CN112760239B - Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof - Google Patents

Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof Download PDF

Info

Publication number
CN112760239B
CN112760239B CN202110293701.9A CN202110293701A CN112760239B CN 112760239 B CN112760239 B CN 112760239B CN 202110293701 A CN202110293701 A CN 202110293701A CN 112760239 B CN112760239 B CN 112760239B
Authority
CN
China
Prior art keywords
seeds
php86
paphiopedilum
hirsutissimum
culture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202110293701.9A
Other languages
Chinese (zh)
Other versions
CN112760239A (en
Inventor
姚娜
曹晓璐
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Research Institute of Forestry of Chinese Academy of Forestry
Original Assignee
Research Institute of Forestry of Chinese Academy of Forestry
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Research Institute of Forestry of Chinese Academy of Forestry filed Critical Research Institute of Forestry of Chinese Academy of Forestry
Priority to CN202110293701.9A priority Critical patent/CN112760239B/en
Publication of CN112760239A publication Critical patent/CN112760239A/en
Application granted granted Critical
Publication of CN112760239B publication Critical patent/CN112760239B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/60—Flowers; Ornamental plants
    • A01G22/63—Orchids
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Dentistry (AREA)
  • Biomedical Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention relates to a glue film bacterium for promoting germination of paphiopedilum hirsutissimum seeds to form seedlings, and a culture method and application thereof, and belongs to the technical field of orchid seedling culture. The invention provides a strain of paphiopedilum hirsutissimum (Tulasnella sp.) Php86 for promoting paphiopedilum hirsutum seeds to germinate and form seedlings, wherein the preservation number of the strain Php86 is CGMCC No. 21082. The glue film bacteria Php86 can remarkably promote paphiopedilum hirsutissimum seeds to develop into healthy seedlings with complete root systems, greenhouse seedling culture or field regression of the paphiopedilum hirsutissimum seeds can be realized, 130d of pollinated seeds and Php86 are symbiotically germinated and rooted to form seedlings, and the seedling formation rate is as high as 9.04%.

