CN101921718B - Dendrobium candidum endophyte with growth promotion function and use thereof - Google Patents
Dendrobium candidum endophyte with growth promotion function and use thereof Download PDFInfo
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Abstract
The present invention discloses a sphingomonas sp. strain with preservation name of Sphingomonas (SH1), preserved in China Centre for Type Culture Collection (CCTCC) in Wuhan University, China, wherein the preservation date thereof is February 4, 2010 with preservation number thereof being CCTCC No: M 2010041. The present invention also discloses a use of the sphingomonas sp. strain applied to promoting growth of dendrobium candidum, comprising the following specific uses: inoculating raw fermenting liquid or fermented liquid of the sphingomonas sp. strain CCTCC No: M 2010041 into a root of a dendrobium candidum seedling so as to promote the growth of the dendrobium candidum seedling.
Description
Technical field
The present invention relates to a kind of natural medicinal plant endogenetic bacteria---dendrobium candidum endophyte and uses thereof.
Background technology
Herba Dendrobii belongs to the second largest genus in the orchid family (Orchidaceae) plant---Dendrobium (Dendrobium SW.), it is the superfine product in the stem of noble dendrobium, belong to aerial orchid section herbaceous plant, iron is green gains the name because of epidermis is for it, have nourishing Yin and clearing heat, the beneficial stomach that promotes the production of body fluid, moisten the lung and relieve the cough, make eye bright keep fit, effect such as easing pain and diminishing inflammation; Modern pharmacological research shows, effect such as that the stem of noble dendrobium also has is antitumor, anti-ageing, enhancing body immunizing power, vasodilation and anti-platelet aggregation.Because its great pharmaceutical use makes it become the whole nation and relates to pharmaceutical factory and the healthcare products processing enterprise raw material lifeblood of using stem of noble dendrobium class.But the growth conditions of Herba Dendrobii is very harsh, so natural production is very rare, more excessively excavates for a long time because of among the people, causes wild resource endangered.
China is annual according to statistics is that medicine, the healthcare products output value of raw material surpasses hundred million yuan of hundreds ofs with the stem of noble dendrobium, and quantity every year of the market requirement is all more than millions of kilograms, and in rising trend year by year.The Herba Dendrobii major part of Sheng Chaning is all from artificial culture in the market, and promptly the mode of training through group earlier expands numerous Herba Dendrobii, again through the acclimatization and transplants field.Yet the artificial culture Herba Dendrobii still exists breeding and seedling difficulty, transplant survival difficulty and poor growth Three Difficult Issues, and this is undoubtedly a serious obstruction to the development and use of Herba Dendrobii.
Endophyte of plant is to separate from the process plant tissue of surface sterilization or to obtain from the juice of inside plants, and apparent the going up of plant do not had the microorganism that harm does not promptly cause obvious illness.Endophyte of plant comprises reciprocal sharp altogether (mutualistic) and neutral (neutral) endosymbiontic microorganism.
At present from the various organs (root, stem, leaf, flower, fruit, seed) of a lot of plants, be separated to endophyte.They are existing many bibliographical informations aspect the intravital ubiquity of plant, abundant species diversity and functional diversity.Endophyte interacts with plant through secular, forms complicated relation between them.Have now found that endophyte has the effect of the following aspects to host plant:
(1) promotes the host plant growth, improve host's productivity, can strengthen the host to resistance sick, worm, thereby aspect agriculture production, biological control and the biological control significance arranged;
(2), strengthen the resistance of host plant to environment-stress.As resistance to arid, global warning, saline and alkaline, heavy metal and other toxic substance pollution etc.;
(3), endophyte produces new biologically active substance.Endophyte of plant is as the microorganism under a kind of special habitats, in plant materials, can produce rich and varied, have a multiple bioactive secondary metabolite, people have found from endophyte and have manyly had the important use that various bioactive novel substances show at pharmaceutical sector, aspect agriculture and be worth at present.
