CN115747077B - Phosphorus-dissolving fungus penicillium decumbens X1 and application thereof - Google Patents
Phosphorus-dissolving fungus penicillium decumbens X1 and application thereof Download PDFInfo
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- 230000000593 degrading effect Effects 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 5
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- 229910000399 iron(III) phosphate Inorganic materials 0.000 claims description 5
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- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a phosphorus-dissolving fungus penicillium decumbens X1, the base sequence of which is shown in a sequence table SEQ ID NO. 1; application of phosphorus-dissolving fungus penicillium decumbens X1 in phosphate dissolution; application of phosphorus-dissolving fungus penicillium decumbens X1 in degrading cellulose; the application of phosphorus-dissolving fungus penicillium decumbens X1 in promoting plant growth is provided. The advantages are that: the phosphorus-dissolving fungus penicillium decumbens X1 screened from the soil has strong dissolving capacity for different insoluble phosphates, and can dissolve the insoluble phosphates into soluble effective phosphorus for plant absorption, and besides, the strain can secrete auxin to promote plant growth; the cellulose degrading capability is strong, the enzyme activity of the cellulase is 34.41U, and the high application value is provided for the combined application of the cellulose degrading enzyme and waste straw compost.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to phosphorus-dissolving fungus penicillium decumbens X1 and application thereof.
Background
Phosphorus (P) deficiency is a common phenomenon in agricultural soils worldwide because only a small fraction of inorganic and organic phosphorus will be dissolved in a given time and thus the need for applying phosphate fertilizer is great. However, excessive application of phosphate fertilizer and its low utilization efficiency cause extensive environmental pollution, especially in china. The use of chemicals in agriculture without redness and soaping can present considerable environmental problems, namely water, air and soil pollution. At present, most of phosphorus-dissolving strains are obtained from rhizosphere soil of crops, and some phosphorus-dissolving bacteria are separated from special environments, so that the phosphorus-dissolving strains are better utilized, and the phosphorus-dissolving strains have the characteristic of dissolving phosphorus and adapting to the indigenous environment. Production practice proves that the microbial fertilizer developed by utilizing the excellent phosphorus-dissolving bacteria not only has fertilizer effect, but also can reduce environmental and food pollution and non-renewable energy consumption caused by production and use of pesticides and fertilizers. Organic fertilizers, particularly those containing phosphorus-solubilizing bacteria, i.e., biofertilizers, are extremely promising in terms of reduced use of chemical fertilizers because they combine the advantages of recycling organic waste, introducing beneficial microorganisms, and providing organics.
Disclosure of Invention
The invention aims to provide phosphorus-dissolving fungus penicillium decumbens X1 and application thereof.
The base sequence of the phosphorus-dissolving fungus penicillium decumbens X1 is shown in a sequence table SEQ ID NO.1, and the preservation number is CCTCC M20221087.
Application of phosphorus-dissolving fungus penicillium decumbens X1 in promoting plant growth;
the phosphate is calcium phosphate, aluminum phosphate and/or ferric phosphate.
The application of phosphorus-dissolving fungus penicillium decumbens X1 in degrading cellulose is provided.
The invention provides a phosphorus-dissolving fungus penicillium decumbens X1, the base sequence of which is shown in a sequence table SEQ ID NO. 1; application of phosphorus-dissolving fungus penicillium decumbens X1 in phosphate dissolution; application of phosphorus-dissolving fungus penicillium decumbens X1 in degrading cellulose; the application of phosphorus-dissolving fungus penicillium decumbens X1 in promoting plant growth is provided. The dissolving capacity of the bacterium on 10g/L concentration calcium phosphate is up to 2318.37mg/L, the dissolving capacity on 5.5g/L concentration aluminum phosphate can be up to 387.88mg/L, and the phosphorus dissolving capacity on 10g/L concentration ferric phosphate can be up to 30.76mg/L; the advantages are that: the phosphorus-dissolving fungus penicillium decumbens X1 screened from the soil has strong dissolving capacity for different insoluble phosphates, and can dissolve the insoluble phosphates into soluble effective phosphorus for plant absorption, and besides, the strain can secrete auxin to promote plant growth; the cellulose degrading capability is strong, the enzyme activity of the cellulase is 34.41U, and the high application value is provided for the combined application of the cellulose degrading enzyme and waste straw compost.
