CN114989989A - Phosphate solubilizing fungus for promoting corn growth and screening and identifying method thereof - Google Patents
Phosphate solubilizing fungus for promoting corn growth and screening and identifying method thereof Download PDFInfo
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- CN114989989A CN114989989A CN202210633010.3A CN202210633010A CN114989989A CN 114989989 A CN114989989 A CN 114989989A CN 202210633010 A CN202210633010 A CN 202210633010A CN 114989989 A CN114989989 A CN 114989989A
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- 230000001737 promoting effect Effects 0.000 title claims abstract description 47
- 230000003381 solubilizing effect Effects 0.000 title claims abstract description 41
- 229910019142 PO4 Inorganic materials 0.000 title claims abstract description 38
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- 239000010452 phosphate Substances 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 21
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- 239000011574 phosphorus Substances 0.000 claims abstract description 58
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
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- 229920001817 Agar Polymers 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
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- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
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- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
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- 235000021049 nutrient content Nutrition 0.000 description 1
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- 238000011017 operating method Methods 0.000 description 1
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- 239000002686 phosphate fertilizer Substances 0.000 description 1
- 150000003017 phosphorus Chemical class 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
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- 230000007928 solubilization Effects 0.000 description 1
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Abstract
The invention discloses a phosphate solubilizing fungus for promoting corn growth and a screening and identifying method thereof, and particularly relates to the technical field of microorganisms, wherein the phosphate solubilizing fungus is named as N062, the phosphate solubilizing fungus is specifically named as Aspergillus niger N062 in a classified manner, and the microorganism preservation number is as follows: CCTCC NO: m2022656, the collection address of the strain is: the sequence of the phosphate-solubilizing fungus is shown in SEQ. No1 by China center for type culture Collection. The invention provides a separation and screening method of a fungus for promoting corn growth and phosphate solubilizing, and provides a single colony capable of solubilizing organic phosphorus and inorganic phosphorus. The phosphate solubilizing fungi has phosphate solubilizing and saline-alkali resistance, has good growth promoting effect on corn, has good prospects in the aspects of improving saline-alkali soil and manufacturing phosphate solubilizing growth promoting bacterial manure, and has remarkable differences in root weight, stem leaf weight, SPAD value and nitrogen content through analysis of experimental groups and control groups of corn growth promoting experiments, wherein the SPAD value and nitrogen content of a second leaf and the nitrogen content of a third leaf are obtained through the difference of the nitrogen content of the third leaf.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a phosphorus-solubilizing fungus for promoting corn growth and a screening and identifying method thereof.
Background
Phosphorus is an important element in the life activities of plants such as corn crops and the like, and a large number of reports indicate that the nutrient content of soil can be changed, the contents of organic matters, nitrogen, available phosphorus and available potassium in the soil can be increased and the nutrient utilization rate can be improved in the life activity process of microorganisms. However, most of phosphorus in soil in China is insoluble phosphorus salt which cannot be directly utilized by plants, so that the content of effective phosphorus is lower although the total phosphorus content in the soil is richer. In the past, most of agricultural production increases the available phosphorus content in soil by applying available phosphate fertilizer, however, the saline-alkali stress can accelerate the combination of available phosphorus with iron ions, calcium ions and the like and further convert the available phosphorus into insoluble phosphate, so that the phenomenon that the available phosphorus content is still very low while the available phosphorus pool in soil is increased is caused.
The phosphate solubilizing microorganisms play an important role in the transformation of soil available phosphorus, the saline-alkali tolerant phosphate solubilizing microorganisms can quickly and efficiently transform insoluble phosphorus into available phosphorus which can be absorbed by plants under the saline-alkali stress, and the content of the available phosphorus is increased under the condition that the total content of phosphorus in the soil is not changed. Therefore, finding a microorganism capable of effectively dissolving phosphorus has important significance for popularizing the saline-alkali tolerant phosphorus-dissolving microorganism bacterial fertilizer. The difficulty in finding saline-alkali tolerant phosphate-solubilizing microorganisms is the screening and identification of the microorganisms and the determination of the performance of the microorganisms.
Disclosure of Invention
The invention provides a phosphate solubilizing fungus for promoting corn growth and a screening and identifying method thereof, and solves the problems of microbial strain, screening, identifying and the like of saline-alkali tolerance and phosphate solubilizing.
To achieve the above object: the invention provides the following technical scheme:
a phosphate solubilizing fungus for promoting the growth of corn, which is named as N062, and is specifically classified and named as Aspergillus niger N062, and the microorganism collection number of the phosphate solubilizing fungus is as follows: CCTCC NO: m2022656, the collection address of the strain is: wuhan university, the name of the preservation unit is: china center for type culture Collection.
Preferably, the phosphate solubilizing fungus for promoting the growth of the corn has a sequence shown in SEQ.No 1;
the invention also provides a separation and screening method of the fungi for promoting the growth of the corns and dissolving phosphorus, which comprises the following steps:
s01, collecting 1500g of corn rhizosphere soil sample, packaging with a sterile bag and bringing back to a laboratory;
s02, selecting 10g of soil attached to the corn root system in a super clean bench, adding sterile water to 100ml, putting the soil into a shaking table at 28 ℃/180r for 1h to uniformly disperse bacterial colonies in the soil in the water, and standing for 0.5h to precipitate soil particles;
s03, sucking supernatant water, diluting to 10-2, 10-3, 10-4, 10-5 and 10-6 times, respectively taking 200ul of supernatant water, coating on a PDA culture medium, culturing for 48h, selecting a single colony, purifying for 3-5 times, and preserving at-80 ℃ for later use;
s04, culturing the purified single colony on an inorganic phosphorus plate, standing and culturing for 48 hours at the temperature of 28 ℃, and selecting the single colony generating a transparent aperture;
s05, culturing the single colony with the transparent ring on an organophosphorus plate, and selecting the single colony with the transparent ring to obtain a single colony N062 capable of decomposing both organophosphorus and inorganic phosphorus, and preserving the single colony at-80 ℃ for later use.
The invention also provides a DNA extraction and identification method of the fungi for promoting the growth of the phosphorus-dissolving of the corns, which comprises the following steps:
s01, scraping hyphae to cover the bottom of a 2ml centrifuge tube, adding CTAB 700ul and grinding beads, and grinding for 6 times and 60S/time in a grinding instrument;
s02, putting the mixture into a water bath at 65 ℃ for 1h after grinding uniformly, and shaking for 10S every ten minutes to fully and uniformly mix CTAB and hyphae;
s03, taking out the centrifuge tube to normal temperature, placing for 2 minutes, adding 600 mu l of chloroform (trichloromethane), fully shaking to make CTAB fully contact with chloroform, and standing for 2 minutes at room temperature;
s04, centrifuging at 12000rpm for 15 minutes, carefully sucking 500 mul of supernatant into a new 1.5ml centrifuge tube, adding 500 mul of isopropanol, fully mixing to obtain DNA floccule, and standing at-20 ℃ for about 2 hours;
s05, taking out the DNA preserved in the-20C, centrifuging for 15 minutes at 12000rpm, discarding the supernatant, and precipitating the DNA at the bottom of a centrifugal tube;
s06: adding 500 mul 70% ethanol to wash DNA precipitate, centrifuging at 7500rpm for 5 min, discarding supernatant, and air drying the ethanol washed DNA at room temperature;
s07, dissolving the air-dried DNA with 30 mu l of ddH2O, and storing at-20 ℃ for later use;
s08, identifying the DNA molecule of the phosphate solubilizing fungus strain.
Preferably, the universal primers for identifying the DNA molecules of the phosphate solubilizing fungal strain in the step S08 are primer ITS1 and primer ITS 4;
preferably, the PCR reaction system for identifying the DNA molecule of the phosphate solubilizing fungal strain in step S08 is 2 × PCR Master Mix: 10 ul; the primers ITS 1: 1 ul; the primers ITS 4:1 ul; DNA template: 1 ul; ddH 2O: 7 ul;
preferably, the PCR reaction procedure for identifying the DNA molecule of the phosphate solubilizing fungus strain in the step S08 is pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, and 35 cycles; extending for 10min at 72 ℃; infinity at 4 ℃;
preferably, the invention provides a phosphorus-solubilizing fungus for promoting corn growth, wherein the phosphorus-solubilizing fungus has salt tolerance and can normally grow under the salt stress of 30g/L, the growth starts to be inhibited when the salt concentration is increased to 70g/L, and the growth is obviously inhibited and slow to grow when the salt concentration is 90 g/L;
preferably, the invention provides a phosphorus-solubilizing fungus for promoting the growth of corn, wherein the phosphorus-solubilizing fungus has acid and alkali resistance and can grow well on a culture medium with the pH value of 3-12;
preferably, the invention provides a phosphorus-solubilizing fungus for promoting the growth of corn, wherein the phosphorus-solubilizing fungus can secrete siderophin and improve the iron nutrition condition of plants, and can be verified by a CAS culture medium culture detection method;
preferably, the invention provides a phosphorus-solubilizing fungus for promoting corn growth, the phosphorus-solubilizing fungus has phosphorus solubilization and saline-alkali tolerance and has a good growth promotion effect on corn, the strain has good prospects in saline-alkali soil improvement and phosphorus-solubilizing growth-promoting bacterial manure preparation, and is obtained by analyzing the remarkable differences of root weight, stem leaf weight, SPAD value and nitrogen content between an experimental group and a control group in a corn growth promotion experiment, wherein the difference of the SPAD value and nitrogen content of a second leaf and the nitrogen content of a third leaf is obtained.
The invention effectively solves the problems of phosphorus-understanding fungus screening, identification, performance test and the like, and lays a foundation for producing strains required by saline-alkali-tolerant phosphate-solubilizing microbial fertilizer.
Drawings
FIG. 1 is a growth diagram of N062 plate culture;
FIG. 2 is a salt tolerance test chart of N062;
FIG. 3 is a test chart of acid and alkali resistance of N062;
FIG. 4 is a graph of the phosphorus solubilizing effect of N062 under salt stress;
FIG. 5 is a graph of the phosphorus-solubilizing effect of N062 under alkaline stress;
FIG. 6 is a diagram of the effect of N062 in decomposing inorganic phosphorus under saline-alkali interaction;
FIG. 7 is a diagram of the effect of N062 on the removal of organic phosphorus under saline-alkali interaction;
FIG. 8 is a color reaction chart of N062 on CAS medium;
FIG. 9 is a graph showing the effect of N062 on the growth promoting effect of the Qingnong 11 phenotype;
FIG. 10 is a graph showing the effect of N062 on the SPAD value and nitrogen content of Qingnong 11 leaves;
FIG. 11 is a graph showing the phenotypic growth promoting effects of N062 on Zhengdan 958;
FIG. 12 is a graph showing an embodiment of the effect of N062 on the SPAD value and nitrogen content of Zhengdan 958 leaves;
Detailed Description
The present invention will be further described with reference to the following examples. The following examples are only a few specific examples of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by the design concept should fall within the scope of infringing on the protection scope of the present invention.
The percentages in the following examples are by mass unless otherwise specified.
The invention provides a phosphate solubilizing fungus for promoting corn growth, which is named as N062, is specifically classified and named as Aspergillus niger N062, and has a microorganism preservation number of: CCTCC NO: m2022656, the collection address of the strain is: wuhan university, the name of the depository is: china center for type culture Collection.
The invention also provides a separation and screening method of the fungi for promoting the growth of the corns and dissolving phosphorus, which comprises the following specific steps:
s01, collecting 1500g of corn rhizosphere soil sample, packaging with a sterile bag and bringing back to a laboratory;
s02, selecting 10g of soil attached to the corn root system in a super clean bench, adding sterile water to 100ml, putting the soil into a shaking table at 28 ℃/180r for 1h to uniformly disperse bacterial colonies in the soil in the water, and standing for 0.5h to precipitate soil particles;
s03, sucking supernatant water, diluting to 10-2, 10-3, 10-4, 10-5 and 10-6 times, respectively taking 200ul of the supernatant water, coating the 200ul of the supernatant water on a PDA (potato 200g/L and glucose 20g/L) culture medium, culturing for 48h, selecting a single colony, purifying for 3-5 times, and preserving at-80 ℃ for later use;
s04, culturing the purified single colony on an inorganic phosphorus plate (inorganic phosphorus plate culture medium comprises 10.0g/L of glucose, 0.5g/L of ammonium sulfate, 0.5g/L of yeast extract powder, 0.3g/L of sodium chloride, 0.3g/L of potassium chloride, 0.3g/L of magnesium sulfate, 0.03g/L of ferrous sulfate, 0.03g/L of manganese sulfate, 5.0g/L of calcium phosphate, 15.0g/L of agar and 7.0-7.5 of pH value), culturing at the temperature of 28 ℃ for 48 hours, and selecting the single colony generating a transparent aperture;
s05, culturing the single colony generating the transparent ring on an organic phosphorus plate (the organic phosphorus plate comprises 10.0g/L of glucose, 0.5g/L of ammonium sulfate, 0.5g/L of yeast extract powder, 0.3g/L of sodium chloride, 0.3g/L of potassium chloride, 0.3g/L of magnesium sulfate, 0.03g/L of ferrous sulfate, 0.03g/L of manganese sulfate, 0.2g/L of lecithin, 1.0g/L of calcium carbonate, 15.0g/L of agar and 7.0-7.5 of pH), and selecting the single colony with a transparent aperture to obtain a single colony N062 for decomposing organic phosphorus and inorganic phosphorus for later use at-80 ℃.
The invention also provides a DNA extraction and identification method of the fungi for promoting the growth of the corns and dissolving phosphorus, which comprises the following specific steps:
s01, scraping hyphae to cover the bottom of a 2ml centrifuge tube, adding 700ul CTAB (components: 1mol/L Tris-HCl 100ml/L, 0.5mol/L EDTA-Na 240 ml/L, CTAB 20g/L, PH 8.0) and grinding beads, and grinding in a grinding instrument for 6 times and 60S/time;
s02, putting the mixture into a water bath at 65 ℃ for 1h after grinding uniformly, and shaking for 10S every ten minutes to fully and uniformly mix CTAB and hyphae;
s03, taking out the centrifuge tube to normal temperature, placing for 2 minutes, adding 600 mu l of chloroform (trichloromethane), fully shaking to make CTAB fully contact with chloroform, and standing for 2 minutes at room temperature;
s04, centrifuging at 12000rpm for 15 minutes, carefully sucking 500 mul of supernatant into a new 1.5ml centrifuge tube, adding 500 mul of isopropanol, fully mixing to obtain DNA floccule, and standing at-20 ℃ for about 2 hours;
s05, taking out the DNA preserved by the-20C, centrifuging at 12000rpm for 15 minutes, discarding the supernatant, and depositing the DNA at the bottom of a centrifugal tube;
s06: adding 500 μ l 70% ethanol to wash DNA precipitate (flicking the centrifuge tube to allow the DNA precipitate to float), centrifuging at 7500rpm for 5 min, discarding supernatant, and air drying the ethanol washed DNA at room temperature;
s07, dissolving the air-dried DNA with 30 mu l of ddH2O, and storing at-20 ℃ for later use;
s08, identifying the DNA molecule of the phosphate solubilizing fungus strain.
The following method for identifying the strain DNA molecule of S08 is as follows:
primer of: universal primers ITS1 and ITS4
ITS1：5'-TCCGTAGGTGAACCTGCGG-3'；
ITS4：5'-TCCTCCGCTTATTGATATGC-3'；
The PCR reaction system is as follows: 2 × PCR Master Mix: 10 ul; primer ITS 1: 1 ul; primer ITS 4:1 ul; DNA template: 1 ul; ddH 2O: 7 ul;
the PCR reaction procedure was as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 10min at 72 ℃; infinity at 4 ℃;
and (3) carrying out agarose electrophoresis gel detection on the PCR product, and picking the PCR product with clear bands to be sent to a Senno sequencing company for sequencing.
And sequencing to obtain the DNA fragment with the sequence shown in SEQ.No. 1:
ccgtaggtgaacctgcgcaaggatcattaccgagtgcgggtcctttgggcccaacctcccatccgtgtct attgtaccctgttgcttcggcgggcccgccgcttgtcggccgccgggggggcgcctctgccccccgggcc cgtgcccgccggagaccccaacacgaacactgtctgaaagcgtgcagtctgagttgattgaatgcaatca gttaaaactttcaacaatggatctcttggttccggcatcgatgaagaacgcagcgaaatgcgataactaa tgtgaattgcagaattcagtgaatcatcgagtctttgaacgcacattgcgccccctggtattccgggggg catgcctgtccgagcgtcattgctgccctcaagcccggcttgtgtgttgggtcgccgtccccctctccgg ggggacgggcccgaaaggcagcggcggcaccgcgtccgatcctcgagcgtatggggctttgtcacatgct ctgtaggattggccggcgcctgccgacgttttccaaccattctttccaggttgacctcggatcaggtagg gatacccgctgaacttaagcatatcaataa
the invention provides a fungus for promoting corn growth and phosphate solubilizing, which has saline-alkali tolerance and phosphate solubilizing performance and specifically comprises the following components:
salt resistance test
As shown in figure 2, N062 is cultured on PDA culture medium with different salt concentrations (10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, 80g/L, 90g/L and 100g/L) for 96h, N062 can grow normally under 30g/L salt stress, when the salt concentration is increased to 70g/L, the growth is inhibited, when the salt concentration is increased to 90g/L, the growth is obviously inhibited, and the growth is slow.
(II) acid and alkali resistance test
As shown in fig. 3, N062 was cultured for 96 hours on PDA media at various pH (pH 3-12), and it was found that N062 grew well on the media at pH 3-12.
(III) N062 phosphate solubilizing experiment
As shown in FIGS. 4-7, in order to verify the effect of N062 in the removal of organophosphorus and inorganic phosphorus under salt stress, we prepared organophosphorus and inorganic phosphorus culture media with different salt concentrations and pH values for experiments, and found that N062 has better phosphorus removal capability and all generates a transparent chromogenic aperture on the culture medium under salt-alkali stress.
1. Different salt concentrations (10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L) inorganic phosphorus medium (cultured for 48h) and different salt concentrations (cultured for 48h) phosphorus removal effect (FIG. 4 lower)
2. Phosphate solubilizing effect in inorganic phosphorus medium (48 h culture) at different pH (pH 7-12) (upper part of FIG. 5), and in organic phosphorus medium (48 h culture) at different pH (lower part of FIG. 5)
3. The effects of N062 on inorganic phosphorus removal (FIG. 6) and organic phosphorus removal (FIG. 7) under the interaction of different salt contents (10g/L, 20g/L and 30g/L) and different pH values (7-12)
The invention provides a phosphorus-solubilizing fungus for promoting corn growth, which can secrete siderophin and improve the iron nutrition condition of plants, and can be verified by a CAS culture medium culture detection method, and the phosphorus-solubilizing fungus comprises the following specific steps: the strain is placed in CAS blue detection liquid of a CAS culture medium, wherein the CAS blue detection liquid is a compound consisting of chromium azure, hexadecyl trimethyl ammonium bromide and iron ions and is bright blue. When the iron ions in the blue detection plate are extracted by siderophins secreted by N062 cultured on the plate, a chromogenic aperture can be formed around the colonies grown by N062. The experimental result is shown in fig. 8, the siderophins can compete with plant rhizosphere pathogenic microorganisms for iron elements, so that the growth of the pathogenic microorganisms is inhibited, and meanwhile, the plants can improve the iron nutrition condition of the plants by utilizing iron chelated by the siderophins generated by the microorganisms.
The invention provides a phosphorus-solubilizing fungus for promoting corn growth, which has a growth promoting effect on corn and is obtained through a growth promoting experiment of N062 on corn, and the phosphorus-solubilizing fungus specifically comprises the following components:
to verify the growth promoting effect of N062 on corn, we selected zhengdan 958 and qingnong 11 varieties, used potting experiments, and to reduce errors, we used white plastic disks 31.5 × wide by 20.5 × high by 7.8cm, the specific operating method:
1. growth promotion experiment of N062 on Qingnong 11
2.5L of vermiculite and 1.5L of water are added into each tray, Aspergillus niger spores are added into an experimental group (M), Aspergillus niger is not added into a control group (CK), 10 seeds of corn farmers with uniform size and no diseases are sown into each tray, and the rest is managed conventionally.
Plant phenotype growth promoting effect
As shown in FIG. 9, the plant height of the experimental group is 4cm higher than that of the control group, and the number of the third leaves is obviously higher than that of the control group.
Measured data-
TABLE 1 SPAD values and leaf nitrogen contents of the second leaf and the third leaf of the experimental group and the control group
Analysis of data
As shown in fig. 10, the SPAD values and nitrogen contents of the second leaf and the third leaf of the experimental group and the control group were significantly different.
