CN103952341B - A kind of nodule azotobacter strain SCAUs152 and application thereof - Google Patents
A kind of nodule azotobacter strain SCAUs152 and application thereof Download PDFInfo
- Publication number
- CN103952341B CN103952341B CN201410150948.5A CN201410150948A CN103952341B CN 103952341 B CN103952341 B CN 103952341B CN 201410150948 A CN201410150948 A CN 201410150948A CN 103952341 B CN103952341 B CN 103952341B
- Authority
- CN
- China
- Prior art keywords
- strain
- soybean
- scaus152
- sichuan
- rhizobium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- Y02P60/216—
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of nodule azotobacter strain SCAUs152 and application thereof, this bacterial strain system is that separation and purification obtains from fresh soybean nodulation, and the new bacterial strain that belongs to rhizobium (Rhizobium) is. This bacterial strain system is preserved in Wuhan University's Chinese Typical Representative culture collection center on March 14th, 2014, and its deposit number is CCTCCNO:M2014085. This bacterial strain system 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 Phos, organophosphor and molten potassium capability, good wide spectrum soybean nodulation bacteria strain that resistance is stronger. And mate compatibility with the main cultivation beans kind 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 the level of signifiance.
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 rate is low, pollution ecologyEnvironment. 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 ecological environmentWith agriculture sustainable development, it is the effective means of agricultural production cost-saving synergistic. In grain-production, need to make great efforts outSend out the biological nitrogen fixation technology useful to environment. Rhizobium-legume fixed nitrogen system is most effective in biological nitrogen fixation.Legume artificial infection rhizobium 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 in biological nitrogen fixationTypical Representative. Synbiosis between soybean and rihizobium japonicum is in long-term evolution, to interact, select mutuallyResult, be also with environment facies adapt to result. The country of other plantation soybean of the world is in the technology of application rihizobium japonicumOn have very large advantage. But China is the area of origin of soybean, plant the with a long history of soybean, rhizobium and soybean associationWith evolving, make the Indigenous Rhizobia that in soil, nitrogen-fixing efficiency is low there is the stronger compatibility of mating with soybean varieties, thus largeReduce greatly the action effect of high-efficiency nitrogen-fixing rhizobium inoculant. Therefore study and grasp rhizobium and soybean varieties matchingThere is important more practical value.
Improve symbiotic azotification, visible bacterial strain and soybean varieties should be two basic factors. And the colony of rhizobium dividesCloth has geographical limitation, in the screening of rhizobium, should be noted that its corresponding adaptive capacity with area surroundings. Generally, in certain region the most effectively rhizobium often from this area or with the bacterium in conditional likelihood area, this areaStrain. Therefore in the time that rhizobium are chosen seeds, not only to carry out the matching combination research of rhizobium and soybean varieties, also must be heavyDepending on the region of microbial inoculum application.
Soybean is Chinese the fourth-largest crops, is deeply subject to liking of Chinese people, and edible way is varied, of many uses,The important cereal crops of China and oil crops. Therefore carry out good soybean nodulation bacterial strain screening and application technology in ChinaResearch work is very meaningful. Northeast soybean producing region is the maximum soybean of China producing region, and Huang-Huai-Hai is China soybean secondLarge producing region, with the main cultivation soybean varieties in two large producing regions of this maximum and the research of the strain excellent that ecological environment mates relativelyMany. For arid soybean producing region, northwest, the screening of good rihizobium japonicum also has report. For first of China soybeanProducing region: producing region, northeast, the people such as Du Yinghui separate from Heilungkiang and obtain a strain with Soybean Varieties In The Northeast of China matching is good and applicable easternThe strain excellent in backlands district, gets permission patent of invention in June, 2012. For China's South China Acidic Soil, Cao Guiqin etc.People is south China 3 the rhizobium strains of acidproof, resistance to aluminium that lacked the seed selection of phosphorus characteristic of acid red soil, has applied for 3 in December, 2007Individual patent of invention. But what match for the soybean varieties in the soybean producing region, Sichuan in Southern soybean producing region and ecological environment is goodRhizobium are studied rarely seen report.
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, fourRiver Soybean production development is swift and violent, occurs scale, large area contiguous plant, and yield level increases substantially, and becomesOne of Sichuan Province's staple food crop, sown area is the 6th of national rank. The Ministry of Agriculture is at " plant production development the 12Five planning " list one of national three advantages producing region, state in by overlapping human consumption soybean between Sichuan and southwest in (2011-2015)Family the Committee of Development and Reform again Chongqing of Sichuan soybean is included in country 12 staple food crops, Sichuan soybean caused country and Sichuan Province eachThe attention of level government. Sichuan soybean culture mode is mainly and corn intercropping and interplanting plantation, accounts for the whole province's soybean acreageMore than 90%. Therefore, the good rhizobium of inoculation coupling in the Soybean production of Sichuan, the intercropping and interplanting system pair of Sichuan soybean" ammonia checks " of rhizobium nitrogenase activity can also play mitigation. The leading kind of Sichuan interplanting soybean is " southern beans 12 "" tribute is selected No. 1 ". The soybean essentialspecies of Sichuan intercropping and interplanting is implanted in hills area, Sichuan again, the resourceful ground, west of climbing of photo-thermalDistrict is mainly taking 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 fourThe good rihizobium japonicum of river different ecological environment. For this reason, for the main breed in Sichuan and the main ecology of plantation soybeanEnvironment carries out the screening of soybean strain excellent, give full play to biological nitrogen fixation, improves biological nitrogen fixation in production practicesEffect, thus using of chemical fertilizer reduced, this saves energy resources for preserving the ecological environment, and promoting agricultural can holdSupervention exhibition has 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 is providedSCAUs152 and application thereof.
