CN101519644A - Sinorhizobium sp. and application thereof - Google Patents
Sinorhizobium sp. and application thereof Download PDFInfo
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Abstract
The invention discloses Sinorhizobium sp. and application thereof. The Sinorhizobium sp. NX2004062 has a storage number of CGMCC No.2883. The Sinorhizobium sp. NX2004062 can lead clover plants to safely grow, establish an effective syntaxial system with the clover plants to promote the growth of clover roots, strengthen the photosynthesis of the clover plants, increase the output of clovers on the ground and under the ground and further improve the output and the quality of the clovers; moreover, the Sinorhizobium sp. NX2004062 reduces the fertilizer application in clover production and further reduces the pollution of fertilizers to the environment.
Description
Technical field
The present invention relates to strain root nodule bacterium and an application thereof, particularly a strain and alfalfa plant root can effectively be set up the root nodule bacterium and the application thereof of syntaxial system.
Background technology
Chemical fertilizer occupies an important position on world's agriculture development history, for the stable yields of agricultural, increase production, retrieve a loss and made significant contribution, but simultaneously also because of excessive use and abuse cause soil fertility decline, environmental pollution, to serious problems such as non-target organism influences.Especially in recent years a large amount of uses of nitrogenous fertilizer in the agricultural-food cause pollutions such as nitrite, and the people's life health in serious threat.Along with the progress of social civilization, agricultural sustainable development makes human understanding to resource and environment that new leap arranged, and the use of fertilizer has been proposed safe, effective, environmentally friendly requirement.Countries in the world are all in the use of taking measures to reduce agrochemicals, and strengthen the strictness of agricultural chemicals rate of fertilizer in the agricultural-food production process and product contamination index is controlled, states such as Japan have expressly provided all kinds of pollutant loads in the farm imports have been carried out the strictness restriction.For solve agricultural-food improve output and ensure the quality of products between contradiction, seek and the applying biological factor is improved the concern that the plant nutrition environment more and more has been subjected to national governments, scientific worker and the common people.Greatly develop food crop and soybean crop rotation for many years as America always,, obtained remarkable economical and ecological benefits to reduce the chemical nitrogen fertilizer consumption.
Vinelandii be a class can with the plant harmonious coexistence, promote the probiotic bacteria of plant-growth.Vinelandii just can utilize airborne N easily under the home condition
2Synthetic ammonia.Its institute's fixed nitrogen is except confession self growth needs, and the overwhelming majority changes into the available itrogenous organic substance of crop and supplies with plant materials.People are called biological nitrogen fixation with this process.The biological nitrogen fixation of microorganism is improving the nitrogen nutrition of plant, is saving chemical nitrogen fertilizer, is reducing chemical nitrogen fertilizer to the pollution of environment with safeguard that all there is crucial effect aspect such as the natural eubiosis.Biological nitrogen fixation comprises from growing nitrogen-fixing, association nitrogen fixation and symbiotic nitrogen fixation.Wherein limited in one's ability from growing nitrogen-fixing and association nitrogen fixation, and symbiotic nitrogen fixation can be able to provide a large amount of nitrogen for the mankind, is the focus that people study.According to estimates, annual biological nitrogen fixation provides about 200,000,000 tons of nitrogen for the whole world, wherein 75% is provided by symbiotic nitrogen fixer, has substantially exceeded global technical azotification amount.
Root nodule bacterium and leguminous plants are the strongest systems of effect in the symbiotic nitrogen fixation, and its amount of nitrogen fixation accounts for 65% of all biological amount of nitrogen fixation, and root nodule bacterium can become free nitrogen transformation in the air combined nitrogen for the leguminous plants growth, and this syntaxial system is the result of long-term evolution.Leguminous plants fixed nitrogen is not only as self syntaxial system nutrition, and can be other non-leguminous plant nitrogen is provided, so they play crucial Ecology Action to keeping suitable nitrogenous source in the plant.So the symbiotic nitrogen fixation of development and use root nodule bacterium has far reaching significance.Relevant investigation shows, make leguminous plants have very strong anti-adversity ability with the root nodule bacterium symbiosis, wherein, the quantity of root nodule bacterium becomes positive correlation with nitrogenase activity with fabaceous resistance, there are not root nodule bacterium to exist in the soil of general new growing area, therefore, generally acknowledge that in the world plantation leguminous plants in newly developed area must artificial inoculation can reach symbiotic root nodule bacterium with it.With the alfalfa is example, studies show that, the inoculation root nodule bacterium can make alfalfa all be significantly increased aspect biomass and the plant quality.
