CN101735969B - Bradyrhizobium sp.RY4 strain and application thereof - Google Patents
Bradyrhizobium sp.RY4 strain and application thereof Download PDFInfo
- Publication number
- CN101735969B CN101735969B CN2009102143193A CN200910214319A CN101735969B CN 101735969 B CN101735969 B CN 101735969B CN 2009102143193 A CN2009102143193 A CN 2009102143193A CN 200910214319 A CN200910214319 A CN 200910214319A CN 101735969 B CN101735969 B CN 101735969B
- Authority
- CN
- China
- Prior art keywords
- bacterium
- root nodule
- khuskhus
- liquid
- bacterium liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000589171 Bradyrhizobium sp. Species 0.000 title claims abstract description 7
- 241000894006 Bacteria Species 0.000 claims abstract description 170
- 239000002689 soil Substances 0.000 claims abstract description 24
- 235000007769 Vetiveria zizanioides Nutrition 0.000 claims description 58
- 239000007788 liquid Substances 0.000 claims description 55
- 230000001580 bacterial effect Effects 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 230000001954 sterilising effect Effects 0.000 claims description 14
- 239000002068 microbial inoculum Substances 0.000 claims description 13
- 241000195940 Bryophyta Species 0.000 claims description 12
- 239000003415 peat Substances 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- 239000007224 yma-medium Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 239000010451 perlite Substances 0.000 claims description 7
- 235000019362 perlite Nutrition 0.000 claims description 7
- 238000000926 separation method Methods 0.000 claims description 7
- 238000012549 training Methods 0.000 claims description 7
- 239000010455 vermiculite Substances 0.000 claims description 7
- 235000019354 vermiculite Nutrition 0.000 claims description 7
- 229910052902 vermiculite Inorganic materials 0.000 claims description 7
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 claims description 6
- 230000035784 germination Effects 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 235000011132 calcium sulphate Nutrition 0.000 claims description 3
- 239000001175 calcium sulphate Substances 0.000 claims description 3
- 238000012545 processing Methods 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 2
- 240000005775 Chrysopogon zizanioides Species 0.000 claims 5
- 239000002594 sorbent Substances 0.000 claims 2
- 241000196324 Embryophyta Species 0.000 abstract description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 229910052757 nitrogen Inorganic materials 0.000 abstract description 6
- 241000412329 Stylosanthes hispida Species 0.000 abstract description 4
- 241001052560 Thallis Species 0.000 abstract description 4
- 230000024121 nodulation Effects 0.000 abstract description 4
- 241001465754 Metazoa Species 0.000 abstract description 2
- 108010020943 Nitrogenase Proteins 0.000 abstract description 2
- 241000282414 Homo sapiens Species 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- 244000284012 Vetiveria zizanioides Species 0.000 description 53
- 241000589151 Azotobacter Species 0.000 description 10
- 239000002609 medium Substances 0.000 description 8
- 235000015097 nutrients Nutrition 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 238000003794 Gram staining Methods 0.000 description 5
- 241000237502 Ostreidae Species 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 5
- 235000020636 oyster Nutrition 0.000 description 5
- 239000004576 sand Substances 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000004459 forage Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 241000589180 Rhizobium Species 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- 239000004568 cement Substances 0.000 description 3
- 238000005138 cryopreservation Methods 0.000 description 3
- 238000012856 packing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 235000004936 Bromus mango Nutrition 0.000 description 2
- 244000025254 Cannabis sativa Species 0.000 description 2
- 241001535083 Dialister Species 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000726221 Gemma Species 0.000 description 2
- 101000654381 Homo sapiens Sodium channel protein type 8 subunit alpha Proteins 0.000 description 2
- 240000007228 Mangifera indica Species 0.000 description 2
- 235000014826 Mangifera indica Nutrition 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 102100031371 Sodium channel protein type 8 subunit alpha Human genes 0.000 description 2
- 235000009184 Spondias indica Nutrition 0.000 description 2
- 241000219793 Trifolium Species 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000007621 cluster analysis Methods 0.000 description 2
- DFWFIQKMSFGDCQ-UHFFFAOYSA-N deethylatrazine Chemical compound CC(C)NC1=NC(N)=NC(Cl)=N1 DFWFIQKMSFGDCQ-UHFFFAOYSA-N 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000002151 riboflavin Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 241000589173 Bradyrhizobium Species 0.000 description 1
- 241000441581 Bradyrhizobium sp. Wall9 Species 0.000 description 1
- 241000637848 Cajanus scarabaeoides Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000694589 Gesneria viridiflora Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004178 biological nitrogen fixation Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 235000019784 crude fat Nutrition 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000001863 plant nutrition Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Bradyrhizobium sp.RY4 strain and application thereof. The Bradyrhizobium sp.RY4 strain is preserved at the China center for type culture collection and the preservation number is CCTCC NO:M 209268. When the Bradyrhizobium sp.RY4 is back-jointed to stylosanthes guianensis TPRC2 and TPRC5, the number of root nodules, the dry weight of the root nodule, the height of the strain, the fresh weight of the plant and the nitrogenase activity are greatly increased and obviously higher than those in a control group; and the effect of the RY4 back jointed to the stylosanthes guianensis TPRC2 is better. Thalli are released to the natural environment, the thalli increase the number of the groups of nodule bacteria in soil, contribute to the nodulation and nitrogen fixation of the stylosanthes guianensis, are harmless for human beings, animals and plants, and do not pollute the environment.
