CN102229901A - Inorganic phosphate solubilizing bacteria capable of promoting accumulation of dry matter in eucalyptus - Google Patents
Inorganic phosphate solubilizing bacteria capable of promoting accumulation of dry matter in eucalyptus Download PDFInfo
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Abstract
The invention provides inorganic phosphate solubilizing bacteria, which is a Lysinibacillussphaericus strain P19, with a collection number of CGMCC No.4769. When the inorganic phosphate solubilizing bacteria are inoculated onto eucalyptus, the accumulation of dry matter in eucalyptus can be promoted. The strain P19 has a high eucalyptus growth promoting effect, has high phosphate solubilizing capacity, can improve the soil phosphorus utilization rate of eucalyptus plants considerably, improve the accumulation of the dry matter in the eucalyptus to a great extent, and can achieve a better effect than applying phosphate fertilizer and organic fertilizer.
Description
Technical field
The present invention relates to a strain fusobacterium bacterial strain and an application thereof, related more specifically to the inorganic phosphate solubilizing bacteria that a strain can promote the eucalyptus dry-matter accumulation.
Background technology
The same pine tree of eucalyptus, willow are called as the three big quick growing species of treess in the world together, because it has adaptability widely, and the economic worth height, simultaneously with ecology and social benefit, and be subjected to many national favors.At present, eucalyptus has become the strategic seeds of China south development fast-growing, high-yield woods.Because how wet southern high-temperature is, the fast and easy leaching of organic matter decomposition runs off, and elements such as iron aluminium gather relatively, and soil reacts acid, and the activity of available phosphorus is reduced.On the other hand, plant the influence that causes the serious problem of soil fertility decline because eucalyptus is subjected to connecting, its normal growth to eucalyptus has caused very big influence.Therefore, the validity of raising southern red loam phosphorus is very important.
Phosphate solubilizing microorganism is the important component part of soil microorganisms.Phosphate solubilizing microorganism or phosphate solubilizing bacteria (phosphatesoluble microorganisms, PSMs) be meant the special microbial function monoid of a class that insoluble chemical combination attitude phosphorus can be converted into the simple titanium pigment that plant can absorb in the soil, mainly comprise phosphate-solubilizing bacteria, phosphorus decomposing fungi and phosphorus decomposing actinomycetes
[]There is bigger otherness in the effect of three major types phosphate solubilizing microorganism phosphorus decomposing, and fungi quantity is less than bacterium, but the phosphorus decomposing ability of some fungi is then obviously greater than bacterium.There is the scholar to act on the form difference of phosphorus again, it is divided into inorganic phosphate solubilizing bacteria and organic phosphate solubilizing bacteria according to phosphate solubilizing microorganism.Think to be that the microorganism that simple plant can absorb form phosphorus is referred to as the organophosphorus microorganism with the organophosphorus mineralising of complexity; The inorganic phosphate compounds that plant can be difficult to absorb is converted into the microorganism that can directly absorb form phosphorus, is referred to as inorganic phosphorus microbe.But, much separate inorganic phosphorus microbe and also have the ability of separating organophosphorus simultaneously, therefore, but be difficult to they are got very clear actually, some bacterium only has single phosphorus decomposing ability, and some bacterium has two kinds of phosphorus decomposing abilities simultaneously, and the latter is more satisfactory screening object.
The effective object of phosphate solubilizing bacteria mainly is the phosphorus of the insoluble in the soil, utilize this microorganism functions peculiar own in the soil to improve the content of soil available phosphorus, also have more superiority than traditional method: the existence of (1) phosphate solubilizing microorganism can promote the absorption of plant to other nutritive element.Phosphate solubilizing microorganism is because metabolic, and trace elements such as zinc, copper, calcium improve the nutrition of root system of plant around the adsorbable root system of plant; Secondly, other instrumentality of its excretory also can influence the growth of plant to a certain extent.(2) resistance against diseases of enhancing plant.After some phosphate solubilizing microorganism was inoculated into plant rhizosphere, the edatope that is fit to the there was bred rapidly.Some material of its excretory can suppress the existence and the growth of other pathogenic micro-organism, has the effect of certain antagonism pathogenic bacteria.(3) protect original soil property, reduce environmental pollution and ecological damage.Phosphate solubilizing microorganism substitutes the use of traditional organic fertilizer, not only reduced cost, and microorganism does not produce pollutent, the pollution of yet having avoided soil to organise in process of production.(4) fertilizer efficiency height.The phosphate solubilizing microorganism that screens can directly act in the soil, also can be made into bacterial manure and is manured into soil, and directly insoluble phosphorus is converted into the phosphorus that can absorb, and utilization ratio height in the production process reduces leaching loss and solidification in the chemical fertilizer use.Therefore, seriously lack under the situation of phosphorus at current soil, filter out phosphate solubilizing microorganism efficiently, improve effective utilization of soil phosphorus, reduce the production cost of agroforestry, protection environment and resource have bigger significance of times.
