CN101781629A - Root nodule azotobacter strain RY5 bacterial strain and application thereof - Google Patents
Root nodule azotobacter strain RY5 bacterial strain and application thereof Download PDFInfo
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- CN101781629A CN101781629A CN200910214316A CN200910214316A CN101781629A CN 101781629 A CN101781629 A CN 101781629A CN 200910214316 A CN200910214316 A CN 200910214316A CN 200910214316 A CN200910214316 A CN 200910214316A CN 101781629 A CN101781629 A CN 101781629A
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Abstract
The invention discloses a root nodule azotobacter strain RY5 bacterial strain and an application thereof. The bacterial strain (Bradyrhizobium.sp.RY5) was collected in China Center for Type Culture Collection (CCTCC) on November 13th, 2009, and the collection number is CCTCC.No: M.209269. The root nodule azotobacter strain RY5 has strong acid resistance, and can improve the effective nodule number of Stylosanthes guianensis and realize the high yield of the two Stylosanthes guianensis when being inoculated to the Stylosanthes guianensis (TPRC2 and TPRC5). When the thallus is released to the natural environment, the root nodule population among soils is increased, thereby promoting the nodulation and the nitrogen fixation of the Stylosanthes guianensis, and ensuring no harm to human beings, animals and plants and no pollution to 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, Chinese Academy of Tropical Agricultural Sciences drew from CIAT (CIAT), and planted 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 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.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.
The objective of the invention is to overcome the deficiencies in the prior art, it is RY5 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 in February, 2009 in the Liuzhou hundred canopy stock-farmss (N=24 ° 06.120 ', E=109 ° 18.00 ' H=125m) collects strain khuskhus root nodule of survival still after severe winter.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 RY5 (Bradyrhizobiumsp.RY5), be preserved in Chinese typical culture collection center on November 13rd, 2009, preserving number is CCTCC No:M 209269.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 RY5.
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.
The present invention provides the method for described root nodule azotobacter strain RY 5 bacterial strain tieback khuskhus simultaneously, may further comprise the steps:
(1) the root nodule RY5 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 0.1% (quality volumetric concentration, 0.1g HgCl in the ethanol of 95% (volume by volume concentration)
2/ 100ml water) HgCl
2Solution-treated is used aseptic water washing, is thrown on the germination paper of sterilizing in the culture dish, soaks germination paper with the 0.05mmol/L calcium sulphate soln of sterilization, is placed in 28 ℃ the incubator dark vernalization 3 days, treats that centimetre time shift of seedling length to 4 goes in the husky training of the sterilization case;
(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.The dross situation is checked in the back approximately, and dross then is root nodule bacterium.
It is the microbial inoculum that RY5 prepares that a further object of the invention provides described nodule azotobacter strain.The bacterium that preserves is activated on flat board, treat dull and stereotypedly to go up the finishing touch bacterium and bacterium washed after longer and shake the bacteria suspension that bacterium (28 ± 1 ℃, 180 rev/mins) obtains in the liquid YMA liquid nutrient medium (bacteria containing amount is greater than 1.0 * 10
9Individual) seed dressing, directly tieback is to plant root or mix soil.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 RY5 bacterium.To be the RY5 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 invention has the beneficial effects as follows:
RY5 root nodule bacterium provided by the invention grow fine under acidic conditions, and under aseptic husky training condition the applicating liquid microbial inoculum to make the output of TPRC2, TPRC5 be not connect 2.4 times and 3.7 times of bacterium.Output also is respectively not connect 3.1 times and 2.3 times of bacterium under the field soil condition.The quality that RY5 has acid resistance and high yield relatively is suitable for China southern characteristic of acid red soil area.Use the RY5 root nodule bacterium in above-mentioned area and not only can significantly improve khuskhus output and quality, can also change the sour environment of soil.