Technical scheme of the present invention is as follows:
A kind of nodule azotobacter strain SCAUs152, its Classification And Nomenclature is Rhizobiumsp.SCAUs152, in 2014Be preserved in Wuhan University's Chinese Typical Representative culture collection center on March 14, in, and its deposit number is CCTCCNO: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 varietiesWide accommodation, with producing IAA ability, there is molten Phos, organophosphor and molten potassium capability, resistance is strongerGood wide spectrum soybean nodulation bacteria strain. And mate compatibility with the main cultivation soybean varieties in Sichuan good, do not execute in planted in different ecological areasNitrogenous fertilizer, inoculation nodule azotobacter strain SCAUs152 make soybean yield-increasing more than 23%, and do not inoculate the difference contrasting and reach aobviousWork level.
Brief description of the drawings
Fig. 1 is the colonial morphology of nodule azotobacter strain SCAUs152 on YMA medium.
Fig. 2 is the systematic growth figure of the 16rRNA gene order of nodule azotobacter strain SCAUs152.
Fig. 3 is glnII, atpD, tri-the housekeeping gene joint mappings of recA of nodule azotobacter strain SCAUs152Systematic growth figure.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Separation, purifying and the preservation of embodiment 1 rhizobium
The soybean of planting from the Hills And Low Mountains of Liangshan State of Sichuan Province Yanyuan County, select on healthy and strong plant main root large and fullRed root nodule, by root nodule scrub, under band portion Gen Picai, paper using blots and places it in anhydrous calcium chloride is housedAnd be covered with in the tubule of absorbent cotton. In laboratory, the root nodule gathering is soaked after imbibition with sterilized water, soak with 95% ethanolBubble 30s is to eliminate surface tension, then with 0.1%(m/v) mercuric chloride surface sterilization 5min, then use aseptic water washing6 times, the in the situation that of sterile working, after being clipped broken, single root nodule is being added with Congo red YAM culture medium (sweet mellow wine10g, dusty yeast 0.8g, KH2PO40.25g，MgSO4.7H2O0.2g，CaCl2.6H2O0.1g，NaCl0.1g，1%(m/v) sodium molybdate 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 insulating box of 28 DEG C, cultivate.
After growing bacterium colony, select and do not inhale bacterium colonies red, that form looks like rhizobium in flat board dilution line training from flat boardSupport. 3d observes colonial morphology in left and right, observes 15d left and right always, occurs bacterium colony because slow raw rhizobium need 6～15d.Repeat dilution line and repeatedly separate, until purifying. Whether carry out preliminary judgement according to following two aspects is rhizobium:(1) add the colonial morphology on Congo red YMA medium: do not inhale red, bacterium colony circle, milky, protuberance, limitEdge does not neatly spread, smooth surface, compared with thickness, more moistening. What cultivation 3～5d just grew bacterium colony is fast raw rhizobium,What cultivation 6～15d grew bacterium colony is slow raw rhizobium. (2) cellular morphology: the rhizobium bacterium colony of confirming is marked,Gram's staining is carried out in film-making, and it is little shaft-like and form is consistent that the microscopy result of rhizobium is that cell is, without gemma, in cellNormal containing beta-hydroxy-butanoic acid and in the form of link, Gram-negative (G-). If above-mentioned mark bacterium colony has above-mentioned two Fang MianteLevy, 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 rhizobium, add Congo redYMA medium on cultivate, thalline is not inhaled red, bacterium colony is large, circle, milky, protuberance degree are higher, moistening,More transparent, 3d left and right grows bacterium colony. Be G-through gram stain microscopy, be little shaft-like.
The tieback of embodiment 2 rhizobium and matching test
The tieback test water training method of rhizobium, the soybean varieties of using in test is the leading kind " southern beans 12 " in Sichuan,In illumination chamber, (22～24 DEG C of temperature controls, intensity of illumination 2700～3000 luxs, sunshine-duration 14h) carries out, plantation46d results. After successful with " southern beans 12 " tieback, carry out matching test, also water with other main cultivation soybean varieties againTraining method is carried out, the soybean varieties of using in test: another main cultivation soybean varieties " tribute is selected No. 1 " of intercropping and interplanting, early soybean productThe main cultivation of kind of " river beans 14 ", Panxi Diqu eat soybean varieties " rich morning in spring " raw. Cultivate after 1 week in above-mentioned illumination chamber,Be placed under normal illumination and temperature conditions and cultivate, plantation 41d results. The aseptic micro-nitrogen nutrition liquid of regular replenishment. By root noduleNitrogen-fixing bacteria strain SCAUs152 and above-mentioned soybean varieties form various combination, and hydroponics device adopts the arrow-necked bottle (doctor of 250mlThe glass infusion bottle that institute uses), taking the same kind soybean plant strain of not inoculating nodule azotobacter strain SCAUs152 as contrast.After results, evaluate by root nodule number and the plant dry weight of soybean plant strain 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 fluid nutrient medium, placesOn shaking table, use 120rpm/min rotating speed, 28 DEG C are cultured to exponential phase (about 4d left and right).
