CN104560785A - Corn growth-promoting rhizobacteria YM11 and application thereof - Google Patents

Corn growth-promoting rhizobacteria YM11 and application thereof Download PDF

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CN104560785A
CN104560785A CN201410761430.5A CN201410761430A CN104560785A CN 104560785 A CN104560785 A CN 104560785A CN 201410761430 A CN201410761430 A CN 201410761430A CN 104560785 A CN104560785 A CN 104560785A
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growth
corn
promoting rhizobacteria
potassium
bacterial strain
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葛庆胜
周鹏
谭云飞
黄林
黄克毅
高爱华
张许
曹瑞梅
巫伟
王阳
刘晓丹
于建华
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Zhengzhou Sewage Purification Co Ltd
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Zhengzhou Sewage Purification Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/085—Bacillus cereus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Abstract

The invention relates to corn growth-promoting rhizobacteria YM11 and application thereof. The corn growth-promoting rhizobacteria YM11 are classified and named Bacillus cereus and preserved in China General Microbiological Culture Collection Center on Oct.31, 2014 with the preservation number of CGMCC No.9900. The corn growth-promoting rhizobacteria YM11 can effectively convert insoluble potassium-bearing silicates into soluble sylvites and dissolve organic phosphorus to improve the utilization ratio of fertilizers, are effectively used for planting corns, can promote corn growth and improve the yield, and is a major innovation of microbes and corn planting.

Description

A kind of Rhizosphere of Crops Promoting bacteria YM11 and application thereof
Technical field
The present invention relates to field of agricultural microorganism, particularly a kind of Rhizosphere of Crops Promoting bacteria YM11 and application thereof.
Background technology
Sandstone area permeability is strong, and ventilation is good, and aerobic microorganism activity is preponderated, and can promote organic matter decomposition, organic mineralization quickening.And loosing soil, easy farming.Soil capillarity is strong, and moisture runs fast, has " Evening Tide " phenomenon.The suitable cultivated phase is also long, Yi Limiao; But nutrient content is low, fertilizer conservation poor performance, the crop later stage is de-fertile early ageing easily.At present, this soil major part makes cultivated land utilization, and its availability reaches 92.3%, yields two crops a year, general year per mu yield grain about 600kg.Ameliorative measure targetedly from now on: because of soil plantation, rational use of chemical fertilizer, carries out rational application of fertilizers; Multipath applying organic manure; Carry out farm field and forest network, ensureing under the prerequisite that grain-production grows steadily, economic crops such as development Chinese yam, watermelon etc.
Plant growth-promoting rhizobacteria (Plant Growth Promoting Bacteria, be called for short PGPB) is defined as the free living that the is conducive to plant growth under certain condition bacterium at soil, rhizosphere, root table, phyllosphere.These bacteriums can fixed nitrogen, molten phosphorus, molten iron, and produce plant hormone, as growth hormone, gibberellin, the basic element of cell division and ethene.In addition, they can also improve the resistance of plant, comprise arid, high salt, heavy metallic poison and agricultural chemicals.Therefore be separated from sandstone area and obtain plant growth-promoting rhizobacteria, and crop forms syntaxial system, utilizes and biological prostheticly improves sandstone area, become the focus of current research, but so far there are no being exclusively used in the open report of plant growth-promoting rhizobacteria of corn.
Summary of the invention
For above-mentioned situation, for overcoming the defect of prior art, the object of the present invention is just to provide a kind of Rhizosphere of Crops Promoting bacteria YM11 and application thereof, that is, an object of the present invention is to provide a kind of plant growth-promoting rhizobacteria, another object is to provide the application of this plant growth-promoting rhizobacteria, effectively can solve and slightly solubility is converted into soluble potassium salt containing potassium silicate, separate organic phosphor, improve the availability of fertilizer, promote the growth of corn and improve the problem of output.
The present invention solve technical scheme be that Rhizosphere of Crops Promoting bacteria is Rhizosphere of Crops Promoting bacteria YM11, Classification And Nomenclature be bacillus cereus ( bacillus cereus), on October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No.9900.
Plant growth-promoting rhizobacteria YM11(CGMCC No.9900) bacterium colony is comparatively large, flat, irregular, and milky, opaque, rough surface, like ground-glass appearance or molten wax-like, produces gemma.
