CN101781630A - Root nodule azotobacter strain RY1 bacterial strain and application thereof - Google Patents
Root nodule azotobacter strain RY1 bacterial strain and application thereof Download PDFInfo
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- CN101781630A CN101781630A CN200910214318A CN200910214318A CN101781630A CN 101781630 A CN101781630 A CN 101781630A CN 200910214318 A CN200910214318 A CN 200910214318A CN 200910214318 A CN200910214318 A CN 200910214318A CN 101781630 A CN101781630 A CN 101781630A
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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, 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% 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., 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 becomes the kind of promoting mainly of China south leguminous forage very soon after introduction with the characteristic of its high yield, high-quality and anti-low level management.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 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 Australian Rhizobium Inoculant is based on the efficient bacterial strain that Australian soil and climatope are selected, just can a spot of raising khuskhus output to the weather of China and edatope, can not reach the such high yield effect of Australia.At present China does not also filter out the bacterial strain that is suitable for China's weather edaphic condition, so screen the khuskhus high-efficiency nitrogen-fixing root nodule bacterium that are 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 important practical sense is arranged.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, it is RY1 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 present invention collects February 27 in 2009 (E10652.483N2347.971H146) in wasteland, farmland, Guangxi province Bose City Tianyang County that a strain dross is big, the root nodule of the khuskhus of riotous growth, it is RY1 (Bradyrhizobium sp.RY1) that separation and purifying obtain a kind of nodule azotobacter strain, be preserved in the Chinese typical culture collection center (CCTCC) of Chinese Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University 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 provides the cultural method that described nodule azotobacter strain is 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 the ice bag from and purifying.
Concrete separation and purge process:
Take out the root system of khuskhus and rinse well from ice bag, transfer in the culture dish after the sterilization, root nodule is smashed in sterilization to pieces, dips in to get oyster white juice draw plate with dilution method progressively on ready YMA solid medium.Be inverted in and place incubator (26～30 ℃, dark) cultivation in the freshness protection package drawing a good plate.Note checking on the substratum whether grow bacterium colony in the culturing process, the mark bacterium colony, record form and glossiness are defined as root nodule bacterium.
Get rid of behind the 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 the light inconsistent bacterium colony of refraction, colony colour down choose, on new solid YMA medium, draw plate and 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, and light under refraction, colony colour unanimity till.Undertaken putting under in the YMA inclined-plane behind microscopy and the tieback by the plate after with purifying of purifying step by step and preserve.
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.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.
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, promptly reflect strong like the cavity particle or make thalline in the form of link, Gram-negative (G-), no gemma, thalline is single or paired.
It is the microbial inoculum that RY1 prepares that a further object of the invention provides described nodule azotobacter strain.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 the flat board is washed
9Individual), seed dressing, directly tieback is to plant root or mix soil.For example the peat composed of rotten mosses, vermiculite or perlite etc. are made microbial inoculum use, the preferred 10ml bacterium liquid of the blending ratio/50g peat composed of rotten mosses, 10ml bacterium liquid/15g vermiculite or 10ml bacterium liquid/15g perlite also can to mix the thalline sorbing material.
The invention provides khuskhus and inoculate the method that described nodule azotobacter strain is the RY1 bacterium.To be the RY1 bacterium prepare liquid bacterial agent and can be prior to seeding soak the plant root with the seed soaking of bacterium liquid, before growing seedlings, water in the khuskhus root soil after transplanting by shaking bacterium described nodule azotobacter strain, also can be with bacterium liquid mixings thalline sorbing material mixed imposing in the soil before for example the peat composed of rotten mosses, vermiculite, perlite etc. are made microbial inoculum and transplanted.
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 China's soils in south china, 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.
Description of drawings
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
Dross situation behind 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 the RY1 peat composed of rotten mosses microbial inoculum
Embodiment
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 the big riotous growth of a strain dross inventor's February 27 in 2009 (E10652.483 N23 47.971 H146) in wasteland, farmland, Guangxi province Bose City Tianyang County, 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.
(1) root system that takes out khuskhus from ice bag washes 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 the culture dish after the sterilization, add alcohol-pickled 3 minutes of 95% (volume by volume concentration) after, alcohol is poured out, with aseptic water washing 4～5 times, add 0.1% (quality volumetric concentration, 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 on flame, sterilize), with after the sterilization tweezers and transfering loop root nodule is smashed 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.
The YMA medium prescription is as table 1, and regulating pH value is 6.
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, 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, promptly reflect strong like the cavity particle or make thalline in the form of link, Gram-negative (G-), no gemma, thalline is single or paired;
C. by isolated root nodule bacterium are received on the khuskhus next time at aseptic condition, can dross then be root nodule bacterium.
The morphological specificity of root nodule bacterium RY1:
Root nodule bacterium RY1 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 less single bacterium colony of translucent, little yellow, cement of 1.0～2.0mm appears in finishing touch after the 6th day, sees the bacterium colony of RY1 root nodule bacterium shown in the accompanying drawing 2.
The optimum growing condition of RY1 is: 28 ℃ of temperature, pH=6, rotating speed 180r/min.Can use carbon source and nitrogenous source widely, on the comprehensive extract of various plant origins, can well grow, better on the YMA medium than growth on protein culture 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 the 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 the light inconsistent bacterium colony of refraction, colony colour down choose, on new substratum, draw plate and 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, and light under refraction, colony colour unanimity till.Undertaken putting under in the YMA inclined-plane behind microscopy and the tieback by 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 RY1 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 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 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 culture dish of germination paper, soak germination paper with the 0.05mmol/L calcium sulphate soln of the bacterium of going out, be placed in 28 ℃ the incubator dark vernalization 3 days.When treating seedling length to 4 centimetre seedling is moved in the sand training case of the ready bacterium of going out, stand-by.
C. tieback
From sand training case, choose sturdy seedling be put in the culture dish with step (5) a prepare 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.Pull up seedling after approximately and whether check dross, dross then 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 the 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 phylogeny status 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：
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
PCR?procedure
94 ℃ of pre-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 RYI 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 RY1 and known bacterial strain Bradyrhizobium sp.PAC40 similarity reach 99%, see NCBI comparison chart 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 RY1 root nodule bacterium shown in the accompanying drawing 7 figure.By comparison result and dendrogram as can be known RY1 be the new strain system of the knurl Pseudomonas (Bradyrhizobium) that takes root slowly, called after heat is ground (RY1) No. 1, be preserved in Wuhan University China typical culture collection center (CCTCC) on November 30th, 2009, preserving number is CCTCCNO:M 209265.
The application experiment of embodiment 3 root nodule bacterium RY1
In order to confirm the effect of RY1 to khuskhus, adopt two main khuskhus kinds (TPRC2, TPRC5), with the method for sand training with root nodule RY1 tieback to the khuskhus root, each handles 4 repetitions (processing of bacterium, sand and seedling is tested referring to tieback), not inoculate root nodule is contrast (CK), carries out the tieback contrast.Adopt low nitrogen nutrition liquid to water in the whole process.Influence sees Table 2 and accompanying drawing 8 to the physical signs of khuskhus behind khuskhus TPRC2, the TPRC5 tieback root nodule bacterium RY1.Left side square column (a) is TPRC5 in the accompanying drawing 8, and the right square column (b) is TPRC2 (other histograms does not have direct relation for existing root nodule bacterium results of comparison with the technology of the present invention in the accompanying drawing 8, do not do mark).
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 behind the tieback root nodule bacterium RY1 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.By accompanying drawing 3 as can be seen the root nodule bacterium RY1 output that is applied to TPRC2, TPRC5 all be higher than contrast (comparison is according to exceeding more than 3 times), 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 behind TPRC2, the TPRC5 to the influence of khuskhus physical signs
Research summary by experiment, root nodule bacterium RY1 of the present invention be at present in the sample that gather in the zone of the numerous column with cultivations of flower or grass flowers and plants of China unique one can make two khuskhus principal item TPRC2, TPRC5 can both reach the root nodule bacterium of high yield, the strong dross quantity of dip-dye ability is many behind the present khuskhus inoculation of its property list root nodule bacterium, the dross volume big, strong stress resistance, can make TPRC2, TPRC5 reach high yield simultaneously.See the dross situation behind the accompanying drawing 4 tieback root nodule bacterium RY1.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.
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 that in the resistance experiment root nodule bacterium can make the pH of substratum rise, its pH value of fast more bacterial strain of growing rises also big more.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, drawing 50 μ l bacterial suspension inoculations respectively is 4,4.5,4.8,5,5.5,6,7 (volume is 50ml) to the pH value, 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 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.When pH value greater than 6 the time, growing state descends a little.
Most ofly can make the root nodule bacterium of leguminous plants drosses such as pea and trifolium all very sensitive, and high density NaCl can reduce the number of root nodule bacterium in the leguminous plants inoculum to salt.So the salt tolerance of root nodule bacterium is very important to improving their survival abilities in soil and competitiveness.The experiment of salt tolerant resistance adopts the YM 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 YM 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/L YMA liquid nutrient medium (pH6.0, 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 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 salt concn is greater than 0.08mol/L in the bacterium liquid levels off to 0, just at last on the span of 0～0.05mol/L growing state also be rapid decline.
Described microbial inoculum can after shaking bacterium, dress seed processing, water in the khuskhus root of just having transplanted seedlings with liquid bacterial agent.For example the peat composed of rotten mosses (the 10ml bacterium liquid/50g peat composed of rotten mosses), vermiculite (10ml bacterium liquid/15g vermiculite), perlite sorbent materials such as (10ml bacterium liquid/15g perlite) are made preceding mixed the imposing in the soil of microbial inoculum transplanting also can to mix the thalline sorbing material.
RY1 peat composed of rotten mosses microbial inoculum is applied in the soil influence to khuskhus output:
Prepare aseptic seed and grow seedlings as above husky training. root nodule bacterium are sucked 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, mensuration OD value (go into=600nm), the bacteria containing amount of every seed is greater than 10 when guaranteeing the field inoculation
9Individual., peat composed of rotten mosses pulverizing (crossing the 2mm sieve aperture) back is standby at 40 minutes postcooling of 121 ℃ of sterilizations.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 sample after three months, it the results are shown in accompanying drawing 11, in the accompanying drawing 11 in the accompanying drawing 8 the right square column (a) be TPRC5, left side square column (b) is TPRC2.((other histograms do not have direct relation for existing root nodule bacterium results of comparison with the technology of the present invention in the accompanying drawing 11, do not do mark).
After having used different microbial inoculums as can be seen by accompanying drawing 11 in soil, 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
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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 (9)
1. a root nodule azotobacter strain RY 1 bacterial strain (Bradyrhizobium sp.RY1) is preserved in Chinese typical culture collection center on November 13rd, 2009, and preserving number is CCTCCNO:M 209265.
2. the described root nodule azotobacter strain RY 1 bacterial strain of claim 1 is characterized in that its 16SDNA sequence is shown in SEQ ID NO 1.
3. the cultural method of the described root nodule azotobacter strain RY 1 bacterial strain of claim 1 is characterized in that it being streak culture on 28 ± 1 ℃ of following YMA solid mediums.
4. according to the described cultural method of claim 3, what it is characterized in that described YMA solid medium consists of N.F,USP MANNITOL 10g, K
2HPO
40.25g, MgSO
47H
2O 0.2g, KH
2PO
40.25g, NaCl0.1g, yeast powder 3g, agar 15g.
5. a microbial inoculum is characterized in that it being to be prepared by the described root nodule azotobacter strain RY 1 bacterial strain bacterium of claim 1 liquid mixing thalline sorbent material.
6. according to the described microbial inoculum of claim 5, it 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.
7. a khuskhus is inoculated the method for described root nodule azotobacter strain RY 1 bacterial strain, it is characterized in that bacterial strain washed to dress seed to handle or water with bacterium liquid after liquid nutrient medium is shaking bacterium making microbial inoculum mixed imposing in the soil before khuskhus is transplanted in the khuskhus root or with bacterium liquid mixings thalline sorbing material.
8. method according to claim 7 is characterized in that the every milliliter of bacteria containing amount of concentration 10 of bacterium in the described bacterium liquid
9Individual/ml.
9. method according to claim 7 is characterized in that described seed dressing is treated to usefulness bacterium liquid and soaked seed 45 minutes～50 minutes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103131652A (en) * | 2013-02-16 | 2013-06-05 | 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 |
| CN105950520A (en) * | 2016-07-18 | 2016-09-21 | 武汉市农业科学技术研究院作物科学研究所 | Rhizobium capable of efficiently solubilizing phosphorus and application of rhizobium |
| CN109294962A (en) * | 2018-12-05 | 2019-02-01 | 福建农林大学 | One plant of Cassia rhizobium TXN1 and its application |
| CN114350559A (en) * | 2021-12-29 | 2022-04-15 | 中国热带农业科学院热带作物品种资源研究所 | Salt-tolerant growth-promoting slow-growing rhizobium Liaoning RY6 strain and application thereof |
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| CN100557015C (en) * | 2007-12-07 | 2009-11-04 | 华南农业大学 | A kind of nodule azotobacter strain is BXBL9 and application thereof |
| CN100557014C (en) * | 2007-12-07 | 2009-11-04 | 华南农业大学 | A kind of nodule azotobacter strain is BXYD3 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103131652A (en) * | 2013-02-16 | 2013-06-05 | 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 |
| 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 |
| CN105950520A (en) * | 2016-07-18 | 2016-09-21 | 武汉市农业科学技术研究院作物科学研究所 | Rhizobium capable of efficiently solubilizing phosphorus and application of rhizobium |
| 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 |
| CN109294962A (en) * | 2018-12-05 | 2019-02-01 | 福建农林大学 | One plant of Cassia rhizobium TXN1 and its application |
| CN114350559A (en) * | 2021-12-29 | 2022-04-15 | 中国热带农业科学院热带作物品种资源研究所 | Salt-tolerant growth-promoting slow-growing rhizobium Liaoning RY6 strain 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 |
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