(2) vernalization of seed: select large, the full undamaged soya 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 DEG C 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 arrow-necked bottle (glass infusion bottle that hospital uses) of 250ml. First systemMake aseptic micro-nitrogen nutrition liquid, micro-nitrogen nutrition formula of liquid: 0.46g calcium sulfate, 0.075g potassium chloride, 0.06g magnesium sulfate,0.03g calcium nitrate, 0.075g ironic citrate, 0.136g dipotassium hydrogen phosphate, 1000ml distilled water, 1ml trace unitElement (2.86g boric acid, 0.02g molybdic acid, 0.8g copper sulphate, 0.22gZnSO4 zinc sulfate, 1.81g manganese 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 ox-hidePaper, opens an aperture (diameter 0.6cm) in bottleneck centre, and aperture is cotton beyond the Great Wall, the resistant to elevated temperatures plastics of outer cover one deckFilm, at 121 DEG C of temperature, sterilizing is for subsequent use.
(4) plantation and testing index: vernalization seed is placed in to sterile petri dish, with bacterium immersion bubble 15min in (1),The root of seedling is inserted in hydroponics device aperture with aseptic nipper, every bottle of 1 strain, then every seedling adds bacterium in (1) againLiquid 1ml, seed is good with the cotton plug in original hole around, prevents that dust from falling in bottle, pollutes. Separately establish and do not inoculateThe same product plant of processing is contrast (CK). When plantation, first plant CK. Each processing repeated 3 times. Water culture experiment result rowIn table 1.
Table 1 result shows, described nodule azotobacter strain SCAUs152 and 4 compatibilities of mating for examination soybean varietiesAll good, all show good Noduling ability and symbiotic nitrogen fixation ability; Compare with contrasting of Rhizobium Inoculation not, root nodule is solidNitrogen bacterial strain is the plant dry weight that SCAUs152 can significantly improve each kind soybean, than not inoculating described nodule azotobacter strainThe contrast that is SCAUs152 improves 32%～43%. Visible, described nodule azotobacter strain SCAUs152 is and SichuanThe good wide spectrum bacterial strain that soybean varieties matching is good.
The water culture experiment result of table 1 nodule azotobacter strain SCAUs152
Note: data are three mean values that repeat; * and * represent respectively inoculation process dry weight between corresponding contrast reach 1%, 5% remarkableLevel.
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 greenhouseScope is measured. Taking YMA medium as basal medium, all taking pH7,28 DEG C of YMA flat boards of cultivating 7d as sunProperty contrasts. 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 basal medium of measuring taking YMA medium as resistance to acids and bases, with HC1 andNaOH regulates pH value, and pH value is followed successively by 4.0,5.0,6.0,8.0,9.0,10.0,11.0,12.0. Salt tolerance is surveyedFixed equally taking YMA medium as basal medium, described bacterial strain point is seeded on the flat board that contains NaC1 to NaC1Quality volume fraction be 1%, 2%, 3%, 4% and 5%. The equal 28 DEG C of cultivations 7 of flat board of acid and alkali-resistance, Salt toleranceAfter d, observe and record result. Growth temperature range is measured, and described inoculation, on YMA medium, is established to 5 altogetherTemperature Treatment is cultivated 30d, 10d, 7d, 7d respectively in 4 DEG C, 10 DEG C, 37 DEG C, 40 DEG C biochemical cultivation cases, anotherAt one 60 DEG C of processing, heat shock is processed after 30min, forwards at 28 DEG C and cultivates 7d. Result of the test shows described nodule nitrogen fixationBacterial strain is that SCAUs152 anti-adversity ability is stronger, can on the flat board of pH5～12, grow, but on the flat board of peracid or excessively alkaliBacterium colony is less than the positive control of pH7, illustrates that peracid crosses alkali its growth is had to certain inhibitory action; There is certain salt tolerantAbility can be grown on the YMA of 2%NaCl flat board; Growth temperature range is wide, can be in 4～40 DEG C of temperature rangesGrowth, and this bacterial strain still can survive after 30min is processed in 60 DEG C of heat shocks, illustrates that this bacterial strain can stand the height of short timeTemperature.
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 it is artificialThe phosphate fertilizer this season crop utilization rate applying is for only having 5%～25%, and major part is fixed by soil chemistry very soon and formed to close and holdsState insoluble phosphate. The ecological chain of this " high investment, low output ", not only causes 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 China soluble potassium scarcity of resources, potassium bearing rock withAnd potassium-bearing mineral particle in soil is but very abundant, but potassium in these potassium-bearing minerals is mainly containing potassium silicic acid with slightly solubilitySalt form exists, and can not be directly Crop utilization. Therefore, by functions such as screening high-efficiency nitrogen-fixing, the molten potassium of molten phosphorusProperty microorganism improve the utilization ratio of atmospheric nitrogen and soil phophorus, potassium, be acknowledged as cheapness and have efficiently environment protection significanceBiological control measure.
The growth-promoting ability of described nodule azotobacter strain SCAUs152 is mainly investigated its molten phosphorus ability, molten potassium capability and is dividedSecrete the ability of auximone (IAA).