At present, along with the adjustment of China's agricultural planting structure, crop varieties presents variation, and the cultivated area that wherein has clover, peanut and the legume vegetable crop of nitrogen fixing capacity sharply increases, and presents the situation that develops rapidly.On the other hand, along with the raising of living standards of the people and the continuous reinforcement of quality of life consciousness, people are more and more higher to the specification of quality of livestock product, and the development of the herbvore cattle breeding industry of good quality and high output has become desirability, thereupon, the production of high quality forage also becomes the task of top priority.Under agricultural restructuring and the pulling of herbage produce market demand both at home and abroad, present the situation that develops rapidly based on the herbage industry of alfalfa.Alfalfa is described as " herbage queen ", is the pulse family clover, and its main root can go deep into soil 7-9 centimetre or darker, contains high protein, mineral substance and ten multivitamins.The introduction of good quality and high output alfalfa new variety at present, screening and popularization have been put into country and each province's emphasis agricultural use technology promotion project.But, the root nodule bacterium that are used for alfalfa growing at present come from non-clover host mostly, its anti-adversity ability, colonization ability, competitive nodulation ability, nitrogenase activity and undesirable with the collaborative nitrogen fixation effect of symbiosis of different alfalfa varieties etc. have influenced the production of alfalfa.Therefore root nodule bacterium with high-efficiency nitrogen-fixing effect of screening and clover have vital role for the production of clover.
Summary of the invention
An object of the present invention is to provide a strain bacterium, this bacterial strain can form effective syntaxial system with the root of alfalfa plant.
Bacterial strain provided by the present invention is root nodule bacterium (Sinorhizobium sp.) NX2004062, and deposit number is CGMCC No.2883.
The bacterium colony of this bacterium is circular, and oyster white is translucent, and along with incubation time increases, bacterium colony is constantly expanded projection, neat in edge, and the bacterium colony smooth surface, growth cellular material thereon easily disperses in liquid.Bacterium is shaft-like under the opticmicroscope, the great majority motion, and flagellum Zhousheng, the bacterium size is (0.6-0.8) * 3
-5μ m, the gramstaining poststaining is even, and visible bacterium is dyed redness.
The classification called after China root nodule bacterium (Sinorhizobium sp.) of this bacterial strain, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 19th, 2009 and (be called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCCNo.2883.
Another purpose of the present invention provides the method for a kind of cultivation above-mentioned root nodule bacterium (Sinorhizobium sp.) NX2004062 CGMCC No.2883.
The method of cultivation root nodule bacterium (Sinorhizobium sp.) NX2004062 CGMCC No.2883 provided by the present invention is under 25-30 ℃ condition, cultivates described bacterium with YMA medium; Consisting of of described YMA medium (being yeast water mannite agar substratum): 10g/L N.F,USP MANNITOL, yeast powder 3g/L, 0.25g/LK
2HPO
4, 0.25g/L KH
2PO
4, 0.2g/L MgSO
4-H
2O, 0.1g/L NaCl, 3g/L CaCO
3, all the other are water, the pH value is 7.2.
Described cultural method also can carry out liquid and shake training; Described speed of shaking training can be 120-200rpm, is preferably 200rpm.
Above-mentioned culture temperature is preferably 28 ℃; Incubation time is 36h-48h, preferred 48h.
Another object of the present invention provides a kind of microbial inoculum, and this microbial inoculum can be used for the inoculation of root nodule bacterium in the alfalfa plant.
Microbial inoculum provided by the present invention obtains root nodule bacterium (Sinorhizobium sp.) NX2004062 (deposit number is CGMCC No.2883) with thalline sorbing material mixing.
Wherein, described thalline sorbing material can be the peat composed of rotten mosses or micro mist lime carbonate.The proportioning of (Sinorhizobium sp.) NX2004062 CGMCC No.2883 of root nodule bacterium in the described microbial inoculum and described thalline sorbing material can be (1 * 10
8-5 * 10
9) cfu/g thalline sorbing material, be preferably 5 * 10
9Cfu/g thalline sorbing material, the pH value of microbial inoculum is 6.0-7.4.
When the thalline sorbing material is the peat composed of rotten mosses, bacterium liquid can be made the powder-type microbial inoculum with peat composed of rotten mosses absorption; When the thalline sorbing material is micro mist lime carbonate, can with the bacterial sediment of the centrifugal acquisition of fermented liquid of described bacterium by 1: 1 mass ratio with micro mist lime carbonate is mixed must microbial inoculum.
Last purpose of the present invention provides the method for clover inoculation root nodule bacterium.
The method of clover inoculation root nodule bacterium provided by the present invention has following dual mode:
First kind of inoculation root nodule bacterium method is to soak the seed of clover with the bacteria suspension of root nodule bacterium (Sinorhizobium sp.) NX2004062 (deposit number is CGMCC No.2883), and then sows cultivation.
Wherein, the concentration of described bacteria suspension can be (1 * 10
8-1 * 10
10) cfu/ml, be preferably 1 * 10
10Cfu/ml; The time of described immersion can be 10-30 minute, is preferably 15 minutes.