The physio-biochemical characteristics of plant growth-promoting rhizobacteria YM11 are: Gram-positive, anaerobism, chemoheterotrophy, and catalase is positive, and M.R tests the positive, VP negative, and Starch Hydrolysis is positive, gelatin liquefaction positive, and nitrate reduction is positive, and citrate utilizes negative.
The main nitrogen used when plant growth-promoting rhizobacteria YM11 cultivates includes but not limited to peptone, dusty yeast, alanine, potassium nitrate, ammonium nitrate, ammonium sulfate, urea; The primary carbon source used includes but not limited to glucose, sucrose, fructose, wood sugar, mannitol, lactose, maltose; The inorganic component used includes but not limited to potassium chloride, sodium chloride, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, tricalcium phosphate, calcium chloride dihydrate, bitter salt, seven water and ferrous sulfate.YM11 fermentation at 28 ~ 32 DEG C, can be carried out under the environment of pH5 ~ 9.
Described preserving number is that the Rhizosphere of Crops Promoting bacteria YM11 of CGMCC No.9900 is promoting the application in corn growth;
Described preserving number is the application of Rhizosphere of Crops Promoting bacteria YM11 in plant cultivation or plantation of CGMCC No.9900;
The preferred corn of described plant.
Described plant growth-promoting rhizobacteria YM11 can utilize slightly solubility to contain potassium silicate to grow for potassium source, also have the ability of solve problem insoluble inorganic microcosmic salt, organic phosphor.
Plant growth-promoting rhizobacteria YM11 of the present invention contains potassium silicate for potassium source with slightly solubility, and slightly solubility Phos, organic phosphor are that phosphorus source grows, and are translated into soluble-salt.Under laboratory shake flask condition, the inversion quantity of described plant growth-promoting rhizobacteria YM11 to feldspar in powder reaches 12.78 mgL -1, 127.67 mgL are reached to the inversion quantity of tricalcium phosphate -1, 0.49 mgL is reached to organic phosphor inversion quantity -1.Illustrate that YM11 bacterium has dissolution to feldspar in powder, tricalcium phosphate, organic phosphor, and can soluble-salt be translated into.
Slightly solubility effectively can be converted into soluble potassium salt containing potassium silicate by plant growth-promoting rhizobacteria YM11 provided by the invention, to solve problem insoluble inorganic microcosmic salt, organic phosphor, improve the availability of fertilizer, promote plant root system development and the absorption to fertilizer, increase available potassium in soils, phosphorus content; The present invention is directed to corn and there is good growth-promoting effect, the raising of available potassium in soils, phosphorus content also makes the availability of corn to potassium, phosphate fertilizer higher, being effective to the plantation of corn, promoting the growth of corn and improve output, is that one on microorganism and corn planting is innovated greatly.
Accompanying drawing explanation
Fig. 1 is the bacterium colony figure of bacterial strain YM11 of the present invention;
Fig. 2 represent different liquid amount on bacterial strain YM11 produce IAA affect situation map;
What Fig. 3 represented that different initial pH produces IAA to YM11 bacterial strain affects situation map;
Fig. 4 represent different carbon source on bacterial strain YM11 produce IAA affect situation map;
Fig. 5 represent different nitrogen sources on YM11 bacterial strain produce IAA affect situation map;
Fig. 6 represents the utilization power figure of bacterial strain YM11 to slightly solubility feldspar in powder;
Fig. 7 represents the utilization power figure of bacterial strain YM11 to organic phosphor;
Fig. 8 represents the utilization power figure of bacterial strain YM11 to slightly solubility Phos;
Fig. 9 represents that plantation peanut 30 days is inoculated HS-11 bacterial strain afterwards and affected situation map to soil IAA content.
Embodiment
Below in conjunction with concrete condition, the specific embodiment of the present invention is elaborated.
Biomaterial preservation information: plant growth-promoting rhizobacteria YM11, Classification And Nomenclature is bacillus cereus (Bacillus cereus), on October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, and preserving number is CGMCC No.9900.
