CN101781629B - 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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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, now spread in each province, south China (district).Khuskhus contains crude protein 16.0～20.0% in the dry-matter of full-bloom stage, crude fat 1.8～2.5% is rented fiber 17.0～19.0%, nitrogen-free extract 38.0～44.0%, and coarse ash 8.0～10.0% is a kind of good tropical leguminous forage.(district) economized in southern china such as Guangdong, Guangxi etc., will plant between khuskhus and fruit tree, and except being used for forage grass, the effect that also can play covering, conserves water and soil and improve the soil and foster and apply fertilizer.If with mixed seeding such as graminous pasture such as Herba Setariae Viridis, can build up good artificial pasture and be used for herding.
Khuskhus selects usually that draining is good, soil layer is deep, soil property husky earth or loam plantation preferably.But in lean soil, the khuskhus seedling can not form root nodule, or only has some to be infected the ineffetive nodulation that forms by indigenous root nodule, and leguminous plants knot kind of root nodule bacterium can increase output 15～50% on barren soil.For improving khuskhus dross rate, the fixed nitrogen loam should be used the seed dressing of khuskhus root nodule bacterium, can effectively improve the dross rate of khuskhus.Therefore the screening root nodule bacterium that can make the efficient dross of khuskhus and reach high yield have great importance.
Khuskhus with the characteristic of its high yield, high-quality and anti-low level management become me very soon after introduction the south China leguminous forage promote mainly kind.At the beginning of khuskhus is introduced due to planting site soil in the content of khuskhus original inhabitants bacterium extremely low, khuskhus is nodulation and nitrogen fixation well, the main fertilising acquisition high yield that relies on.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, therefore the khuskhus high-efficiency nitrogen-fixing root nodule bacterium that screening is suitable for the specific region from the indigenous bacterium of the Different Soil conditions of China column with cultivations of flower or grass flowers and plants have important realistic meaning.
The objective of the invention is to overcome the deficiencies in the prior art, a kind of nodule azotobacter strain RY5 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 ice bag together with the root nodule freshness protection package of packing into.Obtain a kind of knurl bacterial strain RY5 (Bradyrhizobium sp.) of taking root slowly through separation and purifying, be preserved in Wuhan University's Chinese Typical Representative culture collection center, Lopa Nationality an ancient woman's ornament mountain, Hubei China province wuchang, wuhan on November 13rd, 2009, preserving number is CCTCC No:M 209269.Its 16sDNA sequence is as shown in SEQ ID NO 1.
Another object of the present invention is to provide the cultural method of described nodule azotobacter strain 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 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 comprise the following steps:
(1) root system of khuskhus is rinsed well, cut in culture dish the root of root nodule band 2mm with 2 grades of water, that surface washing is clean;
(2) with smashing to pieces after the sterilization of the root nodule rinsed well, dip oyster white juice and rule on the YMA solid medium, the plate that pulls is inverted in is placed in 28 ± 1 ℃ of incubator dark in freshness protection package and cultivates;
(3) began to check whether grow bacterium colony on substratum at the 3rd day that cultivates, until about the 15th day, bacterium colony is marked records form and glossiness;
(4) get rid of after non-root nodule bacterium bacterium colony according to the bacterium colony of above-mentioned mark will grow the time phase difference of single bacterium colony more than 3 days bacterium colony and plate on growthhabit, bacterium colony size, bacterium colony transparency, and light under the inconsistent bacterium colony of refraction, colony colour choose, draw plate and also number on new YMA medium.All adopt aforesaid method to carry out purifying to the new bacterium colony of drawing plate each time until the colony growth form on the same plate, bacterium colony size, bacterium colony transparency, and light under refraction, colony colour unanimously till.Plate by purifying step by step after with purifying carries out putting under after microscopy and tieback in the YMA inclined-plane to be preserved.
Described 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.Cultivating 2～4 days colony diameters, to reach 2～4mm be fast-growing Rhizobium, cultivate 5～10 days bacterium colonies just 1mm be living type root nodule bacterium slowly.
