CN101735969A - Bradyrhizobium sp.RY4 strain and application thereof - Google Patents
Bradyrhizobium sp.RY4 strain and application thereof Download PDFInfo
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- CN101735969A CN101735969A CN200910214319A CN200910214319A CN101735969A CN 101735969 A CN101735969 A CN 101735969A CN 200910214319 A CN200910214319 A CN 200910214319A CN 200910214319 A CN200910214319 A CN 200910214319A CN 101735969 A CN101735969 A CN 101735969A
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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:M209268. 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%, nitrogen-free extract 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 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 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.Through separate and purifying to obtain a kind of nodule azotobacter strain be RY4 (Bradyrhizobium sp.RY4), be preserved in Chinese typical culture collection center on November 13rd, 2009, 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 described nodule azotobacter strain is RY4.
Purpose of the present invention is achieved by 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 culture dish 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 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 YMA medium, draw 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, 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 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, 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
-hydroxybutyric acid promptly reflects the particle in strong seemingly cavity or makes thalline in the form of link, Gram-negative (G-), and no gemma, thalline is single or paired.
It is the method for RY4 bacterial strain tieback khuskhus that the present invention provides described 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% (quality volumetric 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 culture dish, 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 sterilization sand and train in the case with the 0.05mmol/L calcium sulphate soln of sterilization;
(3) choose bacterium liquid that sturdy seedling prepares with step (1) from the described husky training case of step (2) 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 described nodule azotobacter strain.The RY4 root nodule bacterium that preserve are activated in the solid plate substratum, and 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.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.Can directly use above-mentioned bacterium liquid liquid bacterial agent, also can be mixed in proportion the thalline sorbing material and make microbial inoculum use, the preferred peat composed of rotten mosses of described thalline sorbing material, vermiculite or perlite etc.; The preferred 10ml bacterium liquid of the described 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 described nodule azotobacter strain is the RY4 bacterium.Described nodule azotobacter strain is the RY4 bacterium by activation, shake bacterium obtains liquid bacterial agent and can dress seed after shaking bacterium 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 made 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, improve and reach 3.9 times, 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 comparison chart
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 concn
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 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.Through separate and purifying to obtain a kind of nodule azotobacter strain be RY4 (Bradyrhizobium sp.RY4), be preserved in Chinese typical culture collection center on November 13rd, 2009, preserving number is CCTCC NO:M 209268.
(1) root system that takes out khuskhus from ice bag washes under tap water and 2～3 times root system is rinsed well, cuts in culture dish 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 culture dish after the sterilization, added after 95% alcohol-pickled 3 minutes, alcohol is poured out,, add 0.1% HgCl with aseptic water washing 4～5 times
2Sterilized 2～3 minutes, 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 tweezers and transfering loop after the sterilization root nodule is smashed to pieces, dipped 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 as 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, 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
-hydroxybutyric acid promptly reflects the particle in strong seemingly cavity or makes thalline in the form of link, Gram-negative (G-), and 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 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, the bacterium on the flat board sticks together into the oyster white band shape mutually 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, can well grow on the comprehensive extract of various plant origins, 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 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 and on new substratum, draw 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, 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 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.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 and be put into the bacterium liquid immersion for preparing with step (5) a in the culture dish 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 determining of classification
In order to determine 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 as follows:
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 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 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 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 RY4 root nodule bacterium shown in the accompanying drawing 7 figure.By comparison result and dendrogram as can be known RY4 be the new strain system of the knurl Pseudomonas (Bradyrhizobium) that takes 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 sand 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), 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 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 the 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.By accompanying drawing 8 as can be seen the root nodule bacterium RY4 output that is applied to TPRC2, TPRC5 all be 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
Research summary by experiment, root nodule bacterium RY4 is the khuskhus rhizobium strains that can form high yield on the relatively poor clayed soil of ventilation property.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.
