CN103571758B - Soil phosphorus-dissolvinfungi fungi inoculant, preparation method and application - Google Patents
Soil phosphorus-dissolvinfungi fungi inoculant, preparation method and application Download PDFInfo
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Abstract
A kind of Soil phosphorus-dissolvinfungi fungi inoculant and preparation method thereof.Get slant strains one, under 25 DEG C of conditions, cultivate 1-2 days; With 15mL sterilized water wash-out spore, prepare spore suspension liquid, draw 10mL spore suspension liquid and be inoculated in the triangular flask of 100mLPD nutrient solution, under 26 DEG C of conditions, 3-5 days is cultivated in concussion, obtains liquid spawn; Liquid spawn is inoculated in first order seed PD nutrient solution by 15-20%, under 27 DEG C of conditions DEG C, cultivates 1-2 days; First order seed PD nutrient solution is inoculated into secondary seed PD nutrient solution by 15-20% and obtains second-class liquid isolate; Second-class liquid isolate and sterilizing grass carbon 1: 4 are adsorbed then to pulverize and obtains spore powder.
Description
(1) technical field
The present invention relates to a kind of Soil phosphorus-dissolvinfungi fungi inoculant, preparation method and application thereof.
(2) background technology
Phosphorus element is the basic substance of all life activity, and it is not only the component of biological heredity information carrier DNA, prior it be the component of bio-energy carrier A TP.Phosphorus element is under-supply, and all life all will lose its original radiance.At agricultural production, available phosphorus is under-supply is still one of main limiting factor of world crops output.And from China's situation, the whole nation has 74% arable soil fix due to soil phosphorus and lack phosphorus.It is reported: since the 1950's, China started to use chemical phosphatic ferfilizer, the insoluble phosphorus that accumulation is stored in soil is up to 6,000 ten thousand tons, therefore, crack and develop soil insoluble phosphorus technical barrier, to the utilization ratio improving soil phosphorus, scientifically subtract and execute phosphate fertilizer, ensure national food production safety, reduce in agricultural-food production process the pollution of periphery ecotope, all significant from prevention and cure heavy metal pollution of soil and widespread pollution from the overuse of fertilizers and pesticides in rural area source.
Because the conversion of soil indissoluble phosphorus is under certain conditions, the biology centered by biological activity drives process, and therefore, in exploitation soil, indissoluble phosphorus is nothing more than two approach:
One, play the molten phosphorus potentiality of plant self, by genetic engineering breeding technology, seed selection phosphorus efficiency utilizes plant variety to develop soil insoluble phosphorus resource.This respect research one of study hotspot having become crop breeding field in recent years.Up to the present, obtained the molten phosphorus gene of plant, can say that this technology comes from theory, a difficult problem for technological layer cracks, its application, the resistance promoted mainly come from the public's doubting to genetically modified crops Product Safety.
Two, by phosphate solubilizing bacteria, insoluble phosphorus in soil is activated out.The research of this respect was never interrupted in decades, and achieved great successes.From the former Russian scholar Meng Jinna (1935) be separated from soil a strain can reduce nucleic acid and Yelkin TTS phosphorus decomposing bacillus megaterium (Bucillus.megateriumvar.phosphaticum) after, in nineteen forty-seven a large amount of production and application, in inoculation soil, improve soil available phosphorus content.The research of China to phosphate solubilizing bacteria starts from nineteen fifty, and the front northeast institute of agricultural sciences isolates the bacillus megaterium decomposing organophosphorus from black soil of Northeast China and podzolic soil in nineteen fifty.In the same year, the Chinese Academy of Sciences (front forestry soil institute) isolates a kind of pseudomonas (Pseudomonas spp.) and also has the ability reduced nucleic acid with Yelkin TTS from black soil of Northeast China.Bacillus megaterium is made microbial inoculum and uses on chernozem and non-chernozem by the northeast institute of agricultural sciences, and different crop yield amplitudes is different, and average volume increase chernozem is 13.5%, and non-chernozem is 11.7%.
