CN115322926B - Rhodococcus pyridine and application thereof in repairing continuous cropping soil - Google Patents
Rhodococcus pyridine and application thereof in repairing continuous cropping soil Download PDFInfo
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- CN115322926B CN115322926B CN202210895512.3A CN202210895512A CN115322926B CN 115322926 B CN115322926 B CN 115322926B CN 202210895512 A CN202210895512 A CN 202210895512A CN 115322926 B CN115322926 B CN 115322926B
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- 241000316848 Rhodococcus <scale insect> Species 0.000 title claims abstract description 65
- 239000002689 soil Substances 0.000 title claims abstract description 38
- 238000009335 monocropping Methods 0.000 title claims abstract description 17
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 title description 26
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 title description 13
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 72
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims abstract description 36
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 claims abstract description 30
- 150000007965 phenolic acids Chemical class 0.000 claims abstract description 28
- 239000003337 fertilizer Substances 0.000 claims abstract description 24
- 230000000813 microbial effect Effects 0.000 claims abstract description 22
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 17
- 239000000126 substance Substances 0.000 claims abstract description 17
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 claims abstract description 16
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 claims abstract description 16
- 229940114124 ferulic acid Drugs 0.000 claims abstract description 16
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 claims abstract description 16
- 235000001785 ferulic acid Nutrition 0.000 claims abstract description 16
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 claims abstract description 16
- 235000004515 gallic acid Nutrition 0.000 claims abstract description 15
- 229940074391 gallic acid Drugs 0.000 claims abstract description 15
- YQUVCSBJEUQKSH-UHFFFAOYSA-N protochatechuic acid Natural products OC(=O)C1=CC=C(O)C(O)=C1 YQUVCSBJEUQKSH-UHFFFAOYSA-N 0.000 claims abstract description 15
- WKOLLVMJNQIZCI-UHFFFAOYSA-N vanillic acid Chemical compound COC1=CC(C(O)=O)=CC=C1O WKOLLVMJNQIZCI-UHFFFAOYSA-N 0.000 claims abstract description 15
- TUUBOHWZSQXCSW-UHFFFAOYSA-N vanillic acid Natural products COC1=CC(O)=CC(C(O)=O)=C1 TUUBOHWZSQXCSW-UHFFFAOYSA-N 0.000 claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 241001478124 Rhodococcus pyridinivorans Species 0.000 claims abstract description 5
- 235000015097 nutrients Nutrition 0.000 claims description 11
- 239000003895 organic fertilizer Substances 0.000 claims description 7
- 239000000725 suspension Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-O pyridinium Chemical compound C1=CC=[NH+]C=C1 JUJWROOIHBZHMG-UHFFFAOYSA-O 0.000 claims description 4
- 239000000839 emulsion Substances 0.000 claims description 2
- 230000006518 acidic stress Effects 0.000 claims 1
- 239000013598 vector Substances 0.000 claims 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 abstract description 16
- 240000003768 Solanum lycopersicum Species 0.000 abstract description 16
- 239000001963 growth medium Substances 0.000 abstract description 13
- 238000000034 method Methods 0.000 abstract description 8
- 229910017053 inorganic salt Inorganic materials 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 4
- 244000005700 microbiome Species 0.000 abstract description 4
- 230000008439 repair process Effects 0.000 abstract description 3
- 238000009629 microbiological culture Methods 0.000 abstract description 2
- -1 phenolic acid compounds Chemical class 0.000 abstract description 2
- 238000004321 preservation Methods 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 18
- 230000015556 catabolic process Effects 0.000 description 13
- 238000006731 degradation reaction Methods 0.000 description 13
- 239000002068 microbial inoculum Substances 0.000 description 13
- 241000196324 Embryophyta Species 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- SIOXPEMLGUPBBT-UHFFFAOYSA-M picolinate Chemical compound [O-]C(=O)C1=CC=CC=N1 SIOXPEMLGUPBBT-UHFFFAOYSA-M 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 210000003608 fece Anatomy 0.000 description 7
- 239000010871 livestock manure Substances 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 235000009048 phenolic acids Nutrition 0.000 description 7
- 230000000593 degrading effect Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 229910052799 carbon Inorganic materials 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000008223 sterile water Substances 0.000 description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 240000002044 Rhizophora apiculata Species 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
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- 238000006467 substitution reaction Methods 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000035558 fertility Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 229910052500 inorganic mineral Inorganic materials 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 239000011707 mineral Substances 0.000 description 2
- 235000010755 mineral Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000006303 photolysis reaction Methods 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000004382 potting Methods 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229910021536 Zeolite Inorganic materials 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
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- 238000000137 annealing Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 230000008236 biological pathway Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 244000053095 fungal pathogen Species 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 239000004021 humic acid Substances 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000010842 industrial wastewater Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000002262 irrigation Effects 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000011392 neighbor-joining method Methods 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 239000000618 nitrogen fertilizer Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000002686 phosphate fertilizer Substances 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 229940072033 potash Drugs 0.000 description 1
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
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- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000010822 slaughterhouse waste Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000004563 wettable powder Substances 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
Classifications
-
- 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
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- 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
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K17/00—Soil-conditioning materials or soil-stabilising materials
- C09K17/14—Soil-conditioning materials or soil-stabilising materials containing organic compounds only
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C2101/00—In situ
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2101/00—Agricultural use
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- 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
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Soil Sciences (AREA)
- Environmental Sciences (AREA)
- Virology (AREA)
- Pest Control & Pesticides (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- General Life Sciences & Earth Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Materials Engineering (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Environmental & Geological Engineering (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
- Soil Conditioners And Soil-Stabilizing Materials (AREA)
Abstract
The invention discloses rhodococcus dipicola (Rhodococcus pyridinivorans) GXUN1036. The strain is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms on the 6 th month 2 of 2022, and has a biological preservation number of: CGMCC No.25002. The strain can degrade various phenolic acid compounds, degrade 2000mg/L of p-hydroxybenzoic acid contained in an inorganic salt culture medium by 99.7% within three days, and can degrade vanillic acid, gallic acid and ferulic acid, so that continuous cropping obstacles caused by phenolic acid substances can be reduced, and the strain has a good repairing effect on tomatoes planted in continuous cropping soil. Meanwhile, the method has a biological repairing effect on continuous cropping obstacles caused by the phenolic acid substances, can be prepared into microbial agents, compound microbial fertilizers and biological organic fertilizers to repair continuous cropping soil, and has good economic value and application prospect.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms and ecological restoration, in particular to rhodococcus pyridine and application thereof.
Background
The continuous cropping obstacle seriously affects the production and sustainable development of crops, and the autotoxic substances generated by plants are main factors causing the continuous cropping obstacle, and enter the soil through plant leaching, root system secretion, plant residues and other ways to affect the growth of the next crop. The plant root system secreted autotoxic substances such as phenolic acid substances like p-hydroxybenzoic acid are common autotoxic substances. Studies show that phenolic acid provides abundant nutrients for pathogenic fungi, and greatly reduces the fertility of soil and the yield and quality of crops.
Phenolic acids are degraded in the environment by both abiotic and biological pathways, the former involving photodecomposition and hydrolysis, the latter being primarily microbial degradation, the rate of photodecomposition degradation being in most cases much lower than biodegradation, and therefore microbial degradation is the primary degradation pathway of phenolic acids in the natural environment.
The research shows that rhodococcus can degrade aliphatic, aromatic, heterocyclic, polycyclic aromatic, alicyclic hydrocarbon, nitrile, cholesterol and other natural organic compounds, so that rhodococcus is one kind of environment treating special bacterium and has excellent application foreground in restoring continuous cropping soil, treating industrial waste water, treating marine petroleum pollution, etc.
Tomatoes are common commercial crops cultivated, and continuous cropping obstacles become main constraint factors affecting tomato yield and quality along with the increase of planting years. Phenolic acid substances are self-toxic substances generated by tomatoes, and comprise vanillic acid, parahydroxybenzoic acid, ferulic acid and the like, and have remarkable inhibition effect on the growth of tomatoes. Biological measures are adopted to promote the degradation of phenolic acid substances in soil, and continuous cropping obstacles can be effectively reduced.