Described sowing cultured method is cultivated the seed of described immersion with the mixture that obtains for above-mentioned arbitrary described microbial inoculum and culture medium mixing.
The method of second kind of clover inoculation root nodule bacterium is with above-mentioned arbitrary described microbial inoculum and culture medium mixing, with the mixture that obtains the seed of clover is cultivated.
Above-mentioned culture medium can be made up of sand, soil and the peat composed of rotten mosses, also can be just by soil constitution, and the mass ratio of described sand, described soil, the described peat composed of rotten mosses is 6:2:1.Described specifically can be that described matrix is mixed with the mass ratio of described microbial inoculum according to 3:1 with the above-mentioned arbitrary described microbial inoculum and the method for culture medium mixing.
In the method for clover inoculation root nodule bacterium of the present invention, described seed can be the chitting piece through sterilization, vernalization.
In the method for clover inoculation root nodule bacterium of the present invention, described clover is specifically as follows alfalfa.
Bacterial strain NX2004062 of the present invention (deposit number is CGMCC No.2883) is to the growth safety of alfalfa plant, can be by setting up effective syntaxial system with alfalfa plant, promote the clover root growth, strengthen its light and effect, improve on the alfalfa ground, underground part output, and then improved the yield and quality of clover.On the other hand, the application of bacterial strain of the present invention has reduced coming into operation of chemical fertilizer in the ALFALFA PRODUCTION, and then has reduced the pollution of chemical fertilizer to environment.Experimental result shows, the alfalfa plant that obtains with strain culturing of the present invention, with or not do not cultivate the alfalfa plant that obtains altogether and compare any bacterium, its over-ground part dry weight amplification is all more than 67%, reach as high as 284%, underground part dry weight amplification reaches as high as 282% all more than 73%, the amplification of dross number all more than 20%, reaches as high as 3700%.Experimental result also further shows, a plurality of alfalfa varieties such as No., bacterial strain of the present invention and middle lucerne, WL232, De Fu, three get profit, CW787 and CW300 all have good symbiotic nitrogen fixation effect.Therefore, bacterial strain of the present invention has that nitrogen fixation effect is good, the field planting ability is strong, to the characteristics of alfalfa plant growth safety, has overcome strong, the relatively poor shortcoming of nitrogen fixation effect of the root nodule bacterium specialization of inoculating on the present ALFALFA PRODUCTION.
The present invention has filtered out optimum medium and the culture condition of bacterial strain NX2004062, and to have prepared viable bacteria amount be 5 * 10
9The nitragin of cfu/g.The preparation of microbial inoculum of the present invention has that technology is simple, fermentation period is short, drop into low and be easy to characteristics such as preservations, meets agricultural products in China the keep the safety in production requirement of standard and the construction needs of nuisanceless, green and Organic farming production base.
Bacterial strain of the present invention and the method for cultivating alfalfa plant for China's alfalfa industry provides reliable material to support and guidance, and are laid a solid foundation for finally setting up the symbiotic specificity appraisement system of rhizobium strains and alfalfa variety.The popularization of bacterial strain of the present invention and use will play a significant role in agricultural sustainable development, Agro-ecology construction of base and development of regional economy, have important economy and social effect.
Description of drawings
Fig. 1 is the electrophorogram as a result of NodA gene among the amplification bacterial strain NX2004062.
Fig. 2 is the electrophorogram as a result of NifH gene among the amplification bacterial strain NX2004062.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Employed reagent among the following embodiment if no special instructions, all can obtain from commercial channels.
The separation of embodiment 1, bacterial strain and evaluation
One, the separation of bacterial strain
Method: from the clover sample plot of Xixia District reed catkins town, Ningxia city town Bei Bao, take the eugonic alfalfa plants of overground part, observe its root and the root nodule of giving birth to, winning color is that peach root nodule places little sampling bottle, and anhydrous sodium sulphate is put into as siccative in the bottle bottom.With the root nodule of taking take back indoor after, with sterile water wash three times, 3% clorox sterilization 5min is used on the surface, and then root nodule is transferred on the thieving paper of sterilization wipe dry with sterile water wash after once.Root nodule is placed in the mortar of sterilization and smashs to pieces, dip in its juice with transfering loop, adopt the method for bacterium line that its on YMA Viola crystallina selection substratum (i.e. adding 100,000/Viola crystallina on the basis of YMA solid medium) is rule, constant temperature culture 24h under 28 ℃ of conditions, select neat in edge, the translucent single colony inoculation of oyster white is to the YMA solid medium.Carry out biology and Physiology and biochemistry macroscopical identification, and a bacterial strain called after NX2004062 that will obtain.