Description
Technical field
The invention belongs to plant nutrition and forage cultivating technical field, be specifically related to a kind of biological nitrogen fixation technology, especially relate to a kind of root nodule bacterium that can make the efficient dross of khuskhus and reach high yield.
Background technology
Khuskhus (Stylosanthes guianensis SW.) has another name called tropical clover, Brazilian clover, originates in South America.Nineteen eighty-two is drawn from CIAT (CIAT) by Chinese Academy of Tropical Agricultural Sciences, and plants experimentally successfully in Hainan, and big area is extended to each province, south China (district) now.Khuskhus contains crude protein 16.0～20.0% in the dry-matter of full-bloom stage; Crude fat 1.8～2.5%; Rent fiber 17.0～19.0%, NFE 38.0～44.0%, coarse ash 8.0～10.0%; Be China main leguminous forage in south, the saying of " north has clover south that the post flower is arranged " is arranged in grass cultivation circle.(district) economized in southern china such as Guangdong, Guangxi etc., with planting between khuskhus and fruit tree, and except being used for forage grass, the effect that also can play covering, conserves water and soil and improve the soil and foster and apply fertilizer.If with mixed seeding such as graminous pasture such as Herba Setariae Viridis, can build up good artificial pasture and be used to herd.
Khuskhus selects usually that draining is good, soil layer is deep, the husky preferably earth of soil property or loam plantation.But in lean soil, the khuskhus seedling can not form root nodule, or only has some to infect the ineffetive nodulation that forms by indigenous root nodule, and leguminous plants knot kind of root nodule bacterium can increase output 15～50% on the barren soil.For improving khuskhus dross rate, the fixed nitrogen loam should be used the seed dressing of khuskhus root nodule bacterium, can effectively improve the dross rate of khuskhus.Therefore the screening root nodule bacterium that can make the efficient dross of khuskhus and reach high yield have great importance.
Khuskhus with the characteristic of its high yield, high-quality and anti-low level management after introduction, become me very soon China south leguminous forage promote mainly kind.The content of khuskhus original inhabitants bacterium is extremely low at the beginning of khuskhus is introduced because in the planting site soil, and khuskhus is nodulation and nitrogen fixation well, and main dependence fertilising obtains high yield.So just can not embody real high yield and anti-low level management.China has once introduced Rhizobium Inoculant from Australia and has been applied to the khuskhus planting site, and the output of khuskhus is improved.But the root nodule bacterium that Australia introduces can not well adapt to the southern khuskhus planting conditions of China, can not reach significant effect of increasing production after being inoculated in khuskhus.At present China does not also filter out the bacterial strain that is suitable for China's weather edaphic condition, so from the indigenous bacterium of the Different Soil conditions of China column with cultivations of flower or grass flowers and plants, screen the khuskhus high-efficiency nitrogen-fixing root nodule bacterium that are suitable for the specific region important practical sense is arranged.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, it is RY4 that a kind of nodule azotobacter strain is provided, a kind of root nodule bacterium that can make the efficient dross of khuskhus and reach high yield.
The inventor is in November, 2008 (E101 from the mango ground of Sichuan Flos Bombacis Malabarici clayed soil.46.137 ' N26.25.276 H1100) collect the root nodules of the flowers and plants of a strain riotous growth, cut the khuskhus over-ground part whole root system is placed on cryopreservation in the ice bag together with the root nodule freshness protection package of packing into.It is RY4 (Bradyrhizobium sp.RY4) that warp separation and purifying obtain a kind of nodule azotobacter strain, is preserved in Chinese typical culture collection center on November 13rd, 2009, and preserving number is CCTCC NO:M 209268.Its 16sDNA sequence is shown in SEQ ID NO 1.
Another object of the present invention provides the cultural method that said nodule azotobacter strain is RY4.
The object of the invention is achieved through following technical proposals:
With the root nodule that collects, cut the khuskhus over-ground part whole root system is placed on cryopreservation in the ice bag together with the root nodule freshness protection package of packing into, in order to follow-up separation and purification process.
Concrete separation and purge process may further comprise the steps:
(1) root system of khuskhus is rinsed well, cut in petridish the root of root nodule band 2mm surface washing is clean with 2 grades of water;
(2) get oyster white juice and on the YMA solid medium, rule smashing to pieces after the sterilization of the root nodule rinsed well, dipping in, be inverted in and place 28 ± 1 ℃ of incubator dark to cultivate in the freshness protection package drawing a good plate;
(3) began to check whether grow bacterium colony on the substratum at the 3rd day that cultivates, until about the 15th day, the bacterium colony mark is come out to write down form and glossiness;
(4) get rid of behind the non-root nodule bacterium bacterium colony according to the bacterium colony of above-mentioned mark will grow growthhabit on the time phase difference of single bacterium colony bacterium colony and the plate more than 3 days, bacterium colony size, bacterium colony transparency, and the light inconsistent bacterium colony of refraction, colony colour down choose, stroke plate is also numbered on new YMA medium.Each time to the new bacterium colony of drawing plate all adopt aforesaid method carry out purifying until the colony growth form on the same block of plate, bacterium colony size, bacterium colony transparency, reach refraction under the light, colony colour unanimity till.Carry out putting under in the YMA inclined-plane behind microscopy and the tieback through the plate after with purifying of purifying step by step and preserve.
