CN116536188A - Mixed flora for promoting soybean growth and application thereof - Google Patents
Mixed flora for promoting soybean growth and application thereof Download PDFInfo
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- CN116536188A CN116536188A CN202310305759.XA CN202310305759A CN116536188A CN 116536188 A CN116536188 A CN 116536188A CN 202310305759 A CN202310305759 A CN 202310305759A CN 116536188 A CN116536188 A CN 116536188A
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- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
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- OQTQHQORDRKHFW-UHFFFAOYSA-L manganese(2+);sulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O OQTQHQORDRKHFW-UHFFFAOYSA-L 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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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
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- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
- A01N63/22—Bacillus
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- 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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- 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
- C12R2001/07—Bacillus
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- 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
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Abstract
The invention discloses a microorganism mixed flora for promoting soybean growth and application thereof. The microbial mixed flora provided by the invention consists of a Bacillus aryabhattai (Priestia sp.) PLR1 strain and a Bacillus berryis (Bacillus sp.) PLR22 strain. According to the invention, two bacillus strains are separated from peanut rhizosphere: the PLR1 and PLR22 strains are studied, and the mixed flora of the PLR1 and PLR22 strains has a synergistic effect on soybean growth, has better effect than the growth promoting effect of a single strain, and can promote plant growth. After being further mixed with rhizobia, the mixed flora can obviously improve the biomass index of soybeans, promote the growth of the soybeans and improve the yield, and has stronger adaptability in the actual soil rhizosphere complex environment; provides more methods and extraction ways for improving the soybean yield in China and solving the development problem of soybean industry, and has important significance.
Description
Technical Field
The invention belongs to the technical field of microorganisms. More particularly, to a mixed flora for promoting soybean growth and application thereof.
Background
The soybean is a leguminous crop with biological nitrogen fixation capability, is an excellent crop rotation and alternate cropping and intercropping crop, and has obvious ecological effects of soil fertility, soil cultivation, water and soil loss reduction, soil multiple cropping index increase and the like.
Symbiosis with rhizosphere beneficial microorganisms is considered as an important way to increase crop yield and develop green agriculture. The nutrient efficiency is improved by inoculating beneficial microorganisms, so that the soybean yield is improved, meanwhile, the necessary way of developing environment-friendly sustainable ecological agriculture is realized, and the ecological and economic significance is realized for agricultural weight-losing synergy and domestic soybean industry development. The effect of single strain on plants in most of the existing researches, but single inoculation can lack advantages in resource competition, so that functional bacteria are eliminated. With the intensive research of symbiotic interaction of beneficial rhizosphere microorganisms and plants, various mixed flora used in plant growth research has also appeared, such as the adoption of Pseudomonas sp H1 and the fermentation broth of bacillus altitudis Bacillus altitudinis Y1 for mixing can promote the growth of soybean plants and increase the population abundance of probiotic microorganisms in soil; bacillus megaterium Bacillus megaterium HZP and Rhizobium were used: the rhizobium astragalosis (Rhizobium astragali) 7653R or the sinorhizobium freudenreichii (Sinorhizobium fredii) strain HH103 is matched for use in soil, so that the growth of leguminous crops is well promoted, the plant growth of the leguminous crops under the condition of limiting nitrogen, phosphorus and other nutrients can be improved, and the crop yield is improved.
It can be seen that the inoculation of beneficial microorganisms has been widely used to promote plant growth and increase soybean yield, whereas mixed flora has advantages over inoculation alone. There is still a lack of mixed flora available to promote plant growth and increase soybean yield. Therefore, in order to improve the soybean yield in China and solve the problem of soybean industry development in China, more mixed flora which is symbiotic with soybean interaction is researched and screened from the nature, and the method has important significance.
Disclosure of Invention
The invention provides a microbial mixed flora for promoting soybean growth, which is used for promoting plant growth and improving soybean yield, and realizing the interaction symbiosis of multiple strains and plants.
It is a first object of the present invention to provide a mixed microbial community that promotes soybean growth.
It is a second object of the present invention to provide the use of a mixed population of microorganisms.
The third object of the invention is to provide a composite microbial agent for promoting soybean growth and/or improving soybean yield.
