CN113956982B - Penicillium and application thereof - Google Patents
Penicillium and application thereof Download PDFInfo
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- CN113956982B CN113956982B CN202111247713.4A CN202111247713A CN113956982B CN 113956982 B CN113956982 B CN 113956982B CN 202111247713 A CN202111247713 A CN 202111247713A CN 113956982 B CN113956982 B CN 113956982B
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- 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
- A01N63/36—Penicillium
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Botany (AREA)
- Medicinal Chemistry (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Environmental Sciences (AREA)
- Fertilizers (AREA)
Abstract
The invention relates to the field of microorganisms, in particular to penicillium and application thereof. The preservation number of the penicillium provided by the invention is CGMCC No.22420. The penicillium can effectively degrade phenolic acid autotoxic substances such as benzoic acid, salicylic acid and p-hydroxybenzoic acid secreted by plant root systems, has an obvious inhibiting effect on soil-borne pathogenic bacteria fusarium oxysporum, can be applied to degrading plant root autotoxic substances such as vanilla, improves the rhizosphere soil flora structure of continuous cropping vanilla, and promotes healthy growth of plants.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to penicillium and application thereof.
Background
The cell tissue castoff and root secretion of the plant root system provide rich nutrition and energy for rhizosphere microorganisms, so that the number and activity of the microorganisms in the plant root system are obviously higher than those of the soil outside the root. The species and amounts of substances such as rhizosphere carbohydrates, amino acids, vitamins, etc. directly affect the species, amounts and activities of rhizosphere microorganisms, and thus the species of plants and the rhizosphere effects are different.
Root exudates of some plants affect the composition of rhizosphere microbial communities, and imbalance of rhizosphere soil microbial systems is an important factor causing the growth of continuous cropping plants to be restrained and frequently caused by soil-borne diseases. Some crops, such as vanilla, pepper, white gourd, melon, banana and the like, can generate root system autotoxic substances such as benzoic acid, salicylic acid or parahydroxybenzoic acid and the like in growth, so that the relative abundance of fusarium oxysporum which is obviously and positively related to the planting age and the root (stem) rot disease incidence index of soil-borne diseases is increased along with the increase of the planting age; the relative abundance of beneficial bacteria which are obviously and inversely related to the planting period and the disease index is reduced, the disease index of soil-borne wilt is increased, the dry weight of the new drawer-type stem is suddenly reduced, plant diseases are aggravated, the growth is slow, and the yield and the quality are reduced.
How to degrade phenolic acid autotoxic substances secreted by root systems, and avoid the problems of unbalanced soil microorganism systems, high incidence of soil-borne diseases and restrained plant growth, has become a bottleneck problem to be solved urgently.
Disclosure of Invention
In view of the above, the present invention aims to provide a penicillium capable of degrading phenolic acid autotoxic substances and uses thereof.
The invention provides penicillium with a preservation number of CGMCC No.22420.
The invention provides a strain which is separated from rhizosphere soil of a vanilla plantation, named as strain FD-21, and a microbial fertilizer of the strain can degrade phenolic acid autotoxic substances such as benzoic acid, salicylic acid and p-hydroxybenzoic acid secreted by root systems of vanilla plants and has an obvious inhibition effect on fusarium oxysporum. The penicillium is preserved in the China general microbiological culture Collection center (CGMCC) at the 5 th month 17 of 2021, and has the address of the North Chen West Lu No. 1, 3 of the Korean area of Beijing city, and the preservation number of CGMCC No.22420.
The invention also provides a microbial agent which is prepared from penicillium with the preservation number of CGMCC No.22420 and auxiliary materials.
The microbial agent provided by the invention is a liquid preparation or powder. The liquid preparation is prepared by inoculating strains into a culture medium and then propagating. The powder is prepared by inoculating strains into a culture medium, and then carrying out propagation and drying. The auxiliary materials in the microbial agent comprise a culture medium. The culture medium is PDF culture medium or other culture medium suitable for penicillin propagation.
The invention also provides a microbial fertilizer which is prepared by fermenting the penicillium with the preservation number of CGMCC No.22420 as a strain.