After the eighties in 20th century, domestic many units have developed the compound phosphate solubilizing bacteria preparation be made up of multiple genus bacillus in succession, but the effect that these microbial inoculums use on producing is unsatisfactory, are mainly manifested in Field information effect unstable.Cause the major cause of its Field information effect instability except the soil suitability difference of these phosphate solubilizing bacterias, the bacterial classification that more importantly these phosphorus decomposing preparations use is all phosphate-solubilizing bacteria, and the fatal defects of such bacterial classification is: in deep fermentation factorial praluction process, due to the subtle change of the fermentation condition such as kind and ratio, temperature, pH value, oxygen-supplying amount, fermentation time of nutritive substance, capital causes such bacterial classification irreversibly to lose phosphorus decomposing gene, lose dissolving P capacity, thus cause its Field information effect unstable.
Applicant utilizes phosphorus decomposing fungi Pingyang penicillium to filter out clone bacterial strain JB201015 (Eupenicillium euglaucum anamorph JB201015), the JB201016 (Eupenicilliumeuglaucum anamorph JB201016) of two strain Pingyang penicilliums, wherein about the Inoculant patent applied for of JB201015 bacterial strain, the patent No. is 201210085390.8,201210085415.4.The bacterial classification that the present invention's " Soil phosphorus-dissolvinfungi fungi inoculant " uses is Pingyang penicillium clone bacterial strain JB201016 (Eupenicillium euglaucum anamorphJB201016), overcome in factorial praluction process, bacterial classification irreversibly can lose the shortcoming of phosphorus decomposing gene, and its effect of solubilizing phosphate is better than JB201015; On Field information, by seed dressing method, make bacterial classification field planting at the rhizosphere of crop, with crop rhizosphere secretory product be carbon source, nitrogenous source growth, breeding, by the Metabolic activity of P-dissolving fungi, by the phosphoric acid salt (PO of indissoluble in crop rhizosphere soils
4 3-), change into soluble phosphate (H
2pO
4 -, HPO
4 2-) absorb for crop.Overcome the shortcoming of phosphate solubilizing bacteria soil suitability difference in the past.
Therefore at this, a kind of method is proposed, effectively can crack and develop the technical barrier that accumulation is stored in the ten thousand tons of insoluble phosphorus of 6000 in Chinese soil, for improving the utilization ratio of Chinese soil phosphate fertilizer, scientifically subtract and execute phosphate fertilizer, ensure national food production safety, to reduce in agricultural-food production process the pollution of periphery ecotope, provide technology and supporting substances from prevention and cure heavy metal pollution of soil and widespread pollution from the overuse of fertilizers and pesticides in rural area source.
(3) summary of the invention
[problem that will solve]
The object of this invention is to provide a kind of soil P-dissolving fungi bacterial strain;
Another object of the present invention is to provide a kind of Soil phosphorus-dissolvinfungi fungi inoculant;
Another object of the present invention is to provide a kind of preparation method of Soil phosphorus-dissolvinfungi fungi inoculant;
Another object of the present invention is to provide a kind of application of Soil phosphorus-dissolvinfungi fungi inoculant.
[technical scheme]
A kind of soil P-dissolving fungi bacterial strain, this bacterial strain is Pingyang penicillium clone bacterial strain JB201016(Eupenicillium euglaucum anamorph JB201016), preserving number is CGMCC N
o7919.
A kind of Soil phosphorus-dissolvinfungi fungi inoculant, comprise the second-class liquid isolate of Pingyang penicillium clone bacterial strain JB201016 and sterilizing grass carbon 1 ︰ 4(weight) adsorb, under the condition of 27 DEG C, under relative air humidity is greater than 85% condition, 2-5 days is cultivated in moisturizing, and drying and crushing obtains spore powder and is Inoculant.
