CN115161406B - Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants - Google Patents

Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants Download PDF

Info

Publication number
CN115161406B
CN115161406B CN202210842260.8A CN202210842260A CN115161406B CN 115161406 B CN115161406 B CN 115161406B CN 202210842260 A CN202210842260 A CN 202210842260A CN 115161406 B CN115161406 B CN 115161406B
Authority
CN
China
Prior art keywords
flora
synthetic
ginseng
soil
stenotrophomonas
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
Application number
CN202210842260.8A
Other languages
Chinese (zh)
Other versions
CN115161406A (en
Inventor
刘宁
穆朋
梁池嘉
刘政波
关一鸣
张悦
张淋淋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute Special Animal and Plant Sciences CAAS
Original Assignee
Institute Special Animal and Plant Sciences CAAS
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Institute Special Animal and Plant Sciences CAAS filed Critical Institute Special Animal and Plant Sciences CAAS
Priority to CN202210842260.8A priority Critical patent/CN115161406B/en
Publication of CN115161406A publication Critical patent/CN115161406A/en
Application granted granted Critical
Publication of CN115161406B publication Critical patent/CN115161406B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/25—Root crops, e.g. potatoes, yams, beet or wasabi
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G7/00—Botany in general
    • A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
    • 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
    • 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
    • A01N63/27—Pseudomonas
    • 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
    • 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
    • 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
    • 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
    • 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
    • C12R2001/38—Pseudomonas
    • C12R2001/39—Pseudomonas fluorescens
    • 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
    • C12R2001/38—Pseudomonas
    • C12R2001/40—Pseudomonas putida

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Environmental Sciences (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Botany (AREA)
  • Genetics & Genomics (AREA)
  • Pest Control & Pesticides (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Ecology (AREA)
  • General Engineering & Computer Science (AREA)
  • Forests & Forestry (AREA)
  • Dentistry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Agronomy & Crop Science (AREA)
  • Biochemistry (AREA)
  • Soil Sciences (AREA)
  • Materials Engineering (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • General Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

The invention provides a construction method of a synthetic flora and application of the synthetic flora in high-quality cultivation of medicinal plants, and relates to the technical field of plant cultivation. The method comprises the following steps: (a) Analyzing metagenomic DNA information of target plant rhizosphere soil sample microorganisms; (b) Determining the relative abundance of the species according to the metagenomic DNA information; (c) And constructing a correlation network among the strains according to the relative abundance of the strains, and selecting a flora with strong positive interaction among the strains as a synthetic flora according to a correlation network analysis chart. The invention provides a method for artificially constructing a flora, the constructed synthetic flora has good colonization effect on ginseng root systems, the flora is more stable, the functions are more comprehensive, the microenvironment of the rhizosphere soil of the cultivated ginseng can be effectively improved, the organic matter content, the total carbon content and the total nitrogen content of the rhizosphere soil are improved, the formation of soil micro-aggregates is promoted, the soil permeability is improved, the disease index of the ginseng is reduced, and the growth and the quality improvement of the ginseng are promoted.

