CN103952341A - Azorhizobium strain SCAUs152 and application thereof - Google Patents
Azorhizobium strain SCAUs152 and application thereof Download PDFInfo
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Abstract
The invention discloses an Azorhizobium strain SCAUs152 and application thereof. The strain is a new strain which is separated and purified from fresh soybean root nodules and belongs to Rhizobium. The strain is collected to China Center for Type Culture Collection of Wuhan University on March 14th, 2014, and the collection number is CCTCC NO.M2014085. The strain is applied to production of Sichuan soybeans. The Azorhizobium strain SCAUs152 is a favorable broad-spectrum Rhizobium japonicum strain with high symbiotic fixation capacity, wide application range of Sichuan soybean species, IAA (indoleacetic acid) secretion capacity, capacities for dissolving inorganic phosphorus, organic phosphorus and potassium and high stress tolerance. The strain has favorable matching affinity with Sichuan dominant bean species, and can enhance the soybean yield by more than 23% in different ecological regions where no nitrogen fertilizer is applied and the Azorhizobium strain SCAUs152 is inoculated, which is much different from the regions where the Azorhizobium strain SCAUs152 is not inoculated.
Description
Technical field
The present invention relates to microorganism field, in particular a kind of nodule azotobacter strain SCAUs152 and application thereof.
Background technology
Chemical nitrogen fertilizer makes a great contribution for improving crop yield, but production cost is high, utilization ratio is low, pollution of ecological environment.Cost is low, amount of nitrogen fixation large in biological nitrogen fixation, fixed nitrogen process is lasting, pollution-free, is conducive to the protection of ecotope and agriculture Sustainable development, is the effective means of agriculture production cost-saving synergistic.The exploitation biological nitrogen fixation technology useful to environment needs to make great efforts in grain-production.Root nodule bacterium-leguminous plants fixed nitrogen system is most effective in biological nitrogen fixation.Leguminous plants artificial inoculation root nodule bacterium are to improve Crops production and quality, an Important Agricultural measure of improving the ecological environment.Rhizobium Inoculation is the biological nitrogen fixation technology of generally acknowledging in the world.The syntaxial system of soybean and rihizobium japonicum is the Typical Representative in biological nitrogen fixation.Syngenetic process between soybean and rihizobium japonicum, is the result that interacts in long-term evolution, selects mutually, is also the result adapting to environmental facies.There is a very large advantage in the country of other manufacturing soybean of the world at the technical of application rihizobium japonicum.Yet, China is the area of origin of soybean, manufacturing soybean with a long history, root nodule bacterium and soybean coevolution, make the Indigenous Rhizobia that in soil, nitrogen-fixing efficiency is low there is the stronger affinity of mating with soybean varieties, thereby greatly reduce the action effect of high-efficiency nitrogen-fixing rhizobium inoculant.Therefore research and grasp root nodule bacterium and soybean varieties matching have important more practical value.
Improve symbiotic nitrogen fixation, visible bacterial strain and soybean varieties should be two basic factors.And the population distribution of root nodule bacterium has geographical limitation, in the screening of root nodule bacterium, should be noted that its corresponding adaptive faculty with area surroundings.In general, in certain region the most effectively root nodule bacterium often from this area or with the bacterial strain in conditional likelihood area, this area.Therefore when root nodule bacterium are chosen seeds, not only to carry out the matching combination research of root nodule bacterium and soybean varieties, also must pay attention to the region of microbial inoculum application.
Soybean is Chinese the fourth-largest farm crop, is deeply subject to liking of Chinese people, and edible way is varied, of many uses, is the important food crop of China and oil crops.Therefore China carry out good soybean nodulation bacterial strain screening and application technical research work very meaningful.Northeast soybean producing region is the maximum soybean of China producing region, and Huang-Huai-Hai is the second largest producing region of China soybean, relatively many with the main cultivation soybean varieties in two large producing regions and the research of the strain excellent that ecotope mates of this maximum.For arid soybean producing region, northwest, the screening of good rihizobium japonicum also has report.The first producing region for China soybean: producing region, northeast, the people such as Du Yinghui are from Heilungkiang separation acquisition one strain and Soybean Varieties In The Northeast of China matching is good and the strain excellent of applicable the Northeast, get permission patent of invention in June, 2012.For China's South China Acidic Soil, the people such as Cao Guiqin are south China 3 the root nodule bacterium strains of acidproof, resistance to aluminium that lacked the seed selection of phosphorus characteristic of acid red soil, have applied for 3 patents of invention in December, 2007.But study rarely seen report for the soybean varieties in the soybean producing region, Sichuan in Southern soybean producing region and the good root nodule bacterium that ecotope matches.