According to research both at home and abroad, in plant seedlings, introduce microorganism and will produce positive influence growth, the metabolism of seedling, can strengthen resistance to biological and the abiotic stress factor.Insert endophyte and also can carry out comprehensive adjustment, make it comparatively fast to adapt to extraneous complex environment plant strain growth and physiological metabolism.J Nowak is called " biotizafion " with this technology, such as Mycorrhizal (mycorrhization), bacteriumization (bacterization).Beneficial microorganism and little numerous seedling are carried out common cultivation, are the brand-new routes of producing high resistance high-quality seedling, and this extensive high-efficiency high-quality cultivation for Chinese medicinal materials has good application prospects.
At present, more about the endogenetic fungus research of orchid in the world, but the research of endogenetic bacteria is less, does not more have the Herba Dendrobii endogenetic bacteria to be used for the research report of short long candidum tissue culturing face.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of Herba Dendrobii endogenetic bacteria---Sphingol single-cell strain, this bacterial strain is used for the seedlings of Dendrobium officinale promotes growth, helps to solve tissue cultured seedling poor growth in the present seedlings of Dendrobium officinale production process, the production cycle is long, cost is higher technical barrier.
In order to solve the problems of the technologies described above, the invention provides a kind of Sphingol single-cell strain, this Sphingol single-cell (Sphingomonas sp.) bacterial strain preservation name is called: Sphingol single-cell SH1, depositary institution: Chinese typical culture collection center, preservation address: Chinese Wuhan University, preservation date: on February 4th, 2010, preserving number: CCTCC NO:M2010041.
The present invention also provides the purposes of above-mentioned Sphingol single-cell strain simultaneously: be used for the seedlings of Dendrobium officinale promotes growth.
Improvement as the purposes of Sphingol single-cell strain of the present invention: Sphingol single-cell CCTCC NO:M2010041 fermenation raw liquid or fermented liquid are inoculated in the base portion of candidum tissue culturing seedling, thereby promote the candidum tissue culturing seedling growth.
Dendrobium candidum endophyte of the present invention is the strain bacterium that the contriver is separated to from the Herba Dendrobii plant, shows that through traditional method evaluation and Molecular Identification it belongs to Sphingol single-cell, with its called after Sphingol single-cell SH1 (Sphingomonas sp.SH1).Be specially:
This dendrobium candidum endophyte separates the root from wild Herba Dendrobii plant, can obtain from wild Herba Dendrobii plant separation and Culture by the following method:
1), with the root of wild Herba Dendrobii after surface sterilization is handled, be cut into small pieces, be inoculated in special NA culture medium flat plate, cultivated 1～3 day down in 28～32 ℃;
This special NA substratum by basic NA substratum and the fresh Herba Dendrobii after grinding form, the composition of basic NA substratum consists of: peptone 10g, extractum carnis 3g, NaCl 5g, agar 15-20g and H
2O1000ml at the fresh Herba Dendrobii that adds on the above-mentioned basic NA substratum after 5g grinds, and regulates pH=7.4～7.6 with the NaOH of 1mol/L, must special NA substratum.
2), picking list bacterium colony is in the special NA culture medium flat plate line purifies and separates with above-mentioned step 1), places 28～32 ℃ to cultivate 1～3 day down;
3), repeat the cultivation of separating for several times purifying, until obtaining pure growth.
Dendrobium candidum endophyte of the present invention---Sphingol single-cell bacterium SH1 (Sphingomonas sp.SH1) has been deposited in Chinese representative microbial preservation center (CCTCC), preserving number: CCTCC NO:M 2010041 on February 4th, 2010.
Dendrobium candidum endophyte of the present invention---Sphingol single-cell SH1 (being designated hereinafter simply as Sphingol single-cell CCTCCNO:M 2010041) has following biological property:
1. colony morphology characteristic: well-grown on basic NA substratum, the bacterial colony feature: bacterium colony is rounded, and yellow is smooth, neat in edge, the bacterium colony umbo is carinate.
2. morphological features: spherical, diameter is 0.5～1.5 μ m, Gram-negative.
3. major physiological biochemical character: this bacterium can be on basic NA substratum, is that 20～40 ℃, pH value are well-grown in 6.0～8.0 the environment in growth temperature.
4. genetics feature: according to " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying, Science Press, 2001), on the morphological specificity of Sphingol single-cell CCTCC NO:M 2010041 of the present invention, with domestic name to separate polyoxyethylene glycol Sphingol single-cell (Sphingomonas macrogoltabidus) the most approaching; 16S rDNA sequential analysis through this Sphingol single-cell CCTCC M2010041 shows that Sphingol single-cell CCTCC M 2010041 should belong to Sphingol single-cell and belong to.
Herba Dendrobii endogenetic bacteria SH1 of the present invention has growth promoting function to candidum tissue culturing seedling, can promote the long increase by 10%～25% of average root of tissue cultured seedling, increase the height 10%～30% of plant, increase aquatic foods kind and the dry weight 5%～20% of plant, can shorten 10～20 days production times of candidum tissue culturing seedling; Nontoxic to people and animals, environment is not polluted.Can in the production of candidum tissue culturing seedling, be applied, produce certain economic benefits.
When Herba Dendrobii endogenetic bacteria of the present invention is used to promote the Herba Dendrobii growth, only needing this Herba Dendrobii endogenetic bacteria---fermenation raw liquid or the fermented liquid of Sphingol single-cell CCTCC NO:M 2010041 are inoculated in the candidum tissue culturing seedling base portion, can obtain to promote the effect of candidum tissue culturing seedling growth.
Embodiment
The isolation cultivation method of embodiment 1, Sphingol single-cell CCTCC NO:M 2010041, carry out following steps successively:
1, gathers the root sample of the Herba Dendrobii plant of wild health in the Tonglu, Zhejiang, fully clean 25KHz ultrasonication 5 minutes with tap water;
2, under aseptic condition, to step 1 gained for the examination material with aseptic water washing 2 times, then successively with the alcohol of volumetric concentration 75% and saturated Eusol to carrying out surface sterilization for the examination material, thereby kill for trying the material surface microorganism;
3, in gnotobasis, with step 2 gained for the examination material with aseptic water washing 3 times, get the aseptic water washing liquid coating that last 1 flushing of 100 μ l produced and be inoculated in the basic NA substratum, 30 ℃ cultivate 24h after, observe and have or not bacterium colony to produce.Verify in view of the above whether this sterilization method all kills for examination material surface microorganism.
If observe conclusion be: do not have bacterium colony to produce, then carry out following step 4; If observe conclusion be: have bacterium colony to produce, then return step 2.
4, the root sample that will handle well is cut into 0.5～1cm segment, inserts in the special NA culture medium flat plate, is positioned over 30 ℃ and cultivates 1～3 day;
This special NA substratum is made up of basic NA substratum and fresh Herba Dendrobii abrasive slag; The composition of basis NA substratum consists of: peptone 10g, extractum carnis 3g, NaCl 5g, agar 18g and H
2O1000ml at the fresh Herba Dendrobii that adds on the above-mentioned basic NA substratum after 5g grinds, and regulates pH=7.4～7.6 with the NaOH of 1mol/L, must special NA substratum.
5, the bacterial classification that grows on the special NA culture medium flat plate of picking above-mentioned steps 4 gained places 30 ℃ to cultivate picking list bacterium colony 1～3 day in special NA culture medium flat plate line purifies and separates;
Repeat aforesaid operations, till obtaining single bacterium colony.
6, single bacterium colony of step 5 picking gained being connected to special NA medium slant is stored in 4 ℃ of refrigerators.
A kind of single bacterium colony of gained is a dendrobium candidum endophyte---Sphingol single-cell SH1 (Sphingomonas sp.SH1), be deposited in Chinese representative microbial preservation center (CCTCC) on February 4th, 2010, preservation address: Chinese Wuhan University, preservation date: on February 4th, 2010, preserving number: CCTCC NO:M 2010041.Its sequence is shown in SEQID NO:1.
The cultivation producing method for seed of embodiment 2, Sphingol single-cell CCTCC NO:M 2010041, carry out following steps successively:
1, Sphingol single-cell CCTCC NO:M 2010041 is transferred to basic NA medium slant, cultivated 2 days for 30 ℃; Get the inclined-plane seed;
Basis NA medium slant prescription is: peptone 10g, extractum carnis 3g, NaCl 5g, agar 18g and H
2O1000ml, pH=7.4～7.6.
2, with transfering loop picking 0.5g step 1 gained slant strains, be inoculated in the 250ml triangular flask that 100ml LB liquid nutrient medium is housed, cultivate 30h with the 200r/m speed oscillation, get Sphingol single-cell CCTCC NO:M2010041 fermenation raw liquid in 28 ℃;
LB liquid culture based formulas is: peptone 10g, yeast extract paste 10g, NaCl 5g and H
2O1000ml, pH=7.4～7.6.
The promotion experiment of 2010041 pairs of candidum tissue culturing seedling growths of embodiment 3, Sphingol single-cell CCTCC NO:M:
Following experiment all with in the candidum tissue culturing substratum of improvement the candidum tissue culturing seedling cultivated as experimental subjects.
The prescription of candidum tissue culturing substratum of improvement is: employings 1/2MS is a minimum medium, adds bananas juice 10%/L, agar 7g/L, and sucrose 20g/L is 5.8 with the NaOH adjustment pH value of 1mol/L.That is: in the 1/2MS of every L, add 0.1L bananas juice, 7g agar and 20g sucrose, and to adjust the pH value be 5.8.
Experiment 1, Sphingol single-cell CCTCC NO:M 2010041 fermenation raw liquids test one to the candidum tissue culturing seedling promotes growth:
Utilize Sphingol single-cell CCTCC NO:M 2010041 fermenation raw liquids of the foregoing description 2 gained, fermenation raw liquid is diluted, make its bacterial concentration be adjusted into 10 with sterilized water
3CFU/ml is inoculated in this dilution secondary fermentation stoste in the candidum tissue culturing substratum of improvement and has cultivated 15 days 25 strain candidum tissue culturing seedling base portions, as experimental group; Be specially: the dilution secondary fermentation stoste that adds 50 μ l at every strain candidum tissue culturing seedling base portion.
The sterilized water of equal volume is replaced above-mentioned dilution secondary fermentation stoste, and all the other contents are identical, as the blank group.
Above-mentioned experimental group and blank group are all cultivated under the intensity of illumination condition of 25 ℃ of constant temperature, whole day 1000lx.
Cultivation results is as follows:
After 60 days as seen, the average upgrowth situation of the candidum tissue culturing seedling of experimental group obviously is better than the blank group, tissue cultured seedling average root length has increased by 20.5% than blank group in the experimental group, plant height has increased by 17.8% than blank group, and tissue cultured seedling plant fresh weight and dry weight have increased by 16.3% and 15.9% than blank group respectively.
Experiment 2, Sphingol single-cell CCTCC NO:M 2010041 fermenation raw liquids test two to the candidum tissue culturing seedling promotes growth:
With the dilution after bacterial concentration by 10
3CFU/ml makes 10 into
1CFU/ml is to have cultivated 30 days candidum tissue culturing seedling as experimental subjects in the candidum tissue culturing substratum of improvement; All the other equivalent experiments 1.
Cultivation results is as follows:
After 60 days as seen, the average upgrowth situation of the candidum tissue culturing seedling of experimental group all obviously is better than the blank group, tissue cultured seedling average root length has increased by 11.3% than blank group in the experimental group, plant height has increased by 14.7% than blank group, and tissue cultured seedling plant fresh weight and dry weight have increased by 10.2% and 9.7% than blank group respectively.
Experiment 3, Sphingol single-cell CCTCC NO:M 2010041 fermented liquids test one to the candidum tissue culturing seedling promotes growth:
Is the membrane filtration of 0.45 μ m with Sphingol single-cell CCTCC NO:M 2010041 fermenation raw liquids of the foregoing description 2 gained with the aperture, must not have fermented liquid.Get this no fermented liquid and be inoculated in the candidum tissue culturing substratum of improvement and cultivated 15 days 25 strain candidum tissue culturing seedlings, as experimental group; Be specially: the no fermented liquid that adds 50 μ l at every strain candidum tissue culturing seedling base portion; After 15 days, add the no fermented liquid of 50 μ l again at every strain candidum tissue culturing seedling base portion.
The sterilized water of equal volume is replaced above-mentioned no fermented liquid, and all the other contents are identical, as the blank group.
Above-mentioned experimental group and blank group are all cultivated under the condition with experiment 1, and cultivation results is as follows:
After 60 days as seen, the average upgrowth situation of the candidum tissue culturing seedling of experimental group is better than the blank group, tissue cultured seedling average root length has increased by 7.8% than blank group in the experimental group, plant height has increased by 7.3% than blank group, and tissue cultured seedling plant fresh weight and dry weight have increased by 6.6% and 6.3% than blank group respectively.
Experiment 4, Sphingol single-cell CCTCC NO:M 2010041 fermented liquids test two to the candidum tissue culturing seedling promotes growth:
Is the membrane filtration of 0.45 μ m with Sphingol single-cell CCTCC NO:M 2010041 fermenation raw liquids of the foregoing description 2 gained with the aperture, must not have fermented liquid.Get this no fermented liquid and be inoculated in the candidum tissue culturing substratum of improvement and cultivated 30 days 25 strain candidum tissue culturing seedlings, as experimental group; Be specially: the no fermented liquid that adds 100 μ l at every strain candidum tissue culturing seedling base portion.
The sterilized water of equal volume is replaced above-mentioned no fermented liquid, and all the other contents are identical, as the blank group.
Above-mentioned experimental group and blank group are all cultivated under the condition with experiment 1, and cultivation results is as follows:
After 60 days as seen, the average upgrowth situation of the candidum tissue culturing seedling of experimental group is better than the blank group, the long average root length of tissue cultured seedling average root has increased by 4.5% than blank group in the experimental group, plant height has increased by 5.3% than blank group, and tissue cultured seedling plant fresh weight and dry weight have increased by 6.1% and 5.3% than blank group respectively in the experimental group.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ ID NO：1
agagtttgat cctggctcag aacgaacgct ggcggcatgc ctaacacatg caagtcgaac 60
gaaggcttcg gccttagtgg cgcacgggtg cgtaacgcgt gggaatctgc ccttaggttc 120
ggaataacag ctggaaacgg ctgctaatac cggatgatat cgcgagatca aagatttatc 180
gcctgaggat gagcccgcgt tggattaggt agttggtggg gtaaaggcct accaagccga 240
cgatccatag ctggtctgag aggatgatca gccacactgg gactgagaca cggcccagac 300
tcctacggga ggcagcagtg gggaatattg gacaatgggc gaaagcctga tccagcaatg 360
ccgcgtgagt gatgaaggcc ctagggttgt aaagctcttt tacccgggaa gataatgact 420
gtaccgggag aataagcccc ggctaactcc gtgccagcag ccgcggtaat acggaggggg 480
ctagcgttgt tcggaattac tgggcgtaaa gcgcacgtag gcggctttgt aagtcagagg 540
tgaaagcctg gagctcaact ccagaactgc ctttgagact gcatcgcttg aatccaggag 600
aggtcagtgg aattccgagt gtagaggtga aattcgtaga tattcggaag aacaccagtg 660
gcgaaggcgg ctgactggac tggtattgac gctgaggtgc gaaagcgtgg ggagcaaaca 720
ggattagata ccctggtagt ccacgccgta aacgatgata actagctgtc cgggcacttg 780
gtgcttgggt ggcgcagcta acgcattaag ttatccgcct ggggagtacg gccgcaaggt 840
taaaactcaa aggaattgac gggggcctgc acaagcggtg gagcatgtgg tttaattcga 900
agcaacgcgc agaaccttac cagcgtttga catggtagga cgacttccag agatggattt 960
cttcccttcg gggacctaca cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1020
tgttgggtta agtcccgcaa cgagcgcaac cctcgccttt agttaccatc atttggttgg 1080
gtactctaaa ggaaccgccg gtgataagcc ggaggaaggt ggggatgacg tcaagtcctc 1140
atggccctta cgcgctgggc tacacacgtg ctacaatggc aactacagtg ggcagcgacc 1200
ctgcgagggc gagctaatcc ccaaaagttg tctcagttcg gattgttctc tgcaactcga 1260
gagcatgaag gcggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1320
aggccttgta cacaccgccc gtcacaccat gggagttgga ttcacccgaa ggcgttgcgc 1380
caacctagca ataggaagca ggcgaccacg gtgggttcag cgactggggt gaagtcgtaa 1440
caaggtagcc 1450
Claims (3)
1. Sphingol single-cell strain, it is characterized in that: this Sphingol single-cell (Sphingomonas sp.) bacterial strain preservation name is called: Sphingol single-cell SH1, depositary institution: Chinese typical culture collection center, preservation address: Chinese Wuhan University, preservation date: on February 4th, 2010, preserving number: CCTCCNO:M2010041.
2. the purposes of Sphingol single-cell strain as claimed in claim 1 is characterized in that: be used for the seedlings of Dendrobium officinale promotes growth.
3. the purposes of Sphingol single-cell strain according to claim 2, it is characterized in that: is that the no fermented liquid of the membrane filtration gained of 0.45 μ m is inoculated in the base portion of candidum tissue culturing seedling with Sphingol single-cell CCTCC NO:M 2010041 fermenation raw liquids or above-mentioned fermenation raw liquid with the aperture, thereby promotes the candidum tissue culturing seedling growth.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101254098A CN101921718B (en) | 2010-03-16 | 2010-03-16 | Dendrobium candidum endophyte with growth promotion function and use thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101254098A CN101921718B (en) | 2010-03-16 | 2010-03-16 | Dendrobium candidum endophyte with growth promotion function and use thereof |
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| Publication Number | Publication Date |
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| CN101921718A CN101921718A (en) | 2010-12-22 |
| CN101921718B true CN101921718B (en) | 2011-12-07 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104830707B (en) * | 2015-01-09 | 2018-01-16 | 江苏大学 | A kind of interior raw Sphingol single-cell for coming from napier grass and application thereof |
| CN105132293B (en) * | 2015-09-25 | 2018-03-09 | 江苏农林职业技术学院 | A kind of Alternaria tenuissima and its application in dendrobium candidum growth-promoting, drought resisting |
| CN105112308B (en) * | 2015-09-25 | 2018-03-09 | 江苏农林职业技术学院 | An a kind of beading are red mould and its in dendrobium candidum growth-promoting and the application in drought resisting |
| CN105219656B (en) * | 2015-09-25 | 2018-03-30 | 江苏农林职业技术学院 | A kind of Fusarium oxysporum and its application in Growth of Dendrobium candidum is promoted |
| CN105112309A (en) * | 2015-09-25 | 2015-12-02 | 江苏农林职业技术学院 | Umbelopsis nana and application thereof in promoting growth of Dendrobium officinale |
| CN112493252B (en) * | 2020-12-29 | 2021-09-21 | 浙江大学 | Sphingomonas cucurbitae and application of fermentation product thereof in preventing and treating rice bacterial diseases |
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| CN101921718A (en) | 2010-12-22 |
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Application publication date: 20101222 Assignee: Deqing Muge Ecological Agriculture Co., Ltd. Assignor: Zhejiang Sci-Tech University Contract record no.: 2017330000019 Denomination of invention: Dendrobium candidum endophyte with growth promotion function and use thereof Granted publication date: 20111207 License type: Common License Record date: 20170328 |
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