CN109576164B - Efficient phosphate-solubilizing fungus rhizopus microsporus and screening method and application thereof - Google Patents
Efficient phosphate-solubilizing fungus rhizopus microsporus and screening method and application thereof Download PDFInfo
- Publication number
- CN109576164B CN109576164B CN201910064466.0A CN201910064466A CN109576164B CN 109576164 B CN109576164 B CN 109576164B CN 201910064466 A CN201910064466 A CN 201910064466A CN 109576164 B CN109576164 B CN 109576164B
- Authority
- CN
- China
- Prior art keywords
- rhizopus microsporus
- pathogenic bacteria
- culture medium
- neau
- soil
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/845—Rhizopus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
A high-efficiency phosphate-solubilizing fungus rhizopus microsporus and a screening method and application thereof belong to the field of microbiology. Aiming at the problems of degrading inorganic phosphorus in soil and promoting crop growth, the invention provides a high-efficiency phosphate-solubilizing fungus Rhizopus microsporus (Rhizopus microsporus) NEAU-8, the preservation number of which is as follows: CGMCC NO. 14635. The screening method comprises the following steps: 1) adding 0.85% volume fraction of physiological saline into wheat rhizosphere soil, enriching and culturing, and standing; 2) taking the supernatant for gradient dilution, taking each diluent, and respectively coating the diluent on an inorganic phosphorus solid culture medium for culture; 3) picking out the microorganisms with the transparent rings on each culture medium, and inoculating the microorganisms to a new inorganic phosphorus solid culture medium for culture; 4) respectively preparing the cultured thalli into spore suspension, inoculating the spore suspension into an inorganic phosphorus liquid culture medium for culturing, measuring the content of soluble phosphorus in a bacterial liquid, and screening to obtain the fungus rhizopus microsporus with high-efficiency phosphate solubilizing effect. The rhizopus microsporus obtained by the invention can be used in agricultural production.
Description
Technical Field
The invention belongs to the field of microbiology, and particularly relates to a high-efficiency phosphate-solubilizing fungus rhizopus microsporus, and a screening method and application thereof.
Background
Phosphorus is one of three essential nutrients for plant growth and development, is the basic constituent element of organic compounds such as genetic substances, energy substances and the like in plant cells, and is essential to participate in photosynthesis and biochemical metabolic processes in vivo of plants, and plays an irreplaceable role in the whole life process of the plants. It is reported that 74% of cultivated land soil in China is deficient in phosphorus, and about 95% of phosphorus in soil exists in the form of ineffective phosphorus, and cannot be absorbed and utilized by plants. Therefore, phosphorus is one of the major elements that limit crop growth and yield. In order to alleviate the influence of phosphorus deficiency on plant growth, the agricultural production mainly depends on applying phosphate fertilizers to increase the supply of phosphorus. However, most of the phosphate fertilizer applied to the soil will react with metal ions in the soil, such as Al3+、Fe3 +、Ca3+And the combination forms insoluble phosphate, so that the utilization rate of phosphorus in the current year is only 10-25%. In order to meet the requirement of crops on phosphorus, a large amount of phosphate fertilizer is often applied in agricultural production, and excessive application of the phosphate fertilizer can cause serious economic loss, so that the diversity of microorganisms in soil is reduced, and environmental problems such as soil hardening, soil fertility decline, environmental pollution, ecological deterioration, agricultural product quality reduction and the like can be caused.
The phosphate solubilizing microorganism can dissolve insoluble phosphorus in soil, convert ineffective phosphorus into available phosphorus which can be absorbed by plants, and stimulate the growth of plants by producing extracellular enzymes and organic acids. The phosphate solubilizing microorganisms can be classified into phosphate solubilizing bacteria, actinomycetes and fungi, wherein the phosphate solubilizing bacteria are more studied, including Enterobacter (Enterobacter), Agrobacterium (Agrobacterium), Erwinia (Erwinia), Pseudomonas (Pseudomonas), Serratia (Serratia), Flavobacterium (Flavobacterium), Bacillus (Bacillus), Micrococcus (Micrococcus), Azotobacter (Azotobacter), Chromobacterium (Chromobacterium), Salmonella (Salmonella), Alcaligenes (Alcaligenes), Arthrobacter (Arthrobacter), Thiobacillus (Thiobacillus), Escherichia (Escherichia); phosphate solubilizing actinomycetes are mainly focused on Streptomyces (Streptomyces); there are few reports on related studies on phosphate-solubilizing fungi, and most of them are Aspergillus (Aspergillus), Rhizopus (Rhizopus), Fusarium (Fusarium), Penicillium (Penicillium), Sclerotium (Sclerotium), and AM mycorrhiza (Arbusular mycorrhiza). Many phosphate solubilizing bacteria and actinomycetes lose their original phosphate solubilizing ability after subculture, and this loss cannot be recovered any more.