One directly under little ancestor crop before the soybean of Sichuan, plants scatteredly, and output is lower, and cultivated area is less.In recent years, Soybean production development in Sichuan is swift and violent, occurs mass-producing, big area contiguous plant, and yield level increases substantially, and has become one of Sichuan Province's staple food crop, and sown area is the 6th of national rank.The Ministry of Agriculture overlaps human consumption soybean between by Sichuan and southwest and lists one of national three advantages producing region in < < plant production development the 12 planning > > (2011-2015), National Development and Reform Committee includes Chongqing of Sichuan soybean in again country's 12 staple food crops, and Sichuan soybean has caused the attention of country and Sichuan Province governments at all levels.Sichuan soybean culture mode is mainly and the plantation of corn intercropping and interplanting, accounts for the more than 90% of the whole province's soybean acreage.Therefore, the good root nodule bacterium of inoculation coupling in the Soybean production of Sichuan, the intercropping and interplanting system of Sichuan soybean can also play mitigation to " ammonia checks " of root nodule bacterium nitrogenase activity.The leading kind of Sichuan interplanting soybean is " southern beans 12 " and " tribute is selected No. 1 ".The soybean essential species of Sichuan intercropping and interplanting is implanted in hills area, Sichuan again, and the resourceful Panxi Diqu of photo-thermal mainly be take early soybean or eat soybean raw as main.Also do not filter out at present with the main cultivation soybean varieties in Sichuan and mate, be applicable to the good rihizobium japonicum of Sichuan different ecological environment.For this reason; for the main breed in Sichuan and the main ecological of manufacturing soybean, carry out the screening of soybean strain excellent; give full play to biological nitrogen fixation; improve the effect of biological nitrogen fixation in production practice; thereby reduce using of chemical fertilizer; this is for preserving the ecological environment, and save energy resource, promotes agricultural sustainable development and have important more practical value.
Summary of the invention
Technical problem to be solved by this invention is for the deficiencies in the prior art, and a kind of nodule azotobacter strain SCAUs152 and application thereof are provided.
Technical scheme of the present invention is as follows:
A nodule azotobacter strain SCAUs152, its Classification And Nomenclature is Rhizobiumsp.SCAUs152, on March 14th, 2014, is preserved in Wuhan University's Chinese Typical Representative culture collection center, its deposit number is CCTCC NO:M2014085.
Described nodule azotobacter strain SCAUs152 is applied to the production of Sichuan soybean.
Nodule azotobacter strain SCAUs152 of the present invention is that a strain symbiotic nitrogen fixation ability is strong, to Sichuan soybean varieties wide accommodation, with producing IAA ability, there is molten inorganic phosphorus, organophosphorus and molten potassium capability, good wide spectrum soybean nodulation bacteria strain that resistance is stronger.And mate affinity with the main cultivation soybean varieties in Sichuan good, in planted in different ecological areas, nitrogen fertilizer application, inoculation nodule azotobacter strain SCAUs152 do not make soybean yield-increasing more than 23%, and do not inoculate the difference contrasting and reach conspicuous level.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of nodule azotobacter strain SCAUs152 in YMA medium.
Fig. 2 is the phylogeny figure of the 16rRNA gene order of nodule azotobacter strain SCAUs152.
Fig. 3 is glnII, the atpD of nodule azotobacter strain SCAUs152, the phylogeny figure of tri-housekeeping gene joint mappings of recA.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Separation, purifying and the preservation of embodiment 1 root nodule bacterium
The soybean of planting from the Hills And Low Mountains of Liangshan State of Sichuan Province Yanyuan County, select the root nodule of large on healthy and strong plant main root and full redness, by root nodule scrub, under band portion Gen Picai, paper using blots and places it in and Calcium Chloride Powder Anhydrous is housed and is covered with in the tubule of absorbent cotton.In laboratory, the root nodule gathering is soaked after imbibition with sterilized water, alcohol immersion 30s with 95% is to eliminate surface tension, then with 0.1%(m/v) mercuric chloride surface sterilization 5min, use again aseptic water washing 6 times, the in the situation that of aseptic technique, after being clipped broken, single root nodule is being added with Congo red YAM substratum (N.F,USP MANNITOL 10g, yeast powder 0.8g, KH
2pO
40.25g, MgSO
4.7H
2o0.2g, CaCl
2.6H
2o0.1g, NaCl0.1g, 1%(m/v) Sodium orthomolybdate 2ml, 1%(m/v) boric acid 2ml, 1%(m/v) Congo red 2.5ml, pH6.8-7.0, agar 18～20g, water 1000ml) upper line, in the thermostat container of 28 ℃, cultivate.