Summary of the invention
The invention provides the inorganic phosphate solubilizing bacteria of a strain, can promote the accumulation of eucalyptus dry-matter.
Inorganic phosphate solubilizing bacteria of the present invention be fusobacterium (
Lysinibacillus sphaericus) bacterial strain P19, the preserving number of described bacterial strain P19 is CGMCC No.4769.
Fusobacterium of the present invention (
Lysinibacillus sphaericus) bacterial strain P19, being deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on April 21st, 2011, it is called for short CGMCC, and the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is: CGMCC No. 4769.
Bacterial strain P19 of the present invention can be applicable to inoculate eucalyptus.
The concrete steps of described inoculation are as follows:
⑴ insert liquid nutrient medium with bacterial classification point, and through the 48h shaking culture, culture temperature is 28 ℃; Described liquid nutrient medium is formulated as: every liter of liquid nutrient medium contains extractum carnis 3.0 g/L, peptone 5.0 g/L, sodium-chlor 10.0 g/L, pH 7.2-7.4;
⑵ go into blood counting chamber with the bacterium drop, counts under opticmicroscope, draws bacterial concentration;
⑶ dilute bacterium liquid with ultrapure water;
⑷ apply bacterial strain concentration at the eucalyptus rhizosphere is 0.5 * 10
6Cfu/ml-1.5 * 10
6The bacterium liquid of cfu/ml.
Bacterial strain P19 of the present invention is stronger to the eucalyptus growth-promoting effect, has phosphorus decomposing ability efficiently.
Remarkable advantage of the present invention: bacterial strain P19 of the present invention can significantly improve the utilization ratio of eucalyptus plant to soil phosphorus, largely improves the accumulation of eucalyptus dry-matter, and its effect ratio is used phosphate fertilizer and fertilizer better effects if.
Description of drawings
Fig. 1 cultivates the available phosphorus content analysis in the inorganic phosphate solubilizing bacteria P19 cycle;
Fig. 2 cultivates the organic acid total amount the inorganic phosphate solubilizing bacteria P19 cycle to change.
Embodiment
Experimental technique among the following embodiment if no special instructions, is ordinary method.
Embodiment 1: the acquisition of inorganic phosphate solubilizing bacteria bacterial strain P19
One,The separation screening process of inorganic phosphate solubilizing bacteria bacterial strain P19 is as follows:
(1) field acquisition eucalyptus rhizosphere soil.Soil is taken from Yongan, Fujian Province state-owned forest farms, is located in the Yong'an City outskirts of a town, and longitude and latitude is E117 ° of 23'18 ", N25 ° of 56'27 ".
(2) inorganic phosphate solubilizing bacteria separates
1. the preparation of rhizosphere soil suspension liquid: in sterilisable chamber, accurately take by weighing fresh pedotheque 5 g, put into the 250 ml triangular flasks that 95 ml sterilized waters are housed, put on the shaking table vibration 20 min, microorganism cells is disperseed, leave standstill 20-30 s, 10
-1Diluent; By 10 times of methods of progressively diluting, serial dilution makes 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, 10
-9Etc. a series of dilution bacterium liquid, be used for the mensuration of inorganic phosphate solubilizing bacteria
[3]
2. the separation of inorganic phosphate solubilizing bacteria: when adopting the dilution plate streak method to separate inorganic phosphate solubilizing bacteria, adopt 10
-4, 10
-5, 10
-6Extent of dilution, the dilution bacterium liquid of respectively getting 100ul with pipettor is dull and stereotyped central in inorganic phosphorus, and is coated with on flat board evenly with connecing collarium.Each sample is established 3 repetitions.Microbial culture 7d observes the colony growth situation, calculates the bacterial strain number of the inorganic phosphate solubilizing bacteria with phosphate solubilization simultaneously.Separate inorganic phosphate solubilizing bacteria and adopt the inorganic phosphorus substratum.
3. the plate screening of inorganic phosphate solubilizing bacteria: with the bacterium colony that occurs obvious molten phosphorus circle or transparent circle on the transfering loop picking substratum, with the method line purifying of line continuously, inorganic phosphate solubilizing bacteria is cultivated 7 d in 28 ℃, obtains the bacterial strain of individual plant tool phosphorus decomposing ability.Measure the colony diameter and the transparent circle diameter of inorganic phosphate solubilizing bacteria simultaneously, calculate transparent circle and colony diameter ratio, tentatively conclude bacterium colony phosphorus decomposing effect.The inorganic phosphorus decomposing bacterial strain of purifying is inoculated on the slant medium, cultivates preservation and place 4 ℃ of refrigerators standby.
