CN116121147A - Chenopodium ambrosioides seed endophytic Larimol agrobacterium and application thereof - Google Patents
Chenopodium ambrosioides seed endophytic Larimol agrobacterium and application thereof Download PDFInfo
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Images
Classifications
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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
- C12N1/20—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
- 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
Abstract
The invention discloses a strain of chenopodium ambrosioides seed endophytic Larimol agrobacteriumAgrobacterium larrymoorei) HLF14 with a preservation number of CGMCC No.26648 in China general microbiological culture Collection center; the strain has stronger capabilities of phosphate dissolution and IAA production, has the capabilities of nitrogen fixation and cadmium resistance, can obviously promote plant growth and enhance cadmium resistance by inoculating HLF14 under cadmium stress, and has important significance in the combined repair of heavy metal contaminated soil by plants and microorganisms.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and in particular relates to seed endophytic bacteria Agrobacterium tumefaciens @Agrobacterium larrymoorei) HLF14 and its use in promoting plant growth and repair of heavy metal pollution.
Background
With the development of modern human industry and agriculture, heavy metal pollution of soil caused by human activities such as mining and smelting, chemical fertilizer and pesticide use, farmland pollution irrigation and the like is more serious. Heavy metals have bioaccumulation, transmissibility along the food chain, and seriously threaten agricultural production, food safety, and human health. In the current problem of heavy metal pollution of soil, cadmium pollution is most prominent, cadmium in the soil has stronger mobility and is easy to be absorbed and enriched by grain crops, and the national soil pollution condition investigation publication in 2014 indicates that the total exceeding rate of soil in China is 16.1%, wherein the exceeding rate of cadmium is 7.0%. Therefore, the remediation of heavy metal pollution of soil is receiving extensive attention from various communities.
In all restoration methods, the plant restoration is mainly to enrich heavy metals in soil by planting super-accumulated plants, and compared with the traditional physicochemical method, the method has the advantages of low cost, environmental protection, simplicity, high efficiency and the like. However, in practical application, phytoremediation is generally limited by slow growth of super-accumulated plants, types and forms of soil heavy metals, and the like, and the soil heavy metal pollution is aged longer and has lower efficiency by singly using the phytoremediation. Therefore, the improvement of the phytoremediation efficiency becomes an important point of research, endophytes can colonize plants through various ways, promote plant growth through various ways such as biological nitrogen fixation, phosphate dissolution, plant hormone production and the like, and can also enhance the resistance and enrichment capacity of the plants to heavy metals. For example, liu et al research shows that the bacillus cereus PE31 of the cadmium super accumulation phytost Liu Nasheng has excellent IAA and siderophore production, phosphate dissolution, cadmium resistance and other capacities, and inoculation of PE31 remarkably improves the content of nutrient substances such as effective potassium, phosphorus, organic matters and the like in soil, so that the biomass of pokeberry and the cadmium content in tissues are improved. The endophyte-plant combined repair technology can effectively improve the repair efficiency of plants on heavy metals in soil, and has better application prospect.
As a plant reproductive organ, a certain number of endophytes are colonized in seeds. Research shows that partial seed endophytes can be vertically transmitted to next generation plants, and under heavy metal stress, the seed endophytes can promote plant growth, enhance heavy metal resistance of host plants and absorb and enrich. For example, truyes and the like find that the endophytic bacillus bacteria in the seeds of the grass fine and weak glume in the cadmium/nickel polluted rice field have stronger capability of producing siderophores, IAA and the like, can obviously promote the fine and weak glume seedling, and enhance the cadmium resistance; also, li et al separate Pseudomonas K03, K04 and N05 with nitrogen fixation, phosphate dissolution, IAA production, heavy metal tolerance and other characteristics from tobacco seeds, can promote the growth of tobacco in lead contaminated soil, and improve the enzyme activity of root systems. Therefore, the super-accumulated endophyte of the plant seeds in the polluted environment has wide application prospect in improving the repair of plants and microorganisms.
Disclosure of Invention
The invention provides a seed endophytic bacterium HLF14 which is separated from super-accumulated plant chenopodium ambrosioidesDysphania ambrosioides L.) seeds, classified and named as Agrobacterium moriAgrobacterium larrymoorei) The microbial strain is preserved in China general microbiological culture collection center (CGMCC) at the 2 nd month 21 of 2023, and the preservation number is CGMCC No.26648, and the preservation address is: the institute of microorganisms of national academy of sciences of China, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing.