After growing bacterium colony, from flat board, select and do not inhale the bacterium colony as root nodule bacterium in red, form and at flat board, dilute streak culture.3d observes colonial morphology in left and right, observes 15d left and right always, because slow raw root nodule bacterium need 6～15d, occurs bacterium colony.Repeat dilution line repeatedly separated, until purifying.Whether be root nodule bacterium: (1) adds the colonial morphology in Congo red YMA medium if according to following two aspects, carrying out preliminary judgement: do not inhale redly, bacterium colony circle, oyster white, protuberance, neat in edge do not spread, smooth surface, compared with thickness, more moistening.What cultivate that 3～5d just grows bacterium colony is fast raw root nodule bacterium, and what cultivate that 6～15d grows bacterium colony is raw root nodule bacterium slowly.(2) cellular form: the root nodule bacterium bacterium colony of confirming is marked, and gramstaining is carried out in film-making, it is little shaft-like and form is consistent that the microscopy result of root nodule bacterium is that cell is, without gemma, normal containing beta-hydroxy-butanoic acid and in the form of link in cell, Gram-negative (G-).If above-mentioned mark bacterium colony has above-mentioned two aspect features, the test tube slant of this bacterium colony access YMA medium is cultivated and preserved.
The nodule azotobacter strain SCAUs152 that the separation and purification of the present embodiment obtains is fast raw root nodule bacterium, cultivate adding in Congo red YMA medium, thalline is not inhaled red, and bacterium colony is large, circle, oyster white, protuberance degree are higher, moistening, more transparent, grows bacterium colony about 3d.Through gram stain microscopy, be G-, be little shaft-like.
The tieback of embodiment 2 root nodule bacterium and matching test
The tieback test water training method of root nodule bacterium, the soybean varieties of using in test is the leading kind " southern beans 12 " in Sichuan, in illumination chamber, (22～24 ℃ of temperature controls, intensity of illumination 2700～3000 luxs, sunshine duration 14h) carries out, plantation 46d results.After successful with " southern beans 12 " tieback, carry out matching test with other main cultivation soybean varieties again, also by water culture, carry out the soybean varieties of using in test: the main cultivation of another main cultivation soybean varieties " tribute is selected No. 1 " of intercropping and interplanting, early soybean kind " river beans 14 ", Panxi Diqu eat soybean varieties " rich morning in spring " raw.In above-mentioned illumination chamber, cultivate after 1 week, be placed under normal illumination and temperature condition and cultivate, plantation 41d results.The aseptic micro-nitrogen nutrition liquid of regular replenishment.Nodule azotobacter strain SCAUs152 and above-mentioned soybean varieties are formed to various combination, and hydroponics device adopts the narrow-necked bottle (glass infusion bottle that hospital uses) of 250ml, and the same kind soybean plant strain of not inoculating nodule azotobacter strain SCAUs152 of take is contrast.After results, by root nodule number and the plant dry weight of soybean plant strain, evaluate the effect of inoculation that nodule azotobacter strain is SCAUs152.Tieback test is consistent with making and implantation methods that the bacterium liquid of matching test is cultivated the vernalization hydroponics device of seed.
(1) bacterium liquid is cultivated: nodule azotobacter strain SCAUs152 is inoculated in YMA liquid nutrient medium, places on shaking table and use 120rpm/min rotating speed, 28 ℃ are cultured to logarithmic phase (about 4d left and right).
(2) vernalization of seed: select large, the full undamaged soybean seeds of grain, with 95%(m/v) alcohol soaks 5min, removes alcohol, adds 0.1%(m/v) mercuric chloride solution surface sterilization 5min.Finally use sterile water wash 4～6 times, each 5min.28 ℃ of vernalization, treat that main root grows to 2～3cm left and right, sowing when fibrous root does not grow.
(3) making of hydroponics device: make hydroponics device with the narrow-necked bottle (glass infusion bottle that hospital uses) of 250ml.First make aseptic micro-nitrogen nutrition liquid, micro-nitrogen nutrition liquid formula: 0.46g calcium sulfate, 0.075g Repone K, 0.06g magnesium sulfate, 0.03g nitrocalcite, 0.075g ironic citrate, 0.136g dipotassium hydrogen phosphate, 1000ml distilled water, 1ml trace element (2.86g boric acid, 0.02g molybdic acid, 0.8g copper sulfate, 0.22gZnSO4 zinc sulfate, 1.81g manganous sulfate, adding distil water is to 1000ml).The micro-nitrogen nutrition liquid preparing is injected in the bottle after cleaning, and bottleneck covers one deck kraft paper, in bottleneck centre, opens an aperture (diameter 0.6cm), and aperture is cotton beyond the Great Wall, the resistant to elevated temperatures plastics film of outer cover one deck, and at 121 ℃ of temperature, sterilizing is standby.
(4) plantation and testing index: vernalization seed is placed in to sterile petri dish, with bacterium immersion bubble 15min in (1), with aseptic nipper, the root of seedling is inserted in hydroponics device aperture, every bottle of 1 strain, then every seedling adds bacterium liquid 1ml in (1) again, seed is good with the cotton plug in original hole around, prevents that dust from falling in bottle, pollutes.Separately establishing the same product plant of not inoculating processing is contrast (CK).During plantation, first plant CK.Each is processed and repeats 3 times.Water culture experiment the results are shown in table 1.