(1) ability of molten organophosphor and Phos
By molten phosphorus circle method. Organic phosphorus sources is lecithin, and inorganic phosphorous sources is calcium phosphate (Ca3(PO4)2), aluminum phosphate（AlPO4.2H2O), ferric phosphate (FePO4.2H2O), be commercially available AR.
Measure the culture medium Meng Jinna culture medium of dissolved organic phosphorus ability, formula (g/L): 10g glucose, 0.5g(NH4)2SO4，0.3gNaCl，0.3gKCl，0.03gFeSO4.7H2O，0.03gMnSO4.4H2O, 0.2g ovum phosphorusFat, 5gCaCO3, 0.4g dusty yeast, 20g agar, 1000ml distilled water, pH value 6.8～7.0. Wherein lecithinBy 75% alcohol heating for dissolving, separately sterilizing, is down flat plate after the culture medium that is cooled to 60 DEG C of left and right mixes with sterilizing.
Measure the culture medium PKO culture medium that dissolves Phos ability, formula (g/L): 10g glucose, on 3.0gState inorganic phosphorous sources material, 0.5g (NH4)2SO4，0.2gNaCl，0.2gKCl，0.03gMgSO4.7H2O，0.03gMnSO4，0.003gFeSO4.7H2O, 0.5g dusty yeast, 20g agar, 1000ml distilled water, pH value 6.8～7.0.Wherein calcium phosphate, aluminum phosphate, ferric phosphate pulverized after 300 mesh sieves independent hot air sterilization with mortar, fell with sterilising tempMix and be down flat plate to the culture medium of 60 DEG C of left and right, stand-by. Bacterial classification preparation and some inoculation method are tested with embodiment 3 resistance,Repeat 3 times. Whether whether after 28 DEG C of incubators cultivation 7d, observe bacterial strain grows and has molten phosphorus to iris out now. Result showsDescribed nodule azotobacter strain SCAUs152 shows as and does not grow on aluminum phosphate flat board, to calcium phosphate, ferric phosphate and ovumPhosphatide all has certain solvability, molten phosphorus loop diameter and the bacterium colony on lecithin, calcium phosphate, ferric phosphate flat board, measuredDiameter ratio is respectively 1.20,1.13,1.12.
(2) molten potassium capability
Measure the culture medium (g/L) of molten potassium capability: 5.0g sucrose, 2.0gNa2HPO4，0.05gMgSO4.7H2O，0.05gFeCl3，0.1gCaCO3, 5g feldspar in powder, 20.0g agar, distilled water 1000ml. Wherein feldspar in powder, warpGrind to form powdery, cross 300 mesh sieves, wash away water-soluble K, Si plasma by deionized water, dry in the shade. Hot air sterilization separatelyAfter, after mixing, the culture medium that is cooled to 60 DEG C of left and right with sterilizing is down flat plate, and stand-by. Bacterial classification preparation and some inoculation method are with moltenThe mensuration of phosphorus ability, repeats 3 times. After 28 DEG C of incubators cultivation 7d, observe bacterial strain and whether grow, because this culture medium is with potassiumFeldspar powder is unique potassium source, if growth just shows that bacterial strain has molten potassium capability. This result of the test shows, described nodule nitrogen fixationBacterial strain be SCAUs152 taking feldspar in powder normal growth on the flat board in unique potassium source, show that SCAUs152 has oneFixed molten potassium capability.
(3) mensuration of plant auxin secretion ability
Adopt the ability of colorimetric method for determining rhizobium plant auxin secretion (IAA), measure culture medium and adopt the firm of improvementArnotto fluid nutrient medium, culture medium composition: 0.5gK2HPO4.3H2O、0.2gMgSO4.7H2O、0.1gNaCl、1gYeast extract, 10g sweet mellow wine, 10ml0.25%(m/v) Congo red, 1gNH4NO3, 100mgL-tryptophan, 1000Ml distilled water, pH7.0. Color solution formula: 0.5MFeCl31ml, dense H2SO430ml, distilled water 50ml.
Inoculation, in filling the triangular flask of 50ml culture medium, is placed in to rotating speed 125rpm/min, 28 DEG C of temperatureShaking table is cultivated, and repeats for 3 times, cultivates after 12d, and get rhizobium suspension 100 μ l and be placed on white plastic color board,Add the color solution of 100 μ l, after 15min, observe change color. Pink is positive, represents that bacterial strain can producing IAA,Pink color is more deeply felt and is shown that producing IAA ability is larger; Colourless negative, represent that bacterial strain can not producing IAA. ThanIn look liquid, add respectively the 10mg/L(CK1 of equivalent), 30mg/L(CK2), 50mg/L(CK3) IAA does positiveThe comparison of pink color depth is carried out in contrast. Result shows, the colorimetric of described nodule azotobacter strain SCAUs152 is anti-Should be pink, and the pink degree of depth is between two positive control CK1 and CK2, and nodule azotobacter strain is describedThe amount of SCAUs152 producing IAA is between 10～30mg/L.