Table 1 is for examination soil labile organic matter
Soil Organic carbon (g/kg) Full phosphorus (g/kg) Rapid available phosphorus (mg/kg) Full potassium (g/kg) Available potassium (mg/kg) pH(H2O)
Sandstone area 1.91 0.29 3.44 19.56 20.42 7.39
The physio-biochemical characteristics of table 2 YM11 bacterial strain
Project Result Project Result
Gram’s staining + Starch Hydrolysis +
Aerobic is tested Anaerobism Gelatin liquefaction +
Catalase test - Nitrate reduction +
Methyl red (M.R) reacts + Citrate utilizes -
V-P tests +
Note :+: positive reaction;-: negative reaction
In concrete enforcement, first prepare following medium:
LB medium: peptone 10g, yeast extract 5g, sodium chloride 10g, agar 20g, distilled water 1000ml, pH 7.0-7.2,121 DEG C of sterilizings, 20min;
LB liquid nutrient medium: do not add agar, other condition is the same;
Meng Jinna medium: glucose 10.0g, (NH 4) 2sO 40.5g, MgSO 47H 2o 0.3g, NaCl 0.3g, KCl 0.3g, FeSO 40.03g, MnSO 4h 2o 0.03g, distilled water 1000ml, 115 DEG C of sterilizing 30min;
Organic phosphor liquid nutrient medium: 1000ml Meng Jinna medium adds 0.4g yeast extract, then add 0.2g solubility lecithin;
Inorganic phosphorus bacteria medium (PKO medium): tricalcium phosphate 5g, glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, bitter salt 0.3g, potassium chloride 0.3g manganese sulphate 0.03g, green vitriol 0.03g, distilled water 1000ml, pH7.0 ~ 7.2,121 DEG C of sterilizings, 20min;
Potassium bacterium liquid nutrient medium: sucrose 10.0g, yeast extract 0.5g, (NH 4) 2sO 41.0g, Na 2hPO 42.0g, MgSO 47H 2o 0.5g, CaCO 31.0g, feldspar in powder 1.0g, distilled water 1000mL, 121 DEG C of sterilizings, 20min;
Minimal medium: ammonium sulfate 2.0g; Sodium dihydrogen phosphate 0.5g; Dipotassium hydrogen phosphate 0.5g; Epsom salt 0.2 g; Calcium chloride dihydrate 0.1g, distilled water 1000mL, pH 7.0,121 DEG C of sterilizings, 20min.
The sand taked from the North China Wheat and maize rotation nutrition of the Ministry of Agriculture of Zhengzhou City and fertilising scientific observation experiment station is taken the triangular flask that l0g is placed in 250 ml filling 100 ml aqua sterilisas, in shaking table, 30 DEG C, 150rmin -1vibration 20min, leaves standstill 10min, obtains soil bacteria suspension.Containing several plant growth-promoting rhizobacteria in this soil bacteria suspension, be applied to LB medium after adopting dilution method dilution, flat board is inverted, in 30 DEG C, after cultivating 24h in insulating box, the single bacterium colony of the dissimilar typical case of picking, after dull and stereotyped purifying, 4 DEG C to be kept at LB inclined-plane stand-by.
Plant (corn) the growth-promoting bacterium that can secrete heteroauxin is filtered out again below by qualitative determination and quantitative assay.
qualitative determination: by the microbionation after separation and purification in adopting the LB liquid nutrient medium containing L-Trp (100 mg/L), 30 DEG C, 180 rmin -11d cultivated by shaking table.Getting 50 μ L bacteria suspensions drips on whiteware plate, adds 50 μ L Salkowski color solution (50mL 35%HClO simultaneously 4+ 1mL 0.5M FeCl 3).To the color solution of 50 μ L 50 mg/L heteroauxins be added as positive control.Whiteware plate is observed after room temperature lucifuge places 30 min, and the color person of reddening represents and can secrete heteroauxin.
quantitative assay: carry out quantitative assay to the bacterium of the producing IAA that primary dcreening operation obtains, condition of culture is the same.First by the OD600 value of spectrophotometry bacteria suspension, then by bacteria suspension with 10000 rmin -1centrifugal 10 min get supernatant and add equal-volume Salkowski color solution, and lucifuge leaves standstill 30min, measures its OD 530value.Calculate bacteria concentration OD 600when value is 1, the content of heteroauxin in unit volume zymotic fluid.The drafting of calibration curve adopts analytically pure heteroauxin gradient dilution to prepare.
The product IAA bacterium obtained is carried out the Screening test of phosphorus decomposing situation, strains tested is inoculated in the 250 mL triangular flasks filling 50mL Phos liquid nutrient medium (PKO medium), 30 DEG C, 200 rmin -1after cultivating 72h, get the culture fluid 20mL cultivating 72h, the centrifugal 20min of 6000r/min, gets supernatant ultraviolet specrophotometer and measures wherein phosphorus content.