Table 1 result shows, described nodule azotobacter strain SCAUs152 with 4 for examination soybean varieties to mate affinity all good, all show good Noduling ability and symbiotic nitrogen fixation ability; Compare with contrasting of Rhizobium Inoculation not, nodule azotobacter strain SCAUs152 can significantly improve the plant dry weight of each kind soybean, than the contrast of not inoculating described nodule azotobacter strain SCAUs152, improves 32%～43%.Visible, described nodule azotobacter strain SCAUs152 is the good wide spectrum bacterial strain good with Sichuan soybean varieties matching.
The water culture experiment result of table 1 nodule azotobacter strain SCAUs152
Note: data are three mean values that repeat; * and * represent that respectively inoculation processing reaches 1%, 5% conspicuous level with the dry weight between corresponding contrast.
The anti-adversity ability of nodule azotobacter strain SCAUs152 described in embodiment 3
The anti-adversity ability of described nodule azotobacter strain SCAUs152 has mainly been carried out to acid and alkali-resistance, salt tolerant and growth greenhouse scope to be measured.Take YMA medium as basic medium, all with pH7,28 ℃ of dull and stereotyped positive contrasts of YMA of cultivating 7d.By the YMA slant culture of above-mentioned nodule azotobacter strain SCAUs152 with sterilized water scrape wash stand-by.Adopt some inoculation method, repeat for 3 times.The basic medium that the YMA medium of take is measured as resistance to acids and bases, regulates pH value with HC1 and NaOH, and pH value is followed successively by 4.0,5.0,6.0,8.0,9.0,10.0,11.0,12.0.Salt resistant test be take YMA medium equally as basic medium, and described bacterial strain point is seeded on the flat board that contains NaC1, and the quality volume fraction of NaC1 is 1%, 2%, 3%, 4% and 5%.After the equal 28 ℃ of cultivation 7d of flat board of acid and alkali-resistance, Salt tolerance, observe and record result.Growth temperature range is measured, described inoculation, in YMA medium, is established to 5 Temperature Treatment altogether, in 4 ℃, 10 ℃, 37 ℃, 40 ℃ biochemical cultivation cases, cultivate 30d, 10d, 7d, 7d respectively, another process 60 ℃ at heat shock process after 30min, forward at 28 ℃ and cultivate 7d.Test-results shows that described nodule azotobacter strain SCAUs152 anti-adversity ability is stronger, can on the flat board of pH5～12, grow, but peracid or cross the positive control that bacterium colony on the flat board of alkali is less than pH7, illustrates that peracid crosses alkali its growth is had to certain restraining effect; There is certain salt resistance ability, can on the YMA of 2%NaCl flat board, grow; Growth temperature range is wide, can in 4～40 ℃ of temperature ranges, grow, and this bacterial strain still can survive after 30min is processed in 60 ℃ of heat shocks, illustrates that this bacterial strain can stand the high temperature of short period of time.
The growth-promoting ability of nodule azotobacter strain SCAUs152 described in embodiment 4
The soil tillage of China 74% lacks phosphorus, and in soil, more than 95% phosphorus difficulty is directly absorbed by plant.And the phosphate fertilizer this season crop utilization rate manually applying is for only having 5%～25%, major part is fixed by soil chemistry very soon and is formed to close and holds state insoluble phosphate.The ecological chain of this " high investment, low output ", not only cause the waste of a large amount of phosphorus resources, and the environmental pollution that consumption of natural resource causes also increases the weight of day by day.Although the soluble potassium scarcity of resources of China, the potassium-bearing mineral particle in potassium bearing rock and soil is very abundant, but potassium in these potassium-bearing minerals is mainly to exist containing potassium silicate form with insoluble, can not be directly Crop utilization.Therefore, by functional microbials such as screening high-efficiency nitrogen-fixing, the molten potassium of molten phosphorus, improve the utilising efficiency of atmospheric nitrogen and soil phosphorus, potassium, be acknowledged as cheapness and have efficiently the biological control measure of environment protection significance.
The growth-promoting ability of described nodule azotobacter strain SCAUs152 is mainly investigated the ability of its molten phosphorus ability, molten potassium capability and plant auxin secretion (IAA).
(1) ability of molten organophosphorus and inorganic phosphorus
By molten phosphorus circle method.Organic phosphorus sources is Yelkin TTS, and inorganic phosphorous sources is calcium phosphate (Ca
3(PO
4)
2), aluminum phosphate (AlPO
4.2H
2o), tertiary iron phosphate (FePO
4.2H
2o), be commercially available analytical reagent.
Measure the substratum Meng Jinna substratum of dissolved organic phosphorus ability, formula (g/L): 10g glucose, 0.5g (NH
4)
2sO
4, 0.3gNaCl, 0.3gKCl, 0.03gFeSO
4.7H
2o, 0.03gMnSO
4.4H
2o, 0.2g Yelkin TTS, 5gCaCO
3, 0.4g yeast powder, 20g agar, 1000ml distilled water, pH value 6.8～7.0.75% alcohol heating for dissolving for Yelkin TTS wherein, sterilizing separately, is down flat plate after the substratum that is cooled to 60 ℃ of left and right mixes with sterilizing.