In the present invention, the fermented substrate comprises feces of livestock or poultry.
The invention also provides a preparation method of the microbial fertilizer, which comprises the following steps:
step 1, culturing penicillium with the preservation number of CGMCC No.22420 in a PDA liquid culture medium, and suspending the penicillium in sterile water to obtain bacterial suspension;
step 2, inoculating the bacterial suspension into feces of livestock or poultry, and performing solid fermentation at the temperature of less than or equal to 50 ℃ until the quantity of the penicillium reaches 10 8 And cfu/g or more to obtain the microbial fertilizer.
In the step 1, the culture conditions comprise 28-35 ℃, the oscillation speed is 160-220 r/min, and the culture time is 72-120 h. In some embodiments, the incubation time is 96 hours. The PDA liquid medium comprises 200g/L of water and potatoes and 20g/L of glucose.
In the step 2, the volume-mass ratio of the bacterial suspension to the feces is (1 mL-2 mL): 10g.
The fermentation further comprises the steps of after-ripening and drying; the after-ripening condition comprises that the material is placed for 3 to 5 days under indoor or outdoor rain shielding condition, and the drying condition comprises that the material is dried at 45 to 60 ℃ until the water content is less than 30 percent.
In the invention, the manure of livestock or poultry is at least one selected from cow manure, pig manure, sheep manure, chicken manure, duck manure and goose manure. In some embodiments, the livestock or poultry manure is at least one of decomposed cow manure, pig manure, sheep manure, chicken manure, duck manure, and goose manure. In the invention, the mass fraction of organic matters in the decomposed livestock or poultry feces is more than or equal to 35%, the mass fraction of organic nitrogen is 1.0-2.0%, and the mass fraction of water is 25-30%.
The preservation number is CGMCC No.22420, the microbial agent and the microbial fertilizer are applied to improving soil continuous cropping obstacle.
In the invention, the improvement of soil continuous cropping obstacle comprises: degrading root system autotoxic substances, inhibiting pathogenic bacteria and/or promoting plant growth.
In the invention, the soil continuous cropping obstacle is a continuous cropping obstacle of vanilla, pepper, white gourd, melon or banana. In the invention, the effect of the penicillium with the preservation number of CGMCC No.22420 for improving the soil continuous cropping obstacle is verified by taking vanilla as an example.
In the present invention, the root system autotoxic substance is a phenolic acid autotoxic substance, and in some embodiments, the autotoxic substance includes benzoic acid, salicylic acid, or parahydroxybenzoic acid.
The invention also provides a method for improving soil continuous cropping obstacle, which comprises applying the microbial fertilizer disclosed by the invention or using the microbial fertilizer prepared by the preparation method disclosed by the invention.
The invention provides penicillium with a preservation number of CGMCC No.22420. The penicillium can effectively degrade phenolic acid autotoxic substances such as benzoic acid, salicylic acid and p-hydroxybenzoic acid secreted by plant root systems, has an obvious inhibiting effect on soil-borne pathogenic bacteria fusarium oxysporum, can be applied to degrading plant root autotoxic substances such as vanilla, improves the rhizosphere soil flora structure of continuous cropping vanilla, and promotes healthy growth of plants.
Description of biological preservation
Biomaterial FD-21, classification nomenclature: penicillium sp.2021, 5 months and 17 days are deposited at the China general microbiological culture Collection center (CGMCC) with the address of Beijing, chaoyang, no. 1, xili, 3, china academy of sciences microbiological study, and the deposit number of CGMCC No.22420.
