CN103642733B - Achromobacter marplatensis and application thereof - Google Patents

Abstract

The invention discloses achromobacter marplatensis TNSC2011with a preservation number of CGMCC (China General Microbiological Culture Collection) No.8192 and an application thereof in sugar beet cultivation. Taxonomic status of the obtained bacterial strain is primarily determined by carrying out determination and phylogenetic analysis of morphological characteristics, physiological-biochemical characteristics and a 16 SrDNA sequence on the obtained bacterial strain; meanwhile, characteristics such as bacteriostasis rate, growth promotion and stability of fermentation liquor of the bacterium TNSC2011 are deeply researched, so that crop growth can be effectively promoted; slightly-soluble phosphate can be converted into soluble phosphate which can be biologically utilized, so that photosynthesis is improved, and the achromobacter marplatensis is proved to be an excellent endophyte with the serial number of TNSC2011; the endophyte is applied on sugar beet cultivation to obtain a good effect, so that technical effect is outstanding and application value is wide.

Description

Raw Promoting bacteria and application thereof in a kind of beet

Technical field

The present invention relates to agriculture technical field of microbe application, specifically, the present invention relates to the technical field of raw Promoting bacteria and application thereof in a kind of beet.

Background technology

Endophyte is prevalent in plant materials, forms harmonious symbiote, produce a large amount of secondary metabolism active substance with host.It is reported, endophyte can strengthen host disease resistance, improve plant productivity, degeneration-resistant pest-resistant, there is the characteristics such as herbicidal activity.Therefore, endophyte can be used as the potential factor in biological growth-promoting, in the ecological agriculture and biological pesticide development, have important purposes.Endophytic bacterium ( endophytic bactena) promote that its host plant grows by different approaches, its effect with plant rhizosphere pre biooxidation like ( pGPR), plant growth-promoting rhizobacteria ( plant growth-promoting rhizobacteria, be called for short PGPR), survive in plant rhizosphere, root table, can promote directly or indirectly or regulating plant and plant growth.Endophytic bacterium can supply the utilization of its host plant by the nitrogen fixed in air; Synthesis siderophore can dissolve and absorb its host plant of iron supply in soil and utilize; Synthesis plant hormone promotes the growth of plant Different growth phases, has the mechanism of dissolved mineral as phosphorus, makes it more be conducive to plant utilization; Act on the relevant enzyme of growth and make growing of its regulating plant.

Beet endophyte ( endophyte) refer to that those certain phases in its life history or whole stage move in the various tissue of healthy beet and organ is inner or the fungi in intercellular substance, bacterium and actinomycetes.Beet endophyte to live in for a long time in the particular surroundings in beet body and with beet coevolution.Beet provides the required energy of growth and nutrition for it on the one hand; On the other hand, endophyte again by self meta-bolites or by means of intracellular signaling effect, beet is had an impact.Research shows that some endophyte of plant has certain growth-promoting functions to its host plant, and the increasing sugar effect on sugar crop rarely has report.

Beet is one of main raw material of the world and China's sugar industry, in China's agriculture production, occupy critical role.In Xinjiang, development beet is produced, and can not only meet the needs that the people consume sugar, promotes the development of light industry and livestock industry, can also make full use of and improve saltings.Current beet sugar industry is one of mainstay industry becoming Economy in Xinjiang.But, current Xinjiang and even national beet per unit area yield improve gradually, in block root, sucrose content but significantly declines, become the bottleneck of restriction China beet sugar industry development, trace it to its cause very complicated, various places reason is not quite similar, but common causes to be breed breeding delayed, excessively use nitrogen, also have local climatic factor.All widely use various types of sugar enriching agent both at home and abroad at present, these sugar enriching agents comprise plant-growth regulator, trace element, microorganism etc., its ultimate principle increasing sugar promotes the growth of beet tails leaf, improve plant rate of photosynthisis, increase dry-matter and sucrose accumulation, strengthen disease resistance, suppress plant above ground portion leaf growth, promote underground part block root growth and Gen Nei sucrose accumulation, reduce nitrogen metabolism and photosynthate consumption, promote that more photosynthate translocation are to root, and accumulate with the form of sugar.

Current, in production, sugar enriching agent remains based on chemical sugar enriching agent.A large amount of use chemical pesticide can destroy the eubiosis, causes agricultural-food pesticidal contamination, not only compromises the health of humans and animals, and causes the population quantity of soil microorganisms to reduce, and soil fertility reduces; Cause pathogenic bacteria on the other hand and produce resistance, make prevention effect difficulty strengthen.Therefore, we reduce chemical pesticide usage quantity to natural, ecological without the biological growth-promoting method endangered in the urgent need to using.Because endophyte is grown surely in inside plants, so beet endophyte is the desirable biological Promoting bacteria of candidate.The research of current endophyte of plant has become the focus of research both at home and abroad, and has no report about the growth-promoting functions of beet endophyte.

Summary of the invention

The present situation of the research of the growth-promoting functions about beet endophyte is had no for prior art, the present invention aims to provide a kind of beet endogenetic bacteria and application thereof, the present invention is based on the relation that endophyte of plant is combined with plant harmony, the microorganism strains of a collection of product IAA is isolated in beet, therefrom filter out the bacterial strain that a strain is numbered TNSC2011, effectively can promote plant growth, insoluble phosphate can be converted into soluble phosphate that can be bioavailable simultaneously, improve photosynthesis.Another object of the present invention is to provide the application of a kind of endophyte in beet growing.By verification experimental verification, adopt beet endogenetic bacteria provided by the invention achromobacter marplatensisbe a kind of method with promotion Sugarbeet Growth raising Sugar Content, obtain good technique effect, as beet growth-promoting endophyte, there is the extensively value be suitable for.

The present invention adopts main technical scheme:

By producing the screening of IAA microorganism in beet body, obtaining the strain number that a strain has a stronger growth-promoting activity is TNSC2011.By carrying out morphological specificity, physio-biochemical characteristics and 16S rDNA sequencing and Phylogenetic Analysis to obtained bacterial strain, tentatively determine its classification position; The characteristic such as bacteriostasis rate, growth-promoting, stability of the fermented liquid of this bacterium TNSC2011 is conducted in-depth research simultaneously, effectively can promote plant growth, insoluble phosphate can be converted into soluble phosphate that can be bioavailable simultaneously, improve photosynthesis, demonstrating and being numbered TNSC2011 is a kind of excellent endophyte, by the application of this endophyte in beet growing is obtained good action, there is outstanding technique effect.

A kind of beet endogenetic bacteria that the present invention specifically provides achromobacter marplatensis, by being separated in beet, screening and cultivating, obtain the microorganism strains of a collection of product IAA, therefrom filter out the bacterial strain that a strain is numbered TNSC2011, through microbiological classification and qualification, belong to achromobacter marplatensis.

Concrete, a kind of beet endogenetic bacteria provided by the invention achromobacter marplatensis, strain number is TNSC2011.This bacterial strain was preserved in budapest treaty microorganism International Depository Authority before the applying date: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101.Preservation date is on September 16th, 2013, and preserving number is CGMCC No. 8192.Be accredited as through microbiology achromobacter marplatensis.This bacterial strain optimum growing condition is: temperature 29.5 DEG C, and substratum adopts tryptose soya agar (TSA) substratum, culture condition: pH7.2, time 48h; This bacterial strain bacterium colony is medium and level and smooth, transparent, white, surface wettability, neat in edge, and growth is very fast; By gramstaining, microscopy is observed, and finds that this bacterial strain is Gram-negative bacteria. be accredited as through microbiology achromobacter marplatensisbacterium, cell is shaft-like, 0.8-1.0um × 1.5-2um, and leather Lan Shi feminine gender (-), does not produce bud and embrace, and bacterium colony is less and level and smooth, transparent, white, surface wettability, neat in edge.According to the 9th edition " uncle Jie Shi systematic bacteriology identification handbook " (" bergey, s Manual of Systematic Bacterio-logy") and " conventional bacterial system identification handbook " morphology mensuration is carried out to TNSC2011 bacterial strain, Physiology and biochemistry detects determines that TNSC2011 bacterial strain is the member in achromobacter.By the comparison of BLAST homology, after the 16S rDNA sequence of bacterial strain TNSC2011 carries out BLAST analysis in ncbi database, constructing system evolutionary tree, this bacterial strain TNSC2011 with achromobacter marplatensisb2(EU150134) being in minimum branch, is its allied species; And then this bacterial strain TNSC2011 is defined as achromobacter marplatensis.

The major nitrogen source that interior raw Promoting bacteria TNSC2011 provided by the invention uses when cultivating includes but not limited to peptone, yeast powder; The primary carbon source used includes but not limited to sucrose, glycerine, N.F,USP MANNITOL, maltose; The inorganic component used comprises and includes but not limited to Repone K, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, hydrogen dipotassium, tricalcium phosphate, Calcium dichloride dihydrate, bitter salt, seven water and ferrous sulfate.Plant growth-promoting rhizobacteria fermentation of the present invention can be carried out under the environment of 20-37 DEG C, pH5.5-9.1.And this interior raw Promoting bacteria TNSC2011 can high yield indolylacetic acid insoluble phosphate can be utilized to grow for phosphorus source.

Beet endophyte TNSC2011 provided by the invention has the ability of producing IAA:

When GPB nutrient solution DFsalts minimal medium (1L): 4.0g KH ₂pO ₄, 6.0g Na ₂hPO ₄, 0.2gMgSO ₄7H ₂o, 2.0g glucose, 2.0g gluconic acid, 2.0g citricacid; Trace elements: 1mg FeSO ₄7H ₂o, 10mgH ₃bO ₃, 11.1mg MnSO ₄h ₂o, 124.6mg ZnSO ₄7H ₂o, 78.22mg CuSO ₄5H ₂o, 10mg MoO ₃; PH7.2; And2.0g (NH4) ₂sO ₄in add L-Trp 100 μ gmL ^-1, all produce a large amount of IAA at 48-120h Promoting bacteria, reach maximum, up to 141.73 μ g(mLOD at the hormone of 96h secretion ₆₀₀unit) ^-1(every OD ₆₀₀the hormone-content of fermented liquid); Growth-promoting endophyte TNSC2011 has the ability of secretion plant hormone GA, all produces a large amount of GA, reach maximum, reach 1531.42 μ g(mLOD at the hormone (GA) of 120h tri-strain bacterium secretion at cultivation 48-120h Promoting bacteria ₆₀₀unit) ^-1.

Further, the invention provides beet endogenetic bacteria achromobacter marplatensisapplication in beet growing.This bacterium TNSC2011 bacterial strain has for beet the effect that growth promoting effects increases sugar content, obtains good technique effect.

Invention further provides bacterial classification beet endogenetic bacteria achromobacter marplatensisthe preservation condition of TNSC2011 CGMCC No. 8192, adopts TSA medium component: TSA Tryptones 17g, soy peptone 3g, glucose 2.5g, sodium-chlor 5g K ₂hPO ₄2.5g PH=7.0 is settled to 1000ml; Culture condition: 28 DEG C, PH=7.0.

By implementing the concrete summary of the invention of the present invention, following beneficial effect can be reached:

The beet endogenetic bacteria that separation screening of the present invention provides achromobacter marplatensistNSC2011 CGMCC No. 8192 is a kind of excellent interior raw Promoting bacteria, this interior raw Promoting bacteria TNSC2011 can high yield indolylacetic acid insoluble phosphate can be utilized to grow for phosphorus source, there is the ability of strong producing IAA and there is the ability of stronger secretion plant hormone GA, by the application in beet growing, obtain the effect that growth promoting effects increases sugar content, obtain good technique effect.

Accompanying drawing explanation

Fig. 1 is shown as beet endogenetic bacteria achromobacter marplatensisthe colonial morphology figure of TNSC2011 CGMCC No. 8192.

Fig. 2 is shown as beet endogenetic bacteria achromobacter marplatensisthe growth curve chart of TNSC2011 CGMCC No. 8192.

Fig. 3 is shown as beet endogenetic bacteria achromobacter marplatensisthe 16S rDNA phylogeny tree graph of TNSC2011 CGMCC No. 8192.

Fig. 4 is shown as beet endogenetic bacteria achromobacter marplatensisthe effect diagram of TNSC2011 CGMCC No. 8192 pairs of beet endogenous hormones indole acetic acid (IAA).

Fig. 5 is shown as beet endogenetic bacteria achromobacter marplatensisthe effect diagram of TNSC2011 CGMCC No. 8192 pairs of beet endogenous hormones abscisic acid (ABA).

Fig. 6 is shown as beet endogenetic bacteria achromobacter marplatensisthe effect diagram of TNSC2011 CGMCC No. 8192 pairs of beet endogenous hormones gibberellic acid (GA).

Fig. 7 is shown as beet endogenetic bacteria achromobacter marplatensisthe effect diagram of TNSC2011 CGMCC No. 8192 pairs of beet endogenous hormones zeatin riboside (ZR).

Fig. 8 is shown as beet endogenetic bacteria achromobacter marplatensistNSC2011 CGMCC No. 8192 grows the graph of a relation with molten phosphorus.

Fig. 9 is shown as beet endogenetic bacteria achromobacter marplatensisthe graph of a relation of the molten phosphorus of TNSC2011 CGMCC No. 8192 and pH.

Embodiment

, for embodiment, the present invention is described below, but the present invention is not limited to following embodiment.

The main raw and auxiliary material, reagent and the plant and instrument that relate in the present invention:

The substratum that the present invention selects and other material: DFsalts minimal medium (1L): 4.0g KH ₂pO ₄, 6.0g Na ₂hPO ₄, 0.2gMgSO ₄7H ₂o, 2.0g glucose, 2.0g gluconic acid, 2.0g citricacid; Trace elements: 1mg FeSO ₄7H ₂o, 10mgH ₃bO ₃, 11.1mg MnSO ₄h ₂o, 124.6mg ZnSO ₄7H ₂o, 78.22mg CuSO ₄5H ₂o, 10mg MoO ₃; PH7.2; And2.0g (NH ₄) ₂sO ₄in add L-Trp 100 μ gmL ^-1.0.7mmolL ^-1phosphoric acid buffer (pH6.9): use 0.2606gL ^-1na ₂hPO ₄12H ₂o adjusts 0.1092gL ^-1naH ₂pO ₄2H ₂o to pH6.9.

Key instrument and reagent: TG328A type analysis balance, AB204-N type electronic balance, PHS-3TC(0.01 level) precise digital display acidometer, 101-2 type loft drier, UV-2100 spectrophotometer, HYG-II rotary type constant temperature speed governing shaking flask cabinet, LS-B50L vertical pressure steam sterilizer, Bechtop, 3K15 tabletop refrigerated centrifuge; Glycerine, extractum carnis, yeast extract paste, sodium-chlor, sodium hydroxide, glucose, magnesium sulfate, zinc sulfate, ferrous sulfate, SODIUM PHOSPHATE, MONOBASIC, Sodium phosphate dibasic, potassium primary phosphate, agar, sucrose, clorox, PCR premixed liquid (TaKaRa Biotechnology), all the other reagent are analytical pure.

The all raw and auxiliary materials selected in the present invention, and the spawn culture method selected is all well known selecting, the % related in the present invention is weight percentage, unless otherwise indicated.

embodiment one: beet endogenetic bacteria achromobacter marplatensis the separation of TNSC2011 CGMCC No. 8192, screening and qualification

(1) Isolation and screening of bacterial classification

Isolate by gradient dilution method the bacterium that IAA is produced in many strains from healthy beet, therefrom filter out the bacterial strain that a strain is numbered TNSC2011, effectively can promote plant growth, insoluble phosphate can be converted into soluble phosphate that can be bioavailable simultaneously, improve photosynthesis.By applying in beet growing, there is outstanding growth-promoting functions.

This bacterial strain bacterium colony is less and level and smooth, transparent, white, surface wettability, neat in edge, and growth is very fast; By gramstaining, microscopy is observed, and finds that this bacterial strain is Gram-negative bacteria. be accredited as through microbiology achromobacter marplatensisbacterium, cell is shaft-like, 0.8-1.0um × 1.5-2um, and leather Lan Shi feminine gender (-), does not produce bud and embrace, and bacterium colony is medium and level and smooth, opaque, white, surface wettability, neat in edge.See accompanying drawing 1.This bacterium is strictly aerobic, oxidase positive; Glucose, wood sugar can not be utilized to produce acid, can not hydrolyzed starch.According to the 9th edition " uncle Jie Shi systematic bacteriology identification handbook " (" bergey, s Manual of Systematic Bacterio-logy") and " conventional bacterial system identification handbook " morphology mensuration is carried out to TNSC2011 bacterial strain, Physiology and biochemistry detects determines that TNSC2011 bacterial strain is the member in achromobacter.By the comparison of BLAST homology, after the 16S rDNA sequence of bacterial strain TNSC2011 carries out BLAST analysis in ncbi database, constructing system evolutionary tree, this bacterial strain TNSC2011 with achromobacter marplatensisb2(EU150134) being in minimum branch, is its allied species; And then this bacterial strain TNSC2011 is defined as achromobacter marplatensis.

Carry out morphology mensuration by above-mentioned to TNSC2011 bacterial strain, and detect the shape of bacterial strain TNSC2011, size, biochemical reactions, TNSC2011 Biological Characteristics of Strain of the present invention is as shown in table 1.

Table 1: the physio-biochemical characteristics of bacterial strain TNSC2011

(2) PCR amplification endophyte 16S rDNA sequence and order-checking thereof

Single bacterium colony of picking a small amount of TNSC2011 bacterium, put into the EP pipe filling 25 μ L sterilized waters, 100 DEG C are boiled 8-10 min, put into rapidly mixture of ice and water 5 min afterwards.Centrifugal 10000 r/min, 5 min, 4 DEG C of preservations, the used time gets supernatant.PCR increases achromobacter marplatensis16S rDNA sequence.

Primer 1 (27F): 5 '-TCCTCCGCTTATTGATATGC-3 ';

Primer 2 (1492R): 5 '-CAAACTTGGTCATTAGAGGA-3 '.

PCR amplification reaction system is 50 μ L, and containing 24 μ L premix Taq, primer 1 is 1 μ L, and primer 2 is 1 μ L, template 2 μ L, sterilized water 22 μ L.Amplification condition: 94 DEG C of 4 min; 94 DEG C of 30 s, 55 DEG C of 90s, 72 DEG C of 30s, 30 circulations; 72 DEG C of 7min.Through 1% agarose gel electrophoresis isolation identification, PCR product directly carries out two-way order-checking to amplified production (about 1500 bp), and the gene order of t bacteria NSC2011 is see attached gene order table.

(3) 16S rDNA sequence alignment and Phylogenetic Analysis

Nucleotide sequence in the 16S rDNA sequence and GenBank database that obtain checking order carries out BLAST analysis, therefrom obtain close 16S rDNA sequence, with ClustalX software and MEGA 4.1Neighbor-joining method constructing system evolutionary tree, see accompanying drawing 3.After the 16S rDNA sequence of TNSC2011 is carried out BLAST analysis in ncbi database, constructing system evolutionary tree.Shown in accompanying drawing 3, bacterial strain TNSC2011 with achromobacter marplatensisb2(EU150134) between, evolutionary distance is the shortest, is achromobacter marplatensisallied species.

In sum, in conjunction with Morphologic Characteristics and the physio-biochemical characteristics of TNSC2011, beet endogenetic bacteria is determined that it is achromobacter marplatensistNSC2011 CGMCC No. 8192.

embodiment two: beet endogenetic bacteria achromobacter marplatensi s the somatomedin of TNSC2011 CGMCC No. 8192

But should according to beet endogenetic bacteria provided by the invention achromobacter marplatensistNSC2011 CGMCC No. 8192 characteristic determines its concrete somatomedin, bacterial strain TNSC2011 inoculation culture.The results are shown in Table 2.

Table 2: the impact that temperature, pH, salt, microbiotic grow bacterial strain TNSC2011

Temperature (DEG C)	4	15	25	30	32	35
							Growing state	—	+	+	+++	++++	+++
Temperature (DEG C)	38	45	50
							Growing state	++	-	—
pH	1	2	3	4	5	6
							Growing state	—	—	—	—	+	++
pH	7	8	9	10
							Growing state	+++	+	—	—
NaCl concentration	0.5％	1％	2％	3％	4％	5％
							Growing state	+	++	+++	+++	+++	+
NaCl concentration	6％	7％	8％	9％	10％
							Growing state	—	—	—	—	—
Penbritin μ g/ml	50	60	70	80	90	100
							Growing state	—	—	—	—	—	—

2.6.1 growth curve and culture condition

According to such as upper type will achromobacter marplatensis tNSC2011 CGMCC No. 8192cultivate, the culture condition of bacterial classification is: substratum is LB liquid nutrient medium, culture condition: pH7.0, and cultivate 16h under temperature 35 DEG C of conditions, its growth curve is see accompanying drawing 2.

Table 3:pH optimum result

	6.0	6.5	7.0*	7.5	8.0
						TNSC2011	0.351	0.672	0.897	0.668	0.025

Drawn by table 3, the most applicable somatomedin of bacterial strain TNSC2011.

Show that TNSC2011 be 20h, the most suitable growth pH as the best incubation time of seed is 7.0 by above result.

embodiment three: beet endogenetic bacteria achromobacter marplatensis the Study on Fermentation of TNSC2011 CGMCC No. 8192

Realize fermenting by the optimization of carbon and nitrogen sources, pH value etc. and obtain the object of preparation.

(1) determination of medium pH

By pH value 6.0,6.5,7.0,7.5,8.0, at 120 r/min, temperature 35 DEG C of condition bottom fermentations, draw the most suitable growth.

(2) optimization of Carbon and nitrogen sources

Seed liquid nutrient medium: Semen Maydis powder 2.0%, yeast extract paste 0.5%, corn steep liquor 7.5%, KH ₂pO ₄0.1%, K ₂hPO ₄0.1%, MgSO ₄7H ₂o0.05%, the basis of seed culture medium is continued optimize;

A. carbon source optimizing:

Carbon source is respectively glucose, sucrose, Semen Maydis powder, Zulkovsky starch, and other compositions are KH ₂pO ₄0.1%, K ₂hPO ₄0.1%, MgSO ₄7H ₂o0.05% PH7.0;

B. nitrogenous source optimization:

Nitrogenous source is respectively peptone, yeast extract paste, (NH ₄) ₂sO ₄, other compositions are tested with carbon source optimizing.Test is 3 repetitions.

Detected by different carbon source and nitrogenous source OD value, draw the suitableeest Carbon and nitrogen sources of bacterial strain.

OD value: get fermented liquid 0.5mL, adds 2.5M HCL1mL, adds 13.5mL deionized water, and mixing, with spectrophotometer, measures under 600nm wavelength.

(3) fermentation technology optimization result

3.1. fermention medium carbon and nitrogen sources optimum result

Table 4: test design optimizes optimum carbon source-OD value in fermention medium

	Glucose	Sucrose	Semen Maydis powder	Zulkovsky starch
					TNSC2011	0.679	0.845	0.912	0.973*

Table 5: test design optimizes optimum nitrogen source-OD value in fermention medium

	Peptone	Yeast extract paste	（NH 4） 2 SO 4
				TNSC2011	0.785	0.861*	0.633

According to table 4,5, above result determination optimum carbon source is Zulkovsky starch, and optimum nitrogen source is yeast extract paste.

3.2. 10L ferment tank result

In the substratum being main raw material with corn steep liquor and Semen Maydis powder, fermentation condition is: temperature 32 DEG C, rotating speed 250rpm, and ventilation 1:0.3, the time, it the results are shown in as following table 6 at 24h.

Table 6:10L fermenting experiment result

Bacterial strain	Fermentation time	Extension rate	OD 600nm
				TNSC2011	24h	30	0.583

Fermentation ends rear plate count results shows, almost all survives, and through frozen dried, thalline survival rate is higher than 80%.

embodiment four: beet endogenetic bacteria achromobacter marplatensis tNSC2011 CGMCC No. 8192 secretes the test of plant hormone

Raw Promoting bacteria secretion plant hormone IAA(indolylacetic acid in beet) characteristic in table 8, growth-promoting endophyte TNSC2011 has the ability of producing IAA, when adding L-Trp 100 μ gmL in DFsalts minimal medium substratum ^-1, all produce a large amount of IAA at 48-120h Promoting bacteria, reach maximum, up to 141.73 μ g(mLOD at the hormone of 96h secretion ₆₀₀unit) ^-1(every OD ₆₀₀the hormone-content of fermented liquid).

In beet, the characteristic of raw Promoting bacteria secretion plant hormone GA is in table 9, growth-promoting endophyte TNSC2011 has the ability of secretion GA, all produce a large amount of GA at cultivation 48-120h Promoting bacteria, reach maximum at the hormone (GA) of 120h tri-strain bacterium secretion, reach 1531.42 μ g(mLOD ₆₀₀unit) ^-1, along with the prolongation of incubation time, GA output is on a declining curve, cultivates 144h Promoting bacteria GA output and declines to some extent.

Beet pot experiment: the Dual culture of endophyte and thinning beet test tube: 1. bacterial classification preculture: endogenetic bacteria nutrient solution is diluted to 1.64 × 106 CFUml-1 respectively.2. the preculture of Sugarbeet: take out seed from beet kind ball, after carrying out surface sterilization, proceeds to seedling in MS substratum by seed, culture temperature 25 DEG C, and light application time 12h, proceeds to when being cultured to height of seedling about 2cm in Dual culture base.3. the screening of Dual culture base: by MS(I) substratum based on substratum, prepare 1/2MS(II respectively), l/3MS(III), l/4MS(IV) substratum of 4 kinds of vegetative gradients, often kind of substratum additional saccharose 10 gL-1, agar 1.0 gL-1, pH 5.8.4. Dual culture: Dual culture base central authorities bacterial classification pre-culture solution 10mL being accessed own sterilizing, the Sugarbeet (E+) that every bottle of 5 strain access growing ways are consistent.Meanwhile, often kind of substratum all establishes blank (namely E-only connects test-tube plantlet and do not connect bacterium), and often kind is treated to 3 bottles.

Transplant experiment: together taking out together with culturing bottle with the Sugarbeet (E+) of endophyte Dual culture and blank (E-) of about 30d will be cultivated respectively, after room temperature condition lower refining seedling 1-2 week, take out after cleaning attachment substratum and carry out respectively transplanting in aseptic basin alms bowl.After cultivating for some time, measure its increment and transplanting survival rate.This test adopts pot-culture method to carry out in the culturing room of Artificial Control temperature, illumination and humidity.By soil disinfection (121 DEG C, 2h), load the plastic tub of surface sterilization, every basin dress sterilized soil 0.8kg, base fertilizer is that every kilogram of soil executes N 0.02g, P ₂o ₅0.015g, K ₂o 0.020g.Beet adopts plastic nutritional alms bowl to plant.Nutritive medium is Hoagland nutritive medium.Intensity of illumination 14000-15000lx between incubation period, illumination every day 12h, diurnal temperature 20-31 DEG C, humidity 60%-80%.After transplanting, 30d measures beet plant height, root length, fresh weight, dry weight, the number of blade.The mensuration of Sugarbeet Growth index: respectively after Dual culture for some time, thinning beet is taken out from basin, clean surface attachment material, and use filter paper suck dry moisture, by fresh weight, the dry weight of the every strain thinning beet of electronics balance measurement, measure plant height with ruler and write down the number of blade, often organizing and measure 20 strains altogether, record also processes with statistical method, the significance of difference of relatively more each group.

The test-results of beet shows, in table 7, compared with the control, different endophyte fermented liquid has different impacts to Sugarbeet Growth.Endophyte fermented liquid improves 55.4%, 59.0 %, 100%, 210.3%, 50.0% respectively to the plant height of beet, fresh weight, dry weight, the number of blade, and difference reaches conspicuous level.

Table 7: endophyte is on the impact of thinning beet

Table 8: endophyte is IAA output in DFsalts minimal medium substratum

Mean ± standard error from three separate experiments

In beet, the characteristic of raw Promoting bacteria secretion plant hormone ABA is in table 10, growth-promoting endophyte TNSC2011 has the ability of secretion ABA, all produce a large amount of ABA at 48-120h Promoting bacteria, the hormone (ABA) secreted at 120h bacterial strain TNSC2011 reaches maximum, reaches 538.15 μ g(mlOD ₆₀₀unit) ^-1, along with the prolongation of incubation time, ABA output is on a declining curve, cultivates 144h Promoting bacteria ABA output and reduces.

In beet, the characteristic of raw Promoting bacteria secretion plant hormone ZR is in table 11, growth-promoting endophyte TNSC2011 has the ability of secretion ZR, all produce a large amount of ZR at cultivation 48-120h Promoting bacteria, reach maximum at the hormone (ZR) of 96h tri-strain bacterium secretion, reach 117.89 μ g(mlOD ₆₀₀unit) ^-1, along with the prolongation of incubation time, ZR output is on a declining curve, cultivates 144h Promoting bacteria ZR output and declines all to some extent.

Table 9: endophyte is GA output in DFsalts minimal medium substratum

Mean ± standard error from three separate experiments

Table 10: endophyte is ABA output in DFsalts minimal medium substratum

Mean ± standard error from three separate experiments

Table 11: endophyte is ZR output in DFsalts minimal medium substratum

Mean ± standard error from three separate experiments

In beet, the characteristic of raw Promoting bacteria secretion vitamin V b2 is in table 12, and growth-promoting endophyte TNSC2011 has the ability of secretion Vb2, all produces a large amount of Vb2, reach maximum, reach 2.45 × 10 at the hormone of 96h tri-strain bacterium secretion at cultivation 48-120h Promoting bacteria ^-3ppm, along with the prolongation of incubation time, this bacterium Vb2 output is on a declining curve, cultivates 144h Promoting bacteria Vb2 output and declines to some extent.

In beet, the characteristic of raw Promoting bacteria secretion vitamin V c is in table 13, growth-promoting endophyte TNSC2011 has the ability of secretion Vc, a large amount of Vc is all produced at cultivation 48-120h Promoting bacteria, maximum is reached at the hormone of 96h secretion, for 1.29ppm, along with the prolongation of incubation time, Vc output is on a declining curve, cultivates 144h Promoting bacteria Vc output and reduces.

table 12: endophyte existstSA vb2 output in substratum

Mean ± standard error from three separate experiments

table 13: endophyte existstSA vc output in substratum

Mean ± standard error from three separate experiments

Beet hormone in vivo content after interior raw Promoting bacteria process

After interior raw Promoting bacteria process beet, plant endogenous hormones content characteristic is see accompanying drawing 4, Fig. 5, Fig. 6, Fig. 7, and after interior raw Promoting bacteria process, beet endogenous hormones compared with the control, and four kinds of sugar beet plants endogenous hormones output all have significant difference.

After the process of interior raw Promoting bacteria bacterial strain, the change of the plant endogenous hormones content such as beet tumor growth element (IAA), zeatin (ZR), Plant hormones regulators,gibberellins (GA), dormin (ABA) is as Fig. 4, Fig. 5, Fig. 6, Fig. 7.Result of study shows, growth hormone, zeatin, Plant hormones regulators,gibberellins etc. after the process of interior raw Promoting bacteria bacterial strain in beet body grow the content of relevant endogenous hormones than the height contrasted with Promoting plant growth, after endophyte fills with root, Plant hormones regulators,gibberellins (GA) in beet body is on a declining curve, peak value is reached, than contrast raising 22.35 times during 5d; Growth hormone IAA is in the trend of falling after rising, and after filling root, 15d reaches peak value, than contrast raising 1.59 times; Zeatin (ZR) is in the trend of falling after rising, and after filling root, 20d reaches peak value, than contrast raising 1.06 times; Dormin (ABA) is in bimodal trend, and after filling root, 25d reaches peak value, than contrast reduction by 0.53 times.

Forefathers' research shows, Plant hormones regulators,gibberellins can promote the growth of overground part cauline leaf, lateral bud, breaks seed dormancy, and induced flowering.IAA is not only combined by proton pump on plasma membrane and makes it activation, change intracellular environment cause cell wall sugar to dissolve and plasticity-increase to increase cell volume and promotion NRA(nitrate reductase activity), the synthesis of protein, increase cell volume and quality to reach the effect of growth-promoting; And, the more important thing is and tryptophane (Trp) analogue is detoxified, alleviate its toxic action; In addition, plant defenses enzyme can also be suppressed, as the activity of chitinase (ehitinase), beta-1,3-glucanase (β-1,3-polyglucosanase) etc.; And AAB has restraining effect to plant materials.Hormone such as the IAA content that host plant is relevant with growth after the process of interior raw Promoting bacteria bacterial strain from the above experimental results increases, and suppresses the hormone of plant-growth such as ABA content to decline.

embodiment five: beet endogenetic bacteria achromobacter marplatensi s the impact of TNSC2011 CGMCC No. 8192 on phosphorus decomposing

By the original preservation bacterial strain of the endophyte screened in PKO nutrient solution 30 DEG C of activation, 200rmin ^-1shaking culture 12h, is diluted to 108CFUml with sterilized water ^-1as seed.2ml seed is inoculated, to inoculate 2ml sterilized water in contrast in 200ml nutrient solution.Every 24h measures 1 endophyte dissolving P capacity, until cultivate 7d.Endophyte fermentation broth sample 10000rmin ^-1centrifugal 20min, measures supernatant liquor pH value with pH meter.Titanium pigment molybdenum antimony resistance colorimetric method (λ=690nm) measures.The making of P typical curve, by 100mgL ^-1mother liquor dilution for series concentration, the method identical with test sample product measures light absorption value.The pH value of nutrient solution pH instrument measures ( sartoriuspD20).The phosphate solubilization of interior raw growth-promoting bacterial strain is determined under liquid culture condi.Result is see accompanying drawing 8, Fig. 9, and bacterial strain has dissolved metals ability, and amount of phosphorus dissolved reaches 189.22 μ gmL ^-1, dissolved metals Ca ₃(PO ₄) ₂ability is better.The pH value of endophyte dissolving P capacity and substratum has certain relation.Compared with the control, interior raw Promoting bacteria can remarkable molten phosphorus.

sequence table

SEQUENCE LISTING

 

<110> Microorgan Application Inst., Xinjiang Agricultural Academy

<120> TNSC2011 16SrDNA primer and 16SrDNA sequence

Raw Promoting bacteria and application thereof in <130> beet

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> bacterial strain TNSC2011 16SrDNA upstream primer sequence

<400> 1

agagtttgat cmtggctcag 20

<210> 2

<211> 22

<212> DNA

<213> bacterial strain TNSC2011 16SrDNA downstream primer sequence

<400> 2

tacggytacc ttgttacgac tt 22

<210> 3

<211> 1435

<212> DNA

<213> Achromobacter marplatensis TNSC2011

<400> 3

taatacgggc tgcattacca tgcaagtcga acggcagcac ggacttcggt ctggtggcga 60

gtggcgaacg ggtgagtaat gtatcggaac gtgcctagta gcgggggata actacgcgaa 120

agcgtagcta ataccgcata cgccctacgg gggaaagcag gggatcgcaa gaccttgcac 180

tattagagcg gccgatatcg gattagctag ttggtggggt aacggctcac caaggcgacg 240

atccgtagct ggtttgagag gacgaccagc cacactggga ctgagacacg gcccagactc 300

ctacgggagg cagcagtggg gaattttgga caatggggga aaccctgatc cagccatccc 360

gcgtgtgcga tgaaggcctt cgggttgtaa agcacttttg gcaggaaaga aacgtcatgg 420

gctaataccc cgtgaaactg acggtacctg cagaataagc accggctaac tacgtgccag 480

cagccgcggt aatacgtagg gtgcaagcgt taatcggaat tactgggcgt aaagcgtgcg 540

caggcggttc ggaaagaaag atgtgaaatc ccagagctta actttggaac tgcattttta 600

actaccgagc tagagtgtgt cagagggagg tggaattccg cgtgtagcag tgaaatgcgt 660

agatatgcgg aggaacaccg atggcgaagg cagcctcctg ggataacact gacgctcatg 720

cacgaaagcg tggggagcaa acaggattag ataccctggt agtccacgcc ctaaacgatg 780

tcaactagct gttggggcct tcgggccttg gtagcgcagc taacgcgtga agttgaccgc 840

ctggggagta cggtcgcaag attaaaactc aaaggaattg acggggaccc gcacaagcgg 900

tggatgatgt ggattaattc gatgcaacgc gaaaaacctt acctaccctt gacatgtctg 960

gaattccgaa gagatttgga agtgctcgca agagaaccgg aacacaggtg ctgcatggct 1020

gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca acccttgtca 1080

ttagttgcta cgaaagggca ctctaatgag actgccggtg acaaaccgga ggaaggtggg 1140

gatgacgtca agtcctcatg gcccttatgg gtagggcttc acacgtcata caatggtcgg 1200

gacagagggt cgccaacccg cgagggggag ccaatcccag aaacccgatc gtagtccgga 1260

tcgcagtctg caactcgact gcgtgaagtc ggaatcgcta gtaatcgcgg atcagcatgt 1320

cgcggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccatgg gagtgggttt 1380

taccagaagt agttagccta accgcaaggg gggcgatacc acggtagatt caggt 1435

 

Claims (2)

1. a beet endogenetic bacteria achromobacter marplatensistNSC2011, is characterized in that, described beet endogenetic bacteria achromobacter marplatensisthe culture presevation number of TNSC2011 is CGMCC No. 8192.

2. a beet endogenetic bacteria as claimed in claim 1 achromobacter marplatensisthe application of TNSC2011 in beet growing.