The invention also aims to provide a new application of the seed endophytic bacterium Agrobacterium tumefaciens HLF14, namely application of the seed endophytic bacterium Agrobacterium tumefaciens HLF14 in promoting plant growth and bioremediation of cadmium pollution, and the seed endophytic bacterium Agrobacterium tumefaciens HLF14 has nitrogen fixation, cadmium resistance, stronger phosphate dissolving and IAA producing capabilities, and can promote the growth of host plants and enhance cadmium resistance under cadmium stress.
In order to achieve the above object, the present invention adopts the following technical scheme:
1. will be collected from the sea of people in the county of Jing City, yunnan provinceSuper-accumulated plant chenopodium ambrosioides on slag pile in heavy metal pollution area of ZhenyikacunD. ambrosioides) Airing the plant sample in sunlight until seeds naturally fall off, and then placing the seed sample in a refrigerator at 4 ℃ for storage;
2. accurately weighing 1g of seeds, cleaning with tap water, sterilizing the surfaces, and placing the seeds on sterile filter paper to absorb water; grinding the surface sterilized seed mixed sterile quartz sand into powder, fully mixing with 9mL of sterile water, coating the mixture on an LB culture medium after gradient dilution, culturing for 10 days at 37 ℃, observing every other day, picking up when colonies grow out, separating and purifying to obtain a plurality of endophytic bacterial strains, and preparing the endophytic bacterial strains into bacterial suspension;
3. inoculating the separated endophytic strain to an NBRIP solid culture medium to determine the phosphate dissolving capacity of the strain by hydrolysis circle;
4. selecting a bacterial strain with stronger phosphorus dissolving capability, storing the bacterial strain on an LB inclined plane for standby, and naming the bacterial strain as HLF14;
5. identification of Strain HLF14
(1) Morphological characterization of strain HLF 14: the bacterial colony is in a milky round bulge on the LB culture medium, the edge is neat, the surface is moist and smooth, gram staining is negative, and the bacterial colony is in a rod shape and has no spores;
(2) Molecular identification: using MoBio PowerSoil ® Extracting total DNA of the strain by using a DNA kit, detecting, sending to a sequencing company for sequence determination, and comparing a sequencing result with a sequence on NCBI; combining morphological characteristics and molecular identification results, and finally identifying the strain as the Agrobacterium tumefaciens @Agrobacterium larrymoorei) The method comprises the steps of carrying out a first treatment on the surface of the The culture mediums used for preserving and activating the strain are LB culture mediums;
6. the invention carries out potting test on seed endophytic bacteria Larimol agrobacterium HLF14 separated from chenopodium ambrosioides, discusses the influence of the seed endophytic bacteria Larimol agrobacterium HLF14 on the growth of corn and cadmium stress resistance, namely, under the cadmium stress, carries out research on the influence of seed endophytic bacteria Larimol agrobacterium HLF14 on the growth of corn, and provides bacterial strain and theoretical research basis for the combined repair of seed endophytes and plants.
Compared with the prior art, the invention has the following beneficial effects:
the seed endophytic bacterium A.lardii HLF14 provided by the invention is derived from super-accumulated plant chenopodium ambrosioides, has nitrogen fixation, cadmium resistance, strong phosphate dissolving and IAA producing capacity, can obtain a large amount of bacterial suspension through simple liquid fermentation, is easy to obtain thalli, has low cost and has the potential of commercial application; the inoculated strain HLF14 can obviously promote the growth of host plants under cadmium stress, improve the viability of the host plants and enhance the cadmium resistance of the host plants, and has great significance for restoring heavy metal contaminated soil.
Drawings
FIG. 1 shows colony morphology of the seed endophytic bacterium Agrobacterium tumefaciens HLF14 on LB medium;
FIG. 2 is a photograph of gram stain of seed endophytic bacterium Agrobacterium tumefaciens HLF14;
FIG. 3 is a standard operating curve for a phosphorus standard solution;
FIG. 4 shows the results of determination of the ability of Agrobacterium tumefaciens HLF14 to dissolve phosphorus;
FIG. 5 shows the results of determination of the nitrogen fixation capacity of Agrobacterium tumefaciens HLF14
FIG. 6 is a standard working curve of IAA standard solution;
FIG. 7 is a graph showing the results of an IAA producing capacity assay of Agrobacterium tumefaciens HLF14;
FIG. 8 shows the results of a determination of the cadmium tolerance of Agrobacterium tumefaciens HLF14
FIG. 9 is a graph showing the effect of inoculated (E+) and uninoculated (E-) Agrobacterium HLF14 on maize growth under cadmium stress.
Detailed Description
The technical scheme of the present invention will be described in further detail with reference to specific embodiments and drawings, but the present invention is not limited to the following technical scheme. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Those skilled in the art can refer to various common specifications, technical literature or related specifications, manuals, etc. before the filing date of the present invention;
example 1: isolation, screening and identification of seed endophytic bacterium Agrobacterium tumefaciens HLF14
(1) Super-accumulated plant chenopodium ambrosioides collected from slag piles in heavy metal contaminated areas of Areca village in Sharp county of TriJingjing, yunnan provinceD. ambrosioides) Airing the plant sample in sunlight until the seeds can naturally fall off, and then placing the seed sample in a refrigerator at 4 ℃ for storage;
(2) 1g of seeds are weighed accurately, cleaned by tap water and surface sterilized according to the following procedures: rinsing with 75% ethanol for 2 times, each time for 2min, washing with sterile water for 5 times, and drying with sterile filter paper; grinding sterilized quartz sand into powder, mixing with 9mL sterilized water to obtain 10 -1 Gradient diluting the mixed solution to 10 -2 、10 -3 Respectively coating 0.1mL of the diluted solution on LB culture medium (10 g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar, 1L of distilled water and pH of 7.0-7.2), culturing for 10 days at 37 ℃, observing every other day, picking up when colonies grow out, separating and purifying to obtain a plurality of endophytic bacterial strains, and preparing the endophytic bacterial strains into bacterial suspension;
inoculating the separated endophytic strain to NBRIP solid culture medium (glucose 10.0g, magnesium chloride hexahydrate 5.0g, magnesium sulfate heptahydrate 0.25g, potassium chloride 0.2g, ammonium sulfate 0.1g, tricalcium phosphate 5.0g, agar 15g, distilled water 1L, pH 6.8-7.0) to determine phosphorus dissolving capacity of the strain;
the strain with the strongest phosphate solubilizing ability was selected and stored on an LB slope for later use, and the endophytic bacterial strain was designated HLF14.
(5) Identification of Strain HLF14
(1) Morphological characterization of strain HLF 14: the bacterial colony is in a milky round bulge on the LB culture medium, the edge is neat, the surface is moist and smooth, gram staining is negative, and the bacterial colony is in a rod shape and has no spores (figures 1 and 2);
(2) molecular identification: extracting total DNA of the strain by using MoBio PowerSoil DNA kit, detecting, delivering to sequencing company for sequence determination, sequencing the result shown in SEQ ID NO. 1, and sequencing the result with NCBI upper sequenceComparing; combining morphological characteristics and molecular identification results, and combining the molecular identification results with the Agrobacterium tumefaciensAgrobacterium larrymoorei) The homology reaches 100%, and the strain is finally identified as the Agrobacterium tumefaciens @Agrobacterium larrymoorei）。
Example 2: determination of the phosphorus-solubilizing Capacity of seed endophytic bacterium Agrobacterium tumefaciens HLF14
Inoculating seed endophytic bacteria Agrobacterium tumefaciens HLF14 into an NBRIP liquid culture medium at 37 ℃ and 180rpm for 15 hours in a shaking table in an inoculation amount of 4%, and simultaneously inoculating the same amount of LB liquid culture medium as a blank control, wherein three replicates are arranged in each group of treatment, and the seed endophytic bacteria Agrobacterium tumefaciens HLF14 is cultured on the shaking table at 30 ℃ and 180rpm for 6 days; the method for measuring the content of soluble phosphorus by adopting a molybdenum-antimony colorimetric method (the wavelength of a spectrophotometer is 700 nm) comprises the following specific steps:
(1) centrifuging the fermentation liquor at 10000rpm for 15min, placing 1mL of supernatant into a 50mL colorimetric tube with a plug, adding 2 drops of 2, 6-dinitrophenol indicator, adjusting the pH to slightly yellow with 10% sodium hydroxide or 5% dilute sulfuric acid, adding 5mL of molybdenum-antimony color development inhibitor, and adding deionized water to a volume of 50mL;
(2) standing for 30min, performing color comparison under 700nm of a spectrophotometer, and simultaneously measuring a blank control group;
(3) drawing a phosphorus standard curve at the same time in an experiment, respectively sucking 5mg/L of phosphorus standard solution 0, 2, 3, 4, 5, 6 and 10mL into a 50mL colorimetric tube with a plug, adding 2 drops of a 2, 6-dinitrophenol indicator, regulating the pH value to be slightly yellow by using 10% sodium hydroxide or 5% dilute sulfuric acid, adding 5mL of a molybdenum-antimony color-developing resistant agent, adding deionized water to a constant volume of 50mL to obtain 0, 0.2, 0.3, 0.4, 0.5, 0.6 and 1.0mg/L phosphorus standard series solution, standing for 30min, carrying out colorimetry at 700nm by a spectrophotometer, drawing a standard working curve (figure 3), substituting the absorbance value measured in the step (2) into the standard working curve, and obtaining the phosphorus dissolving amount of the A. Larmor HLF14 in an NBRIP culture solution to be 229.058 mg/L, wherein 238.61% (figure 4) is increased compared with a control group, which shows that the A. Larmor HLF14 has stronger phosphorus dissolving capacity, can convert inorganic phosphorus into organic phosphorus, enable plants to absorb more barren tailings and promote the growth of plants, especially in a barren plant growth area and other nutrition-poor areas.