Consisting of of YMA solid medium (being yeast water mannite agar substratum): 10g/L N.F,USP MANNITOL, yeast powder 3g/L, 0.25g/L K
2HPO
4, 0.25g/L KH
2PO
4, 0.2g/L MgSO
4-H
2O, 0.1g/L NaCl, 3g/L CaCO
3, the 10g/L agar powder, all the other are distilled water, the pH value transfers to 7.2.
Two, the evaluation of bacterial strain
1, micro-and ultra micro morphologic observation
Bacterial strain NX2004062 is cultivated on the YMA solid medium, and observed and recorded.
After cultivating 48h, bacterium colony is circular, and oyster white is translucent, and along with incubation time increases, bacterium colony is constantly expanded projection, neat in edge, and the bacterium colony smooth surface, growth cellular material thereon easily disperses in liquid.It is shaft-like observing bacterium under the opticmicroscope, the great majority motion, and flagellum Zhousheng, the bacterium size is (0.6-0.8) * 3
-5μ m, the gramstaining poststaining is even, and visible bacterium is dyed redness.
2, Physiology and biochemistry is identified
Bacterial strain is carried out Physiology and biochemistry identify that qualification result is as shown in table 1.
Table 1, bacterial strain NX2004062 certified variety and result
| Test item | NX2004062 | Reference culture | Test item | NX2004062 | Reference culture |
| Gramstaining | - | - | Tyrosine produces melanochrome | - | - |
| Mobility | - | - | The starch hydrolysis | - | - |
| Catalase | + | + | The hippurate hydrolysis | - | - |
| The V-P reaction | - | - | The Citrate trianion hydrolysis | - | - |
| Egg yolk reaction | - | - | Nitrate reduction | - | - |
| 5% sodium-chlor | + | + | Produce indoles | - | - |
| 7% sodium-chlor | - | - | Tryptophane takes off amine | - | - |
| 10% sodium-chlor | - | - | Casein decomposes | - | - |
| Glucose produces acid | - | - | Tyrosine decomposes | - | - |
| The D-wood sugar produces acid | + | + | Litmus milk | + | + |
| Pectinose produces acid | + | + | Gelatin hydrolysis | + | + |
| N.F,USP MANNITOL produces acid | - | - | Nitrogen fixing capacity | + | + |
| Glucose produces melanochrome | - | - | Viola crystallina detects | + | + |
Annotate: "+" expression positive findings; "-" expression negative findings.
3, the nodA gene of pcr amplification symbiosis dross Box and NifH gene are identified
Use the root nodule bacterium Auele Specific Primer, nodA gene and the NifH gene of the symbiosis dross Box of bacterial strain NX2004062 carried out pcr amplification.The primer sequence of amplification NodA gene is as follows: nodA-1:5 '-TGCRGTGGAARNTRNNCTGGGAAA-3 ' (sequence 1), nodA-2:5 '-GGNCCGTCRTCRAAWGTCARGTA-3 ' (sequence 2); The primer sequence of amplification NifH gene is as follows: nifH-1:5 '-AAGTGCGTGGAGTCCGGTGG-3 ' (sequence 3), nifH-2:5 '-GTTCGGCAAGCATCTGCTCG-3 ' (sequence 4).
Bacterium liquid with bacterial strain NX2004062 is template, carries out pcr amplification with above-mentioned primer respectively, and the result as depicted in figs. 1 and 2.Fig. 1 is the amplification (swimming lane 1 is the amplification of bacterial strain NX2004062, and swimming lane 2 is marker) of NodA gene; Fig. 2 is the amplification (swimming lane 1 is marker, and swimming lane 2 is the amplification of bacterial strain NX2004062) of NifH gene.The size that the result shows the NodA gene fragment that obtains of amplification near 700bp about, consistent with purpose segment size, the size of the NifH gene fragment that amplification obtains near 700bp about, big or small consistent with the purpose segment.Employed among Fig. 1 and Fig. 2 is identical marker.
Above-mentioned experimental result shows that comprehensively this root nodule bacterium cell does not have gemma, is generally bacillus, does not move, and is aerobic.Gram-negative is utilized multiple kinds of carbohydrate, produces mucus outside a large amount of born of the same parents when growing on the carbohydrate containing substratum usually.Do not utilize Citrate trianion, be dissolved in chloroform.Do not generate 3-ketone group glucoside.Slow liquefy gelatin.Not caseinhydrolysate and agar.Bacteria colony white or colourless.Do not utilize Mierocrystalline cellulose and starch.Can be with ammonium salt, nitrate and most of amino acid as nitrogenous source.The catalase reacting positive.
The Physiology and biochemistry proterties detected result of above bacterial strain NX2004062 is compared with the root nodule bacterium reference culture, retrieve according to the key in " general bacterium authentication method commonly used " and " rhizobium ", determine that the corresponding proterties with root nodule bacterium of Physiology and biochemistry proterties of the bacterial strain NX2004062 that this experiment detects is coincide.The amplification of while NodA gene and NifH gene, further confirm the biochemical identification result, identify that bacterial strain NX2004062 is root nodule bacterium, its classification called after China root nodule bacterium (Sinorhizobium sp.), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on January 19th, 2009 and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC No.2883.
