CN113403224B - Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof - Google Patents

Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof Download PDF

Info

Publication number
CN113403224B
CN113403224B CN202110606574.3A CN202110606574A CN113403224B CN 113403224 B CN113403224 B CN 113403224B CN 202110606574 A CN202110606574 A CN 202110606574A CN 113403224 B CN113403224 B CN 113403224B
Authority
CN
China
Prior art keywords
strain
acinetobacter
mrp20
cadmium
oleaginous
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
CN202110606574.3A
Other languages
Chinese (zh)
Other versions
CN113403224A (en
Inventor
王秀荣
王幼娟
罗莎莉
陆星
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.)
South China Agricultural University
Original Assignee
South China Agricultural University
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 South China Agricultural University filed Critical South China Agricultural University
Priority to CN202110606574.3A priority Critical patent/CN113403224B/en
Publication of CN113403224A publication Critical patent/CN113403224A/en
Application granted granted Critical
Publication of CN113403224B publication Critical patent/CN113403224B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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
    • A01G22/40—Fabaceae, e.g. beans or peas
    • 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
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
    • A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Abstract

The invention relates to a strain MRP20 of Acinetobacter oleaginous (Acinetobacter oleivorans) and application thereof, wherein the strain is preserved in the microorganism strain preservation center of Guangdong province at 5-11 of 2021, and the preservation number is GDMCC No:61653. the strain has the characteristics of acid resistance, cadmium resistance, phosphorus dissolution, siderophore production and auxin secretion. Inoculating the soybean with the Acinetobacter oleaginous MRP20 can improve the biomass of the soybean, chlorophyll SPAD value and phosphorus content; but also can overcome the inhibition effect of cadmium on soybean growth, in particular to the inhibition effect of cadmium on soybean root growth, and has high application value.