It is a third object of the present invention to provide a method for promoting soybean growth and increasing soybean yield.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a microorganism mixed flora for promoting soybean growth, which consists of a Bacillus aryabhattai (Priesiasp.) PLR1 strain and a Bacillus subtilis (Bacillus sp.) PLR22 strain. The bacillus aryasis PLR1 strain is preserved in the microorganism strain collection of Guangdong province at the 12 th month of 2022 and the preservation number is GDMCC No:63090; the bacillus belgium PLR22 strain was stored in the Guangdong province microorganism strain collection at 26 days of 12 months of 2022, and the storage number is GDMCC No:63091. according to the invention, two bacillus PLR1 and PLR22 strains are obtained from peanut rhizosphere of the Shaoguan market starter Jiang Ouhuang She Wu village through artificial separation and purification, and the two strains have the characteristics of nitrogen fixation, phosphorus dissolution and siderophore production. The research of the invention shows that compared with a control group, under the condition that the usage amount and the concentration are consistent, the effect of the mixed flora is obviously superior to that of single bacteria, and better effect can be achieved by adopting less usage amount; the mixed flora formed by the bacillus aryabhattai PLR1 strain and the bacillus berryis PLR22 strain has a synergistic effect on soybean growth, is superior to the growth promoting effect of a single strain, and has stronger adaptability in actual soil environment.
Preferably, the volume ratio of the bacillus aryabhattai PLR1 strain to the bacillus behenii PLR22 strain is 1:1.
Since a large number of bacillus and rhizobia bacteria are co-propagated in the leguminous plant rhizosphere microecological system, they form a symbiotic relationship with leguminous plants during long-term evolution. Therefore, the invention further researches that the mixed inoculation of the slow rooting tumor bacteria (Bradyrhizobium sp.) on the soybeans can improve the action effect of the mixed bacteria of the bacillus aryabhattai and the bacillus bailii, improve the biomass index of the soybeans, and show that the mixed bacteria of the bacillus aryabhattai, the bacillus bailii and the slow rooting tumor bacteria can better interact with the soybeans, promote the growth and improve the yield.
Further, the mixed flora also contains Bradyrhizobium sp.
More preferably, the Bradyrhizobium sp is Bradyrhizobium BDYD1, which has been deposited at the university of armed chinese culture collection at month 28 of 2007 under the accession number cctccc No. M207121.
Particularly, as the interaction research of bacillus and rhizobia is carried out, the slow rhizobia BDYD1 adopted by the invention can promote nodulation and fix nitrogen, and is a result of early development of the subject group of the inventor (Chinese patent application number CN 200710032271.5); meanwhile, the invention also adopts a slow rhizobium (Bradyrhizobium sp.) strain BXYD3 for verification (Chinese patent application number: CN 200710032269.8), the rhizobium BXYD3 has been preserved in the China center for type culture collection of Wuhan university, 7 months and 28 th in 2007, and the preservation number is CCTCC NO: M207123. The BXYD3 and the mixed flora can be used for further improving the action effect of the mixed flora, and the consistency is achieved.
More preferably, the volume ratio of the Bacillus aryabhattai PLR1 strain, the Bacillus bailii PLR22 strain and the Rhizobium chromene is 1:1:2.
Thus, the following applications of the mixed flora are within the scope of the present invention:
use of mixed flora in promoting soybean growth or in the preparation of a formulation for promoting soybean growth.
Use of mixed flora for increasing soybean yield or for preparing a formulation for increasing soybean yield.
The invention also provides a composite microbial inoculum for promoting the growth of soybeans and/or improving the yield of the soybeans, which comprises the mixed microbial flora: bacillus aryabhattai PLR1 strain and Bacillus bailii PLR22 strain, or Bacillus aryabhattai PLR1 strain, bacillus bailii PLR22 strain and Rhizobium chronicum.
Preferably, the composite microbial inoculum is formed by mixing bacterial liquid of a bacillus aryabhattai PLR1 strain and bacterial liquid of a bacillus berryis PLR22 strain, wherein the volume ratio is 1:1, or is formed by mixing bacterial liquid of a bacillus aryabhattai PLR1 strain, bacterial liquid of a bacillus berryis PLR22 strain and bacterial liquid of a Rhizobium chronicum, and the volume ratio is 1:1:2.
Preferably, the total concentration of the composite microbial agent is not less than 2×10 9 CFU/mL。
The composite microbial inoculum is prepared by inoculating Bacillus aryabhattai (Priestia aryabhattai) PLR1 strain and Bacillus bailii (Bacillus velezensis) PLR22 strain into LB culture solution, and culturing to bacterial solution OD 600 The bacterial cells are collected by centrifugation and resuspended by low-phosphorus low-nitrogen nutrient solution, and the bacterial liquid of the two resuspended bacterial strains is prepared into mixed bacterial agent according to the volume ratio of 1:1, and the total concentration of the bacterial agent is not lower than 2 multiplied by 10 9 CFU/mL；
Or inoculating Bacillus aryabhattai (Priestia aryabhattai) PLR1 strain and Bacillus bailii (Bacillus velezensis) PLR22 strain and Brevibacterium sp BDYD1 strain respectively to LB culture solution, and culturing to bacterial solution OD 600 About 1.0, and centrifugally collecting thalliAnd re-suspending with low-phosphorus low-nitrogen nutrient solution, and preparing into mixed microbial inoculum according to the volume ratio of 1:1:2, wherein the total concentration of the microbial inoculum is not lower than 2 multiplied by 10 9 CFU/mL。
The invention also provides a method for promoting soybean growth and improving soybean yield, which adopts the composite microbial inoculum to treat soybean plants.
The invention has the following beneficial effects:
the invention provides a microorganism mixed flora for promoting soybean growth, which consists of a bacillus aryabhattai (Priestia aryabhattai) PLR1 strain and a bacillus berryis (Bacillus velezensis) PLR22 strain. According to the invention, two bacillus strains are separated from peanut rhizosphere: the research shows that the mixed flora consisting of the PLR1 strain and the PLR22 strain has synergistic effect on soybean growth, the effect is better than the growth promoting effect of a single strain, and the plant growth can be promoted. Further mixing with slow rooting tumor (Bradyrhizobium sp.) can promote the action effect of the mixed flora of the Bacillus aryabhattai PLR1 strain and the Bacillus berensis PLR22 strain, and shows that the mixed flora of the Bacillus aryabhattai, the Bacillus berensis and the slow rooting tumor can better perform symbiotic interaction with soybean, promote growth and improve yield. The mixed flora and the composite microbial inoculum thereof provided by the invention can obviously improve the biomass index of soybeans, promote the growth of the soybeans and improve the yield, have stronger adaptability in the actual soil rhizosphere complex environment, provide more methods and extraction ways for improving the soybean yield in China and solving the problem of soybean industry development in China, and have important significance.
Drawings
FIG. 1 is a graph showing the results of nitrogen fixation tests of PLR1 and PLR22 strains.
FIG. 2 shows the developmental trees of PLR1 (A) and PLR22 (B) strains.
FIG. 3 is a graph showing the results of changes in leaf SPAD values and plant dry weight after inoculation with PLR1 and/or PLR22 strains.
FIG. 4 is a graph showing the results of changes in flowering phase soybean plant growth phenotype (A), leaf SPAD value (B), plant height (C), plant biomass (D), plant nitrogen content (E), and plant phosphorus content (F) in different mixed bacteria treatment groups.
FIG. 5 shows the variation of root volume (A), root surface area (B), and average root diameter (C) of flowering soybean plants with different mixed bacterial treatment groups.
FIG. 6 shows the variation of the pod stage soybean plant growth phenotype (A), plant biomass (B) and yield (C) in different treatment groups.
In the figure: different lowercase letters indicate significant differences between different inoculation treatments (P < 0.05).
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.
The formula of the low-nitrogen low-phosphorus nutrient solution adopted in the following examples is as follows: 1.0mM KNO 3 ，0.08mM Fe-Na-EDTA，0.25mM K 2 SO 4 ，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(NH 4 ) 6 Mo 7 O 24 ·4H 2 O，20×10 -3 mM H 3 BO 3 ，5×10 -2 mM KH 2 PO 4 。
The medium formulation used in the following examples was as follows:
LB solid medium: 5.0g of yeast extract, 10g of peptone, 10g of sodium chloride, 15g of agar, fixing the volume to 1L, adjusting the pH to 7.0, and sterilizing at 121 ℃ for 20min.
Nitrogen-free solid medium: 10g of glucose, 0.2g of dipotassium hydrogen phosphate, 0.2g of sodium chloride, 0.2g of magnesium sulfate, 0.2g of potassium sulfate, 5g of calcium carbonate and 0.5g of yeast, fixing the volume to 1L, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min.
Inorganic phosphorus medium (PKO): 10g of glucose, 0.5g of ammonium sulfate, 0.3g of potassium chloride, 0.3g of manganese sulfate heptahydrate, 5g of tricalcium phosphate, 30mg of ferrous sulfate heptahydrate, 30mg of manganese sulfate tetrahydrate, fixing the volume to 1L, adjusting the pH to 7.0, and sterilizing at 121 ℃ for 20min.
MKB liquid medium: 5g of casein amino acid, 2.5g of potassium dihydrogen phosphate, 0.2g of magnesium sulfate heptahydrate, 15mL of glycerol, constant volume to 1L, adjusting pH to 7.0, and sterilizing at 121 ℃ for 20min.