Disclosure of Invention
Aiming at the problems of degrading inorganic phosphorus in soil and promoting crop growth, the invention provides a high-efficiency phosphate-solubilizing fungus Rhizopus microsporus (Rhizopus microsporus) NEAU-8, the preservation number of which is as follows: CGMCC NO. 14635.
The invention also provides a screening method of the rhizopus microsporus, which comprises the following steps:
1) taking rhizosphere soil of wheat, adding 0.85% of normal saline for enrichment culture, and standing for 15-30 min; the ratio of the soil to the normal saline is 1:9 (g/mL);
2) then taking 1mL of supernatant for gradient dilution, taking the diluent of each dilution, and respectively coating the diluent on an inorganic phosphorus solid culture medium for culture;
3) picking out the microorganisms with the transparent rings on each culture medium, and inoculating the microorganisms to a new inorganic phosphorus solid culture medium for culture;
4) then, respectively preparing the cultured thalli into spore suspensions, inoculating the spore suspensions into an inorganic phosphorus liquid culture medium for culturing, and screening to obtain the fungus rhizopus microsporus with high-efficiency phosphorus dissolution by measuring the content of soluble phosphorus in a bacterial liquid.
Further defined, the enrichment culture in the step 1) refers to shaking culture at 150r/min for 90-120min in a shaking table at 28 ℃.
Further defined, the step 2) of gradient dilution means that the supernatant is diluted to 10 of the original volume respectively-1、10-2、10-3、 10-4、10-5、10-6。
Further defined, the formulation of the inorganic phosphorus solid medium in the step 2) is as follows: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, MgSO4.7H2O 0.3g，FeSO4.7H2O 0.03g，MnSO4.4H20.03g of O, 10g of calcium phosphate, 18g of agar and 1000mL of deionized water, and the pH is adjusted to 7.0.
Further defined, the inoculation in the step 4) is to inoculate the spore suspension into the inorganic phosphorus liquid culture medium according to 1 percent (volume fraction); the culture is carried out for 7d under the condition of shaking culture at the temperature of 28 ℃ and at the speed of 150 r/min.
The invention also provides application of the rhizopus microsporus in inhibiting the generation of pathogenic fungi.
Further defined, the pathogenic fungi are cucumber wilt pathogen (Fusarium oxysporum), corn large spot pathogen (Exserohilum turcum), cucumber brown spot pathogen (Corynebacterium cassiacola), cucumber anthracnose pathogen (Colletotrichum orbiculare), corn small spot pathogen (Helminthosporium maydis), rice sheath blight pathogen (Rhizoctonia solani), maize Curvularia pathogen (Curvularia lunata), soybean root rot pathogen (Rhizoctonia solani Kuhn) or soybean sclerotium pathogen (Sclerotinia sclerotiorum).
The invention also provides application of the rhizopus microsporus in promoting crop growth.
Further, the rhizopus microsporus with spore concentration of 1.0 x 10 in soil is mixed with soil and used for cultivating corn or wheat seeds3cfu/g～1.0×105cfu/g, optimum spore concentration 1.0 × 104cfu/g。
Advantageous effects
The rhizopus microsporus NEAU-8 separated from the rhizosphere soil of wheat has stronger phosphate-solubilizing capability to calcium phosphate and zinc phosphate, and the corresponding phosphate-solubilizing capability is 403mg/L and 507mg/L respectively. Potted plant experiments show that the spore liquid of the strain can obviously promote the growth of wheat and corn. The rhizopus microsporus NEAU-8 strain and the microbial inoculum thereof provided by the invention have no pollution and public nuisance in the using process, can reduce the using amount of chemical fertilizers, and have stable hereditary characters of phosphate-solubilizing fungi, are not easy to lose the phosphate-solubilizing capability in subculture generally, provide excellent strain resources for developing microbial fertilizers, and have good application and development prospects.
The strain Rhizopus microsporus NEAU-8 provided by the invention is separated from wheat rhizosphere soil in corridor city of Hebei province, is preserved in China general microbiological culture collection center (CGMCC for short) in 2017 and 11 and 14 days, and has the preservation address as follows: no. 3 Xilu No.1 Beijing, Chaoyang, and the preservation number is CGMCC NO. 14635.
Drawings
FIG. 1 is a characteristic of the colony of the strain NEAU-8 on PDA medium.
FIG. 2 is a graph showing the effect of decomposing calcium phosphate when the strain NEAU-8 is cultured in a shake flask.
FIG. 3 is a phosphorus standard curve.
FIG. 4 shows the antagonistic effect of the strain NEAU-8 on pathogenic fungi, with the control of pathogenic fungi in the left plate of each picture, the upper right plate of each picture inoculated with the strain NEAU-8 and the lower plate inoculated with the pathogenic fungi. Pathogenic fungi: a cucumber brown spot pathogen (Corynespora cassiicola), b cucumber anthracnose pathogen (Colletotrichum orbiculosum), c soybean sclerotium pathogen (Sclerotinia sclerotiorum), d cucumber wilt pathogen (Fusarium oxysporum), e corn northern leaf blight (Exserohilum turcicum), f soybean root rot pathogen (Rhizoctonia solani Kuhn), g corn Curvularia (Curvularia lunata), h rice wither pathogen (Rhizoctonia solani), i corn microsporum maydis (Helminthosporium maydis)
FIG. 5 is a graph showing the growth promoting effect of the strain NEAU-8 on potted maize.
FIG. 6 is a graph showing the results of the effect of the strain NEAU-8 on the growth of potted wheat.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
The Rhizopus microsporus NEAU-8 of the present invention is simply referred to as strain NEAU-8 and Rhizopus microsporum NEAU-8 in the following examples or drawings.
The plate refers to a solid medium, which is a term commonly used in the art.
PDA medium, PDB medium and other chemical reagents or instruments, which are conventional reagents or instruments without specific instructions, are commercially available.
Example 1 isolation and characterization of Rhizopus microsporus (Rhizopus microsporus) NEAU-8.
1. Isolation and cultivation of the Strain
Rhizopus microsporus NEAU-8 is obtained by screening wheat rhizosphere soil in corridor city of Hebei province. The phosphate solubilizing bacteria are screened by adopting an inorganic phosphorus solid culture medium: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, MgSO4.7H2O 0.3g，FeSO4.7H2O 0.03g，MnSO4.4H20.03g of O, 10g of calcium phosphate, 18g of agar and deionized water are added to the volume of 1000mL, and the pH value is adjusted to 7.0.
1) Adding 0.85% volume fraction of physiological saline into wheat rhizosphere soil, performing enrichment culture, and standing for 15-30 min; the ratio of the soil to the physiological saline was 1:9 (g/mL). The method comprises the following specific steps: sieving a proper amount of wheat rhizosphere soil with a 20-mesh sieve, adding the wheat rhizosphere soil into a mortar for full grinding, weighing 10g of ground wheat rhizosphere soil, pouring the ground wheat rhizosphere soil into a 250mL conical flask, simultaneously adding 90mL of 0.85% volume fraction normal saline, carrying out shaking culture in a shaking table at the temperature of 28 ℃ for 90-120min at the speed of 150r/min, preferably 120min, and taking out the wheat rhizosphere soil and standing the wheat rhizosphere soil for 30min at room temperature.
2) Then taking 1mL of supernatant for gradient dilution, taking the dilution of each dilution, respectively coating the dilution on an inorganic phosphorus solid culture medium for cultureCultivating; the method comprises the following specific steps: 1mL of the supernatant was collected and diluted to 10 volumes of the original volume according to the dilution plating method-1、10-2、10-3、10-4、10-5、10-6Then, 200. mu.L of each dilution was applied to the inorganic phosphorus solid medium uniformly, and the plate was repeated 2 times at each dilution. All plate markers were sealed with plastic wrap and inverted in a 28 ℃ incubator for culture.
3) In the culture process, the microorganism with the transparent ring on each flat plate is picked out, cultured in a new inorganic phosphorus solid culture medium, marked with strains and preserved at 4 ℃. Activating the strains with better phosphate-solubilizing ability obtained by primary screening, and carrying out inverted culture in a constant-temperature incubator at 28 ℃ for 5 days.
4) Then, respectively preparing the cultured thalli into spore suspensions, inoculating the spore suspensions into an inorganic phosphorus liquid culture medium for culturing, and screening to obtain the fungus rhizopus microsporus with high-efficiency phosphorus dissolution by measuring the content of soluble phosphorus in a bacterial liquid. The method comprises the following specific steps: then the activated strain is transferred to a PDA large test tube inclined plane to be cultured in a constant temperature incubator at 28 ℃, the cultured strain is washed by sterile water after 7 days to be prepared into spore suspension, and the spore concentration is counted by a blood counting chamber. Inoculating the spore suspension into inorganic phosphorus liquid culture medium (formula: glucose 10g, ammonium sulfate 0.5g, sodium chloride 0.3g, potassium chloride 0.3g, MgSO 2)4.7H2O 0.3g，FeSO4.7H2O 0.03g，MnSO4.4H20.03g of O, 10g of calcium phosphate and 1000mL of deionized water, wherein the pH value is adjusted to 7.0), inoculating sterile distilled water with the same volume in a control group, carrying out shake cultivation at 28 ℃ for 7d at 150r/min, taking samples every 1d, centrifuging, measuring the content of soluble phosphorus in a supernatant by a molybdenum-antimony colorimetric resistance method, continuously measuring, determining the decomposition condition of the test strain on the insoluble inorganic phosphorus, and determining that the higher the content of the soluble phosphorus in the test strain exceeds that in the control group, the better the decomposition effect of the strain on the inorganic phosphorus is, thereby obtaining the strain with better phosphorus dissolving effect. The purified strain was designated as NEAU-8 and stored in an ultra-low temperature refrigerator at-80 ℃ with 20% glycerol.
2. And (5) identifying the strain.
(1) Morphological characteristics: as shown in figure 1, rhizopus microsporus NEAU-8 is cultured on PDA culture medium at 28 deg.C for 5 days, the colony has rapidly spread and spread over the whole plate, the hypha is gray, flocculent, loose in texture, and has radially arranged villiform white hypha on the periphery, and the back of the culture medium is not discolored (no pigment is generated). Growth temperature range: 15-48 ℃; growth pH range: 3-8; growth NaCl range: 3 to 10 percent.
(2) Sequencing ITS rDNA sequence: inoculating Rhizopus microsporus NEAU-8 on a PDA plate, culturing in a biochemical incubator at 28 deg.C for 5-7 days, scraping the mycelia into a sterilized mortar when the mycelia amount is sufficient, adding liquid nitrogen, grinding into powder, and extracting fungus DNA by CTAB (CetylltriethylLamium bromide) method.
The universal primers ITS1/ITS4 amplify the sequence of the 5.8Sr DNA-ITS region of the test fungus. The sequence is as follows:
the ITS1 primer sequence is: 5'-TCCGTAGGTGAACCTGCGG-3'
The ITS4 primer sequence is: 5'-TCCTCCGCTTATTGATATGC-3'
A50-microliter reaction system is selected, and the components are shown in the following table 1:
TABLE 1PCR amplification System
The procedure used for the reaction: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s, extension at 72 ℃ for 1min for 28 cycles, and final extension at 72 ℃ for 5 min. The amplification products were detected by electrophoresis on a 1% agarose gel, 1 XTAE electrophoresis buffer, and the amount of the sample was 5. mu.L. And observed by a gel imaging system. And recovering and purifying the amplification product by using a gel recovery kit. The specific operation is carried out according to the kit instructions.
The gel recovery product was sequenced by DNA sequencing Inc. (Jilin province, Kuume Biotech). Inputting the obtained ITS rDNA sequence into GenBank to obtain accession number. Comparative analysis was performed using Blast software.
The ITS rDNA sequence is shown in SEQ ID NO.1, and the sequence has been registered with NCBI and has the registration number of MF 945552. Sequence alignment analysis is carried out by a BLAST program, and the result shows that the similarity of the ITS rDNA sequence of the strain and Rhizopus microsporus CBS 130158 reaches 100 percent.
The isolated strain was identified as Rhizopus microsporus NEAU-8 by combining morphological characteristics of the thallus and ITS rDNA (shown in SEQ ID No: 3) sequence sequencing. The strain is preserved in China general microbiological culture collection center (CGMCC for short) in 2017, 11 months and 14 days, and the preservation number is CGMCC NO. 14635.
Example 2. determination of the phosphate solubilizing ability of Rhizopus microsporus NEAU-8.
The phosphate solubilizing capability of phosphate solubilizing fungi Rhizopus microsporus NEAU-8 is measured by using an inorganic phosphorus liquid culture medium, the Rhizopus microsporus NEAU-8 cultured on a slant is inoculated into a PDB liquid culture medium (a potato culture medium without agar) to serve as a seed solution, the seed solution is subjected to shaking culture for 12 hours in a constant temperature shaking table at the temperature of 28 ℃ and the speed of 200r/min, then a cultured bacterium solution is inoculated into an inorganic phosphorus liquid culture medium triangular flask according to the inoculation amount (v/v) of 1 percent, a control group is inoculated into a PDB liquid culture medium without inoculation according to the inoculation amount (v/v) of 1 percent and is subjected to shaking culture for 7 days in the constant temperature shaking table at the temperature of 28 ℃ and the speed of 200 r/min. The phosphorus dissolving effect is shown in figure 2, and it can be seen from the figure that the liquid culture medium inoculated with the rhizopus microsporus NEAU-8 is clear and transparent, the precipitate completely disappears, while the liquid culture medium in the control group is turbid, and the precipitate is not dissolved, which indicates that the rhizopus microsporus NEAU-8 obtained by screening has strong phosphorus dissolving effect.
The content of available phosphorus in the supernatant is measured by molybdenum-antimony colorimetry. Respectively adding 100mg/L standard phosphorus solution with corresponding volume into a volumetric flask, adding 2 drops of 2, 6-dinitrophenol as an indicator, adjusting the pH value by using dilute sulfuric acid and 10% NaOH (g/v) solution, adding 5ml of molybdenum-antimony anti-color-developing agent, fixing the volume to scale, enabling the standard phosphorus concentration to be 0, 0.2, 0.4, 0.6, 0.8 and 1.0mg/L respectively, shaking up, reacting for 30min at room temperature (about 25 ℃), carrying out color comparison at 720nm by using a UV-1601PC ultraviolet-visible spectrophotometer, drawing a standard curve according to the result, and calculating the corresponding effective phosphorus content according to the standard curve. Centrifuging the fermentation liquor at 8000r/min for 10min, taking the supernatant in a volumetric flask of 2.5-5.0 ml to 50ml, and carrying out color comparison according to the method.
The phosphorus standard curve is shown in FIG. 3, and the equation of the phosphorus standard curve is: and y is 0.4405x +0.0484, and the correlation coefficient can reach 0.9993. Wherein y is OD720And x is the concentration (mg/L) of the standard phosphorus solution, and the phosphorus dissolving capacity of the phosphorus dissolving bacteria can be calculated according to the standard curve equation. The concentration of the available phosphorus in the blank control solution is 87.4mg/L, the pH value is 5.82, the content of the available phosphorus in the rhizopus microsporus solution is 403.2mg/L, and the pH value is 2.08.
Example 3. use of Rhizopus microsporus (Rhizopus microspors) NEAU-8 for inhibiting the production of pathogenic fungi.
The method comprises the steps of taking the center of a culture dish containing a PDA solid culture medium as an original point, inverting a cultured fungus cake to be tested in the culture dish at a radius which is 1/2 away from the original point, inverting the fungus cake of fungi at a symmetrical position on the other side of the original point, taking the culture dish which is not inoculated with the fungus to be tested as a contrast, repeating each combination for three times, culturing in an incubator at 28 ℃, observing the size of colonies of the pathogenic fungi and the fungus to be tested, the change condition of the distance between two colonies, the existence and the size of an antibacterial zone at intervals, and marking on a flat plate by using a marker pen to observe the change condition. And measuring the width of the bacteriostatic zone and the distance between the two fungus cakes, and calculating the bacteriostatic rate.