In recent years, the measure of repairing continuous cropping soil by microorganisms attracts attention. Bacterial manure is one of common products for restoring soil, is a preparation for restoring and improving soil fertility by using the growth and metabolism activities of microorganisms and ensuring plant growth by interacting with plants, and is widely used in agriculture.
Disclosure of Invention
In view of the above prior art, the present invention provides a rhodococcus dipicola (Rhodococcus pyridinivorans) GXUN1036 strain that degrades phenolic acids. The rhodococcus pyridinium GXUN1036 is separated from mangrove rhizosphere soil, can degrade various phenolic acid substances such as p-hydroxybenzoic acid, vanillic acid, gallic acid, ferulic acid and the like, and can repair the soil where the phenolic acid is produced by continuous cropping.
The invention provides a microbial inoculum containing the rhodococcus pyridine-philic GXUN1036.
The rhodococcus pyridine-like GXUN1036 can be used for microbial agents, compound microbial fertilizers and application of biological organic fertilizers in repairing phenolic acid continuous cropping obstacles.
Specifically, the invention relates to the following technical scheme:
the invention separates a bacterial strain capable of degrading p-hydroxybenzoic acid from the rhizosphere soil of the mangrove forest in North sea. The strain can degrade 2000mg/L of p-hydroxybenzoic acid, vanillic acid, gallic acid and ferulic acid in inorganic salt ion culture medium.
The invention provides rhodococcus pyridine (Rhodococcus pyridinivorans) GXUN1036, which is preserved in China general microbiological culture Collection center (CGMCC) for 2 days of 6 months of 2022, and has the biological preservation number of: CGMCC No.25002.
The bacterial colony and the bacterial body of the rhodococcus pyridine GXUN1036 strain are characterized in that:
the lawn grown on the LB plate is raised, is pink, has opaque colonies, has a smooth surface and Xu Guangze edges, and can grow on an inorganic salt solid medium with parahydroxybenzoic acid as a sole carbon source; the scanning electron microscope result shows that the thallus is in a bar shape, bulges and has no flagella, the length of the thallus is 300-781 nm, and the width of the thallus is 3-5 nm.
The formula of the LB culture medium is as follows: yeast extract 5g tryptone 10g sodium chloride 10g agar 20g distilled water 1000mL, pH 7.0.
The bacterial fertilizer containing the rhodococcus pyridinphilia GXUN1036 comprises a microbial agent, a compound microbial fertilizer and a biological organic fertilizer, and also belongs to the protection scope of the invention.
The bacterial fertilizer comprises rhodococcus pyridinphilia GXUN1036 and other functional strains, carriers, fertilizers and organic fertilizers;
the bacterial fertilizer can comprise auxiliary materials such as humic acid, biochar, animal manure, straws of various crops, rice straws, peanut hulls and the like besides rhodococcus pyridinium GXUN1036. The microbial agent may also include a carrier, which may be a solid carrier or a liquid carrier. The solid support may be selected from mineral materials, plant materials or polymeric compounds; the mineral material can be one or more of clay, talcum powder, kaolin, zeolite and diatomite; the plant material may be at least one of corn flour, soy flour or starch; the polymer compound may be polyvinyl alcohol. The liquid carrier may be an organic solvent, vegetable oil or water;
the bacterial fertilizer can comprise fertilizer such as simple substance fertilizer and compound fertilizer such as nitrogen fertilizer, phosphate fertilizer, potash fertilizer and the like besides rhodococcus pyridine-philic GXUN 1036;
the bacterial manure can comprise organic fertilizers composed of various animals, plant residues and metabolites besides rhodococcus pyridinium GXUN1036, such as human and animal manure, straw, animal residues and slaughterhouse waste;
in the bacterial manure, rhodococcus pyridinphilis GXUN1036 can be in the form of cultured living cells, fermentation broth of the living cells, filtrate of cell culture or mixture of cells and filtrate.
The bacterial manure can be in the form of liquid, emulsion, suspending agent, granule, powder, wettable powder or water dispersing agent.
Furthermore, the invention also provides a preparation method of the rhodococcus pyridine GXUN1036 viable bacteria agent, which comprises the following steps:
inoculating rhodococcus pyridine-philic GXUN1036 into a fermentation medium, and culturing at 30-37 ℃ for 72 hours to obtain rhodococcus pyridine-philic GXUN1036 fermentation liquor;
diluting rhodococcus pyridine-philic fermentation liquor to 5-10% with sterile water to obtain live bacterial agent;
preferably, the live bacteria number of the live bacteria agent of the rhodococcus pyridinphilia strain GXUN1036 is 2 to 3 multiplied by 10 9 cfu/mL。
Preferably, the fermentation medium is formulated as yeast extract 5g tryptone 10g sodium chloride 10g distilled water 1000mL, pH 7.0.
The application method of the live bacteria preparation of the rhodococcus pyridine-philic GXUN1036 strain comprises the following steps:
the live bacteria agent of the rhodococcus pyridine GXUN1036 strain is diluted to 5-10% by adding sterile water, and the seedling is dipped in root or irrigated to plant rhizosphere.
The application of the rhodococcus pyridinphilia GXUN1036 or the microbial inoculum containing the rhodococcus pyridinphilia GXUN1036 in any one of the following 1-4 also belongs to the protection scope of the invention:
1. application of promoting plant growth in phenolic acid environment;
2. the application of the bacterial manure for phenolic acid soil remediation is prepared;
3. application of phenolic acid substances in degradation soil;
4. can be used for degrading autotoxic substances produced by crops and relieving continuous cropping obstacle.
In the above application, the phenolic acid substance includes: p-hydroxybenzoic acid, vanillic acid, gallic acid, ferulic acid.
In the above application, the crop plant-generated autotoxic substances include: p-hydroxybenzoic acid, vanillic acid, gallic acid, ferulic acid.
The invention has the beneficial effects that:
the rhodococcus pyridine-philic GXUN1036 can degrade plant autotoxic substances such as p-hydroxybenzoic acid, vanillic acid, gallic acid, ferulic acid and the like, after being cultured for 3 days, the degradation rates of the p-hydroxybenzoic acid, the vanillic acid, the gallic acid and the ferulic acid are respectively 99.7%, 98.8%, 90.1% and 15.4%, and the live bacteria preparation can be used for degrading the autotoxic substances generated by phenolic acid soil tomatoes, so that continuous cropping obstacles are reduced, tomato growth is promoted, and tomato yield is improved.
Drawings
FIG. 1 is a scanning electron microscope (c) showing the growth of Rhodococcus pyridine-philic on LB (a) and p-hydroxybenzoic acid as the sole carbon source media in example 1;
FIG. 2 is an adjacent junction tree of the strain constructed based on the 16S rRNA gene sequence in example 1;
FIG. 3 shows the growth and degradation of p-hydroxybenzoic acid by Rhodococcus pyridine-philic in example 2;
FIG. 4 shows the degradation of vanillic acid, gallic acid and ferulic acid by Rhodococcus pyridine-philic bacteria in example 2;
FIG. 5 is the values of stem height and stem thickness for tomato growth in the tomato potting experiment of example 3;
figure 6 is a graph showing the fresh and dry weight values of tomato growth in the tomato potting experiment of example 3.
Detailed Description
The invention is further described below with reference to the drawings and specific examples. The following examples are preferred embodiments of the present invention, but are not intended to limit the scope of the present invention in any way. The invention is mainly described by the strains and based on the application ideas of the strains, the simple parameter substitutions in the embodiments cannot be described in the examples, but are not limited by the examples, and any other changes, modifications, substitutions, combinations and simplifications which do not deviate from the spirit and principle of the invention should be regarded as equivalent substitutions and are included in the scope of the invention. It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
The inorganic salt culture medium used in the invention is as follows: k (K) 2 HPO 4 0.05g、MgSO 4 0.02g、CaCl 2 0.01g、FeCl 3 0.001g、MnSO 4 0.001g、NH4NO 3 0.1g、KH 2 NO 3 0.05g of NaCl 0.02g, adjusting the pH to 7.0, adding 2g of p-hydroxybenzoic acid and 1000mL of distilled water.
The nutrient medium is as follows: yeast extract 5g tryptone 10g sodium chloride 10g distilled water 1000mL, pH 7.0.
The plate solid medium was 1.5% agar added to the corresponding medium.
EXAMPLE 1 isolation and identification of Rhodococcus pyridine-philic GXUN1036
1. Screening and isolation of strains utilizing parahydroxybenzoic acid
2g of mangrove sediment is weighed and placed in a 10mL centrifuge tube, and 2mL of sterile water and a glass strain (sterilization) shaker are added for shaking for 10min to prepare a suspension. Diluting the suspension into 10 by ten times dilution method -3 、10 -4 、10 -5 100 mu L of each diluted suspension is respectively coated on an inorganic salt culture medium containing parahydroxybenzoic acid, the culture is carried out for 7d in a light-proof incubator at 30 ℃, the strain capable of growing on a solid culture medium taking parahydroxybenzoic acid as a sole carbon source is observed and selected, the strain is purified for multiple times by a three-area line separation method until a single colony is separated from the culture medium, and a strain degrading parahydroxybenzoic acid is separated and named GXUN1036.
2. Morphological observation and identification of strain GXUN1036
(1) The method comprises the following steps: the activated strain is cultured for 3 days on an LB plate and a solid inorganic salt plate containing 2000mg/L of parahydroxybenzoic acid at a constant temperature and in a dark place at 30 ℃, the growth condition of the strain is observed, and the cell morphology of the bacteria is observed by a scanning electron microscope. Extracting genome DNA of the strain GXUN1036 by using a bacterial genome extraction kit, and amplifying 16S rRNA genes of the strain GXUN1036 by PCR, wherein the amplified upstream primer sequence is as follows: 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), the downstream primer sequence is: 1492R (5'-GGTTACCTTGTTACGACTT-3'), the total reaction system was 25. Mu.L, PCR premix (2 XTaq Master Mix) 12.5. Mu.L, ultrapure water (ddH) 2 O) 10.5. Mu.L of each of the upstream and downstream primers was 1. Mu.L of the amplified template. The set reaction procedure is: pre-denaturation at 94℃for 5min, denaturation at 95℃for 10s, annealing at 55℃for 20sExtending at 72 ℃ for 1.5min, 32 cycles in total, and extending at 72 ℃ for 10min. The PCR products were detected by agarose (1%) gel electrophoresis, and amplified products meeting the expected target band size were picked up by Marker bands and sent to the Probiotics (Shanghai) Co., ltd for sequencing. In EZbiocloudhttp：//eztaxon-e.ezbiocloud.net)The 16S rRNA gene sequence of GXUN1036 is compared with other standard strains on a website to obtain the 16S rRNA of the strain similar to GXUN1036, a phylogenetic tree is constructed in MEGA6 software by using a Neighbor-joining method, and 1000 times of repeated verification is carried out by using a Bootstrap method (Bootstrap).
(2) Results:
as shown in FIG. 1, the rhodococcus pyridine is grown on LB (a) and p-hydroxybenzoic acid as the only carbon source culture medium (b), and the scanning electron microscope image (c) is obtained;
as shown in FIG. 2, the contiguity tree of the strain constructed based on the 16S rRNA gene sequence;
as shown in the above figure, strain GXUN1036 has 100% identity with Rhodococcus pyridinivorans.
EXAMPLE 2 degradation Performance test of Rhodococcus pyridine-philic GXUN1036 Strain
(1) The method comprises the following steps: and 4 phenolic acids, namely p-hydroxybenzoic acid, vanillic acid, gallic acid and ferulic acid, are selected as degradation target products, and the capacity of GXNU1036 for degrading the phenolic acids is measured. The 4 phenolic acids are respectively added into 20mL of inorganic salt liquid culture medium, the final concentration of the phenolic acids in the culture medium is 2g/L, 200 mu L of bacterial suspension with OD600 value of 0.6 is taken and inoculated into the culture medium, and the culture medium is subjected to shaking culture at a constant temperature of 30 ℃ and 200rmp for 72 hours. And (3) placing the fermentation broth in a refrigerator at the temperature of minus 80 ℃ for 3 hours, and performing freeze drying treatment for 72 hours by a freeze dryer. Weighing the freeze-dried product, adding ten times of methanol, performing ultrasonic treatment for 10min, filtering the extracted product with 0.45 μm organic filter membrane, and measuring residual concentrations of p-hydroxybenzoic acid, vanillic acid, gallic acid and ferulic acid at 254nm, 290nm, 311nm and 270nm of ultraviolet spectrophotometer. The degradation rate was calculated using the standard curve for each phenolic acid. Each experimental group was set with 3 replicates and samples of unvaccinated bacterial fluid served as blank.
(2) Results:
as shown in fig. 3, rhodococcus pyridine is grown and p-hydroxybenzoic acid is degraded;
as shown in the graph, the average degradation rate of the rhodococcus pyridine is greater than 99% when the rhodococcus pyridine is degraded to hydroxyl.
As shown in fig. 4, rhodococcus pyridine is used for degrading vanillic acid, gallic acid and ferulic acid;
as shown in the graph, the degradation rates of the paravanillic acid, the gallic acid and the ferulic acid are respectively 98.8%, 90.1% and 15.4%.
EXAMPLE 3 preparation of live bacterial preparation of Rhodococcus Pyridinii GXUN1036 Strain
(1) Activating strains: rhodococcus pyridine GXUN1036 was transferred to LB medium plates and cultured at 30℃for 72 hours for activation.
(2) Preparation of fermentation liquor: the activated rhodococcus pyridinphilius GXUN1036 lawn is scraped by an inoculating loop and inoculated into 200mLLB liquid culture medium for culturing at 30 ℃ for 72h.
(3) Preparation of a live bacterial strain agent: diluting the fermentation liquor by 5-10%, wherein the viable count of the viable bacteria agent of the rhodococcus pyridinphilippica GXUN1036 is 2-3 multiplied by 10 9 cfu/mL。
Example 4 repair of P-hydroxybenzoic acid soil tomato growth by Rhodococcus Pyridinophilum GXUN1036 inoculant
(1) The method comprises the following steps: the control group was filled with nutrient soil into a flowerpot, and the experimental group added p-hydroxybenzoic acid to the nutrient soil so that the final concentration of p-hydroxybenzoic acid was 10g/kg, and then filled into the flowerpot. The test adopts a root irrigation treatment method, and seedlings growing in a seedling raising tray are transferred into a flowerpot filled with nutrient soil. The test had the following 5 treatments: t1, 70g of normal nutrient soil is irrigated with 10% sterile water; t2, 70g 10g/kg P-hydroxybenzoic acid nutrient soil irrigates 10% rhodococcus picolinatus GXUN1036 microbial inoculum; t3, 70g 10g/kg p-hydroxybenzoic acid nutrient soil irrigates 5% rhodococcus picolinatus GXUN1036 microbial inoculum; t4, 70g of normal nutrient soil is irrigated with 10% rhodococcus picolinae GXUN1036 microbial inoculum. T5, 70g of normal nutrient soil is irrigated with 10% sterile water. The test is carried out in a greenhouse at a temperature of 24-28 ℃, RH of 75-90% and photoperiod of 12h. Each treatment was 3 strains and the test was repeated 3 times. Fresh weight, dry weight, plant height and stem thickness of tomato were measured 28d after inoculation.
(2) Results
As shown in FIG. 5, 10g/kg of the rhodococcus picolinate soil drenching 10% of the rhodococcus picolinate GXUN1036 microbial inoculum, 10g/kg of the rhodococcus picolinate soil drenching 5% of the rhodococcus picolinate GXUN1036 microbial inoculum, 10% of the rhodococcus picolinate GXUN1036 microbial inoculum and 10g/kg of the rhodococcus picolinate soil drenching 10% have significant differences in plant height and stem thickness, indicating that the rhodococcus picolinate GXUN1036 microbial inoculum can restore tomatoes stressed by the rhodococcus picolinate soil.
As shown in FIG. 6, 10g/kg of the p-hydroxybenzoic acid soil drenching 10% of the rhodococcus picolinae GXUN1036 microbial inoculum, 10g/kg of the p-hydroxybenzoic acid soil drenching 5% of the rhodococcus picolinae GXUN1036 microbial inoculum, 10% of the p-hydroxybenzoic acid soil drenching 10% of the rhodococcus picolinae GXUN1036 microbial inoculum and 10% of the p-hydroxybenzoic acid soil drenching 10% are significantly different in fresh weight and dry weight, indicating that the rhodococcus picolinae GXUN1036 microbial inoculum can restore tomatoes stressed by the p-hydroxybenzoic acid soil.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (9)
1. Rhodococcus dipicola (Rhodococcus pyridinivorans) GXUN1036 with biological deposit number: CGMCC No.25002.
2. A microbial agent comprising the rhodococcus pyridine-philic strain GXUN1036 of claim 1.
3. A compound microbial fertilizer comprising rhodococcus pyridinphilia strain GXUN1036 of claim 1.
4. A bioorganic fertilizer comprising the rhodococcus pyridinphilia strain GXUN1036 of claim 1.
5. The microbial agent of claim 2, wherein the microbial agent comprises rhodococcus pyridinium strain GXUN1036 and other functional strains and vectors.
6. A compound microbial fertilizer according to claim 3, characterized in that it comprises rhodococcus pyridinphilia strain GXUN1036 and other functional strains and nutrients.
7. The bio-organic fertilizer according to claim 4, wherein the bio-organic fertilizer comprises rhodococcus pyridinphilia strain GXUN1036 and other functional strains and organic fertilizers.
8. The microbial agent, the compound microbial fertilizer and the bio-organic fertilizer according to claim 2 to 4, wherein the microbial agent, the compound microbial fertilizer and the bio-organic fertilizer are in a liquid state, an emulsion state, a suspension state, a granular state, a powdery state and a water-dispersed state.
9. The use of the microbial agent, the compound microbial fertilizer, the bio-organic fertilizer according to any one of claims 3 to 8, which is at least one of the following (b 1) to (b 3):
(b1) The damage of soil phenolic acid stress to plants is relieved, wherein the phenolic acid is p-hydroxybenzoic acid, vanillic acid, gallic acid and ferulic acid;
(b2) The microbial agent, the compound microbial fertilizer and the bio-organic fertilizer are used as phenolic acid soil plants, wherein the phenolic acid is p-hydroxybenzoic acid, vanillic acid, gallic acid or ferulic acid;
(b3) The physical and chemical properties of continuous cropping phenolic acid soil are improved, wherein the phenolic acid is p-hydroxybenzoic acid, vanillic acid, gallic acid or ferulic acid.
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| CN108546658A (en) * | 2018-04-10 | 2018-09-18 | 暨南大学 | One plant of phosphorus-solubilizing bacteria and its compound microbial inoculum and application with DEHP degradation bacterias |
| CN112063567A (en) * | 2020-09-24 | 2020-12-11 | 自然资源部第三海洋研究所 | Rhodococcus pyridinivorans and application thereof in production of PHBV |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108546658A (en) * | 2018-04-10 | 2018-09-18 | 暨南大学 | One plant of phosphorus-solubilizing bacteria and its compound microbial inoculum and application with DEHP degradation bacterias |
| CN112063567A (en) * | 2020-09-24 | 2020-12-11 | 自然资源部第三海洋研究所 | Rhodococcus pyridinivorans and application thereof in production of PHBV |
Non-Patent Citations (1)
| Title |
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| Comparative genomics reveals response of Rhodococcus pyridinivorans B403 to phenol after evolution;Fang Peng等;Appl Microbiol Biotechnol;第106卷(第7期);第2751-2761页 * |
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