CN102399698A - Separation and application of verticillium spp. fungus capable of promoting cymbidium wenshanense tissue cultured seedling growth - Google Patents
Separation and application of verticillium spp. fungus capable of promoting cymbidium wenshanense tissue cultured seedling growth Download PDFInfo
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- CN102399698A CN102399698A CN2010105777225A CN201010577722A CN102399698A CN 102399698 A CN102399698 A CN 102399698A CN 2010105777225 A CN2010105777225 A CN 2010105777225A CN 201010577722 A CN201010577722 A CN 201010577722A CN 102399698 A CN102399698 A CN 102399698A
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Abstract
The invention relates to separation and application of a verticillium spp. fungus capable of promoting cymbidium wenshanense tissue cultured seedling growth, and belongs to the field of plant protection and biotechnology. A production strain of the verticillium spp. fungus is a verticillium spp. strain having a preservation number of china general microbiological culture collection center (CGMCC) No.3927. The verticillium spp. fungus has a substantial characteristic of promoting cymbidium wenshanense tissue cultured seedling growth. In the invention, a cymbidium wenshanense tissue cultured seedling growth is inoculated with separated symbiotic fungi through two methods, and the separated symbiotic fungi are subjected to preliminary identification according to morphological characteristics, wherein through the combination with molecular biology technologies such as internal transcribed spacer (ITS) expansion, cloning sequencing and the like, it is determined that selected symbiotic fungi capable of promoting cymbidium wenshanense tissue cultured seedling growth are the verticillium spp. fungi. In the invention, the problem that an orchidaceous tissue cultured seedling transplanting survival rate is low is solved partly. The separation and application of the verticillium spp. fungus capable of promoting cymbidium wenshanense tissue cultured seedling growth produce promotion effects on orchid industry future development.
Description
Technical field:
Blue tissue cultured seedling growth has promoter action to the red post of mountain of papers to the invention relates to the mycorhiza bacterium that from orchid root, is separated to, and belongs to plant protection and biological technical field.
Background technology:
The orchid family (Orchidaceae) plant is commonly called as orchid, and the whole world has 700 to belong to kind more than 20000 and a large amount of mutation approximately, mainly is distributed in the torrid zone, subtropics and area, temperate zone, and especially the torrid areas in South America and Asia is many.Orchid can give birth to blue, epiphytic orchid and saprophytic blue 3 types according to the difference of growing environment with being divided into, as a kind of important ornamental flower and resources of medicinal plant, huge economic is arranged on flowers and natural drug industry and utilize potential.Along with the fast development of the modernization of Chinese medicine and orchid industry, the demand of orchid is increased rapidly.But the orchid seed is tiny, only the undifferentiated proembryo of tool; Root is thick, and the root hair is undeveloped.Seed can not normally be sprouted under field conditions (factors), and root can not satisfy the demand of plant materials to moisture and nutritive substance, makes the artificial culture of orchid quite difficult.In recent years; The tissue rapid propagation technology has been applied to orchid, has realized the industrial seedling rearing of orchid, but the tissue cultured seedling transplanting survival rate is very low; Poor growth; Be difficult to big area and promote, its major cause is that orchid can't form mycorhiza with relevant fungi in a short time, and mycorrhizal fungi has great significance for orchid.Research shows, have only fungi and orchid seed or root to form symbiotic mycorhiza relation under the state of nature after, orchid could normally sprout and grow.
Mycorhiza is the symbiosis association of formed mutual reciprocity and mutual benefit between one type of fungi and the root system of plant in the soil.Frank in 1885 has proposed " mycorhiza (Mycorrhiza) " this term and has described the symbiosis association of plant and fungi, and mycorhiza research begins to develop rapidly thus.Link has at first confirmed to exist fungi in the orchid root, and afterwards, orchid and mycorhiza relation are confirmed, and have carried out broad research.Bernard reported first in 1903 relation of fungi and germination of arethusa seeds.Burgeff draws fungi and the research of the orchid family seed; The orchid family seed is sprouted under the infecting of fungi; He separates and has named many fungies; Developed the most important theories of digestion fungi, carried out the research of the relation between the orchid family mycorrhizal fungi and the orchid afterwards both at home and abroad in succession in a large number as orchid growing nutrient source.
China's orchid mycorhiza bacterium researchdevelopment is very fast, and rhizoma Gastrodiae mycorhiza bacterium successfully has been applied to gastrodia seed germination and artificial culture.But still there are a lot of orchid mycorhiza bacterium to need research; Understand kind and the characteristics of fungal component in depth, inquire into the interaction mechanism between orchid and fungi, screen good orchid symbiosis fungi; Development the orchid family microbial inoculum has great importance for the batch production production that realizes orchid.
Summary of the invention:
Main purpose of the present invention is that solution the orchid family tissue cultured seedling transplanting survival rate is low, filters out the fungal component that helps the orchid growth.
The fungi that the present invention adopts is Verticillium (Verticillium tenerum), depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. No. 3, No. 1 institute in North Star West Road, Chaoyang District, city; Preservation date: on 06 17th, 2010; The numbering CGMCC No.3927 that preservation is registered on the books.
Fungal bacterial strain Verticillium of the present invention (Verticillium spp.) bacterial strain separates from orchid root inner tissue, through the cultivation proterties, and morphology and Molecular Identification, this bacterial strain is Verticillium (Verticillium spp.).This bacterial strain has following characteristic: (1) this bacterial strain can be observed conidiophore colyliform branch at microscopically, and the conidiogenous cell base portion slightly expands; (2) this bacterial strain can promote the growth of orchid tissue cultured seedling.
The present invention is achieved in that and separates fungi in the orchid root, the fungi that is separated to is carried out identification of morphology, and effective symbiosis fungi is screened in its inoculation of carrying out the orchid tissue cultured seedling, and effective strain is carried out ITS section cloning and sequencing.
The isolation and identification method of Verticillium of the present invention (Verticillium spp.):
1, separation of mycorhiza bacterium and purifying
PDA solid culture based formulas is: 20% yam, 2% glucose, 2% agar powder.
Take off healthy and strong root segment from the orchid plant, rinse well, it is for use to put into beaker.On the Bechtop that has carried out sterilization and disinfected, the root segment in the beaker with 70% alcohol disinfecting 30s, is soaked 1min with 0.1% mercuric chloride solution again, use at last sterile water wash 3-5 time.Under aseptic condition, smash tissue to pieces, pour sterilized water into, draw hypha body at microscopically in the PDA substratum with liquid-transfering gun, 24 ℃ of constant temperature dark culturing after bacterium colony grows up to, repeat to get the colony edge purifying of partly transferring.
2, the morphology of isolated strains is identified
Bacterial strain behind the purifying was cultivated 5-7 days under 25 ℃ constant temperature; Be placed on the slide glass the colony edge mycelia that takes a morsel with tweezers or dissecting needle; Slide is made in KOH solution with 3% and the dyeing of 0.05% sarranine dye liquor, through its morphological specificity of microscopic examination, under the preliminary evaluation isolated strains.
3, the Molecular Identification of isolated strains
3.1DNA extraction
With mycelium inoculation in liquid PDA; Be positioned on the shaking table 25 ℃ of conditions and cultivated 10 days, blot mycelia moisture with thieving paper and take by weighing weight, change in the mortar of precooling; With liquid nitrogen it fully is ground to Powderedly, adds 1mlDAN and extract damping fluid (100mM Tris-HCL PH8.0; 100mM EDTA; 250mMNaCl), 20%SDS, 5M NaCl, 10%CTAB, abundant mixing, insulation is 60 minutes under 60 ℃ of conditions, 10; 000rpm got supernatant in centrifugal 5 minutes, added isopyknic saturated phenol, and the centrifuging and taking supernatant adds isopyknic 25: 24: 1 saturated phenol: chloroform: primary isoamyl alcohol; The centrifuging and taking supernatant adds isopyknic 24: 1 chloroform: the primary isoamyl alcohol concussion mixes, the centrifuging and taking supernatant, and the ice absolute ethyl alcohol that adds 2 times of volumes places-20 ℃ of refrigerators; Deposit D AN, centrifugal, keep deposition, and with 70% ice ethanol rinsing 2 times; Natural air drying or vacuum-drying add TE damping fluid dissolving DAN, place-30 ℃ for use.
3.2 the 5.8S rDNA-ITS of isolated strains clone and order-checking
Carry out DNA cloning with ITS1 and ITS4 primer.Amplified reaction carries out on PCR appearance Perkin Elmer, and the PCR reaction system is 50uL.Following PCR circulation: 94 ℃ of preparatory sex change 3min, circulate 1 time; 94 ℃ of sex change 1min, 51 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations; Last 72 ℃ are extended 10min.The PCR product detects with 1% agarose gel electrophoresis.Pcr amplification product carries out purifying with the test kit that the precious biology in Dalian provides, and purified product can directly check order, subsequent use.Plasmid vector pGEM-T-easy and the ligation of PCR purified product, conversion, are chosen the male recon at last and are served Hai Shenggong and carry out dna sequencing after recombinant plasmid PCR through red-hickie screening.
3.3 sequential analysis
Utilize software analysis such as Bioxm, DNAMAN and BLAST, carry out homology relatively with the known array that lands among the GenBank.
Verticillium of the present invention (Verticillium spp.) fungi promotes the blue tissue cultured seedling growth test of the red post of mountain of papers
1, test materials is prepared
The matter that fetches earth is soil preferably, sterilization; Select the blue tissue cultured seedling of the red bamboo of mountain of papers for supplying the examination material, carry out inoculation experiments with isolating fungi in the orchid root material.
2, TP
2.1 mix the soil inoculation, to kind cultured inoculation is arranged in the soil of the blue tissue cultured seedling of the red post of mountain of papers, carry out symbiosis culture; And the orchid of being planted with the soil that does not connect bacterium is as contrast, and repeated inoculation 3 times is observed the leaf look of orchid seedling after 2 months; Growing way, the root growth situation filters out effective bacterium.
2.2 infect the blue tissue cultured seedling root of the red post of mountain of papers with strain separated, the one all backs, inoculation back of preserving moisture in sterilization soil, are carried out symbiosis culture to the orchid seed; And the orchid of being planted with the soil that does not connect bacterium is as contrast; Repeated inoculation 3 times, the leaf look of observation orchid seedling after 2 months, growing way; The root growth situation filters out effective bacterium.
3, test-results
The blue tissue cultured seedling symbiosis culture of the red post of table 1 Verticillium (Verticillium spp.) bacterial strain and mountain of papers situation
Can find out that from table 1 the blue tissue cultured seedling surviving rate of the red post of the mountain of papers of Verticillium (Verticillium spp.) inoculation is higher, mix soil inoculation and bacterial strain and invade the average survival of root and reached 85.00% and 86.11%.The tissue cultured seedling transplanting survival rate is played apparent in view promoter action.
After 2 months,, find that the leaf look and the growing way of the blue tissue cultured seedling of the red post of mountain of papers of Verticillium (Verticillium spp.) inoculation all obviously will be compared the good of photograph through screening; Blue seedling leaf look greener; Well developed root system grows fine, and filters out the fungal component that helps the orchid growth.
Description of drawings
Fig. 1 is the cultivation proterties figure of Verticillium (Verticillium spp.) bacterial strain on the PDA substratum;
Fig. 2 is that Verticillium (Verticillium spp.) inoculation has effect of inoculation and map in the blue tissue cultured seedling soil of the red post of mountain of papers to kind;
Fig. 3 is the effect of inoculation figure that Verticillium (Verticillium spp.) bacterial strain infects the blue tissue cultured seedling root of the red post of mountain of papers;
Fig. 4 is the inoculation map that Verticillium (Verticillium spp.) bacterial strain infects the blue tissue cultured seedling root of the red post of mountain of papers.
Embodiment:
The separation of fungal component:
PDA solid culture based formulas is: 20% yam, 2% glucose, 2% agar powder.
Take off healthy and strong root segment from the orchid plant, rinse well, it is for use to put into beaker.On the Bechtop that has carried out sterilization and disinfected, the root segment in the beaker with 70% alcohol disinfecting 30s, is soaked 1min with 0.1% mercuric chloride solution again; Use at last sterile water wash 3-5 time, under aseptic condition, smash tissue to pieces; Pour sterilized water into, draw hypha body at microscopically in the PDA substratum with liquid-transfering gun, 24 ℃ of constant temperature dark culturing; After bacterium colony grows up to, repeat to get the colony edge purifying of partly transferring.
The identification of morphology of isolated strains: the bacterial strain behind the purifying was cultivated 5-7 days under 25 ℃ constant temperature; Be placed on the slide glass the colony edge mycelia that takes a morsel with tweezers or dissecting needle; Slide is made in KOH solution with 3% and the dyeing of 0.05% sarranine dye liquor; Through its morphological specificity of microscopic examination, under the preliminary evaluation isolated strains.
The screening of effective strain:
To kind cultured inoculation is arranged in the soil of the blue tissue cultured seedling of the red post of mountain of papers, carry out symbiosis culture, and the orchid of being planted with the soil that does not connect bacterium observes the leaf look of orchid seedling as contrast after 2 months, growing way, the root growth situation filters out effective bacterium;
Infect the blue tissue cultured seedling root of the red post of mountain of papers with strain separated, the one all backs, inoculation back of preserving moisture in sterilization soil, are carried out symbiosis culture to the orchid seed; And the orchid of being planted with the soil that does not connect bacterium is observed the leaf look of orchid seedling, growing way as contrast after 2 months; The root growth situation filters out effective bacterium.
The Molecular Identification of bacterial strain:
The extraction of isolated strains DNA: with mycelium inoculation in liquid PDA; Be positioned on the shaking table 25 ℃ of conditions and cultivated 10 days; Blot mycelia moisture with thieving paper and take by weighing weight; Change in the mortar of precooling, with liquid nitrogen it fully is ground to Powderedly, add 1mlDAN and extract damping fluid (100mM Tris-HCL PH8.0; 100mM EDTA; 250mM NaCl), 20%SDS, 5M NaCl, 10%CTAB, abundant mixing, insulation is 60 minutes under 60 ℃ of conditions, 10; 000rpm got supernatant in centrifugal 5 minutes, added isopyknic saturated phenol, and the centrifuging and taking supernatant adds isopyknic 25: 24: 1 saturated phenol: chloroform: primary isoamyl alcohol; The centrifuging and taking supernatant adds isopyknic 24: 1 chloroform: the primary isoamyl alcohol concussion mixes, the centrifuging and taking supernatant, and the ice absolute ethyl alcohol that adds 2 times of volumes places-20 ℃ of refrigerators; Deposit D AN, centrifugal, keep deposition, and with 70% ice ethanol rinsing 2 times; Natural air drying or vacuum-drying add TE damping fluid dissolving DAN, place-30 ℃ for use.
The 5.8S rDNA-ITS clone and the order-checking of isolated strains: carry out DNA cloning with ITS1 and ITS4 primer.Amplified reaction carries out on PCR appearance Perkin Elmer, and the PCR reaction system is 50uL.Following PCR circulation: 94 ℃ of preparatory sex change 3min, circulate 1 time; 94 ℃ of sex change 1min, 51 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations; Last 72 ℃ are extended 10min.The PCR product detects with 1% agarose gel electrophoresis.Pcr amplification product carries out purifying with the test kit that the precious biology in Dalian provides, and purified product can directly check order, subsequent use.Plasmid vector pGEM-T-easy and the ligation of PCR purified product, conversion, are chosen the male recon at last and are served Hai Shenggong and carry out dna sequencing after recombinant plasmid PCR through red-hickie screening.Check order and classify as:
GCTAACCGGTCTGTACACTCATAACCCTTTGTGACCATTATACCTAGTTGCTTCGGCGGCGCGCCTCCGGGCGTGCCCGCCGGCCAATTATAGTCTCTTAGTTGTGAATCGACGTTACATCTGAGTGTTCTAAGCGAACTGTTAAAACTTTCAACAACGGATCTCTTGGCTCCAGCATCGATGAAGAACGCAGCGAAACGCGATATGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCTTCCAGTATCCTGGGAGGCATGCCTGTCCGAGCGTCGTTTCAACCCTCGAGCCCCCGTGGCCCGGTGTTGGGGACGCTGCCCAGCTACCTGGGCAGCCCCCCTAAATGCAGTGGCGGTCCCGCAAGTCCCAGCCCTTTGCGTAGTAATATCATTCTCGCTCTGGACGGCTCGCGGGCTTACCAGCCTCGAAACCCCCTACAAAGACCGCCCCGGCGGCAACGGTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAGCCGGGGGAGGAA
Utilize software analysis such as Bioxm, DNAMAN and BLAST, carry out homology relatively with the known array that lands among the GenBank.Through comparison, this isolated strains belongs to Verticillium (Verticillium spp.) bacterial strain fungi.
Promptly get symbiosis fungal bacterial strain of the present invention.
The nucleotides sequence tabulation:
GCTAACCGGTCTGTACACTCATAACCCTTTGTGACCATTATACCTAGTTGCT
TCGGCGGCGCGCCTCCGGGCGTGCCCGCCGGCCAATTATAGTCTCTTAGTT
GTGAATCGACGTTACATCTGAGTGTTCTAAGCGAACTGTTAAAACTTTCAA
CAACGGATCTCTTGGCTCCAGCATCGATGAAGAACGCAGCGAAACGCGATA
TGTAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATT
GCGCCTTCCAGTATCCTGGGAGGCATGCCTGTCCGAGCGTCGTTTCAACCC
TCGAGCCCCCGTGGCCCGGTGTTGGGGACGCTGCCCAGCTACCTGGGCAG
CCCCCCTAAATGCAGTGGCGGTCCCGCAAGTCCCAGCCCTTTGCGTAGTAA
TATCATTCTCGCTCTGGACGGCTCGCGGGCTTACCAGCCTCGAAACCCCCT
ACAAAGACCGCCCCGGCGGCAACGGTTGACCTCGGATCAGGTAGGAATAC
CCGCTGAACTTAAGCATATCAATAGCCGGGGGAGGAA
Claims (3)
1. symbiosis fungi that can promote the blue tissue cultured seedling growth of the red post of mountain of papers, it is characterized in that: this symbiosis fungal bacterial strain is Verticillium ((Verticillium spp.), preserving number CGMCC No.3927.
2. the separation method of the said Verticillium of claim 1 (Verticillium spp.) bacterial strain; It is characterized in that; After the orchid root tissue disinfection finished, smash tissue to pieces under the aseptic condition, pour sterilized water into; Observe hypha body earlier at microscopically, draw hypha body with liquid-transfering gun and to the PDA substratum, cultivate.Its PDA culture medium prescription is: yam 20.0%, glucose 2.0%, agar 2.0%.
3. the application of the described Verticillium of claim 1 (Verticillium spp.) bacterial strain in promoting the blue tissue cultured seedling growth of the red post of mountain of papers.
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| CN108048334A (en) * | 2017-12-22 | 2018-05-18 | 中国林业科学研究院林业研究所 | 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 |
| CN108048334B (en) * | 2017-12-22 | 2021-01-08 | 中国林业科学研究院林业研究所 | Establishment method of symbiotic system of gloeosporium fungi for promoting germination of cymbidium and cattleya seeds |
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