Description

Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an acinetobacter oleaginous strain MRP20 and application thereof.
Background
Cadmium is not an element necessary for plant growth. When the content in the plant body exceeds a certain concentration, the plant is seriously damaged, such as the symptoms of plant wilting, yellowing, weaker growth and the like. The plant root system can easily absorb cadmium and then transport the cadmium to the overground part, so that the plant is poisoned, the absorption of nutrient elements is affected, and the plant growth is inhibited. Cadmium also damages root structure, thereby affecting plant nutrient absorption.
Phosphorus is one of the most important essential nutrients for plants, and is a macronutrient essential for key metabolic processes such as plant cell division, energy generation, macromolecular biosynthesis, membrane integrity, signal transduction, photosynthesis, and the like. It also plays a role in respiration of plants and nitrogen fixation of leguminous crops. Although the soil contains a large amount of phosphorus compounds, the plant can actually utilize very little soluble phosphorus. Because most of the phosphorus in the soil exists in a poorly soluble form, and only the phosphate form of the phosphorus source is absorbed by the plant.
Microorganisms are closely related to plants, and microorganisms either positively or negatively affect the growth and development of plants. Under the influence of plant root system secretion, microorganisms in soil, plant rhizosphere and root system establish stable symbiotic relation, and some bacteria can infect plant root system and colonize inside the plant. Chinese patent No. 104974962A discloses a strain of Acinetobacter calcoaceticus which can effectively reduce the pH of the solution and dissolve insoluble phosphorus in the solution to promote the growth of tomato and eggplant plants. But the strain is not cadmium-resistant and cannot solve the technical problem that plants are stressed by cadmium poison.
Disclosure of Invention
The invention aims to provide a novel Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 which is symbiotic with plants and can solve the technical problems of low phosphorus stress and cadmium poison stress of the plants. The strain has the characteristics of secreting auxin, acid resistance, cadmium resistance, phosphorus dissolution and siderophore production. Inoculating the acinetobacter oleaginous to the soybean can improve the biomass (dry weight) of the soybean, remarkably improve the chlorophyll SPAD value and the phosphorus content, and promote the growth of soybean roots; but also can overcome the inhibition effect of cadmium on soybean growth, in particular to the inhibition effect of cadmium on soybean root growth.
The invention aims to provide a strain MRP20 of Acinetobacter oleaginous (Acinetobacter oleivorans).
Another object of the invention is to provide the use of Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 for the cultivation of cadmium-tolerant plants.
Another object of the invention is to provide the use of Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 for reducing the inhibition of cadmium in plant growth.
Another object of the invention is to provide the use of Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 for promoting the synthesis of chlorophyll from plant leaves.
Another object of the invention is to provide the use of Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 for promoting phosphorus uptake in plants.
Another object of the invention is the use of Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 for the preparation of growth promoters, cadmium-toxic microbial pesticides, chlorophyll biosynthesis promoters or phosphorus absorption promoters suitable for plants.
Another object of the invention is to provide the use of Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 for the solubilization of poorly soluble phosphorus.
Another object of the present invention is to provide a soybean planting method.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the applicant team obtains a strain with the characteristics of secreting auxin, acid resistance, cadmium resistance, phosphorus dissolution and siderophore production from the corn rhizosphere of about village in the North of Shaoguan city starter Jiang Ou through artificial separation and purification, can obviously improve soybean biomass, phosphorus content and leaf SPAD value, and enables the soybean to be cadmium resistant. Sequencing results of the strain were completed by Guangzhou Rui Bo biotechnology Co., ltd, and sequencing results of 16S rDNA thereof were subjected to Blast multiple sequence alignment analysis in NCBI database, thereby determining that the isolated strain was a novel strain of Acinetobacter oleaginous (Acinetobacter oleivorans), designated MRP20. The microorganism strain is preserved in Guangdong province at 5.11 of 2021 with the preservation number of GDMCC No:61653.
the morphological characteristics of strain MRP20 are as follows: MRP20 grows at an optimum temperature of 37 ℃, forms approximately round, smooth, protruding and milky colonies on LB plate medium, is rod-shaped under an optical microscope, and is dyed into red and gram-negative bacteria.
Research shows that the Acinetobacter oleaginous strain MRP20 remarkably promotes soybean to absorb phosphorus and improves the cadmium-resistant capability of soybean. The invention thus claims:
the application of the Acinetobacter oleaginous strain MRP20 in the aspect of cultivating cadmium-toxicity-resistant plants, in particular to the cultivation of large leguminous plants with cadmium toxicity resistance, and the inhibition effect of cadmium on root growth of the large leguminous plants is overcome.