Description

Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof
Technical Field
The invention relates to the technical field of orchid seedling culture, and particularly relates to a strain of paphiopedilum hirsutissimum which promotes paphiopedilum hirsutissimum seeds to germinate and form seedlings, and a culture method 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. All wild orchids are currently listed in International trade convention on endangered species of wild animals and plants (CITES). Paphiopedilum (Paphiopedilum) is the most distinctive group of orchids and has high ornamental value. Paphiopedilum has now become one of the endangered plant species due to destruction and excessive collection of the natural habitat.
In order to realize the recovery and conservation of wild paphiopedilum populations, paphiopedilum seed sterile germination research is carried out, and large-scale propagation of paphiopedilum is expected to be realized through seed sterile germination and tissue culture, but paphiopedilum tissue culture is still in a starting research stage, so that the problems of low survival rate of tissue culture seedlings after bottle-out transplantation, poor stress resistance and the like exist, and the field regression of populations is limited; therefore, the method simulates seed germination in a natural environment, and seedlings are obtained through symbiotic germination of seeds and mycorrhizal fungi, so that the method becomes an important technical approach for obtaining a large number of paphiopedilum seedlings for field regression. However, because the mature period of the paphiopedilum seeds is long, the difference of the suitable sowing periods among different species is large, the symbiotic germination technical system of paphiopedilum seeds is not complete, the reported symbiotic germination is mostly from the development stage to the protocorm stage, and the reports of quickly and stably obtaining a large number of rooted seedlings through symbiotic germination are few.
Paphiopedilum hirsutissimum is overground or epiphytic on stone, is mainly distributed from north to west of Guangxi, southwest of Guizhou and southeast of Yunnan in China, and has higher ecological value and ornamental value because sepals and petals are purple to purple brown. The symbiotic germination efficiency of the seeds of the paphiopedilum is low, and most researches ignore the influence of the maturity of the seeds on the germination efficiency.
Disclosure of Invention
The invention aims to provide a strain of paphiopedilum hirsutissimum for promoting germination of paphiopedilum hirsutissimum seeds to form seedlings, and a culture method and application thereof. The glue film bacterium Php86 can remarkably promote paphiopedilum hirsutissimum seeds to develop into healthy seedlings with complete root systems, and can realize greenhouse seedling culture or field regression of paphiopedilum hirsutissimum seeds.
The invention provides a strain of Achillea Wilsonii (Tulasnella sp.) Php86 for promoting germination of paphiopedilum hirsutum seeds to form seedlings, wherein the preservation number of the Achillea Wilsonii Php86 is CGMCC No. 21082.
The invention also provides a culture method of the metlbostemma strain Php86 in the technical scheme, which comprises the following steps: the Mucor Php86 is inoculated in the culture medium for culture.
The invention also provides application of the glue film bacterium Php86 in promoting germination of paphiopedilum hirsutissimum seeds to form seedlings.
The invention also provides application of the colletotrichum Php86 in promoting rooting of paphiopedilum hirsutissimum seeds.
The invention also provides application of the glue film bacteria Php86 in promoting greenhouse seedling culture or field regression of paphiopedilum hirsutissimum seeds.
The invention also provides a symbiotic germination method of the glue film bacteria Php86 and paphiopedilum hirsutissimum in the technical scheme, which comprises the following steps:
co-culturing seeds of paphiopedilum hirsutissimum with hymenophora Php 86; the seeds of paphiopedilum hirsutissimum are harvested more than 110 days after pollination.
Preferably, the seeds of paphiopedilum hirsutissimum are harvested 130d or more after pollination.
Preferably, the co-culturing comprises: seeds of paphiopedilum hirsutissimum are sowed in a culture medium, inoculated with mucilaginosus Php86 and subjected to symbiotic germination culture.
Preferably, the culture medium comprises OMA plate medium; the OMA plate medium included 3.0g/L oat flour and 7.0g/L agar.
Preferably, the co-cultivation conditions include: the illumination period is 16h and 8 h; the illumination intensity is 1500-2000 lx; the temperature is 23-27 ℃.