In the confronting culture, the rhizopus microsporus NEAU-8 has inhibition effect on various pathogenic fungi in a laboratory, shows good bacteriostatic activity and has broad spectrum of bacteriostatic spectrum. The inhibition rate of the pathogenic bacteria of cucumber wilt is the largest and can reach 100%, and the inhibition rates of 9 pathogenic fungi are respectively cucumber wilt pathogenic bacteria, corn large spot pathogenic bacteria, cucumber brown spot pathogenic bacteria, cucumber anthracnose pathogenic bacteria, corn small spot pathogenic bacteria, rice sheath blight pathogenic bacteria, corn curvularia pathogenic bacteria, soybean root rot pathogenic bacteria and soybean sclerotium pathogenic bacteria (Table 2). Rhizopus microsporus NEAU-8 expands by sporulation in large quantities and inhibits the growth of the pathogenic fungi tested by matrix competition (FIG. 4). The rhizopus microsporus NEAU-8 shows antagonism to various disease fungi in a flat plate antagonism experiment, so that the rhizopus microsporus NEAU-8 can be applied to soil, plays a role in inhibiting certain disease fungi in the soil and provides biocontrol protection for crops during the growth period of the crops.
TABLE 2 inhibition of pathogenic fungi%
Example 4. use of Rhizopus microsporus (Rhizopus microsporus) NEAU-8 for promoting crop growth.
First, the growth promoting effect of Rhizopus microsporus (Rhizopus microsporus) NEAU-8 on corn.
Activating Rhizopus microsporus NEAU-8 twice on PDA culture medium, inoculating on PDA large test tube slant, culturing at 28 deg.C, collecting mycelium and spore mycelium with sterile water after 7 days, breaking with magnetic stirrer, and diluting with sterile water to 1.0 × 104cfu/mL for use. 1mL of spore suspension is added into each gram of soil of the treatment group and mixed uniformly, and the final spore concentration is 1.0X 104cfu/g, sterile water as control. Well wrapping the intact and plump corn seeds with a wet towel, moisturizing and accelerating germination at the temperature of 28 ℃, and sowing when 80% of the seeds are exposed to white. Selecting corn seeds with consistent growth vigor and showing white color, gently clamping the corn seeds by using forceps, placing the corn seeds in a basin containing 1kg of soil, placing 3 seeds in each basin, and repeating the treatment for 4 times. After 30 days, the plant height, root length, fresh weight and dry weight of the seedlings and roots of the maize were measured, respectively. The results are shown in FIG. 5 and Table 3.
TABLE 3 Effect of the strains NEAU-8 spore fluid on the growth of potted maize
As can be seen, the concentration of the spores of the fungus Rhizopus microsporus NEAU-8 was 1.0X 10 compared to the control4The growth promoting effect on the corn is remarkable when cfu/g is adopted, the plant height is increased by 24.1%, the root length is increased by 34.8%, the fresh weight of the seedlings is increased by 67.1%, the dry weight of the seedlings is increased by 38.4%, the fresh weight of the roots is increased by 69.0%, and the dry weight of the roots is increased by 71.1%.
Secondly, the growth promoting effect of Rhizopus microsporus (Rhizopus microsporus) NEAU-8 on wheat.
Rhizopus microsporus NEAU-8 is activated twice on PDA culture medium and inoculated in PDA large scale experimentCulturing on tube slant at 28 deg.C for 7 days, diluting with sterile water to 1.0 × 104cfu/mL for use. 1mL of spore suspension is added into each gram of soil of the treatment group and mixed uniformly, and the final spore concentration is 1.0X 104cfu/g, sterile water as control. Well wrapping the perfect and full wheat seeds with a wet towel, moisturizing and accelerating germination at the temperature of 28 ℃, and sowing when 80% of the seeds are exposed to white. Selecting the exposed white wheat seeds with consistent growth vigor, lightly clamping the seeds by using forceps, placing the seeds in a basin containing 1kg of soil, placing 10 seeds in each basin, and repeating the treatment for 4 times. After 30 days, the plant height, root length, fresh weight and dry weight of seedlings and roots of wheat were measured, respectively. The results are shown in FIG. 6 and Table 4.