(3) quantitative assay of inorganic phosphorus decomposing fungi degradation inorganic phosphorus ability: the inorganic phosphorus liquid nutrient medium that will not phosphorate is sub-packed in the 150 ml triangular flasks, and every bottle 30 ml accurately adds 0.250 g Ca
3(PO
4)
2, the sterilization back adds 1ml has activated 24 h with the beef extract-peptone liquid nutrient medium bacterium liquid to be measured, establishes 3 repetitions.Put 28 ℃ of full temperature shaking culture case 160 r
Min
-1Cultivate 5d, strain cultured solution, centrifugal 20 min of 10000 rpm, supernatant liquor adopt molybdenum antimony resistance colorimetric method to measure its phosphorus content, measure the pH value of nutrient solution simultaneously with pH meter, to add 1ml beef extract-peptone liquid nutrient medium as reference liquid.
(4) the nutrient solution available phosphorus content is measured---molybdenum antimony resistance colorimetric method.
Get the supernatant liquor 200 μ ls of strain cultured solution after centrifugal and add in the 50 ml volumetric flasks, add water to 15-20ml, add 12,4-dinitrophenol indicator is regulated pH value to solution with dilute alkaline soln and diluted acid (sulfuric acid) and is little yellow.Add the anti-developer of 5 ml molybdenum antimony with transfer pipet, the water constant volume shakes up.Behind 30 min, with 700 nm wavelength colorimetrics, be reference liquid with blank test solution on spectrophotometer, transfer absorption value to zero, measure the absorption value of liquid to be measured then, find the amount of phosphorus dissolved that shows liquid on working curve, it is stable that color can keep in 8 h.
Beef-protein medium
[4]: extractum carnis 3.0 g/L, peptone 5.0 g/L, sodium-chlor 10.0 g/L, agar 18.0 g/L, pH 7.2-7.4.
Inorganic phosphorus substratum (Meng Jinna basic medium)
[5 ~ 7]: glucose 10.0 g/L, (NH
4)
2SO
40.5 g/L, MgSO
47H
2O 0.3 g/L, NaCl 0.3 g/L, KCl 0.3 g/L, FeSO
47H
2O 0.03 g/L, MnSO
44H
2O 0.03 g/L, Ca
3(PO
4)
25.0 g/L, yeast extract 0.5g/L, agar 18.0 g/L, pH 7.2-7.4.
(5) interpretation of result
1. inorganic phosphate solubilizing bacteria treadmill test
Filter out 30 strains on the inorganic phosphorus flat board and have the inorganic phosphate solubilizing bacteria of obvious transparent circle.Screening obtains bacterial strain P19, and its transparent circle is 1.770cm with the ratio of colony diameter, has stronger phosphorus decomposing ability from the treadmill test analysis.
2. inorganic phosphate solubilizing bacteria thalline absorbs phosphorus content
Further measure the phosphorus content that inorganic phosphate solubilizing bacteria is absorbed by thalline self in the process of dissolving insoluble phosphorus, show bacterial strain P19, absorbing phosphorus content is 9.651 μ g/ml, and from the Analysis of phosphorus contents of thalline itself as can be known, bacterial classification P19 has the ability of stronger absorption phosphorus.
3. inorganic phosphate solubilizing bacteria dissolved available phosphorus content
Measure and find that inorganic phosphate solubilizing bacteria bacterial strain P19 dissolved available phosphorus content is 235.624 μ g/ml, and contrast only 5.481 μ g/ml, the phosphorus decomposing ability of inorganic phosphate solubilizing bacteria bacterial strain P19 is 42.99 times of contrast.Bacterial strain P19 has stronger phosphorus decomposing ability.
4. inorganic phosphate solubilizing bacteria medium pH value
The medium pH value of further measuring inorganic phosphate solubilizing bacteria bacterial strain P19 is 4.32, and contrast is 6.73.The medium pH value comparison of bacterial strain P19 is according to having descended 35.8%.The result shows that there are correlationship in amount of phosphorus dissolved and pH value, this and Chen Yang etc.
[8]Result of study is consistent, shows that bacterial strain P19 has stronger molten phosphorus ability.