The invention provides a strain of glue film bacteria for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings. The glue film bacterium Php86 can remarkably promote paphiopedilum hirsutissimum seeds to develop into healthy seedlings with complete root systems, and can realize greenhouse seedling culture or field regression of paphiopedilum hirsutissimum seeds. Test results show that symbiotic germination tests are carried out on paphiopedilum hirsutissimum seeds and hymenophora Php86 at different periods, seeds 120d and above can symbiotically germinate with Php86 and grow roots to form seedlings after pollination, wherein after the seeds 130d symbiotically germinate and root with Php86 to form seedlings, the seedling formation rate is up to 9.04 percent and is obviously higher than that of non-symbiotic germination on a RE1 culture medium; the efficiency of Php86 in promoting seedling formation gradually decreased with the increase of seed maturation time, but healthy seedlings with intact root systems could still be obtained above non-symbiotic germination on RE1 medium.
Biological preservation Instructions
Mucor pulmonale (Tulasnella sp.) Php86 is deposited in China general microbiological culture Collection center (CGMCC) at 03.12.2020, with the unit of CGMCC for short, and the address of No.3 Ceh West Lu 1 of the Kyoho area in Beijing, and the number of the deposited microorganism is CGMCC No.21082 at the institute of microbiology of China academy of sciences.
Drawings
FIG. 1 is a colony morphology and hyphal microstructure of Php86 provided by the present invention;
FIG. 2 is a phylogenetic tree of Mucor based on rDNA-ITS sequence according to the present invention;
FIG. 3 shows the efficiency of mycorrhizal fungi strain Php86 in promoting symbiotic germination of paphiopedilum hirsutum provided by the present invention;
FIG. 4 shows symbiotic germination and mycelial mass formation of paphiopedilum hirsutissimum provided by the present invention.
Detailed Description
The invention provides a strain of Achillea Wilsonii (Tulasnella sp.) Php86 for promoting germination of paphiopedilum hirsutum seeds to form seedlings, wherein the preservation number of the Achillea Wilsonii Php86 is CGMCC No. 21082. The jelly membrane fungus (Tulasnella sp.) Php86 is preserved in China general microbiological culture Collection center (CGMCC) in 12 months and 03 days in 2020, and is addressed to Xilu No.1 Beichen of Indormitory, Beijing, and institute of microbiology, China academy of sciences. The invention separates roots of adult plants of Paphiopedilum (paphiopediluum rhizotomatum) to obtain a strain of glued membrane fungus Php 86. The bacterial colony is light yellow brown, the edge is radial, and hyphae sink and grow; the hyphae have septa, branches are nearly right-angled, and the branches are slightly contracted. By ITS sequencing, the ITS sequence of the Mycoplasma capsulatum Php86 is shown in SEQ ID NO. 1: TTAACACCCGCATGTGCACTCCTTAACACAATTACACACCTGTGAACCTCGAACCGTGTCTGTGTGCTGATCCCGTAGGGAGATGCGTGCGCGCGCGTTCTTACTTTACAAAACCCTGTTAACTAAAGCTCTAGAAAGTGTTGGTTAATAAAAAACAACCATCAGCAACGGATCTCTTGGCATTCCAATCGATGAAGAACGTAGCGAATTGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCTCTGGTATTCCGGAGGGCATGCCCGGTTGAGTGTCATGAATATCTCAACTCTAACGTTTTGTTAACGGTCTTGTGGGTGAAGCCTGTGGTCTAGCGAGATCGCAGGTTGTAATCTTTAGGACTGAAGTGTTAGAGATTGGACTTGAGTTTTGTTGGCCCGCTGGGTTGACTGACTCGAAATTGATTAGCGATGCGTGATCCATTGGGTCCATCTCGGCGTGATAAGTTGATCGCTGTAAAGGAACCATGTGGGTGTGGCTTGCTTCTAATCGTCTTAGGACAGCTT phylogenetic tree is shown in FIG. 2. The Mucor rhinelloides Php86 has a relatively distant relationship with the published Mucor rhinelloides fungi and is a potential new species of Mucor.
The invention also provides a culture method of the metlbostemma strain Php86 in the technical scheme, which comprises the following steps: the Mucor Php86 was inoculated into the culture medium for culture. In the present invention, the medium is preferably a PDA medium. In the present invention, the time for the culture is preferably 20 days.
The invention also provides application of the glue film bacterium Php86 in promoting germination of paphiopedilum hirsutissimum seeds to form seedlings. The prior art is to the symbiotic culture of paphiopedilum hirsutissimum, and it is limited to obtain the seedling efficiency of taking root, and it is not clear yet to the best seeding period of seed, and this application glued membrane fungus Php86 can obtain healthy seedling through symbiotic culture with paphiopedilum hirsutissimum, is applied to subsequent greenhouse seedling or field regression, can solve aseptic seeding can not obtain the seedling or the seedling that obtains is poor to environmental suitability, technical problem such as adverse resistance low.
The invention also provides application of the colletotrichum Php86 in promoting rooting of paphiopedilum hirsutissimum seeds.
The invention also provides application of the colletotrichum Php86 in promoting greenhouse seedling raising or field returning of paphiopedilum hirsutissimum seeds.
The invention also provides a symbiotic germination method of the glue film bacteria Php86 and paphiopedilum hirsutissimum in the technical scheme, which comprises the following steps:
co-culturing seeds of paphiopedilum hirsutissimum with hymenophora Php 86; the seeds of paphiopedilum hirsutissimum are harvested for more than 120 days after pollination.
In the present invention, the seeds of paphiopedilum hirsutissimum are preferably seeds harvested 130d or more after pollination, and more preferably seeds harvested 130d after pollination.
