CN1252247C - Oxalic acid penicillium, bacterial preparation thereof and its preparation method, and the application of the preparation in dephosphorization, prophylaxis and soil fertility enhancement - Google Patents
Oxalic acid penicillium, bacterial preparation thereof and its preparation method, and the application of the preparation in dephosphorization, prophylaxis and soil fertility enhancement Download PDFInfo
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Abstract
本发明涉及一种微生物、微生物菌剂及其应用，特别是涉及一种草酸青霉菌(Penicillium oxalicum)、草酸青霉菌菌剂及其制备方法、以及草酸青霉菌在制备植物解磷防病并提高土壤肥力菌剂的应用，尤其是能产生水溶性物质草酸和柠檬酸(2-羟基-1，2，3-丙烷三羧酸)的草酸青霉菌及其制剂、菌剂的制备方法和应用。本发明同时具有解磷和防病作用，在解磷方面，与现有国内使用的磷细菌相比，其解磷效果稳定；在防病方面，与现有国内使用的磷细菌相比，其有独特的防病作用。本发明可使有效磷的含量提高135.32ppm；在PDA培养基上经过对多种重要植物病原菌进行测定，发现本发明菌株对它们有显著抑制作用，本发明在我国为首次报道。The present invention relates to a kind of microorganism, microbial inoculum and application thereof, in particular to a kind of Penicillium oxalicum (Penicillium oxalicum), Penicillium oxalicum inoculum and preparation method thereof, and Penicillium oxalicum in preparing plant phosphorus solubilizing disease prevention and improving Application of soil fertility bacterial agent, especially Penicillium oxalicum capable of producing water-soluble substances oxalic acid and citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid), preparation method and application of the bacterial agent. The present invention has phosphorus-dissolving and disease-preventing effects at the same time. In terms of phosphorus-dissolving, compared with existing domestically used phosphorus bacteria, its phosphorus-dissolving effect is stable; in terms of disease prevention, compared with existing domestically used phosphorus bacteria, its It has a unique disease prevention effect. The present invention can increase the content of available phosphorus by 135.32ppm; after measuring a variety of important plant pathogenic bacteria on the PDA medium, it is found that the strain of the present invention has a significant inhibitory effect on them, and the present invention is the first report in my country.
Description
Technical field
The present invention relates to a kind of microorganism, microbiobacterial agent and application thereof, particularly relate to the application of a kind of penicillium oxalicum (Penicillium oxalicum), penicillium oxalicum bacteria agent and preparation method thereof and penicillium oxalicum at the preparation phosphorus decomposing diseases prevention and the microbial inoculum of increasing soil fertility, especially can produce water-soluble substances oxalic acid and citric acid (2-hydroxyl-1, the preparation method and the application of penicillium oxalicum 2,3-tricarballylic acid) and microbial inoculum thereof, microbial inoculum.
Background technology
Phosphorus is one of essential nutritive element of plant, and full phosphorus concentration on average is about 0.05% in 0.02%～0.5% scope in the soil.But the phosphorus in the soil more than 95% is invalid form, can not be the plant absorbing utilization, and therefore, the arable soil of China 74% lacks phosphorus.The phosphate fertilizer of this season crop applying, its utilization ratio are 5%～25% only, and the phosphorus during major part is manured into soil is fixed to and can not utilizes attitude.Therefore, the effectuation process of phosphorus element in the research soil, to increase soil fertility, save energy, the agricultural spending of reduction and environmental protection all had extremely important meaning.Twentieth century thirties, USSR (Union of Soviet Socialist Republics) is separated to phosphorus decomposing bacillus megaterium (B.megatherium phospherium) from soil, and the fifties, Sperber affirmed the effect of soil bacteria in the plain mineralising of phosphorus publishing an article on " nature " magazine; 50 to the sixties, and China has carried out the research of phosphate-solubilizing bacteria, and tests and use on big face; Continue after practical application in, most examples show that phosphorus bacteria has phosphorus decomposing and production-increasing function, but wild effect is arranged.This phenomenon has many reports abroad, therefore, and to the research of phosphate solubilizing microorganism with to utilize be the problem of being paid close attention to both at home and abroad always.Since the nineties, external continuously to the phosphorus decomposing fungi studies show that its phosphorus decomposing efficient and stability all are better than phosphate-solubilizing bacteria, and begun to develop to the