CN108048334A - It is a kind of to promote the blue screening of Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system method for building up - Google Patents
It is a kind of to promote the blue screening of Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system method for building up Download PDFInfo
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Abstract
Promote the blue screening of Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system method for building up the invention discloses a kind of, establish symbiosis using Tcs1 bacterial strains and following any one or more Orchid Seeds, promote germination of arethusa seeds：Sclerophyll orchid species, Bowring cattleya YK seeds, Bowring cattleya YD seeds.Symbiotic germination experiment is carried out using Tulasnella fungi and Orchid Seeds on synthetic medium, and the Orchid Seeds control group with not connecing bacterium is compared, screening can efficiently promote the mycorrhizal fungi of seed sprouting and suitable symbiotic culture medium extensively.Effective symbiotic effects in germination of arethusa seeds stage are obtained, and germination of arethusa seeds rate is improved using symbiotic germination culture technique, establish the syntaxial system of orchid and mycorrhizal fungi.
Description
Technical field
The present invention relates to a kind of biotechnology more particularly to a kind of glue for effectively facilitating sclerophyll orchid and being sprouted with Bowring cattleya seed
The screening of film Pseudomonas fungi and syntaxial system method for building up.
Background technology
Orchid (Orchidaceae) belongs to Angiospermae Monocotyledonae, and there are about 700 categories for whole world distribution
More than 35000 kinds (Dressler, 1993；Cribb, 2003), number of species is only second to feverfew, and come second big section (youth
Pattern forever, 1994).Its shape is graceful, bright-colored, great ornamental value and medical value.
There are two distinguishing features for orchid：
First, the embryo of Orchid Seeds is only made of tens cells, and differentiation is incomplete, is sprouted naturally difficult.
2nd, orchid is nearly all mycorrhizal plants, and under natural growth conditions, no matter greenery orchid is also that non-greenery are blue,
- seed sprout stage or cannot carry out photoautotrophic period-all must be relied on partially or completely and fungi in its history of life
Normal activities could be completed by establishing symbiosis.In the case where no mycorrhizal fungi symbiosis participates in, the kind of orchid
Nutrition supply will all be affected in son sprouting and ontogenetic process, can not complete normal growth and development.Therefore, deeply
Carry out the research of orchid symbiosis mycorrhiza fungi become particularly important content in the numerous correlative studys of orchid (Xu Lu,
2015)。
Mycorhiza (Mycorrhiza) is that natural plant is formed in long-term survival processes with fungi common evolutionary
The fungi that participation mycorhiza is formed usually is called mycorrhizal fungi (Mycorrhiza) by a kind of symbiosis of generally existing.From
Ressiek in 1847 has found that Bird's Nest orchid root memory after the mycelium of life, constantly there is fungi and different orchid symbiosis
Report, the mycorhiza that orchid and mycosymbiosis are formed also independently is divided into Orchid Mycorrhizae due to its feature is unique
(Orchid mycorrhiza)(Harley,1989).Substantially, for almost all of orchid all with mycosymbiosis, mycorhiza is common
Raw relation is almost sprouted the orchid family to the entire history of life yielded positive results, largely grown up from seed with orchid and is planted
Object still need to mycorrhizal fungi provide growth needed for carbohydrate and organic matter (such as amino acid, vitamin etc.) (Chen Xinqi etc.,
2003；Smith and Read,2008).Under primary border, one plant of orchid may have more than one fungi to participate in forming mycorhiza
Homobium, a kind of fungi also can establish symbiosis with more than one orchid.It is known that in the life cycle of orchid, it is different
Fungi may be asked to participate in realizing plant life history, and mycorhiza colonizes species of the process depending on fungi and orchid, certain
Fungi can establish symbiosis (Bonnardeaux et al., 2007) with the orchid of different stages of growth.
Result of study shows that Orchid Mycorrhizae is of great significance for orchid：
1st, seed is promoted to sprout and provides necessary nutrient for it；
2nd, needed nutrient matter is provided for adult orchid growth and development；
3rd, absorption of the orchid to inorganic ions such as P is promoted；
4th, the disease-resistant and anti-adversity ability of host plant is improved；
5th, there is ecological significance.In view of important meaning of the mycorrhizal fungi that is shown of early-stage study result to orchid
Justice, the research for carrying out Coupling effects therebetween are extremely urgent.