In the present invention, the co-cultivation preferably comprises: seeds of paphiopedilum hirsutissimum are sowed in a culture medium, inoculated with mucilaginosus Php86 and subjected to symbiotic germination culture. In the present invention, the medium preferably comprises an OMA plate medium; the OMA plate medium preferably includes 3.0g/L oat flour and 7.0g/L agar.
In the present invention, the co-cultivation conditions preferably include: the illumination period is 16h and 8 h; the illumination intensity is 1500-2000 lx; the temperature is 23-27 ℃.
The RE1 culture medium is a culture medium which has a good promoting effect on the germination of paphiopedilum seeds. In the embodiment of the invention, a negative control (OMA culture medium) and a positive control (a non-symbiotic germination culture medium, here, RE1) are arranged, 130d of paphiopedilum hirsutissimum seeds after pollination are cultured on the OMA culture medium for 90d, only protocorms which germinate to the 2 nd stage can be observed, and seedlings cannot be formed by germination, which indicates that Php86 has a promotion effect on the germination of the seeds to form the seedlings; protocorms and seedlings can be formed by culturing for 90 days on an RE1 culture medium, but the seedlings are slimy and difficult to root; in the invention, Php86 has the highest symbiotic germination efficiency with 130d paphiopedilum hirsutissimum seeds after pollination, the seedling formation rate can reach 9.04%, and compared with non-symbiotic germination on a RE1 culture medium, the seedling root system has better development, and can be used for greenhouse seedling culture or field regression.
The present invention provides a strain of paphiopedilum hirsutissimum for promoting germination of paphiopedilum hirsutissimum seeds to form seedlings, a culture method thereof and applications thereof.
Example 1
Materials and methods
Material
Mycorrhizal fungi: isolated from the roots of adult greenhouse-cultivated plants of the rhizome Paphiopedilum (paphiopediluum rhizotomatosum).
Paphiopedilum capsule: paphiopedilum hirsutissimum (paphiopediococcum hirsutissimum) plants are cultivated in scientific research greenhouses of China forestry scientific research institute, self-pollination is carried out in the initial flowering period, and capsules are respectively collected at 110d, 120d, 130d, 140d, 150d, 160d, 170d and 180d after pollination for symbiotic germination of seeds.
Isolation of the Strain
The paphiopedilum root fungi is separated by adopting a tissue section method. Collecting healthy and plump paphiopedilum root segments, washing off attachments in flowing water, scraping down villi of root quilt with a blade, making a bare-handed slice, observing the fungal colonization condition under a microscope, and carrying out surface disinfection on the root segments near the slice if fungal colonization exists: soaking in 75% ethanol for 30s, washing with sterile water for 1 time, sterilizing the surface of NaClO solution containing 1% of available chlorine for 5min, washing with sterile water for 5 times, and drying with sterile filter paper. Cutting the root segment into thin slices with thickness of about 0.5mm with sterile blade, spreading on PDA culture medium, culturing at 25 deg.C in dark, picking edge hyphae when hyphae grow out from the root segment, transferring to PDA culture medium for purification, inoculating the purified hyphae on slant of PDA test tube, and storing at 4 deg.C.
Molecular characterization of strains
Inoculating the separated strain to a PDA culture medium paved with cellophane, culturing for 20d, scraping a small amount of hyphae by using a sterile gun head, and extracting DNA by a CTAB method for PCR amplification. The amplification primers were ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO.2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO. 3). A40. mu.L PCR amplification reaction system was used: template 2.0. mu.L, 10 XEx Taq Buffer 4.0. mu.L, dNTP mix 3.0. mu.L, primers (10. mu. mol/L) each 0.4. mu.L, TaKaRa Ex Taq 0.3. mu.L, ddH2O make up to 40. mu.L. PCR amplification procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 95 ℃ for 40s, annealing at 56 ℃ for 40s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min. And (3) carrying out electrophoresis detection on the PCR product, then delivering the PCR product to Shanghai biological engineering (Shanghai) corporation for bidirectional sequencing, splicing the sequences, then comparing the sequences in an NCBI database, selecting the sequences with higher similarity for molecular identification, and constructing a phylogenetic tree by using a Maximum Likelihood Method (ML).
Symbiotic germination of seeds
Symbiotic germination of seeds: wiping the surface of a mature uncracked capsule with 75% ethanol, transferring to an ultra-clean bench, treating the surface of the pericarp with 75% ethanol for 30s, washing with sterile water for 5 times, drying the water by a filter paper, splitting the pericarp with a sterile scalpel, taking out the seed, uniformly sowing on an OMA plate culture medium (OMA: 3.0g/L oat flour +7.0g/L agar, natural pH value), and inoculating 1 bacterial block with the diameter of 0.5cm in the center of the culture medium.