The product IAA bacterium obtained is carried out the Screening test of phosphorus decomposing situation, strains tested is inoculated in the 250 mL triangular flasks filling 50mL organic phosphor liquid nutrient medium, 30 DEG C, 200 rmin -1after cultivating 72h, get the culture fluid 20mL cultivating 72h, the centrifugal 20min of 6000r/min, gets the content that supernatant ultraviolet specrophotometer measures wherein phosphorus.
The product IAA bacterium obtained is carried out the Screening test of potassium decomposing situation, strains tested is inoculated in the 250 mL triangular flasks filling 50mL potassium bacterium liquid nutrient medium, 30 DEG C, 200 rmin -1after cultivating 72h, get the culture fluid 20mL cultivating 72h, the centrifugal 20min of 6000r/min, gets supernatant flame spectrophotometer and measures wherein K +content.
Secretion heteroauxin can be filtered out by measuring above, separating organic phosphor, separating Phos, the bacterial strain that ability of dissolving potassium is strong, called after YM11.As shown in Figure 1, the bacterium colony that this bacterial strain is formed is comparatively large, flat, irregular, and milky is opaque, rough surface, like ground-glass appearance or molten wax-like, produces gemma.As shown in Figure 6, bacterial strain YM11 for potassium source grows, and is translated into soluble potassium salt with slightly solubility feldspar in powder.Under laboratory shake flask condition, the inversion quantity of described plant growth-promoting rhizobacteria YM11 to feldspar in powder reaches 12.78mgL -1.Illustrate that YM11 bacterium has dissolution to feldspar in powder, with slightly solubility feldspar in powder for potassium source grows, and be translated into soluble potassium salt.As shown in Figure 7, bacterial strain YM11 grows for phosphorus source with the organic phosphor being difficult to utilize, and is translated into available phosphorus.Under laboratory shake flask condition, the inversion quantity of described plant growth-promoting rhizobacteria YM11 to solubility lecithin reaches 0.49mgL -1.Illustrate that YM11 bacterium has transformation to solubility lecithin, grow for phosphorus source with the organic phosphor being difficult to utilize, and be translated into available phosphorus.As shown in Figure 8, bacterial strain YM11 for phosphorus source grows, and is translated into solubility microcosmic salt with slightly solubility Phos.Under laboratory shake flask condition, the inversion quantity of described plant growth-promoting rhizobacteria YM11 to tricalcium phosphate reaches 127.67mgL -1.Illustrate that YM11 bacterium has dissolution to tricalcium phosphate, with slightly solubility Inorganic phosphate for phosphorus source grows, and be translated into solubility microcosmic salt.
Said method is screened isolated bacterial strain, the handsome biotechnology Co., Ltd order-checking through Shanghai, according to the sequencing result of 16SrDNA, analyze in http://www.ncbi.nlm.nih.gov online query, utilize Blast software to carry out tetraploid rice with other 16S rDNA sequence in GenBank, select the 16SrDNA systematic evolution tree of the sequence MEGA version 3 software building YM11 of close sequence and YM11.According to the physiological and biochemical property of this bacterial strain, be accredited as bacillus cereus (Bacillus cereus).By this bacterial strain on October 31st, 2014 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number CGMCC No.9900.
This bacterial classification is Gram-positive, sporiferous irregular shaft-like.Bacterium colony is comparatively large, flat, irregular, and milky is opaque, rough surface, like ground-glass appearance or molten wax-like, produces gemma.Anaerobism, chemoheterotrophy.Optimum growth temperature is 30 DEG C.Catalase is positive, and nitrate reduction is positive.Producing IAA ability is strong, reaches 29.21 μ gmL -1, with slightly solubility feldspar in powder for potassium source grows, and be translated into soluble potassium salt, utilize phosphorus-containing matter to grow for phosphorus source with slightly solubility Phos and difficulty, and be translated into available phosphorus, there is nitrogen fixing capacity.
aerobic is tested
Sterilized LB medium is poured in 3 sterilized test tubes, greatly about 2/3 place, on aseptic operating platform, the bacterial strain YM11(CGMCC No.9900 cultivated with transfer needle picking inclined-plane), percutaneous puncture-inoculation (must be punctured at the bottom of pipe) in above-mentioned medium.30 DEG C of cultivations, respectively 3 days to 7 days observed results.Be aerobic bacteria agar column surface-borne person, as being anaerobic bacteria or facultative anaerobe along the raw elder of puncture line.Result of the test shows, and bacterial strain YM11 bacterium colony is along agar column superficial growth, and also having colony growth in puncture line, is anaerobism.
catalatic mensuration
Clean slide drips 1 3%H 2o 2, get bacterial strain YM11 LB slant culture 1 ring that 18 ~ 24 h cultivate, at H 2o 2in smear, if there is bubble to produce, be positive, otherwise be feminine gender.
Result of the test display bacterial strain YM11 is that catalase is positive.
methyl red test (M.R test)
A. medium and reagent: peptone 5g, glucose 5g, sodium chloride 5g, distilled water 1000mL, regulate pH7.0 ~ 7.2, packing test tube, often pipe fills 4 ~ 5 mL, 121 DEG C of sterilizing 20 min.Reagent: methyl red 0.1g, 95 % alcohol 300 mL, distilled water 200 mL.
B. Spawn incubation and result observe inoculating strain YM11(CGMCC No.9900) in above-mentioned culture fluid, cultivate l ~ 2 day for 30 DEG C.In culture fluid, add several methyl red reagent, as culture fluid presents redness, for methyl red is positive, yellow is negative (methyl red color change interval 4.4 redness ~ 6.0 is yellow).
Result of the test display bacterial strain YM11 is that M.R is positive.
second phthalein carbinol methine test (VP test)
A. the same methyl red test of medium.
B. Spawn incubation and result are observed inoculation and are cultivated same methyl red test.When doing VP test, get culture fluid (about 2mL) and mix with 40 %NaOH phases of equivalent, add a small amount of creatine, after 2 ~ 5 min that fully vibrate, as culture fluid occurs red, be the VP positive.
Result of the test display bacterial strain YM11 is that VP is negative.
starch Hydrolysis is tested
A. medium and reagent add the soluble starch of 0.2% in meat soup peptone agar, and packing triangular flask, 121 DEG C of sterilizing 20min are for subsequent use.Road Ge Shi iodine liquid: crystalline flake of iodine 1g, potassium iodide 2g, first uses a small amount of (3 ~ 5mL) distilled water to dissolve potassium iodide, now adds the crystalline flake of iodine, after iodine dissolves completely, be diluted with water to 300mL.
B. Spawn incubation and result are observed and are got YM11 bacterial classification point and be connected on flat board, cultivate 2 ~ 4 days for 30 DEG C, after forming bacterium colony, flat board drips road Ge Shi iodine liquid, to be paved with periphery of bacterial colonies for degree, dull and stereotyped in blue, and periphery of bacterial colonies is irised out existing if any water white transparency, illustrate that starch is hydrolyzed.The size of the size general remark hydrolyzed starch ability of transparent circle.
Result of the test display bacterial strain YM11 is that Starch Hydrolysis is positive.
gelatin hydrolysis is tested
A. medium and reagent peptone 5g, gelatin 120g, distilled water 1000mL.Regulate pH7.2 ~ 7.4, packing test tube, medium height is about 4 ~ 5cm, 121 DEG C of sterilizing 20min.
B. Spawn incubation and result observation with puncture method inoculating strain YM11 in test tube central authorities.Cultivate one month in 30 DEG C of incubators, observe gelatin and whether liquefy.
Result of the test display bacterial strain YM11 is gelatin liquefaction positive.
nitrate reduction test
A. medium and reagent nitrate liquid nutrient medium: peptone 10g, KNO 31g, distilled water 1000mL, pH7.0 ~ 7.4.Ge Lisishi (Gries) reagent: A liquid: sulfanilic acid 0.5g, spirit of vinegar (about 10%) 150mL; B liquid: Cai's amine 0.1g, distilled water 20mL, spirit of vinegar (about 10%) 150mL.Diphenylamines reagent: diphenylamines 0.5g is dissolved in the l00mL concentrated sulfuric acid, uses 20mL distilled water diluting.
B. Spawn incubation and result are observed and are inoculated in nitrate liquid nutrient medium by bacterial strain YM11, cultivate 1,3,5 day for 30 DEG C.In white porcelain dish aperture, pour a little culture fluid into, then drip 1 reagent A and B liquid wherein respectively, when culture fluid become pink, rose, orange or brown etc. time, indicate that nitrite exists, be that nitrate reduction is positive, otherwise be feminine gender.