Glued membrane bacterium is a kind of mycorrhizal fungi being proved earliest with orchid symbiosis, belongs to Basidiomycota
(Basidiomycota), Tulasnella (Tulasnella).Temperate zone, Perenniporia martius cymbidium variety in, glued membrane bacterium
Category be most common mycorrhizal fungi (Rasmussen, 1995；Yuan et al.,2010).2012, Martos etc. passed through tune
It looks into the mycorrhizal fungi of 77 kinds of orchids on La Reunion islands (Indian Ocean), it is found that Tulasnella (Tulasnella) can be with
88% orchid symbiosis (Martos, 2012).Tulasnella Distribution of fungi is in extensive range, in the whole world from the torrid zone to temperate zone
Almost be found in each ecosystem (Cruz et al., 2012,2014,2016；Hu Tao, 2006；Wu Jingyu,
2009)。
The seed of orchid is small, lacks endosperm and other nutritive issues, thus lack with seed sprout relevant enzyme,
The substances such as vitamin and inorganic ions, seed cannot be supported to sprout (Arditti and by using seeds self nutrition
Ghani, 2000).All the time, researcher all constantly studies Orchid Seeds symbiotic germination mechanism, it is intended to protect
Precious, Rare, Endangered cymbidium variety.It can be sprouted to find out with the fungal species of Orchid Seeds symbiosis with improving Orchid Seeds
Hair rate, it is numerous that quickening orchid utilizes the mode of generative propagation to expand, and researcher attempts to use various Orchid Seeds and fungi
Special culture medium or training method carry out cultured in vitro, simulate habitat characteristics, symbiosis is established in vitro
(Arditti et al., 1990；Rasmussen, 1995).
In the prior art, to understand orchid and the mechanism of mycosymbiosis relation, researcher is attempted in vitro
Under the conditions of by the orchid structure symbiosis artificial with the mycorrhizal fungi for separating acquisition.In past symbiosis research, use more
Two kinds of culture substrate structure：One kind is to utilize culture substrate made of Fagaceae bark or leaf；Another kind of is to utilize
Oat-agar cultures base (Oatmeal agar medium, OMA).
20th century, the nineties were to the early 21st century, and the researcher in China is mostly using bark and leaf culture from wild gold
Line lotus (Anoectochilus roxburghii), dendrobium candidum (Dendrobium candidum) and HERBA DENDROBII (D.
Nobile in root), acquisition is separated in dendrobium hancockii Rolfe (D.hancockii) and nervate twayblade herb (Liparis nervosa) protocorm
Fungi, utilize method and sieve river stem of noble dendrobium (D.lohohens), long Soviet Union's stem of noble dendrobium (D. brymerianun), the iron sheet of bacterium leaf culture
The seed of the plants such as the stem of noble dendrobium, Rhizoma Gastrodiae carries out mixing bacterium sowing, finds halimasch (Armillariella mellea), Mycena
The fungies energy such as (Mycena sp.), angle mycorhiza Pseudomonas (Ceratorhiza sp.), Rhizoctonia (Rhizoctionia sp.)
Symbiosis is established with cymbidium seed and effectively improves germination rate, while it has also been found that promotes the mycorrhizal fungi that seed is sprouted might not
Can promote plant seedling growth (Guo Shunxing and Xu Jintang, 1991；Guo is along magnitude, 2000a；2000b, Xu Jintang and Fan Li,
2001)；In the recent period, Guo Shitan etc. does symbiotic culture medium matter development Rhizoctonia fungi and cymbidium mastersii with robur bark
(Cymbidium mastersii) seed symbiosis culture experiment, it is found that the rhizoctonia from Chunlan and Chinese cymbidium root can be carried significantly
The high sub- germination rate of heavy snow orchid species, and inquired into mycorhiza of the mycorrhizal fungi of cymbidium mastersii adult plants with promoting seed symbiotic germination
Single-minded sex chromosome mosaicism (Guo Shitan etc., 2012) between fungi.A variety of results of study show have between orchid and mycorrhizal fungi
There is diversity simultaneously, there are still complicated specificities between the orchid and mycorrhizal fungi of different stages of growth.
Oat-agar cultures base has that nutrient concentration is controllable, simple operation and other advantages, and in Orchid Mycorrhizae fungi and orchid family
During vegetable seeds symbiotic germination, how much very crucial the nutrient of culture medium is, and fungi tends to carry out saprophytic when nutrition is more than needed
Life without with plant symbiosis, when nutrition is excessively barren and can not meet seed sprout and growth needed for (Beyrle, 1991,
1995).Earlier than 1998, Li Lubin was filtered out to Chunlan and blue with leaf pocket in the experiment of China blue mycorrhizal fungi tieback for the country
The suitable oat-agar cultures base concentration of (Paphiopedilum hirsutissimum) seed symbiotic germination (Li Lubin,
1998).Researcher utilizes the advantages of oat-agar cultures base, gradually mostly establishes symbiosis using this kind of culture medium, so as into one
Step carries out the further investigation on VA Mycorrhizal Fungi and orchid symbiosis.
With the intensification recognized Orchid Mycorrhizae fungi, to explore the cell of " fungi-orchid " symbiosis and dividing
Subbase plinth, researchers are mostly co-cultured using culture medium, such as Zhao Mingming is by wax shell Pseudomonas (Sebacina sp.) fungi
With Seeds of Dendrobium Candidum in oat-agar cultures base symbiotic germination, structure SSH libraries are Orchid Seeds and mycorrhizal fungi
The Molecular level study of symbiotic germination lay a good foundation (Zhao Mingming and Guo Shunxing, 2012；Zhao et al.,2013).In mistake
In the foreign study gone, Stewart etc. is successfully belonged to Epulorhiza category fungi and beautiful phoenix flower using oat-agar cultures base
(Habenaria), a variety of cymbidium seeds structure symbiosis of Spiranthes (Spiranthes brevilabris), seed germination rate
It significantly improves (Stewart et al., 2002,2003,2006).In recent years, external correlative study was more employs oat
Agar medium, such as fly blue (Oncidium sphacelatum) seed of swallow and be isolated from Coppensia doniana orchids
Angle load bacterium (Ceratobasidium sp.) co-culture, be isolated from down the beautiful born of the same parents away from blue (Anacamptis laxiflora)
Glued membrane bacterium (Tulasnella calospora) infects tongue epidendrum Serapias vomeracea, eulophia genus
The separated Epulorhiza category fungi in (Eulophia epidendreae) root (Epulorhiza sp.) and more arteries and veins punctatelip coelogyne herbs
(Coelogyne nervosa) in oat-agar cultures matrix exosymbiosis, successfully establishes symbiosis (Valadares
Et al., 2012,2014；Perotto et al., 2014；Sebasti á n et al., 2014；Sathiyadash et
Al., 2014).
At present, it is true to there are no the Tulasnella that Bowring cattleya and sclerophyll orchid species can while efficiently be promoted to sprout in the prior art
The screening of bacterium and the report of corresponding syntaxial system method for building up.
The content of the invention
The object of the present invention is to provide a kind of screenings for promoting the blue Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll
And syntaxial system method for building up.
The purpose of the present invention is what is be achieved through the following technical solutions：
The present invention's promotes the screening of the blue Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system to establish
Method establishes symbiosis using Tcs1 bacterial strains and following any one or more Orchid Seeds, promotes Orchid Seeds
It sprouts：
Sclerophyll orchid species, Bowring cattleya YK seeds, Bowring cattleya YD seeds；
The Latin of the sclerophyll orchid is named as Cymbidium mannii, the Latin of the Bowring cattleya is named as
Cattleya hybrida, the YK and YD are two kinds of Bowring cattleya；
The Tcs1 bacterial strains are U.S. spore Tulasnella strain, and the Latin of U.S. spore glued membrane bacterium is named as：Tulasnella
Calospora, the bacterial strain submit preservation, preservation in China Committee for Culture Collection of Microorganisms's common micro-organisms center
Center number of registering on the books is：CGMCC No.14794.
As seen from the above technical solution provided by the invention, promotion sclerophyll orchid provided in an embodiment of the present invention and Ka Te
The screening of Tulasnella fungi and syntaxial system method for building up that orchid species is sprouted, use Tulasnella on synthetic medium
Fungi and Orchid Seeds carry out symbiotic germination experiment, and the Orchid Seeds control group with not connecing bacterium is compared, sieve
Choosing can efficiently promote the mycorrhizal fungi of seed sprouting and suitable symbiotic culture medium extensively.Obtain germination of arethusa seeds
Effective symbiotic effects in stage, and germination of arethusa seeds rate is improved using symbiotic germination culture technique, establish orchid
Syntaxial system with mycorrhizal fungi is to carry out the key link of Intergrown molecular mechanism study and the Reproduction of orchid, right
Effective protection of orchid is of great significance.
Specific embodiment
The technical solution in the embodiment of the present invention is clearly and completely described below, it is clear that described embodiment
Only part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiment of the present invention, the common skill in this field
Art personnel all other embodiments obtained without making creative work belong to the protection model of the present invention
It encloses.
The present invention's promotes the screening of the blue Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system to establish
Method, preferable specific embodiment are：
Symbiosis is established using Tcs1 bacterial strains and following any one or more Orchid Seeds, promotes orchid kind
Son is sprouted：
Sclerophyll orchid species, Bowring cattleya YK seeds, Bowring cattleya YD seeds；
The Latin of the sclerophyll orchid is named as Cymbidium mannii, the Latin of the Bowring cattleya is named as
Cattleya hybrida, the YK and YD are two kinds of Bowring cattleya；
The Tcs1 bacterial strains are U.S. spore Tulasnella strain, and the Latin of U.S. spore glued membrane bacterium is named as：Tulasnella
Calospora, the bacterial strain submit preservation, preservation in China Committee for Culture Collection of Microorganisms's common micro-organisms center
Center number of registering on the books is：CGMCC No.14794, preservation time is：On November 14th, 2017, preservation address are：Beijing
The institute 3 of city Chaoyang District North Star West Road 1.
The symbiosis is established in oat-agar cultures base.
Symbiosis settling time is sclerophyll orchid species in the oat-agar cultures base of 2.0g/L with Tcs1 bacterial strains
21 days, sclerophyll orchid seed germination rate was 92.05 ± 2.45% after co-culturing 60 days；
Symbiosis is built in the oat-agar cultures base that the Bowring cattleya YK seeds are 12.0g/L with Tcs1 bacterial strains in concentration
It it is 12 days between immediately, Bowring cattleya YK seed germination rates are 87.13 ± 3.40% after co-culturing 60 days；
Symbiosis is built in the oat-agar cultures base that the Bowring cattleya YD seeds are 12.0g/L with Tcs1 bacterial strains in concentration
It it is 14 days between immediately, Bowring cattleya YD seed germination rates are 75.63 ± 5.41% after co-culturing 60 days.
Germination rate is extremely low, it is necessary to which mycorrhizal fungi participates in its entire growth and development under field conditions (factors) for Orchid Seeds
Journey.The present invention carries out symbiotic germination experiment on synthetic medium using Tulasnella fungi and Orchid Seeds, and with not
The Orchid Seeds control group for connecing bacterium is compared, and screening can efficiently promote the mycorhiza of germination of arethusa seeds true extensively
Bacterium and suitable symbiotic culture medium.Effective symbiotic effects in germination of arethusa seeds stage are obtained, and utilize symbiotic germination
Culture technique improves germination of arethusa seeds rate, and the syntaxial system for establishing orchid and mycorrhizal fungi is to carry out symbiosis point
The key link of sub- mechanism study and the Reproduction of orchid is of great significance to effective protection of orchid.
Specific embodiment：
1st, test material
(1) vegetable material：Two of sclerophyll blue (Cymbidium mannii) and Bowring cattleya (Cattleya hybrida)
The seed of kind (YK, YD).
(2) fungal material：7 plants of Tulasnella Orchid Mycorrhizae fungi (Tulasnella of gained are tested for many years in this laboratory
Spp.), it is shown in Table 1.
2nd, test method (Orchid Seeds and Tulasnella mycosymbiosis sprouting test)
(1) configuration of culture medium
The configuration of symbiotic culture medium (oat medium OMA)：Oatmeal is taken to wear into powdery, takes the oat of three gradients respectively
Powder is made into the solution of 0.5g/L, 1.0g/L, 2.0g/L, 3.0g/L, 4.0g/L, 8.0g/L, 12.0g/L OMA.Deionized water
1h is boiled, is filtered, agar 8.0g, filtrate adds deionized water to be settled to 1000mL, high-temperature sterilization.
The configuration of fungi culture medium：200g peeled potatoes are cut into the fritter of 0.5~1cm square, add deionized water
After 800mL boils 30min, filtrate is obtained with 4~5 layers of filtered through gauze, and after adding in 20g glucose and 8g agar in filtrate, then
1000mL is settled to deionized water, and after high-temperature sterilization, streptomycin sulphate is added in when temperature is down to 50 degrees Celsius to final concentration
50mg/L。
(2) activation and identification of Tulasnella fungi
7 plants of Orchid Mycorrhizae fungies of gained are tested for many years in this laboratory.Picking hypha,hyphae is in PDA culture medium, 28 DEG C
7~10d of constant temperature light culture.Morphological feature observation is carried out to the fungi after activation；The mycelium inoculation at picking bacterial strain edge is in being covered with
In the PDA culture medium of cellulose acetate film, 28 DEG C of constant temperature 7~10d of light culture are fully ground after liquid nitrogen flash freezer, utilize OMEGA
Kit extracts DNA, PCR specific amplified ITS areas, and PCR amplification primer is ITS1F：5′- TCCGTAGGTGAACCTGCGG-3′；
ITS4R：5′-TCCTCCGCTTATTGATATGC-3′；PCR product is sent to company and is sequenced, by sequential file software
After DNAMAN is proofreaded, pass through online BLAST (http://www.ncbi.nlm.nih.gov) carry out sequence alignment.