OMA control: OMA plate medium was inoculated with only seeds and no fungi.
RE1 control: RE1 was selected as a control for seed sterile germination: RE medium +200ml/L coconut juice, RE1 plate medium only inoculated with paphiopedilum hirsutissimum seeds, not inoculated with fungi.
RE culture medium: NH (NH)4NO3400mg/L，(NH4)2SO4150mg/L，Ca(NO3)2150mg/L，Mg(NO3)2·6H2O 100mg/L，KNO3300mg/L，KH2PO4300mg/L，Na2EDTA37.3mg/L，FeSO4·7H2O27.8 mg/L, mature banana homogenate 100mg/L, activated carbon 2.0g/L, fructose 20.0g/L, distilled water to 1000ml, agar 16.0 g/L.
Seedling medium MS 2: 1/4MS +7.5g/L sucrose +6.0g/L +0.1g/L activated carbon. Each treatment was repeated 3 times.
Symbiotic germination culture conditions: the illumination period is 16h/8h (light/dark), the illumination intensity is 1500-2000 lx, and the temperature is 25.0 +/-2.0 ℃.
Symbiotic germination efficiency assessment
Paphiopedilum seeds germinate and are 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, level: the appearance of primary tissues; 4, level: differentiating to obtain leaves; and 5, stage: the leaves continue to elongate, and true roots grow out to form seedlings.
Counting the germination conditions of seeds: the number of ungerminated, germinated only (g), protocorm (p) and seedling(s) was counted in stages 0, 1, (2+3) and (4+5), respectively, and the sum of the number of stages was the total number of seeds sown (t). Protocorm formation rate (P) and seedling formation rate (S) were calculated as follows, respectively: p is 100 Xp/t, S is 100 Xs/t. The statistical time is 90 days after sowing.
The percentage results were square root transformed by arcsine and then tested by one-way variance (ANOVA) and Duncan (Duncan) multiple ranges at P <0.05 using SPSS 16.0.0 software and expressed as mean ± standard deviation.
Fungus specific staining
Fixing protocorms in stage 3 of symbiotic germination in FAA solution at 4 deg.C for at least 24h, sucking off the fixing solution, and washing with distilled water. Adding 10% (W/V) KOH solution into the washed protocorm, autoclaving at 121 deg.C for 20min, neutralizing with 2% (V/V) HCl for 5min, transferring the protocorm into staining solution (10% (V/V) pekoe black ink and 3% (V/V) acetic acid), boiling for 30min, removing the staining solution, adding lactic acid, decolorizing, and storing. The colonization condition of the mycelium pellet is observed under an Olympus BX41 microscope, and a picture is taken by an E3CMOS06300KPA microscope imaging system.
Results and analysis
Isolation and identification of mycorrhizal fungi
1 mycorrhizal fungus Php86 was isolated from roots of paphiopedilum rhizogenes. Culturing on PAD medium for 20d (FIG. 1, Php86 colony morphology and hypha microstructure; wherein, a: Php86 colony morphology and b: Php86 hypha microstructure), the colony is light yellow brown, the edge is radial, hypha sinks to grow, a small amount of aerial hypha is generated after culturing for 40d, and the growth rate of the colony is 0.73 +/-0.07 mm/d.
The ITS sequence alignment result shows that the fungus with the highest similarity with the strain is Tulasnellaceae clone PM15(GQ241830) of paphiopedilum harderianum root non-cultured Muscovieae. Phylogenetic analysis shows (figure 2, a phylogenetic tree of a part of the Mucor based on rDNA-ITS sequences, wherein the value of a branch point is the support rate obtained by repeating 1000 times of estimation by a Bootstrap method, and only shows that the Bootstrap is more than or equal to 50 percent), Php86 has a far-away genetic relationship with published Mucor fungi and is a potential new species of the Mucor.
The invention provides a Mucuna fungus capable of effectively promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, the difference between the colony morphology of the Mucuna fungus and the reported mycorrhizal fungus capable of promoting paphiopedilum seeds to symbiotically germinate is larger, and the ITS sequence phylogenetic analysis shows that the strain has a far distant genetic relationship with common species of the Mucuna fungus and is a potential new fungus.
Influence of mycorrhizal fungi on germination and seedling formation of paphiopedilum seeds in different periods
And (3) symbiotically germinating paphiopedilum seeds and Php86 at different periods after pollination, simultaneously inoculating the paphiopedilum seeds on an OMA culture medium and an RE1 culture medium to serve as controls, and counting protocorm formation rate and seedling formation rate after culturing for 90 days. FIG. 3 is a graph showing the results of the mycorrhizal fungi strain Php86 on the efficiency of promoting symbiotic germination of paphiopedilum hirsutissimum; wherein, a: the formation rate of symbiotic germination protocorms of paphiopedilum hirsutissimum seeds with different maturity; b: the formation rate of symbiotic germination seedlings of paphiopedilum hirsutissimum seeds with different maturity is increased; c: php86 promotes efficiency in formation of protocorms and seedlings of paphiopedilum hirsutissimum seeds at 130d after pollination.