2. Growth promotion experiment of N062 on Zhengdan 958
2.5L of vermiculite and 1.5L of water are added into each tray, and the phosphate solubilizing effect is realized based on Aspergillus niger, so that 1g of calcium phosphate is added into each treatment, Aspergillus niger spores are added into an experimental group (M), Aspergillus niger is not added into a control group (CK), the additives are fully mixed with the vermiculite, 10 corn Zhengdan 958 seeds with uniform size and no diseases are planted into each tray, and the rest is managed conventionally. After the corn plants are planted for 14 days, the root weight (g), the stem leaf weight (g), the chlorophyll SPAD value and the leaf nitrogen content (mg/g) of the corn plants in the experimental group are observed and measured to be obviously improved.
Plant phenotype growth promoting effect
As shown in FIG. 11, the plant height, root length and third leaf length of the experimental group are all superior to those of the control group
Measured data-
TABLE 2 root weight, Stem leaf weight, Total weight of Experimental group and control group
TABLE 3 SPAD values and leaf nitrogen contents of the second leaf and the third leaf of the experimental group and the control group
Analysis of data
As shown in fig. 12, the root weight, stem leaf weight, SPAD value and nitrogen content of the experimental group and the control group were significantly different, wherein the SPAD value and nitrogen content of the second leaf and nitrogen content of the third leaf were significantly different.
3. The embodiment shows that N062 has the capabilities of phosphate solubilizing and saline-alkali resistance and has a good growth promoting effect on corn, and the strain has good prospects in the aspects of improving saline-alkali soil and preparing phosphate solubilizing growth promoting bacterial manure.
While the invention has been described above with reference to an embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In particular, the various features of the embodiments disclosed herein may be used in any combination, provided that there is no structural conflict, and the combinations are not exhaustively described in this specification merely for the sake of brevity and conservation of resources. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.
Sequence listing
<110> Qingdao university of agriculture
<120> a phosphate solubilizing fungus for promoting corn growth and screening and identifying method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 590
<212> DNA
<213> Aspergillus niger
<400> 1
ccgtaggtga acctgcgcaa ggatcattac cgagtgcggg tcctttgggc ccaacctccc 60
atccgtgtct attgtaccct gttgcttcgg cgggcccgcc gcttgtcggc cgccgggggg 120
gcgcctctgc cccccgggcc cgtgcccgcc ggagacccca acacgaacac tgtctgaaag 180
cgtgcagtct gagttgattg aatgcaatca gttaaaactt tcaacaatgg atctcttggt 240
tccggcatcg atgaagaacg cagcgaaatg cgataactaa tgtgaattgc agaattcagt 300
gaatcatcga gtctttgaac gcacattgcg ccccctggta ttccgggggg catgcctgtc 360
cgagcgtcat tgctgccctc aagcccggct tgtgtgttgg gtcgccgtcc ccctctccgg 420
ggggacgggc ccgaaaggca gcggcggcac cgcgtccgat cctcgagcgt atggggcttt 480
gtcacatgct ctgtaggatt ggccggcgcc tgccgacgtt ttccaaccat tctttccagg 540
ttgacctcgg atcaggtagg gatacccgct gaacttaagc atatcaataa 590
Claims (10)
1. A phosphate solubilizing fungus for promoting the growth of corn, wherein the phosphate solubilizing fungus is named as N062, the phosphate solubilizing fungus is named as Aspergillus niger N062 in a specific classification, and the microorganism preservation number is as follows: CCTCC NO: m2022656, the collection address of the strain is: china center for type culture Collection.
2. The fungi for promoting the growth of the maize and dissolving phosphorus according to claim 1, wherein the fungi for dissolving phosphorus has the sequence shown in SEQ.No 1.
3. A separation and screening method of phosphate solubilizing fungi for promoting corn growth is characterized by comprising the following steps:
s01, collecting 1500g of corn rhizosphere soil sample, sealing the corn rhizosphere soil sample by using a sterile bag and bringing the corn rhizosphere soil sample back to a laboratory;
s02, selecting 10g of soil attached to the root system of the corn in a super clean bench, adding sterile water to 100ml, putting the soil into a shaker at the temperature of 28 ℃/180r for 1h to uniformly disperse bacterial colonies in the soil in the water, and standing for 0.5h to precipitate soil particles;
s03, sucking supernatant water, diluting to 10-2, 10-3, 10-4, 10-5 and 10-6 times, respectively taking 200ul of supernatant water, coating on a PDA culture medium, culturing for 48h, selecting a single colony, purifying for 3-5 times, and preserving at-80 ℃ for later use;
s04, culturing the purified single colony on an inorganic phosphorus plate, standing and culturing for 48 hours at the temperature of 28 ℃, and selecting the single colony generating a transparent aperture;
s05, culturing the single colony with the transparent ring on an organic phosphorus plate, selecting the single colony with the transparent ring to obtain the single colony which can decompose both organic phosphorus and inorganic phosphorus, and preserving the single colony at-80 ℃ for later use.