Drawings
FIG. 1 is a photograph of colony morphology of P.praecox-dissolving fungus recumbent penicillium;
FIG. 2 is a morphological image of the invention under a Penicillium decumbens microscope of a phosphorus-dissolving fungus;
FIG. 3 P.decumbens X1 phylogenetic tree;
FIG. 4 is a graph showing the effect of the phosphorus-dissolving fungus of the invention on the degradation of cellulose by Penicillium decumbens.
Detailed Description
Example 1 screening and identification of phosphorus-solubilizing fungal strains
1. Screening
The screening method of the phosphorus-dissolving fungus strain adopts a plate dilution method and comprises the following steps:
samples were collected from the rhizosphere of lespedeza in itong city in northeast. 10g of soil sample are weighed into a 250ml conical flask and 9 are addedShaking with 0g water for 30min, standing for 10min, sucking 1ml supernatant with a pipette into a test tube filled with 9ml distilled water, and diluting to 10 -2 And diluting 1ml to a new test tube containing 9ml of distilled water to 10 -3 And so on to dilute to 10 -4 、10 -5 、10 -6 、10 -7 Taking 100uL of bacterial solutions with different concentrations to a solid phosphorus-dissolving culture medium, culturing for 5-7d in a constant temperature incubator at 28 ℃, selecting a single colony plate with a large transparent ring on a plate of a primary screening culture medium, streaking the single colony plate to the screening culture medium for re-screening until single colonies with the large transparent ring appear, sealing the single colony plate in glycerol, and preserving the single colony plate at-40 ℃;
the solid phosphorus-dissolving culture medium comprises the following components: the water content per kiloliter is 10g of glucose and (NH) 4 )SO 4 0.5g，MgSO 4 ·7H 2 O 0.3g，NaCl 0.3g，FeSO 4 ·7H 2 O 0.03g，MnSO 4 ·4H 2 O 0.03g ，KCl 0.3g，Ca(PO 4 ) 2 5g, (solid medium plus 18g agar powder), ph=7.
Colony morphology: the bacterial colony is dried, opaque, the aerial hypha is flocculent, the spore-forming surface is dark green, the back surface is light yellow, the growth is rapid, and the bacterial strain is penicillium after dyeing and observing by a microscope.
2. Molecular characterization
Molecular identification is carried out on the phosphorus-dissolving fungi obtained by screening, and the molecular identification is carried out according to the following steps: single colonies of the selected strains were picked up and inoculated into liquid PDA medium, shake-cultured at 28℃with a 160r/min shaker, the culture broth (containing the thallus) was taken out at 3d, the samples were sent to Jilin Kumei Biotechnology Co., ltd for sequence sequencing, and the sequencing results were BLAST in NCBI database for sequence analysis and homology comparison.
Phylogenetic tree of phosphorus-dissolving fungi was drawn using MEGA software, see fig. 3, to determine the species of the strain. The result shows that the confidence coefficient of the sequence and the 18S rDNA gene sequence of the penicillium recumbens is up to 97%, and the penicillium recumbens belongs to penicillium by the morphological characteristics of bacterial colony and the characteristics under a staining microscope, thus the penicillium recumbens is @ recumbentPenicilium decumbens) Strains.
Selected solutionsThe sequence of the phosphorus fungus penicillium decumbens is shown in a sequence table SEQ ID NO. 1. Screening out a phosphorus-dissolving fungus strain named as penicillium decumbens X1 and Latin namePenicillium decumbens sp.The strain is preserved in China Center for Type Culture Collection (CCTCC) No. M20221087 at 7 and 11 of 2022.
EXAMPLE 2 Effect of Penicillium decumbens X1 Strain on the lytic potential of different poorly soluble phosphates
Inoculating the selected phosphorus-dissolving fungi into potato dextrose culture medium sterilized at 115 ℃ for 20min, wherein the potato dextrose culture medium comprises the following main components: boiling 20% potato in distilled water for 30min,2% glucose, and distilled water; culturing in a shaker at 28deg.C at 160r/min for 2d to logarithmic phase to obtain bacterial liquid;
inoculating the obtained bacterial liquid into a liquid inorganic phosphorus-dissolving culture medium containing 10g/L calcium phosphate, 5.5g/L aluminum phosphate and 10g/L ferric phosphate according to the inoculation amount of 2%, culturing for about 13 days, and measuring the content of soluble phosphorus in the culture medium by adopting a molybdenum-antimony colorimetric method; the specific method comprises the following steps:
1. reagent preparation:
molybdenum antimony stock solution: 153mL of concentrated sulfuric acid (analytically pure, density 1.84 g/mL) was measured, slowly added to 400mL of distilled water, stirred continuously, and cooled. Weighing ground ammonium molybdate [ (NH) 4 ) 6 Mo 7 O 24 ·H 2 O, analytically pure]10g was dissolved in 300mL of water at about 60℃and cooled. The sulfuric acid solution was then slowly poured into the ammonium molybdate solution. Then 0.5% of potassium antimonate [ KSbOC ] is added 4 H 4 O 6 ·1/2H 2 O, analytically pure]100mL of solution is cooled, diluted to 1000mL by adding water, shaken well and stored in a brown reagent bottle, and the stock solution contains 1% of ammonium molybdate and 2.75mol/L of sulfuric acid.
Molybdenum-antimony color development resisting agent: 1.50g of ascorbic acid is weighed and dissolved in 100mL of molybdenum-antimony storage solution, and the solution has a short effective period and is suitable for being used along with the preparation.
5mg/L phosphorus standard solution: 0.4394g of potassium dihydrogen phosphate (KH) dried at 50 ℃and a process for preparing the same 2 PO 4 . Analytically pure), 100mL of water, 5mL of concentrated sulfuric acid (preservative), and water to a volume of 1L at a concentration of 100mg/L of phosphorus(P) the solution can be stored for a long period of time. 10mL of the above solution was pipetted into a 200mL volumetric flask and water was added to scale to a concentration of 5mg/L of phosphorus (P) standard solution, which was not suitable for long-term storage.
2. Drawing a standard curve:
accurately sucking 5mg/L of phosphorus (KH) 2 PO 4 ) And (3) adding 0, 2,4, 6, 8 and 10mL of standard solution (molar concentration) into a 50mL volumetric flask, simultaneously adding a blank solution with the same volume as that of a sample solution used for color development measurement, adding 2-3 drops of a dinitrophenol indicator, regulating the solution to be yellowish, regulating the solution to be colorless by using 1mol/L sulfuric acid (or hydrochloric acid) solution, regulating the solution to be yellowish by using 1mol/L sodium hydroxide solution, accurately adding 5mL of molybdenum-antimony anti-color developing agent, shaking uniformly, and adding water to fix the volume to obtain a standard solution series with the phosphorus (P) content of 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/L respectively. Shaking, standing at room temperature above 15deg.C for 30 min. The absorbance was measured at a wavelength of 700nm, and a standard curve was drawn with the absorbance as the ordinate and the phosphorus concentration (mg/L) as the abscissa.
3. And (3) measuring the content of soluble phosphorus in the bacterial liquid:
a. centrifuging (12000 r/min, 5 min) 4mL of the bacterial liquid to obtain supernatant
b. Taking a proper amount of supernatant (the first preparation requires fumbling the addition amount according to the phosphorus dissolving amount of the strain) into a 50mL volumetric flask, diluting with water to about 3/5 of the total volume, and adding 2 drops of 2, 4-dinitrophenol indicator
c. Adjusting the solution to slightly yellow by using 1mol/L sodium hydroxide solution and 1mol/L sulfuric acid solution
d. Accurately adding 5mL of molybdenum-antimony color-developing resisting agent, shaking, adding water to constant volume, standing at room temperature above 15deg.C for 30min
e. Zeroing by using blank (blank culture medium without inoculating bacteria), reading absorbance OD700nm, substituting OD value into phosphorus standard curve formula to obtain x value, and multiplying x value by dilution multiple to obtain phosphorus dissolving amount (mg/L)
Results: the phosphorus-dissolving fungus of the invention is added, in a liquid inorganic phosphorus culture medium, the dissolving capacity of the phosphorus-dissolving fungus for 10g/L concentration calcium phosphate is up to 2369.51mg/L, the dissolving capacity for 5.5g/L concentration aluminum phosphate is up to 387.88mg/L, and the phosphorus-dissolving capacity for 10g/L concentration ferric phosphate is up to 30.76mg/L.
EXAMPLE 3 Effect of Penicillium decumbens X1 Strain on cellulose degradation Capacity
Single bacterial colony of the phosphorus-dissolving fungus strain is picked up and is sprayed onto a sodium carboxymethyl cellulose solid culture medium, and the main components of the culture medium comprise: 1% sodium carboxymethyl cellulose, 1% peptone, 0.5% yeast extract, 0.5% NaCl,0.1% monopotassium phosphate, 0.05% magnesium sulfate, 1.8% -2% agar, culturing for 3-5 days, performing cover dyeing for 15 min by using 0.1% Congo red staining solution, discarding the upper layer staining solution, eluting for 15 min by using NaCl eluent (1 mol/L), discarding the eluent, and obtaining uncolored transparent rings (figure 4), and recording the diameter (D) of the transparent rings and the diameter (D) of the colonies respectively.
Inoculating the fermentation broth of the Penicillium decumbens X1 strain into 100mL liquid culture medium containing 1% microcrystalline cellulose, and performing shake culture at 28 ℃ for 7d; after the completion of the culture, the enzyme activity was measured by a dinitrosalicylic acid method (DNS method).
When the phosphorus-dissolving fungi grows for 4 days on a carboxymethyl cellulose sodium culture medium flat plate, the diameter of a transparent ring is 2.7cm, the diameter of a bacterial colony is 1.7cm, and the enzyme activity of cellulase produced by the strain is 34.41U and has a certain cellulose degradation capability.
Example 4 example of the Penicillium decumbens X1 Strain promoting plant growth
Selecting rice seeds with consistent size and plump grains, treating the rice seeds with 75% alcohol for 5min, washing the rice seeds with sterile water for 3.4 times, soaking the rice seeds with 5% sodium hypochlorite for 2 min, repeatedly washing the rice seeds with sterile water, and washing the rice seeds with 5% sodium thiosulfate to remove residual sodium hypochlorite to obtain sterile seeds. Soaking in water for 24 hr in dark, spreading soaked filter paper for 72 hr, and accelerating germination until all the materials are white. After germination, the rice is transferred to a seedling raising basin to be cultured until the rice is three leaves and one heart (about 15 days), and then transferred to different treated soil to carry out rice potting experiments.
2 groups of treatment groups are arranged in the experiment and are blank treatment groups (CK) respectively; penicillium decumbens X1 treatment group (X1): 20mL of the bacterial liquid is poured into 1kg of soil and evenly mixed.
Measurement of rice growth index (results are shown in tables 1 and 2):
measuring the distance from the root neck of the rice to the top of the rice by using a ruler for seedling growth; the root length is measured from the root neck to the root bottom by a ruler. And (3) slowly washing the plant to be detected with clear water, airing in a shade, cutting the plant from the root neck, and respectively measuring the fresh weights of the rice plant and the root by using an electronic balance. And (3) placing the fresh plants into a 105 ℃ oven for de-enzyming for 30min, then placing the plants into a 70 ℃ oven for drying to constant weight, taking out the plants, and respectively weighing the plants and the dry weight of roots by an electronic balance. The leaf width and stem thickness of rice were measured using a vernier caliper.
Measurement of physiological index of rice (the result is shown in Table 3):
SPAD values of rice leaves were determined using a chlorophyll meter SPAD-520 Plus. The activity of peroxidase in rice was measured by the guaiacol method. And determining the content of phosphorus in rice plants by adopting a molybdenum-antimony colorimetric method. The content of soluble sugar was determined by anthrone method. The content of soluble protein was determined using coomassie brilliant blue.
As can be seen from the data of the tables, the invention relates to P-lytic bacteria Penicillium decumbensPenicilium decumbens) The X1 has good promotion effect on rice, can promote the absorption of phosphorus by the rice, and helps the rice to resist lodging.
The above description of the embodiments is only for aiding in the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that various modifications and adaptations of the invention can be made without departing from the principles of the invention and these modifications and adaptations are intended to be within the scope of the invention as defined in the following claims.
Claims (5)
1. Penicillium decumbens strain of phosphorus-dissolving fungusPenicillium decumbens) X1, the preservation number is CCTCC NO: M20221087.
2. The use of a phosphorus-dissolving fungus, penicillium decumbens X1, according to claim 1, for promoting rice growth.
3. Use of a strain of the phosphorus-dissolving fungus penicillium decumbens X1 as claimed in claim 1 for dissolving phosphate.
4. A use according to claim 3, characterized in that: the phosphate is calcium phosphate, aluminum phosphate and/or ferric phosphate.
5. The use of a strain of the phosphorus-dissolving fungus penicillium decumbens X1 as defined in claim 1 for degrading cellulose.
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| CN102061267A (en) * | 2010-11-26 | 2011-05-18 | 东北林业大学 | Engonus fungus with high enzyme activity and high capacity of efficiently degrading straw cellulose in northeast China region |
| CN103045484A (en) * | 2011-10-11 | 2013-04-17 | 济南圣泉集团股份有限公司 | Penicillium strain producing cellulase and application in cellulose enzymatic hydrolysis thereof |
| CN103045683A (en) * | 2011-10-11 | 2013-04-17 | 济南圣泉集团股份有限公司 | Comprehensive utilization method of lignocellulose biomass |
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