The preparation method of described Soil phosphorus-dissolvinfungi fungi inoculant, specifically comprises the following steps:
A. getting preserving number is CGMCC N
othe conidium of Pingyang penicillium clone bacterial strain JB201016 of 7919 cultivates 1-2 days under 25 DEG C of conditions; Use 15mL sterilized water wash-out spore again, be prepared into spore suspension liquid, draw 10mL spore suspension liquid and be inoculated in the triangular flask of 100mLPD nutrient solution, under 26 DEG C of conditions, 3-5 days is cultivated in concussion, obtains liquid spawn;
B. liquid spawn and first order seed PD nutrient solution are inoculated in first order seed PD nutrient solution by 15-20% (volume), under 27 DEG C of conditions, cultivate 1-2 days;
C. again first order seed PD nutrient solution and secondary seed PD nutrient solution after cultivating are inoculated in secondary seed PD nutrient solution by 15-20% (volume), 26-27 DEG C of condition bottom fermentation 3 days, acquisition second-class liquid isolate;
D. second-class liquid isolate and sterilizing grass carbon 1: 4 (weight) are adsorbed, under the condition of 27 DEG C, under relative air humidity is greater than 85% condition, 2-5 days is cultivated in moisturizing, and drying and crushing obtains spore powder.
Described Soil phosphorus-dissolvinfungi fungi inoculant, the mycelium of Pingyang penicillium clone bacterial strain JB201016 second-class liquid isolate reaches 3 × 10
8more than cfu/ml; The conidium amount of sterilizing grass carbon reaches 3 × 10
9cfu/g.
Further, described Soil phosphorus-dissolvinfungi fungi inoculant is as the application of seed dressing.
Further, described Soil phosphorus-dissolvinfungi fungi inoculant is as the application of processing soil treatment agent.
Pingyang penicillium clone bacterial strain JB201016(Eupenicillium euglaucum anamorphJB201016) screening method:
A. thecaspore one transfering loop that preserving number is Pingyang penicillium Eupenicillium euglaucum of CGMCC3.4396 is got, put into sterilized water, utilize eddy mixer to shake 1 minute, make thecaspore be evenly dispersed in sterilized water, be prepared into thecaspore suspension;
B. get the thecaspore suspension that 0.1ml is above-mentioned, be uniformly coated on water agar plate;
C. water agar plate is placed in picking monospore under microscope, is inoculated on the first selective medium flat board in the mode of multiple point, in 25-27 DEG C, cultivates 10-15 days;
D. picking has the conidium of clear molten phosphorus circle bacterium colony on selective medium flat board, be inoculated on the second selective medium flat board, 10-15 days is being cultivated equally in 25-27 DEG C, then the conidium that acquisition one strain has the bacterium colony of maximum molten phosphorus circle is screened, through being accredited as new bacterial strain, called after JB201016, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.
Described first selective medium is made into by the secondary calcium phosphate of the sodium-chlor of the ferrous sulfate of the yeast powder of the Repone K of the magnesium sulfate of the ammonium sulfate of the glucose of 10g/L, 0.5g/L, 0.1g/L, 0.2g/L, 0.5g/L, 0.002g/L, 0.2g/L, 5g/L, 20g/L agar, and the recycling hydrochloric acid soln of 0.1mol/L and the sodium hydroxide solution adjust ph of 0.1mol/L are 7.0.
Described second selective medium is made into by the secondary calcium phosphate of the sodium-chlor of the ferrous sulfate of the yeast powder of the Repone K of the magnesium sulfate of the ammonium sulfate of the maltose of 10g/L, 0.5g/L, 0.1g/L, 0.2g/L, 0.5g/L, 0.002g/L, 0.2g/L, 5g/L, 20g/L agar, and the recycling hydrochloric acid soln of 0.1mol/L and the sodium hydroxide solution adjust ph of 0.1mol/L are 7.0.
Described concussion condition is 120 revs/min in step.The mycelium 3 × 10 of the liquid spawn obtained
7more than cfu/ml; The mycelium 3 × 10 of second-class liquid isolate
8more than cfu/ml; The conidium amount of sterilizing grass carbon reaches 3 × 10
9cfu/g.