Example 3: determination of the Nitrogen fixation Capacity of seed endophytic bacterium Agrobacterium mori HLF14
Inoculating Agrobacterium tumefaciens HLF14 to solid Abbe culture medium (mannitol 10g, dipotassium hydrogen phosphate 0.2g, sodium chloride 0.2g, magnesium sulfate heptahydrate 0.2g, calcium carbonate 5g, calcium sulfate 0.1g, agar 15g, distilled water 1L, pH 7.0), inoculating three points on each culture medium, repeating three times, and culturing at 30 ℃ constant temperature for 5 days; if the strain can grow on the culture medium, the strain is proved to have nitrogen fixation capacity, otherwise, the strain has no nitrogen fixation capacity.
The results are shown in FIG. 5, from which it can be seen that Agrobacterium tumefaciens HLF14 can grow on Abbe's medium, demonstrating its ability to fix nitrogen, while Agrobacterium tumefaciens HLF14 can fix N in air 2 Is converted into effective nitrogen, which is helpful for the absorption of nitrogen by plants and further promotes the growth of plants.
Example 4: determination of IAA-producing ability of seed endophytic bacterium Agrobacterium mori HLF14
Culturing Agrobacterium tumefaciens HLF14 in LB liquid medium (containing L-tryptophan 0.5 mg/mL) at 30deg.C on shaking table at 150rpm for 48h, centrifuging 2mL of bacterial liquid at 12000rpm for 15min, removing precipitate, adding Salkowski's reaction solution (1 mL of 0.5mol/L ferric chloride, 49mL of 35% perchloric acid) in equal amount into 1mL of supernatant, reacting for 30min in dark place, measuring absorbance at 530nm, repeating each group three times, and zeroing by using the same treatment as the control without inoculating bacterial medium; IAA standard solutions of concentrations 0, 2.5, 5, 10, 20, 40, 60, 80, 100mg/L were subjected to absorbance values and standard curves were drawn using the methods described above (FIG. 6), and absorbance values of the experimental group inoculated with Agrobacterium tumefaciens HLF14 were substituted into the standard curves to obtain an IAA yield of Agrobacterium tumefaciens HLF14 of 51.738mg/L (FIG. 7), with Agrobacterium tumefaciens HLF14 having a strong ability to synthesize phytohormone IAA, capable of stimulating plant cell growth and proliferation, and regulating vital activities of plants.
Example 5: determination of cadmium tolerance of seed endophytic bacterium Agrobacterium tumefaciens HLF14
Shaking culture of seed endophytic bacteria Agrobacterium Larimolus HLF14 in LB liquid medium at 37deg.C and 180rpm for 15 hr, inoculating Cd with 2% inoculum size 2+ Shake culturing in LB liquid medium with concentration of 0, 25, 50, 100, 150mg/L at 37deg.C at 180rpm for 60 hr, measuring absorbance at 600nm, repeating each concentration for three times, and zeroing with blank LB liquid medium with concentration without bacteria as control;
as shown in FIG. 8, agrobacterium tumefaciens HLF14 can tolerate up to 100mg/L Cd 2+ Has potential application value of promoting the growth of host plants and restoring heavy metals in heavy metal polluted environments.
Example 6: effect of seed endophytic bacterium Agrobacterium mori HLF14 on corn growth under heavy metal stress
This example is intended to demonstrate the promoting effect of Agrobacterium tumefaciens HLF14 on plant growth and heavy metal tolerance in heavy metal contamination; corn is used as a raw materialZea mays L.) are test plants, the experimental procedure is as follows:
preparation of a suspension of Agrobacterium tumefaciens HLF 14: inoculating Agrobacterium tumefaciens HLF14 into 100mL LB liquid medium, culturing for 15h in a constant temperature shaking table at 37 ℃ and 180rpm, subpackaging the bacterial liquid with a sterile 50mL centrifuge tube under sterile conditions, centrifuging at 5000rpm for 10min, discarding the supernatant, re-suspending the bacterial cells with sterile water, and adjusting the absorbance at 600nm to 0.8 to prepare HLF14 bacterial suspension;
seed soaking method inoculation of a lagomorphic agrobacterium HLF14 and germination: randomly selecting corn seeds, firstly soaking the corn seeds in an ethanol solution with the volume concentration of 75% for 2min, washing the corn seeds with sterile water for 3 times, then soaking the corn seeds in a NaClO solution with the effective chlorine concentration of 5% for 1min, washing the corn seeds with sterile water for 5 times, placing the corn seeds on sterile filter paper to absorb water, and sterilizing the corn seeds on the surface for later use. Soaking corn seeds with sterilized surfaces in the HLF14 bacterial suspension for (E+), colonizing for 5h, soaking the seeds in sterile water with the same volume as a control group (E-), placing the soaked seeds in soil after high-pressure sterilization (124 ℃ for 30 min), germinating at room temperature (18-25 ℃) and 60% relative humidity, wherein the soil formula is V Garden soil : V Canadian mudCarbon moss : V Perlite =7:2:1, sterile water was poured once every 3 days in order to retain moisture.
Potting experiment: after germination of the maize seeds for 7d, 5 seedlings of the same growth vigor were selected for each group (E+/E-) and transplanted into pots (10.2X5.5 cm,1 seedling/pot), each pot containing 700g of autoclaved (124 ℃ C., 30 min) post-cultivation substrate (V) Garden soil : V Sphagnum canadensis : V Perlite =7:2:1, containing 15mg/L Cd 2+ ) The method comprises the steps of carrying out a first treatment on the surface of the Randomly arranging flowerpots, placing the seedlings at room temperature, culturing the seedlings with light and dark circulation illumination for 14/10h, pouring sterile water once every 2-3 days, and pouring 50mL of sterile water into each pot (preferably, pouring water through soil without overflowing the bottom of the pot); 7 days after implantation, 20mL of Agrobacterium tumefaciens HLF14 bacterial suspension (OD) was irrigated to the roots of the E+ group plants 600 =0.8), roots of E-group plants were irrigated with equal amounts of sterile water; the growth condition of the corn is closely observed in the potting process, the corn is harvested after 25 days, and the plant height, root length, fresh weight and dry weight of the corn are measured.
As shown in figure 9, under cadmium stress, the growth promoting effect of the seed endophytic bacterium Agrobacterium tumefaciens HLF14 on maize seedlings is obvious, and the plant height, the overground and underground fresh weight and the overground dry weight of the E+ group maize are all obviously higher than those of the E-group (p <0.05, t test), which indicates that the seed endophytic bacterium Agrobacterium tumefaciens HLF14 can effectively promote the growth of host plants in heavy metal polluted environments, enhance the heavy metal resistance of the host plants, and have good growth promoting effect and heavy metal restoration potential.
The results of the above examples show that the seed endophytic bacterium Agrobacterium mori HLF14 obtained by separation in the invention has strong capabilities of dissolving and dissolving phosphorus and producing IAA, nitrogen fixation and cadmium resistance, has good promotion effect on the growth of corn seedlings under heavy metal stress, and enhances the heavy metal resistance.
Claims (3)
1. Chenopodium ambrosioides seed endophytic Larimol agrobacteriumAgrobacteriumlarrymoorei) HLF14 with the preservation number of CGMCC No.26648 in China general microbiological culture Collection center.
2. Use of a chenopodium ambrosioides seed endophytic agrobacterium strain HLF14 according to claim 1 for promoting plant growth and bioremediation of heavy metal pollution.
3. The use according to claim 2, characterized in that: the Agrobacterium tumefaciens HLF14 has the capabilities of phosphate solubilizing, IAA producing, nitrogen fixation and cadmium resistance.
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