Measure the substratum PKO substratum that dissolves inorganic phosphorus ability, formula (g/L): 10g glucose, the above-mentioned inorganic phosphorous sources material of 3.0g, 0.5g (NH
4)
2sO
4, 0.2gNaCl, 0.2gKCl, 0.03gMgSO
4.7H
2o, 0.03gMnSO
4, 0.003gFeSO
4.7H
2o, 0.5g yeast powder, 20g agar, 1000ml distilled water, pH value 6.8～7.0.Wherein calcium phosphate, aluminum phosphate, tertiary iron phosphate pulverized 300 mesh sieves with mortar and separately after dry sterilization, and the substratum of being down to 60 ℃ of left and right with sterilising temp mixes and is down flat plate, stand-by.Bacterial classification preparation and some inoculation method, with embodiment 3 resistance tests, repeat 3 times.Whether whether after 28 ℃ of incubators cultivation 7d, observe bacterial strain grows and has molten phosphorus to iris out now.Result shows that described nodule azotobacter strain SCAUs152 shows as and do not grow on aluminum phosphate flat board, calcium phosphate, tertiary iron phosphate and Yelkin TTS are all had to certain dissolving power, and molten phosphorus loop diameter and the colony diameter ratio on Yelkin TTS, calcium phosphate, tertiary iron phosphate flat board, measured are respectively 1.20,1.13,1.12.
(2) molten potassium capability
Measure the substratum (g/L) of molten potassium capability: 5.0g sucrose, 2.0gNa
2hPO
4, 0.05gMgSO
4.7H
2o, 0.05gFeCl
3, 0.1gCaCO
3, 5g feldspar in powder, 20.0g agar, distilled water 1000ml.Wherein feldspar in powder, through grinding to form powdery, crosses 300 mesh sieves, with deionized water, washes away water-soluble K, Si plasma, dries in the shade.Separately after dry sterilization, after mixing, the substratum that is cooled to 60 ℃ of left and right is down flat plate with sterilizing, and stand-by.Bacterial classification preparation and some inoculation method, with the mensuration of molten phosphorus ability, repeat 3 times.After 28 ℃ of incubators cultivation 7d, observe bacterial strain and whether grow, because this substratum be take feldspar in powder as unique potassium source, if growth just shows that bacterial strain has molten potassium capability.This test-results shows, described nodule azotobacter strain SCAUs152 take feldspar in powder normal growth on the flat board in unique potassium source, shows that SCAUs152 has certain molten potassium capability.
(3) mensuration of plant auxin secretion ability
Adopt the ability of colorimetric method for determining root nodule bacterium plant auxin secretion (IAA), measure the Congo red liquid nutrient medium that substratum adopts improvement, substratum forms: 0.5gK
2hPO
4.3H
2o, 0.2gMgSO
4.7H
2o, 0.1gNaCl, 1g yeast extract paste, 10g N.F,USP MANNITOL, 10ml0.25%(m/v) Congo red, 1gNH
4nO
3, 100mgL-tryptophane, 1000ml distilled water, pH7.0.Color solution formula: 0.5MFeCl
31ml, dense H
2sO
430ml, distilled water 50ml.
Inoculation, in filling the triangular flask of 50ml substratum, is placed in to rotating speed 125rpm/min, and the shaking table that temperature is 28 ℃ is cultivated, repeat for 3 times, cultivate after 12d, get root nodule bacterium suspension 100 μ l and be placed on white plastic color board, the color solution that adds 100 μ l, observes colour-change after 15min.Pink is positive, represents that bacterial strain can producing IAA, and pink color is more deeply felt and shown that producing IAA ability is larger; Colourless negative, represent that bacterial strain can not producing IAA.In color solution, add respectively the 10mg/L(CK1 of equivalent), 30mg/L(CK2), 50mg/L(CK3) IAA carries out the comparison of pink color depth as positive control.Result shows, the colorimetric reaction of described nodule azotobacter strain SCAUs152 is pink, and the pink degree of depth is between two positive control CK1 and CK2, illustrates that the amount of nodule azotobacter strain SCAUs152 producing IAA is between 10～30mg/L.
Amplification and the Phylogenetic Analysis of the 16SrRNA gene of nodule azotobacter strain SCAUs152 and other housekeeping genes glnII, atpD, recA described in embodiment 5
Extract the total DNA of bacterial strain, with primer shown in table 2, respectively above-mentioned 4 genes are carried out to pcr amplification, Bio-RAD MyCyclerTM instrument for PCR reaction, pcr amplification product is delivered to the mensuration that handsome company carries out sequence after detecting on 1.0% agarose gel electrophoresis.The gene amplification primer of this research is listed in table 2.With software DNAman6.0, carry out the calculating of gene order similarity.
