CN101781630B - Root nodule azotobacter strain RY1 bacterial strain and application thereof - Google Patents
Root nodule azotobacter strain RY1 bacterial strain and application thereof Download PDFInfo
- Publication number
- CN101781630B CN101781630B CN 200910214318 CN200910214318A CN101781630B CN 101781630 B CN101781630 B CN 101781630B CN 200910214318 CN200910214318 CN 200910214318 CN 200910214318 A CN200910214318 A CN 200910214318A CN 101781630 B CN101781630 B CN 101781630B
- Authority
- CN
- China
- Prior art keywords
- bacterium
- bacterial strain
- root nodule
- khuskhus
- 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
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a root nodule azotobacter strain RY1 bacterial strain and an application thereof. The bacterial strain (Bradyhizobium sp.RY1) is obtained from gathering fresh root nodules in main Stylosanthes guianensis planting areas in China and separating and purifying in a laboratory, the partial sequence of 16S rDNA of the bacterial strain is measured, and the bacterial strain is determined to be a new bacterial strain of Bradyrhizobium by the phylogenesis analysis and is named as Re Yan 1 (RY1). By the potted planting and the inoculation test in the laboratory, the nodule azotobacter strain RY1 bacterial strain is proven to have the characteristic of enabling the two main Stylosanthes guianensis varieties (TPRC2 and TPRC5) in China to realize the high-efficiency nodulation and achieve the high yield. Compared with the inoculation-free process, the inoculation with the root nodule can improve the yield of the Stylosanthes guianensis by more than 2 times, and can be used as one of conventional Stylosanthes guianensis culturing measures for planting the Stylosanthes guianensis.
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 since CIAT (CIAT) by Chinese Academy of Tropical Agricultural Sciences, and plants experimentally successfully in Hainan, now spread in each province, south China (district).Khuskhus contains crude protein 16.0～20.0% in the dry-matter of full-bloom stage, crude fat 1.8～2.5% is rented fiber 17.0～19.0%, nitrogen-free extract 38.0～44.0%, and coarse ash 8.0～10.0% is a kind of good tropical leguminous forage.(district) economized in southern china such as Guangdong, Guangxi etc., will plant 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 for herding.
Khuskhus selects usually that draining is good, husky earth or the loam plantation preferably of the deep soil property of soil layer.But in lean soil, the khuskhus seedling can not form root nodule, or only has some to be infected the ineffetive nodulation that forms by indigenous root nodule, and leguminous plants knot kind of root nodule bacterium can increase output 15～50% on 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 becomes the kind of promoting mainly of south China leguminous forage very soon after introduction with the characteristic of its high yield, high-quality and anti-low level management.At the beginning of khuskhus is introduced due to planting site soil in the content of khuskhus original inhabitants bacterium extremely low, khuskhus is nodulation and nitrogen fixation well, mainly relies on fertilising to obtain high yield.So just can not embody real high yield and anti-low level management.China had introduced Rhizobium Inoculant from Australia and had been applied to the khuskhus planting site afterwards, and the output of khuskhus is improved.But because the Rhizobium Inoculant of Australia is based on the soil of Australia and the high efficient strain that climatope is selected, arrive the weather of China and the raising khuskhus output that edatope just can be a small amount of, can not reach the such High Yielding Effect of Australia.At present China does not also filter out the bacterial strain that is suitable for China's weather edaphic condition, therefore the khuskhus high-efficiency nitrogen-fixing root nodule bacterium that screening is suitable for the specific region from the indigenous bacterium of the Different Soil conditions of China column with cultivations of flower or grass flowers and plants have important realistic meaning.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of nodule azotobacter strain RY1 is provided, a kind of root nodule bacterium that can make the efficient dross of khuskhus and reach high yield.
The present invention collects February 27 in 2009 (E10652.483N2347.971H146) in wasteland, farmland, Guangxi province Bose City Tianyang County that a strain dross is large, the root nodule of the khuskhus of riotous growth, separation and purifying obtain a kind of knurl bacterial strain RY1 (Bradyrhizobium sp.) of taking root slowly, be preserved in Hubei China Wuhan University's Chinese Typical Representative culture collection center, Lopa Nationality an ancient woman's ornament mountain, province wuchang, wuhan (CCTCC) on November 13rd, 2009, preserving number is CCTCC NO:M 209265.Its 16sDNA sequence is as shown in SEQ ID NO 1.
Another object of the present invention is to provide the cultural method of described nodule azotobacter strain RY1.The present invention is the root nodule that collects, cut the khuskhus over-ground part with whole root system together with root nodule pack into freshness protection package be placed on the follow-up preservation back-up of low temperature in ice bag from and purifying.Concrete separation and purge process: the root system that takes out khuskhus from ice bag is rinsed well, transfers in the culture dish after sterilization, and root nodule is smashed in sterilization to pieces, dips oyster white juice and draw plate with dilution method progressively on ready YMA solid medium.The plate that pulls is inverted in is placed in incubator (26～30 ℃, dark) cultivation in freshness protection package.Note checking on substratum whether grow bacterium colony, the mark bacterium colony records form and glossiness, is defined as root nodule bacterium in culturing process.
Get rid of after non-root nodule bacterium bacterium colony according to the bacterium colony of above-mentioned mark will grow the time phase difference of single bacterium colony more than 3 days bacterium colony and plate on growthhabit, bacterium colony size, bacterium colony transparency, and light under the inconsistent bacterium colony of refraction, colony colour choose, draw plate and numbering on new solid YMA medium.All adopt aforesaid method to carry out purifying to the new bacterium colony of drawing plate each time until the colony growth form on the same plate, bacterium colony size, bacterium colony transparency, and light under refraction, colony colour unanimously till.Plate by purifying step by step after with purifying carries out putting under after microscopy and tieback in the YMA inclined-plane to be preserved.
Described root nodule bacterium 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.Cultivating 2～4 days colony diameters, to reach 2～4mm be fast-growing Rhizobium, cultivate 5～10 days bacterium colonies just 1mm be living type root nodule bacterium slowly.
Its thalli morphology: root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain beta-hydroxy-butanoic acid, namely reflect strong particle like the cavity or make thalline in the form of link, Gram-negative (G-), without gemma, thalline is single or paired.
A further object of the invention is to provide the microbial inoculum that described nodule azotobacter strain RY1 prepares.Shake the bacteria suspension that bacterium obtains in the liquid YMA medium (bacteria containing amount is greater than 1.0 * 10 by the bacterium on flat board is washed
9Individual), seed dressing, directly tieback is to plant root or mix soil.Also can mix thalline sorbing material such as the peat composed of rotten mosses, vermiculite or perlite etc. and make microbial inoculum use, the preferred 10ml bacterium liquid of blending ratio/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite.
The invention provides the method for the described nodule azotobacter strain RY1 bacterium of khuskhus inoculation.Described nodule azotobacter strain RY1 bacterium prepares liquid bacterial agent and can be prior to seeding waters in the khuskhus root soil with the seed soaking of bacterium liquid, the front immersion plant root of growing seedlings, after transplanting by shaking bacterium, also such as the peat composed of rotten mosses, vermiculite, perlite etc. of bacterium liquid mixings thalline sorbing material can be made to mix before microbial inoculum is transplanted to impose in soil.
The present invention gets beneficial effect: the invention provides a kind of high performance column flowers and plants root nodule bacterium bacteria agent RY1, can be widely used in south China soil, and can make two khuskhus kind TPRC2, TPRC5 reach high yield.Contrast with not connecing the bacterium plant, volume increase is compared volume increase with local other indigenous bacterium and is also reached more than 80% more than 3 times.