TABLE 4 Effect of NEAU-8 spore fluid strains on growth of potted wheat
| Plant height (cm) | Root length (cm) | Weight of Miao (g) | Miao gan weight (g) | Fresh weight of root (g) | Root weight (g) | |
| CK | 46.03±2.86 | 18.53±1.95 | 1.662±0.203 | 0.149±0.014 | 0.695±0.022 | 0.053±0.012 |
| Strain NEAU-8 | 57.45±2.83 | 24.51±2.46 | 2.366±0.311 | 0.197±0.023 | 0.996±0.036 | 0.095±0.021 |
As can be seen, the concentration of the strain was 1.0X 10 as compared with the control4The growth promoting effect on wheat is remarkable when cfu/g is adopted, the plant height is increased by 24.8%, the root length is increased by 32.3%, the fresh weight of the seedling is increased by 42.4%, the dry weight of the seedling is increased by 32.2%, the fresh weight of the root is increased by 43.3%, and the dry weight of the root is increased by 79.2%.
Nucleotide sequence listing
<110> northeast university of agriculture
<120> efficient phosphate solubilizing fungus rhizopus microsporus and screening method and application thereof
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> ITS1 primer
<400> 1
tccgtaggtg aacctgcgg 19
<210> 2
<211> 20
<212> DNA
<213> ITS4 primer
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 647
<212> DNA
<213> ITS rDNA
<400> 3
gttggcactt tactgggatt tacttctcag tattgtttgc ttctatactg tgaacctctg 60
gcgatgaagg tcgtaactga ccttcgggag agactcagga catataggct ataatgggta 120
ggcctgttct ggggtttgat cgatgccaat caggattacc tttcttcctt tgggaaggaa 180
ggtgcctggt accctttacc atataccatg aattcagaat tgaaagtata atataataac 240
aacttttaac aatggatctc ttggttctcg catcgatgaa gaacgtagca aagtgcgata 300
actagtgtga attgcatatt cgtgaatcat cgagtctttg aacgcagctt gcactctatg 360
gatcttctat agagtacgct tgcttcagta tcataaccaa cccacacata aaatttattt 420
tatgtggtga tggacaagct cggttaaatt taattattat accgattgtc taaaatacag 480
cctctttgta attttcatta aattacgaac tacctagcca tcgtgctttt ttggtccaac 540
caaaaaacat ataatctagg ggttctgcta gccagcagat attttaatga tctttaacta 600
tgatctgaag tcaagtggga ctacccgctg aacttaagca tatcaaa 647
Claims (5)
1. Efficient phosphate-solubilizing fungus rhizopus microsporus (A. sp.) (Rhizopus microsporus) NEAU-8, accession number: CGMCC NO. 14635.
2. Use of rhizopus microsporus as claimed in claim 1 for inhibiting the production of pathogenic fungi.
3. Use according to claim 2, characterized in that the pathogenic fungus is cucumber wilt pathogenic bacteria (c: (a)Fusarium oxysporum) Corn plaque pathogenic bacteria: (A), (B), (C)Exserohilum turcicum) Cucumber brown spot pathogenic bacteria: (Corynespora cassiicola) Cucumber anthracnose pathogen: (Colletotrichum orbiculare) Corn plaque pathogenic bacteria (A), (B), (C)Helminthosporium maydis) Pathogenic bacteria of rice sheath blight (A)Rhizoctonia solani) Curvularia lunata pathogen: (Curvularia lunata) Pathogenic bacteria of soybean root rot: (Rhizoctonia solani Kuhn) Or pathogenic bacteria of soybean sclerotium: (Sclerotinia sclerotiorum)。
