CN102021129B - Arthrobacterglobiformis CNA9 and application thereof - Google Patents
Arthrobacterglobiformis CNA9 and application thereof Download PDFInfo
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Abstract
The invention provides athrobacterglobiformis CNA9 which can be obtained from the separation in a wheat experimental field. The strain has the preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.4217 and stable capability of producing IAA (Indoleacetic Acid) and has the highest yield of producing the IAA of 109g/ml when being cultured in a culture medium providing precursor substance typtophan. Through detection experiment results of greenhouses and fields, the CNA9 has remarkable growth-promoting effect for plants. The invention can provide a novel excellent strain for researching and developing microbiological fertilizers.
Description
Technical field
Microorganism field of the present invention is specifically related to a kind of spherical Arthrobacter and application thereof.
Background technology
Owing to chemical pesticide under the cropping pattern extensive in the traditional agriculture production, sterilant, the excessive use of growth regulator; Make environment bear immense pressure; Then the soil erosion that causes, Soil degradation, problems such as agricultural product security also badly influence people's production and life.Along with the development of economic society, people make that to the increase of the high-quality agricultural-food demand of high safety the demand of the biogenic preparation of high ambient avidity is got over vigorous, particularly huge to the demand of microbial fertilizer and microbial source medicament in agriculture prodn simultaneously.
At present, international biogenic preparation generally is divided into: animal, plant, mikrobe.But in China's agricultural practical application, can carry out botanical pesticide, microbial pesticide and the microbial source fertilizer of large-scale industrial production.Microbial growth regulator is a kind of between between microbial pesticide and fertilizer, is to utilize the physiologically active substance of mikrobe to wait to regulate growth and development of plant, is of value to plant-growth and output.This quasi-microorganism bacterium in rhizosphere that normally grows nonparasitically upon another plant, be called the short living bacterium of plant rhizosphere (plant growth promoting rhizobacteria, PGPR).
Produce a kind of as in short the livings bacterium (PGPR) of plant rhizosphere of IAA bacterium,, produce physiological process and metamorphosis that plant hormones such as IAA and GA on a small quantity influence plant when utilizing plant metabolism to produce secretory product attached to root system of plant or leaf surface.Be embodied in and can directly promote the elongation of root, thus increased with soil in the chance that contacts of nutritive substance; Can improve the content of Endogenous IAA in the plant materials; Inducing plant defence expression of gene, it is disease-resistant to improve plant materials, resistance such as drought resisting.At present, studying more in the world is azospirillum (Azospirillumbrasilense), pseudomonas putida (Pseudomonas putida) and root nodule bacterium (Bradyrhizobium japonicum).Spaepen in 2008 is through the change effect of root morphology of experiment proof Brasil diazotrophic spirillum, shows as the formation that IAA that this bacterium produces has significantly promoted the wheat root hair.Pseudomonas putida has not only played promoter action in the cymbidium seed symbiotic germination, and can under greenhouse experiment, significantly promote the output of tomato.B.Ali in 2009 etc. are from plant rhizosphere, and isolated five kinds are produced the IAA bacterium in the phyllosphere, and they can make, and plant height improves 29.16% on the wheat seedling, and tiller number can improve 97.35%, and spike length improves 25.2%, and seed weight improves 13.7%.Other has the product IAA bacterium of the directly short fruit of coming into force to also have to plant in the existing report: xanthomonas maltophilia, wax shape bacillus, dark green trichoderma, Ai Xishi Pseudomonas, micrococcus sp, Staphylococcus etc.The spherical pole bacterial classification of mentioning among the present invention was published an article on Nature by people such as Katznelson in 1962 and is pointed out that it produces IAA and GA characteristic, but the Application Research of relevant this Pseudomonas bacterial classification on farm crop do not appeared in the newspapers.
Summary of the invention
The object of the present invention is to provide new bacterial strain of the spherical Arthrobacter of a strain and application thereof.
Bacterial strain CNA9 of the present invention is that wheat experiment Tanaka separation obtains from Huantai County, Shandong Province; In on October 11st, 2010 at China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City; Institute of Microorganism, Academia Sinica; Postcode 100101) preservation, the spherical Arthrobacter CNA9 of classification called after, preserving number is CGMCCNo.4217.
Can separating through following method that the CNA9 bacterial strain is concrete obtains:
Tanaka gathers soil sample from the wheat experiment; The soil of getting 5g is dissolved in the triangular flask of 45ml SPSS; Static 1min behind 28 ℃, 120rpm vibration 15min; The supernatant of getting 100 μ l joins in the Eppendorf pipe that 900 μ l saline water are housed, and carries out the continuous gradient dilution, promptly is diluted to 10
-2, 10
-3, 10
-4Doubly.Get above soil suspension-s and 10 respectively
-2, 10
-3, 10
-4Times soil suspension-s diluent 100 μ l are at NA substratum (Carnis Bovis seu Bubali cream 5g/L, peptone 10g/L, NaCl 5g/L; Agar 15g/L) even coated plate on the flat board dries up in the Bechtop, and each concentration repeats 3 times; Cultivate 36h in 28 ℃ of incubators, behind the single bacterium colony line of the aseptic toothpick picking bacterium purifying, be inoculated into (peptone 5.0g/L in the 500ml volume triangular flask that contains the 150mlDF+Trp substratum; Yeast extract 1.5g/L, Carnis Bovis seu Bubali cream 1.5g/L, NaCl 5.0g/L; Tryptophane 0.5g/L) cultivates after 7 days for 28 ℃ in the test tube; The centrifugal 5min of 12000rpm gets the 1ml supernatant in test tube, adds 50 μ l Solution I (10mM phosphoric acid) and 2ml Solution II (1ml 0.5M FeCl
3Be dissolved in 50ml 35%HClO
4), in room temperature, react 25min behind the mixing, exhibit red is for producing the IAA bacterial strain.According to shade bacterial strain is carried out classification, " +++" color is the darkest, and " ++ " takes second place, "+" once more.Carried out preliminary screening according to the depth of color to producing the IAA bacterium, the bacterial strain that obtains is carried out the trial inspection of the short fruit of coming into force in greenhouse again, it is good and produce the high bacterial strain CNA9 of IAA amount that finishing screen is selected short natural disposition shape.
CNA9 has following characteristic:
It is an aerobic bacteria, and nitrate reductase, glucose utilization, fructose utilization, seminose utilization, starch hydrolysis, urase, gelatine liquefication react positive, sucrose utilization, tartrate utilization, esterase and H
2The S reaction is negative.
(the GenBanK accession number: EU596424.1) similarity is the highest, and homology reaches 100% with genus arthrobacter (Arthrobacter Sp.W17) the 16S rDNA that delivers at present for the 16S rDNA sequence of CNA9 bacterial strain of the present invention.Comprehensive physiological and biochemical property identify with 16S rDNA Sequence Identification CNA9 be spherical Arthrobacter (Arthrobacter globiformis).
Short living test shows that this product IAA bacterial strain CNA9 has stable short living ability, can significantly improve living weight and cucumber, wheat and the corn yield of milpa.
The present invention also provides the microbial inoculum that contains the CNA9 bacterial strain.
The greenhouse detects in the test; Detected the short of this bacterial strain with corn and cucumber as plant indicator respectively and come into force really, the result shows that CNA9 all shows the significant short fruit of coming into force, and wherein the dry weight and fresh weight of plant seedlings of corn and cucumber has increased by 32.53% respectively; 32.86% and 75.76%, 210%; The land for growing field crops is detected in the test, soybean, and the effect of increasing production of wheat and corn yield is respectively 8～12%, and 10～15% and 10～13%.The research and development that the present invention can be microbial-bacterial fertilizer provide a strain novel strain excellent.
Description of drawings
Fig. 1 is the daily variation of CNA9 flora quantity.
Fig. 2 produces the daily variation of IAA amount for CNA9.
Fig. 3 is short the come into force fruit of CNA9 under the greenhouse experiment to corn.
Fig. 4 is short the come into force fruit of CNA9 under the greenhouse experiment to cucumber.
Embodiment
Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
The evaluation of embodiment 1CNA9 bacterial strain
1.1 Physiology and biochemistry is measured
The bacterial strain that is separated to (preserving number is CGMCC No.4217) is identified that the Physiology and biochemistry method for measuring is according to standard method (Institute of Microorganism, Academia Sinica's division bacteria group, 1978; The elegant pearl in east, Cai Miaoying etc., 2001), identification of indicator is with reference to the bibliographical information of Yi-Guang Chenet al. (2009), Dorothy Jones and Ronald M.Keddie (2006), G.S.N.Reddyet al. (2002) etc.Shown in table 1 (part Physiology and biochemistry proterties).
Table 1 bacterial strain Physiology and biochemistry is identified
Annotate: "+" expression is positive, and "-" expression is negative, and a little less than " W " expression, " NA " is expressed as the acquisition data.
1.216S rDNA sequential analysis
With bacterial strain CNA9 be inoculated in shake in the liquid LB substratum training 24h after, get 1mL bacterium liquid in the 1.5mL centrifuge tube, collect thalline behind the centrifugal 3min of 12000r/min;
Thalline is added 567 μ L TE damping fluids shake abundant suspension cell; The SDS that adds 30 μ L 10%, 3 μ L 20mg/mL Proteinase K voltage regulator tubes and abundant mixing, 37 ℃ of water-bath 1h, every separated 20min puts upside down mixing once;
The NaCl mixing that adds 100 μ L 5M, (10%/0.7M) solution gently mixes, and 65 ℃ of water-bath 20min whenever put upside down mixing once at a distance from 4 minutes to add 80 μ L CTAB/NaCl;
Chloroform/the primary isoamyl alcohol (24: 1) that adds 750 μ L, the centrifugal 5min of 12000rpm behind the mixing;
Get phenol/chloroform/primary isoamyl alcohol (25: 24: 1) that supernatant adds 750 μ L, the centrifugal 5min of 12000rpm behind the mixing;
Get chloroform/primary isoamyl alcohol (24: 1) that supernatant adds 750 μ L, the centrifugal 5min of 12000rpm behind the mixing; Water is transferred to new centrifuge tube, add the Virahol of 0.6 times of volume, mixing gently, the centrifugal 15min of 13000rpm;
Remove supernatant, add the 1mL absolute ethyl alcohol, light mixed, the centrifugal 5min of 12000rpm;
Remove supernatant, Bechtop dries up, and adds 200 μ L TE solution dissolution precipitations (can under 50 ℃ of water bath condition hydrotropy);
The RNaseA that adds 1 μ L 20mg/mL, 37 ℃ of water-bath 2h; The phenol that adds 200 μ L: chloroform: primary isoamyl alcohol (25: 24: 1), the centrifugal 5min of 12000rpm behind the mixing;
The water intaking phase adds the chloroform of 200 μ l: primary isoamyl alcohol (24: 1), mixing, the centrifugal 5min of 12000r/min; The water intaking phase adds the NaAc (pH5.2) of 20 μ L 3M, and mixing adds the long-pending absolute ethyl alcohol of diploid, mixing gently, the centrifugal 15min of 13000rpm;
Remove supernatant, add 1mL70% ethanol, light mixed, the centrifugal 5min of 12000rpm; Remove supernatant, Bechtop dries up, and adds 50 μ L TE solution dissolution precipitations and obtains the CNA9 genomic dna.
Pcr amplification obtains CNA916S rDNA.Wherein, primer sequence is shown in SEQ ID No.2 and SEQ ID No.3.
50 μ L reaction systems are: two steaming aqua sterilisa 41.2 μ L, 10 * PCR damping fluid, 5.0 μ L, 10mM dNTP 2 μ L, each 0.5 μ L of upstream and downstream primer (10 μ mol/L), Taq enzyme 1U, template 1 μ L; PCR reaction conditions: 94 ℃ of preparatory sex change 10min; 94 ℃ of 40s of major cycle, 56 ℃ of 40s, 72 ℃ of 1min circulate 34 times; Extend 72 ℃ of 10min eventually; 4 ℃ of preservations.
Pcr amplification product reclaims back by Sinogenomax Co., Ltd. order-checking with the PCR product that is obtained with Gel Extraction Kit (OMEGA) with 1% agarose gel electrophoresis analysis, and its nucleotide sequence is shown in SEQ ID No.1.Sequencing result is compared on NCBI after handling with DNAman, and that homology is the highest is genus arthrobacter (Arthrobacter Sp.W17), (GenBanK accession number: EU596424.1).
Embodiment 2CNA9 bacterial strain produces the IAA amount and the strain growth situation detects
2.1 the cultivation of bacterial strain:
To detect among the substratum DF+Trp in IAA in the inoculation after the activation on the LB substratum, in 28 ℃, 170r/min shakes and accompanies 8 days.Therefrom get quantitative bacterium liquid every day and survey flora number and the amount of producing IAA.
2.1.1 the flora number is measured:
Get bacterium liquid stoste and detect down absorbancy in spectrophotometer 600nm, the flora number along with cultivating increasing of fate, needs dilution stoste to certain multiple, makes absorbance under the 600nm between 0.1-1, guarantees its accuracy, and the result is as shown in Figure 1.
The result shows that flora quantity reached the highest at the 2nd day, reached 10
9CFU/mL, along with the growth of time, quantity reduces successively, in the time of the 8th day, has reduced to the level when rigidly connecting kind basically, thinks that flora accomplishes a life cycle basically.
2.1.2 produce the mensuration of IAA amount:
Get DF+Trp bacterium liquid stoste 1.5mL in centrifuge tube; The centrifugal 5min of 12000rpm gets the 1mL supernatant in test tube, adds 50 μ L Solution I and 2mL Solution II reaction solution; In room temperature, react 25min behind the mixing; Detect 530nm place absorbancy, comparison and calculating IAA output on typical curve, the result is as shown in Figure 2.
The result shows, produces the IAA amount and increases suddenly at the 2nd day, thinks to begin a large amount of synthesis secretion IAA after bacterial strain reaches logarithmic phase, and along with the growth of time, the IAA accumulation volume changes little later on, and the highest IAA output reaches 109g/mL.
Wherein, the making of typical curve: take by weighing the pure article of 0.01g IAA (Sigma) and be dissolved in the 1mL absolute ethyl alcohol, draw 100 μ l behind the mixing and add deionized water and be diluted to 1mL, draw 100 μ l behind the mixing again and add deionized water and be diluted to 1mL, 100 μ g/mL IAA mother liquors.Prepare the following concentration gradient IAA aqueous solution then respectively: 5,10,15,20,25,30 μ g/mL.Do blank with deionized water.Detection different concns IAA solution is light absorption value at the 530nm place, draws IAA content standard curve.
The preparation of embodiment 3CNA9 microbial inoculum
3.1 actication of culture
Use the LB substratum, culture medium prescription is: yeast extract 10g/L, peptone 5g/L, NaCl 10g/L, pH 7.0-7.2.CNA9 is inoculated on the LB culture medium slant with spherical Arthrobacter (Arthrobacter globiformis), cultivates 24 hours for 28 ℃.
3.2 culture of seed liquid
Seed culture is with improvement 523 substratum, and culture medium prescription is: sucrose 10g/L, KH
2PO
42.0g/L, yeast extract paste 10g/L, CaCO
35.0g/L, NaCl 0.2g/L, MgSO4.7H
2O 0.3g/L, pH7.0-7.2.The single bacterium colony of spherical Arthrobacter (Arthrobacterglobiformis) CNA9 that the picking activation is good is mixed with 10 with SPSS
8The bacteria suspension of CFU/mL, the inoculum size with 1% are inoculated in improvement 523 liquid nutrient mediums, and 28 ℃ of shaking table concussions are cultivated, and rotating speed is 150rpm, and incubation time is 28h.
3.3 ferment tank
Fermention medium consists of: glucose 8g/L, yeast extract paste 3g/L, NaCl 1.5g/L, KH
2PO
40.5g/L, K
2HPO
40.5g/L, MgSO
4.7H
2O 0.2g/L, (NH
4)
2SO
45g/L, pH 7.0-7.2.PM air flow volume), tank pressure 1.2-1.8F/cm insert cultured seed liquid in the fermentor tank with 2% inoculum size, 28 ℃, stirring velocity is 160rpm, and air flow is 1: 0.7-0.9 (fermentating liquid volume:
2Fermentation 54h, the bacterium amount reaches 5-10 * 10
8CFU/mL obtains the microbial inoculum of spherical Arthrobacter (Arthrobacter globiformis) CNA9 bacterial strain.
The short fruit of coming into force to corn in the embodiment 4CNA9 bacterial strain greenhouse detects
The CNA9 bacterial strain is shaken training 3 days in ruling activation on the LB solid medium after 2 days, being inoculated in the 500ml volumetrical triangular flask that contains 150ml DF+Trp substratum.On spectrophotometer, detect its IAA output.
Choose sound corn seed, immerse the 30s that sterilizes in 70% alcohol, with aseptic water washing 3 times, seed is put into big petridish with two-layer binding up with gauze, clear water soaks, and makes gauze surface form water membrane.Put into 28 ℃ of incubators, cultivate about 18h and treat that seed shows money or valuables one carries unintentionally, it is subsequent use to choose the consistent seed that germinates.
Get 10
7CFU/mL shakes 3 days CNA9 bacterium liquid of training and mixes in the sterilization sand, and every basin dress 6kg sterilization is husky, establishes the clear water contrast simultaneously.Totally two groups of processing are handled 10 repetitions for every group.Hot-house culture is watered every other day, waters the Hoagland nutritive medium after the week weekly one time, treats to gather in the crops plant after four weeks, measures its plant height, and root is long, dry weight and fresh weight of plant seedlings.Wherein, the long result of root has carried out the classification comparison process with root system scanning analysis system (WinRHIZO50).
Result such as Fig. 3 and table 3, shown in 4, plant height increases by 16.56%; Fresh weight increases by 32.86%; Dry weight increases by 32.53%; Inoculated in the maize root system of CNA9 bacterial strain, diameter has increased by 85.60% greater than the total length comparison of the root of 0.3cm according to long; The root difference with insignificance of 0～0.3cm possibly be that the part of in operating process, losing is uncertain because this part root is too thin.
The CNA9 bacterial strain is to the short fruit of coming into force of milpa under table 3 greenhouse experiment
The short fruit of coming into force to cucumber in the embodiment 5CNA9 bacterial strain greenhouse detects
To showing money or valuables one carries unintentionally, method is with embodiment 4 with cucumber seeds vernalization.Choose the consistent seed that germinates, put into growth cabinet (illumination 12h, dark 12h; 26 ℃, relative humidity is 80%) grow seedlings, after first cotyledon grows, choose the consistent cucumber seedling of growing way again and be transplanted in the flowerpot in greenhouse, the husky and vermiculite mixture of every basin dress 900g sterilization.If clear water contrasts and sprays totally two processing of bacterium liquid, each handles 4 repetitions.Wherein bacterium liquid is handled operation as follows: treat that cucumber seedling length begins to spray CNA9 bacterium liquid one time after rough leaf grows, spray bacterium liquid week about once later on, every day, equivalent was watered; Two processing during this time all spray the equivalent sterilant simultaneously, and results are measured the plant height of cucumber respectively after one month; Stem is thick; The number of blade, leaf area, dry weight and fresh weight of plant seedlings.Plant height increases by 138.90%, and stem slightly increases by 43.21%, and the number of blade increases by 70%, and leaf area increases by 255.23%, and fresh weight increases by 210%, and dry weight increases by 75.76%.Shown in Fig. 4 and table 5.
CNA9 is to the short fruit of coming into force of cucumber under table 5 greenhouse experiment
The short fruit of coming into force to wheat, corn, soybean in the embodiment 6CNA9 bacterial strain detects
After cultivating 3 days 300 times of CNA9 bacterial strain fermentation liquor dilutions, wheat, corn and soybean are dressed seed; Carry out the field and increase the short fruit mensuration that comes into force; Each crop is provided with four repetitions; Output is measured in the results back, and CNA9 is respectively 8～12% to soybean, wheat and corn yield increasing effect, and 10～15% and 10～13%.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from know-why of the present invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (5)
1. the new bacterial strain CNA9 of a spherical Arthrobacter (Arthrobacter globiformis), its preserving number is CGMCC No.4217, and preservation date is on October 11st, 2010, and depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center.
2. the microbial inoculum that contains the said bacterial strain of claim 1.
3. the application of the said bacterial strain of claim 1 in IAA produces.
4. the application of the said bacterial strain of claim 1 in promoting plant-growth.
5. application according to claim 4 is characterized in that said plant is selected from mung bean, corn, cucumber, soybean or wheat.
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| CN102643766B (en) * | 2012-04-10 | 2013-09-11 | 中国农业大学 | Nourishing arthrobacter Ar13 and application thereof as plant growth-promoting rhinoacteria |
| BR112019023237A2 (en) * | 2017-05-09 | 2020-06-02 | Taxon Biosciences Inc. | PLANT GROWTH PROMOTION MICROBES, COMPOSITIONS, AND USES |
| CN108060110B (en) * | 2018-02-27 | 2018-12-18 | 华南农业大学 | A kind of Arthrobacter strain and its application |
| CN109536402B (en) * | 2018-11-12 | 2021-02-02 | 北京林业大学 | Arthrobacter with carbon sequestration capacity and application thereof |
| CN116769679B (en) * | 2023-08-17 | 2023-11-10 | 中国科学院烟台海岸带研究所 | Arthrobacter JY3-2 for producing acid and application thereof |
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