Example 1 isolation and functional analysis of strains
1. Isolation and purification of strains
The sample of this example was derived from the rhizosphere of the Shaoguan market starter Jiang Ouhuang She Wu village planted peanut. 5g peanut root is cut, the surface soil is brushed off by a small brush, the surface soil is rinsed for a plurality of times by sterile water until no soil is attached, and the surface soil is placed in a triangle bottle with the serial number of 250mL and containing 100mL of sterile water, a shaking table at the temperature of 37 ℃ for 30min, and a shaking table is arranged. 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 ℃.
Through the separation, 8 different strains are screened from peanut rhizosphere separation, preliminary screening of a nitrogen fixation qualitative test is further carried out, the nitrogen fixation effect of two strains is better than that of other strains, the strains are named PLR1 strain and PLR22 strain respectively, and the two strains are used for the following study.
2. Characterization of strains
(1) Bacterial strain nitrogen fixation qualitative test: PLR1 and PLR22 strain spot plates are respectively inoculated on nitrogen-free solid culture medium, the constant temperature culture is carried out at 28 ℃, bacterial colonies are observed and photographed in 2-3d, and the strains can grow on the nitrogen-free culture medium, so that the strains have nitrogen fixing capability.
The results are shown in FIG. 1, and show that both PLR1 strain and PLR22 strain have strong nitrogen fixation capacity.
(2) Quantitative determination test of dissolved phosphorus: 1mL of the bacterial heavy suspension is absorbed and inoculated into a 50mL centrifuge tube with 20mL of PKO liquid culture medium, each bacterial strain is repeated three times, the CK control group is inoculated with equal amount of sterile water, and the inoculated bacterial heavy suspension is placed on a shaking table at 28 ℃ for shaking culture at 180r/min for 7 days. 1200r/min, centrifuging for 5min, sucking 1mL supernatant in a 25mL volumetric flask, adding two drops of dinitrophenol indicator, regulating the solution to be yellowish with 2M NaOH, adding 5mL molybdenum-antimony anti-chromogenic solution, fixing the volume to 25mL with secondary water, reacting for 30min, measuring the absorbance value at 880 by using an enzyme-labeling instrument, and obtaining the corresponding phosphorus concentration (mg/L) through a standard curve.
The results are shown in Table 1 below, which show that PLR1 and PLR22 exhibit strong phosphorus dissolving ability, and are soluble to 33.04.+ -. 5.50 and 47.98.+ -. 4.21mg/L of phosphorus, respectively.
(3) Iron production carrier capacity assay: 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.
The results are shown in Table 1 below, which shows that PLR1 and PLR22 strains have a strong siderophore production capacity up to 26.42.+ -. 0.18 and 24.00.+ -. 1.10, respectively.
TABLE 1 quantitative determination of dissolved phosphorus test results
Example 2 identification of strains
The PLR1 and PLR22 are respectively inoculated on LB solid medium for culture, and researches show that the optimal growth conditions of the strain are as follows: after culturing at 37℃for 5-7 days at pH 7.0, the isolated and purified strains PLR1 and PLR22 were observed for single colony status, mainly including colony size, color, colony surface status, colony edge status, and the like. And the bacterial strain in the logarithmic growth phase is stained by smear and then the form of the bacterial strain is observed by an optical microscope.
The results showed that PLR1 formed nearly round, smooth, protruding, pale yellow colonies on LB plate medium, short bars under light microscope, gram staining as purple, gram positive bacteria.
PLR22 forms a nearly circular shape on LB plate medium, the surface of the colony is rugged, the colony is white, the colony is short rod-shaped under an optical microscope, and gram staining is purple, gram positive bacteria.
And inoculating PLR1 and PLR22 strains into an LB culture medium, culturing at 37 ℃ for 24 hours at 180r/min, extracting total DNA of the strains by adopting a bacterial total DNA extraction kit, sequencing by a biological company, performing Blast sequence comparison analysis on the obtained strain sequences in an NCBI database, and constructing a phylogenetic evolutionary tree by using MEGA 7.0.
As a result, as shown in FIG. 2, the highest 16S rRNA gene homology with PLR1 strain in the database was Priestia aryabhattai, and the similarity was 100%. The highest 16S rRNA gene homology with PLR22 strain was Bacillus velezensis with 100% similarity. The PLR1 strain and the PLR22 strain isolated in this example were confirmed to belong to the strains of Bacillus aryabhattai (Priestia aryabhattai) and Bacillus bailii (Bacillus velezensis) in the genus Bacillus (Bacillus sp.) by the above morphological identification and molecular biological identification results of bacteria.