4. A DNA extraction and identification method for promoting the growth of phosphate-solubilizing fungi of corn is characterized by comprising the following steps:
s01, scraping hypha to cover the bottom of a 2ml centrifuge tube, adding CTAB 700ul and grinding beads, and grinding for 6 times and 60S/time in a grinding instrument;
s02, putting the mixture into a water bath at 65 ℃ for 1h after grinding uniformly, and shaking for 10S every ten minutes to fully and uniformly mix CTAB and hyphae;
s03, taking out the centrifuge tube to normal temperature, placing for 2 minutes, adding 600 mu l of chloroform (trichloromethane), fully shaking to make CTAB fully contact with chloroform, and standing for 2 minutes at room temperature;
s04, centrifuging at 12000rpm for 15 minutes, carefully sucking 500 mul of supernatant into a new 1.5ml centrifuge tube, adding 500 mul of isopropanol, fully mixing to obtain DNA floccule, and standing at-20 ℃ for about 2 hours;
s05, taking out the DNA preserved by the-20C, centrifuging at 12000rpm for 15 minutes, discarding the supernatant, and depositing the DNA at the bottom of a centrifugal tube;
s06: adding 500 mul 70% ethanol to wash DNA precipitate, centrifuging at 7500rpm for 5 min, discarding supernatant, and air drying the ethanol washed DNA at room temperature;
s07, dissolving the air-dried DNA with 30 mu l of ddH2O, and storing at-20 ℃ for later use;
s08, identifying the DNA molecule of the phosphate solubilizing fungus strain.
5. The method for extracting and identifying DNA of phosphorus solubilizing fungi for promoting corn growth according to claim 4, wherein the universal primers identified by the DNA molecules of the phosphorus solubilizing fungi strain of step S08 comprise primer ITS1 and primer ITS 4.
6. The method for extracting and identifying DNA of phosphate solubilizing fungi for promoting corn growth according to claim 5, wherein the PCR reaction system for DNA molecular identification of the phosphate solubilizing fungi strain in the step S08 is 2 XPCR Master Mix: 10 ul; the primers ITS 1: 1 ul; the primers ITS 4:1 ul; DNA template: 1 ul; ddH 2O: 7 ul.
7. The method for extracting and identifying DNA of phosphorus-solubilizing fungi for promoting corn growth according to claim 6, wherein the PCR reaction procedure for identifying the DNA molecules of the phosphorus-solubilizing fungi strain in the step S08 is pre-denaturation at 95 ℃ for 10 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 10min at 72 ℃; infinity at 4 ℃.
8. The fungus for promoting the growth of corns and dissolving phosphorus according to claim 2, wherein the fungus for dissolving phosphorus has salt tolerance and can normally grow under the salt stress of 30g/L, when the salt concentration is increased to 70g/L, the growth is initially inhibited, and when the salt concentration is 90g/L, the growth is obviously inhibited and the growth is slow; the phosphate-solubilizing fungi have acid and alkali resistance and grow well on a culture medium with pH of 3-12.
9. The fungus for promoting the growth of phosphorus and improving the plant nutrition of corn according to claim 2, wherein the fungus for promoting the growth of phosphorus and improving the corn is capable of secreting siderophins and improving the plant iron nutrition, and can be verified by a CAS culture medium culture detection method.
10. The fungus for promoting the growth of corns and dissolving phosphorus as claimed in claim 2, wherein the fungus for dissolving phosphorus has the capability of dissolving phosphorus and resisting saline and alkali, has good growth promoting effect on corns, has good prospects in improving saline and alkali soil and preparing bacterial manure for promoting the growth of phosphorus, and is obtained by analyzing the difference between root weight, stem leaf weight, SPAD value and nitrogen content of an experimental group and a control group in a corn growth promoting experiment, wherein the difference between the SPAD value and nitrogen content of a second leaf and the nitrogen content of a third leaf is significant.
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