The 16SrRNA gene of nodule azotobacter strain SCAUs152 and other housekeeping genes glnII described in embodiment 5,The amplification of atpD, recA and Phylogenetic Analysis
Extract bacterial strain total DNA, respectively above-mentioned 4 genes are carried out to pcr amplification with primer shown in table 2, PCR reactsUse Bio-RADMyCyclerTM instrument, pcr amplification product send after detecting on 1.0% agarose gel electrophoresisCarry out the mensuration of sequence to handsome company. The gene magnification primer of this research is listed in table 2. With software, DNAman6.0 entersThe calculating of row 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
Taking 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 ultra-pure water and complements to50 μ l. PCR reaction condition: 95 DEG C of denaturation 5min; 95 DEG C of sex change 1min, 56 DEG C of annealing 30s, 72 DEG C are extended 1Min, circulates 30 times; 72 DEG C of final 10min that extend. Amplified production detects the knot of rear handsome company order-checking as stated aboveFruit is as SEQIDNo1.
SEQIDNo1 sequence:
CGCCGGGGCGGTGAGCCTTACCATGCAGCACGAGCGCCCCGCGCAGGGGAGCGGCAGACGGGTGAGTAACGCGTGGGAACGTACCCTTTTCTACGGAATAACCCAGGGAAACTTGGACTAATACCGTATGTGCCCTTCGGGGGAAAGATTTATCGGAAAAGGATCGGCCCGCGTTGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCCATAGCTGGTCTGAGAGGATGATCAGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTCACCGGTGAAGATAATGACGGTAACCGGAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGTTCGGATTTACTGGGCGTAAAGCGCACGTAGGCGGATCGATCAGTCAGGGGTGAAATCCCAGAGCTCAACTCTGGAACTGCCTTTGATACTGTCGATCTGGAGTATGGAAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGAAGGCGGCTCACTGGTCCATTACTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGTTAGCCGTCGGGCAGTATACTGTTCGGTGGCGCAGCTAACGCATTAAACATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCCCTTGACATGTCCGGCTACTTGCAGAGATGCAAGGTTCCCTTCGGGGACCGGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGGGACTGCCGGTGATAAGCCGAGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGGGCTGGGCTACACACGTGCTACAATGGTGGTGACAGTGGGCAGCGAGACAGCGATGTCGAGCTAATCTCCAAAAGCCATCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGA GTTGGTTTTACCCGAAGGTAGTGCGCTAACCGCAAGGTGGCAGCTATCCAGCGCAGTACGCGG
The sequence results of gained is compared at the state-run biological information of U.S. center (NCBI), select similitude highType strain is as reference bacterial strain, phylogenetic tree construction. With the adjacent method in Mega5 software (Neighbor-joining)Carry out the structure of 16SrRNA 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),Its 16SrRNA gene order and type strain are RhizobiumgrahamiiCCGE502T, similarity is the highest, for99.2%。
(2) association system of multidigit point gene sequence is grown the structure of tree
Select 3 sites for further determining more accurately the classification position of described fast raw rhizobium SCAUs152, alternativeHousekeeping gene atpD, recA and glnII sequence are carried out association system and are grown the structure of setting.
Amplification is primer recAF1, recAR1 for recA, primer atpDF3 and atpDR for atpD, glnII primer GSII-5And GSII-6, primer sequence is as shown in table 2. Reaction system is 50 μ l, and reactant liquor is composed as follows: (50 μ l) for reaction systemFor: 2 × PCRMix25 μ l; The each 0.5 μ l of the forward primer of 10mM and reverse primer; DNA profiling 1 μ l; ddH2O23 μ l. (1) the pcr amplification reaction program of recA and atpD is identical: 95 DEG C of denaturation 5min; 94 DEG C of sex change 45s,59 DEG C of annealing 45s, 74 DEG C are extended 1.5min, circulate 30 times; 74 DEG C of final 6min that extend. (2) glnII amplification barPart: 92 DEG C of denaturation 3min; 94 DEG C of sex change 1min, 55 DEG C of annealing 1.5min, 72 DEG C are extended 2min, circulation 30Inferior; 72 DEG C of final 10min that extend. Amplified production send the order-checking of handsome company after detecting as stated above, each gene is enteredRow two, to order-checking (sequence of positive and negative primer), then splices positive and negative primer sequence with DNAman6.0 software,Remove obtain respectively after the sequence of positive and negative primer atpD sequence size be 495nt, sequence results as SEQIDNo2, glnIISequence size be 638nt, sequence results as SEQIDNo3, recA sequence size is that 499nt, sequence results are as SEQIDNo4。
SEQIDNo2 gene order:
ATCGGCGAGCCGGTCGACGAAGCCGGTCCGCTGGTTACCTCGGGCAAGCGCGCCATCCACCAGGACGCACCTGCCTACGTCGACCAGTCGACGGAAGCACAGATCCTCGTCACCGGCATCAAGGTCGTCGACCTGCTCGCACCTTACGCAAAGGGCGGCAAGATC GGCCTGTTCGGCGGCGCCGGCGTCGGCAAGACCGTTCTGATCATGGAACTGATCAACAACGTCGCCAAGGCGCACGGTGGTTACTCGGTATTCGCAGGCGTGGGTGAACGTACCCGCGAAGGCAACGACCTCTACCACGAAATGATCGAATCCGGCGTCAACAAGCATGGCGGCGGCGAAGGCTCCAAGGCTGCGCTCGTTTACGGCCAGATGAACGAACCGCCGGGCGCCCGCGCTCGCGTCGCTCTGACCGGTCTGACGATCGCCGAAGACTTCCGCGACAAGGGCCAGGACGTTCTGTTCTTCGTCGACAACATCTTCCGCTTTACC
SEQIDNo3 gene order:
CGATGGATACACCCCGGTGCCGAACCTTCGCGGCAAGACGCAGGTCAAGGAATTCGCGGAATTCCCGACGCTCGAGCAGCTGCCGCTGTGGGGCTTCGATGGCTCGTCCACGATGCAGGCCGAAGGCCGCAGCTCGGATTGCGTGCTGAAGCCTGTCGCCGTTTATCCGGACCCGGCTCGTACCAACGGCGTGCTCGTCATGTGCGAAGTCATGATGCCTGATGGTGTCACCCCGCATCCGTCCAACGCCCGCGCCACCATCCTTGACGATGAAGGCACCTGGTTCGGCTTCGAGCAGGAATACTTCTTCTACCAGAACGGCCGTCCGCTCGGCTTCCCCGAGCAGGGCTACCCGGCTCCGCAGGGCCCGTACTACACCGGCGTTGGATACTCCAACGTTGGCGACGTCGCTCGCGAGATCGTCGAAGAACACCTCGACCTGTGCCTGGCTGCCGGTATCAACCACGAAGGCATCAATGCCGAAGTGGCCAAGGGCCAGTGGGAATTCCAGATCTTCGGCAAGGGCTCCAAGCGCGCCGCTGACGAAATCTGGATGGCTCGCTACCTGCTTCAGCGCCTGACCGAAAAGTACGGCATCGACATCGAGTATCATTGCAAGCCGCTCGGCGACACCGACT
SEQIDNo4 gene order:
CAAAAGCAAGGCACTTGAAGCGGCACTCTCACAGATCGAGCGGTCGTTCGGCAAGGGCTCTATCATGAAGCTCGGCTCGAATGAGAGCGTGGTCGAGATCGAGACCGTTTCGACGGGCTCTCTCAGCCTGGATATCGCGCTCGGGATCGGCGGCCTTCCCAAGGGGCGTATCATCGAAATTTACGGGCCGGAAAGCTCGGGCAAGACGACGCTGGCGCTGCAGACGATCGCGGAAGCGCAGAAGAAGGGCGGCATCTGCGCCTTCGTCGACGCCGAGCACGCGCTCGATCCTGTCTACGCACGCAAGCTGGGCGTCGATCTGCACAATCT CCTGATCTCGCAGCCGGATACCGGTGAGCAGGCGCTCGAAATCACCGACACGCTGGTTCGCTCCGGCGCCGTCGACGTGCTGGTCGTGGATTCGGTCGCGGCGCTGACGCCGCGCGCCGAAATCGAAGGCGAGATGGGCGACAGCCTGCCGGGCCTGCAGGCGCGACTG
The sequence results of gained is compared at the state-run biological information of U.S. center (NCBI), select and nodule nitrogen fixationBacterial strain is the pattern of the kind that atpD, the recA of SCAUs152 and the sequence similarity degree of tri-site housekeeping genes of glnII are highBacterial strain is as the reference bacterial strain of contributing. 3 genes (atpD, glnII and recA) association system is grown the structure of tree: firstBy the sequence of atpD, glnII, a recA3 housekeeping gene respectively with the corresponding gene sequences of reference bacterial strain, use MEGA5Comparison, has one's hair trimmed taking minimum length as standard, the sequence after having one's hair trimmed is saved as to FASTA form, rear three genes of having one's hair trimmedSequence length is respectively 443nt, 501nt, 411nt. Open by 3 sequence assemblies together with notepad form, useThe structure that adjacent method (Neighbor-joining) in MEGA5 software carries out association system grows 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 reference bacterial strain use corresponding with it respectivelyDNAman6.0 software carries out similar calculating, finds atpD, recA and the glnII of nodule azotobacter strain SCAUs152Three the highest similar type sepecies are respectively: Rhizobiumgrahamii, Rhizobiumetli, Rhizobiumgrahamii,Similarity is respectively 95.6%, 89.8%, 88.1%, three site housekeeping gene sequence similarity degree is not high, and four genes(comprising 16SrRNA gene) type sepecies that similarity is the highest are also inconsistent, although nodule azotobacter strain SCAUs152The dendrogram (Fig. 2) of 16SrRNA gene order, and atpD, glnII, a recA3 housekeeping gene associating sequenceDendrogram (Fig. 3) show, the type strain R.grahamiiCCGE of nodule azotobacter strain SCAUs152 and rhizobium502TOn same branch node, but especially housekeeping gene is combined Sequence clustering figure apart from each other, thereby determines that root nodule is solidNitrogen bacterial strain is that SCAUs152 is the new bacterial strain system of rhizobium (Rhizobium).
Embodiment 6 Field inoculation effects
Soybean major producing area, Sichuan is hills area, Sichuan, and soybean varieties is Sichuan main breed " southern beans 12 ", mainly doesFor summer soybean and autumn soybean varieties. The habitat difference of the resourceful Panxi Diqu of photo-thermal and hills area is large, Panxi Diqu masterWill be taking early soybean as main, introduce a fine variety in recent years the main cultivation soybean product that soybean " so No. 8 " and " rich morning in spring " they are this ecotopeKind. The Field inoculation effect test of described bacterial strain is selected soybean main producing region, typical Sichuan---Yucheng District, Ya'an, SichuanHills area, and Panxi Diqu---carry out Panzhihua City Renhe District.