Thalli morphology: mark is tentatively confirmed as the root nodule bacterium bacterium colony, and therefrom picking lawn smear, carry out gramstaining, observes under 100 power microscopes.Root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain
-hydroxybutyric acid namely reflects the particle in strong seemingly cavity or makes thalline in the form of link, Gram-negative (G-), and without 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, comprises the following steps:
(1) separation and purification root nodule RY5 is out washed in the liquid YMA medium with sterilized water, with sealed membrane sealing, 180r/min shakes the OD value that bacterium was measured bacterium liquid in 3 days afterwards in shaking table, gets bacterium liquid greater than 0.6 the time as OD;
(2) Stylo seed is soaked in the ethanol of 95% (volume by volume concentration) after with 0.1% (mass body volume concentrations, 0.1g HgCl
2/ 100ml water) HgCl
2Solution-treated is used aseptic water washing, is thrown on the germination paper of sterilizing in culture dish, soaks germination paper with the 0.05mmol/L calcium sulphate soln of sterilization, is placed in the incubator of 28 ℃ dark vernalization 3 days, treats that seedling grows to 4 centimetres of time shifts and enters in the husky training of sterilization case;
(3) choosing sturdy seedling from the described husky training case of step (2) plants after 15 minutes in the flowerpot of filling sterilization sand with the bacterium liquid immersion that step (1) prepares, every seedling adds bacterium liquid l～2ml, guarantees that the bacterium amount that connects of every seedling reaches 1.0 * 10
9More than individual.Approximately check the dross situation after surrounding, dross is root nodule bacterium.
A further object of the invention is to provide the microbial inoculum that described nodule azotobacter strain RY5 prepares.The bacterium that preserves is activated on flat board, after dull and stereotyped upper finishing touch bacterium is longer, bacterium is washed and shake the bacteria suspension that bacterium (28 ± 1 ℃, 180 rev/mins) obtains in 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 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 is to provide the method for the described nodule azotobacter strain RY5 bacterium of khuskhus inoculation.Described nodule azotobacter strain RY5 bacterium prepares liquid bacterial agent and can be prior to seeding waters in the khuskhus root soil with the seed soaking of bacterium liquid, the front immersion plant root of growing seedlings, after transplanting by shaking bacterium, also such as the peat composed of rotten mosses, vermiculite, perlite etc. of bacterium liquid mixings thalline sorbing material can be made to mix before microbial inoculum is transplanted to impose in soil.
The 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 is respectively also not connect 3.1 times and 2.3 times of bacterium under the field soil condition.The quality that RY5 has acid resistance and a high yield relatively is suitable for the characteristic of acid red soil area of south China.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.
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 growing states of TPRC5 after 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 RY5 peat composed of rotten mosses microbial inoculum
The growing state of Figure 12 RY5 in different salt concn
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 ice bag together with the root nodule freshness protection package of packing into.Through separating and purifying obtains a kind of knurl bacterial strain RY5 (Bradyrhizobium sp.) of taking root slowly, be preserved in Chinese Typical Representative culture collection center on November 13rd, 2009, preserving number is CCTCC NO:M 209269.
(1) root system that takes out khuskhus from ice bag rinses under tap water to be rinsed root system well 2～3 times, 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 culture dish after sterilization, added after 95% alcohol-pickled 5 minutes, alcohol is poured out, with aseptic water washing 4～5 times, add 0.1% (mass body volume concentrations, 0.1g HgCl
2/ 100ml water) HgCl
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 sterilize on flame), with tweezers and transfering loop after sterilization, root nodule is smashed to pieces, dipped oyster white juice and rule on ready YMA solid medium.The plate that pulls is inverted in is placed in incubator (28 ± 1 ℃, dark) cultivation in freshness protection package.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 substratum at the 3rd day that cultivates, until about the 15th day, bacterium colony is marked records form and glossiness.
Determine whether to be root nodule bacterium from following 3 in culturing process:
A. colonial morphology: the translucent or White-opalescent point-like thing later stage bacterium colony of root nodule bacterium early growth period bacterium colony water sample be circle, oyster white, translucent, neat in edge, cement more or less.Cultivating 2～4 days colony diameters, to reach 2～4mm be fast-growing Rhizobium, cultivate 5～10 days bacterium colonies just 1mm be living type root nodule bacterium slowly.
B. thalli morphology: mark is tentatively confirmed as the root nodule bacterium bacterium colony, and therefrom picking lawn smear, carry out gramstaining, observes under 100 power microscopes.Root nodule bacterium are the dialister bacterium of (0.5～0.9) * (1.2～0.3) μ m.In often contain
-hydroxybutyric acid namely reflects the particle in strong seemingly cavity or makes thalline in the form of link, Gram-negative (G-), and without gemma, thalline is single or paired.