Three, the culture condition of bacterial strain gropes
Bacterial strain NX2004062 is distinguished streak inoculation in LB, YMA, YEM, PDA, Jin Shi B, BPY, 523, sucrose protein culture medium (sucrose 2.0%, peptone 0.5%, K
2HPO
40.05%, MgSO
47H
2O 0.025%, distilled water 1000ml, agar 17.0g), YGM (glucose 2.5%, yeast extract paste 2.0g, K
2HPO
40.25g, KH
2PO
40.25g, MgSO
47H
2O 0.1g, MnSO
40.15g, NaCl 0.05g, FeSO
47H
2O 0.005g, agar 17.0g, distilled water 1000ml) on totally 9 kinds of substratum, cultivate 24h～48h down at 28～30 ℃, observe the growing state of bacterium.The result shows that bacterial strain NX2004062 all can grow, and shows the pyorrhea shape respectively on 9 kinds of culture medium flat plates.But the growing state on the different substratum there are differences, and bacterium colony is expanded soon on YMA, YEM, 523 substratum, and the bacterium amount is big.
Bacterial strain NX2004062 is inoculated in respectively in above-mentioned 9 kinds of liquid nutrient mediums, shakes training at 28 ℃ of following liquid, the speed that liquid shakes training is 200rpm, measures growth curve, OD respectively
600The variation of value and pH value.3 repetition are established in experiment, and comprehensive every result shows that the YMA liquid nutrient medium is suitable for liquid and shakes training, and bacterial strain NX2004062 shakes to train to 16h in the YMA nutrient solution and enters logarithmic phase, and the 20h bacterium is measured and reached 10
8Cfu/ml, be 36h-48h the period that its bacterium amount peaks, the bacterium amount is 10
12Cfu/ml.
YMA liquid nutrient medium (being yeast water mannite agar substratum) composed as follows: 10g N.F,USP MANNITOL, yeast powder 3g, 0.25g K
2HPO
4, 0.25g KH
2PO
4, 0.2g MgSO
4-H
2O, 0.1g NaCl, 3g CaCO
3, 10g agar powder, 1L distilled water, pH value 7.2.
One, the preparation of the microbial inoculum of bacterial strain NX2004062
1, the cultivation of bacterium
The bacterial strain NX2004062 streak inoculation of cryopreservation on the YMA medium inclined-plane, is cultivated 48h down in 30 ℃ and carried out actication of culture; Bacterial strain NX2004062 is seeded to (liquid amount that is nutrient solution YMA is 30%) in the 1000ml triangular flask that the 300mlYMA liquid nutrient medium is housed with the 1-2% inoculum size again, carrying out shaking table cultivates, wherein, culture temperature is 28 ℃, initial pH is 6.0, shaking table speed is 200rpm, and shaking the training time is 48h, obtains bacterium liquid.
2, prepare microbial inoculum respectively in accordance with the following methods:
(1) bacterium liquid that above-mentioned cultivation is obtained and peat composed of rotten mosses mixing are made the powder-type microbial inoculum with bacterium liquid with peat composed of rotten mosses absorption, and wherein, Chinese root nodule bacterium in the microbial inoculum (Sinorbizobium sp.) NX2004062 (deposit number is CGMCCNo.2883) is 5 * 10 with the proportioning of the peat composed of rotten mosses
9The cfu/g peat composed of rotten mosses.Detect living bacteria count, the pH value in the microbial inoculum and the bacterium rate of mixing by plate count.3 repetitions are established in experiment, and the result takes the mean.The viable bacteria amount that records in the microbial inoculum equals 5 * 10
9Cfu/g, the pH value is 6.8.
(2) the bacterium liquid that above-mentioned cultivation is obtained carries out centrifugal, obtain bacterial sediment, then with bacterial sediment and micro mist lime carbonate (1 μ m＜d＜5 μ m) according to 1: 1 ratio mixing of mass ratio, make microbial inoculum, wherein the bacterium of Chinese root nodule bacterium (Sinorhizobium sp.) NX2004062 (deposit number is CGMCC No.2883) amount is 5 * 10 in the microbial inoculum
9Cfu/g micro mist lime carbonate.Detect living bacteria count, the pH value in the microbial inoculum and the bacterium rate of mixing by plate count.3 repetitions are established in experiment, and the result takes the mean.The viable bacteria amount that records in the microbial inoculum equals 5 * 10
9Cfu/g, the pH value is 6.8.