(6) other performance
1. bacterial strain P19 is to the dissolving vigor of tertiary iron phosphate, secondary calcium phosphate, aluminum phosphate: preparation inorganic phosphorus liquid nutrient medium, use tertiary iron phosphate (12.088 g/L), secondary calcium phosphate (11. 096 g/L), aluminum phosphate (8.406 g/L) to substitute phosphorus source material in the former substratum, other components unchanged simultaneously respectively
[9]Pack in the 150mL Erlenmeyer flask 121 ℃ of sterilization 20min into by the liquid amount of every bottle of 30mL.1ml is drawn with beef extract-peptone liquid nutrient medium activation 48h with liquid-transfering gun in the cooling back, and concentration is adjusted to 10 approximately
9The P19 bacterium liquid of cfu/mL inserts respectively in above-mentioned 3 kinds of different phosphate sources material culture mediums, establishes 3 repetitions, simultaneously to the different phosphate sources material culture medium respectively with do not connect bacterium shake the bottle as blank.Place shake-flask culture 5 d under 28 ℃, 160 r/min conditions, centrifugal 20 min of 10000 rpm, supernatant liquor adopt molybdenum antimony resistance colorimetric method to measure its phosphorus content.
2. the bacterial strain P19 cycle is cultivated the available phosphorus content analysis: preparation inorganic phosphorus liquid nutrient medium, the inorganic phosphorus liquid nutrient medium after will sterilizing is respectively packed in the 150ml triangular flask.The P19 bacterium liquid that pipettes 1ml respectively with liquid-transfering gun is put 28 ℃ of full temperature shaking culture case 160 r/min and is cultivated 7d in triangular flask, takes out three of each sample respectively at 1d, 2d, 3d, 5d, 7d and repeats index of correlation and measure.Get the content that 5ml carries out available phosphorus in the centrifugal survey nutrient solution, adopt molybdenum antimony resistance colorimetric method to measure its phosphorus content.
3. the mensuration of organic acid total amount: accurately take by weighing 4 g sodium hydroxide and be settled to 1000 ml with distilled water, demarcate with 0.05 mol/L oxalic acid of accurately preparation, the demarcation concentration of sodium hydroxide solution is 0.081967mol/L.With 2. 10 times of the nutrient solution dilution of strain cultured solution after centrifugal of step, accurately draw 10 mL in triangular flask with transfer pipet, add 2 of instructions phenolphthalein solutions, with demarcating good sodium hydroxide titrating solution (0.081967mol/L) titration, titration to solution shows pink.Calculate total acid content in the nutrient solution according to the volumeter of used sodium hydroxide solution, total acid content is in oxalic acid.Per 1 ml sodium hydroxide titrating solution is equivalent to 3.688525mg oxalic acid (C
2H
2O
4).
4. interpretation of result
A, inorganic phosphate solubilizing bacteria are to the dissolving vigor of aluminum phosphate
Find that by liquid culture there is some difference to the dissolving vigor of aluminum phosphate for contrast and bacterial strain P19.Contrast is 6.769 vg.ml
-1, bacterial strain P19 is 7.341 vg.ml
-1, comparison is according to having increased by 8.45%, and bacterial strain P19 has stronger dissolving phosphoric acid aluminium ability.
B, inorganic phosphorus decomposing bacterial strain are to the dissolving vigor of tertiary iron phosphate
Bacterial strain P19 is 21.650 vg.ml to the dissolving power of tertiary iron phosphate
-1, and the tertiary iron phosphate dissolving power of contrast only is 15.211 vg.ml
-1Therefore, the comparison of the ability of bacterial strain P19 dissolving phosphoric acid iron is according to having increased by 42.33%, and bacterial strain P19 has the ability of stronger dissolving phosphoric acid iron.
C, inorganic phosphorus decomposing bacterial strain are to the dissolving vigor of calcium monohydrogenphosphate
Bacterial strain P19 is 281.489vg.ml to the dissolving power of calcium monohydrogenphosphate
-1, and the calcium monohydrogenphosphate dissolving power of contrast only is 28.947 vg.ml
-1Therefore, bacterial strain P19 dissolving phosphoric acid calcium monohydrogen phosphate ability is 9.72 times that contrast.Medium pH value is measured demonstration, and blank pH value is 6.47, and bacterial strain P19pH value is 5.05, and comparison is according to having descended 21.9%.Show that bacterial strain P19 has stronger dissolving phosphoric acid calcium monohydrogen phosphate ability.
D, inorganic phosphate solubilizing bacteria cycle are cultivated the available phosphorus content analysis
Along with the propelling of incubation time, available phosphorus content peak value occurs in the nutrient solution about the 3rd day, downward trend then occurs, presents the trend (Fig. 1) of rising subsequently again.This may be because in the initial culturing process, and bacterial strain performance phosphate solubilization is short through the content of available phosphorus in the nutrient solution.But in long culturing process, bacterial strain self growth has consumed the part available phosphorus in the solution, thalline is converted into structure phosphorus in the thalline with part phosphorus, and this also matches with the thalline of this experimental study self can absorb a part of phosphorus in the phosphorus decomposing process result.And P19 reaches vertex the 3rd day phosphorus decomposing ability, and in continuation culturing process subsequently, the phosphorus decomposing ability obviously descends, analyze reason and have 2 kinds: thalli growth has utilized phosphorus source unique in the substratum
［ 13 ］And the precipitation again of titanium pigment, revert to insoluble state.
E, inorganic phosphate solubilizing bacteria cycle are cultivated the organic acid macroanalysis
Along with the propelling of incubation time, organic acid content presents the trend (Fig. 2) that rises gradually in the nutrient solution.The phosphorus decomposing effect and its acid production of bacterial strain are closely bound up.Organic acid content has reacted the phosphorus decomposing effect of nutrient solution to a certain extent.
Two, bacterial strain P19 identifies
The opticmicroscope of inorganic phosphate solubilizing bacteria bacterial strain P19 is identified: bacterial classification is made slide glass, examine under a microscope also and carry out preliminary evaluation with reference to " the outstanding Bacteria Identification of uncle ".
[1] strains tested: P19.
[2] Gram-negative, thalline are the shuttle shape, the two ends taper, and size is (5 ~ 10) μ m * 1 μ m, common is spheroid to free person, has the gram-positive particle to exist sometimes in the thalline.No brood cell, atrichia, nutritional requirement is higher.Well-grown on blood agar, after cultivating in 48 hours, colony diameter 1～2mm, irregular cycle, slightly protruding, grey, luminous, transparent.The biochemical reaction torpescence, any carbohydrate of the most nonfermented of bacterial strain, minority weak fermentation reaction can occur to glucose, fructose.The indoles and the DNA enzyme test positive, catalase-positive does not reduce nitrate, does not grow in 20% bile, and lipase test feminine gender, main metabolites are butyric acid.Therefore, judging that bacterial strain P19 is fusobacterium (Lysinibacillus), is strict obligatory anaerobic bacteria.
Inorganic phosphate solubilizing bacteria bacterial strain molecular classification is identified:
⑴ strains tested: P19.
⑵ substratum: bacterial classification point is inserted liquid nutrient medium, and through the 48h shaking culture, culture temperature is 28 ℃.Described liquid nutrient medium is formulated as: extractum carnis 3.0 g/L, peptone 5.0 g/L, sodium-chlor 10.0 g/L, pH 7.2-7.4.
⑶ primer
Utilize bacterial 16 S rDNA special primer F27 and R1492 to carry out pcr amplification
[14], this primer most 16S rDNA of strains tested sequence that can increase, primer sequence is as follows:
Primer l(F27)：5ˊ-AGA GTT TGA TCA TGG CTC AG-3ˊ
Primer2(R1492)：5ˊ-TAG GGT TAC CTT GTT ACG ACT T-3ˊ；
Test method
1. the extraction of bacteria total DNA
Strains tested is activated with the beef extract-peptone liquid nutrient medium, approximately 24-48 h.Adopt centrifugal column type bacterial genomes DNA extraction test kit (giving birth to worker's biotechnology company limited available from Shanghai) to extract total DNA, step is as follows:
A. get 1.5 m1 bacterial culture fluids in a sterilization EP pipe, centrifugal 1 min of 10 000 rpm removes supernatant liquor, collects thalline.
B. add 200 μ l damping fluid GA in bacterial sediment, vibrating to thalline thoroughly suspends.
C. in pipe, add 20 μ l Proteinase K solution, mixing.
D. add 220 μ l damping fluid GB, 15 s that vibrate place 10 min for 70 ℃, and solution becomes is limpid, and are brief centrifugal to remove the globule of cap wall.
E. add 220 μ l dehydrated alcohols, mixing 15 s that fully vibrate, the brief centrifugal globule with cap wall.
F. the previous step gained is all added among the adsorption column CB3 (adsorption column is put into collection tube), centrifugal 30 s of 12000 rpm outwell waste liquid, and CB3 puts into collection tube with adsorption column.
G. add 500 μ l damping fluid GD in adsorption column CB3, the centrifugal 30s of 12000 rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column.
H. add 700 μ l rinsing liquid PW in adsorption column CB3, the centrifugal 30s of 12000 rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column.
I. add 500 μ l rinsing liquid PW in adsorption column CB3, the centrifugal 30s of 12000 rpm outwells waste liquid, and CB3 puts into collection tube with adsorption column.
J. adsorption column CB3 is put into collection tube, centrifugal 2 min of 12000 rpm outwell waste liquid, place room temperature to place several 8 min adsorption column CB3, thoroughly to dry rinsing liquid remaining in the sorbing material.
K. adsorption column CB3 is changed in the clean centrifuge tube, to the middle part of adsorption film unsettled 100 μ l elution buffer TE, room temperature is placed 5 min, and centrifugal 2 min of 12000 rpm collect solution in the centrifuge tube, and-20 ℃ of preservations are standby.