Description

Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants
Technical Field
The invention relates to the technical field of ginseng cultivation, in particular to a construction method of a synthetic flora and application of the synthetic flora in high-quality cultivation of medicinal plants.
Background
Ginseng (Panax Ginseng c.a.mey) is a perennial herb of the genus Ginseng of the family araliaceae, is a traditional rare medicinal material, is known as "the king of Baicao" and "the first of Baiyao", and has an application history of over four thousand years in china. The ginseng contains ginsenoside, alkaloid, glycoside, polysaccharide and polypeptide, and has pharmacological effects of regulating nerve, resisting cancer, resisting aging, resisting diabetes, regulating immunity, resisting thrombosis, etc. Meanwhile, the ginseng also contains various microelements required by human bodies, so that the ginseng is used as a plant resource for both medicine and food, meets the requirements of human on healthy diet, and has extremely high medicinal value.
Ginseng is mainly distributed in northeast China, after 90 th century in 20 th century, the ginseng is obtained gradually from wild under-forest digging to artificial cultivation along with the increasing of market demand, the cultivation modes are divided into modes of forest planting, non-forest planting, under-forest nursing and the like, and farmland planting belongs to one of the modes of non-forest planting, and soil improvement is carried out by utilizing traditional farmland, so that the problem of forest cutting is effectively solved, but the problems of acidification, hardening, low organic matter content, poor nutrient availability, structural damage of microbial community and the like of cultivated ginseng are caused due to the fact that fertilizer and pesticide are applied in a large amount in the process of farmland planting, and the problem of continuous cropping obstacle of ginseng is caused.
Rhizosphere microorganisms are an integral part of the whole life cycle of plants, possess far more genes than plant genomes, are known as "secondary genomes" of plants, and are also major drivers of soil mass circulation and energy flow. The rhizosphere microorganisms influence the formation and stability of the soil aggregate structure, and play an important role in improving the content of soil organic matters, improving the soil nutrient circulation, promoting the growth and development of plants, enhancing the stress resistance of plants and the like. Previous studies have focused on the plant growth promoting effects of single microorganisms such as Bacillus subtilis (Bacillus subtilis), trichoderma harzianum (Trichoderma harzianum), pseudomonas fluorescens (Pseudomonas fluorescens), and the like. However, most single strains are affected by environmental conditions, interactions among microorganisms and other factors, so that the growth promoting function of the single strains is weak, the colonization and the stability are poor, and therefore, the microbial agents mainly containing the single microorganisms are still limited and restricted to a certain extent during application.
The synthetic microbial community (Synthetic microbial communities) is called SynComs for short, is an emerging cross field of synthetic biology and microbiology, and is characterized in that a multi-strain coexistence system is designed and constructed by utilizing the interaction between microorganisms, so that the technical barrier of stable colonization of functional microorganisms is broken through, the functional complementation of various microorganisms is realized, and the method has the characteristics of stronger stability, environmental friendliness and the like. At present, the invention is particularly proposed in view of the fact that the content of the synthetic microbial community related to ginseng planting is still freshly reported.
Disclosure of Invention
The invention aims to provide a construction method of a synthetic flora, the synthetic flora and application of the synthetic flora in high-quality cultivation of medicinal plants. The invention provides a novel method for artificially constructing a flora, the synthetic flora constructed by the method has good colonization effect on ginseng root systems, the flora is more stable, the rhizosphere microenvironment of the cultivated ginseng soil can be improved, the contents of organic matters, ammonium nitrogen, quick-acting phosphorus and quick-acting potassium in the rhizosphere soil are increased, the absorption and utilization of nutrients and the accumulation of effective components of ginseng are promoted, the disease index of the ginseng is reduced, and the high-quality ginseng cultivation technology is realized.
In one aspect, the invention provides a method of constructing a synthetic flora, comprising the steps of:
(a) Analyzing the information of the micro-organism metagenome DNA in the rhizosphere soil sample of the target plant;
(b) Determining the relative abundance of the species from the genomic DNA information;
(c) And constructing a correlation network among the strains according to the relative abundance of the strains, and selecting a flora with strong positive interaction among the strains as a synthetic flora according to a correlation network analysis chart.
The inventors of the present invention found that the cause of low microbial colonization is related to interactions between the flora, and thus, aimed at this disadvantage, a synthetic flora technical approach was adopted. The invention utilizes synthetic biology and microbiology to construct a synthetic flora with interaction and complementary functions, and the selected community is based on the analysis of a correlation interaction network, and the synthetic community is constructed according to the superposition and synergy of the functions of microorganisms and the complementary functions.
In one embodiment, in step (c), constructing an interaction relationship of the microorganism from the relative abundance of the species by a biological big data platform; preferably, the biological big data platform is the Person gene cloud platform (https:// www.genescloud.cn /).
In one embodiment, the method further comprises the steps of carrying out functional identification on the constructed synthetic flora, and selecting strains with excellent functions for compounding in an equal amount mixing mode to obtain the optimal synthetic flora.
In one embodiment, the genomic DNA information is sequence information of a gene fragment that is genomic DNA of the rhizosphere soil microorganism; the abundance of the strain is the relative abundance of the strain.
In one embodiment, the medicinal plant is a plant of the family araliaceae; preferably a ginseng plant.
In one embodiment, the synthetic microbial flora comprises at least 3 of the following microbial species: polyborogreedy (Variovorax boronicumulans), stenotrophomonas rhizophila (Stenotrophomonas rhizophila), stenotrophomonas maltophilia (Stenotrophomonas maltophilia), pseudomonas putida (Pseudomonas putida), chitin-phagol (Chitinophaga niastensis), and Pseudomonas fluorescens (Pseudomonas fluorescens).
In one embodiment, the synthetic microbial flora consists of the following 6 microbial species: polyborogreedy (Variovorax boronicumulans), stenotrophomonas rhizophila (Stenotrophomonas rhizophila), stenotrophomonas maltophilia (Stenotrophomonas maltophilia), pseudomonas putida (Pseudomonas putida), chitin-phagol (Chitinophaga niastensis), and Pseudomonas fluorescens (Pseudomonas fluorescens).
In the invention, the synthetic flora constructed by the 6 microorganism strains has multiple functions of fixing nitrogen, dissolving phosphorus and potassium, producing auxin and siderophores, and has complementary functions. The invention selects the optimal synthetic community, and inoculates the optimal synthetic community to the ginseng root system to enable the ginseng root system to successfully colonise. The colony colonisation ability of the synthetic flora constructed by the 6 microorganism strains is obviously enhanced (the colonisation rate of the strain is high).
In another aspect, the present invention provides a microbial composition comprising the aforementioned synthetic microbial flora, preferably, the microbial composition is obtained by mixing equal amounts of the microbial species;
more preferably, the composition is one of a tablet, granule, powder, solution, suspension or emulsion.
In another aspect, the invention provides the use of said synthetic flora or a microbial composition comprising said synthetic flora for improving soil and/or promoting the growth and high quality cultivation of medicinal plants; preferably, the medicinal plant comprises ginseng.
In one embodiment, the improvement of the soil comprises increasing the pH, organic matter, total carbon and total nitrogen content of the ginseng rhizosphere soil, facilitating the formation of soil micro-agglomerates and improving the permeability of the soil.
In one embodiment, the synthetic flora or the microbial composition containing the synthetic flora can increase the total root length, total root surface area, total root volume, average root diameter and root tip number of the ginseng root system, increase the total saponin content, and promote the growth, appearance and internal quality of the ginseng.
In another aspect, the invention provides the use of said synthetic flora or said microorganism for controlling diseases of ginseng.
In one embodiment, the invention provides that said synthetic flora or said microorganism is capable of reducing the index of the condition of ginseng. This shows that the synthetic flora or the microorganism containing the same can resist diseases and improve the seedling storage rate.
In another aspect, the present invention provides a method for promoting ginseng growth and high quality ginseng cultivation, the method comprising applying an effective amount of the synthetic flora or the microbial composition to the plant or to the surrounding environment of the plant; preferably, the plant is a ginseng plant.
In the present invention, the synthetic flora or the microbial composition has the functions of fixing nitrogen, dissolving phosphorus, dissolving potassium, producing auxin and siderophore, and can be used for cultivation of medicinal plants such as ginseng, etc.
In one embodiment, the microbial composition comprises a component obtained from a strain of the population of microorganisms or a combination thereof; the component comprises a supernatant obtained from a strain of the population of microorganisms and/or a derivative thereof.
The beneficial effects are that:
the invention takes interaction of rhizosphere microorganisms as a core, takes functional superposition complementation as a basis to manually construct communities, so that the communities stably colonize on root systems, and fundamentally solves the defects of poor colonization and stability and single function in practical application;
the construction method of the synthetic flora is simple and efficient, and can construct the synthetic flora with good stability and excellent functions; the composition formed by the strains of the synthetic flora according to a specific proportion can play a synergistic advantage effect as a biological agent;
the invention has wide application prospect in improving rhizosphere soil microenvironment, promoting ginseng growth and improving quality, and has important significance for realizing green sustainable development of medicinal plant cultivation industries such as ginseng and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a network diagram of interaction of microorganisms with rhizosphere of mountain ginseng under forests provided by the embodiment of the invention;
FIG. 2 shows the nitrogen fixation capacity of the microorganism according to the embodiment of the invention;
FIG. 3 shows the phosphate solubilizing ability of the microorganism according to the embodiment of the present invention;
FIG. 4 shows the potassium decomposing ability of the microorganism provided by the embodiment of the invention;
FIG. 5 shows the ability of a microorganism to produce auxin according to an embodiment of the present invention;
FIG. 6 shows the capacity of the microorganism to produce siderophores according to the embodiment of the present invention;
FIG. 7 shows the result of colonization of ginseng root systems (16S rRNA gene copy number per gram of root system) by artificial synthetic flora (SynComs) provided by the examples of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 Strain information
(1) Available routes for strains:
Figure GDA0003821213650000061
EXAMPLE 2 synthetic flora constructed based on microbial interactions
Inputting 164 strains into a website (https:// www.genescloud.cn /) according to the relative abundance of the strains in metagenome data for correlation interaction network analysis, obtaining 5 core communities of module 2, module4, module1, module 5 and module 3 according to the constructed microorganism interaction relationship, and preliminarily selecting bacterial communities of module1 and module_4 with strong positive interaction among the strains as synthetic bacterial communities (as shown in figure 1)
The Module1 is a synthetic community composed of herbal genus (Herbiconiux ginsengi), streptomyces avermitilis (Streptomyces scabiei), arthrobacter (Arthrobacter oryzae), arthrobacter citricola (Janibacter melonis), nocardia (Nocardioides allogilvus), arthrobacter nicotinovorans (Paenarthrobacter nicotinovorans), bacillus megaterium (Bacillus megaterium), micro Bai Huanglian mold (Streptomyces albidoflavus), rhodococcus equi (Rhodococcus hoagii), cronobacter surimi (Cronobacter turicensis), rhodococcus (Rhodococcus qingshengii).
Module4 is a synthetic community composed of a group of boro-bulimia (Variovorax boronicumulans), stenotrophomonas rhizophila (Stenotrophomonas rhizophila), stenotrophomonas maltophilia (Stenotrophomonas maltophilia), pseudomonas putida (Pseudomonas putida), chitin-phaga (Chitinophaga niastensis) and pseudomonas fluorescens (Pseudomonas fluorescens).
EXAMPLE 3 microbial function identification assay
3.1 identification and analysis of rhizosphere microorganism function of Ginseng radix
Each strain in the synthetic communities model 1 and model 4 constructed according to the interaction relationship between strains is subjected to function identification analysis, and the specific operation is as follows:
(1) Nitrogen fixation function:
inoculating bacterial liquid preserved at 80deg.C into 20mL TSB liquid culture medium, activating, and standing at 180r.min -1 The bacterial liquid OD 600 grows to about 0.8 in a shaking table at 28 ℃. And (3) dripping 10 mu L of bacteria on a prepared nitrogen fixation solid culture medium (Haibo) in an ultra-clean workbench, inverting all the plates, culturing for 3-7 d at 28 ℃, and observing the growth condition of colonies in the plates and whether transparent oily colonies appear or not at any time during the culturing period, wherein the transparent oily colonies represent the strains with the nitrogen fixation capacity (as shown in figure 2).
(2) Phosphate dissolving function:
taking 10 mu L of fungus liquid drop on a prepared inorganic phosphorus solid culture medium (sea bo) and organic phosphorus solid culture medium (sea bo) in an ultra-clean workbench, inverting all the plates, culturing for 3-7 d in a constant temperature incubator at 28 ℃, observing the growth condition of colonies in the plates and the generation of phosphate solubilizing rings at any time during the culturing period, wherein the generation of phosphate solubilizing rings represents that the strain has phosphate solubilizing capability (as shown in figure 3).
(3) Potassium decomposing function:
in the ultra clean bench 10L fungus liquid drop on the improved solid culture medium (Alatine), all the plates are inverted, in the 28 deg.C constant temperature incubator culture for 3-7 d, at any time during the culture period the colony growth condition and the potassium-dissolving ring generation are observed, and the potassium-dissolving ring generation is representative of the strain possessing potassium-dissolving capacity (as shown in figure 4).
(4) Auxin (IAA) function: inoculating 10 mu L of bacterial liquid into 1mL of Kings liquid culture medium containing 0.2g/L tryptophan in a super clean bench, and culturing for 3d; after the strain is cultured for 3d, centrifuging for 10min at 12000r/min, placing 800 mu L of supernatant after centrifugation on a white porcelain plate, adding an equal volume of Salkowski reagent colorimetric solution, and covering the reaction in a dark place for 30min by using tin foil paper; after the reaction, the result of the observation is shown as pink, which indicates that the strain can secrete IAA, and the darker the pink color indicates that the strain has a stronger ability to secrete IAA (see FIG. 5).
(5) Function of siderophore: in the ultra clean bench, 10L of fungus liquid is dropped on the prepared CAS detection plate (Haibo), all plates are inverted, and are cultured for 3-7 d in a constant temperature incubator at 28 ℃, during the culture period, the growth condition of the colony in the plates and the generation of halation yellow circle or not are observed at any time, and the generation of halation yellow circle represents that the strain has the capability of producing iron carrier (as shown in figure 6).
According to the functional analysis of each microorganism with nitrogen fixation, phosphorus dissolution, potassium dissolution, and production of auxin and siderophores, module4 was finally selected as a synthetic community (synComs) with stable interaction and comprehensive and complementary functions, as shown in Table 1.
TABLE 1 identification analysis of the rhizosphere microorganism function of Ginseng radix
Figure GDA0003821213650000091
EXAMPLE 4 Artificial synthetic flora (SynComs) colonization
4.1 preparation of bacterial liquid:
inoculating bacterial liquid of each strain preserved at-80deg.C into 20mL TSB liquid culture medium, activating, and standing at 180r.min - 1 Culturing in a shaking table at 28 ℃ for 3-7 days until bacteria liquid is turbid; absorbing 2 mu L of turbid bacteria liquid, measuring a light absorption value (OD value) at a wavelength of 600nm of a spectrophotometer, adjusting the OD value of all bacteria liquid to 0.2 by using sterile water, and mixing the bacteria liquid in an equal volume ratio to obtain the mixed bacteria liquid.
4.2 colonization of ginseng root systems by synthetic flora (synComs):
the farmland soil was put into pots with a diameter of 18cm, each pot containing 1.5kg of soil. The annual ginseng seedlings which are consistent in size, undamaged in spores and disinfected on the surfaces are prepared, the ginseng seedlings are soaked for 30min in advance by using fluorescent pseudomonas treatment and Syncoms mixed bacteria liquid treatment, then transplanting is carried out, 5 plants are planted in each pot, sterile water is inoculated in a contrast mode, the fluorescent pseudomonas treatment and the Syncoms mixed bacteria liquid treatment are repeated for 3 times, and 25ml of single bacteria and mixed bacteria liquid are respectively taken for root irrigation treatment after the planting is finished. Root irrigation treatment is carried out every 14d, 28d and 56d during ginseng growth, 84d ginseng growth is harvested, and FastDNA is used TM The SPIN Kit (MP Biomedicals, LLC, USA) extracts genomic DNA of the microorganism of the ginseng root system, and the 16S rRNA gene copy number in each gram of root system (as shown in figure 7) shows that the bacterial strain has strong colonization rate, and the colonization rate of Syncoms is obviously higher than that of single bacteria and controls.
The results show that the colonization rate of each strain constituting the artificial synthetic flora is obviously higher in the root system of the ginseng.
Example 5 Effect of synthetic flora (SynComs) on farmland soil
The ginseng seedlings treated with the control, single-fungus treatment and syncooms mixed fungus liquid were taken out, the soil adhering to the root system of ginseng was removed, the rhizosphere soil of ginseng was taken out with a small brush, and the pH, total carbon, total nitrogen content and soil aggregate structure of the rhizosphere soil were measured (table 2). The pH of the rhizosphere soil of the ginseng treated by Syncoms is increased, and the contents of organic matters, total carbon and total nitrogen are increased compared with the control and single bacteria treatment; at the same time, the SynComs treatment significantly promoted soil formation at 0.25-2mm agglomerate size fractions, while decreasing <0.25mm agglomerate size fractions.
TABLE 2 influence of Syncoms on rhizosphere soil of cultivated ginseng in farmlands
Figure GDA0003821213650000101
The results show that the artificial synthetic flora can improve the permeability of farmland soil, thereby improving the structure of farmland soil, promoting the mineralization of nutrients of farmland soil and improving the fertility of farmland cultivated ginseng soil.
Example 6 Effect of synthetic flora (SynComs) on root growth and quality of Ginseng radix
Taking out the ginseng seedlings treated by the control, the single bacteria and the Syncoms mixed bacteria respectively, washing the roots with sterile water, measuring the root morphology of the ginseng by a root system scanner (WINRIZO) and weighing; the root system of ginseng was then dried at 65℃to constant weight, and the dry weight of the root and the total saponin content were measured (Table 3). Compared with the control and single bacteria treatment, the total root length, total root surface area, total root volume, average root diameter and root tip number of the ginseng root system after the Syncoms treatment are obviously increased, and the fresh root weight and dry root weight are increased; compared with the control and single bacteria treatment, the total saponin content of the ginseng root system after the Syncoms treatment is obviously improved.
TABLE 3 influence of Syncoms on the growth and quality of cultivated ginseng in farmlands
Figure GDA0003821213650000111
The result shows that Syncoms has remarkable promoting effect on the growth of ginseng root system, can improve the morphology of ginseng root system and improve the quality of ginseng.
Example 6 influence of synthetic flora (Syncoms) on disease resistance of Ginseng radix
And taking out the ginseng seedlings treated by the control, the single bacteria and the Syncoms mixed bacteria respectively, washing off root soil, and investigating the disease condition of the ginseng according to the disease index. Morbidity = total number of disease/number of plants x 100, disease index = [ Σ (number of disease at each stage x representative value)/(total number of disease x highest disease stage representative value) ]x100 (see table 4). Compared with the control and single bacteria treatment, the ginseng seedling storage rate of Syncoms treatment is improved, and meanwhile, the root system morbidity and disease index are reduced.
TABLE 4 influence of Syncoms on disease occurrence in cultivated-field ginseng
Figure GDA0003821213650000112
The result shows that SynComs has disease resistance and can obviously reduce the morbidity and the disease index.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (5)

1. A synthetic flora, characterized in that the synthetic flora consists of the following microbial species: poly boro greedy phageVariovorax boronicumulans) Stenotrophomonas rhizophilaStenotrophomonas rhizophila) Stenotrophomonas maltophiliaStenotrophomonas maltophilia) Pseudomonas putida @Pseudomonasputida) Chitin-binding bacteriaChitinophaga niastensis) Pseudomonas fluorescens @Pseudomonas fluorescens)；
Poly boro greedy phageVariovorax boronicumulans) The preservation number of the strain is CGMCCNo.1.12091 and stenotrophomonas rhizophilaStenotrophomonas rhizophila) The preservation number of the strain is CGMCC No.1.15515, stenotrophomonas maltophilia(Stenotrophomonas maltophilia) The preservation number of the pseudomonas putida is CGMCCNo.1.8094Pseudomonas putida) The preservation number of the bacterial strain is CGMCCNo.1.8092 and chitin-phage is%Chitinophaga niastensis) The deposit number of the fluorescent pseudomonas is bio-093731Pseudomonas fluorescens) The preservation number of the product is CGMCC No.1.7375.
2. A microbial composition comprising the synthetic flora of claim 1, wherein the microbial composition is obtained by mixing equal amounts of the microbial species.
3. The microbial composition of claim 2, wherein the composition is one of a tablet, granule, powder, solution, suspension, or emulsion.
4. Use of the synthetic flora according to claim 1 or the microbial composition according to claim 2 or 3 for improving the rhizosphere soil of cultivated ginseng in farmlands and/or promoting the growth of root systems of ginseng and increasing the total saponin content of root systems of ginseng.
5. A method of promoting root growth and increasing total saponins content of a ginseng root system, the method comprising applying an effective amount of the synthetic flora of claim 1 or the microbial composition of claim 2 or 3 to ginseng or to the surrounding environment of ginseng.
CN202210842260.8A 2022-07-18 2022-07-18 Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants Active CN115161406B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210842260.8A CN115161406B (en) 2022-07-18 2022-07-18 Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210842260.8A CN115161406B (en) 2022-07-18 2022-07-18 Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants

Publications (2)

Publication Number Publication Date
CN115161406A CN115161406A (en) 2022-10-11
CN115161406B true CN115161406B (en) 2023-04-21

Family

ID=83495101

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210842260.8A Active CN115161406B (en) 2022-07-18 2022-07-18 Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants

Country Status (1)

Country Link
CN (1) CN115161406B (en)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4147575A1 (en) * 2015-02-09 2023-03-15 BioConsortia, Inc. Agriculturally beneficial microbes, microbial compositions, and consortia
US10851399B2 (en) * 2015-06-25 2020-12-01 Native Microbials, Inc. Methods, apparatuses, and systems for microorganism strain analysis of complex heterogeneous communities, predicting and identifying functional relationships and interactions thereof, and selecting and synthesizing microbial ensembles based thereon
US20200164005A1 (en) * 2017-02-23 2020-05-28 Intercept Pharmaceuticals, Inc. Pharmaceutical compositions of a bile acid derivative and microbiome and uses thereof
CN112679280A (en) * 2020-12-22 2021-04-20 吉林农业大学 Preparation method of efficient biocontrol growth-promoting ginseng compound microbial fertilizer
CN112694996A (en) * 2021-01-13 2021-04-23 南昌大学 Method for artificially constructing plant growth-promoting complex functional flora
CN113736685B (en) * 2021-07-05 2023-05-09 西北农林科技大学 Construction method of composite flora

Also Published As

Publication number Publication date
CN115161406A (en) 2022-10-11

Similar Documents

Publication Publication Date Title
CN105936881B (en) One kind is for alignic thermophilic sugared bacillus and its application method of degrading
CN103396971B (en) Burkholderia cepacia and application thereof
CN103484396B (en) New strain of streptomyces thermocarboxydus and application thereof
CN102174403B (en) Application of waste compost composite bacterial agent to improving drought resistance of lawn grass
CN102925385A (en) Compound microorganism bacterium agent for treating poultry excrement to prepare composts and preparation method of compound microorganism bacterium agent
CN111944716B (en) Special compound microbial agent for tobacco seedling culture and preparation method and application thereof
CN103931664A (en) Low-temperature resisting method for festuca arundinacea by using complex microbial inoculants
CN105255770A (en) Bacillus safensis and application thereof
CN107418906B (en) A kind of composite bacteria agent and its application improving color green pepper yield and anti-root rot
CN111662837B (en) Bacillus atrophaeus and application thereof
CN105838627B (en) It is a kind of prevent and treat tobacco bacterial wilt mould xfsf-8 bacterial strain and application
CN107760630B (en) Bacillus methylotrophicus B18, microbial inoculum and application thereof
CN110184225A (en) One plant of rhizosphere growth-promoting endophytic bacteria PHE-2 and its application with PAHs degradation capability
CN109321500A (en) One bacillus amyloliquefaciens bacterial strain and its application in prevention and treatment Oil Tea Anthracnose evil
CN109673469A (en) A kind of preparation method with growth promotion seedling medium
CN105439657A (en) Preparation method for strawberry dedicated anti-continuous cropping biological organic fertilizer
CN116463220B (en) Dark-color DSE fungus for promoting blueberry growth and application thereof
CN115161406B (en) Construction method of synthetic flora and application of synthetic flora in high-quality cultivation of medicinal plants
CN107056351A (en) A kind of fowl and animal excrement continuously ferments the method for compost
CN102329739B (en) Aspergillus versicolor for fermenting Jatropha cake to manufacture biological bacterial manure
CN107032872A (en) The orchid soft rot organic fertilizer and preparation method prepared by raw material of Chinese medicine dreg
CN105274031A (en) Sphingomonas sp. and its application
CN115717115A (en) Cellulose streptomyces LQS-2 for preventing and treating root rot of traditional Chinese medicinal materials, microbial inoculum and application
CN104498413A (en) Functional vegetable seedling biological matrix containing bacillus subtilis G10 and preparation method thereof
CN109810918B (en) Bacillus atrophaeus with effect of preventing wolfberry leaf blight, biological agent and application of biological agent

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