CN105861330B - A kind of bacterial strain of the promotion with leaf pocket orchid plant strain growth and its application - Google Patents
A kind of bacterial strain of the promotion with leaf pocket orchid plant strain growth and its application Download PDFInfo
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- CN105861330B CN105861330B CN201610387325.9A CN201610387325A CN105861330B CN 105861330 B CN105861330 B CN 105861330B CN 201610387325 A CN201610387325 A CN 201610387325A CN 105861330 B CN105861330 B CN 105861330B
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Abstract
The invention belongs to plant growth-promoting bacteria technical fields, and in particular to be a kind of bacterial strain of the promotion with leaf pocket orchid plant strain growth and its application.A kind of bacterial strain of the promotion with leaf pocket orchid plant strain growth, taxology are named as glue film bacterium A6-2 (Tulasnella sp.), and Guangdong Province's Culture Collection is preserved on May 12nd, 2016, and deposit number is GDMCC No.60034.The bacterial strain of promotion of the invention with leaf pocket orchid plant strain growth is promoting with the application in leaf pocket orchid plant strain growth.Band leaf pocket orchid plant strain growth is promoted using method provided by the invention, it is easy to operate, low in cost.The present invention gives birth to important economic value for the artificial breeding with leaf pocket orchid.
Description
Technical field
The invention belongs to plant growth-promoting bacteria technical fields, and in particular to be a kind of bacterium of the promotion with leaf pocket orchid plant strain growth
Strain and its application.
Background technique
Paphiopedilum (Paphiopedilum) plant, flower-shape is peculiar, and pattern is gorgeous, be the favorite orchid of people it
One, often pocket orchid is referred to as " slippers are blue ", " celestial being is carried out blue " to people.In recent ten years, due to wildness smuggling and habitat destruction etc.,
The quantity of Wild Paphiopedilum is sharply reduced, distributed area gradually atrophy, and many types have arrived the edge of extinction.
In order to alleviate the acquisition pressure to Wild plant, endangered species is protected, and meet people to cypripedium
Demand, domestic and foreign scholars study the artificial propagation of pocket orchid plant in large quantities.Currently, sterile the broadcasting of part pocket orchid species class
Kind and tissue culture technique have succeeded, but slow growth after aseptic seedling cultivation, and survival rate is low, restricts the artificial breeding of pocket orchid
It educates.Under field conditions (factors), the Seedling Stage that mycorrhizal fungi can help plant to spend fragility grows up to plant, improves plant and improves nutrient
Absorption efficiency and adverse circumstance resistance.But because cypripedium mycorrhizal fungi is separately cultured difficulty, still lack with more aobvious
The growth-promoting bacterial strain of work.Therefore, screening and cultivate to have promotes the bacterial strain of pocket orchid plant strain growth to restore tool to the cultivation of pocket orchid and population
It is significant.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The object of the present invention is to provide a kind of bacterial strain of the promotion with leaf pocket orchid plant strain growth and its applications.
Strains A 6-2 provided by the invention be from band leaf pocket orchid root it is isolated, through morphology and molecular biology identification
For the Tulasnella (Tulasnella sp.) of Basidiomycotina glue film Cordycepps, Guangdong Province is preserved on May 12nd, 2016
Culture Collection (abbreviation GDMCC, address: 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, the micro- life in Guangdong Province
Object research institute, postcode 510075), deposit number is GDMCC No.60034.
The present invention also provides the strains A 6-2 to promote with the application in leaf pocket orchid plant strain growth.
Compared with prior art, the invention has the following beneficial technical effects:
1. strains A 6-2 of the invention with leaf pocket orchid plant co-culture can promote seedling development, grow can be carried out it is photosynthetic
The blade of effect cultivates seedling replanting to matrix, and seedling can survive and well-grown.
2. band leaf pocket orchid breeding efficiency and process are promoted using method provided by the invention, it is easy to operate, low in cost.This
Invention gives birth to important economic value for the artificial breeding with leaf pocket orchid.
Preservation information explanation
Glue film bacterium A6-2 (Tulasnella sp.), deposit number are GDMCC No.60034, and the deposit date is 2016 5
The moon 12, depositary institution are Guangdong Province's Culture Collection (GDMCC), and preservation address is Xianlie Middle Road, Guangzhou City 100
Number 5 building, the building of compound the 59th.