Said bacterial strain colonial morphology: the translucent or White-opalescent point-like thing later stage bacterium colony of root nodule bacterium early growth period bacterium colony water sample be circle, oyster white, translucent, neat in edge, cement more or less.Cultivate 2～4 days colony diameters and reach 2～4mm and give birth to the type root nodule bacterium for fast, cultivate 5～10 days bacterium colonies just 1mm be living type root nodule bacterium slowly.
Thalli morphology: mark is tentatively confirmed as the root nodule bacterium bacterium colony, and therefrom picking lawn smear carries out gramstaining, under 100 power microscopes, observes.Root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain
-hydroxybutyric acid; Promptly reflect the particle in strong seemingly cavity or make thalline in the form of link; Gram-negative (G-); No gemma, thalline is single or paired.
It is the method for RY4 bacterial strain tieback khuskhus that the present invention provides said nodule azotobacter strain simultaneously, may further comprise the steps:
(1) the root nodule RY4 that separation and purification is come out washes in the liquid YMA medium with sterilized water, seals with sealing film, and 180r/min shakes bacterium is measured bacterium liquid after 3 days OD value in shaking table, when OD gets bacterium liquid greater than 0.6 the time;
(2) the khuskhus seed is soaked the back with 0.2% (mass and size concentration, 0.2g HgCl in the ethanol of 95% (volume by volume concentration)
2/ 100ml water) HgCl
2Handle, use aseptic water washing, be thrown on the germination paper of sterilizing in the petridish, soak germination paper, be placed in 28 ℃ the incubator dark vernalization 3 days, treat that centimetre time shift of seedling length to 4 goes into that sterilization is husky trains in the case with the 0.05mmol/L calcium sulphate soln of sterilization;
(3) from the said husky training case of step (2), choose bacterium liquid that sturdy seedling prepares with step (1) and soak that plantation is in the flowerpot of filling sterilization sand after 15 minutes, every seedling adds bacterium liquid 1～2ml, guarantees that the bacterium that connects of every seedling is measured and reaches 1.0 * 10
9More than individual.
It is the microbial inoculum that RY4 prepares that a further object of the invention provides said nodule azotobacter strain.In the solid plate substratum, 4～5 bacterium colonies begin to grow, and the bacterium colony on the plane is chosen a washing-round with transfering loop go in the ready liquid YMA medium with the RY4 root nodule bacterium activation of preserving.Above-mentioned bacterium liquid 180r/min in shaking table is shaken bacterium obtained every milliliter of bacteria containing amount greater than 1.0 * 10 after (28 ± 1 ℃) in 3～5 days
9Individual above bacterium liquid.Above-mentioned bacterium liquid liquid bacterial agent be can directly use, also can microbial inoculum use, the preferred peat composed of rotten mosses of said thalline sorbing material, vermiculite or perlite etc. be processed by proportional mixing thalline sorbing material; The preferred 10ml bacterium liquid of the said ratio/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite.
A further object of the invention provides khuskhus and inoculates the method that said nodule azotobacter strain is the RY4 bacterium.Said nodule azotobacter strain is the RY4 bacterium through activation, shake bacterium obtains liquid bacterial agent and can after shaking bacterium, dress seed and handle or water in the khuskhus root of just having transplanted seedlings with liquid bacterial agent, also can be with bacterium liquid mixed imposing in the soil before for example the peat composed of rotten mosses, vermiculite perlite etc. are processed microbial inoculum and transplanted by specified proportion mixings thalline sorbing material.
The present invention gets beneficial effect: RY4 khuskhus root nodule is the efficient root nodule that separates in the autohension soil; The edatope that is suitable for viscosity; Use the output that has greatly improved TPRC2 after this khuskhus microbial inoculum; Raising reaches 3.9 times, and the TPRC5 khuskhus is effectively increased production, and amount of increase in production is slightly smaller than TPRC2.
Description of drawings
Fig. 1 root nodule bacterium RY4 tieback check figure
Fig. 2 root nodule bacterium RY4 bacterium colony figure
Fig. 3 root nodule bacterium RY4 gramstaining picture
Dross situation behind Fig. 4 tieback root fungus RY4
Fig. 5 RY4 agarose gel electrophoresis figure
Fig. 6 NCBI compares figure
Fig. 7 RY4 root nodule bacterium cluster analysis figure
Fig. 8 tieback RY4TPRC2, TPRC5 dry weight figure
Fig. 9 RY4 microbial inoculum imposes on soil to TPRC2, TPRC5 yield effect variation diagram
The growth curve of Figure 10 RY4 under different PH conditions
The growth curve of Figure 11 RY4 under different salt concentration
Embodiment
Below in conjunction with specific embodiment and accompanying drawing further explain the present invention.
Acquisition and the cultivation of embodiment 1 root nodule bacterium RY4
The inventor's in November, 2008 (E101 from the mango ground of Sichuan Flos Bombacis Malabarici clayed soil.46.137 ' N26.25.276 H1100) collect the root nodules of the flowers and plants of a strain riotous growth, cut the khuskhus over-ground part whole root system is placed on cryopreservation in the ice bag together with the root nodule freshness protection package of packing into.It is RY4 (Bradyrhizobium sp.RY4) that warp separation and purifying obtain a kind of nodule azotobacter strain, is preserved in Chinese typical culture collection center on November 13rd, 2009, and preserving number is CCTCC NO:M 209268.
(1) root system that from ice bag, takes out khuskhus washes under tap water to be rinsed root system well 2～3 times, cuts in petridish fresh root complete, root nodule band 2mm that color is dark red with 2 grades of water that surface washing is clean with scissors; This process will be guaranteed the complete of root nodule surface.
(2) root nodule of rinsing well is transferred in the petridish after the sterilization, added after 95% alcohol-pickled 3 minutes, alcohol is poured out, with aseptic water washing 4～5 times, the HgCl of adding 0.1%
2Sterilized 2～3 minutes, rapidly with HgCl
2Pour out and add sterilized water; Shift the Bechtop operation;, more than 6 times sterilized water is poured out with aseptic water washing, chosen in the petridish after root nodule is transferred to sterilization and (can on flame, sterilize); Tweezers and transfering loop with after the sterilization are smashed root nodule to pieces, dip in and get oyster white juice and on ready YMA solid medium, rule.Be inverted in and place incubator (28 ± 1 ℃, dark) cultivation in the freshness protection package drawing a good plate.
YMA medium is filled a prescription like table 1:
Table 1 YMA (Yeast Mannitol Agar) culture medium prescription (L
-1)
(3) began to check whether grow bacterium colony on the substratum at the 3rd day that cultivates, until about the 15th day, the bacterium colony mark is come out to write down form and glossiness.
In culturing process, determine whether to be root nodule bacterium from following 3:
A. colonial morphology: the translucent or White-opalescent point-like thing later stage bacterium colony of root nodule bacterium early growth period bacterium colony water sample be circle, oyster white, translucent, neat in edge, cement more or less.Cultivate 2～4 days colony diameters and reach 2～4mm and give birth to the type root nodule bacterium for fast, cultivate 5～10 days bacterium colonies just 1mm be living type root nodule bacterium slowly.
B. thalli morphology: mark is tentatively confirmed as the root nodule bacterium bacterium colony, and therefrom picking lawn smear carries out gramstaining, under 100 power microscopes, observes.Root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain
-hydroxybutyric acid; Promptly reflect the particle in strong seemingly cavity or make thalline in the form of link; Gram-negative (G-); No gemma, thalline is single or paired.
C. through isolated root nodule bacterium are received on the khuskhus at aseptic condition next time, can dross then be root nodule bacterium.
The morphological specificity of root nodule bacterium RY4:
Root nodule bacterium RY4 is a kind of of knurl Pseudomonas of taking root slowly; Cultivate the vestige that is coated with along the first stroke after 3 days and begin to have the banded translucent projection of water sample; Began obviously and independently became gradually independent bacterium colony by the 4th day with rearward projection; The more single bacterium colony of translucent, little yellow, cement of 1.0～2.0mm appears in finishing touch after the 5th day, because colony growth is very fast, it is banded that the bacterium on the flat board sticks together into oyster white each other after 7 days.See the bacterium colony of RY4 root nodule bacterium shown in the accompanying drawing 2.
RY4 number optimum growing condition is: 28 ℃ of temperature; PH=4.5～6; It is former former with nitrogen that rotating speed 180r/min. can use carbon widely, on the comprehensive extract of various plant origins, can well grow, better than growth on protein culture medium on the YMA medium.
The physiological property of root nodule bacterium RY4:
Root nodule bacterium RY4 is purple through gramstaining at microscopically, and to be shaped as rhabdos be Gram-negative bacteria.See the gramstaining of root nodule bacterium RY4 shown in the accompanying drawing 3 picture.
(4) get rid of behind the non-root nodule bacterium bacterium colony according to the bacterium colony of above-mentioned mark will grow growthhabit on the time phase difference of single bacterium colony bacterium colony and the plate more than 3 days, bacterium colony size, bacterium colony transparency, and the light inconsistent bacterium colony of refraction, colony colour down choose on new substratum stroke plate and also number.Each time to the new bacterium colony of drawing plate all adopt aforesaid method carry out purifying until the colony growth form on the same block of plate, bacterium colony size, bacterium colony transparency, reach refraction under the light, colony colour unanimity till.Carry out putting under in the YMA inclined-plane behind microscopy and the tieback through the plate after with purifying of purifying step by step and preserve.
(5) tieback checking khuskhus root nodule bacterium
A. the preparation of bacterium liquid
The root nodule RY4 that separation and purification is come out washes in the ready liquid YMA medium with sterilized water, seals with sealing film, and 180r/min shakes bacterium is measured bacterium liquid after 3 days OD value in shaking table, and (every ML bacterium liquid bacteria containing amount is more than 1.0 * 10 greater than 0.6 the time as OD
9Individual) promptly get bacterium liquid, can be used for tieback.Said liquid YMA medium is in the substratum of table 1, agar to be removed, and the adjusting pH value is 6.
B. the preparation of aseptic seedling
Selected khuskhus seed number grain soaked 5 minutes in 95% ethanol, took out the back at 0.2% HgCl
2In handled 5 minutes, with aseptic water washing 5～10 times, come the bacterium of going out then, put well in the petridish of germination paper, the went out 0.05mmol/L calcium sulphate soln of bacterium of usefulness soaks germination paper, is placed in 28 ℃ the incubator dark vernalization 3 days.Move into seedling in the sand training case of the ready bacterium of going out when treating seedling length to 4 centimetre, for use.