2. pcr amplification
(1) the PCR reaction system is 25 μ l
10×PCR Buffer 2.5 μl
DNTP (concentration 2.5 mmol/L) 2.5 μ l
Mg
2＋ 3.25 μl
Primer 1 (50 μ mol) 1.0 μ l
Primer 2 (50 μ mol) 1.0 μ l
Template DNA 2.0 μ l
Tag enzyme (1 U/ μ l) 1.0 μ l
Mend ddH
2O is to cumulative volume 25.0 μ 1.
(2) PCR reaction conditions
95 ℃ of sex change 5 min
95 ℃ of sex change 1 min
56.0 ℃ renaturation 50 s
33 circulations
72 ℃ are extended 1 min
72 ℃ are extended 10 min.
(3) the PCR product detects
Pcr amplification product is through 0.8% agarose gel electrophoresis (containing nucleic acid dye 0.5 μ l/ml), and electrolytic solution is 0.5 * TAE, with the size and the relative position of Marker indication bands of a spectrum, observes and Taking Pictures recording in the ultraviolet gel imaging system then.
The DNA sequencing:
3. occur the directly order-checking of the purified back of pcr amplification product of bright band under the gel imaging system, order-checking is finished by the biochemical biotechnology in Shanghai company limited.
4. 16S rDNA sequential analysis
The 16S rDNA sequence (shown in SEQ ID NO.1) of bacterial strain is carried out BLAST comparison on the U.S.'s state-run bioinformation center NCBI webpage, find with Genbank in the highest correlated series of homology, similar bacterial strain is
Lysinibacillus sphaericus(accession number is FJ738141), similarity is 99%, determines tentatively that for this reason bacterial strain P19 is fusobacterium (Lysinibacillus).
Embodiment 2: bacterial strain P19 is to the influence of eucalyptus seedling growth
For examination soil is black peat soil (building new flowers market from Foochow), and the pH value is 7.23, available phosphorus 5 mgkg
-1Fertilizer is built new flowers market available from Foochow, for trying the eucalyptus seedling from Fujian forestry scientific research institute, No. 5 clones of alpine ash.If it is blank, execute P contrast (KH
2PO
4), processing such as fertilizer (nitrogen 4%, phosphorus 3%, potassium 4%, organic 30%, moisture 25%, humic acid 10%) and single bacterium P19.The inoculum size that bacterial strain P19 is inoculated in eucalyptus is every basin 50 ml nutrient solution/(1 * 10
6CFUml
-1), three day time repeated 3 times, handled as blank with distilled water.Execute the fertile every basin KH of being of P
2PO
45g adorns native 5 kg, 3 repetitions.Fertilizer is every basin 20 g, adorns native 5 kg, 3 repetitions.This is tested on April 25th, 2010 and goes to the nursery inoculation, gathers in the crops behind the eucalyptus growth 132d, measures plant plant height, thick, the bright quality of stem, dry weight.30 min that complete under 105 ℃ are dried to constant weight for 75 ℃, and the plant sample is used H after pulverizing
2SO
4-H
2O
2Disappear and boil, molybdenum antimony resistance colorimetric method is measured phosphorus content.
P19 is inoculated in eucalyptus with bacterial strain, and concrete steps are as follows:
⑴ insert liquid nutrient medium with bacterial classification point, and through the 48h shaking culture, culture temperature is 28 ℃.Described liquid nutrient medium is formulated as: extractum carnis 3.0 g/L, peptone 5.0 g/L, sodium-chlor 10.0 g/L, pH 7.2-7.4.
⑵ go into blood counting chamber with the bacterium drop, counts under opticmicroscope, draws bacterial concentration.
⑶ be diluted to 1.0 * 10 with bacterium liquid with ultrapure water
6Cfu/ml.
⑷ apply bacterial strain concentration at the eucalyptus rhizosphere is 1.0 * 10
6The bacterium liquid 50ml of cfu/ml, three day time repeated 3 times, handled as blank with distilled water.
Adopt the photosynthetic instrument of GFS-3000, set light intensity 800 μ molm
-2S
-1, 25 ℃ of temperature, humidity 70% is controlled temperature (TA), relative humidity (RH) and CO simultaneously
2Concentration (CA) external environment when measuring is consistent.From the top, select plant 5-6 sheet mature leaf, measure eucalyptus Net Photosynthetic Rate under the different treatment
[15 ~ 16]After mensuration finishes, immediately blade is taken back the laboratory, adopt ethanol-acetone 1:1 hybrid system to measure the chlorophyll content of plant leaf
[17]Accurately take by weighing 0.1g, plant leaf is cut into tiny strip with scissors, the test tube of packing into, each is handled and repeats 3 times.Add the 20ml mixed solution, sealing places dark place lixiviate chlorophyll.After waiting to soak 24 h, more in contrast, survey its light absorption value at 645 nm and 663nm wavelength place, try to achieve chlorophyllous concentration, and bring formula into and obtain chlorophyll a, chlorophyll b, chlorophyllous content with ultraviolet spectrophotometer with reference.Calculation formula: a
645=82.04ca+9.27cb; a
663=82.04ca+9.27cb, unit are g.L
-1Chlorophyll content=C*V/ sample quality m.