Detailed description of the invention
Fig. 1 is the molten bacterium colony shape of glue film bacterium A6-2 (Tulasnella sp.) of the invention on PDA solid medium
State；
Fig. 2 is the hypha form of glue film bacterium A6-2 (Tulasnella sp.) of the invention；
Fig. 3 is the hypha form Torulose cell form of glue film bacterium A6-2 (Tulasnella sp.) of the invention；
Fig. 4 is the microbial inoculum of glue film bacterium A6-2 (Tulasnella sp.) preparation of the invention to plant strain growth comparison diagram；
Explanation in relation to appended drawing reference:
CK is the plant being not used after microbial inoculum transplanting of the invention；A6-2 is using glue film bacterium A6-2 of the invention
Plant after the microbial inoculum transplanting of (Tulasnella sp.) preparation.
Specific embodiment
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
Material and reagent as used in the following examples, are commercially available unless otherwise specified.Following implementations
Experimental method used in example is conventional method unless otherwise specified.
Embodiment 1: the separation identification and culture of mycorrhizal fungi
1.1 separation and pure medium
Separating mycorrhizal fungi culture medium is potato dextrose medium (PDA), potato 200g, agar 6g, glucose
To 1000ml, PH is natural for 15g moisturizing.According to conventional biology test method, isolation medium is configured, 121 DEG C of high pressures are damp and hot to go out
Bacterium 20min is cooled to 45 DEG C or so to culture medium, streptomysin (100ug/ml) and penicillin (100ug/ml) is added, after shaking up
Prepare plate.
The separation of 1.2 mycorrhizal fungis and purification process
Mycorrhizal fungi separates according to conventional organization partition method: taking the Lanzhou-Xinjiang fresh feeding root of band leaf pocket, is chosen at Leica microscope
Lower observation selection has the root segment of hypha body, rinses well under flowing water, handles 8min, sterile water respectively with the rinsing of 75% alcohol
It rinses 1-2 times, then sterilizes 8min, aseptic water washing 7-8 times with 10%NaClO.Pretreated root is cut into the small of 1-2mm
The material handled well, is then placed on PDA plate, 24 DEG C of constant temperature incubation 3-5d, it is seen that the bacterium grown out of cortical cell by section
Filament length enters in culture medium.Bacterial strain is separated and purified using mycelia tip picking method, then is connected in test tube and saves.
The Morphological Identification of 1.3 mycorrhizal fungis
Observe the colony characteristics of bacterial strain, including colony shape, size, color, the speed of growth and mycelia, sporophore and spore
Form etc..Observation discovery is in PDA culture medium, and for bacterium colony at milky, early stage mycelia is close to culture basal growth, part intrusion culture
In base, edge is irregular；Later period can produce a large amount of aerial hyphaes, and form fine granularity hypha body, and bacterium colony is at iron brown；Mycelia
Culture dish can be covered with by growing the about 2.5d or so that is exceedingly fast；Mycelia has every rectangular branch, nearly bifurcation has every Hyphal anastomosis journey
Degree is high；Beads shape mycelia is more, oblong；Do not generate spore.
Method for identifying molecules: the collection of mycelia: strain inoculated to PDA liquid medium is shaken into bacterium 7-10 days (200rpm, 25
DEG C), mycelia is collected by filtration in filter paper, extracts for genome.Genome DNA extraction uses CTAB method.
The PCR amplification of ITS section: the amplification of rDNA ITS section uses universal primer ITS1 (5 '
TCCGTAGGTGAACCTGCGG3 '), ITS4 (5 ' TCCTCCGCTTATTGATATGC3 ').The group of PCR reaction system become 10 ×
Buffer 5ul, 10mmol/L NTP (Mg+) 1ul, each 1ul of 10u mol/L primer, (final concentration is in 80ng/L by template DNA 1ul
Left and right), Taq enzyme 1U (0.25ul) is settled to 50ul with sterile pure water.PCR reaction uses BIARAD Engine type PCR instrument, follows
Ring parameter are as follows: 95 DEG C of initial denaturation, 4min, be denaturalized 95 DEG C, 40s, anneal 56 DEG C, 40s, extend 72 DEG C, 45s；72 DEG C of filling-in,
8min.35 circulations are carried out altogether.
Product send to Beijing AudioCodes biotechnology Co., Ltd and (the use of instrument being ABI 3730XL) is sequenced.Institute
It obtains sequence results and carries out comparison by Internet using PRIMER3 software, and be aided with artificial check and correction, determine the reliability of sequence.Obtain sequence
It is listed in BLAST on NCBI.
Sequencing result is as shown in sequence 1 in sequence table.Sequencing result is compared online in GenBank, the bacterial strain with
The similitude of Tulasnella fungi (Tulasnella sp.) is higher, determines that the bacterial strain being separated to is Tulasnella fungi, by it
It is named as strains A 6-2.
Embodiment 2: the preparation and application of microbial inoculum
The preparation of 2.1 liquid bacterial agents and application
Using PDA liquid medium (potato 200g, glucose 15g moisturizing to 121 DEG C of high pressure moist heat sterilizations of 1000mL)
20min is cooled to room temperature to culture medium.Bacterium 20d or so is shaken with 160-220rpm in shaking table after inoculating strain A6-2, dilutes bacterium solution
200-300 times, root dipping or pouring root, about 60d pouring root 1 time are carried out to the tissue-cultured seedling transplanted in the recent period.Tissue-cultured seedling bottle outlet requires to be root long
At least in 2cm or more, 3 plant with blade can just be transplanted.
The preparation of 2.2 solid fungicides and application
By cultivation matrix (pine bark about 2m2Size: perlite 4:1) be dipped into it is wet in 121 DEG C of high pressures after complete water saturation
Heat sterilization 20min, is cooled to room temperature to culture medium.In 30 DEG C of placement 25d or so after the microbial inoculum of inoculating strain A6-2 preparation, until
Until observing after bark has milky, tissue-cultured seedling is transplanted after the matrix that will carry disease germs packing.
Transplanted seedling about 180d, random each selection 30 after transplanting of microbial inoculum (control) is not applied to application microbial inoculum (A6-2) and
Strain carry out the number of blade, most come into leaves, radical, longest root and fresh weight measure, the results are shown in Table 1.
The microbial inoculum of glue film bacterium A6-2 (Tulasnella sp.) preparation of the invention of table 1 is to band leaf pocket orchid biomass
Influence
Note: different lowercases indicate 0.05 horizontal upper significant difference after data；Different capitalizations indicate that 0.01 is horizontal
On significant difference.
As shown in Table 1, the plant leaf of the microbial inoculum of glue film bacterium A6-2 (Tulasnella sp.) preparation of the invention is applied
How long out than the 1-2 piece leaf for not applying microbial inoculum, how long root goes out 2-3 item, and fresh weight obviously increases 1 times or more.
In conclusion microbial inoculum prepared by glue film bacterium A6-2 (Tulasnella sp.) of the invention can dramatically increase plant
Leaf growth, promote the elongation of root, increase the accumulation of biomass.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (1)
1. a kind of bacterial strain of the promotion with leaf pocket orchid plant strain growth is promoting with the application in leaf pocket orchid plant strain growth, feature exists
In taxology is named as glue film bacterium (Tulasnella sp.) A6-2, is preserved in Guangdong Province microorganism on May 12nd, 2016
Culture Collection Center, deposit number are GDMCC No.60034.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107125005B (en) * | 2017-06-12 | 2020-03-13 | 贵州省林业科学研究院 | Mycorrhizal seedling raising method for paphiopedilum harderi |
| CN107746811B (en) * | 2017-10-30 | 2020-04-03 | 广西壮族自治区农业科学院花卉研究所 | Strain for promoting growth of paphiopedilum hirsutissimum plants and application thereof |
| CN110408551A (en) * | 2019-08-21 | 2019-11-05 | 中国科学院昆明植物研究所 | One plant of U.S. spore glue film bacterium QS104 and its application and the method for promoting pocket orchid Aseptic Seedling Growth |
| CN110760448B (en) * | 2019-11-08 | 2022-03-22 | 广西壮族自治区农业科学院 | Strain for promoting growth of paphiopedilum hirsutissimum plant and application thereof |
| CN112646734B (en) * | 2020-12-31 | 2022-11-29 | 广西壮族自治区林业科学研究院 | Orchid mycorrhizal fungus PF06 and application thereof |
| CN112795489B (en) * | 2020-12-31 | 2022-11-29 | 广西壮族自治区林业科学研究院 | Orchid mycorrhizal fungus PF02 and application thereof |
| CN112592838B (en) * | 2020-12-31 | 2022-11-29 | 广西壮族自治区林业科学研究院 | Orchid mycorrhizal fungus PF07 and application thereof |
| CN112760239B (en) * | 2021-03-19 | 2022-05-13 | 中国林业科学研究院林业研究所 | Mucocel bacterium for promoting paphiopedilum hirsutissimum seeds to germinate and form seedlings, and culture method and application thereof |
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| CN104719166A (en) * | 2015-03-31 | 2015-06-24 | 中国科学院昆明植物研究所 | Method for promoting growth and multiplication of aseptic seedlings of paphiopedilum armeniacum |
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| 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 |
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