Fig. 1 root nodule bacterium RY1 tieback check figure
Fig. 2 root nodule bacterium RY1 bacterium colony figure
Fig. 3 root nodule bacterium RY1 gramstaining picture
Description of drawings
Dross situation after Fig. 4 tieback root fungus RY1
Fig. 5 RYI agarose gel electrophoresis figure
Fig. 6 NCBI comparison chart
Fig. 7 RY1 root nodule bacterium cluster analysis figure
Fig. 8 tieback RY1TPRC2, TPRC5 change of production figure
Fig. 9 RY1 aciduric bacteria liquid OD value
Figure 10 RY1 OD of anti-NaCl value
Figure 11 uses change of production figure after RY1 peat composed of rotten mosses microbial inoculum
Further describe the present invention below in conjunction with specific embodiments and the drawings.
Acquisition and the cultivation of embodiment 1 root nodule bacterium RY1
Collect the root nodule of the khuskhus of a larger riotous growth of strain dross inventor's February 27 in 2009 (E10652.483N2347.971H146) in wasteland, farmland, Guangxi province Bose City Tianyang County, cuts the khuskhus over-ground part whole root system is placed on cryopreservation in ice bag together with the root nodule freshness protection package of packing into.
Embodiment
(1) root system that takes out khuskhus from ice bag rinses under tap water 2～3 times, and root system is rinsed well, with scissors, fresh root complete, root nodule band 2mm that color is dark red is cut, and is with 2 grades of water, that surface washing is clean in culture dish; This process will be guaranteed the complete of root nodule surface.
(2) root nodule of rinsing well is transferred in culture dish after sterilization, after adding alcohol-pickled 3 minutes of 95% (volume by volume concentration), alcohol is poured out, with aseptic water washing 4～5 times, add 0.1% (mass body volume concentrations, 0.1g HgCl
2/ 100ml water) HgCl
2 Aqueous solution sterilization 2～3 minutes is rapidly with HgCl
2Pour out and add sterilized water, shift the Bechtop operation, with aseptic water washing more than 6 times, sterilized water is poured out, choose in the culture dish after root nodule is transferred to sterilization and (can sterilize on flame), with the sterilization after tweezers and transfering loop root nodule is smashed to pieces, dip oyster white juice and rule on ready YMA solid medium.The plate that pulls is inverted in is placed in incubator (28 ± 1 ℃, dark) cultivation in freshness protection package.
The YMA medium formula is as table 1, and regulating pH value is 6.
Table 1YMA (Yeast Mannitol Agar) culture medium prescription (L
-1)
(3) began to check whether grow bacterium colony on substratum at the 3rd day that cultivates, until about the 15th day, bacterium colony is marked records form and glossiness.
Determine whether to be root nodule bacterium from following 3 in culturing process:
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.Cultivating 2～4 days colony diameters, to reach 2～4mm be fast-growing Rhizobium, 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, carry out gramstaining, observes under 100 power microscopes.Root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain beta-hydroxy-butanoic acid, namely reflect strong particle like the cavity or make thalline in the form of link, Gram-negative (G-), without gemma, thalline is single or paired;
C. by isolated root nodule bacterium are received on khuskhus next time at aseptic condition, can dross be root nodule bacterium.
The morphological specificity of root nodule bacterium RY1:
Root nodule bacterium RY1 is a kind of of Bradyrhizobium, 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, after the 6th day, the less single bacterium colony of translucent, little yellow, cement of 1.0～2.0mm appears in finishing touch, sees the bacterium colony of RY1 root nodule bacterium shown in accompanying drawing 2.
The optimum growing condition of RY1 is: 28 ℃ of temperature, pH=6, rotating speed 180r/min.Can use widely Carbon and nitrogen sources, can well grow on the comprehensive extract of various plant origins, better than growth on protein culture medium on YMA medium.
The physiological property of root nodule bacterium RY1
Root nodule bacterium RY1 is purple through gramstaining at microscopically, and to be shaped as rhabdos be Gram-negative bacteria.See the gramstaining of root nodule bacterium RY1 shown in accompanying drawing 3 picture.
(4) get rid of non-root nodule bacterium bacterium colony, according to the bacterium colony of above-mentioned mark will grow the time phase difference of single bacterium colony more than 3 days bacterium colony and plate on growthhabit, bacterium colony size, bacterium colony transparency, and light under the inconsistent bacterium colony of refraction, colony colour choose, draw plate and numbering on new substratum.All adopt aforesaid method to carry out purifying to the new bacterium colony of drawing plate each time until the colony growth form on the same plate, bacterium colony size, bacterium colony transparency, and light under refraction, colony colour unanimously till.Plate by purifying step by step after with purifying carries out putting under after microscopy and tieback in the YMA inclined-plane to be preserved.
(5) tieback checking khuskhus root nodule bacterium
A. the preparation of bacterium liquid
Separation and purification root nodule RY1 is out washed in ready liquid YMA medium with sterilized water, and with sealed membrane sealing, 180r/min shakes the OD value that bacterium was measured bacterium liquid in 3 days afterwards 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) namely get bacterium liquid, can use.Described 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 Stylo seed number soaked 5 minutes in 95% ethanol, after taking out at 0.2% HgCl
2 Middle processing 5 minutes with aseptic water washing 5～10 times, then comes the bacterium of going out, puts well in the culture dish of germination paper, soaks germination paper with the 0.05mmol/L calcium sulphate soln of the bacterium of going out, is placed in the incubator of 28 ℃ dark vernalization 3 days.Treat when seedling grows to 4 centimetres, seedling to be moved in the sand training case of the ready bacterium of going out, stand-by.
C. tieback
Choosing sturdy seedling from sand training case is put in culture dish with step (5) a and prepares to get bacterium liquid immersion 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.Approximately pull up seedling after surrounding and whether check dross, dross is root nodule bacterium.Root nodule RY1 dross after the tieback check is obvious, and provable isolate is pure root nodule bacterium.See the tieback of root nodule bacterium RY1 shown in accompanying drawing 1 assay.
The 16S rDNA sequence order-checking of embodiment 2 root nodule bacterium RY1 and determining of classification
In order to determine the Phylogenetic of root nodule bacterium RY1, the 16SDNA 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.
Upstream primer 35fc:CTKAAGAGTTTGATCMTGGCTCAGATTGAAC;
Downstream primer 1492r:TACGGYTACCTTGTTACGACTT).
Reaction conditions is as follows:
The PCR reaction
Primer：35fc，1492r
PCR recipe：
PCR proCedure
Amplified production assay on 1.0% agarose gel electrophoresis is seen the figure of RYI agarose gel electrophoresis shown in accompanying drawing 5, send Beijing AudioCodes biotech company to check order, and sequencing result is as shown in SEQ ID NO 1.
The sequence results of gained is compared at the state-run bioinformation of U.S. U.S. center (NCBI), find that rhizobium strains RY1 and known bacterial strain Bradyrhizobium sp.PAC40 similarity reach 99%, see NCBI comparison chart shown in accompanying drawing 6.Application software DNAStar and TREECONW carry out cluster to the bacterial strain of surveying, and see the cluster analysis of RY1 root nodule bacterium shown in accompanying drawing 7 figure.By comparison result and dendrogram as can be known RY1 be the new strain of Bradyrhizobium (Bradyrhizobium), called after heat is ground (RY1) No. 1, on November 13rd, 2009 was preserved in Wuhan University's Chinese Typical Representative culture collection center (CCTCC), and preserving number is CCTCCNO:M 209265.
The application experiment of embodiment 3 root nodule bacterium RY1
In order to confirm that RY1 is to the effect of khuskhus, adopt two main khuskhus kinds (TPRC2, TPRC5), with the method for sand training with root nodule RY1 tieback to the khuskhus root, each processes 4 repetitions (processing of bacterium, sand and seedling is tested referring to tieback), not inoculate root nodule as contrast (CK), carry out the tieback contrast.Adopt low nitrogen nutrition liquid to water in whole process.After khuskhus TPRC2, TPRC5 tieback root nodule bacterium RY1, impact sees Table 2 and accompanying drawing 8 on the physical signs of khuskhus.In accompanying drawing 8, left side square column (a) is TPRC5, and the right square column (b) is TPRC2 (in accompanying drawing 8, other histograms for existing root nodule bacterium results of comparison, does not have direct relation with the technology of the present invention, do not do mark).
After tieback root nodule bacterium RY1, the root nodule number of khuskhus TPRC2, TPRC5, root nodule dry weight, plant height, plant fresh weight and nitrogenase activity all increase considerably than the contrast (CK) of correspondence as can be seen from Table 2.
In all physical signs of khuskhus, over-ground part output is to weigh the final index of khuskhus high yield.Can find out output that root nodule bacterium RY1 is applied to TPRC2, TPRC5 all higher than contrast (exceeding more than 3 times than contrast) by accompanying drawing 3, also more high than the output of other bacterial strains.The output of RY1 TPRC2 in the application of TPRC2, TPRC5 is a little more than the output (difference is not remarkable) of TPRC5, and hence one can see that, and root nodule bacterium RY1 is the superior strain that extensively is suitable for TPRC2, TPRC5 khuskhus.
Table 2 root nodule bacterium RY1 tieback after TPRC2, the TPRC5 on the impact of khuskhus physical signs
Research summary by experiment, root nodule bacterium RY1 of the present invention be at present in the sample that the zone of the numerous column with cultivations of flower or grass flowers and plants of China gathers unique one can make two khuskhus principal item TPRC2, TPRC5 can reach the root nodule bacterium of high yield, after the present khuskhus Rhizobium Inoculation of its property list, the strong dross quantity of dip-dye ability is many, the dross volume large, strong stress resistance, can make simultaneously TPRC2, TPRC5 reach high yield.See the dross situation after accompanying drawing 4 tieback root nodule bacterium RY1.When thalline is released in physical environment, harmless to people, animal and plant, do not pollute the environment, increased on the contrary the colony of root nodule bacterium between soil, the nodulation and nitrogen fixation of khuskhus there is the effect of promotion.
The general pH value of soils in south china is low, and khuskhus adapts to southern acid soil except the acidproof system of himself, and root nodule bacterium have also played the effect of balance acid.Find in the resistance experiment, root nodule bacterium can make the pH of substratum rise, and its pH value of faster bacterial strain of growing rises also larger.After each bacterial strain is activated, be inoculated in the YMA liquid nutrient medium, put in constant temperature oscillator and cultivate under 28 ± 1 ℃, treat OD
600Be about 1, drawing respectively 50 μ l bacterial suspension inoculations is 4,4.5,4.8,5,5.5,6,7 (volume is 50ml) to the pH value, put 1 ℃ of 180rpm shaking culture of 28 scholar in 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 processes 3 repetitions.The growing state of RY1 in different PH seen accompanying drawing 9.RY1 can not survive in less than 4 soil at pH value, can grow when PH is between 4.5～4.8 but growing state is not fine, and the growth of root nodule bacterium reaches maximum value when pH value is between 5～6.Greater than 6 the time, growing state descends a little when pH value.