[beneficial effect]
Specifically, advantage of the present invention is as follows:
Strong, the molten phosphorus effect of increasing production of soil suitability of described fungi Inoculant is obvious; Phosphorus decomposing gene can not be lost in process of factory production; The production cost of this fungi Inoculant is low, do not need submerged fermentation, with short production cycle.After acting on the rhizosphere of plant, the phosphoric acid salt of indissoluble in crop rhizosphere soils can be activated, absorb for crop.
(4) accompanying drawing explanation
Fig. 1 is the schema of Inoculant zymotechnique.
(5) embodiment
Soil phosphorus-dissolvinfungi fungi inoculant of the present invention and preparation method thereof, the bacterial classification used is the clone bacterial strain JB201016 (Eupenicillium euglaucum anamorph JB201016) of phosphorus decomposing fungi Pingyang penicillium, this bacterial strain overcomes in factorial praluction process, and bacterial classification irreversibly can lose the shortcoming of phosphorus decomposing gene; On Field information, by seed dressing method, make bacterial classification field planting at the rhizosphere of crop, with crop rhizosphere secretory product be carbon source, nitrogenous source growth, breeding, by the Metabolic activity of P-dissolving fungi, by the phosphoric acid salt (PO of indissoluble in crop rhizosphere soils
4 3-), change into soluble phosphate (H
2pO
4 -, HPO
4 2-) absorb for crop.Overcome the shortcoming of phosphate solubilizing bacteria soil suitability difference in the past.
Know-why:
Phosphorus in soil mainly exists with organophosphorus and inorganic phosphorus two kinds of forms.In calcareous soil, phosphorus mainly exists with calcium phosphate salt form, accounts for the 60-70% of inorganic phosphate.These calcium phosphate salt are mainly with Ca (H
2pO
4)
2, CaHPO
42H
2o, CaHPO
4, Ca
3(PO
4)
2, Ca
4h (PO
4)
32.5H
2o, Ca
5(PO
4)
3oH form exists.According to its chemical property, water-soluble being followed successively by of soil calcium phosphorus: Ca (H
2pO
4)
2> CaHPO
42H
2o > CaHPO
4> Ca
3(PO
4)
2> Ca
4h (PO
4)
32.5H
2o > Ca
5(PO
4)
3oH.Calcium phosphorus existence form in calcareous soil depends on pH value and the CaCO of soil
3content.In the basic conditions, the phosphate fertilizer in being manured into soil is converted primarily into CaHPO
42H
2o and CaHPO
4, a little part changes into Ca
3(PO
4)
2and Ca
4h (PO
4)
32.5H
2o, and under the condition year after year using phosphate fertilizer, Ca
4h (PO
4)
32.5H
2o can not form Ca
5(PO
4)
3oH, and with Ca
4h (PO
4)
32.5H
2there is the several years in the form of O.Under pH < 7.3 condition, CaHPO
42H
2o and CaHPO
4ca (H can be changed into
2pO
4)
2by plant utilization, the pH value reducing calcareous soil contributes to calcium phosphorus forms.
This invention is to be purchased from Pingyang penicillium (numbering: 3.4396) (Eupenicillium euglaucum) is for maternal of China General Microbiological culture presevation administrative center (CGMCC), filter out a strain and utilize the clone bacterial strain JB201016 (Eupenicillium euglaucum anamorphJB201016) that crop rhizosphere secretory product grows and acid producing ability is strong, after in this inoculation to crop rhizosphere soils, significantly can fall the pH value of rhizosphere soil, promote the phosphoric acid salt CaHPO of indissoluble in crop rhizosphere soils
42H
2o and CaHPO
4to Ca (H
2pO
4)
2transform, absorb for crop.
Technological line:
Pass through factorial praluction, expand the mycelium of numerous Pingyang penicillium clone bacterial strain JB201016 (Eupenicilliumeuglaucum anamorph JB201016) and obtain its conidium, conidium is adsorbed in sterilant carriers, is prepared into spore pulvis or careless carbon type pulvis finished product (commodity); By dressing seed or mixing with Nutrition Soil, by the rhizosphere of spore inoculating crop, after molten phosphorus spore germination (soil temperature more than 4 DEG C), by the Metabolic activity of P-dissolving fungi, in activation crop rhizosphere soils, the phosphoric acid salt of indissoluble, absorbs for crop.