C. tieback
From sand training case, choose sturdy seedling and be put into the bacterium liquid immersion for preparing with step (5) a in the petridish 15 minutes; With tweezers seedling is planted in the small flower of filling sterilization sand; The shape that assumes diamond in shape in every basin 4 strains of planting; Every seedling adds bacterium liquid 1～2ml, guarantees that the bacterium amount that connects of every naked seedling reaches 1.0 * 10
9More than individual.Pull up seedling after approximately and whether check dross, dross then is root nodule bacterium.Root nodule RY4 dross after the tieback check is obvious, and provable isolate is pure root nodule bacterium.See the tieback of root nodule bacterium RY4 shown in the accompanying drawing 1 assay.
The 16S rDNA sequence order-checking of embodiment 2 root nodule bacterium RY4 and confirming of classification
In order to confirm the phylogeny status of root nodule bacterium RY4, the 16SrDNA series of the bacterial strain that obtains checks order to separate.At first utilize the test kit of omega company to carry out the extraction of total DNA, utilize primer to carry out the PCR specific amplification then.
Upstream primer 35fc:CTKAAGAGTTTGATCMTGGCTCAGATTGAAC;
Downstream primer 1492r:TACGGYTACCTTGTTACGACTT.
Reaction conditions is following:
The PCR reaction
10×Reaction?Buffer 5.0μL
dNTPs(10mM) 1.0μL
P35fc (25Pmol) primer 0.5 μ L
P1492r (25Pmol) primer 0.5 μ L
Taq archaeal dna polymerase (5u/ μ L) 1.0 μ L
Template DNA 1.0 μ L
Mend ultrapure water to 41 μ L
94 ℃ of preparatory sex change: 3min
94 ℃ of sex change 50s
35 circulations of 56 ℃ of annealing 50s
72 ℃ are extended 60s
72 ℃ of last 5min that extend
Amplified production assay on 1.0% agarose gel electrophoresis is seen the figure of RY4 agarose gel electrophoresis shown in the accompanying drawing 5, send Beijing AudioCodes biotech company to check order, and sequencing result is shown in SEQID NO 1.
The sequence results of gained is compared at the U.S. U.S. state-run bioinformation center (NCBI), find that rhizobium strains RY4 and known bacterial strain Bradyrhizobium sp.Wall9 similarity reach 99%, see the figure of NCBI comparison shown in the accompanying drawing 6.Application software DNAStar and TREECONW carry out cluster to the bacterial strain of being surveyed, and see the cluster analysis of RY4 root nodule bacterium shown in the accompanying drawing 7 figure.Can know that by comparison result and dendrogram RY4 is the new strain system of knurl Pseudomonas (Bradyrhizobium) of taking root slowly, called after heat is ground (RY4) No. 4.
The application experiment of embodiment 3 root nodule bacterium RY4
In order to confirm the effect of RY4 to khuskhus; Adopt two khuskhus kinds (TPRC2, TPRC5); With the method for husky training with root nodule RY4 tieback to the khuskhus root; Each handles 4 repetitions (processing of bacterium, sand and seedling is referring to the tieback experimental procedure), is contrast (CK) not inoculate root nodule, carries out the tieback contrast.Adopt low nitrogen nutrition liquid to water in the whole process.Table 2 and accompanying drawing 8 are seen in physical signs influence to khuskhus behind khuskhus TPRC2, the TPRC5 tieback root nodule bacterium RY4.Left side square column (a) is TPRC2 in the accompanying drawing 8, and left side square column (b) is TPRC5 (other histograms does not have direct relation for existing root nodule bacterium results of comparison with technology of the present invention in the accompanying drawing 8, do not do mark).
Root nodule number, root nodule dry weight, plant height, plant fresh weight and nitrogenase activity by khuskhus TPRC2, TPRC5 behind table 2 and the accompanying drawing 8 visible tieback RY4 all increase considerably than the contrast (CK) of correspondence.In all physical signs of khuskhus, over-ground part output is to weigh the final index of khuskhus high yield.Can find out that by accompanying drawing 8 output that root nodule bacterium RY4 is applied to TPRC2, TPRC5 all is higher than contrast (comparison is according to exceeding more than 2 times).The output of RY4 TPRC2 in the application of TPRC2, TPRC5 is higher than the output of TPRC5, but the yield increased output of same kind internal-phase ratio TPRC5 also exceeds a lot.
Table 2 root nodule bacterium RY4 tieback behind TPRC2, the TPRC5 to the influence of khuskhus physical signs
Sum up through experimental study, root nodule bacterium RY4 is the khuskhus rhizobium strains that on the relatively poor clayed soil of ventilation property, can form high yield.Behind the present khuskhus of its property list inoculation root nodule bacterium under the condition of ventilation property condition difference many, the dross volume of the strong dross quantity of dip-dye ability greatly, strong stress resistance, can make TPRC2, TPRC5 reach high yield simultaneously, see the dross situation behind the accompanying drawing 4 tieback root fungus RY4.When thalline is released in the physical environment, harmless to people, animal and plant, do not pollute the environment, increased the colony of root nodule bacterium between soil on the contrary, the nodulation and nitrogen fixation of khuskhus there is promoted effect.
Said microbial inoculum can be in activation, shake bacterium after with the processing of dressing seed and soak seed of liquid liquid; Before transplanting seedlings, the seedling root soaked in bacterium liquid and carried out the field final singling in about 30 minutes again; Water in khuskhus root of just having transplanted seedlings and rhizosphere soil with liquid bacterial agent.Also can process microbial inoculum mixed imposing in the soil before sowing or transplanting by proportional mixing thalline sorbing material.The preferred peat composed of rotten mosses of said thalline sorbing material, vermiculite or perlite etc.; The preferred 10ml bacterium liquid of the said ratio/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite.
RY4 peat composed of rotten mosses microbial inoculum is applied in the soil influence to khuskhus output:
Prepare aseptic seed and grow seedlings like aforementioned husky training. root nodule bacterium are washed the triangular flask 28 ℃ from solid medium cultivated 96 hours; With sterilized water wash-out thalline; And dilute with sterilized water; With the whirlpool even thalline of device that turns round and round, measure OD value (go into=600nm), the bacteria containing amount that guarantees every seed when the field is inoculated is greater than 10
9Individual.It is subsequent use at 40 minutes postcooling of 121 ℃ of sterilizations the peat composed of rotten mosses to be pulverized (crossing the 2mm sieve aperture) back.Prior to seeding with different bacterium liquid in 10ml bacterium liquid: the ratio of the 50g peat composed of rotten mosses is poured in the peat composed of rotten mosses, and thorough mixing absorbs bacterium liquid by the peat composed of rotten mosses.The preceding microbial inoculum that mixes is mixed of growing seedlings imposes in the soil.The sub-district area is 20m
2, establish four repetitions.Receive appearance, its individual plant hay yield result such as accompanying drawing 9 after three months.Left side square column (a) is TPRC2 in the accompanying drawing 9, and left side square column (b) is TPRC5.(other histograms do not have direct relation for existing root nodule bacterium results of comparison with technology of the present invention in the accompanying drawing 9, do not do mark).
Can know that by accompanying drawing 9 output that TPRC2, TPRC5 khuskhus have been used the RY4 root nodule bacterium is 3.9 times and 2.2 times of plant that do not connect bacterium.
The adaptation experiment of embodiment 5 root nodule bacterium RY4 to soil
Root nodule bacterium, edatope and plant are interactional individual system, the three is united take all factors into consideration the high yield that reaches real and efficient.China's khuskhus mainly is to plant in the wider South China of acid soil distribution.In order to confirm the adaptation situation of RY4, confirm acidproof, the salt tolerance of khuskhus through following method to soil major influence factors (acid-basicity, and saline alkali).For using RY4 khuskhus root nodule bacterium that preferred technical support is provided from now on more scientifically and rationally.
After bacterial strain RY4 activation, wash in the YMA liquid nutrient medium, cultivate down in constant temperature shaking table (28 ± 1 ℃, 180r/min), treat OD
600Be about 1; Drawing 50 μ l bacterial suspension inoculations respectively is in 4,4.5,4.8,5,5.5,6,7 (volume is 50ml) YMA liquid nutrient medium to the pH value; Put 28 ± 1 ℃, 180rpm shaking culture in the constant temperature oscillator; Detect the growing state of bacterium with the variation of nutrient solution opacity, cultivate and use spectrophotometer measurement OD after 72 hours
600Each handles 3 repetitions.The growing state of RY4 in different PH seen accompanying drawing 10.Can be known that by accompanying drawing 10 the RY4 root nodule bacterium can form effective bacterium liquid at PH greater than 4.2 o'clock, the activity of bacterium reaches maximum when the environment pH value is 4.5, and the activity of pH value from 4.5 to 6.5 bacterium is all bigger and pH value is that 4.5 o'clock bacterial concentration is similar.
Explain that the RY4 root nodule bacterium have wider flexibility to the pH value of environment.
The experiment of salt tolerant resistance adopts the YMA liquid nutrient medium that adds NaCl to cultivate root nodule bacterium, and with the OD value of spectrophotometric determination bacterium liquid, the size of OD value can reflect the situation of bacteria growing speed after 3 days.After each bacterial strain is activated, be inoculated in the YMA liquid nutrient medium, put 28 ± 1 ℃ of cultivations down in the constant temperature oscillator, treat OD
600Be about 1; Draw respectively 50 μ l bacterial suspension inoculations to NaCl concentration be 0,0.05,0.08,0.10,0.15,0.20,0.25, in the 0.30mol/LYMA liquid nutrient medium (pH5.5, volume are 50ml); Put 28 ± 1 ℃ of 180rpm shaking culture in the constant temperature oscillator; Detect the growing state of bacterium with the variation of nutrient solution opacity, cultivate and use spectrophotometer measurement OD after 72 hours
600Each handles 3 repetitions.The growing state of RY4 in different salt concentration seen accompanying drawing 11.The RY4 root nodule bacterium grow when salt concn is higher than 0.08mol/L in edatope and have received very big inhibition, can not form concentration greater than 1.0 * 10
9The bacterium liquid of individual/ml, the bacteria containing amount in solution is followed reducing of solution salt concentration and is increased, when salt concn begins to form concentration greater than 1.0 * 10 during for 0.06mol/L
9Effective bacterium liquid of individual/ml.
SEQUENCE?LISTING
< 110>Liu Guodao, Huang Yanxia, Dong Rongshu
< 120>a kind of nodule azotobacter strain is RY4 bacterial strain and application thereof
<130>
<160>1
<170>PatentIn?version?3.3
<210>1
<211>1377
<212>DNA
< 213>artificial sequence
<400>1
caggcttaac?acatgtaagt?cgagcgggcg?tagcaatacg?tcagcggcag?acgggtgagt 60
aacgcgtggg?aacgtacctt?ttggttcgga?acaactgagg?gaaacttcag?ctaataccgg 120
ataagccctt?acggggaaag?atttatcgcc?gaaagatcgg?cccgcgtctg?attagctagt 180
tggtgaggta?atggctcacc?aaggcgacga?tcagtagctg?gtctgagagg?atgatcagcc 240
acattgggac?tgagacacgg?cccaaactcc?tacgggaggc?agcagtgggg?aatattggac 300
aatgggggca?accctgatcc?agccatgccg?cgtgagtgat?gaaggcccta?gggttgtaaa 360
gctcttttgt?gcgggaagat?aatgacggta?ccgcaagaat?aagccccggc?taacttcgtg 420
ccagcagccg?cggtaatacg?aagggggcta?gcgttgctcg?gaatcactgg?gcgtaaaggg 480
tgcgtaggcg?ggtctttaag?tcaggggtga?aatcctggag?ctcaactcca?gaactgcctt 540
tgatactgaa?gatcttgagt?tcgggagagg?tgagtggaac?tgcgagtgta?gaggtgaaat 600
tcgtagatat?tcgcaagaac?accagtggcg?aaggcggctc?actggcccga?tactgacgct 660
gaggcacgaa?agcgtgggga?gcaaacagga?ttagataccc?tggtagtcca?cgccgtaaac 720
gatgaatgcc?agccgttagt?gggtttactc?actagtggcg?cagctaacgc?tttaagcatt 780
ccgcctgggg?agtacggtcg?caagattaaa?actcaaagga?attgacgggg?gcccgcacaa 840
gcggtggagc?atgtggttta?attcgacgca?acgcgcagaa?ccttaccagc?ccttgacatc 900
ccggtcgcgg?actccagaga?cggagttctt?cagttcggct?ggaccggaga?caggtgctgc 960
atggctgtcg?tcagctcgtg?tcgtgagatg?ttgggttaag?tcccgcaacg?agcgcaaccc?1020
ccgtccttag?ttgctaccat?ttagttgagc?actctaagga?gactgccggt?gataagccgc?1080
gaggaaggtg?gggatgacgt?caagtcctca?tggcccttac?gggctgggct?acacacgtgc?1140
tacaatggcg?gtgacaatgg?gatgctaagg?ggcgaccctt?cgcaaatctc?aaaaagccgt?1200
ctcagttcgg?attgggctct?gcaactcgag?cccatgaagt?tggaatcgct?agtaatcgtg?1260
gatcagcacg?ccacggtgaa?tacgttcccg?ggccttgtac?acaccgcccg?tcacaccatg?1320
ggagttggtt?ttacctgaag?acggtgcgct?aaccgaaagg?gggcagccgg?ccacgta 1377
Claims (6)
1. living slowly root nodule bacterium (Bradyrhizobium sp.) RY4 bacterial strain is preserved in Chinese typical culture collection center on November 13rd, 2009, and preserving number is CCTCC NO:M209268.
2. the said living slowly root nodule bacterium RY4 bacterial strain of claim 1 is characterized in that its 16SDNA sequence is shown in SEQ ID NO 1.
3. the method for the said living slowly root nodule bacterium RY4 bacterial strain tieback khuskhus of claim 1 is characterized in that may further comprise the steps:
(1) the living slowly root nodule bacterium RY4 bacterial strain that separation and purification is come out washes in the liquid YMA medium with sterilized water, and 180r/min shakes the OD value of measuring bacterium liquid behind the bacterium in shaking table, when OD gets bacterium liquid greater than 0.6 the time;
(2) be thrown on the germination paper of sterilizing in the petridish after the khuskhus seed sterilization, soak germination paper, place 28 ± 1 ℃ the dark vernalization of incubator, treat that centimetre time shift of seedling length to 4 goes in the husky training of the sterilization case with the 0.05mmol/L calcium sulphate soln of sterilization;
(3) from the said husky training case of step (2), choose the bacterium liquid immersion back plantation that sturdy seedling prepares with step (1), every seedling adds bacterium liquid 1～2ml.
4. microbial inoculum is characterized in that it being that bacterium liquid mixing thalline sorbent material by the said living slowly root nodule bacterium RY4 bacterial strain of claim 1 prepares.
5. according to the said microbial inoculum of claim 4, it is characterized in that said thalline sorbent material is the peat composed of rotten mosses, vermiculite or perlite; Blending ratio is 10ml bacterium liquid/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite.
6. the method for said living the slowly root nodule bacterium RY4 bacterial strain of khuskhus inoculation claim 1 is characterized in that the root nodule bacterium activation shaken to dress seed behind the bacterium to handle or water with bacterium liquid processing microbial inoculum mixed imposing in the soil before the khuskhus transplanting in the khuskhus root or with bacterium liquid mixings thalline sorbing material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102143193A CN101735969B (en) | 2009-12-29 | 2009-12-29 | Bradyrhizobium sp.RY4 strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102143193A CN101735969B (en) | 2009-12-29 | 2009-12-29 | Bradyrhizobium sp.RY4 strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101735969A CN101735969A (en) | 2010-06-16 |
| CN101735969B true CN101735969B (en) | 2012-06-13 |
Family
ID=42460000
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102143193A Expired - Fee Related CN101735969B (en) | 2009-12-29 | 2009-12-29 | Bradyrhizobium sp.RY4 strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101735969B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104604500B (en) * | 2015-01-30 | 2017-03-08 | 杭州师范大学 | A kind of raising Semen sojae atricolor is in the method for salt-soda soil adaptability |
| CN105950520B (en) * | 2016-07-18 | 2019-05-10 | 武汉市农业科学技术研究院作物科学研究所 | A kind of rhizobium of efficient phosphate-solubilizing and its application |
| CN109439590B (en) * | 2018-12-05 | 2020-11-17 | 福建农林大学 | Cassia rhizobium WYS3R1 and application thereof |
| CN109370953B (en) * | 2018-12-05 | 2020-06-16 | 福建农林大学 | Juemina rhizobium JYN6 and application thereof |
| CN110073910A (en) * | 2019-05-22 | 2019-08-02 | 南京农业大学 | A kind of bur clover production method of suitable home mode |
| CN113881601A (en) * | 2021-10-29 | 2022-01-04 | 中国农业科学院农业资源与农业区划研究所 | In-situ efficient compound rhizobium group screening method |
| CN114350559B (en) * | 2021-12-29 | 2023-05-12 | 中国热带农业科学院热带作物品种资源研究所 | Salt-tolerant growth-promoting Liaoning slow rhizobium RY6 strain and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182476A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXYD3 and uses thereof |
-
2009
- 2009-12-29 CN CN2009102143193A patent/CN101735969B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182476A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXYD3 and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| 江木兰 等.大豆—根瘤菌的固氮作用.《中国油料作物学报》.2003,第25卷(第5期),全文. * |
| 祁娟.苜蓿种子内生根瘤菌筛选及其促生能力研究.《甘肃农业大学硕士学位论文》.2006,全文. * |
| 黄艳霞.柱花草根瘤菌的固氮活性研究及抗性筛选.《海南大学硕士学位论文》.2010,全文. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101735969A (en) | 2010-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106987541B (en) | Efficient alfalfa rhizobium with stress resistance and growth promotion performance and application thereof | |
| CN101735969B (en) | Bradyrhizobium sp.RY4 strain and application thereof | |
| CN101748088B (en) | Bacterial strain of root nodule nitrogen-fixing strain series RY3 and application thereof | |
| CN110029077B (en) | Salt-tolerant growth-promoting bacterial strain Y4 and application thereof | |
| CN104263684A (en) | Siderophores-producing bacillus and applications thereof | |
| CN110150013A (en) | A kind of method of chinquapin mycorrhizal seedling raising | |
| CN101824390B (en) | RY2 strain of root nodule nitrogen-fixing bacterial strain system and application thereof | |
| CN107904192B (en) | Rhizobium V9-2 and application thereof | |
| CN107904193B (en) | Rhizobium V14-2 and application thereof | |
| CN116103158A (en) | Disease-resistant growth-promoting mortierella alpina, microbial inoculum containing same and application thereof | |
| CN101781629B (en) | Root nodule azotobacter strain RY5 bacterial strain and application thereof | |
| CN101781630B (en) | Root nodule azotobacter strain RY1 bacterial strain and application thereof | |
| CN111484953A (en) | Bacillus capable of promoting growth and dissolving phosphorus and application thereof | |
| CN109486711B (en) | Rhizobium AXLQ16 and application thereof | |
| CN114350559B (en) | Salt-tolerant growth-promoting Liaoning slow rhizobium RY6 strain and application thereof | |
| CN108118010B (en) | Rhizobium fabae strain Blgs20-1 and application thereof | |
| CN113881606B (en) | Pseudomonas RL-WG26 strain capable of resisting salt and promoting growth and application thereof | |
| CN114891658B (en) | Common vetch root nodule strain HBUJ010033 and application thereof | |
| CN109439589B (en) | Rhizobium YZLH133 and application thereof | |
| CN109355234B (en) | Rhizobium YZM0144 and application thereof | |
| CN109355235B (en) | Rhizobium FAMB126 and application thereof | |
| CN108239615B (en) | Rhizobium fabae strain Bga2-2 and application thereof | |
| CN109337847B (en) | Cassia rhizobium TXR2 and application thereof | |
| CN109370953B (en) | Juemina rhizobium JYN6 and application thereof | |
| CN109439590B (en) | Cassia rhizobium WYS3R1 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: TROPICAL CROP VARIETY RESOURCE INSTITUTE OF CHINES Free format text: FORMER OWNER: LIU GUODAO Effective date: 20130319 Owner name: LIU GUODAO DONG RONGSHU Free format text: FORMER OWNER: HUANG YANXIA DONG RONGSHU Effective date: 20130319 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20130319 Address after: 571737 Institute of tropical crop resources, China Academy of Tropical Agricultural Sciences, Hainan, Danzhou, China Patentee after: Resources Institute of Tropical Crop Varieties of Chinese Academy of Tropical Agricultural Sciences Patentee after: Liu Guodao Patentee after: Dong Rongshu Address before: 571737 Institute of tropical crop resources, China Academy of Tropical Agricultural Sciences, Hainan, Danzhou, China Patentee before: Liu Guodao Patentee before: Huang Yanxia Patentee before: Dong Rongshu |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120613 Termination date: 20151229 |
|
| EXPY | Termination of patent right or utility model |