CN103834579A - Entophytic fungus strain YCEF 199 and application thereof - Google Patents
Entophytic fungus strain YCEF 199 and application thereof Download PDFInfo
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- CN103834579A CN103834579A CN201410071743.8A CN201410071743A CN103834579A CN 103834579 A CN103834579 A CN 103834579A CN 201410071743 A CN201410071743 A CN 201410071743A CN 103834579 A CN103834579 A CN 103834579A
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Abstract
The invention relates to an entophytic fungus strain YCEF 199, an application thereof in promoting plant growth and reducing the heavy metal absorption capacity of tobaccos, and belongs to the fields of microorganisms and an application of the microorganisms. The entophytic fungus strain Pyrenochaetasp. YCEF 199 is collected in China Center for Type Culture Collection of Wuhan University in Wuhan, China, the collection number of the entophytic fungus strain Pyrenochaetasp. YCEF 199 is CCTCC No. M2013596, and the collection time of the entophytic fungus strain Pyrenochaetasp. YCEF 199 is on November 24, 2013. The entophytic fungus strain Pyrenochaetasp. YCEF 199 disclosed by the invention has an obvious growth-promoting effect on the tobaccos or a plant biomass increasing effect, which is called a growth-promoting effect for short.
Description
Technical field
The present invention relates to a kind of endogenetic fungal bacterial strain YCEF199, with and at Promoting plant growth with reduce tobacco and absorb the purposes aspect heavy metal, belong to microorganism and microbe application field.
Background technology
Smoking is the hot issue of domestic and international extensive concern with health, and reducing tobacco product objectionable constituent is focuses of tobacco industry development in science and technology.Heavy metal in tobacco leaf and flue gas is one of most important objectionable constituent, and tobacco diseases also has a strong impact on tobacco production and Flue-cured tobacco Quality in addition.And tobacco agriculture has material impact to tobacco leaf objectionable constituent and quality of tobacco, utilize Agricultural biotechnologies to regulate and control, reduce the background concentration of tobacco leaf objectionable constituent as far as possible, reduce the object of tobacco product to human health risk to reach.
In tobacco, contain detection device for multi metallic elements, as arsenic, cadmium, lead, mercury etc., in aspiration procedure, these heavy metals can enter human body by main flume, and human body is caused to potential harm.Tobacco is the crop extremely strong to cadmium transfer ability, and in tobacco, cadmium content is than high times of the soil of its growth, and cadmium is almost suitable at the various position leaves content of ripe cigarette strain, and minimum in root, in tobacco leaf, the accumulation volume of cadmium accounts for the 79-96% of integral dose.
Endogenetic fungus (endophytic fungi) at least the part of the life history can infect and surely grow in health plant tissue, host is without obvious class fungi (Petrini, 1991 of illness; Wilson, 1995), to be only present in root system of plant different from mycorrhizal fungi, and endogenetic fungus can exist (Faeth & Fagan, 2002) at any histoorgan of the underground and aboveground of plant.Endogenetic fungus is extensively present in plant tissue, is not subject to the impact of external environment, and growth and development of plants, opposing disease and pest etc. are had to biological action widely, simultaneously significant in the widespread use of some plants and farm crop.Along with the lasting deterioration of environmental problem, plant resources is standing acid test, and endogenetic fungus is many-sided on the impact of plant, has become one of hot issue of the outer research of Now Domestic.Development in agriculture production, ecological protection, natural resources of Chinese medicinal materials exploitation, medical and health has important realistic meaning.In recent years, it is found that at plant materials and have useful endogenetic bacteria and endogenetic fungus, preventive and therapeutic effect and the biological Application and Development of endogenous bacterial strain to Plant diseases caused scientist's extensive concern.Plant endogenesis epiphyte is mainly distributed in the seed of plant, flower, stem, in leaf and root system, with the relation of plant be reciprocal symbiosis, and plant endogenesis epiphyte can provide photosynthate and mineral substance, endogenous fungus metabolite can stimulate growing of host plant, improve host plant biology stress with abiotic stress resistance ability, exist material to exchange with the circulation of energy between the two, the energetically growgh promoting effects of plant endogenesis epiphyte to host plant, part is the active substance secreted by plant endogenesis epiphyte or the result of the growth of secondary metabolite to plant.What research was more at present is root nodule symbiote and mycorrhizas homobium, and the symbiote of endogenetic fungus and plant is another manifestation of higher plant and microorganisms symbiosis relation.
The mutual of endogenetic fungus and plant studied the concern that is day by day subject to investigator, and becomes the international focus of endogenetic fungus research field.Tobacco is as the important cash crop of China, and in national economy, in occupation of irreplaceable status, the raising of tobacco production and quality, is the basis that tobacco industry develops in a healthy way.By the interaction of research endogenetic fungus and tobacco, the syntaxial system of research endogenetic fungus and tobacco is significant.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of endogenetic fungal bacterial strain
pyrenochaetasp.YCEF199 and uses thereof.
In order to solve the problems of the technologies described above, the invention provides a kind of endogenetic fungal bacterial strain
pyrenochaetasp.YCEF199, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2013596, November 24 2013 preservation time.
Endogenetic fungal bacterial strain
pyrenochaetasp.YCEF199 is characterized as: this strain growth speed is slower, and on PDA, 25 ℃ of dark colony diameters of cultivating 7 days are 4cm; PDA cultivates white of upper initial stage, and ripe culture is olive colour or grey, and bacterium colony quality is comparatively hard; Aerial hyphae is dense, mycelia have every, colourless, diameter 1.3-1.6 um.Pycnidium, conidium are showed no.
Bacterial strain
pyrenochaetasp.YCEF199 derives from the endogenetic fungus of tobacco, belongs to mycota Fungi, Ascomycota Ascomycota, seat capsule Gammaproteobacteria Dothideomycetes, lattice spore chamber Zoopagales Pleosporales, lattice spore chamber Cordycepps Pleosporaceae, Pyrenochaeta
pyrenochaeta.
The complete sequence of the Internal Transcribed Spacer rDNA of endogenetic fungal bacterial strain YCEF199 is as shown in SEQ ID NO:1.
The present invention also provides above-mentioned endogenetic fungal bacterial strain
pyrenochaetathe purposes of sp.YCEF199: Promoting plant growth or increase biomass.
The growth of promotion plant of the present invention or increase biomass comprise tobacco.
Endogenetic fungal bacterial strain of the present invention
pyrenochaetasp.YCEF199 has obvious promotion growth to tobacco, or increases the biomass of plant, is called for short growth-promoting functions.
Accompanying drawing explanation
Fig. 1 is
pyrenochaetathe colonial morphology of sp.YCEF199 on PDA substratum.
Fig. 2 is
pyrenochaetasp.YCEF199 and cloud and mist 87 are cultivated the photo of two weeks altogether, compared with the control,
pyrenochaetasp.YCEF199 has obvious growth-promoting functions.
Fig. 3 is
pyrenochaetasp.YCEF199 moves into the potted plant growth photo of 2 months, compared with the control,
pyrenochaetasp.YCEF199 has obvious Promoting plant growth effect or increases biomass.
biological preservation statement
Endogenetic fungal bacterial strain
pyrenochaetasp.YCEF199, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2013596, November 24 2013 preservation time.
Embodiment
With reference to above-mentioned accompanying drawing, the specific embodiment of the present invention is elaborated.
Remarks explanation: following all substratum all need to carry out in a conventional manner the sterilising treatment (121 ℃, 0.24 MPa, 20 minutes) of High Temperature High Pressure before using.
embodiment 1, endogenetic fungal bacterial strain
pyrenochaetasp.YCEF199 separates acquisition
2% wort agar substratum (malt extract agar, MEA): leach adding distil water in powder 20g, agar 20g at Fructus Hordei Germinatus and be settled to 1000 mL; Then carry out conventional high-temperature sterilization (1.1 normal atmosphere, 121 ℃ at sterilizing 20min).
Potato dextrose agar (potato dextrose agar, PDA): adding distil water is settled to 1000 mL in potato 200g, glucose 20g, agar 20g; Then carry out conventional high-temperature sterilization (1.1 normal atmosphere, 121 ℃ at sterilizing 20min).
Gather Tennessee86(American Burley tobacco cultivar take Yunnan tobacco kind garden) as vegetable material, the tobacco gathering is clean with tap water rinsing, tobacco rhizome is at 75%(v/v) sterilize 30 seconds in spirituous solution, then at 1%(v/v) the 10 points of kinds of sterilizing in chlorine bleach liquor, rinsed with sterile water 3 times.The fragment of undercut being grown into about 0.6cm by sterile razor blade, be placed on 2% wort agar substratum (MEA) plate containing 50 μ gml-1 penbritins and 50 μ gml-1 Streptomycin sulphates, 25 ℃ of dark cultivations, after mycelia grows, move mycelia top to receive PDA substratum; Separation obtains bacterium colony, 25 ℃ of dark cultivations, and on PDA substratum, continuous three inoculations, confirm as single bacterium colony, do not have other fungies or bacterium jointly to grow, and think to obtain pure culture, called after YCEF199.
Strain number is
pyrenochaetasp.YCEF199, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University, deposit number: CCTCC NO:M 2013596, November 24 2013 preservation time.
embodiment 2, endogenetic fungal bacterial strain
pyrenochaetathe identification of morphology of sp.YCEF199
Of the present invention
pyrenochaetasp.YCEF199(
pyrenochaetasp.YCEF199 CCTCC NO:M 2013596) there is following characteristic:
pyrenochaetathe form of sp.YCEF199 on PDA substratum as shown in Figure 1.The suitable growth temperature of this endogenetic fungus on PDA substratum is 25 ℃, has following characteristics: this bacterial strain belongs to mycota Fungi, Ascomycota Ascomycota, seat capsule Gammaproteobacteria Dothideomycetes, lattice spore chamber Zoopagales Pleosporales, lattice spore chamber Cordycepps Pleosporaceae, Pyrenochaeta
pyrenochaeta.This strain growth speed is slower, and on PDA, 25 ℃ of dark colony diameters of cultivating 7 days are 4cm; PDA cultivates white of upper initial stage, and ripe culture is olive colour or grey, and bacterium colony quality is comparatively hard; Aerial hyphae is dense, mycelia have every, colourless, diameter 1.3-1.6 um.Pycnidium, conidium are showed no.
embodiment 3, endogenetic fungal bacterial strain
pyrenochaetathe Molecular Identification of sp.YCEF199
With the rapid fractionation method extraction DNA of fungal gene.The universal primer ITS-4 (' TCCTCCGCTTATTGATATGC-3 ') of employing ITS amplification and ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') carry out pcr amplification, pcr amplification reaction adopts the reaction system of 50ul: ddH2O39.5ul, 10*PCR Buffer (withMg2+) 5ul, 10mmol/L dNTP 0.4ul, 20 μ mol/L Primer-F 0.8ul, 20 μ mol/L Primer-R 0.5ul, DNA profiling 3ul, 5U/ul Taq polysaccharase 0.8ul.PCR system is: PCR reaction conditions is: 94 ℃ of denaturation 4min, and 94 ℃ of sex change 30s, 59 ℃ of annealing 40s, 72 ℃ are extended 60s, and after 35 circulations, 72 ℃ are extended 10min.Reaction finishes rear use 1.0% agarose gel analysis amplified production.After PCR product reclaims, through the order-checking of Shanghai Sheng Gong biotechnology company limited, obtain its ITS sequence, the complete sequence of the Internal Transcribed Spacer rDNA of endogenetic fungal bacterial strain YCEF199 is as shown in SEQ ID NO:1.Sequencing result carries out homologous sequence search further to determine its classification position in GenBank database.
embodiment 4, endogenetic fungal bacterial strain
pyrenochaetathe growth-promoting functions of sp.YCEF199 to plant
MS substratum (1L): saltpetre 1900mg/L, ammonium nitrate 1650 mg/L, magnesium sulfate 370 mg/L, potassium primary phosphate 170 mg/L, calcium chloride 440 mg/L, manganous sulfate 22.3 mg/L, zinc sulfate 8.6 mg/L, boric acid 6.2 mg/L, potassiumiodide 0.83 mg/L, Sodium orthomolybdate 0.25 mg/L, copper sulfate 0.025 mg/L, cobalt chloride 0.025 mg/L, ferrous sulfate 27.8 mg/L, Na2EDTA37.3 mg/L, glycine 2.0 mg/L, vitamin 0.1 mg/L, pyridoxine hydrochloride 0.5 mg/L, nicotinic acid 0.5 mg/L, inositol 100 mg/L, 30g sucrose, agar powder 8g, H
2o is settled to 1L, pH value 5.8.
(1) seed of selection tobacco bred cloud and mist 87, sterilized water soaks after 48h, rubs kind of a 20 ~ 30min with the hands.70% alcohol sterilizing 1min for seed, 2%(quality %) clorox sterilizing 10min, after aseptic water washing 5 ~ 6 times, put on MS substratum, sprout two weeks (approximately growing to 4 leaves) alternately down in 25 ℃, 16h illumination 80 μ mm-2sec-1 and 22 ℃, 8h are dark, for subsequent use.
(2) select the common cultivation of tobacco seedling in the same size for same strains tested.The tobacco seedling that grows to 4 leaves is transferred to 10*10(centimetre) square 1/2MS substratum on (3 strains/ware), for keeping the good growing way of tobacco, the substratum of cauline leaf part is removed, root is unfolded on 1/2MS substratum.Picking is cultivated the Endophytic Fungal Hyphae of about a week, is placed in 1cm left and right, tobacco seedling root below and carries out common cultivation.Blank does not connect mycelia, and plate sealing is placed under the condition of 25 ℃, 16h illumination 80 μ mm-2sec-1 and 22 ℃, 8h dark and cultivates about two weeks.Remarks explanation: 1/2 MS substratum is macroelement (saltpetre, ammonium nitrate, magnesium sulfate, potassium primary phosphate, calcium chloride) and reduces by half, the MS substratum that all the other constituent contents are constant.
(3) cultivate altogether after two weeks, strains tested shows good growth-promoting effect (as shown in Figure 2).Tobacco seedling is taken out from plate, and blade and root separately, are placed in respectively the centrifuge tube of 1.5ml, dry the precision balance statistics of weighing in baking oven.
Table 1:YCEF199 and cloud and mist 87 are cultivated rear blade biomass statistics altogether
Table 2:YCEF199 and cloud and mist 87 are cultivated back root part biomass statistics altogether
Compare control group, enforcement group is all obviously better than control group in leaf dry weight and root dry weight, and the growth of tobacco is had to just tune effect.Cultivate altogether after two weeks, strains tested shows good growth result.After moving into potted plant 2 months, again biomass is added up.
Table 3:YCEF199 and cloud and mist 87 move into potted plant rear blade dry weight biomass statistics
Above-mentioned experimental result shows, experimental group
pyrenochaetasp.YCEF199 grows fine, and blade is large and thick, and plant is more strong, and potted plant Leaf dry weight and root dry weight data also show,
pyrenochaetasp.YCEF199 has obvious promotion growth to the tobacco of cloud and mist 87 these kinds, or increases the biomass of plant, is called for short growth-promoting functions.
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
<110> China Tobacco Industry Zhejiang Co., Ltd.
<120> endogenetic fungal bacterial strain YCEF199 and uses thereof
<160> 2
<210> 1
<211> 533
<212> DNA
<213> Pyrenochaeta sp.
<400> 1
aggatcatta attgtataac gggggccggc gagggattgc gcacttcggt gcgcctttct 60
tccccgccct gtctgatact acccatgtct tttgcgtacc aattgtttcc tcggtgggct 120
tgcccgccgg ttggacacta caaaaccttt tgtaattgca gtcagcgtca gaaaaacata 180
ataattacaa ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcgaa 240
atgcgataag tagtgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt 300
gcgccccttg gtattccatg gggcatgcct gttcgagcgt catttgtacc ctcaagctct 360
gcttggtgtt gggtgtttgt cccgctttgc gcgtggactc gccttaaagc aattggcagc 420
cggcaatctg gttatagagc gcagcacatt ttgcgcttct tgccatggat gtcggcgtcc 480
atcaagtaca tttttttgct cttgacctcg gatcaggtag ggatacccgc tga 533
Claims (2)
- Endogenetic fungus ( pyrenochaetasp.) bacterial strain YCEF199, is characterized in that: the preservation name of this bacterial strain is called: pyrenochaetasp. YCEF199, depositary institution: Chinese Typical Representative culture collection center, preservation address: Wuhan, China Wuhan University; Preservation date: on November 24th, 2013, deposit number: CCTCC NO:M 2013596.
- 2. the purposes of endogenetic fungal bacterial strain YCEF199 according to claim 1, is characterized in that: this purposes is used for promoting tobacco growing or increases biomass.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249230A (en) * | 2021-05-18 | 2021-08-13 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Endophytic fungus AT177 with stable growth promoting effect and application thereof |
| CN117736945A (en) * | 2024-02-21 | 2024-03-22 | 滨州医学院 | Plant hairy root senescence-resistant bacteria and application thereof |
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| CN1602356A (en) * | 2001-10-16 | 2005-03-30 | Posco公司 | Transgenic organism expressing fungal MRP-like ABC transporters |
| CN102046778A (en) * | 2008-05-29 | 2011-05-04 | 日本烟草产业株式会社 | Bacterium capable of reducing heavy metal content in plant |
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2014
- 2014-02-28 CN CN201410071743.8A patent/CN103834579B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1602356A (en) * | 2001-10-16 | 2005-03-30 | Posco公司 | Transgenic organism expressing fungal MRP-like ABC transporters |
| CN102046778A (en) * | 2008-05-29 | 2011-05-04 | 日本烟草产业株式会社 | Bacterium capable of reducing heavy metal content in plant |
Non-Patent Citations (1)
| Title |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113249230A (en) * | 2021-05-18 | 2021-08-13 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Endophytic fungus AT177 with stable growth promoting effect and application thereof |
| CN113249230B (en) * | 2021-05-18 | 2022-03-18 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Endophytic fungus AT177 with stable growth promoting effect and application thereof |
| CN117736945A (en) * | 2024-02-21 | 2024-03-22 | 滨州医学院 | Plant hairy root senescence-resistant bacteria and application thereof |
| CN117736945B (en) * | 2024-02-21 | 2024-05-07 | 滨州医学院 | Plant hairy root senescence-resistant bacteria and application thereof |
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Inventor after: Xu Qingquan Inventor after: Liu Huabing Inventor after: Zhang Xiaobing Inventor after: He Wenmiao Inventor after: Mu Jirui Inventor after: Zhang Chulong Inventor after: Lin Fucheng Inventor after: Liu Hongyu Inventor after: Liu Ning Inventor after: Feng Xiaoxiao Inventor after: Xia Chen Inventor after: Cheng Changhe Inventor after: Jin Huiqing Inventor before: Xu Qingquan Inventor before: Zhang Xiaobing Inventor before: He Wenmiao Inventor before: Mu Jirui Inventor before: Zhang Chulong Inventor before: Lin Fucheng Inventor before: Liu Hongyu Inventor before: Feng Xiaoxiao Inventor before: Xia Chen Inventor before: Cheng Changhe Inventor before: Jin Huiqing Inventor before: Liu Huabing |
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Free format text: CORRECT: INVENTOR; FROM: XU QINGQUAN ZHANG CHULONG LIN FUCHENG LIU HONGYU FENG XIAOXIAO XIA CHEN CHENG CHANGHE JIN HUIQING LIU HUABING ZHANG XIAOBING HE WENMIAO MU JIRUI TO: XU QINGQUAN ZHANG CHULONG LIN FUCHENG LIU HONGYU LIU NING FENG XIAOXIAO XIA CHEN CHENG CHANGHE JIN HUIQING LIU HUABING ZHANG XIAOBING HE WENMIAO MU JIRUI |
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