CN107475123B - Anoectochilus roxburghii endophytic fungus and application thereof - Google Patents
Anoectochilus roxburghii endophytic fungus and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
Abstract
The invention relates to the technical field of microorganisms, in particular to Anoectochilus formosanus endophytic fungi, which is obtained by separating and purifying endophytic fungi from a Living body of Anoectochilus roxburghii (Wall.) Lindl of Orchidaceae, is classified and named as Chaetomium globosum J9 (CGMCC No. 14128), and has a preservation number of CGMCC No. 14128. The invention further discloses application of the anoectochilus formosanus endophytic fungus J9 in improving biomass of model plant Arabidopsis thaliana and antagonizing Fusarium oxysporum (Fusarium oxysporum) which is a main pathogen of anoectochilus formosanus, and the application value of the strain in improving yield of medicinal materials and enhancing disease resistance of plants in anoectochilus formosanus cultivation is prompted.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to anoectochilus roxburghii endophytic fungi and application thereof.
Background
"Cordyceps militaris is in north and anoectochilus formosanus is in south". Anoectochilus roxburghii is a characteristic Chinese herbal medicine of Fujian, is derived from Anoectochilus Blume plant leaves and Laeliocattleya patens of Orchidaceae (Orchidaceae), is used as a medicine by the whole herb, has the effects of clearing heat and cooling blood, dispelling wind and promoting diuresis, detoxifying and the like (Fujian Chinese herbal medicine standard, Fuzhou: Haifeng Press, 2006: 154), is mainly used for treating hypertension, diabetes, hepatitis, tumors and the like, and has excellent curative effect, so the Anoectochilus roxburghii has the reputations of medicine king, gold grass and the like in folk. In areas such as Fujian, Taiwan area and southeast Asia, Anoectochilus roxburghii is regarded as a rare and famous medicinal material, especially in Taiwan area, and is a food material necessary for food therapy, which is often used as a tonic product by folks.
In recent years, the higher medicinal and health-care values of anoectochilus formosanus attract the attention and research of numerous scholars at home and abroad. A large number of chemical components and pharmacological activity researches show that the anoectochilus formosanus mainly contains lactone glycosides (such as the anoectochilus formosanus glycoside), polysaccharides and flavonoid active ingredients, and particularly contains higher contents of the anoectochilus formosanus glycoside and the polysaccharides; the traditional Chinese medicine composition has biological activities of immunoregulation, blood sugar reduction, liver protection, tumor resistance, oxidation resistance, osteoporosis resistance and the like, and is mainly used for clinically treating tumors, diabetes, hepatitis and hyperuricemia (Zhanghongyan, Panxin, Anoectochilus formosanus chemical components and pharmacological activity research progress. strait pharmaceutical, 2009,21(1): 82-84.). With the continuous deepening of the research on the biological activity of the anoectochilus formosanus and the continuous reflection of the medicinal value of the anoectochilus formosanus, the market demand of the anoectochilus formosanus is also increased year by year, so that the wild anoectochilus formosanus is excessively excavated for a long time, and in addition, the natural reproduction rate of the anoectochilus formosanus is low, the requirements on the ecological environment are strict, the adaptability is poor, the wild resources are sharply reduced, even the wild anoectochilus formosanus is extremely deficient, and.
The anoectochilus formosanus belongs to the cymbidium, the requirement on the growth environment is high, the survival rate of the transplanted tissue culture seedlings is low, and serious plant diseases and insect pests are easily caused in the cultivation process, so that the yield and the quality of the anoectochilus formosanus are influenced. Wherein, the stem rot of anoectochilus formosanus becomes one of the most main diseases of the anoectochilus formosanus in Fujian province (Shao Qing Song, Liu hong Bo, Zhao Xiao Fang, Hu Huai, Li Ming Yan. the biological characteristics of the stem rot of the anoectochilus formosanus and the inhibiting effect of 5 bactericides on the stem rot of the anoectochilus formosanus. Chinese traditional medicine journal, 2014,39(8): 1386-. The stem rot of Anoectochilus roxburghii is caused by Fusarium oxysporum Schl, and pathogenic bacteria invade the stem base of Anoectochilus roxburghii through epidermis, root hair or rhizome, and is one of the most main diseases in artificial cultivation. The pathogenic bacteria can cause harm to the anoectochilus formosanus in different growth and development stages, the harm degree is decreased gradually according to the increase of plant age, and the harm degree is increased according to the increase of concentration and temperature of pathogenic bacteria. When the disease occurs, the base of the plant stem appears yellow brown water stain-like disease spots, and quickly develops to a circle around the stem, and the diseased tissue is rotten, dried, overflowed and shrunk to be linear. The disease condition develops rapidly, seedlings fall down rapidly to die, large-area damping-off occurs, and the disease rate reaches 90% when the seedlings are serious, so that huge economic loss is brought to enterprises and planters. At present, no effective prevention and treatment measures exist, and the large-area artificial cultivation of anoectochilus formosanus is severely limited.
Therefore, how to use a new biotechnology means to effectively improve the yield and quality of anoectochilus formosanus medicinal materials and enhance the disease resistance of plants to promote the large-area standardized planting and industrialization of anoectochilus formosanus becomes a problem which needs to be solved urgently at present.
Orchidaceae plants such as herba Dendrobii and rhizoma Gastrodiae have close relationship with Endophytic fungi (Endophytic fungi). Endophytic fungi play irreplaceable roles in seed germination, tissue culture propagation, later growth and development and other links of orchids (Zhoudping, WushuHangzhou, Jiangzang and Zhang-life. function and application prospect of endophytic fungi of orchids. Shanghai agricultural science and report, 2005,21(3): 110-. From the perspective of mycorrhizal fungi, researchers have screened beneficial strains with significant growth promotion effects on Anoectochilus roxburghii, such as the isolation and biological activity studies of the Rhizopus (Epulorrhiza sp) strains AR-15 and AR-18 (Guo cis star, Chengmei, Yumei, Gloiopeltis roxburghii) Anoectochilus roxburghii mycorrhizal fungi, China journal of pharmacy, 2000,35(7): 443) 445, the lentinula (Mycena sp) strains MF23 and MF24 (Chengmei, Guo cis star, Ronga. four endophytic fungi have effects on the growth and polysaccharide content of Anoectochilus roxburghii sterile seedlings, China journal of pharmacy, 2005,40(1):13-16) and YN-64 (unidentified) (plum aviation. screening of the mycorrhizal fungi that promote the growth of Anoectochilus roxburghii and optimization of inoculation conditions, Fujian agriculture forest (Master thesis), 2014); in addition, mycorrhizal fungi ASF-15 (unidentified and derived from cypripedium margaritacum) and ASF-18 (unidentified and derived from cypripedium calcoelus) which are separated from other orchids and have the function of promoting the growth and development of anoectochilus formosanus (Queen, Junya menxia, Sedum hemsleyanum, Queen, endophytic fungi screening research for promoting the growth and development of anoectochilus formosanus, China pharmaceutical journal, 2009,44(13): 976-. Therefore, the anoectochilus formosanus mycorrhizal fungi are closely related to the quality of the anoectochilus formosanus mycorrhizal fungi, such as the yield of medicinal materials, the content of effective components and the like.
Therefore, the search for new endophytic fungi from Anoectochilus roxburghii, in particular for endophytic fungi capable of improving host biomass and effective component content, is a continuous pursuit of those skilled in the art. At present, no literature reports exist that endophytic fungi which can obviously antagonize pathogenic bacteria of stem rot are separated from anoectochilus roxburghii, and chaetomium globosum which can simultaneously improve host biomass and antagonize pathogenic bacteria of stem rot is not reported.
Disclosure of Invention
The invention aims to provide a novel anoectochilus roxburghii endophytic fungus and provides application thereof.
In the first aspect of the invention, a anoectochilus formosanus endophytic fungus is provided and named as chaetomium globosum J9(Chaetomium globosum).
The Anoectochilus roxburghii endophytic fungi is obtained by separating and purifying endophytic fungi from living bodies of plants of Anoectochilus roxburghii (Wall.) Lindl of Labiatae, and is identified as Chaetomium globosum J9 by microbiological classification. The strain is preserved, the preservation number is CGMCC No.14128, the preservation date is 2017, 5 and 31 months, the preservation unit is China general microbiological culture Collection center (CGMCC), and the address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101.
the microscopic characteristics of the anoectochilus formosanus endophytic fungi are as follows:
the colony diameter is 55mm, the texture is flocculent and light gray, the colony is spread and forms a large amount of olive brown ascochyta in the later period; the back of the colony is brown and has no water-soluble pigment. The shell of the ascon is light to dark brown, spherical or ellipsoidal, and can reach 200-550 mu m; the accessory silk tools are separated, do not branch, have curved or slightly curved top ends and rough walls, the base parts are light to dark brown, the top ends are olive brown, and the width is 2.3-4.0 mu m; early resolution of the ascomycetes; the ascospores are lemon-shaped, nearly spherical, brown and slightly rough in wall, and have the diameter of 6.5-11.4 multiplied by 6.1-8.0 mu m. No vegetative spore-forming structures were observed.
