CN112725191B - Inonotus tumefaciens strain for promoting germination of orchidaceae seeds and application thereof - Google Patents

Abstract

The invention provides a strain of phellinus igniarius (Coriolopsis strumosa) for promoting germination of orchidaceae seeds and application thereof, and belongs to the technical field of symbiotic fungi. The strain Epi910 provided by the invention is from fungi in the tissue culture and germination process of cymbidium sinensis seeds, and is identified as the Inonotus giganteus by combining morphological characteristics of sequencing comparison results of strains ITS, nLSU and the like separated from a culture medium and protocorm. The strain Epi910 is colonized in the seeds and protocorm cells of orchids and cymbidium forbesii in the form of mycelial clusters, and meanwhile, the similar function of the Epi910 and the Mucor is verified through symbiotic germination tests, so that the germination of the seeds of orchids including orchids and cymbidium forbesii can be promoted to form seedlings. The symbiotic germination system of the expanded fomes fomentarius-orchid seeds can be used as a new symbiotic mode except for the Mucor fungi, and provides a new research object for comprehensively analyzing the symbiotic mechanism of the fungi-orchid.

Description

Inonotus tumefaciens strain for promoting germination of orchidaceae seeds and application thereof

Technical Field

The invention belongs to the technical field of symbiotic fungi, and particularly relates to an expanding fomes fomentarius strain for promoting germination of orchidaceae seeds and application thereof.

Background

Orchidaceae (Orchidaceae) plants are one of the more evolved groups of angiosperms, whose seeds are tiny, without endosperm, and only coat an immature embryo with a simple-structured seed coat, lacking the nutrients needed for germination. Under natural conditions, orchidaceae plant seeds need to establish a symbiotic relationship with fungi, and nutrients and signal substances are provided for the orchidaceae plant seeds by the fungi so as to promote germination and seedling formation. The self-reproduction rate is low, and the environment damage of habitats in recent years causes the orchid resources to be more endangered, and the germplasm conservation of the orchid and the ecological restoration of endangered species wild habitats can be effectively realized by screening suitable fungi to carry out the symbiotic germination of seeds.

At present, research on symbiotic fungi of orchids mainly focuses on 3 major groups of tunasellaceae (tulasellaceae), ceratobasidioceae (ceratobasidioceae) and cercosporales (secalinales), and development of new groups of symbiotic fungi or functional research and verification on existing groups of symbiotic fungi have important significance for widening the range of symbiotic fungi of orchids and protecting and cultivating orchids.

The genus Ceriporia (Coriolopsis) belongs to the family of Polyporaceae, and the existing research finds that the fungus contains more lignocellulose degrading enzymes such as laccase, and plays an important role in the aspects of substance circulation and energy flow of forest ecosystem together with other species of Polyporaceae, and does not find that the fungus plays a role in symbiotic germination of orchids.

Disclosure of Invention

In view of the above, the invention aims to provide an erigeron turquoise strain Epi910 for promoting germination of orchidaceae seeds and an application thereof, and the erigeron turquoise strain Epi910 can effectively promote germination of orchids and cymbidium harledebx seeds.

The invention provides an Eleutherococcus expansus (Coriolopsis strumosa) strain Epi910 for promoting germination of orchid seeds, wherein an ITS sequence of the strain Epi910 is shown as SEQ ID NO:1, the nLSU sequence of the strain Epi910 is shown as SEQ ID NO: 2, the sequence of nSSU of the strain Epi910 is shown in SEQ ID NO: 3, the mtSSU sequence of the strain Epi910 is shown in SEQ ID NO: 4, the EF 1-alpha sequence of the strain Epi910 is shown as SEQ ID NO: 5, the sequence of RPB1 of the strain Epi910 is shown in SEQ ID NO: 6, the sequence of RPB2 of the strain Epi910 is shown in SEQ ID NO: shown at 7.

Preferably, when the strain Epi910 is symbiotic with a seed of dendrium arborescens (eridendrim radians) or a seed of Cymbidium bicolor (Cymbidium bicolor), the hyphae of the strain Epi910 colonize inside cells of the seed of dendrium arborescens or the seed of Cymbidium bicolor.

Preferably, the preservation number of the strain Epi910 is CGMCC No. 21081.

The invention provides application of the Inonotus gibbosa strain Epi910 in orchid seed germination.

Preferably, the orchid seeds include tree orchid seeds and cymbidium bicolor seeds.

Preferably, the medium used for germination of the seeds of the orchidaceae plant includes OMA medium.

Preferably, the chaetomium turgidum strain Epi910 is combined with a strain Pap12 of tunica membrana sp for the seed germination of orchids.

The invention provides a phellinus igniarius (Coriolopsis strumosa) strain Epi910 for promoting germination of orchids seeds, and a result of multi-fragment molecular identification shows that the ITS, nLSU, nSSU, mtSSU, EF 1-alpha and RPB2 fragments have higher similarity with the phellinus igniarius except RPB1, and meanwhile, a phylogenetic tree is constructed by combining the ITS and nLSU fragments, and the result shows that the Epi910 and the phellinus igniarius are completely integrated into a branch with the supporting strength of 100 percent, so that the invention preliminarily identifies the strain Epi910 as the phellinus igniarius by combining the characteristics of molecular identification and morphology. The orchid seed symbiotic germination experiment shows that Epi910 can effectively promote germination of cymbidium and cymbidium bicolor seeds, and hyphae colonize seed cells in the form of mycelial clusters when the seed coats of cymbidium bicolor are not cracked. Epi910 can promote germination of cymbidium seeds, but the symbiotic germination efficiency is lower than that of non-symbiotic germination on MS1 culture medium and symbiotic germination with Mucor strain Pap 12; but significantly higher than germination efficiency on non-inoculated OMA medium. The symbiotic germination efficiency of the cymbidium forbesii seeds and the Epi910 is close to the non-symbiotic germination efficiency on an MS1 culture medium, and is obviously higher than the symbiotic efficiency of the jelly fungi strain Pap 12. However, the promoting effect of Epi910 on dendrobium seed germination is not significant, only hyphae are observed to be attached to seed coats in dendrobium symbiotic germination, and hyphae are not observed to colonize inside cells. Therefore, the Inonotus tumefaciens strain Epi910 can effectively promote germination of orchidaceae seeds such as orchids, cymbidium and the like, and has no promotion effect on germination of dendrobium seeds.

The invention provides an application of the Inonotus latus strain Epi910 in orchid seed germination. Experiments prove that the strain Epi910 has selectivity on promoting germination of orchidaceae plant seeds, can promote germination of seeds to seedlings in symbiotic germination of orchida and arundina seeds, and is difficult to form seedlings in symbiotic germination of orchida and dendrobium nobile seeds, and a control strain (jelly fungus) can effectively promote germination of orchida, arundina and dendrobium nobile seeds.

Drawings

FIG. 1 is a colony morphology of Inonotus latreiliana strain Epi 910;

FIG. 2 is a maximum likelihood tree of Inonotus latus and related fungi constructed based on ITS and nLSU sequences, the value at a branch point is the support rate of each node obtained by repeating 1000 times of evaluation by a Bootstrap method, and only Bootstrap is shown to be more than or equal to 50%;

FIG. 3 shows symbiotic germination of different Orchidaceae plants with strain Epi910, wherein FIG. 3A shows symbiotic germination of Epi910 with tree orchid seeds, and formation of mycelial clusters in the seed embryo; FIG. 3B is the colonization of the Epi910 mycelial mass in the dendrina cells; FIG. 3C shows Epi910 colonizing a tree orchid protocorm; FIG. 3D shows Epi910 symbiotically germinating with cymbidium forbesii seeds with mycelial clusters formed in the seed embryo; FIG. 3E shows the colonization of the Epi910 mycelial group in the dendrina cells; FIG. 3F shows the colonization of Epi910 in cymbidium duratum protocorms; FIG. 3G shows that Epi910 hyphae surround the Dendrobium seed; FIG. 3H shows Epi910 symbiotically germinated seedlings with Tulipa; FIG. 3I shows Epi910 symbiotically germinated seedlings with Tulipa; scale 5 m;

FIG. 4 is a comparison of germination results of different strains and Orchidaceae seeds, wherein FIG. 4A is the germination results of different strains and Orchidaceae seeds; FIG. 4B shows germination results of different strains and cymbidium bicolor seeds; FIG. 4C shows germination results of different strains and dendrobium seeds.

Biological material preservation information

The strain of the Inonotus obliquus (Coriolopsis strumosa) is preserved in the China general microbiological culture Collection center (CGMCC) for 2020 and 03 days. The address is No. 3 of Xilu No. 1 of Beijing, Chaoyang, and the microbial research institute of Chinese academy of sciences, the biological preservation number is CGMCC No.21081, and the strain number is Epi 910.

Detailed Description

The invention provides an Eleutherococcus expansus (Coriolopsis strumosa) strain Epi910 for promoting germination of orchid seeds, wherein an ITS sequence of the strain Epi910 is shown as SEQ ID NO:1, the nLSU sequence of the strain Epi910 is shown as SEQ ID NO: 2, the sequence of the nSSU of the strain Epi910 is shown as SEQ ID NO: 3, the mtSSU sequence of the strain Epi910 is shown in SEQ ID NO: 4, the EF 1-alpha sequence of the strain Epi910 is shown as SEQ ID NO: 5, the sequence of RPB1 of the strain Epi910 is shown in SEQ ID NO: 6, the sequence of RPB2 of the strain Epi910 is shown in SEQ ID NO: shown at 7. The comparison result shows that the similarity of the other 6 gene segments except RPB1 with the Inonotus obliquus is the highest. Since the submitting sequence of the Inonotus is mostly concentrated on the ITS and nLSU fragments, the ITS and nLSU fragments are combined to construct a phylogenetic tree, and the result shows that the Epi910 and the Inonotus giganteus are completely integrated into a branch, and the support strength is 100%. Meanwhile, the colony and hypha morphological characteristics of the strain Epi910 are observed, the colony formed by the strain Epi910 on the PDA culture medium is circular, grows quickly, is white in front, has a large amount of aerial hyphae, and is neat in the edge and thin in the hyphae; the back of the bacterial colony is white, and the partial area of the back of the bacterial colony becomes brown along with the extension of the culture time; the growth rate of hyphae is 0.27 + -0.03 mm/h, and the diameter is 2.07 + -0.20 μm. The mycelium is extremely thin on the OMA culture medium, and light brown sclerotium can be produced after culturing for 30-50 days. The strain Epi910 is preliminarily identified to be the Inonotus gibsonii by combining the characteristics of molecules and forms.

In the present invention, when the strain Epi910 is symbiotic with the seeds of cymbidium or cymbidium sinensis, it is preferable that the strain Epi910 is colonized inside the seeds or stem cells of cymbidium or cymbidium sinensis as a mycelial mass. However, when the strain Epi910 is symbiotic with dendrobium seeds, hyphae are not colonized in cells. The strain Epi910 can effectively promote germination of seeds of orchidaceae and cymbidium bicolor, but has an insignificant promoting effect on germination of seeds of dendrobium, which shows that the strain Epi910 has selectivity on promotion of germination of seeds of orchidaceae plants. The strain Epi910 is subjected to biological preservation, and the preservation number is preferably CGMCC No. 21081.

The invention provides an application of the Inonotus latus strain Epi910 in orchid seed germination. The orchid plant seeds preferably include tree orchid seeds and cymbidium harleyanum seeds. When the strain Epi910 and the cymbidium symbiotically germinates, the symbiotic germination lasts for 40-60 d, and leaves stretch and are differentiated into roots to form seedlings. The medium used for germination of the seeds of the orchids preferably comprises OMA medium. Epi910 can promote germination of orchids seeds, but the symbiotic germination efficiency is lower than that of non-symbiotic germination on MS1 medium and symbiotic germination with Mucor (Tulasnella sp.) strain Pap 12; but significantly higher than germination efficiency on non-inoculated OMA medium. When strain Epi910 symbiotically germinates with cymbidium forbesii seeds, seedlings are formed when symbiotically germinates for 180 d. The symbiotic germination efficiency of the cymbidium forbesii seeds and the Epi910 is close to the non-symbiotic germination efficiency on an MS1 culture medium, and is obviously higher than the symbiotic efficiency of the jelly fungi strain Pap 12. Meanwhile, the strain Epi910 cannot grow seedlings in symbiotic germination with dendrobium seeds, while the jelly fungi strain Pap12 can promote germination with dendrobium seeds, differentiate protomeristem and extend partial protocorm leaves.

