CN103642733A - 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 growth-promoting bacterium 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 the interior raw growth-promoting bacterium of a kind of beet and application thereof.

Background technology

Endophyte is prevalent in plant materials, forms harmonious symbiote with host, produces a large amount of secondary metabolism active substances.It is reported, endophyte can strengthen host's disease resistance, improve the productivity of plant, 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, aspect the ecological agriculture and biological pesticide development, has important purposes.Endophytic bacterium ( endophytic bactena) can promote its host plant to grow by different approaches, its effect similar to plant rhizosphere bacterium ( pGPR), plant growth-promoting rhizobacteria ( plant growth-promoting rhizobacteria, be called for short PGPR), survive in plant rhizosphere, root table, promotion directly or indirectly or regulating plant and plant growth.The endophytic bacterium fixedly nitrogen in atmosphere is supplied with its host plant utilization; Synthetic siderophore can dissolve and absorb its host plant utilization of iron supply in soil; Synthetic plant hormone promotes the growth of the different growth phases of plant, has dissolved mineral as the mechanism of 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 fungi, bacterium and the actinomycetes in the various tissues of healthy beet and organ inside or intercellular substance.Beet endophyte live in for a long time in the particular surroundings in beet body and with beet coevolution.Beet provides growth essential energy and nutrition for it on the one hand; On the other hand, endophyte can or exert an influence to beet by means of signal conduction by the meta-bolites of self again.Research shows that some endophyte of plant has certain growth-promoting functions to its host plant, and the effect of increasing on sugar crop sugar rarely has report.

Beet is one of main raw material of the world and China's sugar industry, in China's agriculture production, occupies critical role.In Xinjiang, development beet is produced, and can not only meet the needs of the people to sugar consumption, promotes the development of light industry and livestock industry, can also make full use of and improve saltings.Beet sugar industry has become one of mainstay industry of Economy in Xinjiang at present.Yet, Xinjiang and even national beet per unit area yield improve gradually at present, in piece 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 is breed breeding, lags behind, the excessive nitrogen of using, also has local climatic factor.All be widely used at present various types of sugar enriching agents both at home and abroad, these sugar enriching agents comprise plant-growth regulator, trace element, microorganism etc., it increases sugared ultimate principle is to promote the growth of beet tails leaf, improve plant rate of photosynthisis, increase dry-matter and sucrose accumulation, strengthen disease resistance, suppress plant overground part leaf growth, promote underground part piece root growth and Gen Nei sucrose accumulation, reduce nitrogen metabolism and photosynthate consumption, promote that more photosynthates are transported to root, and accumulate with the form of sugar.

Current, in production, sugar enriching agent remains and take chemical sugar enriching agent as main.A large amount of chemical pesticides that use can destroy the eubiosis, cause agricultural-food pesticidal contamination, have not only damaged the health of humans and animals, and cause that the population quantity of soil microorganisms reduces, and soil fertility reduces; Cause on the other hand pathogenic bacteria and produce resistance, make prevention effect difficulty strengthen.Therefore, we reduce chemical pesticide usage quantity to natural, ecological without the biological growth-promoting method endangering in the urgent need to using.Because endophyte is grown surely in inside plants, so beet endophyte is the desirable biological growth-promoting bacterium of candidate.Endophyte of plant research has at present become the focus of domestic and international research, and the growth-promoting functions of relevant beet endophyte has no report.

Summary of the invention

For prior art, have no the present situation of research of the growth-promoting functions of relevant beet endophyte, the present invention aims to provide a kind of beet endogenetic bacteria and application thereof, the present invention is based on endophyte of plant and the harmonious relation of combining of plant, in beet, isolate the microorganism strains of a collection of product IAA, therefrom filter out the bacterial strain that a strain is numbered TNSC2011, can effectively promote plant growth, insoluble phosphate can be converted into simultaneously can be bioavailable soluble phosphate, 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 that promotes Sugarbeet Growth to improve Sugar Content that has, obtain good technique effect, as beet growth-promoting endophyte, there is extensive and applicable value.

The present invention adopts main technical scheme:

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

A kind of beet endogenetic bacteria that the present invention specifically provides achromobacter marplatensis, by separation in beet, screening and cultivation, obtain the microorganism strains of a collection of product IAA, therefrom filter out the bacterial strain that a strain is numbered TNSC2011, through microbiology classification and identification, 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 the international depositary institution of budapest treaty microorganism 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.Through microbiology, be accredited as achromobacter marplatensis.This bacterial strain optimum growing condition is: 29.5 ℃ of temperature, 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, is 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. through microbiology, be accredited as achromobacter marplatensisbacterium, cell is shaft-like, 0.8-1.0um * 1.5-2um, leather Lan Shi negative (-), does not produce bud and embraces, and bacterium colony is less and level and smooth, is 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 the conventional bacterial system identification handbook > > of < < TNSC2011 bacterial strain is carried out to morphology mensuration, Physiology and biochemistry detects determines that TNSC2011 bacterial strain is the member in achromobacter.By BLAST homology, compare, the 16S rDNA sequence of bacterial strain TNSC2011 is carried out after BLAST analysis in ncbi database, constructing system evolutionary tree, this bacterial strain TNSC2011 with achromobacter marplatensisb2(EU150134), in minimum branch, be its allied species; And then this bacterial strain TNSC2011 is defined as achromobacter marplatensis.

The major nitrogen source that interior raw growth-promoting bacterium TNSC2011 provided by the invention is used while cultivating includes but not limited to peptone, yeast powder; The main carbon source of using includes but not limited to sucrose, glycerine, N.F,USP MANNITOL, maltose; The inorganic component using 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 at 20-37 ℃, under the environment of pH5.5-9.1, carries out.And this interior raw growth-promoting bacterium TNSC2011 can also can utilize insoluble phosphate for growing in phosphorus source by high yield indolylacetic acid.

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

When at 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, 48-120h growth-promoting bacterium, all producing a large amount of IAA, the hormone of secreting at 96h reaches maximum, up to 141.73 μ g(mLOD ₆₀₀unit) ^-1(every OD ₆₀₀the hormone-content of fermented liquid); Growth-promoting endophyte TNSC2011 has the ability of secretion plant hormone GA, cultivation 48-120h growth-promoting bacterium, all produces a large amount of GA, and the hormone (GA) of secreting 120h tri-strain bacterium reaches maximum, reaches 1531.42 μ g(mLOD ₆₀₀unit) ^-1.

Further, the invention provides beet endogenetic bacteria achromobacter marplatensisapplication in beet growing.This bacterium TNSC2011 bacterial strain has the effect that promotes growth to increase sugar content for beet, obtain good technique effect.

The present 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 ℃, PH=7.0.

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

The beet endogenetic bacteria that separation screening of the present invention provides achromobacter marplatensistNSC2011 CGMCC No. 8192 is a kind of good interior raw growth-promoting bacterium, this interior raw growth-promoting bacterium TNSC2011 can also can utilize insoluble phosphate for growing in phosphorus source by high yield indolylacetic acid, the ability and the ability with more intense secretion plant hormone GA with strong producing IAA, by the application in beet growing, obtain the effect that promotes growth to increase 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 affect figure of TNSC2011 CGMCC No. 8192 on beet endogenous hormones indole acetic acid (IAA).

Fig. 5 is shown as beet endogenetic bacteria achromobacter marplatensisthe affect figure of TNSC2011 CGMCC No. 8192 on beet endogenous hormones abscisic acid (ABA).

Fig. 6 is shown as beet endogenetic bacteria achromobacter marplatensisthe affect figure of TNSC2011 CGMCC No. 8192 on beet endogenous hormones gibberellic acid (GA).

Fig. 7 is shown as beet endogenetic bacteria achromobacter marplatensisthe affect figure of TNSC2011 CGMCC No. 8192 on beet endogenous hormones zeatin riboside (ZR).

Fig. 8 is shown as beet endogenetic bacteria achromobacter marplatensisthe graph of a relation of TNSC2011 CGMCC No. 8192 growths and molten phosphorus.

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

Embodiment

Below, for embodiment, the present invention is described, still, the present invention is not limited to following embodiment.

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

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.

All raw and auxiliary materials of selecting in the present invention, and the spawn culture method of selecting is all well known selecting, the % relating in the present invention is weight percentage, unless otherwise indicated except.

embodiment mono-: beet endogenetic bacteria achromobacter marplatensis separation, screening and the evaluation of TNSC2011 CGMCC No. 8192

(1) Isolation and screening of bacterial classification

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

This bacterial strain bacterium colony is less and level and smooth, is 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. through microbiology, be accredited as achromobacter marplatensisbacterium, cell is shaft-like, 0.8-1.0um * 1.5-2um, leather Lan Shi negative (-), does not produce bud and embraces, and bacterium colony is medium and level and smooth, opaque, white, surface wettability, neat in edge.Referring to accompanying drawing 1.This bacterium is strictly aerobic, oxidase positive; Can not utilize glucose, wood sugar 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 the conventional bacterial system identification handbook > > of < < TNSC2011 bacterial strain is carried out to morphology mensuration, Physiology and biochemistry detects determines that TNSC2011 bacterial strain is the member in achromobacter.By BLAST homology, compare, the 16S rDNA sequence of bacterial strain TNSC2011 is carried out after BLAST analysis in ncbi database, constructing system evolutionary tree, this bacterial strain TNSC2011 with achromobacter marplatensisb2(EU150134), in minimum branch, be its allied species; And then this bacterial strain TNSC2011 is defined as achromobacter marplatensis.

By above-mentioned, TNSC2011 bacterial strain is carried out to morphology mensuration, and the shape of bacterial strain TNSC2011, size, biochemical reactions are detected, 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 a small amount of TNSC2011 bacterium of picking, puts into the EP pipe that fills 25 μ L sterilized waters, and 100 ℃ are boiled 8-10 min, rear mixture of ice and water 5 min that put into rapidly.Centrifugal 10000 r/min, 5 min, 4 ℃ of preservations, the used time is got supernatant.PCR amplification achromobacter marplatensis16S rDNA sequence.

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

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

PCR amplification reaction system is 50 μ L, contains 24 μ L premix Taq, and 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 ℃ of 4 min; 94 ℃ of 30 s, 55 ℃ of 90s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 7min.Amplified production (approximately 1500 bp) is through 1% agarose gel electrophoresis isolation identification, and PCR product directly carries out two-way order-checking, and the gene order of t bacteria NSC2011 is referring to attached gene order table.

(3) 16S rDNA sequence alignment and Phylogenetic Analysis

The 16S rDNA sequence that order-checking is obtained and the nucleotide sequence in GenBank database carry out BLAST analysis, therefrom obtain close 16S rDNA sequence, with ClustalX software and MEGA 4.1Neighbor-joining method constructing system evolutionary tree, referring to accompanying drawing 3.The 16S rDNA sequence of TNSC2011 is carried out after BLAST analysis in ncbi database to 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, Morphologic Characteristics and physio-biochemical characteristics in conjunction with TNSC2011, determine that it is beet endogenetic bacteria achromobacter marplatensistNSC2011 CGMCC No. 8192.

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

But should be according to beet endogenetic bacteria provided by the invention achromobacter marplatensistNSC2011 CGMCC No. 8192 characteristics are determined the somatomedin that it is concrete, bacterial strain TNSC2011 inoculation culture.The results are shown in Table 2.

Table 2: temperature, pH, salt, the impact of microbiotic on bacterial strain TNSC2011 growth

Temperature (℃)	4	15	25	30	32	35
							Growing state	—	+	+	+++	++++	+++
Temperature (℃)	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 inciting somebody to action as upper type achromobacter marplatensis tNSC2011 CGMCC No. 8192cultivate, the culture condition of bacterial classification is: substratum is LB liquid nutrient medium, and culture condition: pH7.0 cultivates 16h under 35 ℃ of conditions of temperature, and its growth curve is referring to 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

By table 3, drawn the most applicable somatomedin of bacterial strain TNSC2011.

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

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

Optimization by carbon and nitrogen sources, pH value etc. realizes the object that fermentation obtains preparation.

(1) medium pH determines

Press pH value 6.0,6.5,7.0,7.5,8.0, at 120 r/min, 35 ℃ of condition bottom fermentations of temperature, 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% continues to optimize on the basis of seed culture medium;

A. carbon source optimizing:

Carbon source is respectively glucose, sucrose, and Semen Maydis powder, Zulkovsky starch, 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.

By different Carbon and nitrogen sources OD values, detect, draw the suitableeest Carbon and nitrogen sources of bacterial strain.

OD value: get fermented liquid 0.5mL, add 2.5M HCL1mL, add 13.5mL deionized water, mix, with spectrophotometer, measure under 600nm wavelength.

(3) fermentation technology optimization result

3.1. fermention medium carbon and nitrogen sources optimum result

Table 4: test design is optimized 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 is optimized 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 determines that optimum carbon source is Zulkovsky starch, and optimum nitrogen source is yeast extract paste.

3.2. 10L ferment tank result

Take in the substratum that corn steep liquor and Semen Maydis powder be main raw material, fermentation condition is: 32 ℃ of temperature, and rotating speed 250rpm, 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 survivals, and through frozen dried, thalline survival rate is higher than 80%.

embodiment tetra-: beet endogenetic bacteria achromobacter marplatensis the test of TNSC2011 CGMCC No. 8192 secretion plant hormones

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

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

Beet pot experiment: the common cultivation of endophyte and thinning beet test tube: 1. bacterial classification preculture: endogenetic bacteria nutrient solution is diluted to respectively to 1.64 * 106 CFUml-1.2. the preculture of Sugarbeet: from beet kind ball, take out seed, carry out, after surface sterilization, seed being proceeded to seedling in MS substratum, 25 ℃ of culture temperature, light application time 12h, proceeds in common substratum while being cultured to height of seedling 2cm left and right.3. the altogether screening of substratum: take MS(I) substratum is basic medium, prepares respectively 1/2MS(II), l/3MS(III), l/4MS(IV) and the substratum of 4 kinds of vegetative gradients, every kind of substratum additional saccharose 10 gL-1, agar 1.0 gL-1, pH 5.8.4. cultivate altogether: the common substratum that bacterial classification preculture liquid 10mL is accessed to own sterilizing is central every bottle of consistent Sugarbeet (E+) of 5 strains access growing way.Meanwhile, every kind of substratum is all established blank (E-only connects test-tube plantlet and do not connect bacterium), and every kind is treated to 3 bottles.

Transplant experiment: respectively Sugarbeet (E+) and the blank (E-) cultivated altogether with endophyte of cultivating 30d left and right are together taken out together with culturing bottle, in room temperature condition lower refining seedling 1-2, after week, taking-up is transplanted respectively in aseptic basin alms bowl after cleaning and adhering to substratum.Cultivate after 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 ℃, 2h), pack the plastic tub of surface sterilization into, every basin dress sterilizing soil 0.8kg, base fertilizer is that every kilogram of soil is executed 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 ℃, 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 common cultivation 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, with ruler, measure plant height and write down the number of blade, measure altogether 20 strains for every group, record is also processed with statistical method, relatively the significance of difference of each group.

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

Table 7: the impact of endophyte on 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 growth-promoting bacterium secretion plant hormone ABA is in Table 10, growth-promoting endophyte TNSC2011 has the ability of secretion ABA, 48-120h growth-promoting bacterium, all produce a large amount of ABA, the hormone (ABA) of secreting 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 growth-promoting bacterium ABA yield reducation.

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

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

In beet, the characteristic of raw growth-promoting bacterium secretion vitamin V c is in Table 13, growth-promoting endophyte TNSC2011 has the ability of secretion Vc, cultivation 48-120h growth-promoting bacterium, all produce a large amount of Vc, hormone in 96h secretion reaches maximum, for 1.29ppm, along with the prolongation of incubation time, Vc output is on a declining curve, cultivates 144h growth-promoting bacterium Vc yield reducation.

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

Interior raw growth-promoting bacterium is processed rear beet hormone in vivo content

After interior raw growth-promoting bacterium is processed beet, plant endogenous hormones content characteristic is referring to accompanying drawing 4, Fig. 5, Fig. 6, Fig. 7, and interior raw growth-promoting bacterium is processed rear beet endogenous hormones compared with the control, and four kinds of sugar beet plants endogenous hormones output all have significant difference.

After interior raw growth-promoting bacteria strain is processed, the variation 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, the content that growth hormone after interior raw growth-promoting bacteria strain processing in beet body, zeatin, Plant hormones regulators,gibberellins etc. are grown relevant endogenous hormones with Promoting plant growth is than the height of contrast, at endophyte, fill with after root, (GA) is on a declining curve for Plant hormones regulators,gibberellins in beet body, during 5d, reach peak value, than contrast, improve 22.35 times; Growth hormone IAA is the trend of falling after rising, and after filling with root, 15d reaches peak value, than contrast, improves 1.59 times; Zeatin (ZR) is the trend of falling after rising, and after filling with root, 20d reaches peak value, than contrast, improves 1.06 times; Dormin (ABA) is bimodal trend, and after filling with root, 25d reaches peak value, than contrast, reduces by 0.53 times.

Forefathers study and show, Plant hormones regulators,gibberellins can promote the growth of overground part cauline leaf, lateral bud, breaks seed dormancy, and induced flowering.IAA not only can be combined and be made it activation by proton pump on plasma membrane, changing intracellular environment causes cell wall sugar to dissolve and plasticity-increase increases cell volume and promotes NRA(nitrate reductase activity), the synthesizing of protein, increase cell volume and quality to reach the effect of growth-promoting; And, the more important thing is and make the removing toxic substances of tryptophane (Trp) analogue, alleviate its toxic action; In addition, can also suppress plant defense system enzyme, as chitinase (ehitinase), beta-1,3-glucanase (β-1, activity 3-polyglucosanase) etc.; And AAB has restraining effect to plant materials.After interior raw growth-promoting bacteria strain is processed, the host plant hormone relevant with growth increases as IAA content from the above experimental results, and the hormone that suppresses plant-growth is as the decline of ABA content.

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 screening of activation in PKO nutrient solution 30 ℃, 200rmin ^-1shaking culture 12h, is diluted to 108CFUml with sterilized water ^-1as seed.In 200ml nutrient solution, inoculate 2ml seed, to inoculate 2ml sterilized water in contrast.Every 24h measures 1 endophyte phosphorus decomposing ability, until cultivate 7d.Endophyte fermentation broth sample 10000rmin ^-1centrifugal 20min, measures supernatant liquor pH value with pH meter.Molybdenum antimony resistance colorimetric method for titanium pigment (λ=690nm) is measured.The making of P typical curve, by 100mgL ^-1mother liquor dilution be series concentration, the method identical with test sample product measured light absorption value.PH instrument mensuration for the pH value of nutrient solution ( sartoriuspD20).Under liquid culture condition, measured the molten phosphorus ability of interior raw growth-promoting bacterial strain.Result is referring to accompanying drawing 8, Fig. 9, and bacterial strain has the inorganic phosphorus of dissolving ability, and amount of phosphorus dissolved reaches 189.22 μ gmL ^-1, dissolve inorganic phosphorus Ca ₃(PO ₄) ₂ability is better.The pH value of endophyte phosphorus decomposing ability and substratum has certain relation.Compared with the control, interior raw growth-promoting bacterium can remarkable molten phosphorus.

sequence table

SEQUENCE LISTING

<110> Microorgan Application Inst., Xinjiang Agricultural Academy

<120> TNSC2011 16SrDNA primer and 16SrDNA sequence

Raw growth-promoting bacterium 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 of TNSC2011 number is CGMCC No. 8192.

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

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