The rRNA gene sequence determination result of the anoectochilus roxburghii endophytic fungi comprises an 18S rRNA fragment, complete sequences of ITS1, 5.8S rRNA and ITS2 and a 28S region sequence fragment shown in SEQ ID NO. 1. The sequencing results (SEQ ID NO.1) were aligned on NCBI website (http:// blast. NCBI. nlm. nih. gov/blast. cgi) with 100% similarity to Chaetomium globosum (Chaetomium globosum).
In a second aspect of the invention, the application of the anoectochilus formosanus endophytic fungus in the biomass increase of arabidopsis thaliana is provided.
In a third aspect of the invention, a method for improving the biomass of arabidopsis thaliana is provided, wherein the anoectochilus formosanus endophytic fungi and an arabidopsis thaliana plant are co-cultured.
The culture medium used included:
A) endophytic fungi activation medium: PDA potato solid medium (potato 200g, glucose 20g, deionized water per 1L);
B) arabidopsis tissue culture seedling culture medium: 1/2MS culture medium, pH 5.8;
C) endophytic fungi and arabidopsis co-culture medium: 1/2MS medium, pH 5.8.
Temperature of the culture room: 22 +/-1 ℃;
daily illumination: 16 h;
illuminance: 2000 Ix.
The co-culture specifically comprises the following steps:
A) selecting a small amount of hyphae by using an inoculating needle under the aseptic condition, inoculating the hyphae into a sterilized solid PDA culture medium test tube, and performing activated culture at 28 ℃ for 72 hours by taking the Anoectochilus roxburghii endophytic fungi strain Chaetomium globosum J9;
B) drawing a transverse line at the position of about 7cm wide of the bottom of a culture dish of 1/2MS culture medium, selecting 4d arabidopsis thaliana tissue culture seedlings with basically consistent growth vigor, uniformly picking the seedlings to the position of the drawing line of the culture dish, wherein 6 seedlings in each culture dish are punched by a hole puncher to be 0.5cm
2The bacterial pellet of the chaetomium globosum J9 was placed 3cm from the bottom of the petri dish for co-culture 14 d.
The fourth aspect of the invention provides the application of the anoectochilus formosanus endophytic fungi for antagonizing the pathogenic bacteria of the stem rot.
Preferably, the stem rot pathogenic bacterium is Fusarium oxysporum (Fusarium oxysporum).
In the fifth aspect of the invention, a method for antagonizing pathogenic bacteria of stem rot of anoectochilus formosanus is to co-culture the endophytic fungi of the anoectochilus formosanus and an anoectochilus formosanus plant, wherein the pathogenic bacteria of the stem rot of the anoectochilus formosanus is fusarium oxysporum.
The invention has the advantages that:
the endophytic fungi chaetomium globosum J9 of anoectochilus roxburghii can improve the biomass of a model plant Arabidopsis thaliana and antagonize fusarium oxysporum which is the main pathogenic bacterium of anoectochilus roxburghii, and the potential application value of the strain in improving the yield of medicinal materials and enhancing the disease resistance of plants in the cultivation of the anoectochilus roxburghii is prompted.
Preservation information of biological material sample:
the preservation unit: china general microbiological culture Collection center
Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district
The preservation date is as follows: 31/5/2017
The preservation number is: CGMCC NO.14128
And (3) classification and naming: ball hair shell J9(Chaetomium globosum)
Drawings
FIG. 1 is a morphological diagram (A) and a spore micrograph (B) of the endophytic fungi of Anoectochilus roxburghii according to the invention on a PDA plate culture medium.
FIG. 2 is a morphological diagram of Anoectochilus roxburghii endophytic fungi clump of the invention co-cultured with Arabidopsis thaliana plant for 14 days.
FIG. 3 shows the fresh weight (A) and dry weight (B) of the plant (Control is Control group, J9 is treatment group, J9 is endophytic fungus) after 14 days of co-culture of the Anoectochilus roxburghii endophytic fungus block and the Arabidopsis plant.
FIG. 4 is a morphological diagram of the endophytic fungi Anoectochilus roxburghii antagonistic Fusarium oxysporum of the invention (A and B are J9 treated groups, C and D are blank control groups; A and C are front diagrams, B and D are back diagrams).
Detailed Description
The following examples are provided to illustrate specific embodiments of the present invention.
Example 1: separation and purification of anoectochilus formosanus endophytic fungi
The endophytic fungi of anoectochilus roxburghii is a strain obtained from anoectochilus roxburghii in Jiangxi.
The anoectochilus formosanus endophytic fungi is obtained by separation according to the following steps: washing with tap water for 30min to remove silt, and washing with deionized water for 3 times. The leaves were surface sterilized as follows: 75% ethanol, 30s → 1.3M sodium hypochlorite (3-5% available chlorine), 1min → 75% ethanol, 30s → 3 washes with sterile deionized water. After the residual water was sucked off from the filter paper, the leaves were cut into tissue blocks of 1cm size with a sterilized scalpel and cultured at 28 ℃ on PDA (potato 200g/L, glucose 20g/L, agar, 15g/L) medium containing 100mg/L penicillin. The samples were observed daily for fungal growth. Hyphae grow out after 5-7 days, the tips of the hyphae are picked and transferred to a new PDA culture medium for purification and culture, and are combined according to the shape, and finally the anoectochilus formosanus endophytic fungi strain is obtained. The classified name is Chaetomium globosum J9(Chaetomium globosum), and the preservation number is CGMCCNo.14128.
As shown in FIG. 1A, the colony of the endophytic fungi Anoectochilus roxburghii of the invention grows fast on a PDA culture medium, the diameter of the colony is 55mm under the dark condition of 25 ℃ for 7 days, the texture is flocculent, light gray and spread, and a large amount of olive brown ascocarp is formed in the later period; the back of the colony is brown and has no water-soluble pigment.
As shown in FIG. 1B, the microscopic characteristics of the endophytic fungi of Anoectochilus roxburghii are as follows: the shell of the ascon is light to dark brown, spherical or ellipsoidal, and can reach 200-550 mu m; the accessory silk tools are separated, do not branch, have curved or slightly curved top ends and rough walls, the base parts are light to dark brown, the top ends are olive brown, and the width is 2.3-4.0 mu m; early resolution of the ascomycetes; the ascospores are lemon-shaped, nearly spherical, brown and slightly rough in wall, and have the diameter of 6.5-11.4 multiplied by 6.1-8.0 mu m. No vegetative spore-forming structures were observed.
Example 2: cultivation of Arabidopsis aseptic seedlings
The Arabidopsis seeds of the invention are from the crude drug laboratory of Shanghai second Legionnaire university.
The arabidopsis aseptic seedling is cultured according to the following steps: the arabidopsis seeds are disinfected by a conventional disinfection method: 5min with 75% ethanol, washing with sterile water for 5-6 times, culturing in a refrigerator at 4 deg.C for 3d, inoculating to 1/2MS culture medium, culturing at 25 deg.C under 16h illumination/8 h dark for 5d, and culturing to obtain sterile seedling after seed germination transferring to new 1/2MS culture medium.
Example 3: co-culture of Arabidopsis with endophytic fungi
The arabidopsis aseptic seedling is cultured according to the following steps:
A) taking the anoectochilus roxburghii endophytic fungi strain, picking a small amount of hyphae by using an inoculating needle under the aseptic condition, inoculating the hyphae into a sterilized solid PDA culture medium test tube, and performing activated culture at 28 ℃ for 72 hours;
B) drawing a transverse line at the position of about 7cm wide of the bottom of a culture dish of 1/2MS culture medium, selecting 4d seedlings with basically consistent growth vigor, uniformly picking the 4d seedlings to the position of the drawing line of the culture dish, wherein 6 seedlings are planted on each culture dish, and the seedlings are punched by a hole puncher to be 0.5cm
2The bacterial pellet of the chaetomium globosum J9 was placed 3cm from the bottom of the petri dish for co-culture 14 d. Each treatment was repeated three times.
C) The arabidopsis thaliana plants which are co-cultured for 14d are harvested, the fresh weight of the arabidopsis thaliana plants is weighed by a one-ten-thousandth balance, and then the fresh plants are dried in an oven at 50 ℃ for 8 hours and then the dry weight of the arabidopsis thaliana plants is weighed.
The experimental data of the biomass measurement result of the arabidopsis aseptic seedlings are expressed as mean +/-s.e., statistical processing is carried out by applying SPSS 22.0 software, one-way ANOVA (one-way ANOVA) is adopted, the differences among groups are compared by adopting an LSD-t method, and the test level α is 0.05.
Example 4: endophytic fungi antagonistic fusarium oxysporum
The antibacterial detection method for the anoectochilus roxburghii endophytic fungi antagonistic Fusarium oxysporum comprises the following steps:
A) endophytic fungi activation medium: PDA potato solid medium (potato 200g, glucose 20g, deionized water per 1L);
B) fusarium oxysporum suspension: directly washing the cultured indicator bacteria slant with 5mL of Tween 80, shaking, and diluting the bacterial suspension to 1 × 10 by using a blood cell counting method
4efu/mL Fusarium oxysporum (Fusarium oxysporum). Quickly pouring 100L of bacterial suspension into a PDA culture medium at 40-50 ℃, shaking up, quickly loading into a culture dish, and cooling to obtain a culture medium containing fusarium oxysporum;
C) endophytic fungi fusarium oxysporum experiment: punching the activated endophytic fungi with a puncher to obtain a sample with a diameter of 0.5cm
2The bacterial sheet is placed on a culture medium containing indicator bacteria, the hyphae grow upwards, and 2-3 bacterial blocks are placed in a culture dish of each indicator bacteria. Culturing in 37 deg.C incubator for 5d, observing whether there is bacteriostatic zone, and measuring the diameter of bacteriostatic zone by cross method. Each strain was replicated 3 times.
The results show that Fusarium oxysporum not treated with the fungus of the invention grew rapidly on the PDA morphology as shown in FIGS. 4C and D, filling the entire plate; after the anoectochilus formosanus endophytic fungi is treated, the growth of fusarium oxysporum can be remarkably antagonized, and the mean diameter of the formed inhibition zone is 40 mm.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.
SEQUENCE LISTING
<110> second military medical university of China people liberation army
<120> Anoectochilus roxburghii endophytic fungi and application thereof
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<170>PatentIn version 3.3
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<213> Chaetomium globosum (Chaetomium globosum)
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Claims (8)
1. The anoectochilus formosanus endophytic fungi is characterized in that the anoectochilus formosanus endophytic fungi is classified and named as Chaetomium globosum J9(Chaetomium globosum), and the preservation number is CGMCC No. 14128.
2. The application of the anoectochilus formosanus endophytic fungus of claim 1 in increasing biomass of arabidopsis thaliana.
3. A method for increasing the biomass of Arabidopsis thaliana is characterized in that the method for increasing the biomass of Arabidopsis thaliana is to co-culture the anoectochilus roxburghii endophytic fungus of claim 1 and an Arabidopsis thaliana plant.
4. The method for increasing biomass of arabidopsis thaliana as claimed in claim 3, wherein the culture medium used in the method for increasing biomass of arabidopsis thaliana comprises:
A) endophytic fungi activation medium: PDA potato solid culture medium;
B) arabidopsis tissue culture seedling culture medium: 1/2MS culture medium, pH 5.8;
C) endophytic fungi and arabidopsis co-culture medium: 1/2MS medium, pH 5.8.
5. The method for increasing biomass of Arabidopsis thaliana as claimed in claim 3, wherein said co-cultivation comprises: temperature of the culture room: 22 +/-1 ℃; daily illumination: 16 h; illuminance: 2000 Ix.
6. Use of an endophytic fungus of Anoectochilus roxburghii according to claim 1 for antagonizing pathogenic bacteria of stem rot.
7. The application of the endophytic fungi of Anoectochilus roxburghii for antagonizing pathogenic bacteria of stem rot disease as claimed in claim 6, wherein the pathogenic bacteria of stem rot disease is Fusarium oxysporum.
8. A method for antagonizing pathogenic bacteria of stem rot of Anoectochilus roxburghii is characterized in that the method for antagonizing the pathogenic bacteria of stem rot of Anoectochilus roxburghii is to co-culture the endophytic fungi of Anoectochilus roxburghii of claim 1 and Anoectochilus roxburghii plants, and the pathogenic bacteria of stem rot of Anoectochilus roxburghii is fusarium oxysporum.
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