Thus, PLR1 strain was assigned to Bacillus aryabhattai (Priestia aryabhattai) PLR1 strain, the taxonomic name was Priestia sp., PLR22 strain was assigned to Bacillus bailii (Bacillus velezensis) PLR22 strain, the taxonomic name was Bacillus sp., both strains were stored in the Cantonese microorganism collection at 12 months of 2022, and the storage numbers were GDMCC No:63090 and GDMCC No:63091, deposit address: guangzhou city first middle road No. 100 college No. 59 building 5.
EXAMPLE 3 soybean tieback test of Bacillus
The soybean variety adopted in the study is Yue Chun 03-3, and the sand culture test is designed into a single-factor test, which comprises four inoculation treatments: no inoculation (NM), only inoculation of bacillus aryabhattai PLR1, only inoculation of bacillus beryabhattai PLR22, mixed inoculation of bacillus aryabhattai PLR1 and bacillus beryabhattai PLR22, wherein 4 biological replicates were set for each treatment, and low-phosphorus and low-nitrogen (50 μ M P and 1000 μ M N) treatments were uniformly performed during the planting process. The specific test steps are as follows:
(1) Sterilizing 1.5kg flowerpot (with bottom bracket) with 10% sodium hypochlorite overnight for use;
(2) Selecting full and smooth Yue Chun 03-3 200 grains, sterilizing in a cabinet dryer for 30min by adding 4.2mL hydrochloric acid into 100mL sodium hypochlorite before sowing, opening a super clean workbench after sterilization for 30min, and sealing with a preservative film for later use;
(3) Test sand preparation: sterilizing the weighed sand at 121 ℃ for 40min, taking out, standing for 24h, and sterilizing for the second time. Then standing for a week for standby;
(4) Weighing 375mg of poorly soluble Ca 3 (PO 4 ) 2 (equivalent to 50mg/kg of pure phosphorus) and each basin of sand are fully and uniformly mixed, and seedlings are planted;
(5) Respectively inoculating PLR1 and PLR22 strains into culture bottle containing 250mL LB culture solution, shake culturing at 37deg.C in shaker at 180r/min for about 18 hr, and measuring OD when the bacterial solution is turbid 600 About 1.0, centrifugally collecting thalli, and re-suspending with low-phosphorus low-nitrogen nutrient solution to prepareThe concentration is 2X 10 9 CFU/mL PLR1 and PLR22 single bacterial liquid;
respectively preparing PLR1 and PLR22 bacterial liquid re-suspended by adopting low-phosphorus low-nitrogen nutrient solution according to the volume ratio of 1:1 to obtain the total concentration of 2 multiplied by 10 9 CFU/mL mixed inoculation bacterial liquid;
(6) After soybean seedlings emerge for 7 days, sand on the root surfaces of the seedlings is pulled out by a sterilizing gun head, and inoculating liquid is inoculated into the root of the soybean. Inoculating 5mL of single bacterial liquid or mixed inoculation bacterial liquid to each seedling, and inoculating three times;
(7) The soybeans are randomly placed in a greenhouse. During planting, 150mL of nutrient solution is poured every week, and the basin is moved 2-3 times every week, so that the illumination is uniform. Soybeans were harvested after 30 days of growth, and the biomass of the aerial parts and roots was measured.
The test results are shown in FIG. 3, and the growth of the soybean treated by the mixed inoculation of PLR1 and PLR22 in the seedling stage is obviously better than that of the soybean treated by the CK non-inoculation treatment and the soybean treated by the single inoculation of PLR1 and PLR22, and the results are shown in FIG. 3A.
The inoculation treatment significantly affects the SPAD value of the soybean leaves, and compared with the CK non-inoculation treatment, the SPAD value of the soybean leaves has a trend of increasing, and the SPAD value of the soybean leaves subjected to PLR1+PLR22 mixed inoculation treatment is increased by 20%. Compared with PLR1 single inoculation treatment, the SPAD value of soybean leaves subjected to PLR1+PLR22 mixed inoculation treatment is increased by 22%, and the result is shown in FIG. 3B.
The inoculation treatment significantly affected the biomass of the aerial parts of the soybean plants, and the biomass of the aerial parts of the soybean plants treated by single inoculation of PLR1 and PLR22 tended to increase compared with the CK non-inoculation treatment, and the biomass of the aerial parts of the soybean plants treated by mixed inoculation of PLR1 and PLR22 increased by 27%, and the result is shown in FIG. 3C.
In conclusion, compared with a control group, the effect of the mixed flora is obviously better than that of single bacteria under the condition that the use amount and the concentration are consistent, and better effect can be achieved by adopting less use amount; the mixed flora consisting of the bacillus aryabhattai PLR1 and the bacillus berryis PLR22 has a synergistic effect on the growth of soybeans, has a better effect than the growth promoting effect of a single strain, and can promote the growth of plants.
EXAMPLE 4 Mixed population inoculation experiment
Since a large number of bacillus and rhizobia bacteria are co-propagated in the leguminous plant rhizosphere microecological system, they form a symbiotic relationship with leguminous plants during long-term evolution. Thus, the present invention further investigated the relationship between Bradyrhizobium sp. With mixed flora and soybean plants. The test materials and the culture method adopted in this example are the same as those in example 3, the test is designed as a single-factor experiment, and three inoculation treatments are set: two mixed bacterial groups (+b) without inoculation (CK), with inoculation of PLR1 and PLR22, three mixed bacterial groups (+b+r) with inoculation of PLR1 and PLR22 and slow rooting tumor bacteria BDYD 1. Among them, the bradyrhizobium BDYD1 can promote nodulation and fix nitrogen, and is the result of earlier development of the subject group of the inventor (invention patent application number CN 200710032271.5); each treatment was run in 8 biological replicates and harvested in duplicate. The low-phosphorus and low-nitrogen (50 mu M P and 1000 mu M N) treatment is uniformly carried out in the planting process. And collecting samples about 40 days. The specific test steps are as follows:
(1) 1.0kg of the pot (with the bottom) was sterilized overnight with 10% sodium hypochlorite for use.
(2) Selecting 500 seeds of full and smooth Yue Chun 03-3 soybean seeds, sterilizing in a cabinet dryer for 30min by adding 4.2mL hydrochloric acid into 100mL sodium hypochlorite before sowing, opening and blowing for 2h on an ultra-clean workbench after sterilization, and sealing with a preservative film for later use.
(3) Test sand preparation: sterilizing the weighed sand at 121 ℃ for 40min, taking out, standing for 24h, and sterilizing for the second time. Then the seedlings are placed for one week, and seedlings are planted after one week.
(4) Respectively inoculating PLR1, PLR22 and BDYD1 strains into a culture bottle filled with 250mL LB culture solution, shake culturing at 180r/min in a shaking table at 37deg.C for 18 hr, and measuring OD when the bacterial solution is turbid 600 And (2) obtaining 2×10 bacteria by centrifuging and collecting the bacteria, and re-suspending with low-phosphorus low-nitrogen nutrient solution 9 CFU/mL of single bacterial solutions of PLR1, PLR22 and BDYD 1;
respectively preparing PLR1 and PLR22 bacterial liquid re-suspended by adopting low-phosphorus low-nitrogen nutrient solution according to the volume ratio of 1:1 to obtain the total concentration of 2 multiplied by 10 9 Mixed bacterial solution (+ B) of two strains of CFU/mL;
respectively preparing PLR1, PLR22 and BDYD1 bacterial liquid resuspended by adopting the low-phosphorus low-nitrogen nutrient solution according to the volume ratio of 1:1:2, ensuring the total concentration to be unchanged, and obtaining the concentration of 2 multiplied by 10 9 Mixed bacterial solutions of three strains of CFU/mL (+B+R).
(5) After soybean seedlings emerge for 7 days, sand on the root surfaces of the seedlings is pulled out by a sterilizing gun head, and inoculating liquid is inoculated into the root of the soybean. Each seedling is inoculated with 10mL of single bacterial liquid or mixed bacterial liquid for three times.
(6) The soybeans are randomly placed in a greenhouse. During planting, 150mL of nutrient solution is poured every week, and the basin is moved 2-3 times every week, so that the illumination is uniform. The soybean grows to the flowering period for the first time, and after photographing, the SPAD, the biomass of the overground parts and roots, the nitrogen and phosphorus content and the root system property are measured; the second harvest was performed during the pod stage, and the biomass of the aerial parts and roots, as well as the yield, was measured after photographing.
The sample collection and index measurement method comprises the following steps: the soybeans are grown to harvest in the flowering period. 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. Separating the aerial parts and the root parts of the soybeans, placing the aerial parts into a 105 ℃ oven for enzyme deactivation for 30min, taking out the aerial parts, placing the aerial parts 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.
After harvesting, the roots were rinsed clean with clear water and scanned with a desktop scanner (Epson 1460 XL). Spreading root system in a scanning basin, adding water, spreading the root system without overlapping, scanning, and analyzing the root system characteristics including root volume, root surface area and average root diameter by using a root system analysis software WinRHIZO after scanning.
And (5) drying and weighing the overground parts and the roots of the plants, and grinding the samples. The samples of the overground parts and the root parts are more ground by a gossamling machine, and the samples are less ground by a mortar, so that the loss of the samples in the sample grinding process is reduced. Grinding all samples, and using H 2 SO 4 -H 2 O 2 The digestion liquid is diluted by 4 times, and the nitrogen and phosphorus concentration of the diluted digestion liquid is measured on a flow analyzer.
After the nitrogen and phosphorus concentration is measured and converted, the nitrogen and phosphorus content of the plant is calculated by the following calculation formula:
nitrogen content (mg/plant) =nitrogen concentration of a part of plant (mg/g) ×dry weight (g/plant)
Phosphorus content (mg/plant) =phosphorus concentration (mg/g) of a part of plant x dry weight (g/plant)
The test results show that: during the flowering period, soybean growth was superior to CK-not-inoculated with both PLR1 and PLR22 mixed strains (+b) and PLR1, PLR22, BDYD1 mixed strain inoculated with (+b+r), as shown in fig. 4A.
The inoculation treatment significantly affected the SPAD value of the flowering phase soybean leaves, which increased 45% compared to the CK-not inoculation treatment with +b+r inoculation treatment. The +b+r inoculated soybean leaf SPAD values were increased by 32% compared to +b inoculated treatments, as shown in fig. 4B.
As shown in fig. 4C, the inoculation treatment significantly affected the flowering soybean plant height, and the +b+r inoculation treated soybean plant height increased by 44% compared to CK non-inoculation treatment. The soybean plant height of +b+r inoculation treatment was increased by 38% compared to +b inoculation treatment.
The inoculation treatment significantly affected the flowering soybean plant biomass, with a 25% increase in +b inoculated soybean plant biomass and a 113% increase in +b+r inoculated soybean plant biomass compared to CK non-inoculation treatment. The soybean plant biomass of the +b+r inoculation treatment was increased by 70% compared to +b inoculation treatment, as shown in fig. 4D.
The inoculation treatment significantly affected the nitrogen content of the flowering phase soybean plants, with a 27% increase in nitrogen content of the +b inoculated soybean plants and a 267% increase in nitrogen content of the +b+r inoculated soybean plants compared to the CK non-inoculation treatment. The nitrogen content of the +b+r inoculated soybean plants was increased by 189% as compared to +b inoculated treatments, as shown in fig. 4E.
The inoculation treatment significantly affected the phosphorus content of the flowering soybean plants, with the phosphorus content of the +B inoculation treated soybean plants increased by 57% and the phosphorus content of the +B+R inoculation treated soybean plants increased by 123% compared to the CK non-inoculation treatment. The phosphorus content of the +b+r inoculated soybean plants was increased by 42% as compared to +b inoculated soybean plants, as shown in fig. 4F.
The inoculation treatment significantly affected root system traits of flowering soybean plants, as shown in fig. 5, the +b inoculation treated soybean root volume and average root diameter increased by 31% and 8%, respectively, and the +b+r inoculation treated soybean root volume, root surface area and average root diameter increased by 90%, 54% and 29%, respectively, as compared to CK non-inoculation treatment. The soybean root volume, root surface area and average root diameter of the +b+r seed treatment were increased by 46%, 30% and 19%, respectively, as compared to +b seed treatment.
The pod stage +B inoculation treatment (PLR 1 and PLR22 mixed strain) and +B+R inoculation treatment (PLR 1, PLR22, BDYD1 mixed strain) were better than the CK non-inoculation treatment, as shown in FIG. 6A.
The inoculation treatment significantly affected the pod stage soybean plant biomass, with 86% increase in +b inoculated soybean plant biomass and 122% increase in +b+r inoculated soybean plant biomass compared to CK non-inoculation treatment. Soybean plant biomass was increased by 20% in the +b+r inoculation treatment compared to +b inoculation treatment, as shown in fig. 6B.
The inoculation treatment significantly affected the pod stage soybean yield, with approximately 3-fold increase in biomass for +b inoculated soybean plants and approximately 14-fold increase in biomass for +b+r inoculated soybean plants compared to CK non-inoculation treatment. The biomass of soybean plants inoculated with +b+r was increased approximately 5-fold compared to +b inoculation treatment, as shown in fig. 6C.
In conclusion, research shows that the mixed inoculation of the slow rhizobium sp on the soybeans can improve the action effect of mixed bacteria of the bacillus aryabhattai PLR1 and the bacillus berryis PLR22, improve the biomass index of the soybeans, and show that the mixed bacteria of the bacillus aryabhattai, the bacillus berryis and the slow rhizobium can better interact with the soybean symbiotic, promote the growth and improve the yield.
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.
Claims (10)
1. A mixed microbial flora for promoting soybean growth, comprising a strain PLR1 of Bacillus arvensis (Priestia sp.) and a strain PLR22 of Bacillus berliner (Bacillus sp.); the bacillus aryasis PLR1 strain is preserved in the microorganism strain collection of Guangdong province at the 12 th month of 2022 and the preservation number is GDMCC No:63090; the bacillus belgium PLR22 strain was stored in the Guangdong province microorganism strain collection at 26 days of 12 months of 2022, and the storage number is GDMCC No:63091.
2. the mixed population of claim 1, wherein the volume ratio of bacillus aryabhattai PLR1 strain to bacillus bernatis PLR22 strain is 1:1.
3. The mixed population of claim 1, further comprising Bradyrhizobium sp.
4. The mixed bacterium according to claim 3, wherein said Bradyrhizobium sp.) is Bradyrhizobium BDYD1, which has been deposited at the university of armed forces chinese culture collection at 7.28 at 2007 under the accession number cctccc No. M207121; or BXYD3 strain of Bradyrhizobium sp, which has been deposited at China center for type culture Collection, university of Wuhan, at 7.28 of 2007, with a deposit number of CCTCC NO: M207123.
5. Use of the mixed flora according to any of claims 1-4 for promoting soybean growth or for the preparation of a formulation for promoting soybean growth.
6. Use of a mixed population according to any one of claims 1 to 4 for increasing soybean yield or for the preparation of a formulation for increasing soybean yield.
7. A composite microbial agent for promoting soybean growth and/or increasing soybean yield, comprising the mixed microbial population according to any one of claims 1 to 4.
8. The composite microbial inoculum according to claim 7, which is characterized by being prepared by mixing bacterial liquid of a bacillus aryabhattai PLR1 strain and bacterial liquid of a bacillus berryis PLR22 strain in a volume ratio of 1:1; or is formed by mixing bacterial liquid of a bacillus aryabhattai PLR1 bacterial strain, bacterial liquid of a bacillus berryis PLR22 bacterial strain and bacterial liquid of a bradyrhizobium, and the volume ratio is 1:1:2.
9. The composite microbial agent according to claim 8, wherein the total concentration of the composite microbial agent is not less than 2X 10 9 CFU/mL。
10. A method of promoting soybean growth and increasing soybean yield, comprising treating soybean plants with the composite microbial agent of claim 8 or 9.
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| CA2422343A1 (en) * | 2002-03-27 | 2003-09-27 | Mcgill University | Compositions and methods for increasing plant growth by inoculation with bacillus strains |
| CN101182476A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXYD3 and uses thereof |
| CN101182478A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BDYD1 and uses thereof |
| CN104651286A (en) * | 2015-03-18 | 2015-05-27 | 商丘师范学院 | Soybean root nodule endophyte DEnt1302, preparation method and application of soybean root nodule endophyte Dent1302, fungicide and preparation method of fungicide |
| CN110291059A (en) * | 2017-01-12 | 2019-09-27 | 喜施倍全球股份有限公司 | Microbe soil reinforcing agent |
| CA3149180A1 (en) * | 2019-08-08 | 2021-02-11 | Newleaf Symbiotics, Inc. | Methods and compositions for improving soybean yield |
| CN113923991A (en) * | 2019-06-07 | 2022-01-11 | 巴斯夫欧洲公司 | Novel formulations of microorganisms |
-
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2422343A1 (en) * | 2002-03-27 | 2003-09-27 | Mcgill University | Compositions and methods for increasing plant growth by inoculation with bacillus strains |
| CN101182476A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BXYD3 and uses thereof |
| CN101182478A (en) * | 2007-12-07 | 2008-05-21 | 华南农业大学 | Root nodule azotobacter strain BDYD1 and uses thereof |
| CN104651286A (en) * | 2015-03-18 | 2015-05-27 | 商丘师范学院 | Soybean root nodule endophyte DEnt1302, preparation method and application of soybean root nodule endophyte Dent1302, fungicide and preparation method of fungicide |
| CN110291059A (en) * | 2017-01-12 | 2019-09-27 | 喜施倍全球股份有限公司 | Microbe soil reinforcing agent |
| CN113923991A (en) * | 2019-06-07 | 2022-01-11 | 巴斯夫欧洲公司 | Novel formulations of microorganisms |
| CA3149180A1 (en) * | 2019-08-08 | 2021-02-11 | Newleaf Symbiotics, Inc. | Methods and compositions for improving soybean yield |
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