Detailed Description
The invention provides penicillium and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The screening and separating method for the penicillium with the preservation number of CGMCC No.22420 comprises the following steps:
weighing 5g of vanilla rhizosphere soil, preparing a soil suspension, and placing the soil suspension in a shaking table at 28 ℃ for 30min. Gradient dilution of supernatant soil suspension, taking 10 -3 、10 -4 And 10 -5 Dilutions were pipetted 100 μl each and plated onto PDA plates. Culturing in a 28 ℃ incubator. After obvious colonies are grown, the colony morphology is observed, and single colonies with different morphologies and colors are respectively selected and purified in a PDA culture dish. After continuous purification for 5 times, bacterial colonies are picked up and inoculated into a PDA culture dish for 3-5 days, mycelium blocks are scraped by an inoculating shovel and transferred into an inorganic salt liquid culture medium filled with glass beads, and shaking is carried out for 30min by a 180r/min shaking table, so that bacterial suspension is prepared. Taking 2mL of the bacterial suspension, inoculating to 100mL containing 2.0mmol L -1 Shake culturing in 160r/min and shaking table at 30deg.C in liquid culture medium of inorganic salt of phenolic acid, respectively taking phenolic acid degradation liquid at 0, 12, 24, 36, 48, 60, and 72 hr, taking sterile water as blank control, and measuring phenolic acid content by ultraviolet spectrophotometry. Finally, the high-efficiency strain FD-21 with obvious degradation effect on benzoic acid, salicylic acid and p-hydroxybenzoic acid is screened out. The strain is identified as penicillium by an 18S rDNA method, and researches of a plate counter method show that the strain has a remarkable inhibiting effect on pathogenic bacteria Fusarium oxysporum.
The degradation rate of the strain on phenolic acid autotoxic substances increases along with the extension of the culture time, and the degradation rates of the strain on p-benzoic acid, salicylic acid and p-hydroxybenzoic acid respectively reach 78.87%, 93.62% and 89.15% when the strain is cultured for 72 hours. The microbial organic fertilizer prepared by fermenting the strain is inoculated into the continuous cropping vanilla soil, the degradation rate of phenolic acid substances in the continuous cropping soil is increased along with the extension of the application time, and the degradation rates of benzoic acid, salicylic acid and p-hydroxybenzoic acid in the continuous cropping soil are 67.12%, 92.98% and 77.11% respectively when the continuous cropping vanilla soil is applied for 7 days.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:
example 1: isolation and identification of Penicillium according to the invention
Weighing 5g of vanilla rhizosphere soil, preparing a soil suspension, and placing the soil suspension in a shaking table at 28 ℃ for 30min. Gradient dilution of supernatant soil suspension, taking 10 -3 、10 -4 And 10 -5 Dilutions were pipetted 100 μl each and plated onto PDA plates. Culturing in a 28 ℃ incubator. After obvious colonies are grown, the colony morphology is observed, and single colonies with different morphologies and colors are respectively selected and purified in a PDA culture dish. After continuous purification for 5 times, bacterial colonies are picked up and inoculated into a PDA culture dish for 3-5 days, mycelium blocks are scraped by an inoculating shovel and transferred into an inorganic salt liquid culture medium filled with glass beads, and shaking is carried out for 30min by a 180r/min shaking table, so that bacterial suspension is prepared. Taking 2mL of the bacterial suspension, inoculating to 100mL containing 2.0mmol L -1 And (3) 160r/min of phenolic acid inorganic salt liquid culture medium, shake culturing at 30 ℃ in a shaking table, respectively taking phenolic acid degradation liquid at 0, 12, 24, 36, 48, 60 and 72 hours, taking distilled water as a blank control, and measuring the phenolic acid content by adopting an ultraviolet spectrophotometry. Finally, the high-efficiency strain FD-21 with obvious degradation effect on benzoic acid, salicylic acid and p-hydroxybenzoic acid is screened out. The strain identified by the 18S rDNA method is penicillium, and has remarkable inhibition effect on fusarium oxysporum.
The sequence of the 18s rDNA is shown as SEQ ID NO. 1. The penicillium is preserved in the China general microbiological culture Collection center (CGMCC) at 5-17 of 2021, and has an address of 1 st-Xiyu 3 of the Korean area of Beijing, and a preservation number of 22420.
Example 2: the preparation of the microbial fertilizer
Inoculating penicillium with the preservation number of CGMCC No.22420 into a PDA liquid culture medium with the pH value of 6-8, carrying out shake culture for 96 hours at the temperature of 28-35 ℃ and the rotation speed of 160-220 rpm, centrifuging the thalli, re-suspending the thalli with sterile water, centrifuging the thalli, and suspending the thalli with sterile water with equal volume to obtain a bacterial suspension; the formula of the PDA liquid culture medium is as follows: 200g/L of potato, 20g/L of glucose and the balance of water.
Inoculating the bacterial suspension into the decomposed cow dung (the volume-mass ratio of the bacterial suspension to the decomposed cow dung is 2mL:10 g) for solid fermentation until the quantity of the penicillium reaches 10 8 And (3) stirring the mixture for 1 time per day at the solid fermentation temperature of not higher than 50 ℃ during the period of cfu/g to obtain the microbial fertilizer. Wherein the mass ratio of organic matters in the decomposed cow dung is more than or equal to 35%, the mass ratio of organic nitrogen is 1.0% -2.0%, and the mass ratio of water content is 25% -30%.
And (3) after-ripening the obtained microbial fertilizer for 4 days, and finally evaporating the water content of the microbial fertilizer to be below 30% at the temperature of 45-60 ℃.
The microbial fertilizer after post-ripening contains 10 8 The mass ratio of the total nitrogen, phosphorus and potassium nutrients of the penicillium with the cfu/g is 5-11%, and the mass ratio of the organic matters is 30-40%.
Example 3: the strain of the invention has the capability of degrading phenolic acid autotoxicity
Preparing a bacterial suspension: collecting preserved fungus inclined plane, activating, picking mycelium blocks, inoculating to PDA culture dish, culturing for 3-5 days, scraping mycelium blocks with inoculating shovel, transferring to inorganic salt liquid culture medium filled with glass beads, shaking with 180r/min shaking table for 30min to obtain spore suspension, and regulating spore concentration to 10 7 cfu/mL for use.
Taking 2mL of the prepared bacterial suspension, inoculating to 100mL containing 2.0mmol L -1 Shake culturing in 160r/min and shaking table at 30deg.C in liquid culture medium, taking mycophenolic acid degradation liquid at 0, 12, 24, 36, 48, 60, and 72 hr, taking distilled water as blank, and measuring phenolic acid content by ultraviolet spectrophotometry.
The results show that: the degradation rates of FD-21 on benzoic acid, salicylic acid and p-hydroxybenzoic acid increase with the increase of the culture time, and after 72 hours of culture, the degradation rates are 78.87%, 93.62% and 89.15% respectively.
Example 4: the strain of the invention has the degradation capability on phenolic acid autotoxic substances in continuous cropping soil
1000g of vanilla continuous cropping soil (screened by a 2mm sieve) is taken and added into a 2000ml beaker, the microbial organic fertilizer obtained in example 2 is applied according to the proportion of 10%, 9 times of repetition are carried out for each treatment, the organic fertilizer without inoculating the strain is applied as a control, the mouth of the beaker is covered by a sealing film, the culture is carried out for 7d at 30 ℃ in a light-shielding manner in an incubator, samples are taken for 3d, 5d and 7d after the treatment respectively, and the content of autotoxic substances in the soil is measured. Extracting phenolic acid substances in the soil, and quantitatively analyzing the phenolic acid content change in the soil by adopting high performance liquid chromatography.
The results show that: the degradation rate of phenolic acid substances in the continuous cropping soil is increased along with the extension of time, and the degradation rates of benzoic acid, salicylic acid and parahydroxybenzoic acid in the continuous cropping soil are 67.12%, 92.98% and 77.11% respectively when the microbial organic fertilizer is applied for 7 days.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> national academy of Tropical agriculture for perfumery and beverage institute
<120> Penicillium and use thereof
<130> MP21028434
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 547
<212> DNA
<213> Penicillium spp
<400> 1
ctacctgatc cgaggtcacc tgaaaagatt gattggggtc gccggcgggc gccggccggg 60
cctacagagc gggtgacgaa gccccatacg ctcgaggacc ggacgcggtg ccgccgctgc 120
ctttcgggcc cgccccccgg gagccggggg gcgaagccca acacacaagc cgtgcttgag 180
ggcagcaatg acgctcggac aggcatgccc cccggaatac cagggggcgc aatgtgcgtt 240
caaagactcg atgattcact gaattctgca attcacatta cttatcgcat ttcgctgcgt 300
tcttcatcga tgccggaacc aagagatccg ttgttgaaag ttttaactga tttagctaat 360
ctgctcagac tgcaatcttc agacagagtt caatggtgtc ttcggcgggc gcgggcccgg 420
gggcggatgc cccccggcgg ccgtgaggcg ggcccgccga agcaacaagg tagaataaac 480
acgggtggga ggttggaccc agagggccct cactcggtaa tgatccttcc gcaggtcacc 540
atacgga 547
Claims (9)
1. Penicillium with preservation number of CGMCC No.22420Penicillium sp.）。
2. A microbial agent, which is characterized by being prepared from the penicillium and auxiliary materials in claim 1.
3. A microbial fertilizer, which is characterized by being prepared by fermenting the penicillium strain of claim 1.
4. A microbial fertilizer according to claim 3, wherein the fermented substrate comprises manure from livestock or poultry.
5. The method for preparing the microbial fertilizer according to claim 3 or 4, comprising the following steps:
step 1, culturing the penicillium according to claim 1 in a PDA liquid culture medium, and suspending the bacterial cells in sterile water to obtain bacterial suspension;
step 2, inoculating the bacterial suspension into feces of livestock or poultry, and performing solid fermentation at the temperature of less than or equal to 50 ℃ until the quantity of the penicillium reaches 10 8 And cfu/g or more to obtain the microbial fertilizer.
6. The method of claim 5, further comprising the steps of post-ripening and drying after the fermentation; the post-ripening time is 3-5 days, and the drying conditions comprise drying at 45-60 ℃ until the water content is less than 30%.
7. The method of claim 5, wherein the feces of livestock or poultry are decomposed prior to fermentation.
8. The use of the penicillium according to claim 1, the microbial agent according to claim 3, the microbial fertilizer according to claim 4 or 5, the microbial fertilizer produced by the production method according to any one of claims 6 to 7, for improving vanilla soil continuous cropping obstacle, the improvement of soil continuous cropping obstacle comprising: degrading root system autotoxic substances, inhibiting fusarium oxysporum and/or promoting vanilla growth.
9. A method for improving vanilla soil continuous cropping obstacle, comprising applying the microbial fertilizer of claim 4 or 5, or applying the microbial fertilizer prepared by the preparation method of any one of claims 6-7, wherein the improving vanilla soil continuous cropping obstacle comprises: degrading the root system autotoxic substances, inhibiting fusarium oxysporum and/or promoting plant growth.
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| CN103667158A (en) * | 2013-12-27 | 2014-03-26 | 中国热带农业科学院香料饮料研究所 | Bacillus cereus and application of bacillus cereus |
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| CN106588440A (en) * | 2016-11-25 | 2017-04-26 | 山西省农业科学院农业资源与经济研究所 | Special biological organic fertilizer for cucumis sativus linn succession cropping and preparation method of biological organic fertilizer |
| CN107164234B (en) * | 2017-05-19 | 2020-03-17 | 福建农林大学 | Fungus with strong weed suppression effect screened from passion fruit rhizosphere soil |
| CN108192831B (en) * | 2018-01-25 | 2023-08-04 | 中国药科大学 | Penicillium and application thereof in degrading aromatic compounds |
| CN110241029B (en) * | 2018-09-26 | 2021-04-06 | 西南大学 | Coptis chinensis soil ferulic acid degrading bacterium and application thereof |
| CN111534439B (en) * | 2020-04-03 | 2022-05-17 | 河北科技大学 | Penicillium oxalicum SDF-25 strain and application thereof |
| CN112266881B (en) * | 2020-10-20 | 2021-11-19 | 山东农业大学 | Bacillus amyloliquefaciens and application thereof in preventing and treating apple continuous cropping obstacle |
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| CN103667158A (en) * | 2013-12-27 | 2014-03-26 | 中国热带农业科学院香料饮料研究所 | Bacillus cereus and application of bacillus cereus |
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