Manufacture flow process:
Get thecaspore one transfering loop that preserving number is Pingyang penicillium Eupenicillium euglaucum of CGMCC3.4396, put into sterilized water, eddy mixer is utilized to shake (120 revs/min) 1 minute, make thecaspore be evenly dispersed in sterilized water, be prepared into thecaspore suspension; Get the thecaspore suspension that 0.1ml is above-mentioned, be uniformly coated on water agar plate; Water agar plate is placed in picking monospore under microscope, is inoculated on the first selective medium flat board in the mode of multiple point, in 25-27 DEG C, cultivates 10-15 days; Picking has the conidium of clear molten phosphorus circle bacterium colony on selective medium flat board, be inoculated on the second selective medium flat board, in 25-27 DEG C, cultivate 10-15 days equally, then screen the conidium that acquisition one strain has the bacterium colony of maximum molten phosphorus circle, called after JB201016.
Described first selective medium is made into by the secondary calcium phosphate of the sodium-chlor of the ferrous sulfate of the yeast powder of the Repone K of the magnesium sulfate of the ammonium sulfate of the glucose of 10g/L, 0.5g/L, 0.1g/L, 0.2g/L, 0.5g/L, 0.002g/L, 0.2g/L, 5g/L, 20g/L agar, and the recycling hydrochloric acid soln of 0.1mol/L and the sodium hydroxide solution adjust ph of 0.1mol/L are 7.0.
Described second selective medium is made into by the secondary calcium phosphate of the sodium-chlor of the ferrous sulfate of the yeast powder of the Repone K of the magnesium sulfate of the ammonium sulfate of the maltose of 10g/L, 0.5g/L, 0.1g/L, 0.2g/L, 0.5g/L, 0.002g/L, 0.2g/L, 5g/L, 20g/L agar, and the recycling hydrochloric acid soln of 0.1mol/L and the sodium hydroxide solution adjust ph of 0.1mol/L are 7.0.
Then, the conidium selected is cultivated 1-2 days under 22-27 DEG C of (being preferably 25 DEG C) condition; Use 15mL sterilized water wash-out spore again, be prepared into spore suspension liquid, draw the above-mentioned spore suspension liquid of 10mL and be inoculated in the triangular flask of 100mL PD nutrient solution, under 22-27 DEG C of (being preferably 26 DEG C) condition, 3-5 days is cultivated in concussion, obtains liquid spawn; Liquid bacteria and first order seed PD nutrient solution kind are inoculated in first order seed PD nutrient solution by 15-20% (volume), under 22-30 DEG C of (being preferably 27 DEG C) condition, cultivate 1-2 days.
Again first order seed PD nutrient solution and secondary seed PD nutrient solution after cultivating are inoculated in secondary seed PD nutrient solution by 15-20% (volume), 26-27 DEG C of condition bottom fermentation 3 days, acquisition second-class liquid isolate; Second-class liquid isolate and sterilizing grass carbon 1: 4 (weight) are adsorbed, under the condition of 22-30 DEG C (being preferably 27 DEG C), under relative air humidity is greater than 85% condition, 2-5 days is cultivated in moisturizing, and drying and crushing obtains spore powder.
The mycelium 3 × 10 of wherein above-mentioned obtained liquid spawn
7more than cfu/ml; The mycelium 3 × 10 of second-class liquid isolate
8more than cfu/ml; The conidium amount of sterilizing grass carbon reaches 3 × 10
9more than cfu/g is preferred condition.
