CN103468588A - Tulasnella strain and application thereof in promotion of germination of Dendrobium aphyllum seeds - Google Patents
Tulasnella strain and application thereof in promotion of germination of Dendrobium aphyllum seeds Download PDFInfo
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Abstract
The invention relates to a Tulasnella strain [CGMCC (China General Microbiological Culture Collection Center) No.7552]. According to the invention, the nrDNA ITS (Nuclear Ribonucleic Deoxyribonucleic Acid Internal Transcription Sequence) sequence of the Tulasnella strain is submitted to the database of National Center of Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/), and the sequence number of the strain is KC796463.1. The Tulasnella strain (CGMCC No.7552) is applied to promotion of germination of Dendrobium aphyllum seeds. The Tulasnella strain forms a symbiotic relationship with the protocorm so as to promote the germination of Dendrobium aphyllum seeds and grow into seedlings. Experiments verify that the strain (CGMCC No.7552) not only can remarkably promote the Dendrobium aphyllum seeds to germinate (under the light condition of 97.92+/-0.51% and under the dark condition of 89.34+/-5.89%), but also can remarkably promote formation of protocorm (under the light condition of 91.82+/-3.03% and under the dark condition of 79.90+9.56%) as well as remarkably promote the protocorm to grow to seedlings subsequently.
Description
Technical field
The invention belongs to biological technical field, relate to and a kind ofly can form with the symbiosis of Dendrobium aphyllum (Roxb.) C. E. Fisch． (Dendrobiumaphyllum) protocorm fungal bacterial strain glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) of symbiotic relationship, and, in the application promoted on the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination, relate in particular to and a kind ofly can form symbiotic relationship with the Dendrobium aphyllum (Roxb.) C. E. Fisch． protocorm and promote its seed germination and grow to effective fungi in seedling stage.
Background technology
The seed of orchid is very tiny, and the embryo of ateliosis is only arranged, and seed germination need to rely on specific symbiosis fungi to obtain nutritive substance under field conditions (factors).The sprouting of Orchid Seeds can be cultivated and two kinds of modes of symbiotic germination cultivation by non-symbiosis germination.Although most of orchids can be cultivated by non-symbiosis germination, and there is higher germination rate, but the sprigging that this mode obtains is to occurring in nature, poor growth, the resisting pathogenic microbes ability, survival rate is lower, owing to being difficult to set up symbiotic relationship with the rear fungi contacted, causes follow-up growth seriously to be obstructed simultaneously.The symbiotic germination culture technique refers in specific matrix (substratum) is sowing plant seed and symbiosis fungi simultaneously, the method can improve seed germination rate, seedling the speed of growth and be transplanted to physical environment after survival rate of seedling.Because the symbiotic relationship of Orchid Seeds and fungi has specificity, the symbiosis fungi difference of different Orchid Seeds.Determine and can and promote that with Dendrobium aphyllum (Roxb.) C. E. Fisch． Seed Development symbiotic relationship effective fungi of its sprouting is the key link of cultivating the Dendrobium aphyllum (Roxb.) C. E. Fisch． seedling, is to carry out the basis that the former habitat of Dendrobium aphyllum (Roxb.) C. E. Fisch． returns work.The seedling that symbiotic germination by seed and fungi obtains has environmental compatibility preferably after reverting to natural habitat.Therefore obtain and promote that effective symbiosis fungi of seed germination is the first step of carrying out the Rare and threatened orchid protection.Utilize seed to move ground symbiotic germination technological guide seed germination and become protocorm, the symbiosis fungi separated in protocorm is to obtain to promote the most efficient method of seed germination effective strain.By the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed with separate the fungi obtained carry out the symbiotic germination experiment on artificial medium, screening can promote the effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination, can be High-efficient Production Mycorrhizal seedling prerequisite is provided, for the recurrence of carrying out Dendrobium aphyllum (Roxb.) C. E. Fisch． lays the foundation.At present, in prior art there are no glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) and promoting the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination to grow into purposes aspect seedling and the report of method.
Summary of the invention
The object of the present invention is to provide a kind of glued membrane bacteria strain, and provide this bacterial strain promoting the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination to grow into purposes and the method aspect seedling.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552).
The nrDNA ITS sequence submission U.S. state-run biotechnology information center database of described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) (NCBI, http: //
www.ncbi.nlm.nih.gov/), sequence number is KC796463.1.
The biological property of described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) is: cultivate 7 days its bacterium colonies and be light tan on the PDA flat board, show slightly radial, irregular cycle is dispersed growth, and aerial hyphae is undeveloped, medium sparse; The optical microphotograph Microscopic observation, mycelia tool barrier film, thick 3.5 – 5.0 μ m, most 4.0 μ m, the nearly right angle of branch, the bifurcation contracting of hanging, form barrier film nearby apart from branch; Old hyphal cell wall has the thickening phenomenon; Cultivate more than 2 weeks and start to form catenate chlamydospore, chlamydospore quantity does not wait.
Described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) is in the application promoted on the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination.
As described application, glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552), by with protocorm, forming symbiotic relationship, promotes the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grows up to seedling.
As described application, by by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, respectively cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) is being arranged under 25 ± 2 ℃ of conditions in the artificial culture case, make bacterial strain and protocorm form symbiotic relationship, thereby promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
Described glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) promotes the method for Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination, glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) forms symbiotic relationship with protocorm, promotes the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grows up to seedling.
As described method, by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, respectively cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) is being arranged under 25 ± 2 ℃ of conditions in the artificial culture case, make bacterial strain and protocorm form symbiotic relationship, thereby promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
In order to realize purpose of the present invention, concrete steps provided by the invention are as follows:
1, bacterial classification of the present invention was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 04 28th, 2013, preserved numbering: CGMCC No.7552.The nrDNA ITS sequence of bacterial strain of the present invention has been submitted the U.S. state-run biotechnology information center database (NCBI, http://www.ncbi.nlm.nih.gov/) to, and it is for No. Genbank: KC796463.1.
2, the glued membrane bacterial strain that the present invention is numbered CGMCC No.7552 is cultivated 7 days its bacterium colonies and is light tan on the PDA flat board, shows slightly radial, and irregular cycle is dispersed growth, and aerial hyphae is undeveloped, medium sparse.The optical microphotograph Microscopic observation, mycelia tool barrier film, thick 3.5 – 5.0 μ m, most 4.0 μ m, the nearly right angle of branch, the bifurcation contracting of hanging, form barrier film nearby apart from branch; Old hyphal cell wall has the thickening phenomenon; Cultivate more than 2 weeks and start to form catenate chlamydospore, chlamydospore quantity does not wait.
3, obtain to the effective symbiosis fungi of Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination ITS fragment sequence at the database (NCBI of the U.S. state-run biotechnology information center, http://www.ncbi.nlm.nih.gov/) carry out the BLAST compare of analysis in, itself and GU166410.1Tulasnellacalospora similarity are up to 99%.According to bacterium colony, micro-morphology and molecular biology method, fungi involved in the present invention is accredited as to the Tulasnella fungi.
4, Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and the experiment of glued membrane bacteria strain symbiotic germination
Utilize seed from fungi the symbiotic germination in substratum test to detect the fungi that is separated to and whether there is facilitation effect in the seed germination stage and contrasts the difference of different fungies to seed germination stage facilitation effect.
4.1 will be stored in 4 ℃ of strains testeds in test tube slant, take out, renewed vaccination, on the PDA plate culture medium, is placed in 25 ± 2 ℃ of cultivations in growth cabinet, is activated.When hypha,hyphae covers with culture dish soon, take out as the symbiotic germination material (approximately 7 days).