Most ofly can make the root nodule bacterium of the leguminous plants drosses such as pea and trifolium all very sensitive to salt, and high density NaCl can reduce the number of root nodule bacterium in the leguminous plants inoculum.So the salt tolerance of root nodule bacterium is very important to improving their survival in soils and competitiveness.The experiment of salt tolerant resistance adopts the YM liquid nutrient medium that adds NaCl to cultivate root nodule bacterium, and the size of OD value can reflect the situation of bacteria growing speed afterwards with the OD value of spectrophotometric determination bacterium liquid in 3 days.After each bacterial strain is activated, be inoculated in the YM liquid nutrient medium, put in constant temperature oscillator and cultivate under 28 ± 1 ℃, 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 0.30mol/L YMA liquid nutrient medium (pH6.0, volume are 50ml), put 1 ℃ of 180rpm shaking culture of 28 scholar in 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 processes 3 repetitions.The growing state of RY1 in different salt concn seen accompanying drawing 10.RY1 is quite responsive to salt stress, and the growth of bacterium is very faint when in bacterium liquid, salt concn is greater than 0.08mol/L levels off to 0, just at last on the span of 0～0.05mol/L growing state be also sharply to descend.
Described microbial inoculum can carry out Dressing, water in the khuskhus root of just having transplanted seedlings with liquid bacterial agent after shaking bacterium.Also can mix the sorbent materials such as thalline sorbing material such as the peat composed of rotten mosses (10ml bacterium liquid/50g peat composed of rotten mosses), vermiculite (10ml bacterium liquid/15g vermiculite), perlite (10ml bacterium liquid/15g perlite) and make front mixed the imposing in soil of microbial inoculum transplanting.
RY1 peat composed of rotten mosses microbial inoculum is applied in soil the impact on khuskhus output:
Prepare aseptic seed and grow seedlings as above husky training. root nodule bacterium are sucked 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, mensuration OD value (enter=600nm), when guaranteeing Field inoculation, the bacteria containing amount of every seed is greater than 10
9Individual., with cooling standby after 40 minutes 121 ℃ of sterilizations after peat composed of rotten mosses pulverizing (crossing the 2mm sieve aperture).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 fully mixes and allow the peat composed of rotten mosses absorb bacterium liquid.The front microbial inoculum that mixes is mixed of growing seedlings imposes in soil.The residential quarter area is 20m
2, establish four repetitions.Receive sample after three months, it the results are shown in accompanying drawing 11, in accompanying drawing 11 in accompanying drawing 8 the right square column (a) be TPRC5, left side square column (b) is TPRC2.((in accompanying drawing 11, other histograms for existing root nodule bacterium results of comparison, do not have direct relation with the technology of the present invention, do not do mark).
By accompanying drawing 11 can find out used different microbial inoculums in soil after, difference has appearred in khuskhus over-ground part dry weight.After using RY1, the output of two khuskhus kind TPRC2, TPRC5 all is improved, improves more than 2 times with respect to contrast output.
SEQUENCE LISTING
＜110〉Liu Guodao, Dong Rongshu, Huang Yanxia
＜120〉a kind of root nodule azotobacter strain RY 1 bacterial strain and application thereof
<130>
<160>1
<170>PatentIn version 3.5
<210>1
<211>1349
<212>DNA
＜213〉artificial sequence
<400>1
gtcgagcggg cgtagcaata cgtcagcggc agacgggtga gtaacgcgtg ggaacgtacc 60
ttttggttcg gaacaacaca gggaaacttg tgctaatacc ggataagccc ttacggggaa 120
agatttatcg ccgaaagatc ggcccgcgtc tgattagcta gttggtaggg taatggccta 180
ccaaggcgac gatcagtagc tggtctgaga ggatgatcag ccacattggg actgagacac 240
ggcccaaact cctacgggag gcagcagtgg ggaatattgg acaatggggg taaccctgat 300
ccagccatgc cgcgtgagtg atgaaggccc tagggttgta aagctctttt gtgcgggaag 360
ataatgacgg taccgcaaga ataagccccg gctaacttcg tgccagcagc cgcggtaata 420
cgaagggggc tagcgttgct cggaatcact gggcgtaaag ggtgcgtagg cgggtcttta 480
agtcaggggt gaaatcctgg agctcaactc cagaactgcc tttgatactg aagatcttga 540
gttcgggaga ggtgagtgga actgcgagtg tagaggtgaa attcgtagat attcgcaaga 600
acaccagtgg cgaaggcggc tcactggccc gatactgacg ctgaggcacg aaagcgtggg 660
gagcaaacag gattagatac cctggtagtc cacgccgtaa acgatgaatg ccagccgtta 720
gtgggtttac tcactagtgg cgcagctaac gctttaagca ttccgcctgg ggagtacggt 780
cgcaagatta aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 840
taattcgacg caacgcgcag aaccttacca gcccttgaca tgtccaggac cggtcgcaga 900
gatgtgacct tctcttcgga gcctggaaca caggtgctgc atggctgtcg tcagctcgtg 960
tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ccgtccttag ttgctaccat 1020
ttagttgagc actctaagga gactgccggt gataagccgc gaggaaggtg gggatgacgt 1080
caagtcctca tggcccttac gggctgggct acacacgtgc tacaatggcg gtgacaatgg 1140
gatgcgaagg ggtaacccct agcaaatctc aaaaagccgt ctcagttcgg attgggctct 1200
gcaactcgag cccatgaagt tggaatcgct agtaatcgtg gatcagcacg ccacggtgaa 1260
tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagttggtt ttacctgaag 1320
acggtgcgct aacccgcaag ggaggcagc 1349
Claims (8)
1. knurl bacterial strain RY1 (Bradyrhizobium sp.) of taking root slowly is preserved in Chinese Typical Representative culture collection center on November 13rd, 2009, and preserving number is CCTCC NO:M209265.
2. the cultural method of the described knurl bacterial strain RY1 of taking root slowly of claim 1, it is characterized in that on 28 ± 1 ℃ of lower YMA solid mediums streak culture.
3. cultural method according to claim 2, is characterized in that the every liter of substratum that consists of of described YMA solid medium contains N.F,USP MANNITOL 10g, K
2HPO
40.25g, MgSO
47H
2O0.2g, KH
2PO
40.25g, NaCl0.1g, yeast powder 3g, agar 15g.
4. a microbial inoculum, it is characterized in that being obtained by the described knurl bacterial strain RY1 bacterium liquid mixing thalline absorbent preparation of taking root slowly of claim 1.
5. microbial inoculum according to claim 4, is characterized in that described 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 the described knurl bacterial strain RY1 of taking root slowly of khuskhus inoculation claim 1 is characterized in that bacterial strain is washed and is shaking OD after bacterium in liquid nutrient medium and carry out Dressing greater than the bacterium liquid of 0.6 o'clock or water greater than the bacterium liquid of 0.6 o'clock with OD making microbial inoculum in the khuskhus root or with OD greater than bacterium liquid the mixings thalline sorbing material of 0.6 o'clock and mixing before khuskhus is transplanted and impose on soil.
7. method according to claim 6, is characterized in that in described bacterium liquid, bacteria containing amount is 10
9Individual/ml.
8. method according to claim 6, is characterized in that described Dressing is for soaking seed 45 minutes～50 minutes with bacterium liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910214318 CN101781630B (en) | 2009-12-29 | 2009-12-29 | Root nodule azotobacter strain RY1 bacterial strain and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200910214318 CN101781630B (en) | 2009-12-29 | 2009-12-29 | Root nodule azotobacter strain RY1 bacterial strain and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101781630A CN101781630A (en) | 2010-07-21 |
| CN101781630B true CN101781630B (en) | 2013-06-12 |
Family
ID=42521784
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910214318 Expired - Fee Related CN101781630B (en) | 2009-12-29 | 2009-12-29 | Root nodule azotobacter strain RY1 bacterial strain and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101781630B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103131652B (en) * | 2013-02-16 | 2014-09-24 | 哈尔滨工业大学华龙科技发展有限公司 | Rhizobium japonicum culture medium and method for preparing liquid rhizobium japonicum agent by adopting rhizobium japonicum culture medium |
| 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 |
| CN109294962B (en) * | 2018-12-05 | 2020-07-31 | 福建农林大学 | Cassia rhizobium TXN1 and application thereof |
| 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 (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
| 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 CN 200910214318 patent/CN101781630B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
| 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 (2)
| Title |
|---|
| Tatiana Krasova-Wade et al..Diversity of indigeneous bradyrhizobia associated with three cowpea cultivars (Vigna unguiculata (L.) Walp.) grown under limited and favorable water conditions in Senegal (West Africa).《African Journal of Biotechnology》.2003,第2卷(第1期),全文. * |
| 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. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101781630A (en) | 2010-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106987541B (en) | Efficient alfalfa rhizobium with stress resistance and growth promotion performance and application thereof | |
| CN106967652B (en) | Rhizobium for promoting growth of common vetch and application of rhizobium | |
| CN101735969B (en) | Bradyrhizobium sp.RY4 strain and application thereof | |
| CN105062935B (en) | A kind of rhizobium and its application | |
| CN110093299A (en) | A kind of salt tolerant growth-promoting bacteria strain D5-2 and its application | |
| CN101748088B (en) | Bacterial strain of root nodule nitrogen-fixing strain series RY3 and application thereof | |
| CN101824390B (en) | RY2 strain of root nodule nitrogen-fixing bacterial strain system and application thereof | |
| CN101781630B (en) | Root nodule azotobacter strain RY1 bacterial strain and application thereof | |
| CN101781629B (en) | Root nodule azotobacter strain RY5 bacterial strain and application thereof | |
| CN109988738A (en) | A kind of salt tolerant growth-promoting bacteria strain B9 and its application | |
| CN107027519A (en) | The mycorrhiza fungi seeding cultivating method of Panzhihua ferfas | |
| CN110029077A (en) | A kind of salt tolerant growth-promoting bacteria strain Y4 and its application | |
| CN110218670A (en) | One plant of selenium oxidized form rhizobium T3F4 and its application with good colonization ability | |
| Iqbal et al. | Algal biofertilizer | |
| CN111269841B (en) | New endophytic fungus TK815 and application thereof | |
| CN114350559B (en) | Salt-tolerant growth-promoting Liaoning slow rhizobium RY6 strain and application thereof | |
| CN109486711B (en) | Rhizobium AXLQ16 and application thereof | |
| CN111484953A (en) | Bacillus capable of promoting growth and dissolving phosphorus and application thereof | |
| CN105779334B (en) | A kind of bacillus WDGJ11 and its application on raising quality of tobacco | |
| CN109355235B (en) | Rhizobium FAMB126 and application thereof | |
| CN104560797B (en) | Separate the actinomyces bacterium WZW03 bacterial strains obtained and its application in terms of continuous cropping obstacle of watermelon is alleviated | |
| CN109439589B (en) | Rhizobium YZLH133 and application thereof | |
| CN109355234B (en) | Rhizobium YZM0144 and application thereof | |
| CN108342337B (en) | Hafnia yusinia YYBIO-001, preparation method and application thereof, and microbial inoculum | |
| CN117645954B (en) | Paenibacillus mucilaginosus MSSW03 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 | ||
| 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: DONG RONGSHU HUANG YANXIA Effective date: 20130319 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20130319 Address after: 571737 Institute of tropical crop resources, China Academy of Tropical Agricultural Sciences, Hainan, Danzhou, China Applicant after: Resources Institute of Tropical Crop Varieties of Chinese Academy of Tropical Agricultural Sciences Applicant after: Liu Guodao Applicant after: Dong Rongshu Address before: 571737 China Agriculture Academy of tropical agriculture, Hainan, Danzhou Applicant before: Liu Guodao Applicant before: Dong Rongshu Applicant before: Huang Yanxia |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130612 Termination date: 20151229 |
|
| EXPY | Termination of patent right or utility model |