(3) disinfection of capsule
The orchid capsule that the florescence is consistent, pollination time is consistent, growing way is consistent on same plant is taken as test material, first
Gently wipe exocarp 3 times with the cotton balls for speckling with 75% alcohol, move into superclean bench after, then with same method wiping 3 times.
(4) co-culture
After fungi activation, card punch takes edge mycelia that a diameter of 0.8cm, the cylindric mycobiotic agar of high about 0.5cm is made
Block is inoculated in processing group；Sterile PDA culture medium, which is beaten, takes onesize agar block, is inoculated in control group.
Seed symbiotic germination：3.5 × 3.5cm filter papers of intermediate void are covered on OMA, cymbidium seed is fabricated to seed
Suspension with blood counting chamber legally constituted authority meter seed density, is uniformly sown into aseptic filter paper on piece.Processing group center inoculated fungi fine jade
Fat block, control group inoculation sterile letheen block, sets 15 wares to repeat, the dark culturing at 23 ± 2 DEG C per concentration cultures.
(5) detect
Stage criterion is sprouted according to Zettler et al. seeds, refers to table 2.Routine observation seed sprouting situation, record
The time and germination rate that seed is sprouted.Pending group of cymbidium seed growth and development randomly selects plant sample in every 2 days to the 3rd stage
Product trypan blue (0.05%Trypan Blue-water solution) colouring method, makes interim tabletting in optical microphotograph
Microscopic observation Fungal colonization situation.Comparison screening can efficiently promote the Tulasnella fungal bacterial strain that orchid family seed is sprouted extensively.
2 seed of table sprouts the stage
3rd, result of the test
(1) identification of Tulasnella fungi
7 plants of Tulasnella fungies show following common cultural characteristic in PDA culture medium used in experiment：Bacterium
Falling canescence or pale yellow from color, the circle-shaped diverging of mycelia journey is grown, the concentric garden bacterium colony ring grain for forming interval and not waiting having,
Bacterium colony surface wettability, the gentle exquisiteness of texture, mycelia sink to growing, and some bacterium have lax white aerial hyphae；It can grow within 7-10 days
The PDA culture medium plate of full diameter 9cm；It is formed without sexual reproduction spore.
7 plants of Tulasnella hypha,hyphaes observe following common trait under an optical microscope used in experiment：Bacterium
Silk have every；In contracting shape of hanging, membrane is more nearby slightly formed away from mycelia branching-point for almost right angle, bifurcation：With by tubbiness cell
The Torulose cell group structure that (asexual chlamydospore) is in series.
PCR qualification results such as the following table 1：
1 VA Mycorrhizal Fungi ITS sequence comparison result of table
(2) Tulasnella fungi is formed with Orchid Seeds homobium
It periodically extracts the orchid material co-cultured with U.S. spore glued membrane bacterium and carries out Trypan Blue experiment, check whether to build
Vertical symbiosis.The result shows that not dyed when seed is not sprouted, illustrate that fungi is useless and successfully infect plant；When seed is sent out
Educate to separate living tissue occur when (the 3rd stage), plant dye blueness, and dyeing part concentrates on the surface texture of plant；Work as ball
Stem is developed to when rough leaf occurs (the 4th stage), inside plants dye blueness, and forms more hypha body internal.
Sclerophyll is blue, and symbiosis settling time is most short in the OMA culture mediums of 2.0g/L with Tcs1 bacterial strains, is 21 days；With
Tss bacterial strains symbiosis settling time in the OMA culture mediums of 2.0g/L is 28 days；With Tco2 bacterial strains in 8.0g/L and
Symbiosis settling time is identical in the OMA culture mediums of 12.0g/L, is 56 days；With Tsk1 bacterial strains 1.0g/L's and 2.0g/L
Symbiosis settling time is identical in OMA culture mediums, is 28 days.Bowring cattleya YK is cultivated with OMA of the Tcs1 bacterial strains in 12.0g/L
Symbiosis settling time is most short in base, is 12 days；Symbiosis is established in the OMA culture mediums of 12.0g/L with Tss bacterial strains
Time is 21 days；Symbiosis settling time is identical in the OMA culture mediums of 12.0g/L with Tco2 bacterial strains, is 23 days；Ka Te
Blue YD and Tcs1 bacterial strains symbiosis settling time in the OMA culture mediums of 12.0g/L is most short, is 14 days；Exist with Tss bacterial strains
Symbiosis settling time is 25 days in the OMA culture mediums of 12.0g/L；With Tco2 bacterial strains in the OMA culture mediums of 12.0g/L
Symbiosis settling time is identical, is 23 days.The result shows that when Tcs1 bacterial strains orchid symbiosis different from 3 kinds is established
Between it is most short.
(3) fungi promotes seed germination rate statistics
Sclerophyll is blue to be co-cultured with 7 plants of mycorrhizal fungis respectively from Bowring cattleya seed under different OMA concentration, is observed after 60 days
And the sprouting situation of seed is recorded, when expanding to 3 stage, it is considered as sprouting.The result shows that Tcs1 strains can be at all 7
Promote 3 kinds of germination of arethusa seeds under OMA experimental concentrations；Tss bacterial strains can be under middle OMA concentration (3.0g/L, 4.0g/L)
Sclerophyll orchid species is promoted to sprout, under high OMA concentration (8.0g/L, 12g/L) Bowring cattleya seed is promoted to sprout；Tco2 bacterial strains can
To promote 3 kinds of germination of arethusa seeds in high OMA concentration (8.0g/L, 12.0g/L)；Tsk1 strains are only capable of in low, middle OMA
Sclerophyll orchid species is promoted to sprout under concentration (1.0g/L, 2.0g/L, 3.0g/L, 4.0g/L).Be inoculated with Tsg1, Tcs2, Tcf1 and
Control group (CK) is not sprouted.
4 plants of mycorrhizal fungis respectively facilitate 3 kinds of germination of arethusa seeds rate significant differences point under different OMA concentration
Analysis, it turns out that：1. Tcs1 bacterial strains promote sclerophyll orchid seed germination rate presence extremely notable in the OMA culture mediums of various concentration
Otherness, sclerophyll orchid species germination rate highest in the OMA of 2.0g/L are 92.05 ± 2.45%；Tcs1 bacterial strains are different dense
It is 12.0g/L's in concentration there are the pole significance difference opposite sex to promote Bowring cattleya YK and YD seed germination rate in the OMA culture mediums of degree
Bowring cattleya YK and YD seed germination rate highests in OMA are respectively 87.13 ± 3.40% and 75.63 ± 5.41%.2. Tsk1 bacterium
It is 3.0g/L in concentration there are the pole significance difference opposite sex that strain promotes sclerophyll orchid seed germination rate in the OMA culture mediums of various concentration
OMA sclerophyll orchid seed germination rate highests, be 76.55 ± 2.31%.3. Tss bacterial strains promote firmly in the OMA of 3.0g/L concentration
Aspidistra seed germination rate highest is 61.17 ± 0.79% (3.0g/L OMA)；Promote Bowring cattleya YK seed germination rates be up to
45.47 ± 1.58% (12.0g/L OMA), it is up to 50.45 ± 0.49% (12.0g/L to promote Bowring cattleya YD seed germination rates
OMA).4. Tco2 bacterial strains promote sclerophyll blue and Bowring cattleya (YK and YD) seed germination rate is relatively low, sclerophyll orchid germination rate highest
For 5.06 ± 0.03% (8.0g/L OMA), Bowring cattleya YK germination rates are up to 19.90 ± 1.66% (12.0g/L OMA), card
Special orchid YD germination rates are up to 25.90 ± 2.11% (12.0g/L OMA)
The advantageous effect that technical solution of the present invention is brought：
Germination rate is extremely low, it is necessary to which mycorrhizal fungi participates in its entire growth and development under field conditions (factors) for Orchid Seeds
Journey.Effective symbiotic effects in germination of arethusa seeds stage are obtained, are the key that carry out Rare and threatened orchid to return ring
Section is of great significance to effective protection of orchid.To establish the syntaxial system of orchid and mycorrhizal fungi, screen
Go out extensively efficiently to promote the mycorrhizal fungi and suitable symbiotic culture medium that seed is sprouted, the present invention is true using Tulasnella
Bacterium establishes symbiosis with Orchid Seeds, and the control group with not connecing bacterium is compared.
This experiment finds that U.S. spore Tulasnella strain Tcs1 can be in whole by counting the germination rates of Orchid Seeds
Promote 3 kinds of germination of arethusa seeds under OMA experimental concentrations；By observing the growing state of fungi and orchid, filter out
Maintain the culture medium of both plant and microorganism growth potential balance：Sclerophyll orchid species and Tcs1 bacterial strains are common in the OMA of 2.0g/L
Raw relation settling time is most short, is 12 days, and germination rate highest, is 92.05 ± 2.45%；Bowring cattleya YK exists with Tcs1 bacterial strains
Sprout time is most short in the OMA that concentration is 12.0g/L, is 12 days, and germination rate highest is 87.13 ± 3.40%；Bowring cattleya YD
With Tcs1 bacterial strains concentration be 12.0g/L OMA in sprout time it is most short, be 14 days, germination rate highest, be 75.63 ±
5.41%.
Tulasnella fungi Tcs1 bacterial strains, through the U.S. spore glued membrane bacterium that ITS is identified with NCBI accession number is FJ613255.1
(Tulasnella calospora) uniformity is 99%, which can establish symbiosis pass by and Bowring cattleya seed blue with sclerophyll simultaneously
System, and can efficiently promote sclerophyll orchid and the sprouting of Bowring cattleya seed.Sclerophyll is blue to grow nonparasitically upon another plant type orchid family epidendrum as one kind, pole
Have ornamental value, and wild sclerophyll orchid be also faced with it is in imminent danger.Bowring cattleya is Bowring cattleya category perennial herb epiphyte, is originated in
It is the treasure in orchid family decorative flower in Central and South America.The present invention Wild Orchids be faced with it is in imminent danger in the case of, sieve
It has selected and promotes the blue symbiotic effects bacterial strain with the efficient Germination And Seedling of Bowring cattleya of sclerophyll and suitable culture medium concentration, can be orchid
The Intergrown molecular mechanism study of section plant provides Technical Reference, and provides guidance for the blue Reproduction with Bowring cattleya of sclerophyll, right
The blue protection with Bowring cattleya species resource of sclerophyll is of great significance.
The key problem in technology point of the present invention：
Tcs1 bacterial strains can establish symbiosis by and Bowring cattleya seed blue with sclerophyll simultaneously, and Orchid Seeds is promoted to sprout
Hair；
Tcs1 bacterial strains can promote 3 kinds of germination of arethusa seeds under whole OMA experimental concentrations；
The symbiosis settling time in the oat-agar cultures base of 2.0g/L is 21 days to sclerophyll orchid species with Tcs1 bacterial strains,
Sclerophyll orchid seed germination rate is 92.05 ± 2.45% after co-culturing 60 days；
When symbiosis is established in the oat-agar cultures base that Bowring cattleya YK seeds are 12.0g/L with Tcs1 bacterial strains in concentration
Between for 12 days, Bowring cattleya YK seed germination rates are 87.13 ± 3.40% after co-culturing 60 days；
When symbiosis is established in the oat-agar cultures base that Bowring cattleya YD seeds are 12.0g/L with Tcs1 bacterial strains in concentration
Between for 14 days, Bowring cattleya YD seed germination rates are 75.63 ± 5.41% after co-culturing 60 days.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art is in the technical scope of present disclosure, the change or replacement that can readily occur in,
It should be covered by the protection scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of claims
Subject to enclosing.
Claims (3)
1. a kind of promote the blue screening of Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll and syntaxial system method for building up,
It is characterized in that, establishes symbiosis using Tcs1 bacterial strains and following any one or more Orchid Seeds, promote orchid
Seed is sprouted：
Sclerophyll orchid species, Bowring cattleya YK seeds, Bowring cattleya YD seeds；
The Latin of the sclerophyll orchid is named as Cymbidium mannii, the Latin of the Bowring cattleya is named as Cattleya
Hybrida, the YK and YD are two kinds of Bowring cattleya；
The Tcs1 bacterial strains are U.S. spore Tulasnella strain, and the Latin of U.S. spore glued membrane bacterium is named as：Tulasnella
Calospora, which submits preservation in China Committee for Culture Collection of Microorganisms's common micro-organisms center, in preservation
Heart number of registering on the books is：CGMCC No.14794.
2. the screening and symbiosis according to claim 1 for promoting the blue Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll
Establishing method, which is characterized in that the symbiosis is established in oat-agar cultures base.
3. the screening and symbiosis according to claim 2 for promoting the blue Tulasnella fungi sprouted with Bowring cattleya seed of sclerophyll
Establishing method, which is characterized in that sclerophyll orchid species and Tcs1 bacterial strains are common in the oat-agar cultures base of 2.0g/L
Raw relation settling time is 12 days, and germination rate is 92.05 ± 2.45%；
When symbiosis is established in the oat-agar cultures base that the Bowring cattleya YK seeds are 12.0g/L with Tcs1 bacterial strains in concentration
Between for 12 days, germination rate is 87.13 ± 3.40%；
When symbiosis is established in the oat-agar cultures base that the Bowring cattleya YD seeds are 12.0g/L with Tcs1 bacterial strains in concentration
Between for 14 days, germination rate is 75.63 ± 5.41%.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536391A (en) * | 2018-12-20 | 2019-03-29 | 沈阳农业大学 | A kind of fungi and its application for promoting iris protocorm Stem nematode |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399698A (en) * | 2010-12-08 | 2012-04-04 | 云南农业大学 | Separation and application of verticillium spp. fungus capable of promoting cymbidium wenshanense tissue cultured seedling growth |
| CN102612965A (en) * | 2012-04-05 | 2012-08-01 | 中国科学院西双版纳热带植物园 | Method for obtaining germination effective symbiotic fungus for Cymbidium bicolor seeds |
| CN102876580A (en) * | 2010-12-08 | 2013-01-16 | 云南农业大学 | Separation and application of Nectria fungi for promoting growth of Cymbidium wenshanense tissue culture plants |
| CN103468587A (en) * | 2013-09-17 | 2013-12-25 | 中国科学院西双版纳热带植物园 | Fungi strain of Tulasnella and application thereof in promotion of germination of Cymbidium mannii seeds |
| WO2015100431A2 (en) * | 2013-12-24 | 2015-07-02 | Symbiota, Inc. | Plants containing beneficial endophytes |
-
2017
- 2017-12-22 CN CN201711407614.1A patent/CN108048334B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399698A (en) * | 2010-12-08 | 2012-04-04 | 云南农业大学 | Separation and application of verticillium spp. fungus capable of promoting cymbidium wenshanense tissue cultured seedling growth |
| CN102876580A (en) * | 2010-12-08 | 2013-01-16 | 云南农业大学 | Separation and application of Nectria fungi for promoting growth of Cymbidium wenshanense tissue culture plants |
| CN102612965A (en) * | 2012-04-05 | 2012-08-01 | 中国科学院西双版纳热带植物园 | Method for obtaining germination effective symbiotic fungus for Cymbidium bicolor seeds |
| CN103468587A (en) * | 2013-09-17 | 2013-12-25 | 中国科学院西双版纳热带植物园 | Fungi strain of Tulasnella and application thereof in promotion of germination of Cymbidium mannii seeds |
| WO2015100431A2 (en) * | 2013-12-24 | 2015-07-02 | Symbiota, Inc. | Plants containing beneficial endophytes |
Non-Patent Citations (7)
| Title |
|---|
| LINDE, CELESTE C.等: "New species of Tulasnella associated with terrestrial orchids in Australia", 《IMA GUNGUS》 * |
| SOLIS, KARINA 等: "Tulasnella tubericola (Tulasnellaceae, Cantharellales, Basidiomycota): a new Rhizoctonia-like fungus associated with mycorrhizal evergreen oak plants artificially inoculated with black truffle (Tuber melanosporum) in Spain", 《PHYTOTAXA》 * |
| XIAO-LAN ZHAO 等: "The colonization patterns of different fungi on roots of Cymbidium hybridum plantlets and their respective inoculation effects on growth and nutrient uptake of orchid plantlets", 《WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY》 * |
| 丁晖 等: "卡特兰与丝核菌共培养体系的建立及卡特兰菌根显微结构的研究", 《菌物系统》 * |
| 安曼云: "云南杓兰菌根真菌组成及共生关系研究", 《广西植物》 * |
| 杨前宇: "兰科菌根真菌多样性研究及其对兰科植物的影响", 《中国博士学位论文全文数据库(电子期刊)农业科技辑》 * |
| 盛春玲 等: "硬叶兰种子的迁地共生萌发及有效共生真菌的分离与鉴定", 《植物生态学报》 * |
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