CN103642734A - Microbacterium maritypicum and application thereof in preventing sugar beet disease-causing organisms - Google Patents

Abstract

The invention discloses microbacterium maritypicum TNJK 2011 with a preservation number of CGMCC (China General Microbiological Culture Collection) No.8193 and an application thereof in preventing sugar beet disease-causing organisms. 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; and meanwhile, characteristics such as bacteriostasis rate, bio-control performance and stability of fermentation liquor of the bacterium TNJK2011 are deeply researched, so that endophytic antagonistic bacteria for sugar beet root rot, leaf spot of sugar beet, damping-off of sugar beet and clamp rot of sugar beet are screened out from the isolated sugar beet endophyte in order to obtain a novel way for preventing main diseases of the sugar beet, and therefore, application value is wide.

Description

Raw microbacterium and for the application of beet biological control of diseases in a kind of beet

Technical field

The present invention relates to agriculture technical field of microbe application, specifically, the present invention relates to a kind of beet endophyte and for the technical field of the application of beet biological control of diseases.

Background technology

A large amount of microorganisms in plant materials, in the process with plant cooperated evolution, owing to living in for a long time plant privileged site, thereby formed a kind of special relation with plant, these microorganisms are amount reproduction and diffusion in plant materials, is expected to become potential microorganism in biological control.Endogenetic bacteria is kind and the maximum microorganism of quantity in plant materials, has wide, the advantage such as colonization ability is strong, protection effect good and proliferation and spreading is fast that distributes in plant body, thereby becomes the Plant diseases biocontrol microorganisms with applications well prospect.

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 rhizosphere bacterium namely often say 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.As the natural forming member of plant microecosystem, endogenetic bacteria can surely be grown for a long time in plant materials, and some endogenetic bacteria can be by producing the antibacterial substances such as microbiotic, hydrolase long-term performance Biocontrol Effect in plant materials; With respect to saprophytic bacteria, PGPR(plant growth-promoting bacteria) etc. the biological and ecological methods to prevent plant disease, pests, and erosion factor, endogenetic bacteria is not subject to the impact of envrionment conditions, can more be conducive to bring into play Biocontrol Effect in plant Colonization inside plants and conduction, become a class on biocontrol of plant disease and had the new resources bacterium of applying potential.Existing investigator is separated to endogenetic bacteria host plant disease to good prevention effect from cotton, paddy rice, tobacco, capsicum, tomato, potatoes and other crops, but has not yet to see beet endogenetic bacteria for the research report of biological control of diseases.

Summary of the invention

For prior art, have no relevant beet endogenetic bacteria for the present situation of the technology of the research report of biological control of diseases, the present invention aims to provide raw microbacterium in a kind of beet and for the application of beet biological control of diseases, the present invention is based on endophyte of plant and the harmonious relation of combining of plant, in beet, isolate the bacterial strain of a collection of endophyte, therefrom filter out the bacterial strain that a strain is numbered TNJK2011, and by utilizing this endophyte that is numbered TNJK2011 effectively to prevent and treat the beet root rot of breeding time, brown spot, damping-off, the diseases such as snake eye disease, the disadvantageous effect that disease is produced beet is reduced to bottom line, be conducive to improve the yield and quality of beet, obtain good technique effect, as thering is extensive and applicable value in beet biological control of diseases.

The present invention adopts main technical scheme:

By the separation screening of beet endophyte, in beet, isolate in a collection of beet raw microorganism strains, through further screening, domestication breeding, obtain a strain and be numbered TNJK2011.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 TNJK2011, biocontrol water, stability are conducted in-depth research simultaneously, from separated beet endophyte, screen root rot of beets, beet cercospora leaf spot, black leg of beet, black leg of beet Endophytic antagonistic bacteria, to obtaining the new way of control beet Major Diseases.By endophyte, effectively prevent and treat the beet all kinds of diseases of breeding time, the disadvantageous effect that disease is produced beet is reduced to bottom line, proved that being numbered TNJK2011 is a kind of good interior raw biocontrol microorganisms, be conducive to improve the yield and quality of beet, fully ensure the sufficient supplies of sugar refinery raw material, for stablizing beet, produce, improve the enthusiasm of peasant planting beet, realize the increasing both production and income in peasant household, sugar refinery, the market competitiveness that strengthens Xinjiang sugar industry has profound significance.By the application in beet growing obtains good action by this endophyte, there is outstanding technique effect.