(1) hills area---in the Field inoculation test of Yucheng District, Ya'an, Sichuan
Two processing are established in this test altogether, and inoculate nodule azotobacter strain SCAUs152 and do not inoculate control treatment (CK),Beans kind is selected Sichuan main breed " southern beans 12 ". In plantation, do not execute any chemical fertilizer. Test in June, 2012～10The moon carries out. By the Rhizobium Inoculant preparing (viable count 6.0 × 108CFU/g microbial inoculum) dress seed with soybean, cave after drying in the shadeBroadcast 6, every nest, final singling 2 strains, community area 4.2m2, nest is apart from 40cm, and line-spacing 35cm, first broadcasts CK when 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 strainHeight, root nodule number, aerial part plant dry weight; Measure output harvest time (131d breeding time). Management is during this time by agricultureThe Routine Management of family plantation soybean 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,Increase by 10.3%, 22.9%, 27.7% than CK respectively, output, than the remarkable increase of CK, increases production 36.6%. Visible, connectThe good rhizobium of planting are more obvious to the facilitation of plant full-bloom stage growth later. Described nodule azotobacter strainSCAUs152 is the good rhizobium 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, inoculation nodule azotobacter strainBe SCAUs152 and do not inoculate contrast (CK) process, establish 3 repetitions. Before plantation, press P2O545kg/ha，K2O45Kg/ha applies fertilizer to the subsoil, and by disposable the putting into of unified standard, phosphate fertilizer is calcium superphosphate, and potash fertilizer is potassium chloride. Test adopt withMachine block design, Ridged plant, 4 ridges, every community, row spacing 60cm, interval 20cm between ridge, the long 5m in ridge. On ridge, plantPlant 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. WillAbove-mentioned nitragin and soybean " so No. 8 " seed dressing, bunch planting after drying in the shade, 5, every nest, final singling 2 strains, same when sowingSample is first broadcast CK. In just pod phase (67d breeding time) sampling of plant, mensuration plant plant height, root nodule number, aerial part are plantedStrain dry weight; Measure output harvest time (98d breeding time). Management is during this time by the Routine Management of local Cotton Varieties by Small Farming Households soybeanCarry out.
The Field inoculation effect of table 4 Panxi Diqu
The processing of Rhizobium Inoculation, at the plant height of first pod phase, plant dry weight, root nodule number all than not inoculating contrast (CK)Obviously increase, increase respectively 20.3%, 35.1%, 59.1%, output, than the remarkable increase of CK, increases 23.1%. It is visible,The main cultivation soybean varieties in described nodule azotobacter strain SCAUs152 and Panxi Area, Sichuan Province " so No. 8 " matching is good,The good rhizobium that are applicable to climbing western ecological environment Soybean production.
Field inoculation experimental study in two typical ecotope, Sichuan is found, inoculates described nodule azotobacter strainAfter SCAUs152, 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 present invention separates the nodule azotobacter strain SCAUs152 of acquisition can be Sichuan soybeanLarge-area applying in production.
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 CCTCCNO:M2014085。
2. the application of nodule azotobacter strain SCAUs152 according to claim 1, is characterized in that, shouldFor the production of Sichuan soybean.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410150948.5A CN103952341B (en) | 2014-04-16 | 2014-04-16 | A kind of nodule azotobacter strain SCAUs152 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410150948.5A CN103952341B (en) | 2014-04-16 | 2014-04-16 | A kind of nodule azotobacter strain SCAUs152 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103952341A CN103952341A (en) | 2014-07-30 |
| CN103952341B true CN103952341B (en) | 2016-05-04 |
Family
ID=51329698
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410150948.5A Expired - Fee Related CN103952341B (en) | 2014-04-16 | 2014-04-16 | A kind of nodule azotobacter strain SCAUs152 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103952341B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048326A (en) * | 2018-01-30 | 2018-05-18 | 内蒙古农业大学 | The device and method of biology in situ directional separation culture plant growth-promoting rhizobacteria |
| CN108558519A (en) * | 2018-06-05 | 2018-09-21 | 福建农林大学 | A kind of soybean mill water culture nutrient solution |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104365418A (en) * | 2014-11-19 | 2015-02-25 | 成都红柿子科技有限公司 | Grape protection bag with antibacterial function |
| CN104673729B (en) * | 2015-03-23 | 2017-10-31 | 河南工业大学 | One plant of phosphorus decomposing rhizobium and its application combined in the endogenous phosphorus load of deposit is reduced with submerged plant |
| CN105950520B (en) * | 2016-07-18 | 2019-05-10 | 武汉市农业科学技术研究院作物科学研究所 | A kind of rhizobium of efficient phosphate-solubilizing and its application |
| CN108251329B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Rhizobium fabae strain Blgs19-2 and application thereof |
| CN108239615B (en) * | 2017-12-26 | 2020-06-16 | 四川农业大学 | Rhizobium fabae strain Bga2-2 and application thereof |
| CN108034603B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Common vetch rhizobium strain VS21-2 and application thereof |