Two, the microbial inoculum that utilizes step 1 to obtain is cultivated 12 alfalfa kinds
The title of 12 alfalfa kinds (being) of using in this experiment is as shown in table 2.These 12 kinds (all from Beijing seed control station) all are in the bigger representational kind of China's cultivated area, all can obtain from commercial channels.Be example with one of them kind " three get profit " below, this experimental technique is described, the experimental technique of all the other kinds is all identical therewith.
" three get profit " seed germination: three seeds of getting profit are soaked 30s in 70% ethanol, rinsed with sterile water 2 times, each 5min.It is immersed among 5% chlorine bleach liquor (NaClO) subsequently, fully shake 3min after, sterilized water washing.Once more seed is placed 70% ethanol to soak 30s, rinsed with sterile water 2 times, each 5min.Soak seed 3h with sterilized water afterwards, every 30min changes sterilized water once.Because the three seed kind skins of getting profit are thinner, in the process that seed disinfection is handled, also finished the vernalization of seed simultaneously and handled the disinfectant seed of just having been sprouted at last.
" three get profit " seed connects bacterium and handles (method of seed soaking): the consistent alfalfa seed of selecting to germinate connects bacterium.According to the described method of step 1 in the experiment one, (concentration of bacterium is 5 * 10 in the bacterium liquid to obtain the bacterium liquid of Chinese root nodule bacterium (Sinorhizobium sp.) CGMCCNo.2883
9Cfu/ml); Soak alfalfa seed 15 minutes with bacterium liquid again, sow cultivation again.
The preparation of clover culture medium: according to sand: the ratio of soil: the peat composed of rotten mosses=6:2:1 (volume ratio) with sand, soil and peat composed of rotten mosses mixing, obtains the clover culture medium.
Microbial inoculum soil treatment and seedling are cultivated: in the plastics group training cup of bore 25cm, add 3/5 volume through the clover culture medium of sterilising treatment (wherein, the mass ratio of sand, soil and the peat composed of rotten mosses is 6:2:1) and the NX2004062 Rhizobium Inoculant of 1/5 volume (according to any microbial inoculum of the described method preparation of step 2 in the experiment one), microbial inoculum and culture medium mixing.With the above-mentioned seed that connects bacterium that obtains, implant in the plastic flowerpot of ready 13cm * 13cm * 13cm, growth under natural condition of field (temperature is that 22 ℃-36 ℃, humidity RH=40%-70%, illumination in 12 hours and 12 hours are alternately dark) is placed it in 15 of every glass of plantations afterwards.
Treat that plant strain growth gathers in the crops behind 90d, with plant dry weight (being output) is main performance assessment criteria, dross number, root length etc. are auxiliary characteristics, respectively each index is carried out data analysis and processing with SAS statistical software and EXCEL software, determine the symbiotic nitrogen fixation effect between NX2004062 rhizobium strains and alfalfa variety.
3 repetitions are established in experiment, and the result takes the mean, with the seed of not taking over what bacterium in contrast.The result is as shown in table 2.
The result shows that behind 12 confession examination alfalfa kind inoculation root nodule bacterium NX2004062, its over-ground part dry weight, underground part dry weight, dross quantity all significantly are better than contrast.Show that NX2004062 is safer to the growth fraction of clover, be the root nodule bacterium that a strain has efficient nitrogen fixation activity, can be by setting up effective syntaxial system with the alfalfa of a plurality of kinds, promote the clover root growth, strengthen its light and effect, improve on the alfalfa ground, underground part output, and then improved the yield and quality of clover.
Table 2,12 alfalfa kinds (being) and root nodule bacterium NX2004062 symbiosis result
Embodiment 3, utilize bacterial strain NX2004062 to cultivate 12 alfalfa kinds
One, the preparation of the microbial inoculum of bacterial strain NX2004062
1, the cultivation of bacterium
With identical described in the embodiment 2.
2, prepare microbial inoculum respectively in accordance with the following methods:
(1) bacterium liquid that above-mentioned cultivation is obtained and peat composed of rotten mosses mixing are made the powder-type microbial inoculum with bacterium liquid with peat composed of rotten mosses absorption, detect living bacteria count, the pH value in the microbial inoculum and the bacterium rate of mixing by plate count.3 repetitions are established in experiment, and the result takes the mean.The viable bacteria amount that records in the microbial inoculum equals 1 * 10
8The cfu/g peat composed of rotten mosses, pH value are 6.8.
Two, the microbial inoculum that utilizes step 1 to obtain is cultivated 12 alfalfa kinds
The title of 12 alfalfa kinds (being) of using in this experiment and source are with identical described in the embodiment 2.Be example with one of them kind " three get profit " below, this experimental technique is described, the experimental technique of all the other kinds is all identical therewith.
" three get profit " seed germination: with identical described in the embodiment 2.
" three get profit " seed connects bacterium and handles (method of seed soaking): the consistent alfalfa seed of selecting to germinate connects bacterium.According to the described method of step 1 in the experiment one among the embodiment 2, (concentration of bacterium is 1 * 10 in the bacterium liquid to obtain the bacterium liquid of Chinese root nodule bacterium (Sinorhizobium sp.) CGMCC No.2883
8Cfu/ml); Soak alfalfa seed 30 minutes with bacterium liquid again, sow cultivation again.
The preparation of clover culture medium: according to sand: the ratio of soil: the peat composed of rotten mosses=6:2:1 (volume ratio) with sand, soil and peat composed of rotten mosses mixing, obtains the clover culture medium.
Microbial inoculum soil treatment and seedling are cultivated: method is with described in the embodiment 2.
Treat that plant strain growth gathers in the crops behind 90d, with plant dry weight (being output) is main performance assessment criteria, dross number, root length etc. are auxiliary characteristics, respectively each index is carried out data analysis and processing with SAS statistical software and EXCEL software, determine the symbiotic nitrogen fixation effect between NX2004062 rhizobium strains and alfalfa variety.
3 repetitions are established in experiment, and the result takes the mean, with the seed of not taking over what bacterium in contrast.
The result shows that behind 12 confession examination alfalfa kind inoculation root nodule bacterium NX2004062, its over-ground part dry weight, underground part dry weight, dross quantity all significantly are better than contrast.
Embodiment 4, utilize bacterial strain NX2004062 to cultivate 12 alfalfa kinds
One, the preparation of the microbial inoculum of bacterial strain NX2004062
1, the cultivation of bacterium
With identical described in the embodiment 2.
2, prepare microbial inoculum respectively in accordance with the following methods:
(1) bacterium liquid that above-mentioned cultivation is obtained and peat composed of rotten mosses mixing are made the powder-type microbial inoculum with bacterium liquid with peat composed of rotten mosses absorption.Detect living bacteria count, the pH value in the microbial inoculum and the bacterium rate of mixing by plate count.3 repetitions are established in experiment, and the result takes the mean.The viable bacteria amount that records in the microbial inoculum equals 1 * 10
9Cfu/g, the pH value is 7.0.
Two, the microbial inoculum that utilizes step 1 to obtain is cultivated 12 alfalfa kinds
The title of 12 alfalfa kinds (being) of using in this experiment and source are with identical described in the embodiment 2.Be example with one of them kind " three get profit " below, this experimental technique is described, the experimental technique of all the other kinds is all identical therewith.
" three get profit " seed germination: with identical described in the embodiment 2.
" three get profit " seed connects bacterium and handles (method of seed soaking): the consistent alfalfa seed of selecting to germinate connects bacterium.According to the described method of step 1 in the experiment one among the embodiment 2, (concentration of bacterium is 1 * 10 in the bacterium liquid to obtain the bacterium liquid of Chinese root nodule bacterium (Sinorhizobium sp) CGMCC No.2883
10Cfu/ml); Soak alfalfa seed 10 minutes with bacterium liquid again, sow cultivation again.
The preparation of clover culture medium: according to sand: the ratio of soil: the peat composed of rotten mosses=6:2:1 (volume ratio) with sand, soil and peat composed of rotten mosses mixing, obtains the clover culture medium.
Microbial inoculum soil treatment and seedling are cultivated: method is with described in the embodiment 2.
Treat that plant strain growth gathers in the crops behind 90d, with plant dry weight (being output) is main performance assessment criteria, dross number, root length etc. are auxiliary characteristics, respectively each index is carried out data analysis and processing with SAS statistical software and EXCEL software, determine the symbiotic nitrogen fixation effect between NX2004062 rhizobium strains and alfalfa variety.
3 repetitions are established in experiment, and the result takes the mean, with the seed of not taking over what bacterium in contrast.
The result shows that behind 12 confession examination alfalfa kind inoculation root nodule bacterium NX2004062, its over-ground part dry weight, underground part dry weight, dross quantity all significantly are better than contrast.
Sequence table
＜110〉China Agricultural University
＜120〉strain root nodule bacterium and an application thereof
<130>CGGNAC92195
<160>4
<210>1
<211>24
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>(4，11，12，14，15，16)
＜223〉n=a or g or c or t, r=g or a
<400>1
<210>2
<211>23
<212>DNA
＜213〉artificial sequence
<220>
<221>misc-feature
<222>(3，9，12，15，20)
＜223〉n=a or g or c or t, r=g or a, w=a or t
<400>2
<210>3
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>3
<210>4
<211>20
<212>DNA
＜213〉artificial sequence
<220>
<223>
<400>4
Claims (9)
1, root nodule bacterium (Sinorhizobium sp.) NX2004062, deposit number is CGMCC No.2883.
2, a kind of method of cultivating the described root nodule bacterium of claim 1 is under 25-30 ℃ condition, cultivates described root nodule bacterium with YMA medium.
3, method according to claim 2 is characterized in that: carry out liquid in the process of described cultivation and shake training; The speed that described liquid shakes training is 120-200rpm, is preferably 200rpm; Described culture temperature is preferably 28 ℃; Incubation time is 36h-48h, is preferably 48h.
4, a kind of microbial inoculum obtains described root nodule bacterium of claim 1 and thalline sorbing material mixing.
5, microbial inoculum according to claim 4 is characterized in that: described thalline sorbing material is the peat composed of rotten mosses or micro mist lime carbonate.
6, microbial inoculum according to claim 5 is characterized in that: the proportioning of (Sinorhizobium sp.) NX2004062CGMCC No.2883 of root nodule bacterium described in the described microbial inoculum and described thalline sorbing material is (1 * 10
8-5 * 10
9) cfu/g thalline sorbing material, be preferably 5 * 10
9Cfu/g thalline sorbing material; The pH value of described microbial inoculum is 6.0-7.4.
7, the method for a kind of clover inoculation root nodule bacterium is to soak the seed of clover with the bacteria suspension of the described root nodule bacterium of claim 1, and then sows cultivation.
8, method according to claim 7 is characterized in that: the concentration of bacterium is (1 * 10 in the described bacteria suspension
8-1 * 10
10) cfu/ml, be preferably 1 * 10
10Cfu/ml; The time of described immersion is 10-30 minute, is preferably 15 minutes.
9, the method for a kind of clover inoculation root nodule bacterium is with arbitrary described microbial inoculum and culture medium mixing among the claim 4-6, with the mixture that obtains the seed of clover is cultivated.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101984067A (en) * | 2010-10-19 | 2011-03-09 | 中国农业大学 | Method of detecting growth promoting effect of plant rhizosphere growth promoting bacteria |
| CN104726365A (en) * | 2015-01-19 | 2015-06-24 | 山西农业大学 | New strain t21 of famous-region astragalus azotobacter and preparation method of fermentation liquor |
| CN105349456A (en) * | 2015-11-12 | 2016-02-24 | 甘肃省科学院生物研究所 | Sinorhizobium sp. and its application |
| CN105838655A (en) * | 2016-06-14 | 2016-08-10 | 农业部环境保护科研监测所 | Low-temperature phosphate solubilization Sinorhizobium fredii and inoculant thereof |
| CN108441438A (en) * | 2018-02-06 | 2018-08-24 | 杭州师范大学 | Rhizobium WYJ-E13 and its application in preparing RADIX CURCUMAE growth promoter |
| CN109943496A (en) * | 2019-01-02 | 2019-06-28 | 甘肃农业大学 | One plant of rhizobium seeks the method with the efficient symbiosis alfalfa variety of specificity |
| CN111960883A (en) * | 2020-07-28 | 2020-11-20 | 沧州市农林科学院 | Special bacterial fertilizer for soybean varieties and preparation method thereof |
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2009
- 2009-04-03 CN CN2009100811778A patent/CN101519644B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101984067A (en) * | 2010-10-19 | 2011-03-09 | 中国农业大学 | Method of detecting growth promoting effect of plant rhizosphere growth promoting bacteria |
| CN104726365A (en) * | 2015-01-19 | 2015-06-24 | 山西农业大学 | New strain t21 of famous-region astragalus azotobacter and preparation method of fermentation liquor |
| CN105349456A (en) * | 2015-11-12 | 2016-02-24 | 甘肃省科学院生物研究所 | Sinorhizobium sp. and its application |
| CN105349456B (en) * | 2015-11-12 | 2020-04-07 | 甘肃省科学院生物研究所 | Sinorhizobium and application thereof |
| CN105838655A (en) * | 2016-06-14 | 2016-08-10 | 农业部环境保护科研监测所 | Low-temperature phosphate solubilization Sinorhizobium fredii and inoculant thereof |
| CN105838655B (en) * | 2016-06-14 | 2019-07-05 | 农业部环境保护科研监测所 | A kind of low temperature Soluble phosphorus Sinorhizobium and its microbial inoculum |
| CN108441438A (en) * | 2018-02-06 | 2018-08-24 | 杭州师范大学 | Rhizobium WYJ-E13 and its application in preparing RADIX CURCUMAE growth promoter |
| CN108441438B (en) * | 2018-02-06 | 2020-10-09 | 杭州师范大学 | Rhizobium WYJ-E13 and application thereof in preparation of curcuma wenyujin growth promoter |
| CN109943496A (en) * | 2019-01-02 | 2019-06-28 | 甘肃农业大学 | One plant of rhizobium seeks the method with the efficient symbiosis alfalfa variety of specificity |
| CN111960883A (en) * | 2020-07-28 | 2020-11-20 | 沧州市农林科学院 | Special bacterial fertilizer for soybean varieties and preparation method thereof |
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| Publication number | Publication date |
|---|---|
| CN101519644B (en) | 2011-05-18 |
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