C. by isolated root nodule bacterium are received on khuskhus next time at aseptic condition, can dross be root nodule bacterium.
The morphological specificity of root nodule bacterium RY5:
Root nodule bacterium RY5 is a kind of of Bradyrhizobium, 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, after the 5th day, the bacterium colony circle of 1.0～2.0mm appears in finishing touch, 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 accompanying drawing 2.
The optimum growing condition of No. RY5 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 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 accompanying drawing 3 picture.
(4) get rid of after non-root nodule bacterium bacterium colony according to the bacterium colony of above-mentioned mark will grow the time phase difference of single bacterium colony more than 3 days bacterium colony and plate on growthhabit, bacterium colony size, bacterium colony transparency, and light under the inconsistent bacterium colony of refraction, colony colour choose and draw plate also number on new substratum.All adopt aforesaid method to carry out purifying to the new bacterium colony of drawing plate each time until the colony growth form on the same plate, bacterium colony size, bacterium colony transparency, and light under refraction, colony colour unanimously till.Plate by purifying step by step after with purifying carries out putting under after microscopy and tieback in the YMA inclined-plane to be preserved.
(5) tieback checking khuskhus root nodule bacterium
A. the preparation of bacterium liquid
Separation and purification root nodule RY5 is out washed in ready liquid YMA medium with sterilized water, and with sealed membrane sealing, 180r/min shakes the OD value that bacterium was measured bacterium liquid in 3 days afterwards in shaking table, and (every ML bacterium liquid bacteria containing amount is more than 1.0 * 10 greater than 0.6 the time as OD
9Individual) namely get bacterium liquid, can 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 Stylo seed number soaked 5 minutes in 95% ethanol, after taking out at 0.1% HgCl
2 Middle processing 5 minutes with aseptic water washing 5～10 times, then comes the bacterium of going out, puts well in the culture dish of germination paper, soaks germination paper with the 0.05mmol/L calcium sulphate soln of the bacterium of going out, is placed in the incubator of 28 ℃ dark vernalization 3 days.Treat when seedling grows to 4 centimetres, seedling to be moved in the sand training case of the ready bacterium of going out, stand-by.
C. tieback
Choose sturdy seedling and be put into the bacterium liquid immersion for preparing with step (5) a in culture dish 15 minutes from sand training case, with tweezers, seedling is planted in the small flower of filling sterilization sand, the shape that assumes diamond in shape in every basin 4 strains of planting, every seedling adds bacterium liquid 1～2ml, guarantees that the bacterium amount that connects of every naked seedling reaches 1.0 * 10
9More than individual.Approximately pull up seedling after surrounding and whether check dross, dross is root nodule bacterium.Root nodule 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 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 Phylogenetic of root nodule bacterium RY5, the 16SDNA series of the bacterial strain that obtains checks order to separate.At first (this method is not conventional method to utilize the test kit of omega company to carry out the extraction of total DNA, with test kit extract be a kind of efficiently, method easily, but compare cost with ordinary method a bit slightly high), then 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
Amplified production assay on 1.0% agarose gel electrophoresis is seen the figure of RY5 agarose gel electrophoresis shown in accompanying drawing 5, send Beijing AudioCodes biotech company to check order, and sequencing result is as shown in SEQ ID NO 1.
The sequence results of gained is compared at the state-run bioinformation of U.S. U.S. center (NCBI), find that rhizobium strains RY5 and known strains A Y628222Bradyrhizobium SP.PAV40 similarity reach 99%, see NCBI comparison chart shown in accompanying drawing 6.Application software DNAStar and TREECONW carry out cluster to the bacterial strain of surveying, and see the cluster analysis of RY5 root nodule bacterium shown in accompanying drawing 7 figure.By comparison result and dendrogram as can be known RY5 be the new strain of Bradyrhizobium (Bradyrhizobium), called after heat is ground (RY5) No. 5.