1, bacterial classification is handled the influence to eucalyptus plant fresh weight, dry weight
The average fresh weight of control treatment individual plant is 51.500g, and the average dry weight of individual plant is 15.400g.It is 107.967g that bacterial strain P19 handles the average fresh weight of individual plant, and the average dry weight of individual plant is 42.300g, and comparison is according to having increased by 109.64% and 174.68% respectively.Execute phosphate fertilizer and fertilizer and handle that the average fresh weight of individual plant is respectively 68.433g and 74.500g, dry weight are respectively 18.700g and 23.533g, it is littler all to inoculate inorganic phosphate solubilizing bacteria P19 biomass, shows that bacterial strain P19 has the ability of stronger promotion plant dry substance accumulation.
2, bacterial classification is handled the influence to the eucalyptus plant height of tree, leading thread
The average height of tree of control treatment nursery stock is 80.767 cm, and leading thread is 0.571 cm.It is that 133.967 cm, leading thread are 1.000 cm that bacterial strain P19 handles the average height of tree, and comparison is according to having increased by 65.87% and 75.13% respectively.Execute phosphate fertilizer and fertilizer and handle that the average height of tree of plant is respectively 86.00cm and 88.933cm, average leading thread are respectively 0.675cm and 0.815cm, it is littler also all to inoculate inorganic phosphate solubilizing bacteria P19 increment, shows that bacterial strain P19 has the ability of stronger promotion plant strain growth.
In the inorganic phosphate solubilizing bacteria with obvious transparent circle that 30 strains screening obtains, the ability that bacterial strain P19 improves the eucalyptus dry-matter accumulation is higher than other bacterial strain.
3, bacterial classification is handled phosphorus content influence in the eucalyptus plant body
Control treatment plant phosphorus content is 1.018 g/kg, and it is 2.133 g/kg that bacterial strain P19 handles phosphorus content, and comparison is according to having increased by 109.53%.Execute phosphate fertilizer and fertilizer processing plant phosphorus content and be respectively 1.5343 g/kg and 1.4187 g/kg, all lower than inoculating strain P19 processing, show that bacterial strain P19 has the ability of stronger promotion plant to the absorption of phosphorus.
4, bacterial classification is handled eucalyptus plant Net Photosynthetic Rate
Control treatment plant Net Photosynthetic Rate is 5.286 CO
2μ mol
m
-2s
-1, it is 10.992 CO that inoculating strain P19 handles the plant Net Photosynthetic Rate
2μ mol
m
-2s
-1, comparison is according to having increased by 107.95%.Execute phosphate fertilizer and fertilizer processing plant Net Photosynthetic Rate and be respectively 9.960 CO
2μ mol
m
-2s
-1With 8.711m CO
2μ mol
m
-2s
-1, all lower than inoculating strain P19 processing, show that bacterial strain P19 has the ability of stronger raising plant photosynthetic rate.
5, bacterial classification is handled the influence to eucalyptus plant chlorophyll content
Control treatment plant chlorophyll content is 7.414 mg/g, and bacterial strain P19 chlorophyll content is 11.089 mg/g, and comparison is according to having increased by 49.57%.Execute phosphate fertilizer and fertilizer processing plant chlorophyll content and be respectively 8.507 mg/g and 8.912 mg/g, all lower than inoculating strain P19 processing, show that bacterial strain P19 has the ability of stronger raising plant chlorophyll content.
Comprehensive above every index, bacterial strain P19 has stronger facilitation effect to the eucalyptus growth, and has efficient phosphorus decomposing ability.
Bacterial strain P19 of the present invention can significantly improve the utilization ratio of eucalyptus plant to soil phosphorus, improves the accumulation of eucalyptus dry-matter largely, and its effect ratio is used phosphate fertilizer and fertilizer better effects if.
Reference:
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＜110〉University Of Agriculture and Forestry In Fujian
＜120〉a kind of inorganic phosphate solubilizing bacteria that promotes the eucalyptus dry-matter accumulation
<160> 1
<210> 1
<211> 1457
<212> DNA
＜213〉fusobacterium (
Lysinibacillus sphaericus) bacterial strain P19
<400> 1
gccccgtgcg tagcctatta tgcagtcgag cgacagagaa ggagcttgct cctttgacgt 60
tagcggcgga cgggtgagta acacgtgggc aacctacctt atagtttggg ataactccgg 120
gaaaccgggg ctaataccga ataatctatt tcacttcatg gtgaaatact gaaagacggc 180
tttggctaac gctataagat gggcaagcgg cgcattagct agttggtgag gtaacggctc 240
accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccaaactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cacaatgggc gaaagcctga 360
tggagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaaactctgt tgtaagggaa 420
gaacaagtac agtagtaact ggctgtacct tgacggtacc ttattagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540
gtaaagcgcg cgcaggcggt cctttaagtc tgatgtgaaa gcccacggct caaccgtgga 600
gggtcattgg aaactggggg acttgagtgc agaagaggaa agtggaattc caagtgtagc 660
ggtgaaatgc gtagagattt ggaggaacac cagtggcgaa ggcgactttc tggtctgtaa 720
ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840
attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960
tcttgacatc ccgttgacca ctgtagagat atggttttcc cttcggggac aacggtgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt gatcttagtt gccatcattt agttgggcac tctaaggtga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatggacgat acaaacggtt gccaactcgc gagagggagc taatccgata 1260
aagtcgttct cagttcggat tgtaggctgc aactcgccta catgaagccg gaatcgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagtc ggtggggtaa ccttttggag ccagccgccg 1440
aaggtggata gaaacga 1457
Claims (3)
1. the inorganic phosphate solubilizing bacteria of a strain is characterized in that: described inorganic phosphate solubilizing bacteria be fusobacterium (
Lysinibacillus sphaericus) bacterial strain P19, preserving number is CGMCC No.4769.
2. the purposes of an inorganic phosphate solubilizing bacteria as claimed in claim 1 is characterized in that: described inorganic phosphate solubilizing bacteria is inoculated in eucalyptus.
3. the purposes of inorganic phosphate solubilizing bacteria according to claim 2, it is characterized in that: the concrete steps of described inoculation are as follows:
⑴ insert liquid nutrient medium with bacterial classification point, and through the 48h shaking culture, culture temperature is 28 ℃; Described liquid nutrient medium is formulated as: extractum carnis 3.0 g/L, peptone 5.0 g/L, sodium-chlor 10.0 g/L, pH 7.2-7.4;
⑵ go into blood counting chamber with the bacterium drop, counts under opticmicroscope, draws bacterial concentration;
⑶ dilute bacterium liquid with ultrapure water;
⑷ apply bacterial strain concentration at the eucalyptus rhizosphere is 0.5 * 10
6Cfu/ml-1.5 * 10
6The bacterium liquid of cfu/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104762240A (en) * | 2015-04-27 | 2015-07-08 | 福建师范大学 | Method for extracting inorganic phosphorite-dissolving bacteria of machilus pauhoi wood soil |
| WO2015114552A1 (en) * | 2014-01-29 | 2015-08-06 | University Of Pretoria | Plant growth promoting rhizobacterial strains and their uses |
| CN106566789A (en) * | 2016-10-27 | 2017-04-19 | 湖北大学 | Lysinibacillus macroides capable of enriching phosphorus and degrading organophosphorus pesticides and application of lysinibacillus macroides |
| CN109576171A (en) * | 2018-11-16 | 2019-04-05 | 广东植物龙生物技术股份有限公司 | Spindle lysine bacillus and its application |
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2011
- 2011-05-21 CN CN2011101320643A patent/CN102229901B/en not_active Expired - Fee Related
Non-Patent Citations (1)
| Title |
|---|
| 《亚热带农业研》 20080531 陈俊蓉等 不同桉树土壤微生物数量的比较 第4卷, 第2期 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015114552A1 (en) * | 2014-01-29 | 2015-08-06 | University Of Pretoria | Plant growth promoting rhizobacterial strains and their uses |
| CN104762240A (en) * | 2015-04-27 | 2015-07-08 | 福建师范大学 | Method for extracting inorganic phosphorite-dissolving bacteria of machilus pauhoi wood soil |
| CN106566789A (en) * | 2016-10-27 | 2017-04-19 | 湖北大学 | Lysinibacillus macroides capable of enriching phosphorus and degrading organophosphorus pesticides and application of lysinibacillus macroides |
| CN106566789B (en) * | 2016-10-27 | 2019-07-05 | 湖北大学 | Lysinibacillus macroides and the application of one plant of rich phosphorus and degrading organic phosphor pesticides |
| CN109576171A (en) * | 2018-11-16 | 2019-04-05 | 广东植物龙生物技术股份有限公司 | Spindle lysine bacillus and its application |
| CN109576171B (en) * | 2018-11-16 | 2021-11-23 | 广东植物龙生物技术股份有限公司 | Lysinibacillus fusiformis and application thereof |
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