that were harvested that year, are similar in size and shape, and have no obvious diseases and insect pests, and put them into 2mL EP tubes for later use.

[0096] ② Preparation of treatment solution: After culturing the growth-promoting bacteria on the PDA plate for 10 days, scrape the hyphae and spores with a sterile blade, add 1 mL of sterile water to one plate and vortex to mix, to obtain a spore-hyphae mixed solution.

[0097] (3) Establishment of co-culture system

[0098] The flue-cured tobacco NC89 seeds were placed in a spore-mycelium mixture and soaked overnight (10 h) at 4°C. Sterile water was used as a control and the seeds were sown in seedling pots (9×9 cm in size). After 2 weeks, the NC89 tobacco seedlings with 4 true leaves were transplanted into nutrient soil, one seed per small pot, 12 pots per treatment, and placed in an artificial climate chamber at 25°C, 10000Lx light for 16h, 22°C, 8h dark, and 70% humidity. After 60-70 days of cultivation, the growth of tobacco was observed, and the treatment group with significantly better growth than the control was selected, photographed and recorded, and relevant growth indicators were statistically analyzed.

[0099] (4) Obtaining strains with good and stable growth-promoting effects

[0100] Based on the plant height, aboveground length, underground length, maximum leaf area, aboveground biomass and underground biomass of NC89 tobacco soil-cultured seedlings, the Indigofera endophytic fungus strains that have a growth-promoting effect on NC89 tobacco soil-cultured seedlings were preliminarily screened out.

[0101] 2.1.3 Endophytic fungal colonization detection

[0102] Trypan blue staining was used to detect the colonization of endophytic fungi in growth-promoting seedlings. Fresh growth-promoting seedlings treated with growth-promoting bacteria were selected, their roots were cut, and they were cut into 1 cm long segments, at least 20 segments, and placed in a 50 mL centrifuge tube. 100 g / L KOH was added to completely soak the root sample, and the KOH solution was poured out after 90 ° C water bath for 90 min. The root was rinsed with tap water 3 times and the water was controlled. 30% H2O2 was added for bleaching for 5 min, and the root system was softened at the same time. After decolorization, it was rinsed with tap water and the water was controlled. Lactic acid was added for acidification at room temperature for 5 min, and the lactic acid was poured out. Trypan blue staining solution (0.05 g trypan blue + 20 mL glycerol + 60 mL lactic acid + 20 mL distilled water) was added, and the root was stained for 3-5 min. After that, it was decolorized with a mixture of acetic acid: glycerol (1:1). After overnight, the colonization of endophytic fungi in the roots of the growth-promoting seedlings was observed under a microscope.

[0103] 2.2 Preliminary screening of growth-promoting endophytic fungi to study the growth-promoting effect of Adenophora indigofera

[0104] 2.2.1 Strain activation

[0105] Use the tip of a sterile pipette to pick up a small amount of initially screened growth-promoting endophytic fungal hyphae from the PDA slant storage tube, inoculate it into the center of the PDA plate, seal it with a single layer of sealing film, and place it in a constant temperature incubator at 28°C for activation culture for 5-7 days for later use.

[0106] 2.2.2 Preparation of strain treatment solution

[0107] When the mycelium of the initially screened growth-promoting endophytic fungi grows to 2 / 3 of the plate, use the thick end of a sterile gun tip as a puncher to punch out 5 fungal agar blocks of the same size, inoculate them into PDA liquid culture medium, and culture them at 28°C and 200 rpm / min for 5 days. Break up the mycelium balls to maintain fungal vitality, and prepare fungal culture fluid for the establishment of a co-culture system of growth-promoting fungi and indigofera cuttings.

[0108] 2.2.3 Cultivation of cutting seedlings of Indigofera tinctoria

[0109] Select 3-year-old fresh indigofera stalks of similar size, remove branches and leaves, retain main branches, cut into short strips with a length of 20 cm and 2 stem nodes in the middle, sharpen one end at 45°, insert the tip into the soil, plant in a flowerpot with a diameter of 50 cm and a height of 45 cm, and the soil height in the pot is 30 cm. After watering appropriately, place in an artificial climate chamber for cultivation, the relative humidity (RH) of the environment is 70%, the CO2 concentration in the air is about 400PPM, the indoor temperature is 25°C, the light is 16h, 10000Lx, 22°C, and the darkness is 8h, and seedling cultivation is carried out; after 30d-40d, the growth of the cutting seedlings is observed, and the cutting seedlings with similar growth and rooting are selected for the establishment of a growth-promoting fungus-indigofera stalk cutting seedling co-cultivation system.

[0110] 2.2.4 Root irrigation of indigofera potted cuttings with fungal culture solution

[0111] Take 4mL of fungal culture solution and dilute it to 50mL with double distilled water, mix with soil (1 pot), use 10×10cm disposable plastic pots, select cuttings of similar size and growth status, and transplant them into the pots treated with soil. The control is replaced with sterile water plus sterile PDA liquid medium. Three replicates are set for each treatment, and two groups of experiments are carried out. After watering appropriately, they are placed in an artificial climate chamber at 25℃, 16h light, 10000Lx, 22℃, 8h dark, and co-cultivated. One group is co-cultivated for 1 month, and the other group is co-cultivated for 4 months. The growth of the indigo cuttings is observed, and the treatment group with significantly better growth than the control is selected, photographed and recorded, and relevant indicators are statistically analyzed.

[0112] 2.2.5 Determination of growth indexes of indigofera potted cutting seedlings

[0113] The growth indicators of potted cuttings of Indigofera ovata were tested, including the maximum leaf area, root number, root length, plant height, and dry and fresh weight of aboveground and underground parts. Four biological replicates and four technical replicates were set for each treatment, and three plants with similar growth were selected as the objects of statistical analysis.

[0114] 2.2.6 Root system scanning analysis of indigofera potted cuttings

[0115] The root system of the indigofera tinctoria seedlings was automatically scanned and analyzed using a root scanner (Top Yunnong, model GXY-A). Indicators such as total surface area, total volume, total root length, number of root tips, average root diameter and number of connections were analyzed to make a detailed evaluation of the growth-promoting effect of the growth-promoting bacteria on the roots of indigofera tinctoria.

[0116] 2.2.7 Field Cultivation of Indigofera tinctoria Cuttings

[0117] The field experiment adopts the random block design in the random arrangement design, and the fertility is not gradient. The field is 32m long and 3.5m wide, and it is divided into 10 blocks of the same size. There is a block on each side of the field as the planting area of ​​the protection row, and the middle is divided into 8 blocks. The numbers 1, 2, 3, and 4 represent four different treatments of CK, MLR15, MLR7, and MLL2, respectively. The lottery method determines the sequence number of different treatments. The number of blocks represents the number of repetitions, and 3 blocks represent 3 repetitions. Each plot is planted with 15 plants, the cutting depth is 5-6cm, the cutting density is 40cm in row spacing, 40cm in plant spacing, and half-hole planting, with a hole spacing of 30-35cm. Build a 75% density shade net 1.5-2m high, one layer. After 40 days of raising the Indigofera tinctoria cuttings, select plants with similar growth and transplant them into the field. Apply growth-promoting bacteria culture solution once in the first month after transplanting and once in the third month. The seedlings are harvested in the fourth month and cultured for a total of four months.

[0118] 2.2.8 Determination of growth indexes of field-grown cuttings of Indigofera ovata

[0119] The growth indices of field cuttings of Indigofera tinctoria were tested, including maximum leaf area, root number, root length, plant height, and fresh and dry weight of aboveground and underground parts. 27 plants with similar growth trends were selected as the objects of statistical analysis.

[0120] 2.3 Study on the broad-spectrum growth-promoting effects of endophytic fungi of Indigofera serrata

[0121] 2.3.1 Plant materials

[0122] The tomato variety was Jinpeng No.1. The tomato was treated and its growth indexes were measured by two methods: seed soaking with a mixture of spores and mycelium and root irrigation with a growth-promoting bacteria culture solution.

[0123] 2.3.2 Seed soaking treatment with spore-mycelium mixture and growth index determination

[0124] Tomato seeds were soaked in a mixture of growth-promoting bacteria spores and mycelium (prepared in the same way as tobacco) at 4°C for 10 h and sown evenly in potting soil in small pots of 10×10 cm, with 4 seeds in each pot distributed in the four corners. Six replicates were set for each treatment, and 12 seeds from three pots with similar growth were selected as the objects of statistical analysis to detect growth-related indicators, including biomass, plant height and root length.

[0125] 2.3.3 Root irrigation with growth-promoting bacterial culture solution and growth index determination

[0126] Tomato seeds were sown in small pots of 10×10 cm, one seed per pot. After 30 days of cultivation, the growth-promoting fungal culture solution (refer to the preparation method of Strobilanthes cusia above) was mixed with water at a ratio of 2:23, and the roots were watered until the tomato seedling soil was soaked, 50 mL per small pot, with sterile PDA medium as the control. Four biological replicates were set for each treatment, and the experiment was repeated 3 times. Three plants with similar growth status were selected as the objects for statistical analysis, and their growth-related indicators were detected (the same as 2.3.2).

[0127] 2.4 Data processing and statistical analysis

[0128] One-way analysis of variance of Graphpad Prism 8.0 software was used for data significance analysis. At the 95% confidence level, the critical difference (CD) was used to compare the means between each treatment. p>0.05 indicates no significant difference, and p<0.05 indicates a significant difference. 0.01<p<0.05 is marked with *, indicating a significant difference between the two groups; 0.001<p<0.01 is marked with **, representing a relatively significant difference between the two comparison groups; 0.0001<p<0.001 is marked with ***, representing a significant difference between the two comparison groups; p<0.0001 is marked with ****, representing a very significant difference between the two comparison groups.

[0129] II. Experimental results

[0130] 1. Screening of growth-promoting strains of endophytic fungi from Strobilanthes cusia

[0131] 1.1 Analysis of the growth-promoting effect of endophytic fungi from Strobilanthes cusia on tobacco tissue culture seedlings

[0132] Given the difficulties in seed breeding and tissue culture seedling preparation of Strobilanthes cusia, and the long growth cycle of cuttings, therefore, tobacco NC89 tissue culture seedlings were first selected as the detection objects, and the plant growth-promoting activities of 70 pre-isolated endophytic fungi from Strobilanthes cusia were evaluated to screen and identify growth-promoting strains. The results showed that some strains of endophytic fungi from Strobilanthes cusia had obvious growth-promoting effects on tobacco seedlings of different ages( Figure 1-4 ), but their growth-promoting effects were related to the age of tobacco seedlings. The results are as follows:

[0133] Observing the growth of 12-day-old tobacco seedlings co-cultured with endophytic fungi from Strobilanthes cusia, it was found that compared with the control, the growth status of MLL20, MLL2, MLR15, MLR20, MLL14, MLR24, MLL23, MLR7, MLR29 and MLL17 was good( Figure 1), and all other strains caused the death of tobacco seedlings. Compared with the control, MLL20 and MLL2 significantly promoted the growth of tobacco leaves; while MLR20, MLR24, MLL23, MLR7, MLR29 and MLL17 promoted the growth of tobacco leaves, but caused the leaves to turn yellow and wilt; the growth of tobacco plants treated with MLR15 and MLL14 was similar to that of the control, with no significant effect ( Figure 1 ).

[0134] The growth of 15-day-old tobacco seedlings co-cultured with endophytic fungi of Indigofera serrata was observed, and it was found that compared with the control, 10 endophytic fungi, MLL20, MLS9, MLL14, MLL3, MLS8, MLR13, MLR24, MLR13, MLL2 and MLR7, significantly promoted the growth of tobacco leaves ( Figure 2 ), and all other strains caused the death of tobacco seedlings. Among them, MLL20, MLS9, MLL14, MLL3, MLS8 and MLR13 not only promoted the growth of tobacco leaves, but also their growth was significantly better than the control; while MLR24, MLR13, MLL2 and MLR7 significantly promoted the growth of tobacco leaves, but caused yellowing of tobacco leaves and inhibited the growth of some seedlings, resulting in different growth-promoting effects ( Figure 2 ).

[0135] The growth of 20-day-old tobacco seedlings co-cultured with the endophytic fungus of Indigofera serrata was observed, and it was found that only the tobacco seedlings treated with MLR24, MLL2, MLR13, MLL20, MLR32 and MLR7 grew well ( Figure 3 ), and all other strains caused the death of tobacco seedlings. Compared with the control, MLL2 not only significantly promoted the growth of tobacco seedlings, but also had a significantly better growth than the control; although MLR24 significantly promoted the growth of tobacco seedlings, it caused yellowing of leaves; the growth of MLR13, MLL20 and MLR32 was similar to that of the control, but MLR13 and MLR32 caused yellowing of tobacco seedlings; MLR7 had different promoting effects, and some seedlings died ( Figure 3 ).

[0136] The growth of 30-day-old tobacco seedlings co-cultured with the endophytic fungus Indigofera serrata was observed, and it was found that only tobacco seedlings treated with MLR15, MLL2, MLR7, MLL20 and MLL5 survived, and compared with the control, they significantly promoted the growth of tobacco seedlings. Among them, MLR15, MLL2, MLR7 and MLL20 significantly promoted the growth of tobacco tissue culture seedlings, especially MLR15, and their growth status was significantly better than the control; MLL20 and MLL5 had a lower promoting effect than MLR15, but their leaf size and growth status were better than the control ( Figure 4 ).

[0137] Six strains of endophytic fungi with obvious growth-promoting effects on tobacco at different seedling ages were selected for preliminary qualitative evaluation of their growth-promoting effects. The results are shown in Table 2. MLL2 has the strongest growth-promoting effect, with growth-promoting effects in four periods; MLR24 has the second best growth-promoting effect, with growth-promoting effects in three periods, and no growth-promoting effect at 30 days of seedling age; MLR15 has no obvious growth-promoting effect at 12 days, but its growth-promoting effect is extremely obvious when the seedling age is 30 days; MLR7 has a more obvious growth-promoting effect at 30 days of seedling age, and also has a lower growth-promoting effect at 12 days and 15 days; MLL14 and MLS9 both have obvious growth-promoting effects at 15 days of seedling age, and no growth-promoting effects at other periods. These results also indicate that the period of strain application is related to its growth-promoting effect. Therefore, subsequent studies will use the above six endophytic fungal strains with strong growth-promoting effects as materials to further detect and evaluate their growth-promoting effects.

[0138] Table 2 Comparative analysis of the growth promotion of endophytic fungi on NC89 tobacco tissue culture seedlings of different seedling ages

[0139]

[0140] Note: “+” represents the degree of growth promotion, “-” represents no growth promotion effect

[0141] The growth of tobacco seedlings co-cultured with six growth-promoting bacteria, MLR24, MLL2, MLR7, MLR15, MLS9 and MLL14, was quantitatively analyzed, and the fresh weight and dry weight of the treated plants were measured. The results showed that compared with the control, the above-mentioned growth-promoting bacteria treatments significantly increased the fresh weight of tobacco seedlings; the fresh weight of seedlings treated with MLR15, MLR24, MLR7, MLL2, MLS9 and MLL14 was 7.8, 3.7, 3, 4, 2.6 and 2 times that of the control, respectively; the dry weight of seedlings treated with MLR15, MLR24, MLR7, MLL2 and MLL14 was significantly or extremely significantly increased, and the dry weight was 9.7, 2, 1.5, 2 and 1.6 times that of the control, respectively. Among them, the growth-promoting effect of MLR15 was the most obvious ( Figure 5 ).

[0142] 1.2 Analysis of the growth-promoting effect of endophytic fungi of Indigofera serrata on tobacco seedlings grown in soil

[0143] Soil-cultured tobacco plants were used as materials to further verify the growth-promoting effects of the six growth-promoting bacteria obtained in the preliminary screening. The plants were treated by soaking the mixture of spores and mycelium. The results showed that compared with the control, the tobacco plants treated with MLR15, MLR24, MLL14 and MLS9 were significantly larger, and their leaf areas were also significantly larger than the control, while MLR7 showed yellowing, which confirmed that the above strains also had a growth-promoting effect on tobacco soil-cultured seedlings ( Figure 6). Three dominant tobacco plants were selected from each treatment to further observe the growth phenotype of the whole plant. The results showed that except for MLR7, the other strains increased the height of tobacco plants to varying degrees and promoted the development of tobacco leaves. The increasing effect of MLR15, MLR24 and MLS9 was more obvious ( Figure 7 ). The largest leaves of three plants in each treatment were further selected to observe their leaf areas. The results showed that compared with the control, the maximum leaf areas of tobacco plants treated with MLR15, MLS9, MLR24, and MLR7 were all increased, among which the leaves of plants treated with MLR15 and MLS9 increased more significantly ( Figure 8 ).

[0144] Further quantitative testing of the above plant growth indicators showed that all six endophytic fungi significantly increased the root fresh weight of tobacco seedlings, among which MLR15 increased the most significantly; MLR15 and MLR24 significantly increased the root dry weight of seedlings, while MLS9, MLL14 and MLR7 increased compared with the control, but there was no significant difference ( Fig. 9 A).

[0145] Meanwhile, MLS9, MLL14, MLR24, MLR15 and MLL2 treatments significantly increased the aboveground fresh weight of tobacco seedlings, of which MLS9, MLL14, MLR24 and MLR15 had extremely significant effects, MLL2 had a significant effect, and MLR7 reduced the aboveground fresh weight of tobacco seedlings, but the difference was not significant. MLL14, MLR24 and MLR15 also increased the aboveground dry weight of tobacco seedlings, but the increase of MLL14 and MLR15 was greater than that of MLR24, while MLR7 reduced the aboveground dry weight of tobacco seedlings ( Fig. 9 B). In addition, MLR15 significantly promoted the maximum leaf area of ​​tobacco seedlings, while other bacteria had no significant effect on this indicator ( Fig. 9 C). MLR15 also significantly increased plant height, MLS9 significantly increased plant root length, and the other strains had no significant effect on plant height and root length ( Fig. 9 D).

[0146] The above results show that the six growth-promoting bacteria screened in this paper also have obvious growth-promoting effects on tobacco soil-cultured seedlings when applied in the form of seed soaking with a mixture of spores and mycelium, among which MLR15 has a stronger growth-promoting effect.

[0147] 1.3 Colonization detection of growth-promoting strains of endophytic fungi of Indigofera serra

[0148] In order to clarify the correlation between tobacco growth promotion and endophytic fungi treatment, trypan blue staining was used to detect fungal colonization in the tissue culture seedlings co-cultured with growth-promoting bacteria. The results showed that after the tobacco tissue culture seedlings were co-cultured with growth-promoting bacteria, a large number of dark blue growth-promoting fungal hyphae were distributed in the roots, and the hyphae morphology was clearly visible, indicating that the above-mentioned growth-promoting bacteria played a growth-promoting role by colonizing in the roots ( Fig.10 A, B).

[0149] 2. Study on the growth-promoting effect of endophytic fungi of Indigofera tinctoria on Indigofera tinctoria

[0150] 2.1 Evaluation of the growth-promoting effect of growth-promoting strains on potted cuttings of Indigofera tinctoria

[0151] After 40 days of cultivation, the roots of the indigofera cuttings were irrigated with a growth-promoting fungal culture solution. After 1 month and 4 months of cultivation, the relevant indicators were measured. The results showed that after 1 month of cultivation, compared with the control, except for a slight increase in plant height, the other changes were not obvious in the indigofera cuttings treated with MLR15, MLR7 and MLR24. After 4 months of cultivation, compared with the control, the growth of the indigofera cuttings treated with MLR15, MLR7 and MLL2 was significantly promoted, especially the length and volume of the root system, while the growth-promoting effect of other strains was not obvious ( Fig.11 ).

[0152] The growth phenotype data of the above-mentioned cuttings were further tested. The results showed that after one month of co-cultivation, compared with the control, MLR15 and MLR7 significantly increased the aboveground dry weight of the indigofera cuttings, while MLR24, MLS9 and MLL2 increased it, but there was no significant difference, and MLL14 had no obvious change; after four months of co-cultivation, MLR15, MLL2 and MLR7 significantly increased the aboveground dry weight of the indigofera cuttings, but other strains reduced this index ( Fig.12 A). After 1 month of co-cultivation, compared with the control, MLR15, MLR7 and MLL2 significantly increased the aboveground fresh weight of the indigofera cuttings. Although MLR24 increased it, there was no significant difference. MLS9 was not significantly different from the control. MLL14 significantly reduced the aboveground fresh weight of the indigofera cuttings. After 4 months of co-cultivation, MLR15 and MLL2 significantly increased the aboveground fresh weight of the indigofera cuttings. Although MLR7 increased it, there was no significant difference. The other strains all reduced this index, and MLR24 was particularly obvious ( Fig.12 B). After one month of co-cultivation, except for MLR24, which significantly increased the root dry weight of the indigofera cuttings, the other strains all reduced this index, but there was no significant difference; after four months of co-cultivation, MLR15, MLL2 and MLR7 significantly increased the root dry weight of the indigofera cuttings, and the other strains had no significant effect ( Fig.12C). After one month of co-cultivation, except for MLS9 and MLL14, which significantly reduced the root fresh weight of the indigofera cuttings, the other strains had no significant effect; after four months of co-cultivation, MLR15, MLL2 and MLR7 significantly increased the root fresh weight of the indigofera cuttings, and the other strains had no significant effect ( Fig.12 D). There was no significant difference in the aboveground length of the plants between the two different co-cultivation times ( Fig.12 E). After one month of co-cultivation, although MLR15 and MLR7 promoted the root growth of the indigofera cuttings, there was no significant difference, and other strains had no significant effect; after four months of co-cultivation, MLR15 and MLR7 significantly promoted the root growth of the indigofera cuttings, and other strains had no significant effect ( Fig.12 F). These results are consistent with their phenotypic data. The above results show that among the growth-promoting strains obtained in this paper, MLR15, MLL2 and MLR7 have good growth-promoting effects on the cuttings of Indigofera tinctoria, especially the effect of increasing the root biomass is very significant.

[0153] To further explore the specific effects of the above growth-promoting strains on the root growth of Indigofera tinctoria, this study conducted root scanning analysis on Indigofera tinctoria cuttings treated with growth-promoting strains for 4 months. The results showed that the root density of Indigofera tinctoria cuttings treated with MLL2, MLR15, MLL14 and MLR7 was significantly greater than that of the control, among which the root systems of MLL2 and MLR15 were denser ( Fig.13 ).

[0154] 2.2 Analysis of colonization of growth-promoting bacteria

[0155] The trypan blue staining method was used to perform morphological detection of the colonization of growth-promoting bacteria in the indigofera cuttings treated with the growth-promoting strains. Fig.14 As shown, compared with the control, a large number of fungal spores were distributed in the roots of the indigofera tinctoria seedlings treated with the growth-promoting bacteria culture solution, and their number was significantly greater than that of the control, indicating that the endophytic fungi had successfully colonized the roots of indigofera tinctoria.

[0156] 3. Test on the broad-spectrum effect of growth-promoting strains of endophytic fungi of Indigofera odorata

[0157] In order to explore whether the above 6 growth-promoting bacteria have a broad-spectrum growth-promoting effect, this paper selected Jinpeng No. 1 tomato as the material. After treating it with fungal culture solution and root irrigation, the plant growth and fungal colonization were tested to evaluate the growth-promoting effect.

[0158] The roots of tomato plants were irrigated with fungal culture solution. After 30 days of cultivation, the growth and phenotypic indicators of tomato plants were tested. The results showed that the root growth of tomato plants treated with MLR15 and MLR7 was significantly better than that of the control ( Fig.15 ); plant growth indicators were measured and it was found that these growth-promoting bacteria had no significant effect on the aboveground biomass of tomato plants compared with the control ( Fig.16A); however, MLR15 and MLR7 significantly increased the fresh weight of tomato plants' roots, and also increased the dry weight of roots, but without significant differences, while MLL14 had no significant effect on tomato growth ( Fig.16 B); MLR15 and MLR7 also significantly increased tomato plant height and aboveground length, but MLR15 had no significant difference in promoting aboveground length. At the same time, these growth-promoting bacteria had no significant effect on tomato root length ( Fig.16 C) The above results indicate that MLR7 and MLR15, when applied in the form of root irrigation with fungal culture solution, can promote tomato growth by increasing tomato biomass, and have a certain broad-spectrum growth-promoting effect.

[0159] The above experimental results prove that the MLR15 screened by the present invention has a good growth-promoting effect on Indigofera tinctoria and has a growth-promoting spectrum for crops such as tomatoes.

[0160] Experimental Example 2 Study on the Metabolic Activity of MLR15

[0161] 1. Experimental Methods

[0162] 1. Plant materials

[0163] The growth-promoting strains screened and identified above were used as materials, and the indigofera cuttings treated with their fermented liquid for 1 month and 4 months were used as plant materials. Three seedlings were selected for each treatment, with each seedling as a group, for a total of 3 biological replicates, and all leaves of each seedling were ground into powder and mixed, and samples were taken separately for later use.

[0164] 2. Sample preparation

[0165] The above plant materials were sampled separately, crushed with a grinder, and passed through a sieve with an inner diameter of 0.425 mm (100 mesh). About 75 mg of the sample dried to constant weight was taken from each repetition of each treatment, accurately weighed, and placed in a 10 mL volumetric flask; an appropriate amount of N, N-dimethylformamide was added, and ultrasonic treatment was continued for 3 hours. After cooling, N, N-dimethylformamide was added to the 10 mL mark, shaken well, and filtered with a 0.22 μm organic filter membrane. The filtrate was taken to obtain the sample solution.

[0166] 3. Preparation of standard curve

[0167] Weigh 2.56mg and 2.48mg of indigo and indirubin standards that have been dried to constant weight, respectively, and place them in a 50mL volumetric flask. Add N,N-dimethylformamide to dissolve and dilute to 50mL to prepare standard stock solutions containing 0.0513mg and 0.0495mg of indigo and indirubin per mL. Then, accurately pipette 2mL of each standard stock solution, place it in a 10mL volumetric flask, add N,N-dimethylformamide to the scale, shake well, and obtain each standard solution. Accurately measure 0.5, 1.0, 2.0, 4.0, 6.0, and 8.0mL of the reference stock solution, respectively, place it in a 10mL volumetric flask, add N,N-dimethylformamide to dilute to the scale, shake well, and obtain standard solutions of different concentrations for the determination of the standard curve. Determine according to the following chromatographic conditions, with the indigo and indirubin content (mg / g) as the horizontal axis and the chromatographic peak area (A) as the vertical axis, and draw the standard curve.

[0168] 4. Determination of indigo and indirubin content

[0169] The content of indigo and indirubin in the above-treated indigo plants was determined by HPLC (high performance liquid chromatography), using an Agilent TC-C18 chromatographic column (4.6 mm × 250 mm, 5 μm), a mobile phase of methanol-water (75:25), a column temperature of 25°C, a flow rate of 1.0 mL / min, a detection wavelength of 290 nm, and an injection volume of 20 μL. Standards of indigo (CAS No.: 482-89-3, B20297) and indirubin (CAS No.: 479-41-4, B20298) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0170] 5. RNA extraction from indigofera leaves

[0171] Total RNA was extracted from the leaves of Indigofera indigofera using the OMEGA total RNA extraction kit (EZNATotal RNA Kit I, R6834-01) according to the instructions. III 1st Strand cDNA Synthesis Kit, 11139ES10), reverse transcribed into cDNA, and stored at -80°C.

[0172] 6. Detection of gene expression related to indigo and indirubin synthesis

[0173] According to the genes and primers in Table 3, qRT-PCR amplification was performed with cDNA as template. The PCR amplification reaction system was 10 μL: 2×Taq PCR Master Mix 5 μL, 0.2 μL of upstream and downstream primers, 1 μL of template, and 3.6 μL of ddH2O. The PCR reaction conditions were: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 90 s, 35 cycles, 72°C extension for another 10 min, and 4°C insulation.

[0174] Table 3 Gene information related to growth and metabolism of Indigofera ovata and qRT-PCR primer sequences

[0175]

[0176]

[0177] 7. Data processing and statistical analysis

[0178] Same as Experimental Example 1.

[0179] 2. Experimental Results

[0180] 1. Effect of growth-promoting bacteria treatment on the indigo content of Indigofera serrata

[0181] The contents of indigo and indirubin in potted cuttings of Indigofera tinctoria treated with growth-promoting bacteria were determined by HPLC. The results showed that after one month of treatment with growth-promoting bacteria, MLR15, MLL2 and MLR24 significantly or extremely significantly increased the content of indigo, while other strains had no significant effect ( Fig.17 ).

[0182] 2. Effects of growth-promoting bacteria treatment on the expression of key genes for indigo synthesis

[0183] The indigofera potted cuttings of 1 month and 4 months old treated in the previous period were used as materials to detect the expression of the key genes for indigo synthesis, ScCYP4501, ScIGP, ScASA1 and ScASB. The results showed that when the growth-promoting bacteria and indigofera were co-cultured for 1 month, MLR15, MLL2 and MLR24 increased the content of indigo, while the gene expression detection results only found that MLL2 upregulated the expression of the ScCYP4501 gene when co-cultured for 1 month ( Fig.18 ), it is speculated that MLL2 promotes indigo synthesis mainly by promoting the metabolic reaction of indole oxidation to indoxyl, while the growth-promoting mechanism of other strains is still unclear.

[0184] The present invention successfully isolated an endophyte of Indigofera tinctoria L. MLR15. Experiments show that MLR15 can effectively promote the growth of Indigofera tinctoria L., and has a certain broad-spectrum growth-promoting effect on crops such as tomatoes. In addition, MLR15 can also promote the increase in the content of indigo, a medicinal active ingredient in Indigofera tinctoria L. Therefore, the MLR15 provided by the present invention can be developed into an active bacterial agent for promoting plant growth and the synthesis of Indigofera tinctoria medicinal ingredients, and has a good application prospect.

Claims

1. An endophytic fungus of Indigofera scabra, characterized in that: The endophytic fungus of Indigofera cyanobacteria is a fungus of the genus Trichoderma (Trichoderma sp.), and the endophytic fungus of Indigofera cyanobacteria is a strain with a preservation number of CGMCC No.41250 preserved by the General Microbiological Center of the China Microbiological Culture Collection Administration.

2. A bacterial agent, characterized in that: It comprises the Indigofera endophytic fungus described in claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is prepared from a spore and hyphae mixed solution or a fungal culture solution of the indigofera endophytic fungus.

4. Use of the endophytic fungus of Indigofera tinctoria according to claim 1, or the fungal agent according to claim 2 or 3 in promoting plant growth.

5. The use according to claim 4, characterized in that: The plant is a Solanaceae plant or a Acanthaceae plant.

6. The use according to claim 5, characterized in that: The plant is tobacco Nicotiana tabacum L., tomato Solanum lycopersicum L. or blueberry Strobilanthes cusia (Nees) Kuntze.

7. The use according to claim 4, characterized in that: The indigofera endophytic fungus or bacterial agent is used for promoting the growth of plant roots.

8. Use of the endophytic fungus of Indigofera tinctoria according to claim 1, or the fungal agent according to claim 2 or 3, in promoting the increase of indigo content in Indigofera tinctoria.

9. The use according to claim 8, characterized in that: The indigofera striata is Strobilanthes cusia (Nees) Kuntze.

10. A method for planting plants, characterized in that: During the plant planting process, the indigo endophytic fungus described in claim 1 or the fungal agent described in claim 2 or 3 is applied.

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