CN104818231A - Antagonistic and growth-promoting pseudomonas producing ACC deaminase and application thereof - Google Patents

Abstract

The invention relates to an antagonistic and growth-promoting pseudomonas FRBN3 producing ACC deaminase and an application thereof. The pseudomonas FRBN3 is obtained through separation on surface of watermelon roots from Hefei city, Anhui province and is named Pseudomonas guariconensis. The pseudomonas FRBN3 is preserved in China General Microbiological Culture Collection Center at Apr.14 2015 and is assigned the accession number CGMCC No.10715. The pseudomonas FRBN3 has an ACC deaminase activity and can highly produce siderophores. The pseudomonas FRBN3 has a certain inhibition effect on all of fusarium oxyspirum, fusarium graminearum, bipolaria maydis and fusarium oxgsporum. A liquid bacterial agent prepared from the pseudomonas FRBN3 can be applied in the field of promoting growth of plants and preventing and treating diseases. The liquid bacterial agent can be used in manners of being diluted with water to form spray for use, being blended with seeds or dipped with roots, or being irrigated on root soil.

Description

A kind of somatotrophic pseudomonas of antagonism and application thereof of producing acc deaminase

Technical field

The invention belongs to plant growth-promoting bacteria technical field, be specifically related to the antagonism growth-promoting pseudomonad strain FRBN3 producing acc deaminase, and the application in Promoting plant growth and controlling plant diseases.

Background technology

Watermelon blight is a kind of main fungal soil-borne disease during watermelon is produced, and this disease all can occur at each growthdevelopmental stage of watermelon, and general time sickness rate, at 10%-30%, up to more than 50% time serious, has a strong impact on watermelon growing and output.Fusarium oxysporum (watermelon specialized form) (Fusarium oxysporum f.sp.niveum) is the main pathogenic fungi causing watermelon blight, its survival time in soil is about 10 years, is that the soil of most destructive force passes one of plant pathogenic microorganisms.At present, using etc. of stock grafting and chemical pesticide is the main prevention and controls of watermelon blight.

Though stock grafting can improve the resistance of watermelon to blight, rootstock seedling will shift to an earlier date breeding, strengthen management; The graft stock of suitable coupling must be selected, stock grafting also can affect Watermelon Fruit mouthfeel, local flavor and quality, the such as hardness of fruit, sugariness etc.

The chemical bactericide being usually used in preventing and treating blight mainly contains octicin solution, Sukeling, hymexazol, Yi Bao, lucky star, the withered spirit of standing grain, anti-wither chemical, thiophanate methyl, derosal etc.Although utilize these chemical bactericides to have instant effect, effective feature to prevent watermelon blight, but Agro-chemicals control will accomplish early medication, medication in time, and repeatedly, a large amount of medication, this will certainly cause the drug-fast increase of pathogenic bacteria, forms the vicious cycle of pesticide abuse.These chemical agent toxicity are comparatively large, are difficult to natural degradation, pollute the environment, the havoc eubiosis, are finally detrimental to health.In addition, watermelon is also a kind of high garden crop needing fertilizer, and the application of a large amount of chemical fertilizer makes the environmental degradation such as soil, water body.Therefore, one of bio-feritlizer and the biological pesticide important directions becoming agricultural sustainable development.

The adverse circumstance environment such as disease and pest, pesticide residual contamination, heavy metal contamination, high temperature, arid, waterlogging, frost causes crop yield seriously to reduce, and quality declines.Effective raising crop disease-resistant evil, anti-adversity ability, increasing crop yield, is significant problem urgently to be resolved hurrily in agricultural sustainable development.Plant can produce a large amount of ethene when being subject to the invasion and attack of the adverse circumstance such as disease, and excessive ethene accumulation that plant-growth can be caused to be obstructed is even dead.Therefore alleviate the accumulation of ethene under adverse environmental factor, the anti-adversity ability of plant can be strengthened, promote the growth of plant.

1-amino-cyclopropane-1-carboxylic acid (ACC, 1-aminocyclopropane-1-carboxylate) is the precursor substance of ethylene synthase in plant materials.Some growth-promoting bacteriums metabolism can produce acc deaminase, and this enzyme can by the precursor of ethene---and ACC changes α-batanone acid and ammonia into, thus reduces the accumulation of ethene in stressful environmental, improves the degeneration-resistant border ability of plant.

Excellent high-efficiency strain is the primary precondition of exploitation microbial preparation.Screening has the alternative chemical pesticide of bacterial strain of acc deaminase and antagonism various plants pathogenic bacteria and the utilization of chemical fertilizer concurrently; strengthen the resistance of plant, Promoting plant growth, preserves the ecological environment; safeguard that agricultural ecological balances, significant to agricultural sustainable development.

Summary of the invention

The object of this invention is to provide and produce the somatotrophic pseudomonas of antagonism of acc deaminase, its application is can Promoting plant growth and controlling plant diseases, has the applications well prospect being developed to bio-feritlizer and biological pesticide.

Pseudomonad strain FRBN3 of the present invention is separated from watermelon root table face, Hefei City, Anhui Province and obtains, Classification And Nomenclature is Pseudomonas guariconensis, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on April 14th, 2015, and preserving number is CGMCC No.10715.

Pseudomonas FRBN3 of the present invention is at beef-protein medium (extractum carnis 3g, peptone 10g, NaCl 5g, agar 18g, H ₂o 1000mL, pH7.2) on can well grow, colony characteristics is light beige, neat in edge, surface wettability, basis of microscopic observation, and Gram-negative, does not produce gemma.This bacterial strain can on the flat board being less than 5%NaCl, pH5-10 and 15-37 DEG C all can well grow.

With the genome DNA of pseudomonas FRBN3 bacterial strain for its 16S rDNA sequence of template amplification, carry out DNA sequencing, obtain the 16S rDNA sequence (see sequence table) that length is 1387bp, the 16SrDNA sequence of pseudomonas FRBN3 bacterial strain is submitted to GenBank database, and acquisition sequence number is KR086404.The representative strain 16S rDNA sequence of the 16S rDNA sequence of pseudomonas FRBN3 bacterial strain and GenBank database being included carries out sequence analysis analysis, and 16S rDNA sequence (sequence number HF674459) homology of result and Pseudomonas guariconensis is up to 99.71%.

Pseudomonas of the present invention has wider antimicrobial spectrum, all has obvious antagonistic action to withered germ of water-melon, fusarium graminearum, southern corn leaf blight, tomato wilt bacterium.

The application of liquid bacterial agent in Promoting plant growth and controlling disease of pseudomonas of the present invention and preparation thereof.This microbial inoculum can be watered spray application or seed dressing or dip in root or water and use in root soil.

The metering of using of pseudomonas liquid bacterial agent of the present invention is 0.5L/ mu ~ 4L/ mu.Liquid bacterial agent is applicable to the multiple soil such as subacidity, neutrality and saline alkali.

Advantageous Effects of the present invention embodies in the following areas:

1. pseudomonas FRBN3 bacterial strain of the present invention can produce acc deaminase, contributes to the accumulation reducing Stress ethylene under adverse environmental factor, improves the anti-adversity ability (disease resistance, anti-saline and alkaline, drought-resistant etc.) of plant, Promoting plant growth; Can siderophore be produced, contribute to improving bacterial strain and pathogenic bacteria and compete ferrite rolling technology, help plant absorption ferrite rolling technology, enhancing plant disease-resistant ability, Promoting plant growth.There is pollution-free, instant effect, do not destroy the advantage of the biological control of the eubiosis.

2. provided by the invention having concurrently is produced acc deaminase and has the pseudomonas FRBN3 of antagonistic action to promote watermelon growing to various plants pathogenic bacteria, control watermelon blight evil.Compared with the control, increase overground part dry weight 41.2%, root dry weight 26% is 64% to watermelon blight prevention effect, has good development prospect.

3. pseudomonas FRBN3 bacterial strain of the present invention all has certain restraining effect to fusarium graminearum, southern corn leaf blight, tomato wilt bacterium, and inhibiting rate reaches 41%, 35%, 40% respectively.

Accompanying drawing explanation

Fig. 1 is the bacterium colony figure of pseudomonas FRBN3 of the present invention;

Fig. 2 is that pseudomonas FRBN3 of the present invention produces siderophore situation (reacting phenomenon being followed successively by stoste, dilution 10 times and dilution 20 times in figure from left to right and contrasting).

Embodiment

Below in conjunction with embodiment, the present invention is further described.

Embodiment 1

A kind of growth-promoting diseases prevention bacterial isolates FRBN3 producing acc deaminase and antagonism various plants pathogenic fungi that has concurrently of the present invention obtains from the watermelon plant root table face separation and purification of AnHui Agriculture University, HeFei City, AnHui Province robust growth, and it is specifically separated, screening method is as follows:

The separation of 1 bacterial strain

10 grams of watermelon roots with micro-surface soil, to remove a large amount of soil be attached to around root system, are then soaked in the triangular flask that 90mL sterilized water is housed, shaking table 160rmin by shake watermelon root ^-1vibration 30min, makes sample suspension, adopts 10 times of gradient dilution methods, be diluted to 10 ^-7, get suitable dilution suspension 0.1mL and be coated on beef-protein medium flat board, cultivate after 3d for 28 DEG C, single bacterium colony that picking colony form is different, repeatedly line point pure after, save backup in 4 DEG C of inclined-planes and 40% sterile glycerol-20 DEG C.

The screening of 2 acc deaminase bacterial strains

The primary dcreening operation of 2.1 acc deaminase active bacterial strains

FRBN3 is inoculated in shaking table shaking culture 24h in beef extract-peptone nutrient solution, 4 DEG C of collected by centrifugation thalline, and with brine 2 times, thalline is resuspended in (glucose 2g in DF basic medium; Gluconic acid 2g; Citric acid 2g; KH ₂pO ₄4.0g; Na ₂hPO ₄6g; MgSO ₄7H ₂o 0.2g; (NH ₄) ₂sO ₄2g; 1mg FeSO ₄7H ₂o and 0.1mL trace element solution, distilled water 1000mL, pH about 7.2.Wherein trace element solution is: boric acid 10mg; MnSO ₄h ₂o 11.19mg; ZnSO ₄7H ₂o 124.6mg; CuSO ₄5H ₂o 78.22mg; MoO ₃10mg is dissolved in 100mL sterilized water.), and be inoculated in DF nutrient solution respectively by 2% inoculum size, ADF nutrient solution (adds the ACC of filtration sterilization in DF nutrient solution, its final concentration is made to be 3mM) and NDF nutrient solution (adding the ammonium sulfate of 0.2% as nitrogenous source in DF nutrient solution), 28 DEG C of 160rpm cultivate 3d, the growing state of record bacterium, measures bacterium liquid OD ₆₀₀light absorption value.Result shows that the growth potential of bacterial strain FRBN3 in ADF nutrient solution is better than the growing way in DF nutrient solution, illustrates that bacterial strain FRBN3 can grow using ACC as only nitrogen source, has certain acc deaminase active.

2.2 strains A CC deaminase activity determinations

By be cultured to logarithmic phase FRBN3 bacterium liquid centrifuge washing 2 times after, to be resuspended in DF basic medium and to be inoculated in ADF nutrient solution (including final concentration is that the ACC of 3mM is as only nitrogen source), 28 DEG C of 160rpm cultivate 3d, to induce the generation of acc deaminase.

4 DEG C of centrifugal 10min of 8000rpm, abandon supernatant, thalline 1mL 0.1molL ^-1, pH 7.6, Tris-HCl is resuspended in 600 μ L 0.1molL after washing centrifugal twice ^-1in Tris-HCl pH 8.5buffer, add 30 μ L toluene, high speed vortex 30s, with broken somatic cells, discharges acc deaminase.Get 100 μ L liquid 4 DEG C to preserve for measuring protein content temporarily, remaining cell suspension is rapidly for measuring acc deaminase activity.Get the cell treatment solution of 200 μ L containing toluene in a new 1.5mL centrifuge tube, add 20 μ L 0.5molL ^-1aCC, tip-tap mixes, and hatches 15min for 30 DEG C, adds 1mL 0.56molL ^-1hCl, mixes with termination reaction.16000rmin ^-1centrifugal 5min removes cell debris.Draw 1mL supernatant in test tube, and add 150 μ L 2,4 dinitrophenyl hydrazines and (be dissolved in 2molL ^-1hCl), vortex mixes, and after 30 DEG C of reaction 30min, adds the NaOH solution of 1mL 2N.Simultaneously to add 1mL 0.1molL ^-1pH8.5Tris-HCl damping fluid is cooked returning to zero for blank tube of above-mentioned same treatment, working sample 540nm absorption value.

The preparation of α-batanone acid and protein standard curve: be 0,0.15 in 1mL concentration respectively, 0.3,0.6,1.2,1.8,2.4,3 μm of ol.mL ^-1α-batanone acid solution in, add the 2,4 dinitrophenyl hydrazine of 0.15mL 0.1%, 30 DEG C of reaction 30min, take out and add 1mL 2N NaOH, measure light absorption value under 540nm wavelength.α-batanone acid typical curve is drawn with light absorption value under the 540nm wavelength of concentration and correspondence thereof.Bacterial protein content adopts Coomassie Brilliant Blue to measure.Join 100mgL ^-1bovine serum albumin be standard female solution, dilution obtain concentration be respectively 0,10,20,40,60,80,100mgL ^-1the each 1mL of protein solution, add 5mL Coomassie brilliant blue G250 staining fluid, after reaction 5min, measure light absorption value under 595nm wavelength.Last then draw protein standard curve according to light absorption value under the 595nm wavelength of protein concentration and correspondence thereof.The activity of acc deaminase is to represent in the amount of the α-batanone acid surveying every milligram of albumen formation per hour in enzyme system, and unit is nmol α-batanone acid .mg ^-1proteinh ^-1.FRBN3 bacterial strain has acc deaminase activity, and enzymic activity is 32.98nmol α-batanone acid .mg ^-1proteinh ^-1.

3 bacterial strain antagonistic properties measure

Bacterial strain adopts conventional dull and stereotyped opposite culture method to the antagonistic properties of various plants pathogenic bacteria.The bacterium cake getting the new pathogenic fungi cultivated with punch tool (d=5mm) is attached to PDA solid plate center, and on the dull and stereotyped surrounding position apart from plate center 3cm, inoculation strains tested FRBN3, each plating bacterial strain 3 times, and 3 dull and stereotyped repetitions are set, not connect the process of bacterium as blank, 28 DEG C of cultivations.

$\%\mathrm{Inhibition}=\left[\frac{(R-r)}{R}\right]\×100---\left(1\right)$

The radius R of fungal lawn is recorded when the fungi of blank covers with flat board, and the radius r of the relative bacterium of fungal colony, calculate inhibiting rate according to formula (1).

Result shows that bacterial strain FRBN3 all has certain restraining effect to withered germ of water-melon, fusarium graminearum, southern corn leaf blight, tomato wilt bacterium, and inhibiting rate reaches 33%, 41%, 35%, 40% respectively.

Embodiment 2

Bacterial strain produces the method mensuration of siderophore ability with reference to king's equality.Bacterial strain FRBN3 is inoculated in (casamino acids 5.0g in MKB liquid nutrient medium; Glycerol 15mL; K ₂hPO ₄2.5g; MgSO ₄7H ₂o 2.5g; Distilled water 1000mL; PH 7.2), 160rmin ^-1cultivate 48h for 28 DEG C.By cultured bacterium liquid 8000rmin ^-1centrifugal 10min, gets 2mL supernatant liquor and is added in equal-volume CAS detection liquid, react lh, compare zeroing with distilled water, measure the light absorption value (being designated as A) at 630nm wavelength place after mixing.Meanwhile, get 2mL CAS and detect liquid and 2mL nonvaccinated MKB liquid nutrient medium supernatant liquor and fully mix, be measured in the same method light absorption value and be reference value (being designated as Ar).A/Ar value <1, then show that bacterial strain can produce siderophore, and A/Ar value is less, shows that siderophore output is larger.General evaluation standard is: 0-0.2, +++ ++; 0.2-0.4, ++++; 0.4-0.6, +++; 0.6-0.8, ++; 0.8-1 ,+.

Table 1 bacterial strain FRBN3 produces siderophore situation

Embodiment 3

To separation screening to the bacterial strain FRBN3 producing acc deaminase and antagonism various plants pathogenic bacteria that has concurrently adopt following methods to identify:

1 morphological specificity

The tool acc deaminase active bacterial strain FRBN3 of acquisition is lined beef-protein medium, cultivates 2 days, be described by bacterial colony feature.Obtain its morphological specificity as follows: bacterium colony ivory buff, moistening, smooth surface, opaque, neat in edge.

Adopt gram staining method in optical microphotograph Microscopic observation the bacterial strain growing to logarithmic phase, find that thalline is shaft-like, without gemma, Gram-negative.

2 physiological and biochemical properties

" common bacteria system identification handbook " with reference to eastern elegant pearl etc. measures the physio-biochemical characteristics of above-mentioned isolated strains.Catalase positive, amylolytic enzyme is negative, and VP is negative, the weak positive of MR, and gelatine liquefication is negative, and energy oxidizing glucose, can utilize the several kinds of carbon source such as glucose, lactose, semi-lactosi, sucrose, rhamnosyl, N.F,USP MANNITOL.Bacterial strain on the flat board being less than 5%NaCl and pH5-10 and 15-37 DEG C all can well grow.

3 16S rDNA sequential analyses

Adopt high salt method bacterial genomes STb gene.With 16S rDNA universal primer 27F (forward primer):

5 '-AGAGTTTGATCCTGGCTCAG-3 '; 1492R (reverse primer):

5 '-TACGGCTACCTTGTTACGACTT-3 ', with the STb gene of isolated strains for template carries out pcr amplification.Pcr amplification uses PTC-200PCR instrument (BIO-RAD, USA) to carry out.Amplification system (25 μ L) is: 10 × Taq DNApolymerase buffer 2.5 μ L, 25mM MgCl ₂1.5 μ L, primer 2 7F and 1492R (10 μMs) each 0.5 μ L, 2.5mM dNTP 2 μ L, 5U/ μ L Taq enzyme 0.15 μ L, template DNA 0.5 μ L, dd H ₂o complements to 25 μ L.PCR reaction conditions is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 55 DEG C of annealing 1min, 72 DEG C extend 1.5min, circulate 30 times; Last 72 DEG C extend 10min.The agarose gel electrophoresis of 0.8% detects PCR primer.The sequencing of PCR primer is completed by Shanghai biotechnology company limited.Sequence assembly adopts DNAMAN software, and sequence alignment is completed online by NCBI and EzTaxon website.Obtain the 16S rDNA sequence (see sequence table) that length is 1387bp, the 16S rDNA sequence of this bacterial strain is submitted to GenBank database, and acquisition sequence number is KR086404; The representative strain 16S rDNA sequence of the 16S rDNA sequence of described pseudomonas and GenBank database being included carries out sequence analysis analysis, and the homology of the 16S rDNA sequence (sequence number HF674459) of result and Pseudomonas guariconensis is up to 99.71%.

In view of above-mentioned morphological specificity, physiological and biochemical property and 16S rDNA sequence homology analysis, the pure culture bacterial strain FRBN3 that the separation and purification of above-mentioned institute obtains is accredited as pseudomonas Pseudomonas guariconensis, and be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC on April 14th, 2015, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preserving number is CGMCCNo.10715.

Embodiment 4 bacterial strain FRBN3 is to the growth-promoting of watermelon and prophylaxis effect

Prepared by liquid bacterial agent: be inoculated in beef extract-peptone nutrient solution by the bacterial strain FRBN3 (preserving number CGMCC No.10715) of above-mentioned separation, after 28 DEG C of 160rpm shaking culture 24-48h, the centrifugal 10min of 8000rpm, after removing fermented liquid supernatant, somatic cells is resuspended in the sterilized water of equal-volume or 3-5 times of volume, be prepared into liquid bacterial agent, make living bacteria count amount in this liquid bacterial agent be 10 ⁷-10 ⁸cfumL ^-1.

This test design 3 process, control group 1: completely blank (CK).Control group 2 (CK-F): every basin applies 5mL Fusarium oxysporum f. sp. niveum spore suspension (10 respectively ⁶individual mL ^-1).Test group (FRBN3-F): every basin applies 2.5mL FRBN3 bacteria suspension respectively, and (viable count is 10 ⁷-10 ⁸cfu ﹒ mL ^-1) and 5mL Fusarium oxysporum f. sp. niveum spore suspension (10 ⁶individual mL ^-1).Watermelon seed after vernalization is planted in the basin being embedded in and 2kg soil is housed, every basin 3 watermelon seeds, the every basin of test group applies 2.5mL FRBN3 bacteria suspension in seed surrounding soil, and the every basin of control group applies equal-volume (2.5mL) water in watermelon seed surrounding soil.When watermelon seedlings grows to 2-3 sheet true leaf, control group 2 (CK-F) and the every basin of test group (FRBN3-F) apply 5mL Fusarium oxysporum f. sp. niveum spore suspension respectively, a part is drenched and is executed around seedling rhizosphere, and a part is sprayed on watermelon plant.Each process arranges 3 repetitions.After outdoor outdoor cultivation 60d, measure fresh weight and the dry weight of watermelon plant.During plant strain growth, timing is observed and records the disease incidence of watermelon plant, statistics disease index and prevention effect.

Watermelon blight disease scale and investigation method as follows: in units of blade classification investigation, 0 grade, do not fall ill; 1 grade, scab accounts for leaf area less than 20%; 2 grades, scab accounts for leaf area 20% ~ 40%; 3 grades, scab accounts for leaf area 40% ~ 60%; 4 grades, scab accounts for leaf area 60% ~ 80%; 5 grades, scab accounts for leaf area more than 80%.

Disease index=Σ (incidence of leaf number × onset grade)/investigation blade amt × the highest onset grade × 100;

Prevention effect %=(contrast disease index-process disease index)/contrast disease index × 100%;

Table 2 bacterial strain FRBN3 is to the growth-promoting effect of watermelon seedling

Table 3 bacterial strain FRBN3 is to the prevention effect of watermelon blight

Test-results is as shown in table 2 and table 3, inoculating strain FRBN3 significantly can increase the overground part fresh weight of watermelon seedling and dry weight and total biomass fresh weight and dry weight, compared with inoculating merely the contrast (CK-F) of pathogenic bacteria, biomass fresh weight and dry weight is made to increase by 31% and 39% respectively.FRBN3 significantly can reduce the disease index of watermelon blight, makes prevention effect be up to 64%.

Sequence table

Claims (5)

1. produce the somatotrophic pseudomonas FRBN3 of antagonism of acc deaminase, it is characterized in that: described pseudomonad strain FRBN3 is separated from watermelon root table face, Hefei City, Anhui Province and obtains, and Classification And Nomenclature is pseudomonas guariconensis, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation date is on April 14th, 2015, and preserving number is CGMCC No. 10715;

The colony characteristics of described pseudomonad strain FRBN3 is light beige, neat in edge, surface wettability, basis of microscopic observation, and Gram-negative, does not produce gemma;

With the genome DNA of described pseudomonas for its 16S rDNA sequence of template amplification, carry out DNA sequencing, obtain the 16S rDNA sequence that length is 1387 bp, see sequence table.

2. produce the purposes of the somatotrophic pseudomonas of antagonism of acc deaminase described in claim 1, it is characterized in that: the application of described pseudomonas in the microbial inoculum preparing Promoting plant growth and controlling disease.

3. the purposes of the somatotrophic pseudomonas of antagonism of product acc deaminase according to claim 2, is characterized in that: the application of described pseudomonas in the microbial inoculum of preparation control withered germ of water-melon, fusarium graminearum, southern corn leaf blight, tomato wilt bacterium.

4. the purposes of the somatotrophic pseudomonas of antagonism of product acc deaminase according to claim 2, is characterized in that: described microbial inoculum is liquid bacterial agent, is applicable to subacidity soil, neutral soil or saline-alkali soil; Concrete using method is be watered spraying execute or dress seed or dip in root or water in the soil of plant root.

5. the purposes of the somatotrophic pseudomonas of antagonism of product acc deaminase according to claim 3, is characterized in that: the metering of using of described liquid bacterial agent is 0.5L/ mu ~ 4L/ mu.