4.2 preparation symbiotic germination substratum: used medium is oat nutrient agar (OMA, 4gL
-1oatmeal+8gL
-1agar, pH=5.6 – 5.8).The substratum prepared is poured on and revolves in mouthful bottle, screw bottle cap, and sterilizing (121 ℃, 20min) rear standby.
4.3 seed sterilizing: the seed that will be stored in before experiment in-20 ℃ takes out, and puts at room temperature 10 hours, makes the seed temperature return to room temperature.A small amount of seed is placed in the kind attached bag of papery, with staple, paper bag is sealed.To fill seed-bearing paper bag and be immersed in distilled water 5 – 10 minutes, gently squeeze out bubble.With tweezers, paper bag is transferred to and filled chlorine bleach liquor's (the available chlorine ionic concn is 1%) and, containing in the beaker of a washing composition, stir gently or shake beaker.After 10 minutes, paper bag is moved to Bechtop, with aseptic nipper, paper bag is transferred in the beaker that fills sterile distilled water, shake gently.Repeat to rinse seed 3 – 4 times.Squeeze out gently unnecessary water in paper bag, by sterile scissors, cut off paper bag.
4.4 sowing and cultivation: the method used according to Dixon (1987) slightly makes an amendment.By aseptic seed and 1gL
-1aseptic agar-agar soln is made into aseptic seed suspension.The aseptic nylon cloth of semicircle that is 6cm at two radiuses of the parallel placement of OMA media surface (aperture is 45 μ m), draw 150 μ l seed suspension with liquid-transfering gun and evenly sow on every nylon cloth.The about 0.5cm of inoculation in the middle of substratum
3the agar block that (1 * 1 * 0.5cm) contains single fungi pure growth, with sealed membrane by the culture dish good seal.Be divided into three groups: one group of inoculation separates the bacterial strain that is numbered CGMCC No.7552 obtained from the Dendrobium aphyllum (Roxb.) C. E. Fisch． protocorm; Other 2 groups are set to contrast, wherein one group of inoculation Tulasnella (Tulasnella) fungi (being numbered Fcb4) from being separated in blue (Cymbidium manni) protocorm of sclerophyll; Another group does not connect bacterium for blank group (CK).Every group is repeated 14 culture dish, 25 ± 2 ℃ of lower constant temperature culture in the artificial culture case, and every group of each 7 culture dish are having cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) respectively.
4.5 detect: after planting detect weekly the seed germination situation, record the time of seed germination and formation protocorm.After cultivating several weeks, while in culture dish, producing a large amount of seedling in the early development stage, whole culture dish are taken out to observed and recorded seed germination and protocorm developmental state under stereoscopic microscope.At Stewart& Zettler (2002) slightly adjusts on the grade scale basis to seed germination and protocorm developmental state, seed germination situation under dark and illumination condition is carried out to hierarchical statistics, and screening can effectively promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and can grow to the symbiosis fungi in seedling stage.
In above-mentioned steps, step 1 provides the bacterial classification material warehouse-in that three test tube slants are cultivated to obtain into an accession designation number according to the requirement of culture presevation unit; The ITS base sequence of gained of simultaneously checking order is uploaded to the software that Genbank provides and submits to and obtain No. Genbank.
The described observation bacterial strain of step 2 micro-morphology is used the inserted sheet culture method, and mold incubator is cultivated 7 to 10 days under 25 ± 2 ℃ of conditions, gets inserted sheet flaking method film-making routinely.Need according to the observation to select the mycelia of different growing stage to observe to measure as: be positioned at the long visible beading chlamydospore of mycelial growth time of inserted sheet bottom, auxiliary microscope micrometer scale can be measured the thickness of mycelia.
In the described Molecular Identification of step 3, adopt the CTAB method to extract fungal DNA, the pcr amplification the primer is ITS1 and ITS4; PCR reaction system (25 μ l) comprising: 2.5 μ l10 * PCR damping fluid, 0.4 μ l dNTP, 1.5 μ l Mg2+, 1.5 μ l ITS1,1.5 μ l ITS4,0.2 μ l Taq enzyme, 15.4 μ l ddH2O, 2 μ l DNA profilings.Amplified reaction carries out on PCR instrument Perkin Elmer, following PCR circulation: 94 ℃ of denaturation 3min, circulate 1 time; 94 ℃ of sex change 1min, 51 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations; Last 72 ℃ are extended 10min.Pcr amplification product is that Shanghai living work biotechnology company limited is checked order.To check order row, submit to the U.S. state-run biotechnology information center database to compare, tentatively confirm the lower status of its classification.
The suspension of described 150 microlitres of step (4.4) is approximately containing 150 Dendrobium aphyllum (Roxb.) C. E. Fisch． seeds, and described culture condition is: intensity of illumination is 2000 – 3000Lx, 25 ± 2 ℃ of temperature, photoperiod 12h/12h L/D.
To determine according to the seed germination situation the described cultivation of step (4.5) several weeks, in this invention, select 8 to 10 weeks, because the seed major part has been sprouted and some has reached the seedling stage; Described seed germination and protocorm developmental state are with reference to Stewart& The method of Zettler (2002) is carried out classification to seed germination and protocorm developmental state, and be divided into 5 stages: it is transparent that the stage 0 is described as embryo, and the kind skin is intact, and seed is not sprouted; Stage 1 is described as the embryo water-swelling; Stage 2 is described as embryo continues to expand, and prominent breaking in the seed coat is considered as sprouting; Stage 3 is described as occurring protomeristem, develops into protocorm; Stage 4 is described as growing the first blade; Stage 5 is described as the continued growth of first blade, elongated.
Reference is: Dixon is orchid growing for pleasure and profit K.1987.Modern, orchid club of south Australia:Inc., Adelaide.;
Stewart?SL,Zettler?LW.2002.Symbiotic?germination?of?three?semi-aquatic?rein?orchids(Habenaria?repens,H-quinquiseta,H-macroceratitis)from?Florida.Aquatic?Botany72:25-35.。
Compared with prior art, the present invention possesses following remarkable excellent benefit:
The sprouting of Orchid Seeds can be cultivated and two kinds of modes of symbiotic germination cultivation by non-symbiosis germination.Although most of orchids can be cultivated by non-symbiosis germination, and there is higher germination rate, but the sprigging that this mode obtains is to occurring in nature, poor growth, the resisting pathogenic microbes ability, survival rate is lower, owing to being difficult to set up symbiotic relationship with the rear fungi contacted, causes follow-up growth seriously to be obstructed simultaneously.The symbiotic germination culture technique refers in specific matrix (substratum) is sowing plant seed and symbiosis fungi simultaneously, the method can improve seed germination rate, seedling the speed of growth and be transplanted to physical environment after survival rate of seedling.Because the symbiotic relationship of Orchid Seeds and fungi has specificity, the symbiosis fungi difference of different Orchid Seeds.Determine and can and promote that with Dendrobium aphyllum (Roxb.) C. E. Fisch． Seed Development symbiotic relationship effective fungi of its sprouting is the key link of cultivating the Dendrobium aphyllum (Roxb.) C. E. Fisch． seedling, is to carry out the basis that the former habitat of Dendrobium aphyllum (Roxb.) C. E. Fisch． returns work.Bacterial strain of the present invention is tested Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different fungi and contrasts is cultivated respectively on oat medium by symbiotic germination, promote the effective strain of Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination by the more successful acquisition of seed germination rate, thereby for utilizing Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and mycosymbiosis to sprout the high effect culture seedling to open up a new way.
The CGMCC No.7552 bacterial strain be separated to the protocorm that the present invention produces from the original place symbiotic germination, the seed that is about to Dendrobium aphyllum (Roxb.) C. E. Fisch． is put into special seed packet, seed packet is placed on to the Proterozoic of Dendrobium aphyllum (Roxb.) C. E. Fisch． plant strain growth, induce seed germination to produce protocorm, to after the protocorm surface sterilization obtained, use artificial culture medium culturing under aseptic condition, induce the endogenetic fungus growth in protocorm, when growing mycelia, carry out the purifying of fungi, obtain pure bacterium colony, and carry out Molecular Identification and the preservation of fungi.This bacterial strain can not only significantly promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination (under illumination condition 97.92 ± 0.51%; Under dark condition 89.34 ± 5.89%), and the formation that can significantly promote protocorm is (under illumination condition 91.82 ± 3.03%; Under dark condition 79.90 ± 9.56%) and significantly promote that protocorm is follow-up and develop into seedling.Illustrate and only have inoculating strain CGMCC No.7552 under illumination condition, could effectively promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed growth to grow the seedling stage.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, reach by the following examples testing data, the present invention is described in more detail.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1:
Utilize seed from fungi the symbiotic germination in substratum test to detect the fungi that is separated to and whether there is facilitation effect in the seed germination stage and contrasts the difference of different fungies to seed germination stage facilitation effect:
1, at first obtain the glued membrane bacteria strain (preserve numbering: CGMCC No.7552):
The seed of Dendrobium aphyllum (Roxb.) C. E. Fisch． is put into to seed packet, seed packet is placed on to the Proterozoic of Dendrobium aphyllum (Roxb.) C. E. Fisch． plant strain growth, induce seed germination to produce protocorm, to after the protocorm surface sterilization obtained, use artificial culture medium culturing under aseptic condition, induce the endogenetic fungus growth in protocorm, when growing mycelia, carry out the purifying of fungi, obtain pure bacterium colony.
By by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, respectively cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) is being arranged under 25 ± 2 ℃ of conditions in the artificial culture case, result shows that this bacterial strain can form symbiotic relationship and significantly promotes the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling with protocorm; Determine the taxonomy of this fungi and be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center by colony characteristics, micromorphology feature and molecular biology method simultaneously.
Classification of fungi is learned determining of status:
Morphological specificity is observed: the glued membrane bacterial strain of the above-mentioned CGMCC of being numbered No.7552 is cultivated 7 days its bacterium colonies and is light tan on the PDA flat board, shows slightly radial, and irregular cycle is dispersed growth, and aerial hyphae is undeveloped, medium sparse.The opticmicroscope feature: mycelia tool barrier film, thick 3.5 – 5.0 μ m, most 4.0 μ m, the nearly right angle of branch, the bifurcation contracting of hanging, form barrier film nearby apart from branch; Old hyphal cell wall has the thickening phenomenon; Cultivate more than 2 weeks and start to form catenate chlamydospore, chlamydospore quantity does not wait.
Molecular biology identification: take out the fungal bacterial strain of preserving, use on Bechtop in liquid potato glucose (PDB) substratum after aseptic inoculation pin picking mycelia is accessed sterilizing.Putting into 25 ± 2 ℃ of concussions of concussion shaking table cultivates.Incubation time, depending on the speed of growth of fungi, is generally 3 – 6 days.The mycelium of the about 100mg of suction filtration, adopt conventional CTAB method to extract DNA, and selecting ITS1 and ITS4 is that primer carries out pcr amplification reaction and amplified production is carried out to the agarose gel electrophoresis detection.The sample that detection is contained to target fragment is checked order.Submit the ITS fragment sequence that obtains to the U.S.'s state-run biotechnology information center database (NCBI, http://www.ncbi.nlm.nih.gov/), and carry out the BLAST comparative analysis, obtain No. GenBank and be: KC796463.1.The fungi (Tulasnellacalospora) that resulting ITS sequence is GU166410.1 to accession number is the most similar, and maximum similarity reaches 99%, and combining form credit category feature is the Tulasnella fungi by this identification of strains.This bacterial classification has adopted the test tube slant method to be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 04 28th, 2013, preserves numbering: CGMCC No.7552, storage life: 30 years.Simultaneously the applicant in laboratory by inoculation in some test tube slants, cultivating 14d in 25 ± 2 ℃ of mold incubators, to be placed on 4 ℃ of refrigerator preservations standby.
2, symbiotic germination substratum
Used medium is oat nutrient agar (OMA, 4gL
-1oatmeal+8gL
-1agar, PH=5.6 – 5.8).The substratum prepared is poured on and revolves in mouthful bottle, screw bottle cap, and sterilizing (121 ℃, 20min) rear standby.
3, strains tested is heavily cultivated
Step 1 is stored in to 4 ℃ of strains testeds in test tube slant and takes out, renewed vaccination, on the PDA substratum, is placed in 25 ± 2 ℃ of cultivations in growth cabinet, is activated.When covering with culture dish soon, takes out as the symbiotic germination material hypha,hyphae.
4, seed sterilizing
The seed that will be stored in before experiment in-20 ℃ takes out, and puts at room temperature 10 hours, makes the seed temperature return to room temperature.A small amount of seed is placed in the kind attached bag of papery, with staple, paper bag is sealed.To fill seed-bearing paper bag and be immersed in distilled water 5 – 10 minutes, gently squeeze out bubble.With tweezers, paper bag is transferred in the beaker that fills chlorine bleach liquor's (the available chlorine ionic concn is 1%) and a washing composition, stirred gently or shake beaker.After 10 minutes, paper bag is moved to Bechtop, with aseptic nipper, paper bag is transferred in the beaker that fills sterile distilled water, shake gently.Repeat to rinse seed 3 – 4 times.Squeeze out gently unnecessary water in paper bag, cut off paper bag by sterile scissors and obtain the sterilizing seed.
5, sowing and cultivation
At Dixon (1987), on method used, slightly make an amendment.By aseptic seed and 1gL
-1aseptic agar-agar soln is made into aseptic seed suspension.The aseptic nylon cloth of semicircle that is 6 ㎝ at two radiuses of the parallel placement of OMA media surface (aperture is 45 μ m), draw 150 μ l seed suspension (approximately containing 150 seeds) with liquid-transfering gun and evenly sow on every nylon cloth.The about 0.5cm of inoculation in the middle of substratum
3the agar block that (1 * 1 * 0.5cm) contains single fungi pure growth, with sealed membrane by the culture dish good seal.The bacterial strain that be numbered CGMCC No.7552 of one group of inoculation from being separated in the Dendrobium aphyllum (Roxb.) C. E. Fisch． protocorm, one group of inoculation separates the bacterial strain that is numbered Fcb4 (Tulasnella sp.) obtained from the blue protocorm of sclerophyll, and control group (CK) is not for connecing bacterium.Every group is repeated 14 culture dish, 25 ± 2 ℃ of lower constant temperature culture in the artificial culture case, and every group of each 7 culture dish are having cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) respectively.Be placed in growth cabinet and cultivate with the sealed membrane sealing.Culture condition is: intensity of illumination is 2000 – 3000Lx, 25 ± 2 ℃ of temperature, photoperiod 12h/12h L/D.
6, detect
After planting detect weekly the seed germination situation, record the time of seed germination and formation protocorm.When cultivation produces a large amount of seedling in the early development stage after several weeks in culture dish is arranged, whole culture dish are taken out, examine under a microscope and record seed germination and protocorm developmental state.At Stewart& Zettler (2002) slightly adjusts on the grade scale basis to seed germination and protocorm developmental state, the seed germination situation is carried out to classification, the ratio (K) of seed germination rate (G), formation protocorm ratio (C) and seed, protocorm or seedling in each sprouting stage under each processing of statistics.G=mean(g/t)±SE,C=mean(c/t)±SE，K=mean(k/t)±SE。Wherein, g is the seed number of sprouting in each culture dish under every kind of processing; C forms the seed number (stages 2,3,4 sum) of protocorm in each culture dish under every kind of processing, k sprouts the quantity of seed, protocorm or the seedling in stage in each in each culture dish under every kind of processing; The seed number of t for sowing in each culture dish; SE is standard error.
In R software (version2.14.2), utilize nonparameter test method Kruskal-Wallis test (KW) and Mann-Whitney U – test (U) to carry out test of significance, α=0.05 to seed germination rate, formation protocorm ratio and each stage seed number ratio of different treatment.
Result shows not connect under the illumination of bacterium control group and dark culturing condition does not all have seed germination, and a seed water-swelling reaches the stage 1 and do not form protocorm; And the CGMCC No.7552 bacterial strain be separated to from the protocorm that the original place symbiotic germination produces can not only significantly promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination (under illumination condition 97.92 ± 0.51%; Under dark condition 89.34 ± 5.89%), and the formation that can significantly promote protocorm is (under illumination condition 91.82 ± 3.03%; Under dark condition 79.90 ± 9.56%) and significantly promote that protocorm is follow-up and develop into seedling.Although the FCb4 bacterial strain can significantly promote seed germination to form protocorm (43.79 ± 20.45%) having under illumination condition, show cessation of growth cessation after forming protocorm, can't continued growth develop into seedling.Illustrate and only have inoculating strain CGMCC No.7552 under illumination condition, could effectively promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed growth to grow the seedling stage.
Claims (8)
1. a glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552).
2. glued membrane bacterium as claimed in claim 1 (Tulasnella) bacterial strain (CGMCC No.7552), the nrDNA ITS sequence that it is characterized in that described bacterial strain (CGMCC No.7552) is submitted the database (NCBI of the U.S. state-run biotechnology information center to, http://www.ncbi.nlm.nih.gov/), sequence number is KC796463.1.
3. glued membrane bacterium as claimed in claim 1 (Tulasnella) bacterial strain (CGMCC No.7552), the biological property that it is characterized in that described bacterial strain (CGMCC No.7552) is: cultivate 7 days its bacterium colonies and be light tan on the PDA flat board, show slightly radial, irregular cycle is dispersed growth, aerial hyphae is undeveloped, medium sparse; The optical microphotograph Microscopic observation, mycelia tool barrier film, thick 3.5 – 5.0 μ m, most 4.0 μ m, the nearly right angle of branch, the bifurcation contracting of hanging, form barrier film nearby apart from branch; Old hyphal cell wall has the thickening phenomenon; Cultivate more than 2 weeks and start to form catenate chlamydospore, chlamydospore quantity does not wait.
4. glued membrane bacterium claimed in claim 1 (Tulasnella) bacterial strain (CGMCC No.7552) is in the application promoted on the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination.
5. application as claimed in claim 4, is characterized in that glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552), by with protocorm, forming symbiotic relationship, promotes the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grows up to seedling.
6. application as claimed in claim 4, it is characterized in that by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, respectively cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) is being arranged under 25 ± 2 ℃ of conditions in the artificial culture case, make bacterial strain and protocorm form symbiotic relationship, thereby promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
7. glued membrane bacterium claimed in claim 1 (Tulasnella) bacterial strain (CGMCC No.7552) promotes the method for Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination, it is characterized in that glued membrane bacterium (Tulasnella) bacterial strain (CGMCC No.7552) and protocorm form symbiotic relationship, promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
8. method as claimed in claim 7, it is characterized in that by Dendrobium aphyllum (Roxb.) C. E. Fisch． seed and different types of fungi co-cultivation in oat medium, respectively cultivation under optical condition (photoperiod is 12/12h L/D) and dark condition (photoperiod is 0/24h L/D) is being arranged under 25 ± 2 ℃ of conditions in the artificial culture case, make bacterial strain and protocorm form symbiotic relationship, thereby promote the Dendrobium aphyllum (Roxb.) C. E. Fisch． seed germination and grow up to seedling.
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