4. Use of rhizopus microsporus as claimed in claim 1 for promoting the growth of crops.
5. The use of claim 4, wherein the rhizopus microsporus has a spore concentration of 1.0 x 10 in soil, and is used for cultivating corn or wheat seeds after being mixed with soil3cfu/g～1.0×105cfu/g。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910064466.0A CN109576164B (en) | 2019-01-23 | 2019-01-23 | Efficient phosphate-solubilizing fungus rhizopus microsporus and screening method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910064466.0A CN109576164B (en) | 2019-01-23 | 2019-01-23 | Efficient phosphate-solubilizing fungus rhizopus microsporus and screening method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109576164A CN109576164A (en) | 2019-04-05 |
| CN109576164B true CN109576164B (en) | 2022-03-04 |
Family
ID=65917867
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910064466.0A Active CN109576164B (en) | 2019-01-23 | 2019-01-23 | Efficient phosphate-solubilizing fungus rhizopus microsporus and screening method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109576164B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101439996A (en) * | 2008-12-18 | 2009-05-27 | 杨雪枣 | Flower-protecting and sweet-enhancing composite biological-organic liquid fertilizer |
| CN104774784A (en) * | 2015-03-02 | 2015-07-15 | 河南科技大学 | Phosphate dissolving bacterium and acquisition method thereof |
| CN107858296A (en) * | 2017-12-15 | 2018-03-30 | 山东农业大学 | The preparation and application of the aspergillus niger and its microbial inoculum of one plant of phosphorus decomposing, potassium decomposing and degraded cellulose |
-
2019
- 2019-01-23 CN CN201910064466.0A patent/CN109576164B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101439996A (en) * | 2008-12-18 | 2009-05-27 | 杨雪枣 | Flower-protecting and sweet-enhancing composite biological-organic liquid fertilizer |
| CN104774784A (en) * | 2015-03-02 | 2015-07-15 | 河南科技大学 | Phosphate dissolving bacterium and acquisition method thereof |
| CN107858296A (en) * | 2017-12-15 | 2018-03-30 | 山东农业大学 | The preparation and application of the aspergillus niger and its microbial inoculum of one plant of phosphorus decomposing, potassium decomposing and degraded cellulose |
Non-Patent Citations (2)
| Title |
|---|
| Characterization of a Thermotolerant Phytase Produced by Rhizopus microsporus var. microspores Biofilm on an Inert Support Using Sugarcane Bagasse as Carbon Source;Vanessa Sayuri Sato等;《Applied Biochemisty and Biotechnology》;20160223;全文 * |
| Innovative biorefinery concept for sugar-based ethanol industries: production of protein-rich fungal biomass on vinasse as an aquaculture feed ingredient;Saoharit Nitayavardhana等;《Bioresour Technol.》;20100716;第101卷(第23期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109576164A (en) | 2019-04-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110591963B (en) | Saline-alkali-tolerant bacillus malassezia and application thereof | |
| CN111100805B (en) | Rice sheath blight disease bactericide prepared by using boron-resistant lysine bacillus as chassis cells | |
| CN111500501B (en) | Streptomyces misonii strain and application thereof in preventing and treating wheat root rot and stem basal rot | |
| CN110066756B (en) | Paenibacillus kribbensis and preparation and application thereof | |
| CN107136122B (en) | Biocontrol microbial inoculum for preventing and treating potato late blight | |
| CN108342330B (en) | Trichoderma longibrachiatum with broad-spectrum antibacterial performance and application thereof | |
| CN112940976B (en) | Marine bacillus megaterium, microbial fertilizer, fermentation broth and application | |
| CN112662585B (en) | Bacillus atrophaeus DX-9 and application thereof | |
| CN110819565A (en) | Salt-tolerant bacillus BW9 with plant pathogenic bacteria inhibiting effect and application thereof | |
| CN109112072B (en) | Application of trichoderma in promoting plant growth | |
| CN108048360B (en) | Bacillus subtilis with dual functions of degrading organic phosphorus and preventing diseases | |
| CN106929433B (en) | Phosphate solubilizing penicillium and application thereof | |
| CN117467571A (en) | Bacillus for degrading nicosulfuron and application thereof | |
| CN114874953B (en) | Peanut rhizosphere biocontrol bacterium-Bacillus beleisi SW-1 and application thereof | |
| CN108130303B (en) | Acidovorax vorax TCP2011036 and application thereof | |
| Nookongbut et al. | Selection of acid tolerant purple nonsulfur bacteria for application in agriculture | |
| CN109576164B (en) | Efficient phosphate-solubilizing fungus rhizopus microsporus and screening method and application thereof | |
| CN110373344A (en) | Carp streptomycete and its application | |
| CN115960766A (en) | Microorganism for preventing and treating bacterial wilt and application thereof | |
| CN112725193B (en) | Trichoderma tomentosum and application thereof | |
| CN110396477B (en) | Trichoderma mutant strain suitable for being compounded with hymexazol to prevent and treat melon blight, fungicide and application thereof | |
| CN113564086A (en) | Rhizobium with phosphorus dissolving function and garden plant growth promoting function and application thereof | |
| Devi et al. | Screening and characterization of plant growth promoting rhizobacteria and their effect on growth and yield of barley (Hordeum vulgare L.) | |
| CN116622576B (en) | Mars agricultural bacterium, composite microbial inoculant and application thereof | |
| CN115851507B (en) | Paenibacillus elgii, microbial inoculum and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |