Dendrobium nobile lindl. endophytic fungus and use thereof

Abstract

Provided are a Dendrobium nobile Lindl. endophytic fungus and a use thereof. The endophytic fungus is isolated from a living body of Dendrobium nobile Lindl., which is a perennial herb of the family Orchidaceae, by using endophytic fungus isolation and purification technology, is classified and named as Phyllosticta fallopiae DNL19, and has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on 27 April 2023 with a strain preservation number of CGMCC No.40583. Also provided is a use of the endophytic fungus, wherein the endophytic fungus can promote the growth of Dendrobium nobile Lindl. and promote the accumulation of dendrobine which is an effective ingredient.

Description

Endophytic fungus of Dendrobium nobile and application thereof Technical Field

[0001] The present invention relates to the field of plant growth and secondary metabolism regulation, in particular to an endophytic fungus of Dendrobium nobile and an application thereof in promoting the growth of Dendrobium nobile and the accumulation of dendrobine content. Background Art

[0002] Dendrobium nobile Lindl. is a perennial herbaceous plant of the genus Dendrobium in the Orchidaceae family, primarily found in Guizhou, Yunnan, Guangxi, Guangdong, and Sichuan provinces. The Chinese Pharmacopoeia states that Dendrobium nobile Lindl. nourishes the stomach and produces fluid, nourishes yin and clears heat. It is used to treat fluid depletion caused by febrile illness, dry mouth and thirst, stomach yin deficiency, loss of appetite and retching, persistent post-illness heat, yin deficiency and excessive fire, and blurred vision. Modern medical research indicates that dendrobine, the active ingredient in Dendrobium nobile Lindl., has analgesic, anti-osteoporotic, anti-tumor, and neuroprotective effects.

[0003] Dendrobium nobile has a low reproduction rate under natural conditions, its wild resources are scarce, and the quality of cultivated varieties is variable. Current approaches to improving the quality of Dendrobium nobile and promoting the accumulation of its active ingredients include breeding new varieties, changing cultivation methods, optimizing fertilization schedules, and adding hormone elicitors. However, these methods suffer from long cycles, limited effectiveness, poor sustainability, and soil compaction.

[0004] Endophytic fungi are fungi or bacteria that live within the tissues and organs of healthy plants at certain or all stages and are commonly found in higher plants. Studies have shown that the interaction between endophytic fungi and host plants is an important pathway for promoting plant growth and secondary metabolism. Endophytic fungi can promote the growth of host plants and the accumulation of secondary metabolic components, which is of great research value. For example, Yang He et al. used the wild soybean endophytic fungi Y6R15 and Y2S2 to treat soybeans and corn through root irrigation and foliar spraying, respectively. They analyzed the changes in dry matter and root indicators of corn and soybean seedlings in the different treatment groups and found that strains Y6R1 and Y2S2 promoted the growth of both corn and soybeans, increasing indicators such as root length, root surface area, root volume, and number of root tips. Lan Taoju et al. screened 32 endophytic fungi isolated from the stems of Bruguiera gymnorrhiza and obtained a dark septate endophytic fungus (DSE) strain with a good growth-promoting effect on Dendrobium officinale, numbered HS40. They found that 6 months after the potted Dendrobium officinale seedlings were inoculated with strain HS40, their plant height, tiller number, total fresh weight, total dry weight and stem polysaccharide content increased by 21.7%, 375.0%, 94.7%, 57.6% and 15.8% respectively compared with the control. Among them, the plant height, total fresh weight, total dry weight and stem polysaccharide content reached extremely significant differences compared with the control group (P<0.01), and the tiller number reached a significant difference (P<0.05). Ye Bingzhu et al. isolated 277 endophytic fungi from Anoectochilus roxburghii and Ludisia orchids and found that two of the strains, J162 and J211, could be co-cultured with Anoectochilus roxburghii, a medicinal plant in southern my country, and could significantly improve the biosynthesis and accumulation of active ingredients such as flavonoids, saponins and polysaccharides in Anoectochilus roxburghii.

[0005] However, there are few reports on the application of endophytic fungi to improve the yield and quality of D. nobile. Therefore, studying the ecosystem formed by D. nobile and its endophytic fungi is of great significance for improving the yield and quality of D. nobile and accelerating the development of the D. nobile industry.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide an endophytic fungus of Dendrobium nobile and an application thereof in view of the deficiencies in the prior art.

[0008] The first aspect of the present invention provides a plant endophytic fungus, which is isolated and purified from the leaves of Dendrobium nobile Lindl, a plant of the genus Dendrobium in the Orchidaceae family, and is classified and named Phyllosticta fallopiae DNL19. The strain has been deposited with the strain deposit number CGMCC No. 40583, the deposit date is April 27, 2023, and the deposit unit is the General Microbiology Center of the China Culture Collection Administration (CGMCC), the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

[0009] The solid culture characteristics of the endophytic fungus described in the present invention are as follows: when cultured in the dark at 25°C on potato dextrose agar (PDA), the strain grows slowly, the colonies are dark gray, the middle circle is white round granular protrusions, and the outer circle is grayish white and flat, as shown in Figure 1.

[0010] The formula of the PDA culture medium of the present invention is: 200g potatoes, 20g glucose, 1000mL distilled water, 15g agar, and natural pH.

[0011] The morphological characteristics of the endophytic fungus described in the present invention under an optical microscope are as follows: the colony is black-purple in color as a whole, the hyphae are short and thin, with many branches, and some hyphae have internal septa visible.

[0012] The morphological characteristics of the endophytic fungus described in the present invention under a scanning microscope are as follows: the hyphae are generally round, with some hyphae occasionally flat, with relatively smooth walls, thin and multi-branched, as shown in FIG2 .

[0013] The ITS amplified base sequence of the endophytic fungus described herein is shown in SEQ ID NO. 1. Sequence alignment results were performed on the NCBI website (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), revealing 99.83% homology with Phyllosticta fallopiae (NR147316). Sequence alignment was performed using MEGA 7.0 software, using the neighbor-joining (NJ) method. One randomly selected sequence was aligned 1000 times. Agarose gel electrophoresis and a phylogenetic tree of the endophytic fungus described herein are shown in Figures 3 and 4.

[0014] The second aspect of the present invention provides the use of the above-mentioned endophytic fungus, including increasing the fresh weight and dendrobine content of Dendrobium nobile.

[0015] The above application can promote the increase of the fresh weight and dendrobine content of Dendrobium nobile by co-cultivating the DNL19 mushroom block with Dendrobium nobile. The co-cultivation requires inoculating the mushroom block to 1 / 2MS and co-cultivating it with the Dendrobium nobile tissue culture seedlings, placing it in a greenhouse at a temperature of 25±1°C, a light / dark cycle of 12 / 12h, and a relative humidity of 60-70%.

[0016] Furthermore, the method is mainly achieved by the following technical scheme: (1) taking the above-mentioned endophytic fungal strain, under sterile conditions, using an inoculation needle to pick a small amount of hyphae, inoculating it into PDA culture medium (formula: 200g potato, 20g glucose, 15g agar, 1000mL distilled water, natural pH), and incubating it in the dark at 25°C and 65% humidity for 7 days; (2) under sterile conditions, using a 5mm punch to punch out the bacterial block at the edge of the colony of the above-mentioned strain, i.e., obtaining the DNL19 bacterial block, transferring the bacterial block to 1 / 2MS culture medium and co-cultivating it with the tissue culture seedlings of Dendrobium nobile, with the bacterial block placed in the middle of the tissue culture seedlings. The inoculated tissue culture seedlings of Dendrobium nobile were placed in a greenhouse at a temperature of 25±1°C, a light / dark cycle of 12 / 12h, and a relative humidity of 60-70% for co-cultivation (cultivation for 60 days), which can significantly increase the fresh weight and dendrobine content of Dendrobium nobile. The formula of 1 / 2MS medium ( / L) is: potassium nitrate 1.9 g, ammonium nitrate 1.65 g, potassium dihydrogen phosphate 0.17 g, magnesium sulfate 0.18 g, calcium chloride 0.33 g, potassium iodide 0.83 mg, boric acid 6.2 mg, manganese sulfate 16.9 mg, zinc sulfate 8.6 mg, sodium molybdate 0.25 mg, copper sulfate 0.025 mg, inositol 0.1 g, cobalt chloride 0.025 mg, glycine 2.0 mg, EDTA sodium salt 37.26 mg, vitamin B1 0.1 mg, ferrous sulfate 15.2 mg, niacin 0.5 mg, vitamin B6 0.5 mg, sucrose 15.0 g, agar powder 7.0 g, distilled water 1000 mL, pH 5.8-6.0.

[0017] The beneficial effects of the present invention are:

[0018] The endophytic fungus DNL19 (Phyllosticta fallopiae) described in the present invention can significantly increase the fresh weight and dendrobine content of Dendrobium nobile by co-cultivation with the plant. This endophytic fungus significantly improves the yield and quality of Dendrobium nobile, bringing broad application prospects for high-yield and high-quality cultivation of Dendrobium nobile. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1: Colony morphology of the endophytic fungus Phyllosticta fallopiae DNL19 on PDA medium;

[0020] Figure 2: Mycelial morphology of the endophytic fungus Phyllosticta fallopiae DNL19 under light and scanning microscopes;

[0021] Figure 3: Gel electrophoresis of the ITS sequence of DNL19 (Phyllosticta fallopiae) amplified using primers ITS5 and ITS4;

[0022] Figure 4: Phylogenetic tree of DNL19 (Phyllosticta fallopiae) based on amplification using ITS4 and ITS5 primers (NJ method);

[0023] Figure 5: Phyllosticta fallopiae endophytic fungus DNL19 (Phyllosticta fallopiae) increases the fresh weight and dendrobine content of Dendrobium nobile;

[0024] Figure 6: Gene expression heatmap of the endophytic fungus DNL19 (Phyllosticta fallopiae) that increases the fresh weight and dendrobine content of Dendrobium nobile;

[0025] Figure 7: The expression results of key genes in which the endophytic fungus DNL19 (Phyllosticta fallopiae) increases the fresh weight and dendrobine content of Dendrobium nobile. DETAILED DESCRIPTION

[0026] The endophytic fungus of the present invention is isolated and purified from the leaves of Dendrobium nobile, a plant of the genus Dendrobium in the Orchidaceae family. The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0027] Example 1:

[0028] The endophytic fungus is isolated and obtained according to the following steps:

[0029] Rinse the root, stem, and leaf samples of Dendrobium nobile with clean water for 5 minutes, then rinse with sterile water, and then use sterile filter paper to absorb the surface moisture. Then, the samples were surface-sterilized using a three-step disinfection method (roots: 75 vol% C2H5OH for 1 minute, 2.5 wt% NaClO for 4 minutes, 75 vol% C2H5OH for 1 minute; stems: 75 vol% C2H5OH for 1 minute, 2.5 wt% NaClO for 3 minutes, 75 vol% C2H5OH for 1 minute; leaves: 75 vol% C2H5OH for 1 minute, 2.5 wt% NaClO for 1 minute, 75 vol% C2H5OH for 30 seconds). After the three-step disinfection method, the samples were rinsed with sterile water five times, and then the surface moisture of the samples was absorbed with sterile filter paper. The roots, stems, and leaves of Dendrobium nobile were cut into tissue blocks using sterile scissors and tweezers. The stems and roots were 0.5 cm long, and the leaves were 0.5 cm × 0.5 cm. Afterwards, traditional separation and culture was carried out. 30 tissue blocks were randomly picked from the roots, stems and leaves of three different tissue parts. Every 4-5 tissue blocks were placed on a plate of PDA culture medium containing penicillin (50 mg / L) and cultured in a constant temperature and humidity incubator at 25°C. The growth of the mycelium was observed regularly. Afterwards, the tip mycelium was picked and transferred to a new PDA culture medium to separate and purify a single colony, and the process was repeated to obtain a purified strain. The purified strain was divided into different morphological types, and the endophytic fungus DNL19 of the present invention was finally obtained. As shown in Figure 1, when cultured in the dark at 25°C on potato dextrose agar medium (PDA), the strain grew slowly, the colony was dark gray, the middle circle was white round granular protrusions, and the outer circle was grayish white and flat, as shown in Figure 1. Under an optical microscope, the colony was black-purple as a whole, the mycelium was short and thin, with many branches, and some mycelium had internal septa visible. Under a scanning microscope, the hyphae are generally round, with some parts occasionally flat, with smooth walls, thin and multi-branched, as shown in Figure 2. The strain was stored at 4°C.

[0030] DNA from an endophytic fungal strain from the roots, stems, and leaves of Dendrobium nobile was extracted and purified using the DNAiso Reagent genomic DNA purification kit. PCR amplification was performed using primers ITS4 (SEQ ID NO. 2: 5'-TCCTCCGCTTTATTGATATGC-3') and ITS5 (SEQ ID NO. 3: 5'-GGAAGTAAAGTCGTAACAAGG-3'). The PCR reaction system (50 μL) consisted of: 2 μL template DNA; 1 μL primer ITS4; 1 μL primer ITS5; 25 μL 2× TapPCR MasterMix (containing dye); and 21 μL ddH2O. PCR cycling conditions included an initial denaturation at 95°C for 3 min; denaturation at 94°C for 40 s; annealing at 52°C for 50 s; extension at 72°C for 1 min; 35 cycles of denaturation-annealing-extension; and a final extension at 72°C for 10 min. The PCR product was tested for quality by 1% agarose gel electrophoresis, and the qualified PCR amplification product was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequence is shown in SEQ ID NO.1.

[0031] Using the sequence obtained from the PCR product as the target sequence, a search for homologous sequences was conducted in the GenBank database at NCBI. The reference sequence most similar to the morphological sequence was downloaded, and phylogenetic analysis was performed using the neighbor-joining method (NJ) to determine the phylogenetic position of the strain to be identified. As shown in Figures 3 and 4, the homology with Phyllosticta fallopiae reached 99.83%. Therefore, the endophytic fungus of the present invention was classified and named Phyllosticta fallopiae DNL19.

[0032] The isolated endophytic fungus from Dendrobium nobile was deposited in the General Microbiology Center of China Culture Collection Administration on April 27, 2023, and was classified and named Phyllosticta fallopiae DNL19, with the deposit number CGMCC No.40583.

[0033] Example 2:

[0034] A small amount of hyphae from a cryovial of DNL19 was aseptically harvested with an inoculating needle and inoculated into PDA medium for activation. The cells were then cultured at 25°C and 65% humidity until maturity. A 5mm borer was used to punch out colony clumps from the edge of the colony. These clumps were then inoculated into 1 / 2 MS medium and co-cultured with tissue-cultured D. nobile seedlings, with the clumps positioned in the center of the seedlings. A control group consisted of blank PDA medium inoculated into the center of the seedlings. The seedlings were cultured in a greenhouse at 25±1°C, with a 12 / 12h light / dark cycle and a relative humidity of 60-70%. Thirty replicates were used. After completion of the incubation period, six vials of sterile tissue-cultured D. nobile seedlings were randomly selected for determination of fresh weight and dendrobium alkaloid content. Dendrobium alkaloid content was determined by gas chromatography. GC test sample solutions were prepared, with three replicates for each sample. A Shimadzu SH-Rtx-1 capillary column (0.25 μm × 0.25 mm × 30 m) and N2 were used as carrier gas, and a Shimadzu 2010 Plus gas chromatograph with flame ionization detector (FID) was used. The gas chromatography conditions were based on the 2020 edition of the Chinese Pharmacopoeia. When determining the correction factor, 1.0 μL of the dendrobium alkaloid reference solution was precisely drawn with a microinjection needle and injected into the gas chromatograph. The components of the total ion chromatogram were extracted by flame ionization detection. The injection was repeated three times in a row, and the peak area of ​​the chromatogram was recorded to calculate the correction factor F. The regression equation of the standard curve of dendrobium alkaloid is Y = 794.74X - 7191.50 (R 2 =0.990), with a linear range of 20-200 μg / L. The results showed that the fresh weight of D. nobile inoculated with DNL19 increased by 11.44% compared to the control group, and the dendrobine content increased by 33.80%, as shown in Figure 5 , indicating that inoculation with DNL19 significantly promoted the growth and dendrobine accumulation of D. nobile.

[0035] Total RNA was extracted from tissue culture seedlings of Dendrobium nobile from the DNL19-treated and control groups using the TaKaRa MiniBEST Universal RNA Extraction Kit. 3 μL of RNA sample was loaded onto a 1.0% agarose gel, electrophoresed at 120 V for 30 min, and visualized using a gel imager. 1 μL of RNA sample was diluted with appropriate amounts of ddH2O, and the OD values ​​were measured at 260 nm and 280 nm. After RNA quality control, transcriptome libraries were constructed and paired-end sequenced using an Illumina Hiseq2000 / 2500. After quality control, the sequencing data were assembled using Trinity software. Genes were then aligned with sequences in five public databases (Swiss-Prot, NR, KEGG, KOG, and Pfam) using a threshold of e ≤ 1e. -10Functional annotation was performed based on sequence similarity, and sequence similarity was compared using the BLAST algorithm. Significance testing of read counts was performed using the negative binomial distribution test in the DEGseq software package, and unigene expression levels were estimated using the basemean value. Differentially expressed genes related to the growth and dendrobium alkaloid metabolism of D. nobile were screened after DNL19 treatment. The differentially expressed genes are shown in Figure 6.

[0036] Furthermore, the reverse transcription system PrimerScript RT kit was used to verify the differential gene expression. The reaction was divided into two steps: ① 10 μL reaction system: 5×gDNA Eraser Buffer 2 μL, gDNA Eraser 1 μL, RNA 2 / c μL (c is RNA concentration, μg·μL) -1 ), DEPC HO to 10 μL, 42°C, 2 min; ② 20 μL reaction system: 10 μL of product from step ①, 1 μL of PrimeScript RT Enzyme Mix I, 1 μL of RT Primer Mix, 4 μL of 5× PrimerScript Buffer 2 (for Real Time), 4 μL of EASY Dilution, 37°C, 15 min, 85°C, 5 s. The reverse transcription product was diluted 2-fold for later use. The CDS sequences of the target genes were aligned by BLAST, and primers were designed using Primer Premier 5.0 software to select appropriate fragments. The primer sequences are shown in Table 1, with β-actin as the internal reference gene. The real-time PCR reaction system consisted of a 10 μL total volume, 5 μL SYBR buffer, 0.4 μL Primer-F, 0.4 μL Primer-R, 3.7 μL RNase-free ddH2O, and 0.5 μL cDNA. The reaction procedure was as follows: 95°C for 30 s; 39 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s; and 65°C for 5 s, followed by 95°C for 5 s. The expression levels of the selected D. nobile genes GLG1, GBSS, HMGCS1, and MNEG_4509 were significantly increased after treatment with the endophytic fungus DNL19, as shown in Figure 7. This indicates that the endophytic fungus DNL19 increases the fresh weight and dendrobine content of D. nobile by upregulating the expression of genes such as GLG1, GBSS, HMGCS1, and MNEG_4509.

[0037] Table 1 Primer sequences

[0038] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. An endophytic fungus of Dendrobium nobile, characterized in that: The endophytic fungus of Dendrobium nobile was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on April 27, 2023, and was classified and named Phyllosticta fallopiae DNL19, with the deposit number CGMCC No.40583.

2. An application of the endophytic fungus of Dendrobium nobile according to claim 1, characterized in that: Specifically, the invention relates to the application of the endophytic fungi of Dendrobium nobile in increasing the yield of Dendrobium nobile and improving the quality of Dendrobium nobile.

3. The use according to claim 2, characterized in that: The application is specifically as follows: inoculating the endophytic fungus cake into the growth culture medium of Dendrobium nobile for co-cultivation.

4. The use according to claim 3, characterized in that: Inoculating the endophytic fungus cake into the growth medium of Dendrobium nobile for co-cultivation specifically comprises the following steps: (1) taking the endophytic fungus species of Dendrobium nobile, picking mycelium with an inoculation needle under sterile conditions, inoculating it into a sterilized PDA medium, and culturing it in the dark at 25° C. and 65% humidity for 7 days; (2) Under aseptic conditions, a sterilized cork punch was used to obtain a fungal block at the edge of the colony, which was the endophytic fungal cake. The fungal cake was inoculated into 1 / 2MS medium for co-cultivation with the tissue culture seedlings of Dendrobium nobile. The fungal block was placed in the middle of the tissue culture seedlings and placed in a greenhouse at a culture temperature of 25±1°C, light / dark 12 / 12h, and a relative humidity of 60-70% for co-cultivation.

5. The use according to claim 4, characterized in that: The PDA medium formula is: 200 g potato, 20 g glucose, 15 g agar, 1000 mL distilled water, natural pH.

6. The use according to claim 4, characterized in that: The formula of 1 / 2MS culture medium is: 1.9g potassium nitrate, 1.65g ammonium nitrate, 0.17g potassium dihydrogen phosphate, 0.18g magnesium sulfate, 0.33g calcium chloride, 0.83mg potassium iodide, 6.2mg boric acid, 16.9mg manganese sulfate, 8.6mg zinc sulfate, 0.25mg sodium molybdate, 0.025mg copper sulfate, 0.1g inositol, 0.025mg cobalt chloride, 2.0mg glycine, 37.26mg EDTA sodium salt, 0.1mg vitamin B1, 15.2mg ferrous sulfate, 0.5mg niacin, 0.5mg vitamin B6, 15.0g sucrose, 7.0g agar powder, 1000mL distilled water, pH 5.8-6.0.

Images