The results show (a and b in figure 3) that the paphiopedilum hirsutum seeds germinate in symbiosis with Php86, and the protocorm forming rate and the seedling forming rate of the seeds at different periods after pollination are different; at 130d after pollination, the efficiency of seed formation to protocorms and seedlings both peaked and then gradually decreased over time. The protocorm forming rate of the seeds inoculated on the OMA culture medium does not change remarkably with different periods after the seeds are pollinated, and the seeds are difficult to germinate to form seedlings.
The paphiopedilum seeds at 130d after pollination and Php86 symbiotically germinate, and compared with germination on an OMA culture medium, the germination of paphiopedilum hirsutum seeds to form protocorms and seedlings can be obviously promoted; compared with germination on an RE1 culture medium, the forming efficiency of the Php86 symbiotic germination protocorm is lower, but the forming efficiency of the rooting seedling is obviously higher than that of an RE1 culture medium and can reach 9.04 percent; the seeds germinated on RE1 medium formed a large number of protocorms, but at the stage of developing into seedlings, the leaves were slender and the root system was underdeveloped (c in FIG. 3).
FIG. 4 shows symbiotic germination and mycelial mass formation of paphiopedilum hirsutum; wherein, a: symbiotic germination of paphiopedilum hirsutissimum and Php 86; b: germination of paphiopedilum hirsutissimum on RE1 medium; c: germination of paphiopedilum hirsutissimum on OMA medium; d: symbiotically germinating rooted seedlings of paphiopedilum hirsutissimum and Php 86; e: germinating paphiopedilum hirsutissimum on RE1 culture medium to obtain young seedling; f: php86 colonizes paphiopedilum protocorm cells.
Php86 symbiotically germinates with 130d pollinated paphiopedilum hirsutissimum seeds, and after 20d sowing, the germinated protocorm can be observed, which is a grade 2 germination stage; after about 30 days of culture, the seed coat is broken and enters a grade 3 germination stage; culturing for about 40 days, forming obvious protrusions at the apical meristem of the protocorm, enabling the base of the protocorm to generate a false root, and entering a 4-stage germination stage; culturing for about 50 days, growing the 1 st true leaf from the opposite side of the scutellum, and entering a grade 5 germination stage; after about 60 days of culture, rooted seedlings (a and d in FIG. 4) were observed, and the rooted seedlings were transferred to MS2 medium for further culture to obtain healthy paphiopedilum hirsutum seedlings. Section and fungal specific staining observations showed that Php86 colonized paphiopedilum-bearing protocorm cells as intracellular mycelial mass (f in fig. 4), with mycelial colonized cells mainly distributed at the base of protocorm. The colonization of Php86 in the form of a mycelial mass within protocorm cells was observed in the present invention by fungus-specific staining, a symbiotic pattern typical of orchidaceae mycorrhiza.
130d postpollination, 90d culture of paphiopedilum hirsutissimum seeds on RE1 medium resulted in protocorm and seedling formation, but seedling was slimy and difficult to root (b and e in FIG. 4). After pollination, 130d paphiopedilum seeds were cultured on OMA medium for 90d, and only protocorms that germinated to stage 2 could be observed, and no seedlings could be germinated (c in FIG. 4).
The natural germination rate of paphiopedilum is low, the aseptic germination is greatly influenced by the maturity of seeds, the germination rate of the seeds 130d after pollination of paphiopedilum with leaves is highest, and then the germination rate gradually decreases along with the maturation of the seeds. In the invention, the Php86 has the highest symbiotic germination efficiency with 130d paphiopedilum hirsutissimum seeds after pollination, the seedling formation rate can reach 9.04%, and compared with non-symbiotic germination on a RE1 culture medium, the seedling root system has better development, and can be used for greenhouse seedling culture or field regression.
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
<110> forestry research institute of China forestry science research institute
<120> paphiopedilum hirsutissimum strain for promoting germination of paphiopedilum hirsutissimum seeds to form seedlings and culture method and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 560
<212> DNA
<213> Mycoplasma gondii (Tulasnella sp.)
<400> 1
ttaacacccg catgtgcact ccttaacaca attacacacc tgtgaacctc gaaccgtgtc 60
tgtgtgctga tcccgtaggg agatgcgtgc gcgcgcgttc ttactttaca aaaccctgtt 120
aactaaagct ctagaaagtg ttggttaata aaaaacaacc atcagcaacg gatctcttgg 180
cattccaatc gatgaagaac gtagcgaatt gcgataagta atgtgaattg cagaattcag 240
tgaatcatcg aatctttgaa cgcaccttgc gccctctggt attccggagg gcatgcccgg 300
ttgagtgtca tgaatatctc aactctaacg ttttgttaac ggtcttgtgg gtgaagcctg 360
tggtctagcg agatcgcagg ttgtaatctt taggactgaa gtgttagaga ttggacttga 420
gttttgttgg cccgctgggt tgactgactc gaaattgatt agcgatgcgt gatccattgg 480
gtccatctcg gcgtgataag ttgatcgctg taaaggaacc atgtgggtgt ggcttgcttc 540
taatcgtctt aggacagctt 560
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tccgtaggtg aacctgcgg 19
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tcctccgctt attgatatgc 20