Described 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 be mixed in proportion the thalline sorbing material and make microbial inoculum mixed imposing in the soil before sowing or transplanting.The preferred peat composed of rotten mosses of described thalline sorbing material, vermiculite or perlite etc.; The preferred 10ml bacterium liquid of the described 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:
Husky as described above training is prepared aseptic seed and is grown seedlings. and 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, mensuration OD value (go into=600nm), the bacteria containing amount of every seed is greater than 10 when guaranteeing the field inoculation
9Individual.It is standby 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 sample after three months, its individual plant hay yield result such as accompanying drawing 9.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 the technology of the present invention in the accompanying drawing 9, do not do mark).
By accompanying drawing 9 as can be known, to have used the output of RY4 root nodule bacterium be 3.9 times and 2.2 times of plant that do not connect bacterium for TPRC2, TPRC5 khuskhus.
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 acid soil South China distributed more widely.In order to determine the adaptation situation of RY4, determine acidproof, the salt tolerance of khuskhus by the 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.By accompanying drawing 10 as can be known, the RY4 root nodule bacterium can form effective bacterium liquid at PH greater than 4.2 o'clock, and 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.
Illustrate that the RY4 root nodule bacterium have wider adaptability 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 concn seen accompanying drawing 11.The RY4 root nodule bacterium grow when salt concn is higher than 0.08mol/L in edatope and have been subjected to 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 (10)
1. a nodule azotobacter strain is RY4 bacterial strain (Bradyrhizobium sp.RY4), is preserved in Chinese typical culture collection center on November 13rd, 2009, and preserving number is CCTCCNO:M209268.
2. the described nodule azotobacter strain of claim 1 is the RY4 bacterial strain, it is characterized in that its 16SDNA sequence is shown in SEQ ID NO 1.
3. the described nodule azotobacter strain of claim 1 is the cultural method of RY4 bacterial strain, it is characterized in that it being to smash to pieces after the root nodule sterilising treatment that will collect to obtain to such an extent that juice is rule to be placed in 28 ± 1 ℃ of incubator dark on the YMA solid medium and cultivated.
4. be the cultural method of RY4 bacterial strain according to the described nodule azotobacter strain of claim 3, it is characterized in that may further comprise the steps:
(1) root system of khuskhus is rinsed well, the root of root nodule band 2mm is cut rinsed well;
(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, the bacterium colony mark is come out to write down form and glossiness at the 3rd day that cultivates;
(4) get rid of non-root nodule bacterium bacterium colony, according to the bacterium colony of 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, the plate behind the purifying carries out putting under in the YMA inclined-plane behind microscopy and the tieback and preserves step by step.
5. be the cultural method of RY4 bacterial strain according to the described nodule azotobacter strain of claim 4, it is characterized in that the described sterilization of step (2) is to adopt 95% alcohol-pickled back aseptic water washing, adds 0.1% HgCl
2Sterilization.
6. be the cultural method of RY4 bacterial strain according to the described nodule azotobacter strain of claim 4, what it is characterized in that described YMA solid medium consists of N.F,USP MANNITOL 10g, K
2HPO
40.25g, MgSO
47H
2O0.2g, KH
2PO
40.25g, NaCl0.1g, yeast powder 3g, agar 15g.
7. method that the described nodule azotobacter strain of claim 1 is a RY4 bacterial strain tieback khuskhus is characterized in that 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, 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) will the khuskhus seed be thrown on the germination paper of sterilizing in the culture dish after the sterilization, soak germination paper, place 28 ± 1 ℃ the dark vernalization of incubator, 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) choose the bacterium liquid immersion back plantation that sturdy seedling prepares with step (1) from the described husky training case of step (2), every seedling adds bacterium liquid 1～2ml.
8. microbial inoculum is characterized in that being is that the bacterium liquid mixing thalline sorbent material of RY4 bacterial strain prepares by the described nodule azotobacter strain of claim 1.
9. described according to Claim 8 microbial inoculum 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.
10. a khuskhus is inoculated the method that described nodule azotobacter strain is the RY4 bacterial strain, it is characterized in that root nodule bacterium activation shaken to dress seed behind the bacterium to handle or water with bacterium liquid making microbial inoculum mixed imposing in the soil before khuskhus is transplanted in the khuskhus root or with bacterium liquid mixings thalline sorbing material.
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