Raw microbacterium in a kind of beet that the present invention specifically provides microbacterium maritypicum, by separation in beet, screening and cultivation, obtain the microorganism strains of a collection of interior life, therefrom filter out the bacterial strain that a strain is numbered TNJK2011, through microbiology classification and identification, belong to interior raw microbacterium microbacterium maritypicum.

Concrete, raw microbacterium in a kind of beet provided by the invention microbacterium maritypicum, strain number is TNJK2011.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. 8193.This bacterial strain optimum growing condition is: 32 ℃ of temperature, and substratum adopts tryptose soya agar (TSA) substratum, culture condition: pH7.2, time 48h; This bacterial strain bacterium colony is large and level and smooth, is transparent, yellow, surface wettability, neat in edge, and growth comparatively fast; By gramstaining, microscopy is observed, and finds that this bacterial strain is gram-positive microorganism, is accredited as through microbiology microbacterium maritypicumbacterium.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 < < TNJK2011 bacterial strain is carried out to morphology mensuration, Physiology and biochemistry detects determines that TNJK2011 bacterial strain is microbacteriummember in genus.By BLAST homology, compare, the 16S rDNA sequence of bacterial strain TNJK2011 is carried out after BLAST analysis in ncbi database, constructing system evolutionary tree, this bacterial strain TNJK2011 with microbacterium maritypicumDSM12512 in minimum branch, is its allied species; And then this bacterial strain TNJK2011 is defined as microbacterium maritypicum.

Interior raw growth-promoting bacterium provided by the invention microbacterium maritypicumthe major nitrogen source of using when TNJK2011 cultivates includes but not limited to peptone yeast powder; The main carbon source of using includes but not limited to sucrose, seminose, glycerine, 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 Antagonistic Fungi TNJK2011 can effectively suppress root rot, brown spot, damping-off, snake eye disease pathogen bacteria growing.

Further, the invention provides raw microbacterium in a kind of beet microbacterium maritypicumtNJK2011 CGMCC No. 8193 is in the application for beet biological control of diseases.By this t bacteria NJK2011 bacterial strain is had to the root rot of inhibition, brown spot, damping-off, the effect of snake eye disease pathogen bacterium for beet, obtain good technique effect.

The present invention further provides raw microbacterium in beet microbacterium maritypicumthe preservation condition of TNJK2011 CGMCC No. 8193, adopts TSA medium component: TSA Tryptones 17g, soy peptone 3g, glucose 2.5g, sodium-chlor 5g K ₂hPO ₄2.5g PH=7.2 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:

Raw microbacterium in the beet that separation screening of the present invention provides microbacterium maritypicumtNJK2011 CGMCC No. 8193 is a kind of good interior raw Antagonistic Fungis, there is outstanding biological and ecological methods to prevent plant disease, pests, and erosion characteristic, this interior raw growth-promoting bacterium TNJK2011 root rot of beets, beet cercospora leaf spot, black leg of beet, black leg of beet Endophytic antagonistic bacteria, to obtaining the new way of control beet Major Diseases.By endophyte, effectively prevent and treat the diseases such as the beet root rot of breeding time, brown spot, damping-off, snake eye disease, the disadvantageous effect that disease is produced beet is reduced to bottom line, proved that being numbered TNJK2011 is a kind of good interior raw biocontrol microorganisms, obtain good technique effect, be conducive to improve the yield and quality of beet, fully ensure the sufficient supplies of sugar refinery raw material, for stablizing beet, produce, improve the enthusiasm of peasant planting beet, realize the increasing both production and income in peasant household, sugar refinery, the market competitiveness that strengthens Xinjiang sugar industry has profound significance.

Accompanying drawing explanation

Fig. 1 is shown as raw microbacterium in beet microbacterium maritypicumthe colonial morphology figure of TNJK2011 CGMCC No. 8193.

Fig. 2 is shown as raw microbacterium in beet microbacterium maritypicumthe growth curve chart of TNJK2011 CGMCC No. 8193.

Fig. 3 is shown as raw microbacterium in beet microbacterium maritypicumthe 16S rDNA phylogeny tree graph of TNJK2011 CGMCC No. 8193.

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: 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-: raw microbacterium in beet microbacterium maritypicumseparation, screening and the evaluation of TNJK2011 CGMCC No. 8193

(1) Isolation and screening of bacterial classification

From Sugarbeet Tissue, by gradient dilution method, isolate many strains bacterium, adopt dull and stereotyped face-off method, according to having or not inhibition zone to judge whether it has antagonistic action, preliminary screening goes out the bacterial strain cause of diseases such as root rot of beets, brown spot, damping-off, snake eye disease to certain antagonistic ability.To thering is the bacterial strain of antagonistic action, carry out further experiment, filter out the bacterial strain that antagonistic ability is stronger and prepare against subsequent experimental.

By separated in Sugarbeet Tissue, screening with cultivate, obtain the microorganism strains of a collection of endophyte, therefrom filter out the bacterial strain that a strain is numbered TNJK2011, through microbiology classification and identification, belong to Microbacterium microbacterium maritypicum.

Concrete, raw microbacterium in a kind of beet provided by the invention microbacterium maritypicum, strain number is TNJK2011.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. 8193.Through microbiology, be accredited as microbacterium maritypicum.This bacterial strain optimum growing condition is: 32 ° of C of temperature, and substratum adopts tryptose soya agar (TSA) substratum, culture condition: pH7.2, time 48h; This bacterial strain bacterium colony is large and level and smooth, is transparent, yellow, surface wettability, neat in edge, and growth comparatively fast; By gramstaining, microscopy is observed, and finds that this bacterial strain is gram-positive microorganism, and bacterial strain TNSC2011 is Gram-positive, thalline 0.3-0.5 μ m * 0.8-1.7 μ m, and the dull and stereotyped upper 28 ℃ of cultivation 48h bacterium colonies of TSA are yellow, bacterium colony 2-4mm.Through microbiology, be accredited as microbacterium maritypicumbacterium.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 < < TNJK2011 bacterial strain is carried out to morphology mensuration, Physiology and biochemistry detect determine TNJK2011 bacterial strain be Microbacterium ( microbacterium )in member.By BLAST homology, compare, the 16S rDNA sequence of bacterial strain TNJK2011 is carried out after BLAST analysis in ncbi database, constructing system evolutionary tree, this bacterial strain TNJK2011 with microbacterium maritypicumDSM12512 in minimum branch, is its allied species; And then this bacterial strain TNJK2011 is defined as microbacterium maritypicum.

The major nitrogen source that interior raw growth-promoting bacterium TNJK2011 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, seminose, glycerine, 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.Interior raw Antagonistic Fungi fermentation of the present invention can be at 20-37 ℃, under the environment of pH5.5-9.1, carries out.And this interior raw Antagonistic Fungi TNJK2011 can effectively suppress root rot, brown spot, damping-off, snake eye disease pathogen bacteria growing.

By above-mentioned, TNJK2011 bacterial strain is carried out to morphology mensuration, and the shape of bacterial strain TNJK2011, size, biochemical reactions are detected, TNJK2011 Biological Characteristics of Strain of the present invention is as shown in table 1.

Table 1: the physio-biochemical characteristics of bacterial strain TNJK2011

By above-mentioned for raw microbacterium in bacterial classification beet microbacterium maritypicumthe thalli morphology of TNJK2011 CGMCC No. 8193, cultural characteristic are observed and Determination of Physiological And Biochemical Indices: thalli morphology observation, strain culturing observation of characteristics, aerobism and mobility mensuration, growth temperature mensuration, Salt tolerance, Citrate trianion utilization test, catalase test, glycitols fermentation test, nitrate reduction test, Starch Hydrolysis, gelatine liquefication, indole test and H ₂s produces test, all with reference to the method for the conventional bacterial system identification handbook > > of < <, carries out.

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

Single bacterium colony of a small amount of TNJK2011 bacterium of picking, puts into the EP pipe that fills 25 μ L sterilized waters, and 100 ° of C boil 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.

The structure of 16S rRNA gene sequencing and systematic evolution tree thereof: extract according to a conventional method total DNA of bacterial isolates, adopt bacterium universal primer to carry out the pcr amplification of 16S rDNA, design of primers is as follows:

PA: 5′- A G A G T T T GATCCTGGCTCAG- 3′；

PB：5′- AAGGAGGTGATCCAGCCGCA-3′；

Two primer spacings are 1500bp.The reaction system of 50 μ l contains: 10 * PCR damping fluid, 5 μ l, each 20pmol of primer, template DNA (1 000ng/ μ l) 1 μ l, TaqTM (TaKaRa company) 0.5U, dNTP 8 μ l.PCR amplification condition: first at 94 ℃ of denaturation 2min, then 98 ℃ of 10s, 55 ℃ of 30s, 72 ℃ of 1. 5min, circulate 30 times, finally extends 10min at 72 ℃; The purifying of PCR product: get the PCR product of 8 μ l, electrophoresis in 1% sepharose, uses taKaRa PCR Fragment RecoveryKitfrom glue, reclaim object fragment, be dissolved in the high purity water of 20 μ l.To PCR product PA(+) and PB (-) make sequencing primer, measure 16S rDNA sequence, the gene order of t bacteria NJK2011 is referring to attached gene order table.Need to be according to the single-row gene order table of the gene order providing.

(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 Clustal X software and MEGA 4.1Neighbor-joining method constructing system evolutionary tree, referring to accompanying drawing 3; The 16S rDNA sequence of TNJK2011 is carried out after BLAST analysis in ncbi database to constructing system evolutionary tree; Shown in accompanying drawing 3, bacterial strain TNJK2011 with microbacterium maritypicumDSMbetween 12512, evolutionary distance is the shortest, is microbacterium maritypicumallied species.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of TNJK2011, determine that it is Microbacterium microbacterium maritypicum.

By measured 16S rDNA sequence input Genbank, with Blast program, carry out homology comparison, find it with microbacterium maritypicumthe similarity maximum (99%) of the 16S rDNA sequence of DSM 12512 (T), thus further determine that it is microbacterium maritypicumdSM 12512 (T).In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of TNJK2011, determine that it is beet endophyte microbacterium microbacterium maritypicumtNJK2011 CGMCC No. 8193.

embodiment bis-: raw microbacterium in beet microbacterium maritypicumtNJK2011 CGMCC No. 8193 somatomedin

Referring to following mode: but should be according to raw microbacterium in beet provided by the invention microbacterium maritypicumtNJK2011 CGMCC No. 8193 characteristics are determined the somatomedin that it is concrete, bacterial strain TNJK2011 inoculation culture.The results are shown in Table 2.

Table 2: temperature, pH, salt, the impact of microbiotic on bacterial strain TNJK2011 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 if upper type is by beet endophyte microbacterium microbacterium maritypicumtNJK2011 CGMCC No. 8193 cultivates, and the culture condition of bacterial classification is: substratum is LB solid medium, and culture condition: pH7.0 cultivates 28h under 32 ° of C 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
						TNJK2011	0.522	0.794	0.864	0.612	0.001

By table 3, drawn the most applicable somatomedin of bacterial strain TNJK2011.

By above result, draw beet endophyte microbacterium microbacterium maritypicumtNJK2011 CGMCC No. 8193 is 20h as the best incubation time of seed, and the most suitable growth pH is 7.0, and the large volume production gemma time is 24h.

embodiment tri-: raw microbacterium in beet microbacterium maritypicumtNJK2011 CGMCC No. 8193 study on Fermentation

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 200 r/min, 35 ℃ of condition bottom fermentations of temperature, show that the most suitable growth pH value is 7.0.

2. the optimization of Carbon and nitrogen sources

Seed liquid nutrient medium: glucose 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 corn steep liquor, other compositions are KH ₂pO ₄0.1%, K ₂hPO ₄0.1%, MgSO ₄7H ₂o 0.05%, 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	Corn steep liquor
				TNJK2011	0.823	0.617	0.912*

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

	Peptone	Yeast extract paste	（NH 4） 2 SO 4
				TNJK2011	0.726	0.867*	0.584

According to table 4,5, above result determines that optimum carbon source is corn steep liquor, and optimum nitrogen source is yeast extract paste.

3.2 10L ferment tank results

Take in the substratum that corn steep liquor and yeast extract paste be main raw material, fermentation condition is: 32 ℃ of temperature, and rotating speed 200rpm, ventilation 1:0.3, the time is at 24h.It the results are shown in as following table 6:

Table 6:10L fermenting experiment result

Bacterial strain	Fermentation time	Extension rate	OD 600nm
				TNJK2011	24h	30	2.728

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

embodiment tetra-: the separation of beet main pathogenic microbes, purifying and preservation

1. the separation of beet main pathogenic microbes, purifying and preservation

Beet main pathogen bacteria strain is according to the method for document, and the position of being infected disease plant is separated.Choose disease plant in test sample, in the strong intersection sampling of disease, 5% clorox surface sterilization 5min for sample, aseptic water washing 3 times, each 2-5min, is then cut into 0.5-1.0cm section, being inoculated into the PSA preparing cultivates after 7d on culture medium flat plate for trying, record classification and the frequency of occurrences of isolate, preliminary evaluation isolate, after purifying, tube saves backup.

Pathogenic bacteria bacterial strain preservation: adopt monospore or single sclerotium partition method.According to colony morphology characteristic, single bacterium colony that picking obviously separates exists if any single bacterium colony of several different shapes simultaneously, and picking, to corresponding separating plate substratum, carries out nitrogen bacterium colony separation and Culture respectively.To guarantee that isolated bacterium must be pure bacterium.Slant preservation: prepare corresponding medium slant substratum with the centrifuge tube of 1.5ml.Under the inclined-plane normal temperature preparing, place one week, contaminated to guarantee that inclined-plane does not have.The good pure bacterium picking of separation and purification, to inclined-plane, is cultivated one week for 28 ℃.After slant culture grows, 4 ℃ of preservations are placed in inclined-plane.The preservation of glycerine pipe: the glycerine of preparation 20%, after sterilizing, get the centrifuge tube that 0.5ml packs 1.5ml into.The good pure bacterium picking of separation and purification is mixed to being equipped with in the centrifuge tube of glycerine, place-20 ℃ of preservations.

2. isolate Pathogenicity and pathogen identification

By after perlite and vermiculite sterilizing respectively in 1: ratio 1(V/V) mixes, and the diameter that packs in advance sterilization into is standby in the flowerpot of 20 cm.Beet seed after selected is soaked to 10-12 h in clear water, be sowed in flowerpot, 10, every basin, covers sterilization matrix 1.0 cm, cultivates in the greenhouse of 25-32 ℃.Thinning beet keeps matrix moistening before emerging, and until cotyledon, is unearthed after true leaf expansion, suitably reduces irrigation times, keeps dry hypothallus to be no more than 3-5 cm.Pathogen separation thing is cultivated with PSA culture medium flat plate, with sterilized water wash-out spore, prepares spore suspension, and spore suspension concentration is 3.5 * 107 spore ml ^-1.Take the healthy thinning beet plant of above-mentioned seedling age 40d left and right, clean root, adopt and soak root inoculation method, its root system is immersed to 10 min in fungal spore suspension.Using aseptic water logging root as blank.Postvaccinal thinning beet is transplanted in the flowerpot that sterilizing perlite and vermiculite (1: 1) are housed, and in 25-32 ℃ of greenhouse, routine observation records incidence.Test is carried out 3 times, and the 1st every processing 2 strains repeat for 5 times; The 2nd time and the 3rd every processing 10 strains, repeat for 3 times.The isolate obtaining after Pathogenicity is placed in respectively on PSA substratum, cultivates 7d at 28 ℃, observe and cultivate proterties, morphological specificity, and carry out microscopic observation and photographic recording.< < fungi identification handbook > > with reference to Wei Jingchao, this work of Britain C. cloth, the bibliographys such as < < Fusarium > > (Chen Qiyi) are identified.

3. the isolation identification of beet the main pathogenic fungi and Pathogenicity

From beet variety KWS2409 diseased plant, be separated to altogether pine root fungus 25 strains, brown patch germ 21 strains, rhizoctonia solani 17 strains, 9 strains of snake eye germ, to pine root fungus, rhizoctonia solani virulence, adopt potted plant root inoculation method to measure, to brown patch germ, snake eye germ virulence, adopt potted plant blade inoculation method to measure.Result shows: 25 root-rot bacterial strains, 19 strain brown patch germs, 17 strain rhizoctonia solanis, 9 strain snake eye germs have certain virulence to beet leaves, and especially stronger with the virulence of root-rot bacterial strain 1, brown patch germ bacterial strain 2, rhizoctonia solani bacterial strain 3,4 pairs of beets of snake eye germ bacterial strain, the sick level of beet reaches respectively 4.2,4.0,3.8,3.5.Pathogen identification result is as follows.

4. beet the main pathogenic fungi Antagonistic Endophytic bacterial strain screening, evaluation and antagonistic action are measured

Using root-rot bacterial strain 1, brown patch germ bacterial strain 2, rhizoctonia solani bacterial strain 3, snake eye germ bacterial strain 4 as target bacterial strain, be separated to 401 endophytic bacterial controlled effects are carried out to antagonistic action mensuration, there are 71 bacterial strains to have antagonistic action, account for 17.71% of total bacterial strain number, what the strain number of wherein take was TNJK2011 is larger to the main pathogenic fungi inhibition zone radius, especially obvious to the effect of root-rot bacterium, reach 18.9, mm, in Table 7.

Table 7: the endophyte that suppresses beet the main pathogenic fungi

1		2		3		4
								Inhibition zone (mm)	Bacterial strain number	Inhibition zone (mm)	Bacterial strain number	Inhibition zone (mm)	Bacterial strain number	Inhibition zone (mm)	Bacterial strain number
≥5.0	11	≥5.0	10	≥5.0	13	≥5.0	12
								3.0-5.0	15	3.0-5.0	14	3.0-5.0	16	3.0-5.0	15
2.0-3.0	19	2.0-3.0	20	2.0-3.0	19	2.0-3.0	18
								1.0-2.0	23	1.0-2.0	25	1.0-2.0	24	1.0-2.0	22
1.0-0.1	31	1.0-0.1	32	1.0-0.1	29	1.0-0.1	27
								0	278	0	301	0	269	0	271

embodiment tetra-: Antagonistic Fungi microbacterium microbacterium maritypicumtNJK2011 CGMCC No. 8193 the effect that beet seed is germinateed

1. presoaking and germinating test

Use 109 cfumL ^-1seed soaking 3 min, are placed in the culture dish that is lined with moistening filter paper, cultivate 3,5,7 d for 28 ℃, detect bud ratio, and Seed soaking is contrast.

2. the effect that Antagonistic Fungi germinates to beet seed

The beet seed bud ratio of processing 3,5,7 d through microbacterium TNJK2011 bacteria suspension is all apparently higher than control group.Show that certain density microbacterium TNJK2011 bacteria suspension has promoter action to sprouting of beet seed, through the beet seed of endophyte immersion kind emerge early, emerge neat, Ye Senong, table 8.

Table 8: the impact that seed soaking is emerged on beet

Note: different capitalizations and lowercase alphabet show the significance of difference, P=0.05 and P=0.01.

embodiment five: Antagonistic Fungi microbacterium microbacterium maritypicumtNJK2011 CGMCC No. 8193 colonazition

1. endogenetic bacteria colonazition mensuration, separated and evaluation

The screening of anti-Rifampin (Rif) mutant strain: strains tested is proceeded to containing 50 μ gm ^l-1on the NA plate culture medium of Rif, cultivate, the mutant strain of picking growth, then access the NA substratum of same Rif concentration, after subculture 1 time, proceed to the next one and contain Rif, its concentration is followed successively by 100,120,150,180,200,220,240,260,280,300 μ gmL ^-1substratum in, until filter out, containing 300 μ g mL ^-1can stable growth on the NA substratum of Rif, and colonial morphology and mutant strain that the antagonistic action of pathogenic bacteria is remained unchanged.The colonazition of the bacterial strain of different vaccination concentration in beet body measured: by the nutrient solution stoste of the anti-medicine mutant strain of bacterial strain to be measured, (bacteria containing amount is 9.4 * 109 cfumL ^-1) and dilution l0, l02, l03, l04 diluent doubly, water respectively beet root (root scratches with cutter before pouring), 14 d after processing, the colonazition of mensuration bacterial strain, the same treatment that does not connect bacterium of take is blank.The colonazition of the bacterial strain of different vaccination method in beet body measured: adopt injection and the anti-medicine mutant strain of pouring method inoculating strain nutrient solution, take and do not connect bacterium as blank.Surely grow the Isolation and Identification of bacterium: by described method, carry out separation, purifying, substratum used is for containing 300 μ gmL ^-1the improvement PSA substratum of Rif, each dilution gradient is processed and is repeated 5 times, in 28 ℃ of thermostat containers, cultivates 36 h, calculates each ware colony number.Bacterial strain separated, purifying is identified by described method.

2. Antagonistic Fungi colonazition is measured

Containing on the NA flat board of 300 μ g/mL Rif microbacterium TNJK2011 bacteria suspension dilution 10 ⁶after, average colony number is respectively 295, and thalli morphology is consistent with before inoculation, shows the better colonazition of the equal tool of this strain endophyte.Inoculation different concns bacterium liquid has a certain impact to the amount of growing of determining of bacterial strain, and bacterial strain TNJK2011 is minimum, and intrusion concentration is respectively 9.7 * 106cfumL ^-1.Bacterial strain is grown quantity surely increases (table 9) along with the rising of bacterial concentration.Adopt injection and pouring method inoculation Rif resistant strain, all can in beet body, be separated to the TNJK2011 bacterial strain of anti-Rif, and these 2 kinds of methods grow no significant difference between quantity surely, and in blank, do not isolate anti-Rif bacterial strain.Therefore, pouring method is that test method is grown in a kind of desirable determining, in Table 9,10.

Table 9: separated again after different concns bacterium liquid inoculation beet

Note :-: represent not to be separated to; +: represent to be separated to.

Table 10: after bacterium liquid Different treatments, endophyte is separated again

Note :-: represent not to be separated to; +: represent to be separated to.

embodiment tetra-: Antagonistic Fungi microbacterium microbacterium maritypicumtNJK2011 CGMCC No. 8193 field biological and ecological methods to prevent plant disease, pests, and erosion test

1. field efficacy is measured

2006-2008, carries out field efficacy mensuration on Tou Tunhe farm, Xinjiang in continuous 3 years.Choose continuous cropping grave illness beet ground for many years, before beet sowing, with Antagonistic Fungi immersion kind 24h, after emerging, piecemeal root expanding stage, Sugar content accumulated stage are processed for 2 times, and microbacterium is used in every strain microbacterium maritypicumtNJK2011 bacterial strain bacteria suspension (NA medium centrifugal is collected thalline, sterilized water dilution) 10 mL, concentration 5 * 10 ⁷cfumL ^-1, with clear water, to process in contrast, each is processed 3 times and repeats, and each processing area is 22 m ², contrast is scattered between each treatment group.After dispenser, 14d investigation is 1 time, and before results, investigation is the 2nd time, records morbidity strain numbers at different levels, calculates disease index.

Damping-off grade scale is as follows: 0 grade: complete stool is anosis; 1 grade: slightly fall ill, young root or hypocotyl have small brown specks or striped, but do not form the contracting of hanging; 2 grades: moderate is fallen ill, young root or hypocotyl brown striped expand and form the contracting of hanging, and it is long partly that brown is partly less than root; 3 grades: seriously fall ill, it is long partly that browning is partly greater than root, hang contracting part obviously, become chocolate and even black; 4 grades: young root and hypocotyl are withered, blackening is dead.

The sick grade scale of brown spot and snake eye is as follows: 0 grade: anosis or minority strain has minority scab; L level: most plant have minority scab or minority plant to have most scabs; 2 grades: most plant have most scabs, 1/4th following siphonal lobes are due to illness withered; 3 grades: most plant have most scabs, 1/4th to 2/4ths siphonal lobes are due to illness withered; 4 grades: in whole district's group, most plant leafs are due to illness withered.

Root rot grade scale is as follows: 0 grade: piece root growth is normal, intact; 1 grade: there is Minimal change in Gen Weijigen table organization, pathology is not yet invaded and root in-vivo tissue; 2 grades: there is obvious pathology in Gen Weijigen table organization, and vascular bundle is brown, but lignifying not yet; 3 grades: vascular bundle is Vandyke brown, conduit lignifying, fertility is seriously obstructed, and old complaint has part to start to decay, and rotten part accounts for below 10% of piece root; 4 grades: the rotten part of old complaint accounts for 10%～30% of piece root; 5 grades: old complaint rot part account for piece root more than 30% or complete stool due to illness withered.

2. field efficiency test is measured

Table 11 result shows, endophyte TNJK2011 can reduce the disease index of black leg of beet, root rot, brown spot and snake eye disease, and preventive effect is respectively 64.4%, 81.4%, 80.5%, 84.6%.Endophyte has good prevention effect to beet Major Diseases.There is significant difference with contrasting in bacterial strain TNJK2011.

Table 11: the field test preventive effect of endophyte to beet Major Diseases

Note: different capitalizations and lowercase alphabet show the significance of difference, P=0.05 and P=0.01.

sequence table

SEQUENCE LISTING

<110> Microorgan Application Inst., Xinjiang Agricultural Academy

<120> raw microbacterium and for the application of beet biological control of diseases in a kind of beet

<130> TNJK2011 16SrDNA primer and 16SrDNA sequence

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> bacterial strain TNJK2011 16SrDNA upstream primer sequence

<400> 1

agagtttgat cmtggctcag 20

<210> 2

<211> 22

<212> DNA

<213> bacterial strain TNJK2011 16SrDNA downstream primer sequence

<400> 2

tacggytacc ttgttacgac tt 22

<210> 3

<211> 1435

<212> DNA

<213> Microbacterium maritypicum TNJK2011

<400> 3

cgaaatgggg gcgtgcttac catgcaagtc gacggtgaac acggagcttg ctctgtggga 60

tcagtggcga acgggtgagt aacacgtgag caacctgccc ctgactctgg gataagcgct 120

ggaaacggcg tctaatactg gatatgtgac gtgaccgcat ggtctgcgtc tggaaagaat 180

ttcggttggg gatgggctcg cggcctatca gcttgttggt gaggtaatgg ctcaccaagg 240

cgtcgacggg tagccggcct gagagggtga ccggccacac tgggactgag acacggccca 300

gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc tgatgcagca 360

acgccgcgtg agggacgacg gccttcgggt tgtaaacctc ttttagcagg gaagaagcga 420

aagtgacggt acctgcagaa aaagcgccgg ctaactacgt gccagcagcc gcggtaatac 480

gtagggcgca agcgttatcc ggaattattg ggcgtaaaga gctcgtaggc ggtttgtcgc 540

gtctgctgtg aaatccggag gctcaacctc cggcctgcag tgggtacggg cagactagag 600

tgcggtaggg gagattggaa ttcctggtgt agcggtggaa tgcgcagata tcaggaggaa 660

caccgatggc gaaggcagat ctctgggccg taactgacgc tgaggagcga aagggtgggg 720

agcaaacagg cttagatacc ctggtagtcc accccgtaaa cgttgggaac tagttgtggg 780

gtccattcca cggattccgt gacgcagcta acgcattaag ttccccgcct ggggagtacg 840

gccgcaaggc taaaactcaa aggaattgac ggggacccgc acaagcggcg gagcatgcgg 900

attaattcga tgcaacgcga agaaccttac caaggcttga catatacgag aacgggccag 960

aaatggtcaa ctctttggac actcgtaaac aggtggtgca tggttgtcgt cagctcgtgt 1020

cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct cgttctatgt tgccagcacg 1080

taatggtggg aactcatggg atactgccgg ggtcaactcg gaggaaggtg gggatgacgt 1140

caaatcatca tgccccttat gtcttgggct tcacgcatgc tacaatggcc ggtacaaagg 1200

gctgcaatac cgcgaggtgg agcgaatccc aaaaagccgg tcccagttcg gattgaggtc 1260

tgcaactcga cctcatgaag tcggagtcgc tagtaatcgc agatcagcaa cgctgcggtg 1320

aatacgttcc cgggtcttgt acacaccgcc cgtcaagtca tgaaagtcgg taacacctga 1380

agccggtgag cacgtaacct ttttggaggg agccgacgaa tggtgtattt cgata 1435

Claims (3)

1. in a beet, give birth to microbacterium microbacterium maritypicumtNJK2011, is characterized in that, raw microbacterium in described beet microbacterium maritypicumthe culture presevation of TNJK2011 number is CGMCC No. 8193.

2. in a beet as claimed in claim 1, give birth to microbacterium microbacterium maritypicumtNJK2011 is in the application for beet biological control of diseases.

3. in beet as claimed in claim 2, give birth to microbacterium microbacterium maritypicumtNJK2011, in the application for beet biological control of diseases, is characterized in that, raw microbacterium in described beet microbacterium maritypicumtNJK2011 is applied to beet and suppresses the application in root rot, brown spot, damping-off, snake eye disease pathogen bacterium.

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