Description of drawings
Fig. 1 root nodule bacterium RY5 tieback check figure
Fig. 2 root nodule bacterium RY5 bacterium colony figure
Fig. 3 root nodule bacterium RY5 gramstaining picture
TPRC2, two kind khuskhus of TPRC5 growing state behind Fig. 4 tieback root fungus RY5
Fig. 5 RY5 agarose gel electrophoresis figure
Fig. 6 NCBI comparison chart
Fig. 7 RY5 root nodule bacterium cluster analysis figure
Fig. 8 tieback RY5TPRC2, TPRC5 fresh weight figure
Fig. 9 tieback RY5TPRC2, TPRC5 dry weight figure
The growth curve of Figure 10 RY5 root nodule bacterium under different acidic conditions
Figure 11 uses TPRC2, TPRC5 dry weight figure after the RY5 peat composed of rotten mosses microbial inoculum
The growing state of Figure 12 RY5 in different salt concn
Embodiment
Further describe the present invention below in conjunction with specific embodiments and the drawings.
Acquisition and the cultivation of example 1 root nodule bacterium RY5
The inventor in February, 2009 in the Liuzhou hundred canopy stock-farmss (N=24 ° 06.120 ', E=109 ° 18.00 ' H=125m) collects strain khuskhus root nodule of survival still after severe winter.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 RY5 (Bradyrhizobium sp.RY5), be preserved in Chinese typical culture collection center on November 13rd, 2009, preserving number is CCTCC NO:M 209269.
(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 5 minutes, alcohol is poured out,, add 0.1% (quality volumetric concentration, 0.1g HgCl with aseptic water washing 4～5 times
2/ 100ml water) HgCl
2Solution 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 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 1YMA (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 RY5:
Root nodule bacterium RY5 is a kind of of knurl Pseudomonas of taking root slowly, cultivate the vestige that is coated with along the first stroke after 50 hours 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 bacterium colony circle of 1.0～2.0mm appears in finishing touch after the 5th day, the edge is complete, smooth surface, protruding globule shape, the water white transparency degree is high, and viscous secretion is less.See the bacterium colony of RY5 root nodule bacterium shown in the accompanying drawing 2.
RY5 number optimum growing condition is: 28 ℃ of temperature, pH=4.8～6.0, 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 RY5:
Root nodule bacterium RY5 is purple through gramstaining at microscopically, is shaped as little rod-short body, and size is 0.5～0.9 μ m * 1.2～3.0 μ m, and cell is single or paired, and Chang Kejian arranges in groups.Be Gram-negative bacteria.See the gramstaining of root nodule bacterium RY5 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 RY5 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 7.
B. the preparation of aseptic seedling
Selected khuskhus seed number grain soaked 5 minutes in 95% ethanol, took out the back at 0.1% 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 RY5 dross after the tieback check is obvious, and provable isolate is pure root nodule bacterium.See the tieback of root nodule bacterium RY5 shown in the accompanying drawing 1 assay.
The 16S rDNA sequence order-checking of embodiment 2 root nodule bacterium RY5 and determining of classification
In order to determine the phylogeny status of root nodule bacterium RY5, the 16SDNA series of the bacterial strain that obtains checks order to separate.At first utilize the test kit of omega company carry out total DNA extraction (this method is not conventional method, with test kit extract be a kind of efficiently, method easily, have a few high slightly but compare cost with ordinary method), 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
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
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 RY5 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 RY5 and known strains A Y628222 Bradyrhizobium SP.PAV40 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 RY5 root nodule bacterium shown in the accompanying drawing 7 figure.By comparison result and dendrogram as can be known RY5 be the new strain system of the knurl Pseudomonas (Bradyrhizobium) that takes root slowly, called after heat is ground (RY5) No. 5.
The application experiment of embodiment 3 root nodule bacterium RY5
In order to confirm the effect of RY5 to khuskhus, with two khuskhus kinds (TPRC2, TPRC5) with the method for husky training with root nodule RY5 tieback to the khuskhus root, each handles 4 repetitions (processing of bacterium, sand and seedling is tested referring to tieback) is contrast (CK) not inoculate root nodule, carries out the tieback contrast.Adopt low nitrogen nutrition liquid to water in the whole process.Influence sees Table 2 and accompanying drawing 8, accompanying drawing 9 to the physical signs of khuskhus behind khuskhus TPRC2, the TPRC5 tieback root nodule bacterium RY2.Left side square column (a) is TPRC2 in accompanying drawing 8 and 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 accompanying drawing 8,9 and the accompanying drawing 11, do not do mark)
By table 2, accompanying drawing 8 and accompanying drawing 9 as can be seen behind the tieback root nodule bacterium RY5 root nodule number, root nodule dry weight, plant height, plant fresh weight and the nitrogenase activity of khuskhus TPRC2, TPRC5 all increase considerably than the contrast (CK) of correspondence, it is extremely remarkable that difference reaches.Root nodule bacterium RY5 tieback can improve the dross number of khuskhus behind khuskhus TPRC2 and the TPRC5, makes the khuskhus of two kinds can both reach high yield.See the dross situation behind the accompanying drawing 4 tieback root fungus RY5.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.
Table 2 root nodule bacterium RY5 tieback behind TPRC2, the TPRC5 to the influence of khuskhus physical signs
Described microbial inoculum is that bacterium is shaken in the bacterium activation back of preserving in the liquid YMA medium, treats that bacterial concentration reaches 1 * 10
9Individual/as can to use in order to make microbial inoculum during ml.With the bacterium that just shakes out with the sterilized water dilution or directly be used for soaking the khuskhus seed 60 minutes, khuskhus for the plantation of transplanting seedlings soaks root the seedling that brings out under the aseptic condition more than 30 minutes before plantation in above-mentioned bacterium liquid, also seed can be planted behind big Tanaka to water above-mentioned bacterium liquid in the shoot root portion soil (every young plant root waters 5～10ml).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.
RY5 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 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.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 single plant yield result such as accompanying drawing 11.Left side square column (a) is TPRC2 in the accompanying drawing 11, and left side square column (b) is TPRC5.In the field production khuskhus, use the output that makes TPRC2 and TPRC5 after the RY5 Rhizobium Inoculant and be the output that do not connect root nodule bacterium (CK) 3.1 times and 2.1 times.
The adaptation experiment of embodiment 5 root nodule bacterium RY5 to soil
Root nodule bacterium, edatope and plant are interactional individual system, and having only unites the three takes all factors into consideration the high yield that just can reach real and efficient.In order to determine the adaptation situation of RY5, determine acidproof, the salt tolerance of khuskhus by the following method to soil major influence factors (acid-basicity, and saline alkali).For using RY5 khuskhus root nodule bacterium that preferred technical support is provided from now on more scientifically and rationally.
After bacterial strain RY5 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 ℃ 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.RY5 can grow faster in pH value is 4.5～6 environment and form effective bacterium liquid, is that 4.5 growths are best at pH value particularly.See the growth curve chart of accompanying drawing 10RY5 under the different PH condition.The pH value of raising bacterium liquid that can be very fast in the process of growth of bacterium; So RY5 is applied to the double effects that makes the khuskhus volume increase and improve soil acidity is arranged in the southern characteristic of acid red soil of China.Root nodule bacterium RY5 tieback can improve the dross number of khuskhus behind khuskhus TPRC2 and the TPRC5, makes the khuskhus of two kinds can both reach high yield.See the dross situation behind the accompanying drawing 4 tieback root fungus RY5.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 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/L YMA liquid nutrient medium (the pH value is 5.5, volume be 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 12.The RY5 root nodule bacterium grow when salt concn is higher than 0.09mol/L in edatope and have been subjected to very big inhibition, can not form concentration greater than 1 * 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 * 10 during for 0.05mol/L
9Effective bacterium liquid of individual/ml.
SEQUENCE?LISTING
＜110〉Liu Guodao, Dong Rongshu, Huang Yanxia
＜120〉a kind of root nodule azotobacter strain RY 5 bacterial strain and application thereof
<130>
<160>1
<170>PatentIn?version?3.3
<210>1
<211>1374
<212>DNA
＜213〉artificial sequence
<400>1
cttaacacat?gcaagtcgag?cgggcgtagc?aatacgtcag?cggcagacgg?gtgagtaacg 60
cgtgggaacg?taccttttgg?ttcggaacaa?cacagggaaa?cttgtgctaa?taccggataa 120
gcccttacgg?ggaaagattt?atcgccgaaa?gatcggcccg?cgtctgatta?gctagttggt 180
agggtaatgg?cctaccaagg?cgacgatcag?tagctggtct?gagaggatga?tcagccacat 240
tgggactgag?acacggccca?aactcctacg?ggaggcagca?gtggggaata?ttggacaatg 300
ggggcaaccc?tgatccagcc?atgccgcgtg?agtgatgaag?gccctagggt?tgtaaagctc 360
ttttgtgcgg?gaagataatg?acggtaccgc?aagaataagc?cccggctaac?ttcgtgccag 420
cagccgcggt?aatacgaagg?gggctagcgt?tgctcggaat?cactgggcgt?aaagggtgcg 480
taggcgggtc?tttaagtcag?gggtgaaatc?ctggagctca?actccagaac?tgcctttgat 540
actgaagatc?ttgagtccgg?gagaggtgag?tggaactgcg?agtgtagagg?tgaaattcgt 600
agatattcgc?aagaacacca?gtggcgaagg?cggctcactg?gcccggtact?gacgctgagg 660
cacgaaagcg?tggggagcaa?acaggattag?ataccctggt?agtccacgcc?gtaaacgatg 720
aatgccagcc?gttagtgggt?ttactcacta?gtggcgcagc?taacgcttta?agcattccgc 780
ctggggagta?cggtcgcaag?attaaaactc?aaaggaattg?acgggggccc?gcacaagcgg 840
tggagcatgt?ggtttaattc?gacgcaacgc?gcagaacctt?accagccctt?gacatgtcca 900
ggaccggtcg?cagagacgtg?accttctctt?cggagcctgg?aacacaggtg?ctgcatggct 960
gtcgtcagct?cgtgtcgtga?gatgttgggt?taagtcccgc?aacgagcgca?acccccgtcc 1020
ttagttgcta?ccatttagtt?gagcactcta?aggagactgc?cggtgataag?ccgcgaggaa 1080
ggtggggatg?acgtcaagtc?ctcatggccc?ttacgggctg?ggctacacac?gtgctacaat 1140
ggcggtgaca?atgggatgcg?aaggggtaac?ccctagcaaa?tctcaaaaag?ccgtctcagt 1200
tcggattggg?ctctgcaact?cgagcccatg?aagttggaat?cgctagtaat?cgtggatcag 1260
cacgccacgg?tgaatacgtt?cccgggcctt?gtacacaccg?cccgtcacac?catgggagtt 1320
ggttttacct?gaagacggtg?cgctaacccg?caagggaggc?agccggccac?ggta 1374
Claims (10)
1. a root nodule azotobacter strain RY 5 bacterial strain (Bradyrhizobium sp.RY5) is preserved in Chinese typical culture collection center on November 13rd, 2009, and preserving number is CCTCCNo:M 209269.
2. the described root nodule azotobacter strain RY 5 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 5 bacterial strain of claim 1 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 inverted in 28 ± 1 ℃ of incubator dark after ruling and cultivates on the YMA solid medium.
4. according to the cultural method of the described root nodule azotobacter strain RY 5 bacterial 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 ℃ 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. according to the cultural method of the described root nodule azotobacter strain RY 5 bacterial 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. according to the cultural method of the described root nodule azotobacter strain RY 5 bacterial 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. the method for the described root nodule azotobacter strain RY 5 bacterial strain tieback of claim 1 khuskhus is characterized in that may further comprise the steps:
(1) the root nodule RY5 that separation and purification is come out washes in the liquid YMA medium with sterilized water, 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) 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. a microbial inoculum is characterized in that it being to be prepared by the described root nodule azotobacter strain RY 5 bacterial strain bacterium of claim 1 liquid mixing thalline sorbent material.
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 for described root nodule azotobacter strain RY 5 bacterial strain, it is characterized in that the bacterial strain activation, dresses seed to handle or water in the khuskhus root soil or with bacterium liquid mixings thalline sorbing material with bacterium liquid after shaking table shakes bacterium and make microbial inoculum mixed imposing in the soil before khuskhus is transplanted.
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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 |
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| CN108085272A (en) * | 2017-12-04 | 2018-05-29 | 康泰民生环境科学研究院（北京）有限公司 | Rhizobium and its application and microbial inoculum and the method for repairing rare-earth tailing soil or silicon ore deposit tailing waste material |
| CN108085272B (en) * | 2017-12-04 | 2019-03-22 | 康泰民生环境科学研究院（北京）有限公司 | Rhizobium and its application and microbial inoculum and the method for repairing rare-earth tailing soil or silicon mine tailing waste material |
| WO2019109822A1 (en) * | 2017-12-04 | 2019-06-13 | 康泰民生环境科学研究院（北京）有限公司 | Rhizobium and use and bacterial preparation thereof, and method for restoring rare-earth tailing soil or silica ore tailing waste |
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| 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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