| CN108034605B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Rhizobium fabae strain Bdz5-3 and application thereof |
| CN107904191B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Rhizobium V2-2 and application thereof |
| CN108034604B (en) * | 2017-12-26 | 2020-06-30 | 四川农业大学 | Rhizobium JSP2-5 and application thereof in improving tobacco field rotation soil |
| CN108118010B (en) * | 2017-12-26 | 2020-06-09 | 四川农业大学 | Rhizobium fabae strain Blgs20-1 and application thereof |
| CN107904194B (en) * | 2017-12-26 | 2020-06-02 | 四川农业大学 | Common vetch rhizobium strain VS13-1 and application thereof |
| CN108558518A (en) * | 2018-06-05 | 2018-09-21 | 福建农林大学 | A kind of mill water culture nutrient solution and its preparation and application improving soybean yields |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1156179A (en) * | 1996-12-18 | 1997-08-06 | 张龙男 | High-efficiency root-nodule bacteria for increasing yield of leguminous plant |
| CN1951197A (en) * | 2006-11-01 | 2007-04-25 | 沈阳农业大学 | A novel method for controlling soybean root disease by using rhizobia |
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643761B (en) * | 2011-10-08 | 2013-10-23 | 中国农业科学院农业资源与农业区划研究所 | Rhizobium with rice growth promoting function and application thereof |
-
2014
- 2014-04-16 CN CN201410150948.5A patent/CN103952341B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1156179A (en) * | 1996-12-18 | 1997-08-06 | 张龙男 | High-efficiency root-nodule bacteria for increasing yield of leguminous plant |
| CN1951197A (en) * | 2006-11-01 | 2007-04-25 | 沈阳农业大学 | A novel method for controlling soybean root disease by using rhizobia |
| CN101182477A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXBL9 and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| 1株具解磷特性大豆根瘤菌的分离筛选与回接鉴定;韩梅 等;《微生物学杂志》;20100331;第30卷(第2期);51-56 * |
| JQ342895.1;Kang,S.A.等;《genbank》;20120219;全文 * |
| 四川高效大豆根瘤菌的筛选及初步应用研究;周涛 等;《植物营养与肥料学报》;20120930;第18卷(第1期);第228页左栏第4段－右栏第4段,第231页右栏第1-2段和表3-4,第232页左栏第段，右栏第3段, * |
| 黄怀琼 等.四川紫色土壤中土著大豆根瘤菌的资源分布.《西南农业学报》.2000,第13卷(第3期),39-44. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048326A (en) * | 2018-01-30 | 2018-05-18 | 内蒙古农业大学 | The device and method of biology in situ directional separation culture plant growth-promoting rhizobacteria |
| CN108558519A (en) * | 2018-06-05 | 2018-09-21 | 福建农林大学 | A kind of soybean mill water culture nutrient solution |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103952341A (en) | 2014-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103952341B (en) | A kind of nodule azotobacter strain SCAUs152 and application thereof | |
| CN104277994B (en) | One strain Sinorhizobium SCAUs65 and application thereof | |
| CN103952343B (en) | One strain Semen sojae atricolor is raw root nodule bacteria SCAUs36 and application thereof slowly | |
| CN103952344B (en) | One strain high-efficiency nitrogen-fixing rihizobium japonicum SCAUs46 and application thereof | |
| CN104263684B (en) | A kind of product siderophore series bacillus and application thereof | |
| CN106987541A (en) | One plant has degeneration-resistant, growth-promoting performance efficient rhizobium melioti and its application | |
| CN104974962B (en) | One plant of acinetobacter phosphorus decomposing growth-promoting bacterium Y40 and its application | |
| CN109852565A (en) | A kind of salt-soda soil composite modifying agent and its method of administration | |
| CN106967652A (en) | Rhizobium and its application that one plant of promotion Jian Kuo pea grows | |
| CN105274030B (en) | A kind of rhizobium and application thereof | |
| CN109679884A (en) | One plant of efficient Promoting bacteria of corn that can be reduced nitrogen phosphorus fertilizer application and its application | |
| CN102827793A (en) | Medicago pseudomonas strain capable of producing ACC (1-aminocyclopropane-1-carboxylate) deaminase and application of medicago pseudomonas strain | |
| CN105112319A (en) | Pseudomonas phosphate dissolving bacterium Y2, bioorganic fertilizer prepared through using bacterium, and application of fertilizer | |
| CN101519644B (en) | Sinorhizobium sp. and application thereof | |
| CN104705347B (en) | One cultivates peanut Slow_growing rhizobia agent, preparation method and use | |
| CN107904191A (en) | Rhizobium V2 2 and its application | |
| CN107904192A (en) | Rhizobium V9 2 and its application | |
| CN109735468A (en) | One plant with wide spectrum dross characteristic soybean slowly raw rhizobium and its application and with its preparation compound nitragin | |
| CN104593301B (en) | One plant of wall bacillus G1 and its preparation method and application | |
| CN106148250B (en) | One plant of Costa Rica streptomyces actinomyces and application thereof | |
| CN108795797A (en) | Raw enterobacter cloacae and its application in one plant of maize root system | |
| CN104651286A (en) | Soybean root nodule endophyte DEnt1302, preparation method and application of soybean root nodule endophyte Dent1302, fungicide and preparation method of fungicide | |
| CN107602222A (en) | A kind of tealeaves special fertilizer and preparation method thereof | |
| CN102533564B (en) | A kind of screening method of bio-control trichoderma in corn seedling stage root rot period | |
| CN108841761B (en) | Method for promoting growth of clover and/or increasing yield of clover and microbial inoculum used by method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160504 Termination date: 20170416 |