The application experiment of embodiment 3 root nodule bacterium RY5
In order to confirm that RY5 is to the effect of khuskhus, with two khuskhus kinds (TPRC2, TPRC5) with the method for husky training with root nodule RY5 tieback to the khuskhus root, each processes 4 repetitions (processing of bacterium, sand and seedling is tested referring to tieback) not inoculate root nodule as contrast (CK), carries out the tieback contrast.Adopt low nitrogen nutrition liquid to water in whole process.After khuskhus TPRC2, TPRC5 tieback root nodule bacterium RY2, impact sees Table 2 and accompanying drawing 8, accompanying drawing 9 on the physical signs of khuskhus.In accompanying drawing 8 and accompanying drawing 9, left side square column (a) is TPRC2, and left side square column (b) is TPRC5.(accompanying drawing 8,9 and accompanying drawing 11 in other histograms for existing root nodule bacterium results of comparison, there is no direct relation with the technology of the present invention, do not do mark)
Can find out that by table 2, accompanying drawing 8 and accompanying drawing 9 root nodule number, root nodule dry weight, plant height, plant fresh weight and the nitrogenase activity of khuskhus TPRC2, TPRC5 all increases considerably than the contrast (CK) of correspondence after tieback root nodule bacterium RY5, it is extremely remarkable that difference reaches.Root nodule bacterium RY5 tieback can improve the dross number of khuskhus after khuskhus TPRC2 and the TPRC5, makes the khuskhus of two kinds can reach high yield.See the dross situation after accompanying drawing 4 tieback root fungus RY5.When thalline is released in physical environment, harmless to people, animal and plant, do not pollute the environment, increased on the contrary the colony of root nodule bacterium between soil, the nodulation and nitrogen fixation of khuskhus there is the effect of promotion.
Table 2 root nodule bacterium RY5 tieback after TPRC2, the TPRC5 on the impact of khuskhus physical signs
Described microbial inoculum is to shake bacterium after the bacterium activation of will preserve 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.The bacterium that just shakes out is diluted or directly was used for soaking Stylo seed 60 minutes with sterilized water, khuskhus for the plantation of transplanting seedlings soaks with root the seedling that brings out under aseptic condition more than 30 minutes before plantation in above-mentioned bacterium liquid, also seed can be planted after large Tanaka to water above-mentioned bacterium liquid in the shoot root section 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 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 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 soil the impact on khuskhus output:
Prepare aseptic seed and grow seedlings as aforementioned husky training. root nodule bacterium are sucked triangular flask 28 ℃ from solid medium cultivated 96 hours, with sterilized water wash-out thalline, and dilute with sterilized water, with the whirlpool even thalline of device that turns round and round, mensuration OD value (enter=600nm), when guaranteeing Field inoculation, the bacteria containing amount of every seed is greater than 10
9Individual.With cooling standby after 40 minutes 121 ℃ of sterilizations after peat composed of rotten mosses pulverizing (crossing the 2mm sieve aperture).Prior to seeding with different bacterium liquid in 10ml bacterium liquid: the ratio of the 50g peat composed of rotten mosses is poured in the peat composed of rotten mosses, and fully mixes and allow the peat composed of rotten mosses absorb bacterium liquid.The front microbial inoculum that mixes is mixed of growing seedlings imposes in soil.The residential quarter area is 20m
2, establish four repetitions.Receive sample after three months, its single plant yield result such as accompanying drawing 11.In accompanying drawing 11, left side square column (a) is TPRC2, 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 to soil of embodiment 5 root nodule bacterium RY5
Root nodule bacterium, edatope and plant are interactional individual system, and only having unites the three considers the high yield that just can reach real and efficient.In order to determine that RY5 to the adaptation situation of soil major influence factors (acid-basicity, and saline alkali), determines acidproof, the salt tolerance of khuskhus by the following method.For using more scientifically and rationally from now on RY5 khuskhus root nodule bacterium that preferred technical support is provided.
After bacterial strain RY5 activation, wash in the YMA liquid nutrient medium, cultivate in constant-temperature table (28 ± 1 ℃, 180r/min) is lower, treat OD
600Be about 1, drawing respectively 50 μ l bacterial suspension inoculations is in 4,4.5,4.8,5,5.5,6,7 (volume is 50ml) YMA liquid nutrient medium to the pH value, put 1 ℃ of 180rpm shaking culture of 28 scholar in constant temperature oscillator, detect the growing state of bacterium with the variation of nutrient solution opacity, cultivate and use spectrophotometer measurement OD after 72 hours
600Each processes 3 repetitions.RY5 can grow faster in pH value is 4.5～6 environment and form effective bacterium liquid, is particularly that 4.5 growths are best at pH value.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; Therefore RY5 is applied to, the double effects that makes the khuskhus volume increase and improve soil acidity is arranged in the acid red soil of south China of China.Root nodule bacterium RY5 tieback can improve the dross number of khuskhus after khuskhus TPRC2 and the TPRC5, makes the khuskhus of two kinds can reach high yield.See the dross situation after accompanying drawing 4 tieback root fungus RY5.When thalline is released in physical environment, harmless to people, animal and plant, do not pollute the environment, increased on the contrary the colony of root nodule bacterium between soil, the nodulation and nitrogen fixation of khuskhus there is the effect of promotion.
The experiment of salt tolerant resistance adopts the YMA liquid nutrient medium that adds NaCl to cultivate root nodule bacterium, and the size of OD value can reflect the situation of bacteria growing speed afterwards with the OD value of spectrophotometric determination bacterium liquid in 3 days.After each bacterial strain is activated, be inoculated in the YMA liquid nutrient medium, put in constant temperature oscillator and cultivate under 28 ± 1 ℃, treat OD
600Be about 1, draw respectively 50 μ l bacterial suspension inoculations to NaCl concentration be 0,0.05,0.08,0.10,0.15,0.20,0.25, in 0.30mol/L YMA liquid nutrient medium (the pH value is 5.5, volume be 50ml), put 1 ℃ of 180rpm shaking culture of 28 scholar in constant temperature oscillator, detect the growing state of bacterium with the variation of nutrient solution opacity, cultivate and use spectrophotometer measurement OD after 72 hours
600Each processes 3 repetitions.The growing state of RY5 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 subject to very large 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, and begins to form concentration greater than 1 * 10 when salt concn is 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 (5)
1. knurl bacterial strain RY5 (Brabyrhizobium sp.) of taking root slowly is preserved in Chinese Typical Representative culture collection center on November 13rd, 2009, and preserving number is CCTCC No:M209269.
2. the method for the described knurl bacterial strain RY5 tieback khuskhus of taking root slowly of claim 1 is characterized in that comprising the following steps:
(1) the separation and purification knurl bacterial strain RY5 of taking root is slowly out washed in the liquid YMA medium with sterilized water, 180r/min shakes the OD value that bacterium was measured bacterium liquid in 3 days afterwards in shaking table, gets bacterium liquid greater than 0.6 the time as OD;
(2) will be thrown on the germination paper of sterilizing in culture dish after the Stylo seed sterilization, soak germination paper with the 0.05mmol/L calcium sulphate soln of sterilizing, be placed in the dark vernalization of incubator of 28 ± 1 ℃, treat that seedling grows to 4 centimetres of time shifts and enters in the husky training of sterilization case;
(3) choose the rear plantation of bacterium liquid immersion that sturdy seedling prepares with step (1) from the described husky training case of step (2), every seedling adds bacterium liquid 1～2ml.
3. a microbial inoculum, it is characterized in that being obtained by the described knurl bacterial strain RY5 bacterium liquid mixing thalline absorbent preparation of taking root slowly of claim 1.
4. microbial inoculum according to claim 3, 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.
5. the method for the described knurl bacterial strain RY5 of taking root slowly of khuskhus inoculation claim 1 is characterized in that the bacterial strain activation, OD carries out Dressing greater than the bacterium liquid of 0.6 o'clock or waters in the khuskhus root soil greater than the bacterium liquid of 0.6 o'clock with OD or OD is made microbial inoculum greater than bacterium liquid the mixings thalline sorbing material of 0.6 o'clock mix before khuskhus is transplanted and impose on soil after shaking table shakes bacterium.
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| 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 |
| CN108029495A (en) * | 2017-12-27 | 2018-05-15 | 四川农业大学 | A kind of Sichuan province tobacco planting subtracts the method for applying synergy |
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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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| CN101182476A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXYD3 and uses thereof |
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101182476A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXYD3 and uses thereof |
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
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| Tatiana Krasova-Wade et al..Diversity of indigeneous bradyrhizobia associated with three cowpea cultivars (Vigna unguiculata (L.) Walp.) grown under limited and favorable water conditions in Senegal (West Africa).《African Journal of Biotechnology》.2003,第2卷(第1期),13-22. * |
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