Scrophularia ningpoensis endophytic fungus and application thereof in promoting growth of scrophularia

Abstract

The invention discloses a radix scrophulariae endophytic fungus and application thereof in promoting growth of radix scrophulariae, and belongs to the technical field of plant growth promotion. The endophytic fungus is separated from a Scrophularia ningpoensis living body of a scrophulariaceae scrophulariaceae plant by adopting an endophytic fungus separation and purification technology, belongs to fungi of ascomycophyta, Alternaria, Alternaria and Bordetella, is preserved in the China General Microbiological Culture Collection Center on January 23, 2024, is classified and named as Boeremia exigua SNF128, and has the characteristics that the endophytic fungus is named as Scrophularia ningpoensis SNF128; the preservation number of the strain is CGMCC (China General Microbiological Culture Collection The invention also discloses application of the endophytic fungi: the growth of radix scrophulariae tissue culture seedlings is promoted, the dry weight is increased by 62.07%, and the plant height, the root length, the leaf length and the leaf width are respectively increased by 73.29%, 22.41%, 43.37% and 24.02%.

Description

Technical Field

[0001] The present invention relates to the technical field of plant growth promotion, and specifically, to an endophytic fungus of Scrophularia ningpoensis and its application in promoting the growth of Scrophularia ningpoensis. Background Art

[0002] Scrophularia ningpoensis is the dried root of Scrophularia ningpoensis Hemsl. of the genus Scrophularia in the family Scrophulariaceae. It is one of the famous authentic Chinese medicinal materials in Zhejiang, and is one of the varieties included in the Chinese Pharmacopoeia (2025 Edition). It has the effects of cooling blood and nourishing yin, purging fire and detoxifying, etc., and is used for symptoms such as heat entering nutrient blood, crimson tongue with restlessness and thirst, yin injury caused by febrile diseases, consumptive fever with hectic fever, maculae due to warm-toxicity, constipation due to fluid injury, red eyes, diphtheria, scrofula, sore throat, carbuncles and sores. Scrophularia ningpoensis is mainly produced in provinces such as Zhejiang, Anhui, Shaanxi, Sichuan, Hunan, and Hubei. It is a large-scale Chinese medicinal material variety in China, and the market demand is large. At present, the field cultivation of Scrophularia ningpoensis mainly uses asexual reproduction with buds, and this asexual reproduction method is prone to cause the germplasm degradation of Scrophularia ningpoensis, aggravate the occurrence of Scrophularia ningpoensis diseases, and lead to a decrease in the yield of Scrophularia ningpoensis medicinal materials.

[0003] Fungal endophytes are a type of fungi that parasitize inside plants without causing obvious diseases in plants. Endophytic fungi and host plants have gradually formed a mutually beneficial relationship during the long-term co-evolution process. The host plant provides a shelter and a place for survival for the endophytic fungus, while the endophytic fungus can directly or indirectly secrete certain chemical substances to promote the growth of the host or help it resist adverse environmental stresses. Research shows that endophytic fungi play an important role in plant growth and development and secondary metabolism, and they can promote plant growth, accelerate plant secondary metabolism, enhance disease resistance and other stress resistances. Endophytic fungi of plants have become a hot issue in the field of plant microbiology.

[0004] It is reported that endophytic fungi can improve the growth of host plants by producing phytohormones, promoting plant nutrient absorption, enhancing their photosynthesis efficiency, etc. Some scholars reported that Cladosporium sp. and Coniochaeta sp. can colonize the roots of orchid plants and secrete IAA to promote the growth of orchid plants. Phoma herbarum D603 was co-cultured with sterile seedlings of Salvia miltiorrhiza, and it promoted the growth and root development of Salvia miltiorrhiza by producing IAA, promoting nutrient absorption and producing siderophores. The endophytic fungus Phomopsis liquidambri promotes the growth of peanut plants by increasing nodulation and nitrogen assimilation through hydrogen peroxide H2O2 and nitric oxide NO signals. The root endophytic fungus Curvularia geniculata of Parthenium hysterophorus promotes plant growth by dissolving phosphorus. Some scholars reported that the plant height, chlorophyll and soluble sugar content of the endophytic fungus symbiont seedlings of Achnatherum inebrians increased with the prolongation of light time, and were significantly higher than other treatments at a photoperiod of 16 h·d -1 When it was significantly higher than other treatments. Treatment of germinated tomato roots with the spore suspension of the endophytic fungus Fusarium lateritium for 50 d can increase the contents of chlorophyll a and chlorophyll b in tomatoes, increase the biomass of tomato roots, and promote the growth of tomato plants.

[0005] However, there is currently no report on the application of promoting the growth of Scrophularia ningpoensis through its endophytic fungi. SUMMARY OF THE INVENTION

[0006] The purpose of the present invention is to provide an endophytic fungus of Scrophularia ningpoensis and its application in promoting the growth of Scrophularia ningpoensis in view of the deficiencies of the prior art.

[0007] In the first aspect of the present invention, an endophytic fungus of Scrophularia ningpoensis is provided, which belongs to the fungi of Ascomycota, Pleosporales, Didymellaceae, Boeremia, and the endophytic fungus is isolated from the living body of Scrophularia ningpoensis of the genus Scrophularia in the family Scrophulariaceae by using the endophytic fungus isolation and purification technology, and is identified as Boeremia exigua SNF128 of the genus Boeremia by microbial taxonomy. This strain has been deposited, and the deposit number of the strain is CGMCC No. 41086, the deposit date is January 23, 2024, the deposit unit is the China General Microbiological Culture Collection Center (CGMCC), the address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the postal code is 100101.

[0008] The solid culture characteristics of the endophytic fungus described in the present invention are as follows:

[0009] When cultured in the dark at 26 °C on a potato dextrose agar medium (PDA), its colony is entirely black, with white raised hyphae scattered in the center, a grayish blue ring belt covering the outer circle of the colony, and the edge hyphae are dark black, see Figure 1Under the microscope, the hyphae are colorless to dark brown, filamentous, straight or slightly curved, with thin walls, smooth surfaces, and are seen Figure 2 。

[0010] The ITS molecular sequence of the endophytic fungus described in the present invention is shown in SEQ ID NO.1.

[0011] The sequenced sequence was searched for homologous sequences in the NCBI (https: / / www.ncbi.nlm.nih.gov / ) GenBank database and subjected to BLAST alignment for phylogenetic analysis. The MEGA 11.0 software was used to construct a phylogenetic tree to clarify the systematic taxonomic status of the Scrophularia ningpoensis endophytic fungus SNF128. NCBI database alignment found that the sequence homology of SNF128 with Boeremia exigua (MH782559), Boeremia exigua (OP113851), and Boeremia exigua (PP156749) was 100.00%, 100.00%, and 99.56% respectively. Multiple sequence alignment was performed using MEGA11.0, and neighbor-joining (NJ) analysis was used. The alignment was repeated 1000 times to establish a phylogenetic tree of SNF128. It was found that SNF128 and the above 3 fungi clustered in the same terminal branch. The ITS agarose gel electrophoresis pattern and phylogenetic tree of the endophytic fungus described in the present invention are shown in Figure 3 、 Figure 4 。

[0012] In the second aspect of the present invention, there is provided the use of the above-mentioned endophytic fungus in promoting the growth of Scrophularia ningpoensis.

[0013] Specifically: The SNF128 fungus was made into a 5×5 mm fungal cake and inoculated into the medium of Scrophularia ningpoensis tissue culture seedlings, which can increase the dry weight, plant height, root length, leaf length, and leaf width of Scrophularia ningpoensis tissue culture seedlings, thereby promoting the growth of Scrophularia ningpoensis.

[0014] The above application is mainly achieved through the following technical solutions:

[0015] (1) Pick out SNF128 from the cryopreservation tube and inoculate it into PDA medium (formula: 200 g of potatoes, 20 g of glucose, 15 g of agar, 1000 mL of distilled water, pH natural), and incubate it in the dark at 26°C and 65% humidity for 5 - 7 d. After the hyphae cover the plate, it is ready for use.

[0016] (2) Surface sterilize Scrophularia ningpoensis seeds using a three-step disinfection method: 75% ethanol for 1 min, 2.5% sodium hypochlorite for 3 min, and 75% ethanol for 30 s. Wash away the residual ethanol on the surface with sterile water, dry with sterile filter paper, inoculate into MS medium, and culture in a sterile culture room. Culture conditions: 26 ± 1 °C, humidity 65%, light duration 10 h / d, light intensity 1000 Lux. When the seeds germinate and grow into seedlings 2 - 3 cm tall, transplant them into a new MS medium for later use.

[0017] The formula of MS medium is ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, calcium chloride 440 mg / L, magnesium sulfate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate 27.8 mg / L, disodium ethylene diamine tetraacetate 37.3 mg / L, glycine 2 mg / L, vitamin B3 0.5 mg / L, vitamin B6 0.5 mg / L, vitamin B1 0.1 mg / L, sucrose 20 g / L, agar 8.5 g / L, pH 5.7 - 5.8.

[0018] (3) Use a fungal punch to cut out a SNF128 fungal cake, and pick up the fungal block with an inoculation loop and place it in the Scrophularia ningpoensis tissue culture seedling medium for co-culture.

[0019] (4) Culture in a sterile laboratory at a temperature of 26 ± 1 °C, humidity 65%, light duration 16 h / d, and light intensity 1000 Lux. After 1 month, measure the dry weight, plant height, root length, leaf length, leaf width and other indicators of Scrophularia ningpoensis, and it can be clearly observed that SNF128 promotes the growth of Scrophularia ningpoensis tissue culture seedlings ( Figure 5 ).

[0020] The advantages of the present invention are as follows:

[0021] The endophytic fungus Boeremia exigua SNF128 of Scrophularia ningpoensis described in the present invention can promote the growth of Scrophularia ningpoensis after symbiosis with Scrophularia ningpoensis tissue culture seedlings. The dry weight increases by 62.07%, and the plant height, root length, leaf length and leaf width increase by 73.29%, 22.41%, 43.37% and 24.02% respectively. The endophytic fungus of the present invention has an obvious effect on promoting the growth of Scrophularia ningpoensis, bringing broad application prospects to the field of plant growth promotion technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described below in conjunction with the drawings and embodiments;

[0023] Figure 1: Colony morphology of Boeremia exigua SNF128 on PDA solid medium;

[0024] Figure 2 : Mycelial morphology of Boeremia exigua SNF128 under an optical microscope;

[0025] Figure 3 : ITS sequence gel electrophoresis map of Boeremia exigua SNF128;

[0026] Figure 4 : Phylogenetic tree of Boeremia exigua SNF128 based on ITS sequence (NJ method);

[0027] Figure 5 : Physical photos and statistical charts of growth indexes of the growth-promoting effect of Boeremia exigua SNF128 on Scrophularia ningpoensis tissue culture seedlings. Detailed implementation mode

[0028] The effects of the present invention will be further described below in conjunction with specific embodiments.

[0029] In the following embodiments, unless specifically indicated, % all represent volume percentages.

[0030] The endophytic fungus of the present invention is a strain isolated from the living body of Scrophularia ningpoensis produced in Pan'an, Zhejiang.

[0031] Example 1:

[0032] The endophytic fungus is obtained by separation according to the following steps: The collected Scrophularia ningpoensis root tubers are rinsed under tap water for 5 min to remove the surface soil and other impurities, and the surface moisture is blotted dry with sterile filter paper, and surface disinfection treatment is carried out. First, soak in 75% alcohol for 30 s, then rinse with sterile water 3 times, then rinse with 5% sodium hypochlorite for 30 s, and then rinse with sterile water 3 - 4 times. After disinfection, the surface of the sample is blotted dry with sterile filter paper, and the root is cut into small tissue blocks (0.5 cm × 0.5 cm) with a disinfected scalpel. Every 4 tissue blocks are placed in a PDA medium containing sodium penicillin (50 mg·L -1 ). After sealing, culture at 26 °C. After the mycelia grow out from around the tissue blocks, transfer them to a new PDA petri dish, pick the mycelia according to the morphological characteristics and inoculate them into a new PDA petri dish for further culture, number and record. After the culture is completed, observe and select again until the endophytic fungus strain SNF128 of the present invention is isolated. As Figure 1 , its colony is entirely black as a whole, with white raised mycelia scattered in the center, a gray-blue ring belt covering the outer circle of the colony, and the edge mycelia are dark black. Under the microscope (Figure 2 ) The visible hyphae are colorless to dark brown, filamentous, straight or slightly curved, with thin walls and smooth surfaces.

[0033] Take out the cryopreservation tube of the Scrophularia ningpoensis endophytic fungus SNF128 from the refrigerator. In the ultra-clean bench, pick up a little hyphae with an inoculation loop and transfer it onto the PDA medium. Place it in a fungal incubator at a temperature of 26 °C and culture for 5 - 7 days until the hyphae cover the plate, indicating that the activation is completed. Take an appropriate amount of SNF128 fungal hyphae, use a fungal genomic DNA extraction kit, rinse the mycelium 3 times with sterile water, perform freeze-drying, and extract fungal DNA. Use the universal primers ITS4 (5'-GGAAGTAAAAGTCGTAAGG-3', SEQ ID NO.2) and ITS5 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO.3) for PCR amplification. The PCR reaction cycle parameters for the amplification steps are as follows: 1. 95 °C, 3 min, initial denaturation; ........ Figure 3 ) After that, perform sequencing, conduct BLAST alignment in the NCBI database according to the ITS sequence, and construct a phylogenetic tree as shown in Figure 4 . It can be seen that the sequence homology of SNF128 with Boeremia exigua (MH782559), Boeremia exigua (OP113851), and Boeremia exigua (PP156749) is 100.00%, 100.00%, and 99.56% respectively. It belongs to Boeremia exigua SNF128 of the genus Boeremia in the Ascomycota, Pleosporales, Didymellaceae, and the preservation number of the strain is CGMCC No. 41086.

[0034] Example 2:

[0035] Under aseptic conditions, pick out SNF128 from the cryopreservation tube and inoculate it into PDA medium (formula: 200 g of potatoes, 20 g of glucose, 15 g of agar, 1000 mL of distilled water, natural pH). Incubate it in the dark at 26°C and 65% humidity for 5 - 7 days until the mycelium covers the plate. Use a fungal punch to cut out SNF128 fungal cakes with a size of 5 mm × 5 mm for later use. Surface disinfect the Scrophularia ningpoensis seeds using the three-step disinfection method: 75% ethanol for 1 min, 2.5% sodium hypochlorite for 3 min, and 75% ethanol for 30 s. Wash off the residual ethanol on the surface with sterile water, dry it with sterile filter paper, inoculate it into MS medium, and place it in a sterile culture room for cultivation. The cultivation conditions are: 26 ± 1°C, humidity 65%, light duration 10 h / d, and light intensity 1000 Lux. When the seeds germinate and grow into seedlings 2 - 3 cm tall, transplant them into a new MS medium for later use. The formula of MS medium is ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, calcium chloride 440 mg / L, magnesium sulfate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate 27.8 mg / L, disodium ethylenediaminetetraacetate 37.3 mg / L, glycine 2 mg / L, vitamin B3 0.5 mg / L, vitamin B6 0.5 mg / L, vitamin B1 0.1 mg / L, sucrose 20 g / L, agar 8.5 g / L, pH 5.7 - 5.8.

[0036] Use an inoculation loop to pick up the fungal block and place it in the Scrophularia ningpoensis tissue culture seedling medium for co-cultivation. Set up 3 biological replicates for the experiment. Use a punch to cut out PDA blocks of the same size as the control group and place them in a sterile laboratory for cultivation at a temperature of 26 ± 1°C, humidity 65%, light duration 16 h / d, and light intensity 1000 Lux. After 1 month, measure the dry weight, plant height, root length, leaf length, leaf width, and other indicators of Scrophularia ningpoensis. The results show that SNF128 can coexist with Scrophularia ningpoensis tissue culture seedlings for a long time and promote the growth of tissue culture seedlings. The dry weight, plant height, root length, leaf length, and leaf width of Scrophularia ningpoensis tissue culture seedlings in the SNF128 treatment group are 0.047 ± 0.017 g, 7.33 ± 4.80 cm, 4.37 ± 1.21 cm, 27.01 ± 4.23 mm, and 19.05 ± 1.24 mm respectively, which are 62.07%, 73.29%, 22.41%, 43.37%, and 24.02% higher than those of the control group ( Figure 5 ).

[0037] The preferred embodiments of the present invention have been specifically described above. However, the present invention is not limited to the described embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A Scrophularia endophytic fungus, characterized in that, It belongs to the fungi of Ascomycota, Pleosporales, Didymellaceae, Boeremia, and was deposited in the China General Microbiological Culture Collection Center on January 23, 2024. Its taxonomic name is Boeremia exigua SNF128, and the strain preservation number is CGMCC No. 41086.

2. The application of the endophytic fungus of Scrophularia ningpoensis described in claim 1, characterized in that, It is used to promote the growth of Scrophularia ningpoensis tissue culture seedlings.

3. The application according to claim 2, wherein The Scrophularia ningpoensis endophytic fungus promotes the growth of Scrophularia ningpoensis by promoting the increase of dry weight, plant height, root length, leaf length and leaf width of Scrophularia ningpoensis tissue culture seedlings.

4. The application according to claim 3, characterized in that, The specific application is to co-culture the Scrophularia ningpoensis endophytic fungus with Scrophularia ningpoensis tissue culture seedlings.

5. The application according to claim 4, wherein The co-culture conditions are: culturing in a sterile tissue culture room at 26±1°C, with 16 h of light and 8 h of darkness, and the light intensity is 1000 Lux.

6. The application according to claim 4, wherein The co-culture medium is MS medium. The Scrophularia ningpoensis endophytic fungus is inoculated into the MS medium in the form of a fungal cake, and the inoculation amount of the fungal cake is 1 fungal cake / bottle.

7. The application according to claim 6, characterized in that, characterized in that, The formula of MS medium is ammonium nitrate 1650 mg / L, potassium nitrate 1900 mg / L, calcium chloride 440 mg / L, magnesium sulfate 370 mg / L, potassium dihydrogen phosphate 170 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, potassium iodide 0.83 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, ferrous sulfate 27.8 mg / L, disodium ethylenediaminetetraacetate 37.3 mg / L, glycine 2 mg / L, vitamin B3 0.5 mg / L, vitamin B6 0.5 mg / L, vitamin B1 0.1 mg / L, sucrose 20 g / L, agar 8.5 g / L, pH 5.7 - 5.8.

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