Molten phosphorus test:
The molten phosphorus circle of picking JB201015, JB201016 clearly respectively, have the JB201001 of molten phosphorus circle, the conidium without the JB201009 bacterial strain of molten phosphorus circle has carried out quantitative test, the results are shown in Table 1.
| Bacterial strain number | Amount of phosphorus dissolved (P mg/10day) | Account for add-on % |
| Ck | 1.42a | 0.62 |
| JB201016 | 89.3b | 39.17 |
| JB201015 | 85.5c | 37.5 |
| JB201001 | 45.8d | 20.1 |
| JB201009 | 22.3e | 9.8 |
P=0.05
The above results all can determine water-soluble phosphorus, and significant difference.The water-soluble phosphorus wherein measured in CK should be caused by substratum autoclaving.
Concrete implementation result:
Black earth: at Fertilization Level nitrogen (N) 200kg/ha, phosphorus (P
2o
5) 80kg/ha, potassium (K
2o) under 60kg/ha condition, spore pulvis seed dressing (300g/ha), corn yield increasing 10-20.9%; Subtract and execute phosphate fertilizer 1/3, corn yield 10706kg/ha (1ha is 10 mu).
Baijiang soil: at Fertilization Level nitrogen (N) 250kg/ha, phosphorus (P
2o
5) 125kg/ha, potassium (K
2o) under 90kg/ha condition, spore pulvis seed dressing (300g/ha), corn yield increasing 10.1-10.6%; Subtract and execute phosphate fertilizer 1/3, corn yield 12130kg/ha.
Technology controlling and process:
(1) optimum temperuture of temperature---fermentation generally should control at about 26 DEG C.
(2) optimum pH of pH value---fermentation, sometimes also can be slightly high or more lower slightly about 6.5, but pH value should be avoided more than 7.0.
(3) dissolved oxygen---oxyty is the important factor affecting fermenting process.When oxyty drops to below 30% saturation ratio, productive rate sharply declines, and during lower than 10% saturation ratio, then causes irreversible infringement.In general requirement fermentation, dissolved oxygen content is not less than 30% of saturated dissolved oxygen, and ventilating ratio is generally 1: 0.8L/min, and mixing speed should adjust as required in fermentation each stage.
(4) mycelial growth rate---specific growth rate is not less than 0.015h-1.
A series of detailed description listed is above only illustrating for a possible embodiments of the present invention, but this embodiment is also not used to limit the scope of the claims of the present invention, allly do not depart from the skill of the present invention Equivalent embodiments done of spirit or change, such as, Deng the equivalence embodiment of change, within the scope of the claims that all should be contained in this case.
Claims (4)
1. a soil P-dissolving fungi bacterial strain, is characterized in that: this bacterial strain is Pingyang penicillium clone bacterial strain JB201016, and preserving number is CGMCC N
o7919.
2. a Soil phosphorus-dissolvinfungi fungi inoculant, it is characterized in that: by the second-class liquid isolate of Pingyang penicillium clone bacterial strain JB201016 according to claim 1 and sterilizing grass carbon 1:4(weight) adsorb, under the condition of 27 DEG C, under relative air humidity is greater than 85% condition, 2-5 days is cultivated in moisturizing, and drying and crushing obtains spore powder and is Inoculant; The mycelium of described second-class liquid isolate reaches 3 × 10
8more than cfu/mL; The conidium amount of sterilizing grass carbon reaches 3 × 10
9cfu/g.
3. a Soil phosphorus-dissolvinfungi fungi inoculant according to claim 2 is as the application of seed dressing.
4. a Soil phosphorus-dissolvinfungi fungi inoculant according to claim 2 is as the application of processing soil treatment agent.
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| CN102618481A (en) * | 2012-03-28 | 2012-08-01 | 宋丹丹 | Soil phosphate-solubilizing fungal inoculant grain spore powder and preparation method thereof |
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| CN102618481A (en) * | 2012-03-28 | 2012-08-01 | 宋丹丹 | Soil phosphate-solubilizing fungal inoculant grain spore powder and preparation method thereof |
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| 有机磷农药敌百虫多菌株复合系降解菌的降解特性研究;王锐登;《生物技术世界》;20121231(第4期);6-7 * |
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