industrialization direction.People such as Kucey commented the effect of microorganism aspect phosphorus decomposing in 1989.People such as Torre commented the mechanism by fungi mineralising phosphorus element in 1993.Cunningham in 1992 etc. have studied phosphate solubilization and the mechanism than Laplace mould (Penicillium bilaii).In recent years, Vassilev in 1996 etc. did to report to the phosphate solubilization of wrinkle mould Penicilliumrugulosum etc. to radiation mould Penicillium radicum, Reyes in 1999 etc. to penicillium variable Penicillium variabile, Whitelaw in 1999 etc.Calendar year 2001 Reyes etc. has reported the effect and the application prospect of wrinkle mould Penicilliumrugulosum wild strain and UV-mutagenic strain mineralising phosphorus.From above these researchs as can be seen, phosphate solubilizing microorganism research focus is turned to fungi from bacterium, especially the research and development direction to Penicillium notatum develops.Cunningham and Kuiack prove Penicillium notatum phosphorus decomposing Mechanism Study, the generation of citric acid and oxalic acid is the mechanism (" Production of Citric and Oxalic Acidsand Solubilization of Calcium Phosphate by Penicillium bilaii " of this fungi degradation phosphorus, this literary composition is published in Applied and Environmental.Microbiology, May 1992, p.1451-1458; ).1993 Torre etc. have studied the biochemical mechanism and the proof of common mould Penicillium frequentans differentiation ore, the generation of oxalic acid, citric acid and gluconic acid is key factor (" Biochemical mechanisms of stonealteration carried out by filamentous fungi licing in monuments ", this literary composition is published in Biogeochemistry 19:129-147,1993; ).Kucey etc. commented microorganism improve the progress that plant can utilize the phosphorus element (Microbially mediated increases in plant-acailable phosphorus, this literary composition is published in Advances in agronomy, vol, 42,199-228; ).Calendar year 2001 Reyes etc. studied the wild strain of wrinkle mould Penicillium rugulosum and ultraviolet mutagenesis bacterial strain decomposing phosphate rock stone improve plant can utilize phosphorus content (Solubilizaton of phosphate rocks and minerals by a wild-type strainand two UV-induced mutants of Penicillium rugulosum ", this literary composition is published in Soil Biology﹠amp; Biochemistry 33:1741-1747).Nineteen eighty-two, mashed kind and sprout term disease before Windels and Kommedahl utilize penicillium oxalicum Penicillium oxalicum to handle pea seed control pea to emerge (Rhizosphere effects of pea seed treatment with Penicillium oxalicum ", this literary composition is published in Phytopathology 72:198-194).
Abroad, Penicillium bilaii biology, biological chemistry and process study have been carried out, in Australia and Canada's own registration commercialization production and application.
In sum, the fungi or the bacterium that are used for phosphorus decomposing are at present both at home and abroad only mentioned phosphate solubilization, do not relate to its prophylaxis effect; And be used for the useful fungi or the bacterium of diseases prevention, do not see that relating to its phosphorus separates effect yet.New through looking into, the report that China is relevant with this research is not found as yet.
Summary of the invention
The purpose of this invention is to provide a kind of microorganism, microbiobacterial agent and application thereof, particularly relate to a kind of penicillium oxalicum (Penicillium oxalicum), contain the microbial inoculum and preparation method thereof of penicillium oxalicum and by the phosphorus decomposing diseases prevention of penicillium oxalicum preparation and the application of the microbial inoculum of increasing soil fertility.Especially can produce water-soluble substances oxalic acid and citric acid (2-hydroxyl-1,2, the preparation method of the penicillium oxalicum 3-tricarballylic acid) and microbial inoculum thereof, microbial inoculum and application, the present invention has overcome and has been used for the phosphorus decomposing fungi at present or bacterium only relates to phosphate solubilization, do not relate to its prophylaxis effect, or be used for the useful fungi or the bacterium of diseases prevention, and do not see and relate to the shortcoming that its phosphorus is separated the microbial inoculum of effect.Penicillium oxalicum bacteria agent provided by the invention has phosphorus decomposing and prophylaxis effect simultaneously.
The object of the invention is achieved through the following technical solutions:
The microorganism of using
Being used for producing microbial inoculum of the present invention is a kind of new microorganism, belongs to the Penicillium of fungi, penicillium oxalicum bacterial classification (Penicillium oxalicum).Name is called BGP-01, is called for short the BGP-01 bacterium.It can produce water-soluble substances oxalic acid and citric acid (2-hydroxyl-1,2,3-tricarballylic acid), and this water-soluble substances is the main mechanism of non-available phosphorus of dissolving and diseases prevention.The BGP-01 separation does not have the mountain soil of farming activities and the plant rhizosphere on the anthropogenic soil from Hebei province.This bacterial strain is deposited in Chinese microbial preservation center on February 17th, 2003, and preserving number is CGMCC 0891.
The separation method of penicillium oxalicum
Isolating method is dull and stereotyped isolation by dilution method, cultivates with the tricalcium phosphate selective medium, measures and clears up circle, and the applying high voltage liquid chromatography proves that this bacterium can produce water-soluble substances oxalic acid and citric acid (2-hydroxyl-1,2,3-tricarballylic acid).
The BGP-01 bacterial strain has following character
1, morphological specificity
This bacterium conidiophore upright (100-200um), colourless, separation arranged, have brush shape stigma, stigma grow thickly be in the branch top vase-like, the normal chain-like growth of conidium, spherical to oval, colourless to painted, smooth (4.5-6.5 * 3.0-4.0um).
2, bacterium colony situation
This bacterium goes up colony edge white at potato-glucose-nutrient agar (PDA), and is yellowish green to deep green after the sporulation.
The preparation of penicillium oxalicum BGP-01 of the present invention, described preparation can be liquid culture, also can be solid culture, can make to contain carbon source, nitrogenous source and other nutritive medium that is usually used in microorganism culturing in the substratum.Wherein carbon source can be starch, dextrin, glycerine, glucose, sucrose, inositol, N.F,USP MANNITOL etc.Nitrogenous source can be peptone, soyflour, meat extract, wheat skin, urea, corn steep liquor, ammonium salt, nitrate, and other organic salts or inorganic salt nitrogenous compound.Then can suitably add some inorganic salt as for other nutrition sources.The metal-salt of salt, phosphoric acid salt and potassium, calcium, zinc, manganese, iron and so on for example.
Be preferably potato, glucose, agar-agar powder, the PDA substratum of water configuration.Penicillium oxalicum BGP-01 (microbial preservation number for CGMCC No.0891) is inoculated in this substratum.
Culture condition: suitable growth temperature is 20-25 ℃, and suitable pH value is 4.0-8.0, and preferred pH value is 7.0, generally can be in the PDA culture medium culturing.
The penicillium oxalicum BGP-01 bacterial strain of cultivating is stored in-20 ℃ of refrigerators.
The present invention also provides the substratum of another kind of described penicillium oxalicum, is used for industry and cultivates this fungi, and described nutrient media components is: starch, vegetable-protein, vegetation fat, robust fibre are preferably: Semen Maydis powder, soybean cake powder, bran powder, K
2HPO4, KH
2PO4, MgSO4, peanut oil.
The prescription of described each component of substratum comprises: Semen Maydis powder 1.5-4.5%, soybean cake powder 1.5-4.5%, bran powder 1.5-4.5%, K
2HPO
41.9-2.4%, KH
2PO
40.5-1.0%, MgSO
40.005-0.1%, peanut oil 0.2-0.4%, water.
Cui prescription is: Semen Maydis powder 3%, soybean cake powder 3%, bran powder 3%, K
2HPO
42.15%, KH
2PO
40.8%, MgSO
40.075%, peanut oil 0.3%, all the other are water.
A kind of penicillium oxalicum bacteria agent of the present invention: described microbial inoculum comprises penicillium oxalicum BGP-01 and ferment basic thing, sorbent material, weighting material, water.Be used for preventing and treating multiple soil-borne disease and increase soil fertility by dissolving soil non-available phosphorus.
Described weighting material can be selected ground phosphate rock for use.
Described sorbent material can be selected vermiculite, the peat composed of rotten mosses for use.
The preparation method of penicillium oxalicum bacteria agent of the present invention comprises the steps: 1 at least) actication of culture; 2) seeding tank fermentation; 3) produce a jar submerged fermentation; 4) change solid culture over to; 5) collect spore.
Described penicillium oxalicum bacterial preparation process also further comprises: 1) cultivate the inclined-plane bacterial strain and cultivate the seed bacterial strain in the eggplant bottle; 2) stirring behind the adding filler, air feed, fermentation; 3) solid fermentation substrate thing sterilization; 4) evenly add fermenting agent, heat insulating culture; 5) washing filtering is collected spore; 6) add sorbent material, weighting material, fully mixing; 7) check, packing.
Described seeding tank is 3～5% to the penicillium oxalicum bacterial classification inoculation amount of producing jar, is preferably 4%; Initial pH is 7.2～8.0, and preferred pH is 7.5.
Described penicillium oxalicum bacterial preparation process processing condition are:
Described medium pH is 6.6～8.0, is preferably 7;
Culture temperature is 20～25 ℃ in the described fermentor tank, be preferably 23 ℃, pH remains on 6.4～8.0, is preferably 7, and ventilation is 1: 0.5～0.8, be preferably 0.6, tank pressure is 67000～89000Pa, is preferably 80100Pa, and stirring velocity is 220rpm, 72～96 hours, be preferably fermentation in 80 hours and finish.
The stopping composition of described adding is selected from vermiculite, the peat composed of rotten mosses, and optimum ratio is 1: 1.
Penicillium oxalicum bacteria agent of the present invention can use known agricultural chemicals in this technical field general various carriers such as solid phase, liquid phase and emulsifying dispersant, and can make formulation arbitrarily such as granule, pulvis, emulsifying agent, wettable powder, tablet, finish, sprays, smoke substance.Generally speaking, the proportioning of effective constituent is 10-90% in emulsion and wettable powder in each preparation; In pulvis and finish etc. is 0.1-10%; Can increase and decrease in right amount according to different application targets.
The present invention also provides a kind of penicillium oxalicum in increase soil fertility application aspect the microbial inoculum of preparation plant phosphorus decomposing diseases prevention.
The present invention has following beneficial effect and advantage:
Penicillium oxalicum bacteria agent provided by the invention has phosphorus decomposing and prophylaxis effect simultaneously, and being better than present fungi or the bacterium that is used for phosphorus decomposing both at home and abroad has phosphate solubilization, does not relate to its prophylaxis effect; Or be used for the useful fungi or the bacterium of diseases prevention, do not see yet and mention that its phosphorus separates effect.
1) aspect phosphorus decomposing: compare its phosphorus decomposing effect stability with the phosphorus bacteria of existing domestic use.
2) aspect diseases prevention: compare with the phosphorus bacteria of existing domestic use, it has unique prophylaxis effect.
Through mixing the fermentative preparation process with ground phosphate rock, bacterial strain of the present invention has following effect: can make the content of available phosphorus improve 135.32ppm; On the PDA substratum, through multiple important phytopathogen is measured, find that bacterial strain of the present invention has apparent restraining effect to them.
Embodiment
Embodiment 1
The preparation of penicillium oxalicum BGP-01, step is as follows:
With potato 200g, glucose 20g, agar-agar powder 15g, the ratio of water 1000ml is made substratum.Penicillium oxalicum BGP-01 (microbial preservation number for CGMCC No.0891) is inoculated in this substratum culture condition: growth temperature is 23 ℃, and the pH value is 7.0.
The penicillium oxalicum BGP-01 bacterial strain of cultivating is stored in-20 ℃ of refrigerators.
Embodiment 2
The preparation of penicillium oxalicum microbial inoculum
Penicillium oxalicum bacteria agent of the present invention comprises penicillium oxalicum BGP-01 and ferment basic thing, sorbent material, weighting material, water.
Described nutrient media components is: Semen Maydis powder 3%, soybean cake powder 3%, bran powder 3%, K
2HPO
42.15%, KH
2PO
40.8%, MgSO
40.075%, peanut oil 0.3%.
Preparation process is as follows: preparation inclined-plane seed bacterial strain, and preparation eggplant bottle seed bacterial strain, seed tank culture, ferment tank adds stopping composition and cultivates, and collects spore, adds auxiliary agent, check, packing.
Concrete steps are as follows:
1, preparation inclined-plane seed bacterial strain: with embodiment 1, the penicillium oxalicum BGP-01 bacterial strain of PDA culture medium culturing is stored in-20 ℃ of refrigerators, twice of taking-up and activation before using (be about to pure strain and be seeded in the dull and stereotyped cultivation of going up).
2, preparation seed bacterial strain in the eggplant bottle: the bacterial strain of above-mentioned cultivation is cultivated in 250 milliliters of eggplant bottles with the PDA substratum, and culture temperature is 23 ℃, washes with the water bacterium of going out during 96 hours well-growns, as the inoculum of seeding tank.
3, press 2/3 of seeding tank volume and throw substratum, substratum consists of Semen Maydis powder 3%, soybean cake powder 3%, bran powder 3%, K
2HPO
42.15%, KH
2PO
40.8%, MgSO
40.075%, peanut oil 0.3%.Initial pH7.5.
4, inoculate to producing jar from seeding tank, inoculum size 4% begins fermentation.Fermentation condition control: culture temperature is 23 ℃, ventilation is 1: 0.6, tank pressure is 80100Pa, stirring velocity is 220rpm, inoculates after 72 hours and takes a sample once every 4 hours, observes the thalli growth situation, whether pollution is arranged, after waiting to find that spore generates, liquid fermenting is finished, and in vermiculite, the peat composed of rotten mosses, ratio is 1: 1 with the fermented product uniform mixing.Carry out solid fermentation.After treating that spore fully forms, WATER-WASHING METHOD is collected spore.Add the ground phosphate rock stopping composition, check, packing.
Quality test: carry out the mensuration of every index by the relevant regulations of product company standard, the bacterium number reaches 2,000,000,000/gram.
Through mixing fermenting experiment with ground phosphate rock, bacterial strain of the present invention has following effect: can make the content of available phosphorus improve 135.32ppm; On the PDA substratum, through multiple important phytopathogen is measured, find that bacterial strain of the present invention has apparent restraining effect to them.
The available phosphorus content measuring method:
1, soil quick-effective phosphor measuring method---sodium bicarbonate method (according to Olsen et.al., 1954Eestimation of available P in soils by extraction wim sodium bicarbonate.USDAcirc.939.)
2, ground phosphate rock rapid available phosphorus measuring method---the yellow colorimetry of vanadium molybdenum (according to " soil chemical analysis methods " such as Lu Rukun 1999 372 pages)
Measuring method and result to main soil-borne plant pathogen:
Measuring method: adopt dull and stereotyped face-off culture method, according to the power of the big small records bacteriostatic action of inhibition zone.Below be that penicillium oxalicum is to the inhibiting measurement result of main soil-borne pathogen;
| The pathogenic bacteria title | Antibacterial intensity |
| Ultimate corruption is mould | +++ |
| The melon and fruit corruption is mould | +++ |
| Rhizoctonia solani | ++++ |
| Gaeumannomyces graminis | ++++ |
| Fusarium oxysporum | +++ |
| Fusarinm solani | +++ |
| Fusarlum roseum | +++ |
| Verticillium wilt pathogen | +++ |
| Agrobacterium | +++ |
| The tomato wilt bacterium | ++++ |
Annotate: go up table "+" and number represent inhibition strength, be divided into Pyatyi, by " 0 " to " 4 "." 0 " expression unrestraint effect, " 4 " expression suppresses fully.
Claims (8)
1, a kind of penicillium oxalicum (Penicillium oxalicum), its microbial preservation number is CGMCCNo.0891; Described this bacterium conidiophore is upright, highly for 100-200 μ m, colourless, separation arranged, have brush shape stigma, stigma grow thickly be in the branch top vase-like, the normal chain-like growth of conidium, spherical to oval, colourless to painted, smooth, size is 4.5-6.5 * 3.0-4.0 μ m; Colony edge white, yellowish green after the sporulation to deep green.
2, a kind of penicillium oxalicum microbial inoculum comprises the described penicillium oxalicum of claim 1 and fermentation base thing, sorbent material, weighting material and water; The basic thing of described fermentation is Semen Maydis powder, soybean cake powder, bran powder, K
2HPO
4, KH
2PO
4And MgSO
4Described weighting material is a ground phosphate rock; Described sorbent material is vermiculite and peat composed of rotten mosses mixture.
3, according to the penicillium oxalicum microbial inoculum of claim 2, the blending ratio of the described sorbent material vermiculite and the peat composed of rotten mosses is 1: 1.
4,, it is characterized in that and to make granule, pulvis, emulsifying agent, wettable powder, tablet, finish, sprays or smoke substance behind the described microbial inoculum adding auxiliary material according to the described penicillium oxalicum microbial inoculum of claim 2.
5, the method for preparing claim 2 penicillium oxalicum microbial inoculum comprises the steps: the penicillium oxalicum actication of culture with claim 1; The seeding tank fermentation; Produce a jar submerged fermentation; Change solid culture over to; After collecting spore, add sorbent material, weighting material, mixing; Check, packing; Wherein said weighting material is a ground phosphate rock; Described sorbent material is vermiculite and peat composed of rotten mosses mixture;
The method processing condition are: described penicillium oxalicum medium pH is 6.6～8.0; Culture temperature is 20～25 ℃ in the described fermentor tank, and pH remains on 6.4～8.0, and ventilation is 1: 0.5～0.8, and tank pressure is 67000～89000Pa, and stirring velocity is 220rpm, and fermentation in 72～96 hours finishes;
Described seeding tank is 3～5% to the penicillium oxalicum bacterial classification inoculation amount of producing jar, and initial pH is 7.2～8.0.
6, according to the method for claim 5, it is characterized in that: described penicillium oxalicum medium pH is 7; Culture temperature is 23 ℃ in the described fermentor tank, and pH is 7, and ventilation is 1: 0.6, and tank pressure is 80100Pa, and fermentation in 80 hours finishes;
Described seeding tank is 4% to the penicillium oxalicum bacterial classification inoculation amount of producing jar, and initial pH is 7.5.
7, the purposes of the described penicillium oxalicum of claim 1 in plant phosphorus decomposing diseases prevention.
8, the purposes of the described penicillium oxalicum of claim 1 in increasing soil fertility.
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2003
- 2003-02-24 CN CN03105225.8A patent/CN1252247C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9732007B2 (en) | 2008-10-01 | 2017-08-15 | Novozymes Biologicals Limited | Methods and compositions for increasing the amounts of phosphorus available for plant uptake from soils |
| US10035735B2 (en) | 2008-10-01 | 2018-07-31 | Novozymes Biologicals Limited | Methods and compositions for increasing the amounts of phosphorus available for plant uptake from soils |
| US10308561B2 (en) | 2008-10-01 | 2019-06-04 | Novozymes Biologicals Limited | Methods and compositions for increasing the amounts of phosphorus available for plant uptake from soils |
| US11059759B2 (en) | 2008-10-01 | 2021-07-13 | Novozymes Biologicals Limited | Methods and compositions for increasing the amounts of phosphorus available for plant uptake from soils |
| US12172936B2 (en) | 2008-10-01 | 2024-12-24 | Novozymes Biologicals Limited | Methods and compositions for increasing the amounts of phosphorus available for plant uptake from soils |
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