PCR primer used in this test of table 2
Note: Y=CorT, H=A, CorT, R=AorG, S=CorG, K=GorT, N=A, C, GorT, I=inosine, M=AorC, N=anybase..
(1) amplification of 16SrRNA and the structure of phylogenetic tree
Take total DNA as template, with table 2 universal primer P1 and P6 amplification 16SrRNA.PCR reaction system (50 μ l): 2 * PCRMix25 μ l, primer P1 and P6(20 μ M) each 1 μ l, DNA profiling 1 μ l, adds ultrapure water and complements to 50 μ l.PCR reaction conditions: 95 ℃ of denaturation 5min; 95 ℃ of sex change 1min, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times; 72 ℃ of final 10min that extend.Amplified production detects the result of rear handsome company order-checking as stated above as SEQIDNo1.
SEQIDNo1 sequence:
CGCCGGGGCGGTGAGCCTTACCATGCAGCACGAGCGCCCCGCGCAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAACGTACCCTTTTCTACGGAATAACCCAGGGAAACTTGGACTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGAAAAGGATCGGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGATCGATCAGTCAGGGGTGAAATCCCAGAGCTCAACTCTGGAACTGCCTTTGATACTGTCGATCTGGAGTATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATGTCCGGCTACTTGCAGAGATGCAAGGTTCCCTTCGGGGACCGGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGACAGCGATGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTTTTACCCGAAGGTAGTGCGCTAACCGCAAGGTGGCAGCTATCCAGCGCAGTACGCGG
The sequence results of gained is compared at the state-run bioinformation of U.S. center (NCBI), select type strain that similarity is high as reference bacterial strain, phylogenetic tree construction.With the adjacent method in Mega5 software (Neighbor-joining), carry out the structure of 16S rRNA Phylogenetic Tree, the value of bootstrapping (bootstrap) 1000, its phylogenetic tree is shown in Fig. 2.From comparison result and phylogenetic tree, nodule azotobacter strain SCAUs152 belongs to rhizobium (Rhizobium), and its 16SrRNA gene order and type strain are Rhizobium grahamiiCCGE502
t, similarity is the highest, is 99.2%.
(2) association system of multidigit point gene sequence is grown the structure of tree
For further determining more accurately the classification position of described fast raw root nodule bacterium SCAUs152, alternative, select housekeeping gene atpD, recA and the glnII sequence in 3 sites and carry out the structure that association system is grown tree.
Amplification is primer recAF1, recAR1 for recA, primer atpDF3 and atpDR for atpD, and primer GSII-5 and GSII-6 for glnII, primer sequence is as shown in table 2.Reaction system is 50 μ l, and reaction solution is composed as follows: reaction system (50 μ l) is: 2 * PCRMix25 μ l; Each 0.5 μ l of the forward primer of 10mM and reverse primer; DNA profiling 1 μ l; ddH
2o23 μ l.(1) the pcr amplification reaction program of recA and atpD is identical: 95 ℃ of denaturation 5min; 94 ℃ of sex change 45s, 59 ℃ of annealing 45s, 74 ℃ are extended 1.5min, circulate 30 times; 74 ℃ of final 6min that extend.(2) glnII amplification condition: 92 ℃ of denaturation 3min; 94 ℃ of sex change 1min, 55 ℃ of annealing 1.5min, 72 ℃ are extended 2min, circulate 30 times; 72 ℃ of final 10min that extend.Amplified production send the order-checking of handsome company after detecting as stated above, each gene is carried out to two to order-checking (sequence of positive and negative primer), then by DNAman6.0 software splicing for positive and negative primer sequence, remove that to obtain respectively atpD sequence size after the sequence of positive and negative primer be that 495nt, sequence results are as SEQIDNo2, glnII sequence size be 638nt, sequence results as SEQIDNo3, recA sequence size is that 499nt, sequence results are as SEQI D No4.
SEQIDNo2 gene order:
ATCGGCGAGCCGGTCGACGAAGCCGGTCCGCTGGTTACCTCGGGCAAGCGCGCCATCCACCAGGACGCACCTGCCTACGTCGACCAGTCGACGGAAGCACAGATCCTCGTCACCGGCATCAAGGTCGTCGACCTGCTCGCACCTTACGCAAAGGGCGGCAAGATCGGCCTGTTCGGCGGCGCCGGCGTCGGCAAGACCGTTCTGATCATGGAACTGATCAACAACGTCGCCAAGGCGCACGGTGGTTACTCGGTATTCGCAGGCGTGGGTGAACGTACCCGCGAAGGCAACGACCTCTACCACGAAATGATCGAATCCGGCGTCAACAAGCATGGCGGCGGCGAAGGCTCCAAGGCTGCGCTCGTTTACGGCCAGATGAACGAACCGCCGGGCGCCCGCGCTCGCGTCGCTCTGACCGGTCTGACGATCGCCGAAGACTTCCGCGACAAGGGCCAGGACGTTCTGTTCTTCGTCGACAACATCTTCCGCTTTACC
SEQIDNo3 gene order:
CGATGGATACACCCCGGTGCCGAACCTTCGCGGCAAGACGCAGGTCAAGGAATTCGCGGAATTCCCGACGCTCGAGCAGCTGCCGCTGTGGGGCTTCGATGGCTCGTCCACGATGCAGGCCGAAGGCCGCAGCTCGGATTGCGTGCTGAAGCCTGTCGCCGTTTATCCGGACCCGGCTCGTACCAACGGCGTGCTCGTCATGTGCGAAGTCATGATGCCTGATGGTGTCACCCCGCATCCGTCCAACGCCCGCGCCACCATCCTTGACGATGAAGGCACCTGGTTCGGCTTCGAGCAGGAATACTTCTTCTACCAGAACGGCCGTCCGCTCGGCTTCCCCGAGCAGGGCTACCCGGCTCCGCAGGGCCCGTACTACACCGGCGTTGGATACTCCAACGTTGGCGACGTCGCTCGCGAGATCGTCGAAGAACACCTCGACCTGTGCCTGGCTGCCGGTATCAACCACGAAGGCATCAATGCCGAAGTGGCCAAGGGCCAGTGGGAATTCCAGATCTTCGGCAAGGGCTCCAAGCGCGCCGCTGACGAAATCTGGATGGCTCGCTACCTGCTTCAGCGCCTGACCGAAAAGTACGGCATCGACATCGAGTATCATTGCAAGCCGCTCGGCGACACCGACT
SEQIDNo4 gene order:
CAAAAGCAAGGCACTTGAAGCGGCACTCTCACAGATCGAGCGGTCGTTCGGCAAGGGCTCTATCATGAAGCTCGGCTCGAATGAGAGCGTGGTCGAGATCGAGACCGTTTCGACGGGCTCTCTCAGCCTGGATATCGCGCTCGGGATCGGCGGCCTTCCCAAGGGGCGTATCATCGAAATTTACGGGCCGGAAAGCTCGGGCAAGACGACGCTGGCGCTGCAGACGATCGCGGAAGCGCAGAAGAAGGGCGGCATCTGCGCCTTCGTCGACGCCGAGCACGCGCTCGATCCTGTCTACGCACGCAAGCTGGGCGTCGATCTGCACAATCTCCTGATCTCGCAGCCGGATACCGGTGAGCAGGCGCTCGAAATCACCGACACGCTGGTTCGCTCCGGCGCCGTCGACGTGCTGGTCGTGGATTCGGTCGCGGCGCTGACGCCGCGCGCCGAAATCGAAGGCGAGATGGGCGACAGCCTGCCGGGCCTGCAGGCGCGACTG
The sequence results of gained is compared at the state-run bioinformation of U.S. center (NCBI), and the type strain of the kind that selection and atpD, the recA of nodule azotobacter strain SCAUs152 and the sequence similarity degree of tri-site housekeeping genes of glnII are high is as the reference bacterial strain of contributing.3 genes (atpD, glnII and recA) association system is grown the structure of tree: first by the sequence of atpD, glnII, a recA3 housekeeping gene respectively with the corresponding gene sequences of reference bacterial strain, with MEGA5, compare, the minimum length of take is had one's hair trimmed as standard, sequence after having one's hair trimmed is saved as to FASTA form, and rear three the gene order length of having one's hair trimmed are respectively 443nt, 501nt, 411nt.With notepad form, open by 3 sequence assemblies together, with the adjacent method in MEGA5 software (Neighbor-joining), carry out the structure that association system is grown tree, the value of bootstrapping (bootstrap) 1000, atpD, glnII, recA association system are grown tree as shown in Figure 3.
By three gene orders of nodule azotobacter strain SCAUs152 respectively reference bacterial strain corresponding to it with DNAman6.0 software, carry out similar calculating, find the atpD of nodule azotobacter strain SCAUs152, tri-the highest similar type species of recA and glnII are respectively: Rhizobium grahamii, Rhizobium etli, Rhizobium grahamii, similarity is respectively 95.6%, 89.8%, 88.1%, three site housekeeping gene sequence similarity degree are not high, and four genes (comprising 16SrRNA gene) type species that similarity is the highest are also inconsistent, although the dendrogram (Fig. 2) of the 16SrRNA gene order of nodule azotobacter strain SCAUs152, and atpD, glnII, the dendrogram (Fig. 3) of recA3 housekeeping gene associating sequence shows, the type strain R.grahamiiCCGE502 of nodule azotobacter strain SCAUs152 and root nodule bacterium
ton same branch node, but especially housekeeping gene is combined Sequence clustering figure apart from each other, thereby the new bacterial strain that definite nodule azotobacter strain SCAUs152 is rhizobium (Rhizobium) is.
Embodiment 6 Field inoculation effects
Soybean principal producting area, Sichuan is hills area, Sichuan, and soybean varieties is Sichuan main breed " southern beans 12 ", mainly as summer soybean and autumn soybean varieties.The habitat difference of the resourceful Panxi Diqu of photo-thermal and hills area is large, and Panxi Diqu mainly be take early soybean as main, and introducing a fine variety in recent years soybean " so No. 8 " and " rich morning in spring " is the main cultivation soybean varieties of this ecotope.The Field inoculation effect test of described bacterial strain selects that soybean main producing region, typical Sichuan---the hills area of Yucheng District, Ya'an, Sichuan, and Panxi Diqu---carry out Panzhihua City Renhe District.
(1) hills area---the Field inoculation test in Yucheng District, Ya'an, Sichuan
Two processing are established in this test altogether, inoculate nodule azotobacter strain SCAUs152 and do not inoculate control treatment (CK), and beans kind is selected Sichuan main breed " southern beans 12 ".In plantation, do not execute any chemical fertilizer.Tested in June, 2012～October and carry out.By the Rhizobium Inoculant preparing (viable count 6.0 * 10
8cFU/g microbial inoculum) with soybean seed dressing, bunch planting after drying in the shade, 6, every nest, final singling 2 strains, community area 4.2m
2, nest is apart from 40cm, and line-spacing 35cm, first broadcasts CK during sowing, to avoid CK to process the impact that is subject to Rhizobium Inoculation.In plant full-bloom stage (64d breeding time) sampling, measure plant plant height, root nodule number, over-ground part plant dry weight; Measure output harvesting time (131d breeding time).Management is during this time pressed the Routine Management of Cotton Varieties by Small Farming Households soybean and is carried out.
The Field inoculation effect of Yucheng District, table 3 Ya'an (hills area)
The processing of Rhizobium Inoculation, all obviously increases than not inoculating contrast at plant height, plant dry weight, the root nodule number of full-bloom stage, increases by 10.3%, 22.9%, 27.7% respectively than CK, and output is than the remarkable increase of CK, volume increase 36.6%.Visible, the good root nodule bacterium of inoculation are more obvious to the promoter action of plant full-bloom stage growth later.Described nodule azotobacter strain SCAUs152 is the good root nodule bacterium that are applicable to this ecotope (hills area, Sichuan) Soybean production.
(2) the field indirect test of Panxi Diqu
It is local main breed " so No. 8 " that beans kind is selected in this test, establishes altogether two processing, inoculates nodule azotobacter strain SCAUs152 and does not inoculate contrast (CK) and process, and establishes 3 repetitions.Before plantation, press P
2o
545kg/ha, K
2o45kg/ha applies fertilizer to the subsoil, and by disposable the putting into of unified standard, phosphate fertilizer is calcium superphosphate, and potash fertilizer is Repone K.Test adopts randomized block design, Ridged plant, 4 ridges, every community, row spacing 60cm, interval 20cm between ridge, the long 5m in ridge.On ridge, plant two row, line-spacing 40cm, nest is apart from 30cm, and 32 nests are planted on every ridge.Tested in May, 2013～September and carry out.By above-mentioned nitragin and soybean " so No. 8 " seed dressing, bunch planting after drying in the shade, 5, every nest, final singling 2 strains, equally first broadcast CK during sowing.In just pod phase (67d breeding time) sampling of plant, measure plant plant height, root nodule number, over-ground part plant dry weight; Measure output harvesting time (98d breeding time).Management is during this time carried out by the Routine Management of local Cotton Varieties by Small Farming Households soybean.
The Field inoculation effect of table 4 Panxi Diqu
The processing of Rhizobium Inoculation, plant height, plant dry weight, root nodule number in the first pod phase all obviously increase than not inoculating contrast (CK), increase respectively 20.3%, 35.1%, 59.1%, and output, than the remarkable increase of CK, increases 23.1%.Visible, the main cultivation soybean varieties in described nodule azotobacter strain SCAUs152 and Panxi Area, Sichuan Province " so No. 8 " matching is good, is the good root nodule bacterium that are applicable to climbing western ecotope Soybean production.
In Sichuan, the Field inoculation experimental study of two typical ecotope is found, inoculates after described nodule azotobacter strain SCAUs152, and plant dry weight, root nodule number and output is all higher than not inoculating contrast, and has significant effect of increasing production, can increase production more than 23%.Visible, the separated nodule azotobacter strain SCAUs152 obtaining of the present invention can be in the Soybean production of Sichuan large-area applying.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.
Claims (2)
1. a nodule azotobacter strain SCAUs152, is characterized in that, its deposit number is CCTCC NO:M2014085.
2. the application of nodule azotobacter strain SCAUs152 according to claim 1, is characterized in that, is applied to the production of Sichuan soybean.
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