Result of the test bacterial strain display YM11 is that nitrate reduction is positive.
the utilization of citrate
A. medium and reagents citric acid sodium 2g, NaCl 5g, MgSO 47H 2o 0.2g, (NH 4) 2hPO 41g, 1% Bromothymol blue aqueous solution 10mL, agar 20g, distilled water 1000mL, pH6.8-7.0,121 DEG C of sterilizing 20min.
B. Spawn incubation and result are observed and are got children's YM11 strain inoculation in age on inclined-plane, and cultivate 3-7 days for 30 DEG C, medium is positive reaction in alkalescence (blueness) person, and constant person is then negative.
The result of the test display bacterial strain YM11 that citrate utilizes is feminine gender.
In order to verify that plant growth-promoting rhizobacteria YM11 produces ability and the optimum condition of heteroauxin further, below for different pH, liquid amount, different carbon source, the impact of different nitrogen sources exploration on heteroauxin output.
To press 25ml containing L-Trp (100mg/L) LB liquid nutrient medium, 50ml, 75ml, 100ml, 150ml are loaded in the triangular flask of 250mL, by 1%(v/v) inoculum concentration inoculation be in the YM11 of exponential phase after, be placed in 30 DEG C, 180rmin -124 h cultivated by shaking table, measure the amount of producing IAA by the method for quantitative assay.Result as shown in Figure 2, due to bacterial strain YM11(CGMCC No.9900) be anaerobic metabolism, throughput affects bacterial strain and produces the efficiency of IAA, and during 25mL liquid amount, bacterial strain produces IAA amount at most, and afterwards along with liquid amount increases, output is fewer.
LB medium containing L-Trp (100mg/L) is adjusted to respectively different pH(4,5,6,7,8,9,10), getting 50mL is loaded in the triangular flask of 250mL, by 1%(v/v) inoculum concentration inoculation be in the YM11 of exponential phase after, be placed in 30 DEG C, 180rmin -124h cultivated by shaking table, and measure the amount of producing IAA by the method for quantitative assay, result as shown in Figure 3, do not produce IAA when showing that pH is 10, in strong acid and strong base environment, thalline cannot carry out growth metabolism, bacterial classification produces IAA more than sour environment in micro-alkali environment, and the optimal pH of this bacterial classification high yield IAA is 7 ~ 9.
In containing L-Trp (100mg/L) minimal medium, add 1%(w/v respectively) carbon source, carbon source has glucose, wood sugar, sucrose, fructose, mannitol, lactose, maltose, getting 50ml is loaded in the triangular flask of 250ml, by 1%(v/v) inoculum concentration inoculation be in the YM11 of exponential phase after, be placed in 30 DEG C, 180 rmin -124 h cultivated by shaking table, measure the amount of producing IAA by the method for quantitative assay.As shown in Figure 4, this bacterial strain is when supplying wood sugar, and the ability of producing IAA is the strongest, and be secondly fructose, the availability of mannitol is minimum for result.
In containing L-Trp (100mg/L) minimal medium (not comprising ammonium sulfate), add 0.1%(w/v respectively) nitrogenous source, nitrogenous source comprises ammonium nitrate, ammonium sulfate, potassium nitrate, peptone, dusty yeast, alanine, urea etc., get 50ml to be loaded in the triangular flask of 250ml by 1%(v/v) inoculum concentration inoculation be in the YM11 of exponential phase after, be placed in 30 DEG C, 180rmin -124 h cultivated by shaking table, measure the amount of producing IAA by the method for quantitative assay.As shown in Figure 5, when illustrating that getting urea is nitrogenous source, the amount of producing IAA is maximum, is secondly dusty yeast for result.
Bacterial strain YM11 of the present invention has obvious growth promoting function to corn, is described below by pot experiment.
The fresh soil of sand 0 ~ 20cm soil layer under collection natural conditions, cross 5mm sieve, every basin fills native 700g, maize planting, regulate water content to 60% of maxmun field capacity, 30 days post-samplings, after obtaining root system image with root scanner (LA1600+ scanner, Canada) scanning, with root system analysis software (Winrhizo2003b, Canada) related root index analysis is carried out, measure soil IAA content by HPLC method, and measure soil quick-effective phosphor, quick-acting potassium content, plant fresh weight, plant height and full nitrogen, full phosphorus, full potassium content.
Corn seed: corn seed carries out 20% hydrogen peroxide surface sterilization 20min, repeatedly, vernalization 2d, chooses the consistent seed that germinates for subsequent use to aseptic water washing.
Connect bacterium process: YM11 of the present invention is inoculated in LB liquid nutrient medium, 30 DEG C, 180rmin -1shaking table is cultivated, and cultivates bacterium and grows to exponential phase, then by bacteria suspension 10000rmin -1centrifugal 3min, then use sterile water resuspended, centrifugal equally three times, inoculum concentration is 10 8cFUg -1(i.e. every gram of dry ground inoculation 10 8cFUg -1yM11 bacterial classification).
Control treatment: in contrast, soil does not spray YM11 bacterium liquid, adds equivalent sterile water.
The results are shown in following each table:
Table 3 inoculating strain YM11 is on the impact of maize root system
Process Root long (cm) Root surface area (cm 2） Root volume (cm 3） Tip of a root number (individual)
CK 773.33±138.08 115.87±15.67 1.40±0.26 4357.00±967.08
YM11 1571.53±138.37 ** 191.38±13.39 ** 1.98±0.14 ** 5871.20±648.35 *
Note: in same row * indicate significant difference ( p<0.05), * * represent pole significant difference ( p<0.01); Lower same.
Table 4 inoculating strain YM11 is on the impact of milpa
Process Fresh weight (g) Plant height (cm) SPAD Full nitrogen (g/kg) Full phosphorus (g/kg) Full potassium (g/kg)
CK 1.43±0.18 22.37±1.56 21.18±1.51 1.81±0.05 1.99±0.56 14.29±1.84
YM11 2.20±0.23 ** 28.38±3.59 ** 24.48±1.92 * 2.18±0.07 * 3.28±0.36 ** 17.12±0.91 *
Table 5 represents the impact of bacterial strain YM11 on soil quick-effective phosphor available potassium
Process Rapid available phosphorus (mg/kg) Available potassium (mg/kg)
CK 2.24±0.31 35.00±1.73
YM11 3.30±0.10 ** 49.50±6.36 *
As can be seen from Table 4, be vaccinated with the overground part fresh weight of the milpa that YM11 soil-grown goes out, and plant height comparatively CK have obvious growth trend; Because YM11 has the effect of dissolving phosphor and dissolving potassium, rapid available phosphorus and quick-acting potassium content in soil is made to increase (table 5), thus facilitate the absorption (table 5) of plant to elements such as P, K, as can be seen from Table 3, inoculate YM11 process and do not connect bacterium process and contrast, maize root system total length, root surface area, average root diameter and tip of a root number all significantly increase, and facilitate the growth of maize root system; As can be seen from Figure 9, after connecing bacterium process, soil IAA content significantly increases, and exceeds about 1.5 times than control group.Can find out in conjunction with above result, plant growth-promoting rhizobacteria YM11 of the present invention to the growth of foundation, grow there is positive effect, IAA output is high, effectively can promote crop growth, improves output.
Can find out in conjunction with above result, plant growth-promoting rhizobacteria YM3 of the present invention to the growth of foundation, grow there is positive effect, IAA output is high, corn planting can be effective to, promote crop growth, improve output, used at 3 mu of corn fields continuously through 3 years, output all improves more than 10%, and the good of its effect was not expected.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the premise without departing from the principles of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (3)

1. a Rhizosphere of Crops Promoting bacteria YM11, Classification And Nomenclature be bacillus cereus ( bacillus cereus), on October 31st, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is CGMCC No.9900.
2. preserving number according to claim 1 is the application of Rhizosphere of Crops Promoting bacteria YM11 in promotion corn growth of CGMCC No.9900.
3. preserving number according to claim 1 is the application of Rhizosphere of Crops Promoting bacteria YM11 in corn planting of CGMCC No.9900.
CN201410761430.5A 2014-12-13 2014-12-13 Corn growth-promoting rhizobacteria YM11 and application thereof Pending CN104560785A (en)

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CN109679884A (en) * 2019-02-28 2019-04-26 中国农业大学 One plant of efficient Promoting bacteria of corn that can be reduced nitrogen phosphorus fertilizer application and its application
CN112680383A (en) * 2021-01-28 2021-04-20 山西省农业科学院农业环境与资源研究所 Novel phosphorus-dissolving bacteria strain and application thereof

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