The Acinetobacter oleaginous strain MRP20 is applied to weakening the cadmium to inhibit the growth of plants, in particular to the application to promoting the biomass improvement and root growth of large leguminous plants under the stress of cadmium poison or preparing products capable of weakening the cadmium to inhibit the growth of the plants.
Use of a strain MRP20 of acinetobacter oleaginous for promoting synthesis of chlorophyll from plant leaves, or for preparing a product capable of promoting synthesis of chlorophyll from plant leaves, in particular chlorophyll from leaves of large leguminous plants.
Use of a strain of a. Oleaginous MRP20 for promoting phosphorus uptake by plants, in particular by large leguminous plants, or for the preparation of a product capable of promoting phosphorus uptake by plants.
Use of Acinetobacter oleaginous strain MRP20 for dissolving poorly soluble phosphorus, wherein preferably the poorly soluble phosphorus is Ca 3 (PO 4 ) 2 。
The application of the Acinetobacter oleaginous strain MRP20 in preparing growth promoters, cadmium-toxin-resistant microbial pesticides, chlorophyll biosynthesis promoters or phosphorus absorption promoting bactericides applicable to plants.
Wherein, preferably, the plant is a large leguminous plant.
A method for planting soybeans, comprising the step of irrigating soybean seedlings with the bacterial suspension of the acinetobacter oleaphilus strain MRP20 according to claim 1;
particularly preferably, the treatment mode is irrigation;
more preferably, the method specifically comprises the following steps: after soybean seed is grown, the bacterial suspension of the Acinetobacter oleaginous strain MRP20 of claim 1 is irrigated in a cultivation medium, and then the seedlings are irrigated once in 1, 3 and 5 days after seedling transplanting; wherein preferably each time pouringThe irrigation amount is 80-120 mL/seedling; preferably, the bacterial suspension OD 600 0.6 to 1.0.
Wherein, as an alternative embodiment, the dispersion medium of the bacterial suspension is soybean nutrient solution, and the formula is as follows: 2.5mM KNO 3 ，2.5mM Ca(NO 3 ) 2 ·4H 2 O，0.08mM Fe-Na-EDTA，0.25mM K 2 SO4，1mM MgSO 4 ·7H 2 O，4.5×10 -3 mM MnCl 2 ·4H 2 O，0.3×10 -3 mM ZnSO 4 ·7H 2 O，0.16×10 -3 mM CuSO 4 ·5H 2 O，0.16×10 -3 mM(NH4) 6 Mo 7 O 24 ·4H 2 O，20×10 -3 mM H 3 BO 3 ，50×10 -3 mM KH 2 PO 4 。
The invention has the following beneficial effects:
the invention provides a cadmium-resistant growth-promoting Acinetobacter oleaginous strain MRP20 which is symbiotic with plants and can:
(1) Promoting plant growth, promoting plant to absorb phosphorus content, and increasing biomass and chlorophyll SPAD, and is especially suitable for large leguminous plants.
(2) Improving the cadmium-toxicity resistance of plants, overcoming the growth inhibition effect of cadmium on plants, in particular to the growth inhibition effect of cadmium on soybeans.
(3) The strain has the characteristics of secreting auxin, acid resistance, isolation resistance, phosphorus dissolution and siderophore production.
Drawings
FIG. 1 shows the phosphate solubilizing circle of Acinetobacter oleaginous strain MRP20.
FIG. 2 shows that Acinetobacter oleaginous strain MRP20 produces IAA at different tryptophan concentrations.
FIG. 3 shows a quantitative graph of IAA production by Acinetobacter oleaginous strain MRP20, in which letters a, b represent whether there is a significant difference between the average numbers of each sample after analysis of variance, the same letters between groups represent no significant difference between groups (P > 0.05), and the different letters represent significant differences between groups (P < 0.05).
FIG. 4 shows Acinetobacter oleaginous bacteriaCadmium tolerance curve (OD) of strain MRP20 600 Cadmium concentration), the letter combinations a and ab in the figure represent whether there is a significant difference between the average numbers of the samples after analysis of variance, the same letters between groups represent that the difference between groups is not significant (P>0.05 Inter-group letter differences represent significant inter-group differences (P)<0.05)。
FIG. 5 shows the acid resistance curve (OD) of Acinetobacter oleaginous strain MRP20 600 pH), the letter combinations a, ab and b in the figure represent whether there is a significant difference between the average of the samples after analysis of variance, the same letters between groups represent that the difference between groups is not significant (P>0.05 Inter-group letter differences represent significant inter-group differences (P)<0.05)。
FIG. 6 shows the developmental evolutionary tree of Acinetobacter oleaginous strain MRP20.
FIG. 7 shows the change in plant dry weight (A) and SPAD value (B) of soybeans inoculated with Acinetobacter oleaginous strain MRP20 under cadmium toxic stress.
FIG. 8 shows the changes in root length (A), root surface area (B), root diameter (C) and root volume (D) of soybeans inoculated with Acinetobacter oleaginous strain MRP20 under cadmium toxic stress.
FIG. 9 shows the upper cadmium concentration (A) and the root cadmium concentration (B) of soybeans inoculated with Acinetobacter oleaginous strain MRP20 under cadmium toxic stress.
FIG. 10 shows the variation of phosphorus content (A) in the aerial parts and phosphorus content (B) in the roots of soybeans inoculated with Acinetobacter oleaginous strain MRP20 under cadmium toxic stress.
In the figure, 0.01< P <0.05, 0.001< P <0.01, and P <0.001.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1: isolation and purification of strains
1. Isolation and purification of strains
Jiang Ou North of Shaoguan Massa MedicaAbout village, a strain was isolated from the corn rhizosphere of corn (normal sweet 68) planted on cadmium contaminated soil. 5g of corn roots are respectively sheared, the surface soil is brushed off by a small brush, sterile water is used for rinsing for a plurality of times until no soil is attached, and the corn roots are placed in a triangle bottle with the serial number of 250mL and containing 100mL of sterile water, and a shaking table at the temperature of 37 ℃ is used for shaking for 30min. Adding 10-20 glass beads into the conical flask to help break up soil, releasing bacteria from the soil, standing the soil suspension for 10min after vibration to obtain soil suspension, collecting supernatant, and diluting to 10 times of serial gradient concentration -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Then absorbing 0.1mL of diluent, coating on an LB plate, repeating each concentration gradient three times, culturing in an inverted mode at 37 ℃, observing colony growth conditions, picking up monoclonal antibodies with different phenotypes, numbering each colony on the back of a culture medium, recording colony morphology and colony growth time, picking up monoclonal antibodies of each type, shaking the monoclonal antibodies into a 1.5mL centrifuge tube, culturing at 180r/min and 37 ℃, purifying for 3-4 times by streaking through an LB solid culture medium (until the colony morphology is consistent under a microscope, indicating that the purification is finished), and adding sterilized glycerol with the concentration of 25% for later use at-80 ℃.
As a result, 55 strains were isolated from the rhizosphere of maize, and MRP1 to 55 were numbered, which were isolated and purified strains to be tested.
Example 2: strain screening
2.1 Primary screening of Phosphomorph
The screened strains are inoculated on PKO solid culture medium, each flat plate is inoculated three times, the culture is carried out in a 28 ℃ incubator, the outer diameter (D) of the phosphate-dissolving ring of the strain and the colony diameter (D) are measured in the 7D step, and the size of the phosphate-dissolving ring is photographed, so that the phosphate can be dissolved. And comparing the ratio (D/D) to judge the phosphorus dissolving capacity of the strain.
1mL of bacterial solution of the bacterial strain with the phosphate solubilizing ring is absorbed, inoculated into 20mL of LB liquid medium (50 mL of centrifuge tube), cultured for 24h, and OD is measured 600 About 1.0, 1mL of the bacterial heavy suspension is inoculated into 20mL of PKO liquid culture medium (50 mL of centrifuge tube), each bacterial strain is repeated three times, the CK control group is inoculated with equal amount of sterile water, and the mixture is placedCulturing at 28deg.C in 180r/min shaker. Measuring the pH value of PKO liquid culture medium at 7d, sucking 1mL of supernatant (1200R/min, centrifuging for 5 min) to a 25mL volumetric flask for measurement, sucking 5mL of molybdenum-antimony anti-chromogenic liquid, fixing the volume to 25mL with secondary water, measuring absorbance at 880nm after 30min of reaction (to remove the influence of the color of the culture medium, applying 880nm measurement, obtaining a solution of the pH value in a standard curve (y=0.4833x+0.0002 (R 2 =0.9997). The corresponding phosphorus concentration (mg/L) was obtained by conversion.
Results: the ratio of the phosphate solubilizing circles of strain MRP20 was found to be greater than 1.5 (FIG. 1 and Table 1). In the phosphorus-dissolving quantitative test, the strain MRP20 shows strong phosphorus-dissolving capacity and can dissolve 22.61+/-0.58 phosphorus.
TABLE 1
Strain numbering D(cm) d(cm) D/d Concentration of dissolved phosphorus (mg/mL)
MRP20 1.58 0.92 1.73 22.61±0.58
2.2 screening for IAA secretion
Preparing LB liquid culture media with L-tryptophan concentration of 0, 100, 200 and 500mg/L respectively, inoculating the screened strains into the liquid culture media, culturing each strain for 2d at 28 ℃ and 180r/min in three times (taking the same culture media without bacteria as blank control), taking 200 mu L of supernatant in each repetition, adding 200 mu L of Salkowski chromogenic liquid into a 96-hole ELISA plate, taking the same liquid culture media without bacteria and 200 mu L of Salkowski chromogenic liquid as control, and measuring the wavelength at 530nm by using an ELISA reader after placing the strains in dark place at room temperature for 20 min. The corresponding IAA yields were found on the standard curve. IAA yield unit is mg/mL.
Qualitative results showed (FIG. 2) that strain MRP20 had IAA-producing ability, but was weak. MRP20 showed that the release capacity of strain IAA was enhanced with increasing tryptophan concentration in the range of 0-500mg/L tryptophan concentration (FIG. 3).
2.3 screening for iron-producing Carrier Capacity
1mL of bacterial suspension of the strain to be detected is sucked and inoculated into MKB liquid culture medium. Culturing at 28℃and 180r/min for 48h. The culture broth was centrifuged for 10min (1200 r/min), 200. Mu.L of supernatant (200. Mu.L of unvaccinated MKB broth was added for measurement with reference (Ar)) was mixed with CAS detection solution at a ratio of 1:1. After 1h of reaction at normal temperature, the OD value (A) of the wavelength at 630nm is measured by an enzyme-labeled instrument. In the experiment, the CAS broth was blue as compared to the control if no siderophores were produced, and changed to orange if the strain produced siderophores. The ratio of A/Ar is used for representing the relative content of siderophores in a sample, the smaller the value is, the stronger the siderophores producing capacity of the strain is, and the ratio of (Ar-A)/Ar is used for representing the activity unit of siderophores in the sample, the higher the activity unit is, and the stronger the siderophores producing capacity is.
As a result, MRP20 strain was found to have the highest siderophore-producing activity unit among MRP1 to MRP 55 strains, reaching 58.30.+ -. 11.29%.
2.4 cadmium resistance screening
6 LB liquid culture media with cadmium concentration of 0, 4, 8, 12, 16 and 20mg/L are respectively prepared, and sterilized at 121 ℃ for 20 min. 2 96 Kong Miejun cell culture plates were prepared and 200. Mu.L of each of the prepared LB liquid media with different cadmium concentrations was aspirated into the plates. After the strains to be tested are activated and cultured for 24 hours, each strain absorbs 5 mu L of the strain into culture holes with different cadmium concentration values, each strain is cultured for 48 hours at 37 ℃ and 180r/min for 4 times, and the absorbance value of the bacterial liquid at 600nm is measured.
Results: at 0-20mg/L Cd concentration, MRP20 growth rate was almost unchanged and cadmium resistance was very strong (FIG. 4).
2.5 acid resistance screening
6 LB media with pH values of 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0 were prepared respectively, and sterilized at 121℃for 20 min. 2 96 Kong Miejun cell culture plates were prepared and 200. Mu.L of each of the prepared LB liquid media of different pH was aspirated into the plates. After the strains to be tested are activated and cultured for 24 hours, each strain is absorbed into 5 mu L of culture holes with different pH values, each strain is cultured for 48 hours at 37 ℃ and 180r/min for 4 times, and the absorbance value of the bacterial liquid at 600nm is measured.
The strain MRP20 grew most rapidly at a pH of 5.5-7.0 (FIG. 5), indicating that this strain was able to grow well under slightly acidic conditions.
Example 3 identification of strains
The strain MRP20 is identified through comprehensive consideration of various aspects.
3.1 morphological identification of strains
Strain MRP20 was inoculated on LB solid medium for cultivation and observation recording. After 5-7 days of culture under the optimal growth conditions (pH 7.0, temperature 37 ℃), the isolated and purified strain MRP20 is subjected to single colony state observation, which mainly comprises the size, color, colony surface state, colony edge state and the like of the colony. On the other hand, the strain MRP20 in the logarithmic growth phase was stained with a smear and the morphology of the cells was observed with an optical microscope.
On LB plate medium, it forms nearly round, smooth, protruding, milky white colony, rod-like under optical microscope, gram-staining red, gram-negative bacteria.
3.2 molecular characterization
The strain MRP20 was identified and studied continuously through extensive screening and comprehensive consideration. After inoculating the strain in LB culture medium for 24 hours at 37 ℃ and 180r/min, extracting total DNA of the strain by adopting a bacterial total DNA extraction kit, sequencing by a biological company, performing Blast sequence comparison analysis on the obtained strain sequence in NCBI database, and constructing a phylogenetic evolutionary tree by using MEGA7.0 (figure 6).
MRP20 is Acinetobacter oleaginous (Acinetobacter oleivorans) according to the sequencing result, and the 16S rDNA sequence of the MRP20 is shown as SEQ ID NO. 1; and the Acinetobacter oleaginous (Acinetobacter oleivorans) strain MRP20 was deposited with the Guangdong province microorganism strain collection at 2021, 5 and 11 days under the accession number GDMCC No:61653.
EXAMPLE 4 tieback soybean test of Acinetobacter oleaphilus MRP20
4.1 tieback test
The study used a substrate soil potting test. Experiment sets three cadmium concentration groups of 0, 10 and 20mg/kg CdCl respectively 2 ·5/2H 2 O, inoculated with acinetobacter oleaphilus MRP20 and each pot was a low phosphorus treatment, each treatment was repeated 4 times, as follows.
The mixed substrate treatment method of the substrate and the vermiculite comprises the following steps: weighing a non-fat substrate and vermiculite according to the following weight ratio of 3:1 (0.5 kg of mixed soil=0.125 kg of vermiculite+0.375 kg of matrix), sterilizing at 121 ℃ for 40min, repeating sterilizing once after 24h intervals, and standing for one week for later use. Taking a flowerpot with the capacity of 2L, soaking the flowerpot with 10% sodium hypochlorite overnight, fully flushing the flowerpot with clear water for a plurality of times, and air-drying the flowerpot for later use. Weighing quantitative cadmium chloride, dissolving in water, preparing 0, 10, 20mg/kg solution, weighing a certain volume, pouring into mixed matrix, and mixing 125mg Ca into each pot (0.5 kg) 3 (PO4) 2 (equivalent to 50mg/kg of pure phosphorus), each basin is weighed and mixed uniformly, and stands for a week for standby.
Seed was sterilized with hydrochloric acid-sodium hypochlorite to produce chlorine for 4h, with grit: middlings = 1:2, after seedling raising for one week, selecting soybean plants (Brazil 10) with basically consistent growth, and transplanting one plant in each pot. The bacterial suspension is irrigated 1, 3 and 5 days after seedling transplanting, 100mL at a time and 3 times in total.
Preparing a fungus suspension nutrient solution: the strain is inoculated in 250mL LB culture solution, shake-cultured for 18h at about 180r/min in a shaking table at 37 ℃, OD is measured between 0.6 and 1.0 when the bacterial liquid is turbid, the bacterial cells are collected centrifugally and resuspended in low-phosphorus nutrient solution, and the surface matrix of the root of the seedling is pulled out by a sterilizing gun head. And pouring the fungus resuspension nutrient solution into the root, and pouring the fungus along with the nutrient solution into the matrix.
Wherein the formula of the low-phosphorus nutrient solution is 2.5mM KNO 3 ，2.5mM Ca(NO 3 ) 2 ·4H 2 O，0.08mM Fe-Na-EDTA，0.25mM K 2 SO4，1mM MgSO 4 ·7H 2 O，4.5×10 -3 mM MnCl 2 ·4H 2 O，0.3×10 -3 mM ZnSO 4 ·7H 2 O，0.16×10 -3 mM CuSO 4 ·5H 2 O，0.16×10 -3 mM(NH4) 6 Mo 7 O 24 ·4H 2 O，20×10 -3 mM H 3 BO 3 ，50×10 -3 mM KH 2 PO 4 。
4.2 index detection
The soybeans were harvested after 30 days of cultivation. And under the condition of sufficient illumination, measuring the SPAD values of different parts of the plant with three reversed leaves by using a SPAD instrument, and taking an average value. Tap water is washed clean and then is put into a refrigerator, and scanning is performed in time within a week. Scanning the root system by a desk-top scanner (Epson 1460 XL), spreading the root system as much as possible, enabling the root system not to overlap, enabling the root system to be too large to be sheared and scanned, not influencing the scanning result, covering a blue light shielding plate, analyzing the root system characters of each sample root by a root system analysis software WinRHIZO (Regent Instruments Inc., canada) after the scanning is completed,
separating the overground part and the root part of the crop, weighing fresh weights of the overground part and the root part, placing the overground part into a 105 ℃ oven for de-enzyming for 30min, taking out the overground part, placing the overground part and the root part at room temperature for 10min for cooling, and weighing dry weight. The root is scanned by a root system scanner, and is weighed, and the rest parts are dried after fixation, and the dry weight is weighed.
Soaking soybean root in 10mmol/L Na 2 EDTA solution for 5min to remove surface adsorbed Cd 2+ Then washing with secondary water, drying root system and stem and leaf at 75deg.C, weighing dry weight, and measuring phosphorus content and cadmium concentration of aerial parts; and the root cold storage is used for sweeping roots, drying and weighing after sweeping, and measuring the phosphorus content and cadmium concentration of the roots. The determination of phosphorus adopts ultraviolet spectrophotometry, and the determination of cadmiumThe flame absorption method of atomic absorption spectrophotometry is adopted.
The results were as follows:
(1) As the cadmium concentration increases, the dry weight of the plant is obviously reduced, and compared with 0Cd, the biomass of the non-inoculated (CK group) plant is 10mg/kg Cd 2+ And 20mg/kg Cd 2+ Under the conditions (10 Cd and 20Cd groups), the dry weight of the soybean plants inoculated with the strain MRP20 is 10mg/kg Cd, and the dry weight of the soybean plants is reduced by 31 percent and 55 percent respectively 2+ And 20mg/kg Cd 2+ The reduction was 31% and 41%, respectively. At 20mg/kg Cd 2+ Under the conditions, the dry weight of the soybean plants inoculated with strain MRP20 was significantly higher than that of the soybeans without inoculation, by 34%. It was shown that seeding with MRP20 can alleviate cadmium poisoning (FIG. 7A).
(2) At 20mg/kg Cd 2+ Under the condition, the SPAD value of the soybean leaf subjected to MRP20 inoculation treatment is significantly higher than that of the soybean leaf subjected to CK non-inoculation treatment by 20%, which shows that the inoculation of MRP20 can promote the absorption of soybean nitrogen (figure 7B).
(3) At 0mg/kg Cd 2+ Under the condition, the total root length of the soybean inoculation treatment is obviously higher than that of the soybean non-inoculation treatment (CK group), and the root surface area is obviously higher than that of the soybean non-inoculation treatment (CK group), which shows that the inoculation of MRP20 can obviously promote the growth of soybean root systems (figure 8).
(4) The data shows that as cadmium concentration increases, the cadmium concentration in the aerial parts and roots of soybeans increases significantly. 0mg/kg Cd 2+ 、10mg/kg Cd 2+ And 20mg/kg Cd 2+ Under the condition, compared with CK without inoculation, the cadmium concentration of the aerial part of the soybean inoculated with Acinetobacter oleaginous MRP20 has no significant change (FIG. 9A), but is 20mg/kg Cd 2+ Under the conditions, the root cadmium concentration after inoculation is reduced by 60% compared with the CK without inoculation (fig. 9B).
(5) At 0mg/kg Cd 2+ Under the condition, compared with the CK without inoculation, the phosphorus content of the overground part of the soybean treated by inoculating Acinetobacter oleaginous MRP20 is improved by 145 percent. Shows that MRP20 has remarkable phosphate dissolving and growth promoting effects (figure 10).
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
SEQUENCE LISTING
<110> agricultural university of south China
<120> a strain of cadmium-resistant growth-promoting Acinetobacter oleaginous and application thereof
<130> YGZS214949
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1406
<212> DNA
<213> Acinetobacter oleivorans MRP 16S rDNA of 20
<400> 1
tgcagtcgag cggagagagg tagcttgcta ctgatcttag cggcggacgg gtgagtaatg 60
cttaggaatc tgcctattag tgggggacaa catttcgaaa ggaatgctaa taccgcatac 120
gtcctacggg agaaagcagg ggatcttcgg accttgcgct aatagatgag cctaagtcgg 180
attagctagt tggtggggta aaggcctacc aaggcgacga tctgtagcgg gtctgagagg 240
atgatccgcc acactgggac tgagacacgg cccagactcc tacgggaggc agcagtgggg 300
aatattggac aatgggcgga agcctgatcc agccatgccg cgtgtgtgaa gaaggcctta 360
tggttgtaaa gcactttaag cgaggaggag gctactttag ttaataccta gagatagtgg 420
acgttactcg cagaataagc accggctaac tctgtgccag cagccgcggt aatacagagg 480
gtgcaagcgt taatcggatt tactgggcgt aaagcgcgcg taggcggcta attaagtcaa 540
atgtgaaatc cccgagctta acttgggaat tgcattcgat actggttagc tagagtgtgg 600
gagaggatgg tagaattcca ggtgtagcgg tgaaatgcgt agagatctgg aggaataccg 660
atggcgaagg cagccatctg gcctaacact gacgctgagg tgcgaaagca tggggagcaa 720
acaggattag ataccctggt agtccatgcc gtaaacgatg tctactagcc gttggggcct 780
ttgaggcttt agtggcgcag ctaacgcgat aagtagaccg cctggggagt acggtcgcaa 840
gactaaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900
cgatgcaacg cgaagaacct tacctggcct tgacatagta agaactttcc agagatggat 960
tggtgccttc gggaacttac atacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa cccttttcct tatttgccag cgagtaatgt 1080
cgggaacttt aaggatactg ccagtgacaa actggaggaa ggcggggacg acgtcaagtc 1140
atcatggccc ttacggccag ggctacacac gtgctacaat ggtcggtaca aagggttgct 1200
acctagcgat aggatgctaa tctcaaaaag ccgatcgtag tccggattgg agtctgcaac 1260
tcgactccat gaagtcggaa tcgctagtaa tcgcggatca gaatgccgcg gtgaatacgt 1320
tcccgggcct tgtacacacc gcccgtcaca ccatgggagt ttgttgcacc agaagtagct 1380
agcctaactg caaagagggc ggtacc 1406

Claims (9)

1. Acinetobacter oleaginous strainAcinetobacter oleivorans) Strain MRP20, deposited with the collection of microorganisms and cell cultures, cantonese province, under accession number GDMCC No. 5/11 of 2021: 61653.
2. 16S rDNA of the acinetobacter oleaphilus strain MRP20 as claimed in claim 1, characterized in that the sequence is shown in SEQ ID No. 1.
3. Use of the acinetobacter oleaginous strain MRP20 of claim 1 for cultivating cadmium-toxic resistant soybeans.
4. Use of a strain of acinetobacter oleaginous MRP20 as claimed in claim 1 for reducing the growth of cadmium inhibited soybeans or for the preparation of a product capable of reducing the growth of cadmium inhibited soybeans.
5. Use of the acinetobacter oleaphilus strain MRP20 of claim 1 for promoting synthesis of chlorophyll from soybean leaves, or for preparing a product capable of promoting synthesis of chlorophyll from soybean leaves.
6. Use of the acinetobacter oleaphilus strain MRP20 of claim 1 for promoting phosphorus absorption by soybeans or for preparing a product capable of promoting phosphorus absorption by soybeans.
7. Use of the acinetobacter oleaginous strain MRP20 of claim 1 for the preparation of a growth promoter, a cadmium-toxic microbial pesticide, a chlorophyll biosynthesis promoter or a phosphorus absorption promoting microbial agent suitable for soybean.
8. Use of a strain of Acinetobacter oleaginous MRP20 according to claim 1 for dissolving poorly soluble phosphorus, or for the preparation of a product capable of dissolving poorly soluble phosphorus, said poorly soluble phosphorus being Ca 3 (PO 4 ) 2 。
9. A soybean planting method is characterized in that soybean seedlings are irrigated by using the bacterial suspension of the Acinetobacter oleaginous strain MRP20, wherein the amount of each irrigation is 80-120 mL/seedling; the OD600 of the bacterial suspension is 0.6-1.0.
CN202110606574.3A 2021-05-28 2021-05-28 Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof Active CN113403224B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110606574.3A CN113403224B (en) 2021-05-28 2021-05-28 Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110606574.3A CN113403224B (en) 2021-05-28 2021-05-28 Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof

Publications (2)

Publication Number Publication Date
CN113403224A CN113403224A (en) 2021-09-17
CN113403224B true CN113403224B (en) 2023-05-26

Family

ID=77675655

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110606574.3A Active CN113403224B (en) 2021-05-28 2021-05-28 Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof

Country Status (1)

Country Link
CN (1) CN113403224B (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102634465A (en) * 2011-12-27 2012-08-15 西北大学 Acinetobacter with authigenic nitrogen fixation capacity and application thereof
CN109136137A (en) * 2018-09-05 2019-01-04 广西大学 A kind of plant growth-promoting bacterial strain of preventing from heavy metal and its application

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201106759D0 (en) * 2011-04-20 2011-06-01 Exosect Ltd Compositions for growth and vigour in soybean
CN107846838A (en) * 2015-05-01 2018-03-27 靛蓝农业公司 Compound endophyte composition and method for the separation of improved plant trait

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102634465A (en) * 2011-12-27 2012-08-15 西北大学 Acinetobacter with authigenic nitrogen fixation capacity and application thereof
CN109136137A (en) * 2018-09-05 2019-01-04 广西大学 A kind of plant growth-promoting bacterial strain of preventing from heavy metal and its application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Heavy Metals Biosorption by Copper Resistant Bacteria of Acinetobacter sp. IrC2;Irawati, W等;《MICROBIOLOGY INDONESIA》;第19卷(第4期);第163-170页 *
耐镉促生菌株的分离鉴定及对大豆的促生效应;罗莎莉等;《微生物学通报》;第49卷(第8期);第3137-3149页 *

Also Published As

Publication number Publication date
CN113403224A (en) 2021-09-17

Similar Documents

Publication Publication Date Title
CN113234630B (en) Cadmium-resistant growth-promoting microbacterium strain and application thereof
CN106011005B (en) Bacillus amyloliquefaciens T600 and preparation method and application of microbial inoculum thereof
CN115820461B (en) High-yield indoleacetic acid strain JB0319 and application thereof
CN113755382A (en) Bacillus aryabhattai NDFY-1 and application thereof
CN109868242B (en) Salt-tolerant acetoin-producing bacillus subtilis and application thereof
CN115895960A (en) Strain for comprehensive planting and breeding of rice and fish and application thereof
CN110892805B (en) Application of biological stimulin for improving salt tolerance of corn seed germination
CN110257282A (en) A kind of widow oxygen unit cell nitrogen-fixing bacteria strain SI-NJAU-1 and its application
CN113699059B (en) Cadmium-resistant growth-promoting paenibacillus strain and application thereof
CN109735457B (en) Mutant eurotium cristatum and application thereof in preventing and controlling barnyard grass
CN113403224B (en) Cadmium-resistant growth-promoting Acinetobacter oleaginous strain and application thereof
CN114934000B (en) Salt-tolerant bacillus amyloliquefaciens and screening and application thereof
CN113549566B (en) Cadmium-resistant providencia retartii strain and application thereof
CN112458021B (en) Biocontrol pseudomonas fluorescens and culture method and application thereof
CN106011004B (en) Nitrogen-fixing microorganism G96, and preparation method and application of microbial inoculum thereof
CN110452821B (en) Rhizosphere fungus capable of promoting development of adventitious roots and secondary roots of nursery stocks and application thereof
CN111471608B (en) Strain capable of adsorbing manganese and promoting growth of plants and application thereof
CN114561324A (en) Tomato bacterial wilt antagonistic strain and application thereof in prevention and treatment of tomato bacterial wilt
CN116536188B (en) Mixed flora for promoting soybean growth and application thereof
CN116042439B (en) Rhizosphere actinomyces ASG and application thereof in aluminum-resistant tea tree growth promotion
CN114107117B (en) Rice endophyte and application thereof
CN114854640B (en) Pantoea ananatis and application thereof in growth promotion and stress resistance of tomatoes
CN114774311B (en) Flavobacterium and application thereof in promoting crop growth and improving crop cold resistance
CN114891658B (en) Common vetch root nodule strain HBUJ010033 and application thereof
CN114107123B (en) Sword fungus strain and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant