CN102618469A - Rhodobacter capsulatus and application thereof - Google Patents
Rhodobacter capsulatus and application thereof Download PDFInfo
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- CN102618469A CN102618469A CN2012100920315A CN201210092031A CN102618469A CN 102618469 A CN102618469 A CN 102618469A CN 2012100920315 A CN2012100920315 A CN 2012100920315A CN 201210092031 A CN201210092031 A CN 201210092031A CN 102618469 A CN102618469 A CN 102618469A
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- rhodobacter capsulatus
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- 229910052711 selenium Inorganic materials 0.000 claims abstract description 112
- 239000011669 selenium Substances 0.000 claims abstract description 112
- 241000894006 Bacteria Species 0.000 claims abstract description 38
- 239000007788 liquid Substances 0.000 claims description 24
- 235000009024 Ceanothus sanguineus Nutrition 0.000 claims description 23
- 240000003553 Leptospermum scoparium Species 0.000 claims description 23
- 235000015459 Lycium barbarum Nutrition 0.000 claims description 23
- 239000002068 microbial inoculum Substances 0.000 claims description 21
- 239000012528 membrane Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 16
- 238000002360 preparation method Methods 0.000 claims description 9
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- 238000010521 absorption reaction Methods 0.000 abstract description 5
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/12—Technologies relating to agriculture, livestock or agroalimentary industries using renewable energies, e.g. solar water pumping
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses rhodobacter capsulatus and application thereof. The collection number of the rhodobacter capsulatus AYGHJ-16 is CCTCC NO: M 2011377; the rhodobacter capsulatus belongs to photosynthetic bacteria; and through the rhodobacter capsulatus, certain organic small molecules (such as organic acid and amino acid) can be converted into growth-stimulating substances and anti-virus substances by using light energy, so that growth of plants is stimulated, and the disease-resistant immunity of the plants is improved. Moreover, through the photosynthetic bacteria, product feedback inhibition of heterotrophic microbes can be released by using organic acid produced by metabolism of the heterotrophic microbes in soil, so that a large quantity of heterotrophic microbes are propagated, and conversion and absorption of certain inorganic substances in the soil are promoted; and the photosynthetic bacteria have strong nitrogen fixation and carbon fixation effects, so more inorganic substances can be provided for plants. The selenium-rich microbial agent can improve the micro-ecological environment of the soil, so that the disease resistance of the plants and internal absorption of various mineral elements and rare elements (such as selenium) are improved, and the selenium-rich requirements of the plants are met.
Description
Technical field
The present invention relates to red bacterium of a strain pod membrane and application thereof.
Background technology
Selenium is that Sweden chemist Berzelius found in production vitriolic mine tailing in 1817, is considered to a kind of poisonous element at that time.Nineteen fifty-seven, Schwarz etc. prove that first selenium is the essential nutrient element of animal, and people just are familiar with the health protection effect of selenium to some extent.1973, discovery such as Rotmck and confirm that selenium is after the moity of animal and human's body Selenoperoxidase, the importance of selenium has received unprecedented attention.1973, the World Health Organization announced that selenium is the bioelement of needed by human.Medical research in recent years shows that selenium deficiency can cause the generation of more than 40 kind of disease, especially can cause following disease: (1) Keshan disease, Kaschin-Beck disease; (2) various cancers; (3) male fertility and women's P-AH; (4) radiation injury is with old and feeble; (5) immunologic hypofunction and AIDS; (6) heavy metal toxicity and occupational illness; (7) cardiovascular diseases; (8) epidemic hemorrhagic fever.Replenish a certain amount of selenium and can prevent the generation of above-mentioned disease well, guarantee HUMAN HEALTH.
Selenium is the skewness weighing apparatus on earth, and known have more than 40 countries and regions to lack selenium.According to incompletely statistics, the area of China about 72% is in scarce selenium, low selenium band.The deficiency of selenium intake is having a strong impact on the healthy of China's several hundred million populations in the meals; According to Chinese Soclety of Nutrition's report of survey; The selenium intake of China adult every day is merely 26.63 μ g, and the daily intaking amount 50 μ g and the 60 μ g that recommend apart from Chinese Soclety of Nutrition and international selenium association differ greatly.Human body mainly is to take in selenium through meals, is used for seeing that from the benefit selenium of different shape selenium source to human body inorganic selenium has certain toxicity; Specific absorption is low again; And inorganic selenium is converted into organoselenium through biotransformation, and can reduce the toxicity of inorganic selenium, improve the physiologically active and the specific absorption of selenium.Therefore, all mend selenium to organoselenium at present and be regarded as the most effectively means.
Plant selenium source in organic selenium source is better than animality selenium source.In the vegetalitas selenium source, tea rich in selenium is that a kind of ideal is mended one of selenium source.Tea tree is a kind of natural selenium-rich plant, and the tealeaves that originates in rich selenium district is tea rich in selenium, even the tea tree of planting on low selenium soil also can increase selenium method production tea rich in selenium through manual work.Selenium in the tealeaves 80% is the organoselenium to the human body beneficial.Tealeaves is one of the world's three big nourishing drinks, and China people have permanent tea drinking habit, therefore develops tea rich in selenium HUMAN HEALTH is had great significance.The enhancing of Along with people's health care consciousness, people will constantly increase the demand of tea rich in selenium.
China is that low selenium is national, and the place of production of natural selenium-rich tea is fewer, so the selenium content that increases in the selenium approach increase tealeaves through manual work has the certain significance.Tealeaves increases the selenium approach to be had a variety of.In the natural tea rich in selenium garden, because the soil selenium-containing amount is high, the selenium content in the middle of the tealeaves is just high naturally.But range of distribution is limited after all for the high soil of selenium content, is difficult to satisfy the large market demand, needs to adopt manual work to increase selenium.Manual work increases selenium and mainly contains two kinds of approach: the one, and soil spraying selenium is fertile, and the 2nd, carry out the foliage spray of tealeaves.Tea garden soil increase execute selenium fertilizer after, a large amount of selenium can not be well absorbed by tea tree, the selenium content in the tealeaves increases slowly, amplification is very little.Behind the foliage-spray selenium fertilizer; Can improve the selenium content in the tealeaves rapidly, the tealeaves selenium content is directly proportional with the amount of Spraying Se-fertilizer, and blade face spray selenium should note selecting suitable spray selenium concentration and reasonable picking time especially; Be in the exploratory stage, still can not guarantee the utilization ratio of tealeaves the selenium element.
Photosynthetic bacterium (Photosynthetic Bacteria, abbreviation PSB) is a kind of in the mikrobe of waters, is the prokaryotic organism with original luminous energy synthesis system that occur the earliest on the earth, and it extensively is distributed in the anaerobic layer of all kinds of water bodys.Show common about 50 genus of 7 jumpbogroups such as obligate aerobic bacteria that known photosynthetic bacterium exists painted Bacteriaceae, outer sulphur Rhodospirillaceae, purple nonsulfur bacteria, green sulfur bacteria, the thread green bacterium of many cells, screw rod Cordycepps, contains bacteriochlorophyll.Photosynthetic bacterium extensively is present in natural paddy field, lake, rivers, ocean, active sludge and the soil as the important microbe groups of occurring in nature, characteristics such as vitality is extremely strong, nutritional requirement is low, growth and breeding is fast, toxicological harmless property.
Summary of the invention
The purpose of this invention is to provide red bacterium of a strain pod membrane and application thereof.
The red bacterium of pod membrane provided by the invention (Rhodobacter capsulatus) AYGHJ-16 is called for short the red bacterium AYGHJ-16 of pod membrane, is preserved in Chinese typical culture collection center on November 4th, 2011 and (is called for short CCTCC; Address: Chinese Wuhan, Wuhan University; Postcode: 430072), deposit number is CCTCC NO:M 2011377.
The red bacterium AYGHJ-16 of said pod membrane can be used for preparing fermented liquid (rich selenium microbial inoculum).
The present invention also protects a kind of method for preparing fermented liquid (rich selenium microbial inoculum), is the red bacterium AYGHJ-16 of the said pod membrane of fermentation, obtains fermented liquid.
The condition of said fermentation specifically can be: 30 ℃, carbonic acid gas ventilation flow velocity is 4L/min, and intensity of illumination is 1000-3000Lux.
The preparation method of the fermention medium that said fermentation is adopted is specific as follows: get 120mg Sodium Selenite, 40g glucose, 7.5g peptone, 9g urea, 5g yeast decoction, 0.4g NaHCO
3, 1g NH
4Cl, 0.2g K
2HPO
4, 2.0CH
3COONa, 0.2g MgSO
47H
2O, 2.0g NaCl and 10mL trace element solution are with water dissolution and be settled to 1000mL; The preparation method of said trace element solution is following: get 0.5g H
3BO
3, 400mg MnSO
4, 60mgZnSO
47H
2O, 180mg NaMoO
42H
2O and 10mg Ca (NO
3)
22H
2O is with water dissolution and be settled to 250mL.
The pH of said fermention medium specifically can be 6.7-7.2.
More than the fermented liquid (rich selenium microbial inoculum) for preparing of any said method also belong to protection scope of the present invention.
The method of use of said fermented liquid is that said fermented liquid is carried out being sprayed on the tea tree blade face after 30-50 doubly dilutes.
Said fermented liquid can be used for producing tealeaves.
Said fermented liquid can be used for cultivating tea tree.
The present invention also protects a kind of method of producing tealeaves, comprises the step of said fermented liquid being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.Said tea tree specifically can be golden kwan-yin tea tree.
The present invention also protects a kind of method of cultivating tea tree, comprises the step of said fermented liquid being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.Adopt method of the present invention to cultivate tea tree, the selenium content of tealeaves is significantly increased.Said tea tree specifically can be golden kwan-yin tea tree.
The red bacterium AYGHJ-16 of pod membrane provided by the invention belongs to photosynthetic bacterium, can utilize luminous energy, and some organic molecules (like organic acid, amino acid etc.) are converted into growth stimulator and antiviral substance, and the stimulating plant growth improves the anti pathologic immunity power of plant.And; Photosynthetic bacterium can utilize the organic acid that different oxygen animalcule metabolism is produced in the soil; Remove the product feedback inhibition of different oxygen animalcule, make its a large amount of breedings, promoted the conversion and the absorption of some inorganic substance in the soil; Add photosynthetic bacterium itself and can carry out intensive fixed nitrogen and carbon solidification effect, can more inorganics be provided for plant.
Rich selenium microbial inoculum provided by the invention can improve the soil micro-ecosystem environment, thereby improves the resistance against diseases of plant and for the interior suctionization of various mineral elements and rare elements (such as selenium), reach the requirement that improves plant materials richness selenium.
Rich selenium microbial inoculum provided by the invention can further improve the se use efficiency of tea tree, improves the rich selenium efficient of tealeaves, and the ecological tea rich in selenium of scale operation satisfies drinking tea according to amount of people and mends the purpose of selenium health care service.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with repeated experiments three times, results averaged.Gold kwan-yin tea tree: tealeaves institute of Fujian Province's research of agricultural science institute.
The acquisition of embodiment 1, strains A YGHJ-16 and preservation
One, the acquisition of strains A YGHJ-16S
Utilize enrichment to differentiate culture technique with selecting, separation energy tolerates the high density Sodium Selenite, also can transform the photosynthetic bacterium that inorganic selenium is an organoselenium from the bed mud soil of the West Lake, Foochow.Obtain a strain bacterial strain, called after strains A YGHJ-16S.The rich selenium amount of strains A YGHJ-16S is 150ug/ml.
Two, the evaluation of strains A YGHJ-16S
1, morphological specificity
Gram-negative bacteria;
Thalline is quarter butt or ovum type, and size is about (0.6-1.2) μ m-(2.0-2.2) μ m;
Bacterium colony is smooth, mellow and full, projection under illumination condition;
Bacterium colony is red under illumination condition, is colourless under dark condition.
2, characterization of molecules
The sequencing result of 16S rDNA shown in the sequence 1 of sequence table, with the sequence similarity of the red bacterium of pod membrane of GENBANK ACCESSION NO.NR043407 be 98%.
Comprehensive morphological characteristic and characterization of molecules; With reference to " " common bacteria system identification handbook " (the 1st edition) that uncle's Jie Shi systematic bacteriology handbook (the 9th edition) and eastern elegant pearl etc. are write, strains A YGHJ-16S belongs to the red bacterium of pod membrane and belongs to (Rhodobacter capsulatus).
Three, the acquisition of strains A YGHJ-16
Be the bacterium that sets out with strains A YGHJ-16S,, obtain strains A YGHJ-16 through the protoplastis breeding of routine and the high-yield strains of the rich selenium of the further seed selection of DNA shuffling technology.The rich selenium amount of strains A YGHJ-16 is 300ug/ml.
Four, the evaluation preservation of strains A YGHJ-16
The red bacterium of pod membrane (Rhodobacter capsulatus) AYGHJ-16 is called for short the red bacterium AYGHJ-16 of pod membrane, is preserved in Chinese typical culture collection center on November 4th, 2011 and (is called for short CCTCC; Address: Chinese Wuhan, Wuhan University; Postcode: 430072), deposit number is CCTCC NO:M 2011377.
The fermented ingredient of embodiment 2, the red bacterium AYGHJ-16 of pod membrane and the optimization of processing condition
Through single factor and orthogonal test, confirm following substratum composition parameter:
Glucose is the optimum carbon source in the substratum, can obtain high-biomass and selenium content when adding 4-5%;
Peptone and urea are the compound nitrogen source in the substratum, and addition is respectively: peptone 0.5%, urea 0.9%;
Yeast extract paste is the auxiliary nitrogenous source in the substratum, and addition is 0.4%;
Sodium Selenite is that the inorganic selenium source in the substratum is selected, and addition is 120ug/mL.
Through the shake flask fermentation experiment of homogeneous design, confirm following culture condition parameter:
Adopt 250mL to shake bottle, liquid amount 120mL, 28 ℃ of culture temperature, pH5-6, incubation time 5d.
Adopt above-mentioned preferred parameter, the living weight of the red bacterium AYGHJ-16 of pod membrane reaches 37.4g/L, and the total selenium content of thalline enrichment reaches 36.4mg/L.
Embodiment 3, rich selenium microbial inoculum preparation
One, rich selenium microbial inoculum preparation
The preparation method of fermention medium (pH7.0): get 120mg Sodium Selenite, 40g glucose, 7.5g peptone, 9g urea, 5g yeast decoction, 0.4g NaHCO
3, 1g NH
4Cl, 0.2g K
2HPO
4, 2.0CH
3COONa, 0.2gMgSO
47H
2O, 2.0g NaCl and 10mL trace element solution are with dissolved in distilled water and be settled to 1000mL.The inorganic selenium concentration of fermention medium is 120mg/L.In the practical application, fermention medium adopts the pH of 6.7-7.2 all can.
With fermention medium sterilization 20min under 115 ℃.
The preparation method of trace element solution: get 0.5g H
3BO
3, 400mg MnSO
4, 60mg ZnSO
47H
2O, 180mgNaMoO
42H
2O and 10mg Ca (NO
3)
22H
2O is with dissolved in distilled water and be settled to 250mL.
Adopt the 200L fermentor tank, and fermentor tank is transformed as follows: the lamp under water (light mainly provides the necessary light source of the growth of photosynthetic bacterium as the energy) that glows is installed in the fermentor tank bottom in (1); (2) the electric heating constant temperature rod is installed in the fermentor tank bottom; (3) in the fermentor tank bottom two external openings are set, a ventage as supply fermentor tank carbonic acid gas, another is as the opening that adds fermention medium and bacterial classification.
In fermentor tank, add the 160L fermention medium, inoculate the red bacterium AYGHJ-16 of pod membrane then; Fermentation condition: temperature is controlled at 30 ℃, and carbonic acid gas ventilation flow velocity is 4L/min, and intensity of illumination is 1500Lux (1000-3000Lux all can); Bacteria concentration in fermentor tank reaches 5.35 * 10
8During cfu/mL, be warming up to 55 ℃ and handle 45min (impelling cytoclasis), room temperature centrifugal (5000g, 15min) is collected supernatant, is rich selenium microbial inoculum, 4 ℃ of preservations.
Two, the mensuration of selenium rich ability
With 10 times of the rich selenium microbial inoculum dilutions behind 4 ℃ of preservation 3d, draw 3mL and water and be settled to 10mL, as solution to be measured.Adopt the Socryl Blue BRL reduction method to detect the fading time of solution to be measured.
The Socryl Blue BRL reduction method detects principle: under acidic conditions; Selenite radical can with the DAB effect, generate yellow complex (Se-DAB), when certain pH; Extraction with an organic solvent; According to the degree of organic layer colour developing, can carry out spectrophotometry, calculate the content of inorganic selenium according to inorganic selenium content standard opisometer; Organoselenium can not with the DAB complexing, can not generate Se-DAB.
Sodium Selenite: available from Shanghai epoch bio tech ltd.
Concrete grammar (carries out following operation) as follows successively:
(1) get the separating funnel of 5 125ml, add 2.0,4.0,6.0,8.0 respectively, the sodium selenite aqueous solution (1mg/L) of 10.0ml, then all adding distil water to 40ml;
(2) the EDTA-2Na aqueous solution of every funnel adding 2ml 0.2mol/L is regulated pH to 2-3 to cover interfering ion with formic acid;
(3) the DAB aqueous solution of every funnel adding 4ml 5g/L shakes up, the following 60 ℃ of reaction 20min of dark condition;
(4) regulate pH to 7.0 with dense NaOH, every funnel adds 4ml toluene then, fully shakes up, and leaves standstill the 3-4min layering;
(5) organic layer is filtered through absorbent cotton in the cuvette of 1cm, measured solution absorbency (toluene is as blank) in the 420nm place.With light absorption value A is X-coordinate, is ordinate zou drawing standard curve with selenium concentration Y (unit is mg/L).The typical curve function is following: Y=1.8674X+0.0009 (R
2=0.9995).
Rich selenium microbial inoculum with the step 1 preparation replaces sodium selenite aqueous solution to carry out the experiment of above step (1) to (5), and with light absorption value substitution typical curve, the result who obtains is the inorganic selenium concentration in the rich selenium microbial inoculum.
Inorganic selenium concentration in the inorganic selenium concentration-Fu selenium microbial inoculum of the organoselenium concentration=fermention medium in the rich selenium microbial inoculum.
Selenium rich ability (organoselenium transformation efficiency)=(the inorganic selenium concentration of the organoselenium concentration/fermention medium in the rich selenium microbial inoculum) * 100%.
Organoselenium concentration in the rich selenium microbial inoculum is 116.4mg/L, and selenium rich ability is 98.9%.
Three, rich selenium microbial inoculum uses the preparation of liquid
Rich selenium microbial inoculum is diluted with water to 50 times of volumes, is rich selenium microbial inoculum and uses liquid.
The rich selenium test of the tealeaves in embodiment 4, tea place, sub-district
Experimental period is summer.Adopt field district's group arrangement design experiment at random, three groups of experiments are set altogether, every group is adopted 3 experiment sub-districts, and the area of every experiment sub-district is 8m * 2.5m, and every experiment sub-district is evenly planted and is implanted with 100 strains gold kwan-yin tea tree.Experiment is provided with three repetitions, results averaged.
Tested the 1st day, each group is handled respectively as follows:
First group: evenly spray rich selenium microbial inoculum on the tea tree blade face and use liquid, 20L is sprayed in every experiment sub-district;
Second group: evenly spray clear water on the tea tree blade face, 20L is sprayed in every experiment sub-district;
The 3rd group: do not do any processing.
Test and gathered tealeaves on the 18th day and carry out selenium content detection (according to the detection method of GB5009.93-2010).Selenium content in first group of tealeaves is 350 μ g/kg, and the selenium content in second group of tealeaves is 75 μ g/kg (please indicate), and the selenium content in the 3rd group of tealeaves is 50 μ g/kg (please indicate).
After summer tea was used rich selenium microbial inoculum use liquid, effect was obvious, mainly shows as: 1. improve the germination density and the reguarity of summer shoot, increase summer tea output; 2. promote the summer shoot growth, ahead of time the productive life: 3. improve the summer tea quality, optimize the green tea quality biochemical component; 4. reduce the pesticide residue in the tealeaves; 5. increased the selenium content in the tealeaves in the tealeaves.
Claims (9)
1. the red bacterium of pod membrane (Rhodobacter capsulatus) AYGHJ-16, its deposit number is CCTCC NO:M 2011377.
2. the application of the red bacterium AYGHJ-16 of the said pod membrane of claim 1 in producing rich selenium microbial inoculum.
3. the method for the fermented liquid of the red bacterium AYGHJ-16 of the preparation said pod membrane of claim 1 is the red bacterium AYGHJ-16 of the fermentation said pod membrane of claim 1, obtains fermented liquid.
4. the fermented liquid for preparing of the said method of claim 3.
5. the method for use of the said fermented liquid of claim 4 is that said fermented liquid is carried out being sprayed on the tea tree blade face after 30-50 doubly dilutes.
6. the application of the said fermented liquid of claim 4 in producing tealeaves.
7. the application of the said fermented liquid of claim 4 in cultivating tea tree.
8. a method of producing tealeaves comprises the step of the said fermented liquid of claim 4 being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.
9. a method of cultivating tea tree comprises the step of the said fermented liquid of claim 4 being carried out being sprayed on after 30-50 doubly dilutes the tea tree blade face.
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| CN105111019A (en) * | 2015-08-24 | 2015-12-02 | 广东省农业科学院农业资源与环境研究所 | Special olive fertilizer for accelerating roots and retaining fruits |
| CN105861390A (en) * | 2016-05-30 | 2016-08-17 | 天津师范大学 | Rhodobacter capsulatus strain as well as screening method and application thereof |
| CN110241236A (en) * | 2019-06-14 | 2019-09-17 | 湖南农业大学 | For identifying the Specific PCR primers, kit and purposes of Rhodobacter capsulatus |
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| CN1986774A (en) * | 2005-12-23 | 2007-06-27 | 王栋 | Industrial preparing process, fermented liquid and use of spheroidal erythrocyte |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105111019A (en) * | 2015-08-24 | 2015-12-02 | 广东省农业科学院农业资源与环境研究所 | Special olive fertilizer for accelerating roots and retaining fruits |
| CN105861390A (en) * | 2016-05-30 | 2016-08-17 | 天津师范大学 | Rhodobacter capsulatus strain as well as screening method and application thereof |
| CN110241236A (en) * | 2019-06-14 | 2019-09-17 | 湖南农业大学 | For identifying the Specific PCR primers, kit and purposes of Rhodobacter capsulatus |
| CN110241236B (en) * | 2019-06-14 | 2022-11-11 | 湖南农业大学 | Specific PCR primer, kit and application for identifying rhodobacter capsulatus |
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