Claims (9)

1. A strain of glue film bacterium (Tulasnella sp.) Php86 for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings is characterized in that the preservation number of the glue film bacterium Php86 is CGMCC No. 21082; the seeds of paphiopedilum hirsutissimum are harvested over 130 days after pollination.
2. The cultivation method of the glued membrane fungus Php86 as claimed in claim 1, comprising the following steps: the Mucor Php86 is inoculated in the culture medium for culture.
3. Use of the colletotrichum Php86 according to claim 1 for promoting germination of paphiopedilum hirsutissimum seeds to form seedlings.
4. Use of the colletotrichum Php86 according to claim 1 for promoting rooting of paphiopedilum hirsutissimum seeds.
5. The use of the colletotrichum Php86 as claimed in claim 1 for promoting greenhouse seedling culture or field returning of paphiopedilum hirsutissimum seeds.
6. The method for symbiotic germination of the colletotrichum gloeosporioides Php86 and paphiopedilum hirsutissimum of claim 1, comprising the following steps:
co-culturing seeds of paphiopedilum hirsutissimum with hymenophora Php 86; the seeds of paphiopedilum hirsutissimum are harvested over 130 days after pollination.
7. The method of claim 6, wherein the co-culturing comprises: seeds of paphiopedilum hirsutissimum are sowed in a culture medium, inoculated with mucilaginosus Php86 and subjected to symbiotic germination culture.
8. The method of claim 7, wherein said culture medium comprises OMA plate medium; the OMA plate medium included 3.0g/L oat flour and 7.0g/L agar.
9. The method of claim 6, wherein the co-culturing conditions comprise: the illumination period is 16h and 8 h; the illumination intensity is 1500-2000 lx; the temperature is 23-27 ℃.
CN202110293701.9A 2021-03-19 2021-03-19 Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof Active CN112760239B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110293701.9A CN112760239B (en) 2021-03-19 2021-03-19 Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110293701.9A CN112760239B (en) 2021-03-19 2021-03-19 Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof

Publications (2)

Publication Number Publication Date
CN112760239A CN112760239A (en) 2021-05-07
CN112760239B true CN112760239B (en) 2022-05-13

Family

ID=75691298

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110293701.9A Active CN112760239B (en) 2021-03-19 2021-03-19 Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof

Country Status (1)

Country Link
CN (1) CN112760239B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103087927A (en) * 2013-01-21 2013-05-08 北京林业大学 Fungus for promoting symbiotic germination of paphiopedilum hirsutissimum seed and application thereof
CN105861330A (en) * 2016-06-02 2016-08-17 广西壮族自治区农业科学院花卉研究所 Bacterial strain for promoting growth of paphiopedilum hirsutissimum plant and application thereof
CN111793567A (en) * 2020-08-11 2020-10-20 云南大学 Mucoraceae fungus and application thereof in promoting paphiopedilum brandisil seeds to germinate and form seedlings
CN111876336A (en) * 2020-08-11 2020-11-03 云南大学 Mucuna fungus and application thereof in promoting germination of paphiopedilum brandisil seeds to form seedlings

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103087927A (en) * 2013-01-21 2013-05-08 北京林业大学 Fungus for promoting symbiotic germination of paphiopedilum hirsutissimum seed and application thereof
CN105861330A (en) * 2016-06-02 2016-08-17 广西壮族自治区农业科学院花卉研究所 Bacterial strain for promoting growth of paphiopedilum hirsutissimum plant and application thereof
CN111793567A (en) * 2020-08-11 2020-10-20 云南大学 Mucoraceae fungus and application thereof in promoting paphiopedilum brandisil seeds to germinate and form seedlings
CN111876336A (en) * 2020-08-11 2020-11-03 云南大学 Mucuna fungus and application thereof in promoting germination of paphiopedilum brandisil seeds to form seedlings

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Isolation and identification of Rhizoctonia-like fungi from roots of three orchid genera, Paphiopedilum, Dendrobium, and Cymbidium, collected in Chiang Rai and Chiang Mai provinces of Thailand;Sureeporn Nontachaiyapoom，et al;《Mycorrhiza》;20101031;全文 *

Also Published As

Publication number Publication date
CN112760239A (en) 2021-05-07

Similar Documents

Publication Publication Date Title
CN103087927B (en) Fungus for promoting symbiotic germination of paphiopedilum hirsutissimum seed and application thereof
CN107460133B (en) Dark color has every endogenetic fungus HS40 and its application in dendrobium candidum production
CN112725191B (en) Inonotus tumefaciens strain for promoting germination of orchidaceae seeds and application thereof
US20220369648A1 (en) Endophytic falciphora oryzae fo-r20 and its application
CN115537346B (en) Mucillus mucilaginosus for promoting growth and differentiation of sansevieria trifasciata and application thereof
KR20130056585A (en) Plant growth promotion by using bacterial strains isolated from roots of miscanthus sacchariflorus
CN111876336B (en) Mucuna fungus and application thereof in promoting germination of paphiopedilum brandisil seeds to form seedlings
CN109536391B (en) Fungus for promoting growth of protocorm of phalaenopsis and application thereof
CN111084104A (en) Method for rapidly propagating orange-heart ginger flowers
WO2024083061A1 (en) Method for breeding improved variety of forage sorghum-bacillus megaterium symbiont
CN102168106A (en) Transgenic method capable of controlling ALA synthesis in plants and promoting growth and stress resistance
CN103190346A (en) Method for constructing corn reproduction system taking coleoptile section as explant
CN117568190A (en) Strain for promoting germination of edible tulip seeds and method for rapidly breeding seedlings
KR101905674B1 (en) Novel Mycena purpureofusca NIFOS101 strain having excellent germination property of Gastrodia elata seeds and its use
Prasad et al. Pathogenicity and characterization of Rhizoctonia solani Kühn Inciting wet root rot in chickpea
CN101904302B (en) Method for somatic cell embryogeny and plant regeneration of medicinal plant schisandga chinensis baill
CN112760239B (en) Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof
CN114395485B (en) Adhesive film fungus strain TP-2 capable of promoting stem thickness of dendrobium nobile and application
CN114480143B (en) Trichoderma harzianum M6 for preventing and treating sclerotinia sclerotiorum of sunflower and application thereof
CN115287194B (en) Medicinal wild rice endophytic fungi YYA21 and application thereof
CN113249429A (en) Large-scale rice blast resistance identification method suitable for breeding application
CN1582641A (en) Tetraploid transgenic locust and quick cultivation
CN101836591A (en) Method for culturing axillary bud tissue of strawberry
KR102577368B1 (en) Novel Mycena sp. strain having germination property of Gastrodia elata seed and production method of sexual immature rhizomes of Gastrodia elata using the same
CN116849125B (en) Method for in-vitro cultivation of wild tomato polyploid

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant