CN108048334B - Establishment method of symbiotic system of gloeosporium fungi for promoting germination of cymbidium and cattleya seeds - Google Patents

Establishment method of symbiotic system of gloeosporium fungi for promoting germination of cymbidium and cattleya seeds Download PDF

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CN108048334B
CN108048334B CN201711407614.1A CN201711407614A CN108048334B CN 108048334 B CN108048334 B CN 108048334B CN 201711407614 A CN201711407614 A CN 201711407614A CN 108048334 B CN108048334 B CN 108048334B
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seeds
cattleya
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cymbidium
orchid
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CN108048334A (en
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李潞滨
刘蕾
魏莉
王涛
杨前宇
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Research Institute of Forestry of Chinese Academy of Forestry
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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Abstract

The invention discloses a screening and symbiont system establishing method of Mucor fungus for promoting germination of cymbidium harderium and cattleya seeds, which adopts Tcs1 strain to establish symbiotic relationship with any one or more of the following orchid seeds to promote germination of the orchid seeds: cymbidium forbesii seed, cattleya YK seed, cattleya YD seed. Symbiotic germination tests are carried out on the artificial culture medium by adopting the Mucor fungus and the orchid seeds, and compared with an orchid seed control group without inoculation, mycorrhizal fungi capable of widely and efficiently promoting seed germination and a proper symbiotic culture medium are screened. Obtaining effective symbiotic fungi of the orchid seeds in the germination stage, improving the germination rate of the orchid seeds by using a symbiotic germination culture technology, and establishing a symbiotic system of the orchid and mycorrhizal fungi.

Description

Establishment method of symbiotic system of gloeosporium fungi for promoting germination of cymbidium and cattleya seeds
Technical Field
The invention relates to a biotechnology, in particular to a method for establishing a symbiont system of gummy fungus of the genus Mucuna for effectively promoting germination of seeds of cymbidium and cattleya.
Background
Orchidaceae (Orchidaceae) belongs to Monocotyledoneae of angiosperma, and there are approximately over 700 genus 35000 species distributed worldwide (Dressler, 1993; Cribb, 2003), which are second in number to the Compositae family, and in the second major family (Lang-Shu-Yong, 1994). It has beautiful appearance, bright color, high ornamental value and high medicinal value.
Orchids have two distinct characteristics:
first, the embryo of the seed of the Orchidaceae plant is composed of only dozens of cells, and is incompletely differentiated and difficult to germinate naturally.
Secondly, orchidaceae plants are almost mycorrhizal plants, and under the natural growth state, no matter the green-leaved orchid or the non-green-leaved orchid, in the life history of the orchidaceae plants, the orchidaceae plants need to partially or completely rely on establishing a symbiotic relationship with fungi to complete normal life activities. Under the condition of no symbiotic participation of mycorrhizal fungi, the seed germination and the nutrition supply in the individual development process of the orchids are affected, and the normal growth and development can not be completed. Therefore, the intensive development of the orchid symbiotic mycorrhizal fungi has become an important part of many related researches on orchids (alloxylonite, 2015).
Mycorrhiza (Mycorrhiza) is a ubiquitous symbiosis formed by the co-evolution of natural plants and fungi during long-term survival, and the fungi involved in the formation of Mycorrhiza are generally called Mycorrhiza fungi (Mycorrhiza). Since the discovery of the existence of viable mycelium in bird nest orchids by Ressiek in 1847, fungi have been reported to symbiotically form with different orchids, and mycorrhiza formed by the symbiosis of orchids with fungi has also been independently classified as orchids (Orchidmororhiza) due to their unique characteristics (Harley, 1989). Essentially, almost all orchids live symbiotically with fungi, with mycorrhizal symbiosis almost accompanying the entire life history of orchids from seed germination to flowering and fruiting, and most adult orchids still require mycorrhizal fungi to provide the carbohydrates and organic matter (e.g., amino acids, vitamins, etc.) required for growth (Chenxinqi et al, 2003; Smith and Read, 2008). Under a natural environment, one orchid may have more than one fungus participating in the formation of mycorrhizal symbiota, and one fungus may also establish symbiosis with more than one orchid. It is known that during the life cycle of orchids, different fungi may be required to participate in achieving a plant life history, while the process of mycorrhiza colonization depends on the species of fungi and orchids, and that certain fungi may establish symbiosis with orchids at different stages of growth (Bonnardeaux et al, 2007).
Research results show that the orchidaceae mycorrhiza has important significance for orchidaceae plants:
1. promoting the germination of seeds and providing necessary nutrients for the seeds;
2. providing required nutrients for the growth and development of the adult orchids;
3. promoting the absorption of inorganic ions such as P by orchids;
4. improving the disease resistance and stress resistance of host plants;
5. has ecological significance. In view of the significance of mycorrhizal fungi to orchid as shown in the previous research results, the research on the interaction mechanism between the mycorrhizal fungi and orchid is urgent.
Mucor is the first mycorrhizal fungus to be identified as symbiotic with orchid plants, and belongs to Basidiomycota (Basidiomycota) and Mucor (Tulasnella). Of the temperate, tropical and subtropical orchid species, the genus Gliocladium is the most common mycorrhizal fungus (Rasmussen, 1995; Yuan et al, 2010). In 2012, Martos et al found that the genus colletotrichum (Tulasnella) could symbiotically grow with 88% of orchids by investigating mycorrhizal fungi of 77 species of orchids on the island La Reunion (indian ocean) (Martos, 2012). The genus mucilaginosus has a wide distribution range and is found in almost every ecosystem from tropical to temperate zones worldwide (Cruz et al, 2012,2014,2016; Hu pottery, 2006; Wushu, 2009).
Seeds of orchids are tiny, lack endosperm and other vegetative tissues, and therefore lack enzymes, vitamins and inorganic ions, etc. associated with seed germination, and cannot support germination by utilizing the seed's own nutrition (Arditti and Ghani, 2000). Researchers have continuously studied the symbiotic germination mechanism of orchid seeds and tried to protect rare or endangered orchid varieties. In order to detect the kind of fungus which can be symbiotic with the seeds of orchid family plants, to increase the germination rate of seeds of orchid family plants and to accelerate the propagation of orchid plants by sexual reproduction, researchers have tried to culture various seeds of orchid family plants and fungus in vitro in special culture media or culture modes, to simulate the characteristics of habitat and to establish symbiotic relations in vitro (Arditti et al, 1990; Rasmussen, 1995).
In the prior art, in order to understand the occurrence mechanism of the symbiotic relationship between orchids and fungi, researchers try to artificially construct symbiosis between orchids and mycorrhizal fungi obtained by separation under the in vitro condition. In the past symbiotic studies, two types of culture medium constructions were used: one is culture medium prepared from bark or leaf of Fagaceae; another class is the use of oat agar medium (OMA).
In the nineties of the 20 th century to the early stages of the 21 st century, researchers in China often culture fungi separated from the root of wild Anoectochilus roxburghii (Anoectochilus roxburghii), Dendrobium officinale (Dendrobium candididum) and Dendrobium nobile (D.nobile), Dendrobium nobile (D.hancocockii) and serum (Liparis nervosa) protocorm by using bark and leaves, and mixedly sow the fungi with seeds of plants such as Dendrobium loddium (D.lohons), Dendrobium loddium (D.bromerianum), Dendrobium officinale and Gastrodia elata by using a method of fungus leaf culture, and find that fungi such as Armillilia mellea, Agrocybe (Mycena sp.), Rhizopus (Rhizobium (Ceorhiza sp.), Rhizoctonia (Rhizobium sp.) and the like can establish symbiosis with orchid seeds and promote germination and germination of orchid seeds, and seedling growth of plants such as Armillaristolonia officinalis (1991, Xun sinomenia sp), 2001) (ii) a Recently, oak barks are used as symbiotic culture mediums for carrying out symbiotic culture tests of rhizoctonia fungi and Cymbidium major seeds, such as Guoshi, and the like, and the rhizoctonia from roots of Cymbidium and Cymbidium can be found to remarkably improve the germination rate of the Cymbidium major seeds, and the problem of specificity between mycorrhizal fungi of adult plants of the Cymbidium major and mycorrhizal fungi for promoting symbiotic germination of the seeds is discussed (Shioshi, and the like, 2012). Various research results show that the orchid and the mycorrhizal fungi have diversity, and complex specificity still exists between the orchid and the mycorrhizal fungi at different growth stages.
The oat agar culture medium has the advantages of controllable nutrient concentration, simple operation and the like, while in the symbiotic germination process of the orchid mycorrhizal fungi and the orchid seeds, the nutrient of the culture medium is very critical, the fungi tend to carry out saprophytic life without symbiosis with the plants when the nutrition is rich, and the nutrition is too poor to meet the requirements of seed germination and growth (Beyrole, 1991, 1995). In the Chinese orchid mycorrhizal fungi inoculation test, the concentration of an oat agar culture medium suitable for symbiotic germination of cymbidium and Paphiopedilum hirsutissimum seeds is screened out in the Li Lu Bing (Li Lu Bing, 1998) earlier than 1998 in China. Researchers utilize the advantages of the oat agar culture medium and gradually adopt the culture medium to establish symbiosis so as to further carry out deep research on the symbiosis of mycorrhizal fungi and orchids.
With the growing understanding of orchid mycorrhizal fungi, researchers mostly adopt culture media to carry out co-culture for exploring cell and molecular basis of 'fungus-orchid plant' symbiotic relationship, such as Zhaming et al, the ceriporiopsis (Sebacina sp.) fungus and dendrobium officinale seeds are symbiotically germinated on oat agar culture media, SSH library is constructed, and the foundation is laid for the molecular level research of orchid plant seed and mycorrhizal fungus symbiotic germination (Zhao et al, 2012; Zhao et al, 2013). In past foreign research, Stewart and others successfully constructed mycorrhizal fungi and various orchid seeds of jatropha (Habenaria) and Spiranthes (Spiranthes breviatilis) to symbiote by using oat agar medium, and the seed germination rate is obviously improved (Stewart et al, 2002, 2003, 2006). In recent years, many foreign related studies have employed oat agar medium, such as co-culture of delphinium odoratum (Oncidium sphacelatum) seeds with corncob (Ceratobasidium sp) isolated from Coppensis doniana orchid, invasion of the Laelia glauca plant Serapias vomeraceae by Blastomyces (Tulasnella calospora) isolated from the orchid of the Orchidaceae (Anamptia laevigata), symbiosis of mycorrhiza fungi (Epulorrhiza sp) isolated from roots of Eupatorium and Polybola calamus (Coelogyne nervosa), both in vitro, and successful symbiosis of Valadares et al, 20142012, 2014, Perto et al, 2014 et al.
At present, no report exists in the prior art on screening of the jelly fungi capable of simultaneously and efficiently promoting germination of cattleya seeds and establishment of a corresponding symbiont system.
Disclosure of Invention
The invention aims to provide a method for establishing a symbiont system of Mucuna fungi for promoting germination of cymbidium and cattleya seeds.
The purpose of the invention is realized by the following technical scheme:
the method for establishing the symbiont system of the gloeosporium fungi for promoting germination of cymbidium harderium and cattleya seeds adopts a Tcs1 strain to establish symbiotic relationship with any one or more of the following orchid seeds so as to promote germination of the orchid seeds:
cymbidium forbesii seeds, cattleya YK seeds, cattleya YD seeds;
the Latin text of the Cymbidium bicolor is named Cymbidium mannii, the Latin text of the Cattleya hybrida is named Cattleya hybrida, and YK and YD are two varieties of the Cattleya hybrida;
the Tcs1 strain is an Alternaria mellea strain, and the Latin of the Alternaria mellea strain is named as: tulasnella calospora, which has been submitted to the China general microbiological culture Collection center with the accession number: CGMCC No. 14794.
According to the technical scheme provided by the invention, the establishment method of the symbiont system of the jelly fungi for promoting germination of the cymbidium and cattleya seeds provided by the embodiment of the invention adopts the jelly fungi and the orchid seeds to carry out symbiotic germination tests on an artificial culture medium, and compared with an orchid seed control group without inoculation, the mycorrhizal fungi capable of widely and efficiently promoting seed germination and a proper symbiotic culture medium are screened. The method has the advantages that the effective symbiotic fungi in the germination stage of the orchid seeds are obtained, the germination rate of the orchid seeds is improved by utilizing a symbiotic germination culture technology, and the establishment of a symbiotic system of the orchid and mycorrhizal fungi is a key link for developing symbiotic molecular mechanism research and production and propagation of the orchid, and has important significance for effective protection of the orchid.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a method for establishing a symbiont system of Mucuna fungi for promoting germination of cymbidium and cattleya seeds, which comprises the following steps:
the Tcs1 strain is adopted to establish symbiotic relationship with any one or more of the following orchid seeds to promote the germination of the orchid seeds:
cymbidium forbesii seeds, cattleya YK seeds, cattleya YD seeds;
the Latin text of the Cymbidium bicolor is named Cymbidium mannii, the Latin text of the Cattleya hybrida is named Cattleya hybrida, and YK and YD are two varieties of the Cattleya hybrida;
the Tcs1 strain is an Alternaria mellea strain, and the Latin of the Alternaria mellea strain is named as: tulasnella calospora, which has been submitted to the China general microbiological culture Collection center with the accession number: CGMCC No.14794, the preservation time is as follows: 11/14/2017, the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The symbiotic relationship is established in an oat agar culture medium.
The establishment time of the symbiotic relationship of the cymbidium forbesii seeds and the Tcs1 strain in a 2.0g/L oat agar culture medium is 21 days, and the germination rate of the cymbidium forbesii seeds after 60 days of cocultivation is 92.05 +/-2.45%;
the establishment time of the symbiotic relationship between the cattleya YK seeds and the Tcs1 strain in the oat agar culture medium with the concentration of 12.0g/L is 12 days, and the germination rate of the cattleya YK seeds is 87.13 +/-3.40% after 60 days of cocultivation;
the establishment time of the symbiotic relationship between the cattleya YD seeds and the Tcs1 strain in the oat agar culture medium with the concentration of 12.0g/L is 14 days, and the germination rate of the cattleya YD seeds is 75.63 +/-5.41 percent after 60 days of cocultivation.
The germination rate of the orchid seeds is extremely low under natural conditions, and mycorrhizal fungi are needed to participate in the whole growth and development process. The method adopts the Mucor fungus and the orchid seed to carry out symbiotic germination test on an artificial culture medium, and compares the symbiotic germination test with an orchid seed control group without inoculation, and screens the mycorrhizal fungus capable of widely and efficiently promoting the germination of the orchid seed and a proper symbiotic culture medium. The method has the advantages that the effective symbiotic fungi in the germination stage of the orchid seeds are obtained, the germination rate of the orchid seeds is improved by utilizing a symbiotic germination culture technology, and the establishment of a symbiotic system of the orchid and mycorrhizal fungi is a key link for developing symbiotic molecular mechanism research and production and propagation of the orchid, and has important significance for effective protection of the orchid.
The specific embodiment is as follows:
1. test materials
(1) Plant material: two varieties (YK, YD) of Cymbidium goeringii (Cymbidium mannii) and Cattleya hybrida.
(2) Fungus material: 7 strains of the fungus Mycoplasma orchidaceae mycorrhiza (Tulasnella spp.) obtained in the laboratory through many years of experiments are shown in Table 1.
2. Test method (symbiotic germination test of Orchidaceae plant seeds and Mucuna fungus)
(1) Preparation of culture Medium
Configuration of symbiotic medium (oat medium OMA): grinding oatmeal into powder, and respectively preparing the three graded oatmeal powders into solutions of 0.5g/L, 1.0g/L, 2.0g/L, 3.0g/L, 4.0g/L, 8.0g/L and 12.0g/L OMA. Boiling deionized water for 1h, filtering, adding 8.0g of agar, adding deionized water into the filtrate to reach the constant volume of 1000mL, and sterilizing at high temperature.
Preparing a fungus culture medium: 200g of peeled potatoes are cut into small pieces of 0.5-1 cm square, 800mL of deionized water is added and boiled for 30min, 4-5 layers of gauze are used for filtering to obtain filtrate, 20g of glucose and 8g of agar are added into the filtrate, the volume is determined to be 1000mL by using the deionized water, and streptomycin sulfate is added to the filtrate after high-temperature sterilization and the temperature is reduced to 50 ℃ until the final concentration is 50 mg/L.
(2) Activation and identification of Mucuna fungi
7 strains of orchidaceae mycorrhizal fungi obtained by the laboratory through years of experiments. And (3) selecting fungal hyphae to be cultured in a PDA culture medium at a constant temperature of 28 ℃ for 7-10 days in a dark mode. Carrying out morphological characteristic observation on the activated fungi; inoculating hypha at the edge of a strain on a PDA culture medium paved with a cellulose acetate membrane, culturing at a constant temperature of 28 ℃ for 7-10 days in dark, fully grinding after quick freezing by liquid nitrogen, extracting DNA by using an OMEGA kit, specifically amplifying ITS zones by PCR (polymerase chain reaction), wherein a PCR amplification primer is ITS 1F: 5'-TCCGTAGGTGAACCTGCGG-3', respectively; ITS 4R: 5'-TCCTCCGCTTATTGATATGC-3', respectively; the PCR products were sent to the company for sequencing, and after the sequence files were aligned using the software DNAMAN, the sequences were aligned by on-line BLAST (http:// www.ncbi.nlm.nih.gov).
(3) Disinfection of capsules
The capsule of orchid plant with the same flowering phase, pollination time and growth vigor is taken as a test material, the epicarp is lightly wiped with a cotton ball stained with 75% alcohol for 3 times, and the test material is wiped with the same method after being moved into an ultra-clean workbench for 3 times.
(4) Co-cultivation
After fungus activation, a puncher takes edge hypha to prepare a cylindrical fungus agar block with the diameter of 0.8cm and the height of about 0.5cm, and the fungus agar block is inoculated to a treatment group; the same size agar block was punched out of the sterilized PDA medium and inoculated into the control group.
Symbiotic germination of seeds: OMA is paved with a 3.5 × 3.5cm filter paper sheet with a middle hollow hole, orchid seeds are made into seed suspension, the seed density is counted by a blood counting plate method, and the seeds are uniformly sowed on the sterile filter paper sheet. The center of the treatment group was inoculated with fungal agar blocks, and the control group was inoculated with sterile agar blocks, which were plated in 15 dishes per concentration of medium, and incubated in the dark at 23. + -. 2 ℃.
(5) Detection of
According to Zettler et al, the seed germination phase criteria are detailed in Table 2. And regularly observing the germination condition of the seeds, and recording the germination time and the germination rate of the seeds. And (3) growing and developing the seeds of the orchid to be treated to the 3 rd stage, randomly selecting a plant sample every 2 days, dyeing the plant sample by Trypan Blue-water solution (0.05 percent), preparing a temporary pressed sheet, and observing the colonization condition of the fungus under an optical microscope. And (3) contrasting and screening the Mucuna fungus strains which can widely and efficiently promote the germination of the orchid seeds.
TABLE 2 seed Germination stages
Figure GDA0002591440490000061
3. Test results
(1) Identification of Mucor fungi
The 7 strains of the fungus of the genus mucilaginosus used in the tests all showed the following common culture characteristics on PDA medium: the bacterial colonies are grey white or light yellow, hypha processes grow in a circumferential divergent mode, some bacterial colonies can form concentric circular bacterial colony rings with unequal intervals, the surfaces of the bacterial colonies are wet, the texture is mild and fine, hypha can grow in a sinking mode, and some bacteria have loose white aerial hypha; the culture medium plate can grow to be full of PDA culture medium plates with the diameter of 9cm in 7-10 days; no sexual germ spores are formed.
The following common characteristics were observed under an optical microscope for all of the 7 strains of the genus Mycoplasma used in the test: the hypha has a septum; nearly right angle, the branch is slightly contracted, and a diaphragm is formed near the branch point of the hypha: has a structure of moniliform cell group formed by connecting barrel cells (asexual chlamydospores) in series.
The PCR identification results are shown in the following Table 1:
TABLE 1 alignment of ITS sequences of mycorrhizal fungi
Figure GDA0002591440490000071
(2) Mucor genus fungi and orchid seed symbiont forming
And (3) periodically extracting the orchid plant material co-cultured with the gloeosporium amansii to perform a trypan blue staining test, and checking whether a symbiotic relationship is established. The results show that the seeds are not dyed when not germinating, which indicates that the fungi are not used for successfully infecting the plants; when the seeds develop until meristematic tissue appears (stage 3), the plant is stained blue, and the stained parts are concentrated in the surface tissue of the plant; when the corm develops until the first true leaf appears (stage 4), the interior of the plant is stained blue and more mycelial clusters are formed inside.
The establishment time of the symbiotic relationship between the cymbidium bicolor and the Tcs1 strain in 2.0g/L OMA culture medium is the shortest, and is 21 days; the establishment of the symbiotic relationship with the Tss strain in 2.0g/L OMA medium was 28 days; the same time for establishing the symbiotic relationship was 56 days as that for the Tco2 strain in OMA medium of 8.0g/L and 12.0 g/L; the same time for establishment of the symbiotic relationship was 28 days as that of Tsk1 strain in OMA medium of 1.0g/L and 2.0 g/L. The establishment time of the symbiotic relationship between the cattleya YK and the Tcs1 strain in the OMA culture medium of 12.0g/L is the shortest, and is 12 days; the establishment of the symbiotic relationship with the Tss strain in 12.0g/L OMA medium was 21 days; the same time for establishing the symbiotic relationship in 12.0g/L OMA medium as that of Tco2 strain was 23 days; the establishment time of the symbiotic relationship between the cattleya YD and the Tcs1 strains in the OMA culture medium of 12.0g/L is shortest and is 14 days; the establishment of the symbiotic relationship with the Tss strain in 12.0g/L of OMA medium was 25 days; the establishment of the symbiotic relationship was carried out for 23 days in the same manner as that of the Tco2 strain in 12.0g/L of OMA medium. The result shows that the establishment time of the symbiotic relationship between the Tcs1 strain and 3 different orchids is shortest.
(3) Statistics of germination rate of fungus-promoted seeds
The cymbidium sclarea and cattleya seeds were co-cultured with 7 mycorrhizal fungi respectively at different OMA concentrations, and after 60 days, the germination of the seeds was observed and recorded, and when the seeds expanded to the 3 rd stage, the seeds were regarded as germination. The results show that Tcs1 strain can promote germination of 3 orchid seeds at all 7 OMA test concentrations; the Tss strain can promote germination of cymbidium haryngii seeds at medium OMA concentrations (3.0g/L, 4.0g/L) and at high OMA concentrations (8.0g/L, 12 g/L); the Tco2 strain can promote germination of 3 orchid seeds at high OMA concentrations (8.0g/L, 12.0 g/L); tsk1 strain can only promote germination of cymbidium duranum seeds at low and medium OMA concentrations (1.0g/L, 2.0g/L, 3.0g/L, 4.0 g/L). Neither inoculation Tsg1, Tcs2, Tcf1 nor Control (CK) germinated.
The significant difference analysis of the germination rates of 3 orchid seeds promoted by 4 mycorrhizal fungi under different OMA concentrations respectively shows that: firstly, the germination rates of cymbidium bicolor seeds promoted by Tcs1 strains in OMA culture media with different concentrations are very obvious, and the germination rate of the cymbidium bicolor seeds in 2.0g/L OMA is the highest and is 92.05 +/-2.45%; the Tcs1 strain promotes the germination rates of the YK and YD seeds of the cattleya in different concentrations of OMA culture medium to have very significant difference, and the germination rates of the YK and YD seeds of the cattleya in the OMA with the concentration of 12.0g/L are the highest, namely 87.13 +/-3.40% and 75.63 +/-5.41% respectively. ② the Tsk1 strains have very significant difference in promoting the germination rate of cymbidium forbesii seeds in OMA culture media with different concentrations, the germination rate of the OMA cymbidium forbesii seeds with the concentration of 3.0g/L is the highest, and is 76.55 +/-2.31%. ③ Tss strain promotes the highest germination rate of cymbidium forbesii seeds in OMA with the concentration of 3.0g/L, which is 61.17 +/-0.79% (3.0 g/LOMA); the highest germination rate of the cattleya YK seeds is 45.47 +/-1.58% (12.0g/L OMA), and the highest germination rate of the cattleya YD seeds is 50.45 +/-0.49% (12.0g/L OMA). The Tco2 strain promotes the germination rate of cymbidium bicolor and cattleya (YK and YD) seeds to be relatively low, the germination rate of cymbidium bicolor is 5.06 +/-0.03 percent (8.0g/L OMA) at most, the germination rate of YK of cattleya is 19.90 +/-1.66 percent (12.0g/L OMA) at most, and the germination rate of YD of cattleya is 25.90 +/-2.11 percent (12.0g/L OMA) at most
The technical scheme of the invention has the following beneficial effects:
the germination rate of the orchid seeds is extremely low under natural conditions, and mycorrhizal fungi are needed to participate in the whole growth and development process. The method for obtaining the effective symbiotic fungi at the germination stage of the seeds of the orchids is a key link for developing the regression of rare or endangered orchids, and has important significance for the effective protection of the orchids. In order to establish a symbiotic system of orchid and mycorrhizal fungi and screen out mycorrhizal fungi capable of promoting seed germination widely and efficiently and a proper symbiotic culture medium, the invention adopts the Mucuna fungus and orchid seeds to establish a symbiotic relationship and compares the symbiotic relationship with a control group without inoculation.
According to the experiment, the germination rate of the seeds of the orchids is counted, and the result shows that the jelly fish grass strain Tcs1 can promote the germination of the seeds of the 3 orchids under the whole OMA test concentration; by observing the growth of fungi and orchids, a medium is selected that maintains a balanced growth potential of both plants and microorganisms: the establishment time of the symbiotic relationship between the cymbidium forbesii seeds and the Tcs1 strain in 2.0g/L OMA is shortest, namely 12 days, and the germination rate is highest, namely 92.05 +/-2.45%; the germination time of the cattleya YK and Tcs1 strains in OMA with the concentration of 12.0g/L is shortest, 12 days, and the germination rate is the highest, and is 87.13 +/-3.40%; the germination time of the cattleya YD and Tcs1 strains in OMA with the concentration of 12.0g/L is shortest, 14 days, and the germination rate is the highest, and is 75.63 +/-5.41%.
The consistency of the Mucor fungus Tcs1 strain and Muscovitum Engelovii (Tulasnella calospora) with NCBI accession number of FJ613255.1 is 99% through ITS identification, the strain can establish symbiotic relationship with the seeds of cymbidium duranum and cattleya hybrida at the same time, and can efficiently promote germination of the seeds of cymbidium duranum and cattleya hybrida. The cymbidium bicolor is a periphyton type orchid plant in the orchid family and has high ornamental value, and the wild cymbidium bicolor is in imminent danger. Cattleya is a perennial herbaceous epiphyte of cattleya, native to central and south america, and is a treasure in orchidaceous ornamental plants. Under the condition that wild orchids are in imminent danger, the symbiotic fungus strain for promoting the cymbidium goeringii and cattleya to germinate into seedlings efficiently and the appropriate culture medium concentration are screened out, so that the method can provide technical reference for the symbiotic molecular mechanism research of orchids, provide guidance for the production and propagation of the cymbidium goeringii and cattleya, and has important significance for protecting species resources of the cymbidium goeringii and cattleya.
The technical key points of the invention are as follows:
the Tcs1 strain can establish a symbiotic relationship with cymbidium bicolor and cattleya seeds at the same time to promote the germination of the orchidaceae seeds;
the Tcs1 strain promoted germination of 3 orchid seeds at all OMA test concentrations;
the establishment time of the symbiotic relationship of the cymbidium forbesii seeds and the Tcs1 strain in a 2.0g/L oat agar culture medium is 21 days, and the germination rate of the cymbidium forbesii seeds after 60 days of total culture is 92.05 +/-2.45%;
the establishment time of the symbiotic relationship between the cattleya YK seeds and the Tcs1 strain in the oat agar culture medium with the concentration of 12.0g/L is 12 days, and the germination rate of the cattleya YK seeds is 87.13 +/-3.40 percent after 60 days of cocultivation;
the establishment time of the symbiotic relationship between the cattleya YD seeds and the Tcs1 strain in the oat agar culture medium with the concentration of 12.0g/L is 14 days, and the germination rate of the cattleya YD seeds is 75.63 +/-5.41 percent after 60 days of cocultivation.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (3)

1. A method for establishing symbiont system of Mucuna fungus for promoting germination of seeds of cymbidium and cattleya includes such steps as culturing the fungus in the culture medium, culturingTulasnella calospora) The Tcs1 strain establishes symbiotic relationship with any one or more of the following orchid seeds to promote germination of the orchid seeds:
cymbidium forbesii seeds, cattleya YK seeds, cattleya YD seeds;
the Latin of said cymbidium bicolor is namedCymbidium manniiThe Latin nomenclature of said cattleyaCattleya hybridaYK and YD are two varieties of cattleya;
the Tcs1 strain is submitted to the preservation in the China general microbiological culture Collection center, the registration number of the preservation center is: CGMCC No. 14794.
2. The method for establishing a symbiont system of Mucor fungi promoting germination of cymbidium and cattleya seeds as claimed in claim 1, wherein the symbiont relationship is established in oat agar medium.
3. The establishment method of the symbiont system of the jelly fungi for promoting the germination of the cymbidium duranum and the cattleya seeds as claimed in claim 2, wherein the establishment time of the symbiotic relationship between the cymbidium duranum seeds and the Tcs1 strain in a 2.0g/L oat agar culture medium is 21 days, and the germination rate is 92.05 +/-2.45%;
the establishment time of the symbiotic relationship between the cattleya YK seeds and the Tcs1 strain in the oat agar culture medium with the concentration of 12.0g/L is 12 days, and the germination rate is 87.13 +/-3.40%;
the establishment time of the symbiotic relationship between the cattleya YD seeds and the Tcs1 strain in the oat agar culture medium with the concentration of 12.0g/L is 14 days, and the germination rate is 75.63 +/-5.41 percent.
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