Since the strain Pap12 of the colletotrichum (Tulasnella sp.) can effectively promote symbiotic germination and seedling formation of seeds of orchids including orchids, cymbidium and dendrobium, and experiments prove that the colletotrichum fungus and the strain Epi910 of the colletotrichum fungus do not have antagonistic action when symbiotically cultured, the invention provides the application of the strain Epi910 of the colletotrichum fungus, preferably the strain Tulasnella sp, in germination of seeds of orchids. The strain of the glued membrane strain is preferably the strain of glued membrane strain Pap 12.

The invention provides a strain of Inonotus latus for promoting germination of Orchidaceae seeds and its application, which are described in detail in the following examples, but they should not be construed as limiting the scope of the invention.

Example 1

1 materials and methods

1.1 test strains

Strain Epi910 was isolated from cymbidium protocorm during aseptic germination. The denudate seeds cultured on OMA culture medium in 2018 and 9 months germinate earlier than other seeds, the fungi are separated on PDA culture medium by a protocorm tissue section method, the strain Epi910 is obtained by purification, and the denudate seeds are transferred to a PDA test tube for storage at 4 ℃.

The Pap12(GenBank accession: MT804628) fungus of Tulasnella (Tulasnella) is isolated from the roots of Paphiopedilum (Paphiopedilum sp.) and can promote the germination of seeds of Cymbidium (Epidendrum radians), Cymbidium (Cymbidium bicolor) and Dendrobium (Dendrobium sp.).

1.2 plant Material

And respectively collecting the capsules of the cymbidium bicolor, the cymbidium sinensis and the dendrobium nobile for symbiotic germination.

The requirements for harvesting capsules: the seeds are mature, the fruit pods are healthy and not cracked, and no obvious plant diseases and insect pests exist on the surface; after harvesting, the surface was wiped with 75% ethanol, stored at 4 ℃ and sown within 3 d.

1.3 Medium

Fungus culture medium: PDA culture medium, 200.0g/L peeled potato, 20.0g/L glucose and 15.0g/L agar, and natural pH value.

Symbiotic germination culture medium: OMA medium, 3.0g/L oat flour +6.0g/L agar, natural pH.

Non-symbiotic germination culture: MS1 medium, MS +0.5 mg/L6-BA +10.0g/L sucrose +6.0g/L agar, pH 6.3.

1.4 identification of the Strain

1.4.1 fungal DNA extraction: inoculating the fungus to PDA solid culture medium paved with cellophane, culturing for 7d, scraping a small amount of hypha with aseptic toothpick, and extracting fungus genome DNA with plant genome rapid extraction kit (Beijing Ederly Biotech limited) for PCR amplification.

1.4.2PCR amplification and sequencing: a total of 7 gene fragments were amplified (Table 1) with reference to Ji et al (2019) PCR primers and amplification program. The amplification reaction system was run using 40 μ LPCR: mu.L template, 10 XEx Taq buffer 4.0. mu.L, dNTP mix 3.0. mu.L, 0.4. mu.L primers (10. mu. mol/L), TaKaRa Ex Taq 0.3. mu.L, and ddH for the remainder₂O make up to 40. mu.L. The PCR product is subjected to electrophoresis detection and then is delivered to Shanghai biological engineering (Shanghai) corporation for bidirectional sequencing, and 7 gene segments (SEQ ID NO: 1-SEQ ID NO:7) obtained after sequence splicing are subjected to sequence comparison with GenBank.

1.4.3 phylogenetic analysis: and (4) comparing the ITS sequences of the Epi910 in GenBank to determine the genus of the strain. The ITS and nLSU sequences of the same genus dominant species are downloaded from an NCBI nucleic acid database, a phylogenetic tree is constructed by a maximum likelihood method by using megaX software, a TN93+ G + I model is adopted, and Boletopsis leucomelaena is used as an outer cluster.

2 results and analysis

2.1 isolation and colony characterization of Strain Epi910

The germination of the tree orchid seeds polluted by the fungi in the aseptic germination process is relatively regular in the original culture dish, after 80 days of culture, part of protocorms are divided into leaves, the protocorms are sliced by hands, the fungi are found to be colonized in protocorm basal cells, and the fungi specificity staining shows that the fungi are colonized in the tree orchid protocorm cells in the form of mycelial clusters. The strain Epi910 is obtained through separation and purification.

The colony formed by the strain Epi910 on the PDA culture medium is round, grows quickly, is white in front, has a large amount of aerial hyphae, and is neat in the edge and thin (figure 1A); the back of the bacterial colony is white, and the partial area of the back of the bacterial colony becomes brown along with the extension of the culture time; the growth rate of hyphae is 0.27 + -0.03 mm/h, and the diameter is 2.07 + -0.20 μm. The mycelia were very thin on OMA medium, and light brown sclerotia could be produced after culturing for 30-50 days (FIG. 1B).

2.2 Epi910 Strain phylogenetic analysis

The alignment results of the genomic DNAPCR product sequences of strain Epi910 at the NCBI nucleic acid database (table 1) show that all fragments have the highest similarity to chaetomium turgidum except RPB 1. The sequence submitted by the fomes is mostly concentrated in ITS and nLSU fragments, so that a phylogenetic tree (shown in figure 2) is constructed by combining the ITS and nLSU fragments, the result shows that the Epi910 and the expanding fomes are completely integrated into a branch, the supporting strength is 100%, and the strain Epi910 is preliminarily identified to be the expanding fomes.

TABLE 1 alignment of PCR amplified DNA sequences of strain Epi910

Example 2

1. Symbiotic germination of orchid seeds

Removing stem and residual attachments from the collected capsule, washing with tap water for 1min, and transferring into a clean bench after excessive water is absorbed by filter paper. Soaking in 75% ethanol for 30s, washing with sterile water for 3 times, sucking water with filter paper, cutting pericarp with sterile scalpel, picking seeds with forceps, and spreading on culture medium.

Cutting out small pieces of strains Epi910 and Pap12 which are cultured for 7d respectively, and placing the small pieces in the center of the planted OMA culture medium; seeds were simultaneously inoculated on the non-inoculated OMA medium and MS1 medium as controls. Each treatment was inoculated with 10 dishes and 3 capsules were sown per orchid. The culture dish is sealed and cultured in a culture room, the illumination period is 16h/8h (light/dark), and the temperature is 25.0 +/-2.0 ℃. Seeds were observed and photographed weekly and seed germination recorded. Randomly extracting symbiotic-germinating protocorms, carrying out tissue slice culture on a PDA culture medium, separating endophytic fungi amplification ITS fragments, and determining that only purposeful strains colonize in the protocorms and are not polluted by other fungi.

Division of morphological characteristics of orchid seeds in different stages of germination is referred to Stewart & Zettler (2002), and the division is divided into 6 stages: level 0: the seeds are not germinated; level 1: the seed embryo absorbs water and expands, and epidermal hair or rhizoid appears; and 2, stage: the seed embryo continuously expands and the seed coat is cracked; and 3, stage: the appearance of native tissue; 4, level: differentiating to obtain leaves; and 5, stage: the leaves continue to elongate, forming seedlings. Statistical reference for seed germination (Meng et al.2019): the total number of seeds (t) was calculated by counting the number of non-germinating, seed germinating (G), protocorm (P) and seedling (S) with stages 0, 1, (2+3) and (4+5), respectively. The percentages of germinated seeds (G), protocorms (P) and seedlings (S) were calculated as follows: g100 × (G + P + S)/t, P100 × P/t, and S100 × S/t. The statistical time of the dendrobe, the cymbidium and the dendrobium is respectively 60d, 180d and 120d after symbiotic germination.

The results were square root transformed by arcsine and presented as mean ± standard deviation using SPSS 19.0.0 software to perform one-way variance (ANOVA) and Duncan (Duncan) multiple range tests on the data at a P <0.05 level.

1.1 Observation of fungal colonisation sites

Collecting the orchid protocorm which germinates to the 2 nd to 4 th grades to make a free-hand section, and observing the colonized part of hyphae in the protocorm under a microscope.

1.2 fungus-specific staining observations

Referring to Yamamoto et al (2017), specific staining of fungi was performed to observe colonization of fungi inside cells. Collecting the protocorm which germinates to the 0-4 th grade, fixing in FAA solution for at least 24h, taking out the protocorm, and washing with distilled water to remove FAA. And (3) placing the washed protocorm into 10% (W/V) KOH solution, carrying out autoclaving at 121 ℃ for 20min, neutralizing with 2% (V/V) HCl solution for 5min, transferring the protocorm to 10% (V/V) park black ink dyeing solution (10% ink and 3% acetic acid), boiling at 100 ℃ for 30min, sucking the dyeing solution, dehydrating with 100% lactic acid, and storing at 4 ℃. And observing the colonization condition of the mycelium pellet in the protocorm and the colonization condition of the intracellular mycelium pellet under a microscope, and taking a photomicrograph.

2. Symbiotic germination result of orchid seeds and strain Epi910

Epi910 can effectively promote germination of seeds of dendrobii orchid and cymbidium bicolor, but has no significant promotion effect on germination of the seeds of dendrobii orchid. When the seed coat of the tree orchid is not broken, hyphae colonize in the seed cells in the form of mycelial clusters (FIGS. 3A and 3B); only hyphae attachment to the seed coat was observed in symbiotic germination with dendrobii orchid (FIG. 3G), and no hyphae colonization inside the cells was seen.

After the dendranthema hybrida seeds and the Epi910 symbiotically germinate for 10 days, the green and expanded seed embryos can be observed, the seeds germinate to level 1, and hyphae can be observed to be colonized in dendranthema hybrida cells by fungus specific staining (fig. 3A and 3B); after 20 days of inoculation, the seed embryo continuously expands to form protocorm, and the protocorm germinates to 2 grades; the protocorm volume continues to increase at 30d of symbiotic germination and begins to gradually differentiate into primary meristems at the tip, germinating to level 3 (fig. 3C); symbiotic germination is carried out for 40-60 days, leaves stretch and roots are differentiated to form seedlings (figure 3H). Epi910 can promote germination of cymbidium seeds, but the symbiotic germination efficiency is lower than that of non-symbiotic germination on MS1 culture medium and symbiotic germination with Mucor strain Pap 12; but significantly higher than germination efficiency on non-inoculated OMA media (figure 4A).

After the cymbidium forbesii seeds and the Epi910 symbiotically germinate for 50 days, the green and the large of the embryo can be observed; protocorm is formed at about 90 days, scutellum is differentiated from partial protocorm, the protocorm germinates to grade 3, and hyphae can be observed to colonize in the cymbidium forbesii cells (fig. 3D and 3E); at about 120d, the protocorm continued to increase in volume and began to gradually differentiate into primary meristems at the tip, germinating to grade 4 (FIG. 3F); at about 180d, seedlings were formed (fig. 3I). Symbiotic germination efficiency of cymbidium haryngii seeds and Epi910 is close to non-symbiotic germination efficiency on an MS1 culture medium, and is obviously higher than symbiotic germination efficiency of the jelly fish strain Pap12 (fig. 4B).

The dendrobium seeds and the Epi910 are symbiotically germinated for about 30 days, and the green and the swelling of the seed embryos can be observed; at 90 days after inoculation, the seed embryo continuously expands to break through the seed coat and form protocorm in a small amount, hyphae surround the seed/protocorm under the observation of fungus specific staining (figure 3G), and hyphae do not enter cells; meanwhile, seeds germinated in MS1 culture medium and symbiosis with the strain Pap12 germinate to the 4 th to 5 th stages, primary meristem is differentiated, and partial protocorm leaves are elongated. The symbiotic germination efficiency of dendrobium seeds with Epi910 was close to that of non-symbiotic germination on MS1 medium, but significantly higher than that on non-inoculated OMA medium (fig. 4C).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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agagccccat attgttattt attgtcacta cctccccgtg tcgggattgg gtaatttgcg 720

cgcctgctgc cttccttgga tgtggtagcc gtttctcagg ctccctctcc ggaatcgaac 780

ccttattccc cgttacccgt tgaaaccatg gtaggcctct atcctaccat cgaaagttga 840

tagggcagat atttgaatga agcatcgccg gcgcaaggcc atgcgattcg agaagttatt 900

atgaatcacc aatggagcgg cgaaccgcgt tggtttttta tctaataaat acaccccttc 960

cagaagtcgg ggcttgattg catgtattag ctctagaatt accacagtta tccatgtagc 1020

aaggtaccat caaataaact ataactgatt taatgagcca ttcgcagttt cacagtacaa 1080

acttgtttat acttagacat gcatg 1105

<210> 4

<211> 483

<212> DNA

<213> Coriolopsis strumosa

<400> 4

aatcatagaa tttcttaaaa ttcatgatgt cgtaagggaa aataatgata ataccttact 60

atgagtgtcg tccaaatctg gtgccagaag actcggtaag accagagacg caaacgttaa 120

tcatcataaa caggcgtaaa gggtttgtag gcagctttca caagacaatg attacaaaat 180

aaagtggtct aattgaaagc tagaatcaaa aagaggttat agtgtataac gcctagagga 240

gggctgatat ccgtagatcc taggcagaat actaagggcg aaggccactc tccactaatg 300

attgacgctg agaaacgaag gttagggtag gaaataggat tagatacccc ggtactcctt 360

tctgtaaacg atgaatggta gtcattagtt ttataactag agacgaagtt aacacaataa 420

ccattccgcc ttgtgagtac tactgcaaag tagaaaacaa aaaaattagt cggtctcgaa 480

gca 483

<210> 5

<211> 507

<212> DNA

<213> Coriolopsis strumosa

<400> 5

ccaggccgac tgcgctatcc tcatcatcgc tggtggtacc ggcgaattcg aggcaggtat 60

ttccaaggat ggccagaccc gcgagcacgc cctcctcgcc tacactctcg gtgttaggca 120

gcttatcgtc gctgtcaaca agatggacac gaccaaggtg cgtctctagc gctgtgcttt 180

tgattgtcag gttcatcttg actgatgttg gtctgcagtg gtctgaggac cgtttcaacg 240

agatcatcaa ggagacgtcc accttcatca agaaagtcgg cttcaacccg aagtcgatcg 300

cgttcgtccc catctctggc tggcacggtg acaacatgtt ggaggagtcc accaagtaag 360

ttatgcattg tgtatgctcg ccccacctgc tcatattctc ttcagcatgc cctggtacaa 420

gggttggagc agggaggcga agtctggtac cgtcaagggc aagacgctcc tcgatgccat 480

tgacgctatc gagccccccg tccgtcc 507

<210> 6

<211> 1241

<212> DNA

<213> Coriolopsis strumosa

<400> 6

ttcattgtga aagtgaagaa gattttggag tgtatatgtg taaactgtgg tcgtctaaag 60

gctgatagcg tgagtgacct cttctttcgt ccctctcgtt tcgcacccca ccacccaatt 120

ttctgggggc tctctcctgt atcatggaga accccaaacc cgaactgcgt tagttctcga 180

caatcgaggc gttcttccac ccgtgacggg ggccgcgcgc gttttcggtc gcttgatcgc 240

cgcgtggtcc actgcacacg tccggtcatg gttggacacg cggttcgtgg tgaaatgggg 300

gctgagcgag gagtgcggaa aaaacaagag tagttggctc cttgtgggct actctgatgg 360

atggccaact gcacttgctg tgacgcagat tgagtgacat gttgtatgcc tggcacttga 420

gactcggttg tcgtagcccc ctgttgtatc cttttcgctg ttgtactgtc cgttctagat 480

gcagacagca agctgtctcg tcttctgagt ttcactcgct gacatcttga tcttcatgtc 540

tccttgctca cacggccgtg ctcgtcatcg tcttcgtgat gtaagtccga ctctaccttc 600

gcggaccgga ttcggcacat tcgcgacccg aaagcacgta tgcaagccgt ctggaactat 660

tgcaaaagca agatgatttg cgagacggat gaacccaagg aggataacga aggtggtgat 720

ccagaagagc ctaagaaggg ccatggcggt tgtggggcgc atcagcctca aatcaggaag 780

gagggactga agcttttcgt ccaatacaag cgtgggaagg acgaggacga ggtgtgtaat 840

ctgtcgtctt ctcagctcat acctcattca ttgtttgcag gatatgaaga atcttcagcc 900

agacaagcga ttgtttcccc ctcatgaggt ctatactgcg ttgaagaagg tctctgatgc 960

ggaccttcac ctccttggac tctcggtcga ctatgcccga ccagagtgga tgattctgac 1020

tgtgcttccc gtccctcctc cgcctgtaag gccaagtata gcagtggacg gcggaacgat 1080

gcggagtgaa gatgatctga cttacaagtt gggcgacatc atcaaggcct ctgcgaatgt 1140

tcggcgatgt gaacaggagg gagcacccgc tcatgtcatc aatgagtttg agcagttgct 1200

gcaggtgtgt atcgtcctgg agcaactaat aacctgtctg a 1241

<210> 7

<211> 1126

<212> DNA

<213> Coriolopsis strumosa

<400> 7

ttggcgatct cttccgcatg ctcttccgca agcttacaaa ggaagtctac cgttacctcc 60

aaaaggcagg ttccgtttgc ttgacagttt gtctaaccat ttctcaacat catcgcattt 120

agtgcgttga gacccataag gagttcaacc tatcgcttgc agtcaagcac aacaccatta 180

ccaatggtct caagtactcg cttgccacgg gtaactgggg cgaccagaag aagaccatgt 240

cggccaaggc aggggtgtct caagttctta accgttacac ttacgcgtcg acattgtcac 300

atctgcgtcg gtgtaacacg cctctcggtc gtgaagggaa gatcgcgaag cctcgtcagc 360

ttcacaacac tcattgggga atggtatgtc ccgcggaaac tccggaagga caagcatgtg 420

gcctcgtcaa gaacctttcg ctcatgtcct gcatctctgt cggcactctg tcagcccctg 480

tgatcgagtt cttggaggag tggggtcttg aatcgttgga ggagaacgct catgcgtcaa 540

caccgtgtac aaaggtgttc gtgaacggtg tctggatggg tgttcatcga gacccggtca 600

gtctcgtcaa gaccctcaga aagctccgtc gcaaggacga catcaactgc gaggtgtcag 660

tggtccgtga cattcgtgag cgcgagctcc gtttatacac agatgctggg cgcgtatgcc 720

gacctctctt catcgttgag aatcagcaac tcctcgttca gaagagacat atcgagaatc 780

tggtacgtgg caaggacgac cctgacttcg actacacttg ggacaaactc atcaaggagg 840

gtgtcatcga gttgctggat gccgaagaag aggagactgt catgatctgt atgacacctg 900

aagacctgga gaattcgagg cttcaatcca cggggcaaca gatgggagac gacgagaatg 960

aggatccttc tgctcgcctc aaggcgccta ctgtcgcaca tacttggacc cactgcgaga 1020

ttcatccaag tatgatcttg ggcgtttgtg cgagcatcat cccattcccc gatcacaacc 1080

aggttagact gtttgcatat ctttcattgc agtatgctta cacaca 1126

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tccgtaggtg aacctgcgg 19

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcctccgctt attgatatgc 20

<210> 10

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tactaccacc aagatct 17

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gtacccgctg aacttaagc 19

<210> 12

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccaagcttga attcgtagtc atatgcttgt ctc 33

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cccgtgttga gtcaaatta 19

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cagcagtcaa gaatattagt caatg 25

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gcggattatc gaattaaata ac 22

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gcyccygghc aycgtgaytt yat 23

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

achgtrccra taccaccsat ctt 23

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gaktgtcckg gwcattttgg 20

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cngcdatntc rttrtccatr ta 22

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gaygaymgwg atcayttygg 20

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cccatrgctt gyttrcccat 20

Claims (5)

1. A strain Epi910 of Elsinoe exarata (Coriolopsis strumosa) for promoting germination of Orchidaceae seeds, wherein an ITS sequence of the strain Epi910 is shown as SEQ ID NO:1, the nLSU sequence of the strain Epi910 is shown as SEQ ID NO: 2, the sequence of the nSSU of the strain Epi910 is shown as SEQ ID NO: 3, the mtSSU sequence of the strain Epi910 is shown in SEQ ID NO: 4, the EF1 alpha sequence of the strain Epi910 is shown as SEQ ID NO: 5, the sequence of RPB1 of the strain Epi910 is shown in SEQ ID NO: 6, the sequence of RPB2 of the strain Epi910 is shown in SEQ ID NO:7 is shown in the specification;

the preservation number of the strain Epi910 is CGMCC No. 21081.

2. The strain Epi910 of Eremothecium expansum for promoting germination of Orchidaceae seeds of claim 1, wherein when the strain Epi910 is symbiotic with a tree or hardy orchid seed, hyphae of the strain Epi910 colonize inside the cells of the tree or hardy orchid (Epidenrum radians) seed or the hardy orchid (Cymbidium bicolor) seed.

3. Use of the strain of fomes expansa Epi910 according to claim 1 or 2 for germination of orchid seeds, which are tree and hardleaf orchid seeds.

4. The use according to claim 3, wherein the culture medium used for germination of the seeds of the Orchidaceae plant comprises OMA medium.

5. Use according to claim 3 or 4, characterized in that the strain Eleutherococcus expansus Epi910 is combined with the strain Pap12 (Tulasnella sp.) for seed germination in Orchidaceae.

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