CN105296380A

CN105296380A - Complex soil remediation agent and application thereof to remedying greenhouse soil

Info

Publication number: CN105296380A
Application number: CN201510439946.2A
Authority: CN
Inventors: 王心歌; 张松林; 韩友青; 苏玲
Original assignee: Xinjiang Huisen Biotechnology Co ltd
Current assignee: Xinjiang Huisen Biotechnology Co ltd
Priority date: 2015-07-23
Filing date: 2015-07-23
Publication date: 2016-02-03
Anticipated expiration: 2035-07-23
Also published as: CN105296380B

Abstract

The invention discloses a complex soil remediation agent and application thereof to remedying greenhouse soil. Bacillus subtillis XHS0035Kc with a number of CGMCC NO.9434, geotrichum candidum XHS0030B with a number of CGMCC NO.9435 and streptomyces microflavus XHSOO32Xh with a number of CGMCC NO.9432 are obtained by means of separating and screening and are mixed with 20%-25% of rhodopseudomonas palustris according to proportions of 20%-25%, 15%-20% and 30%-35%, and strain mixed liquid is sufficiently adsorbed in carriers and is mixed with accessories, so that the complex soil remediation agent can be prepared by means of fermentation. The complex soil remediation agent and the application have the advantage that outstanding technical effects can be realized by the complex soil remediation agent for remedying the greenhouse soil.

Description

A kind of composite soil reparation group agent and the application in warmhouse booth soil remediation thereof

Technical field

The present invention relates to agriculture technical field of microbe application, specifically, the soil remediation group agent that the bacterial classification that the present invention relates to the screening of seed selection voluntarily prepares compound and the technical field applied in warmhouse booth soil remediation.

Background technology

Along with the development of industrialized agriculture, chemical fertilizer, agricultural chemicals and other chemical substance are more and more in farmland accumulation, cause the various direct seed output and quality problem affecting agricultural-food of soil compaction, disease, this problem has become the difficult problem that plant husbandry is badly in need of capturing.At present, the company of more domestic process contaminated soils, physical method and the chemical process of adopting is administered more, wherein, and physical method many employings burning method, landfill method process pollutent, easy spoiled soil structure, and treating processes is loaded down with trivial details, cost is higher; Chemical process many employings extraction processs etc., owing to using the cause of chemical reagent, the method easily causes the secondary pollution of environment.

The input of China environmental protection industry in soil remediation field is in the trend of increase always, and China's cultivated land resource is relatively in short supply, ends the cultivated area 18.31 hundred million mu in the whole nation in 2006, accounts for 12.71% of area.Eleventh Five-Year Plan is pointed out " China soil, fresh water, the energy, Mineral resources and environmental aspect have formed serious restriction to Economic development ".According to the data that national environmental protection portion issues for 2006; contaminated arable land, the whole nation about has 1.5 hundred million mu; pollute to irrigate and pollute 3,250 ten thousand mu, arable land; solid waste is stored up and is taken up an area and ruin 2,000,000 mu, field; add up to and account for more than 1/10 of cultivated area; every year because soil pollution causes grain drop in production 2,500,000,000 kilograms, direct economic loss is more than 20,000,000,000 yuan.

In contaminated soil, the soil pollution of a class warmhouse booth is had especially to cause our concern.Abuse chemical fertilizer, agricultural chemicals cause warmhouse booth soil compaction, and the crop products produced, with pesticide residue, brings great threat to the physical and mental health of the people.Microbiological method is adopted to repair contaminated soil, pollutent in soil utilizes own metabolism to transform into water and carbonic acid gas by the microbes with degradation capability, have simple to operate, processing cost is low, the advantages such as non-secondary pollution are a kind of health, environmentally friendly Biological clean technology.Although have many about studying the report of organic materials on the impact of soil property and crop yield large Tanaka, but about from warmhouse booth soil, separation screening can improve the bacterial classification of warmhouse booth soil, and the subtilis utilizing seed selection voluntarily to screen domestication to obtain, geotrichum candidum and streptomyces microflavus and other bacterial classifications are prepared soil-repairing agent according to the blending of bacterial classification, suitability and security basic demand and strain properties and be have not been reported for the research improveing warmhouse booth soil.

Summary of the invention

Merge with other bacterial classifications are compatible the state of the art preparing warmhouse booth soil-repairing agent about screening subtilis, geotrichum candidum and streptomyces microflavus as excellent species from warmhouse booth soil mutually for having no in prior art, the present invention aims to provide a kind of composite soil reparation group agent and the application in warmhouse booth soil remediation thereof.The present invention by isolating a collection of microorganism strains in warmhouse booth soil, therefrom separate the subtilis being numbered XHS0035Kc, be numbered the geotrichum candidum of XHS0030B and the streptomyces microflavus of XHS0032Xh, and by utilizing the subtilis being numbered XHS0035Kc, be numbered the geotrichum candidum of XHS0030B and prepare the agent of composite soil reparation group with compatible the fusion mutually of the streptomyces microflavus of XHS0032Xh and Rhodopseudomonas palustris, for improvement and activating soil, objectionable impurities in degraded soil, solve the salinification problem of soil, solve soil nutrient in actual facility booth unbalance, salinification, desertify or the serious present situation that hardens, improve the growing environment of farm crop in booth, improve the aspects such as crop quality and all there is important effect, the composite soil reparation group agent of comprehensive utilization preparation is reduced to bottom line for the disadvantageous effect of Vegetable produce, be conducive to the yield and quality improving vegetables, obtain good technique effect, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, reduce the aspects such as pesticide residue and there is vital role, there is extensively actual value simultaneously in warmhouse booth soil remediation.

The present invention adopts main technical scheme:

By the separation screening of microbial strains, a collection of microorganism strains is isolated in warmhouse booth soil, through further separations, screening, seed selection, domestication, acquisition be numbered XHS0035Kc subtilis, be numbered the geotrichum candidum of XHS0030B and be numbered the streptomyces microflavus of XHS0032Xh.Measure and Phylogenetic Analysis by carrying out morphological specificity, physio-biochemical characteristics and 16srDNA sector sequence, rDNAITS section DNA sequence dna and 16srDNA sector sequence to obtained bacterial strain, tentatively determine its classification position.Simultaneously, the subtilis of XHS0035Kc will be numbered, the geotrichum candidum being numbered XHS0030B prepares warmhouse booth soil-repairing agent with compatible the fusion mutually of the streptomyces microflavus being numbered XHS0032Xh and Rhodopseudomonas palustris, assess according to weight ratio, to be numbered the subtilis 20%-25% of XHS0035Kc, be numbered the geotrichum candidum 15%-20% of XHS0030B, the streptomyces microflavus 30%-35% and the Rhodopseudomonas palustris 20%-25% that are numbered XHS0032Xh mix, bacterial classification mixed solution is fully adsorbed in carrier, and mix mutually with auxiliary material, the agent of composite soil reparation group is prepared by utilizing fermentation technique.Utilize the composite soil reparation group agent of preparation can improve the quality of warmhouse booth soil, improvement and activating soil, objectionable impurities in degraded soil, solve the salinification problem of soil, solve soil nutrient in actual facility booth unbalance, salinification, desertify or the serious present situation that hardens, improve the growing environment of farm crop in booth, improve crop quality, comprehensively the disadvantageous effect for Vegetable produce is reduced to bottom line, be conducive to the yield and quality improving vegetables, obtain good technique effect, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, reduce the aspects such as pesticide residue and there is vital role, there is the extensively using value of reality simultaneously in warmhouse booth soil remediation.

The present invention specifically provide be numbered XHS0035Kc subtilis, be numbered the geotrichum candidum of XHS0030B and be numbered the streptomyces microflavus of XHS0032Xh, by being separated in warmhouse booth soil, screening, tame and cultivating, obtain a collection of bacterium microbe bacterial strain, therefrom filter out the subtilis being numbered XHS0035Kc, through microbiological classification and qualification, belong to subtilis (BacillusSubtilis); Be numbered the geotrichum candidum of XHS0030B, through microbiological classification and qualification, belong to geotrichum candidum (GeotrichumCandidum); Be numbered the streptomyces microflavus of XHS0032Xh, through microbiological classification and qualification, belong to streptomyces microflavus (Streptomycesmicroflavus).

Described, the invention provides the subtilis (BacillusSubtilis) that strain number is XHS0035Kc.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 July 10th, 2014, and preserving number is CGMCCNo.9434.BacillusSubtilis is accredited as through microbiology.This bacterial strain optimum growing condition is: temperature 28 DEG C, 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; Shaft-like, Gram-positive, strictly aerobic, chemoheterotrophy, catalase is positive, M.R and V-P tests the positive, and Starch Hydrolysis is positive, gelatin liquefaction positive, and nitrate reduction is positive, and Citrate trianion utilizes positive; BacillusSubtilis bacterium is accredited as through microbiology.According to the 9th edition " uncle Jie Shi systematic bacteriology identification handbook " (" Bergey, sManualofSystematicBacterio-logy ") and " conventional bacterial system identification handbook " morphology mensuration is carried out to XHS0035Kc bacterial strain, Physiology and biochemistry detects determines that XHS0035Kc bacterial strain is the member in subtilis (BacillusSubtilis).By the comparison of BLAST homology, after the 16SrRNA sequence of strain X HS0035Kc carries out BLAST analysis in ncbi database, constructing system evolutionary tree, strain X HS0035Kc and 30334-31756BacillusSubtilisKCTC1028 is its allied species, thus according to 16SrDNA the sequencing results, combining form and physio-biochemical characteristics, be accredited as subtilis (BacillusSubtilis) by strain X HS0035Kc.

This strain X HS0035Kc is at tryptose soya agar (TSA) substratum well-grown, and the major nitrogen source when identification plate test proves XHS0035Kc cultivation 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.

Described, the invention provides the geotrichum candidum (GeotrichumCandidum) that strain number is XHS0030B.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 July 10th, 2014, and preserving number is CGMCCNo.9435.This bacterial strain optimum growing condition is: temperature 25 DEG C, and substratum adopts potato dextrose agar (PDA) substratum, culture condition: pH5-7, time 24h-48h.This bacterial strain produces white, in lint shape or powdery film, has fungal filament, some branches, more or less, modes of reproduction is fragmentation to tabula, and the arthrospore of formation is single or connect chaining, spore is in oval or circular, and bacterium colony is planar diffusion, and growth is fast, flat, front oyster white, back side khaki color, diameter 40-70mm, short flannel shape or be bordering on powdery, has concentric turns can radioactive rays, have in umbo.Can protolysate, wherein most can liquefy gelatin, peptonized milk, minority can only peptonized milk, can not liquefy gelatin, is accredited as geotrichum candidum (GeotrichumCandidum) through microbiology.With reference to " Fungal identification handbook ", the qualification of system Physiology and biochemistry is carried out to numbering XHS0030B bacterial strain, detect through Physiology and biochemistry and determine that numbering XHS0030B bacterial strain is the member in Geotrichum (Geotrichum).By the comparison of BLAST homology, after the ITSrDNA sequence nucleotide sequence of strain X HS0030B carries out BLAST analysis in ncbi database, constructing system evolutionary tree, this is numbered strain X HS0030B and GeotrichumcandidumisolateL11B and is in minimum branch, be its allied species, its homology is 94%, and then this strain X HS0030B is defined as Geotrichumcandidum, through said system taxonomy qualification classification, demonstrate geotrichum candidum provided by the invention (GeotrichumCandidum) and have certain physio-biochemical characteristics difference and the otherness of molecular level with common geotrichum candidum, according to bacterial classification Analysis of The Physiological And Biochemical Properties, the comprehensive identification of molecular level analysis and systematics, although the bacterial classification being numbered XHS0030B has obvious difference with common geotrichum candidum in physio-biochemical characteristics and molecular level, geotrichum candidum (GeotrichumCandidum) is accredited as from taxonomy.

Described, the invention provides the streptomyces microflavus (Streptomycesmicroflavus) that strain number is XHS0032Xh.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 July 10th, 2014, and preserving number is CGMCCNo.9432.Streptomycesmicroflavus is accredited as through microbiology.This bacterial strain optimum growing condition is: temperature 30 DEG C, and substratum adopts ISP2 substratum, pH7.2, time 4d; This bacterial strain substrate mycelium multi-branched, does not rupture, and aerial hyphae is enriched, and spore chain is straight and long, and occasionally have curve, spore is column; Bacterial strain is well-grown on most substratum, and on gas Gao Shi Starch Agar, aerial mycelium is yellow to almond in vain, pink blue time ripe; Substrate mycelium is yellow, and soluble pigment tinges; Gas silk pink or pink straw grass look, hair shape and powdery; Base silk is good, colourless or yellow, the careless look of straw, light brown or micro-tawny slightly, can not lysochrome; Streptomycesmicroflavus bacterium is accredited as through microbiology.With reference to " Fungal identification handbook ", the qualification of system Physiology and biochemistry is carried out to numbering XHS0032Xh bacterial strain, detect through Physiology and biochemistry and determine that numbering XHS0032Xh bacterial strain is the member that strepto-belongs in (Streptomyces).By the comparison of BLAST homology, after the 16srDNA sequence nucleotide sequence of strain X HS0032Xh carries out BLAST analysis in ncbi database, constructing system evolutionary tree, this is numbered strain X HS0032Xh and StreptomycessilaceusCB5G6 and is in minimum branch, be its allied species, its homology is 95%, and then this strain X HS0032Xh is defined as Streptomycesmicroflavus, through said system taxonomy qualification classification, demonstrate streptomyces microflavus provided by the invention (Streptomycesmicroflavus) and have certain physio-biochemical characteristics difference and the otherness of molecular level with common streptomyces microflavus, according to bacterial classification Analysis of The Physiological And Biochemical Properties, the comprehensive identification of molecular level analysis and systematics, although the bacterial classification being numbered XHS0032Xh has obvious difference with common geotrichum candidum in physio-biochemical characteristics and molecular level, streptomyces microflavus (Streptomycesmicroflavus) is accredited as from taxonomy.

Further, the invention provides be numbered XHS0035Kc subtilis, be numbered the geotrichum candidum of XHS0030B and the application of the streptomyces microflavus of XHS0032Xh in warmhouse booth soil remediation.The soil that the agent of composite soil reparation group is applied to improvement warmhouse booth is prepared by utilizing XHS0035Kc bacterial strain, XHS0030B bacterial strain and XHS0032Xh bacterial strain, unbalance, the salinification of soil nutrient in objectionable impurities effectively in improvement and activating soil, degraded soil, solution actual facility booth, to desertify or the serious present situation that hardens, improve the growing environment of farm crop in booth, improve crop quality, in the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, minimizing pesticide residue etc., there is vital role, obtain significantly good technique effect.

Meanwhile, the present invention specifically provides the preparation method of a kind of composite soil reparation group agent, and concrete preparation method's step is as follows:

(1) inoculate: preparation subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434, geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435 and the solid medium preparing streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432 and Rhodopseudomonas palustris four kinds of bacterial classifications, strictly aseptic technique is carried out after sterilizing, flat board is forwarded to from inclined-plane, the subtilis being numbered XHS0035Kc is cultivated 3 days at temperature 28 DEG C, the geotrichum candidum being numbered XHS0030B is cultivated 3 days at temperature 25 DEG C, the streptomyces microflavus of XHS0032Xh is cultivated 3 days at temperature 30 DEG C, Rhodopseudomonas palustris is temperature 32 DEG C-36 DEG C, intensity of illumination is cultivate 3 days under 3500-4500 lux.

The substratum of described subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434 is tryptose soya agar (TSA) substratum, preparation method is: Tryptones 15g, soy peptone 5g, NaCI5g, agar 20g, moisturizing is to 1000mL, pH7.2, packing, sterilizing, can prepare tryptose soya agar (TSA) substratum.

The substratum of described geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435 is PDA substratum, preparation method is: potato is cleaned peeling, gets 200 grams and be cut into small pieces, add water 1000 milliliters, after boiling half an hour, supply moisture.In filtrate, add 10 grams of agar, to boil after dissolving with sucrose 20 grams, supply moisture, packing, sterilizing, PDA solid medium can be prepared.

The substratum of described streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432 is ISP2 solid medium, preparation method is: yeast leaching powder 4.0g, Fructus Hordei Germinatus leaching powder 10.0g, glucose 4.0g, agar 20.0g, moisturizing is to 1000mL, pH7.2, packing, sterilizing, can prepare ISP2 solid medium.

The medium preparation method of described Rhodopseudomonas palustris is: Sodium Propionate 3.0g, ammonium sulfate 2.0g, potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.2g, sodium-chlor 0.5g, calcium chloride 0.1g, yeast extract paste 1.0g, moisturizing is natural to 1000mL, pH, packing, sterilizing, can prepare.。

(2) one-level is cultivated: from the solid medium step (1), picking list bacterium colony is forwarded to and is equipped with in the 50mL Erlenmeyer flask of liquid nutrient medium, aseptic technique, be numbered the subtilis of XHS0035Kc temperature 28 DEG C, 120r/min cultivates 24h-48h, be numbered the geotrichum candidum of XHS0030B temperature 25 DEG C, 120r/min cultivates 24h-48h, the streptomyces microflavus of XHS0032Xh is temperature 30 DEG C, 120r/min cultivates 24h-48h, Rhodopseudomonas palustris is temperature 32 DEG C-36 DEG C, intensity of illumination is 3500-4500 lux, 120r/min cultivates 24h-48h.

(3) second order fermentation: above-mentioned steps one-level is cultivated strain inoculation in the 500mL Erlenmeyer flask filling liquid nutrient medium, aseptic technique, the subtilis being numbered XHS0035Kc cultivates 24h-48h at temperature 28 DEG C, 120r/min, the geotrichum candidum being numbered XHS0030B cultivates 24h-48h at temperature 25 DEG C, 120r/min, the streptomyces microflavus of XHS0032Xh cultivates 24h-48h at temperature 30 DEG C, 120r/min, Rhodopseudomonas palustris temperature 32 DEG C-36 DEG C, intensity of illumination be 3500-4500 lux, 120r/min cultivates 24h-48h.

(4) compatibility of strain fermentating liquid: assess according to weight part ratio, the subtilis second order fermentation liquid 20%-25% of prepared by optional step (3) be numbered XHS0035Kc, the geotrichum candidum second order fermentation liquid 15%-20% being numbered XHS0030B, the streptomyces microflavus second order fermentation liquid 30%-35% being numbered XHS0032Xh and Rhodopseudomonas palustris 20%-25% second order fermentation liquid mix, and prepare the mixed solution of composite bacteria.

(5) auxiliary material is added: be fully adsorbed in carrier by composite bacteria mixed solution prepared by step (4), carrier selects warmhouse booth soil; Auxiliary material adopts urea and calcium superphosphate, and wherein the weight ratio of urea and calcium superphosphate is 1:2; The consumption of carrier and auxiliary material is the mixing of 1:1.5 proportioning according to weight ratio, add water to water content 40-70%, then every cubic metre of carrier and auxiliary material mixing materials add composite bacteria mixed solution 100-300g prepared by above-mentioned steps (4) by volume, and mix, prepare warmhouse booth soil-repairing agent.

Further, the invention provides the application of the above-mentioned composite soil reparation group agent of application in warmhouse booth.Subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434 is obtained by utilizing screening and separating domestication voluntarily, geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435 and prepare streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432, by practice and Rhodopseudomonas palustris phase test for fusion, facts have proved that the composite soil reparation group agent of preparation is applied to the soil of improvement warmhouse booth, improvement and activating soil effectively, objectionable impurities in degraded soil, solve soil nutrient in actual facility booth unbalance, salinification, desertify or the serious present situation that hardens, improve the growing environment of farm crop in booth, improve crop quality, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, reduce the aspects such as pesticide residue and there is vital role, obtain significantly good technique effect.

The present invention further provides the application method of above-mentioned composite soil reparation group agent: composting at air themperature 20-30 DEG C; Composting carries out under covering straw screen or mat, and composting starts turning in latter every 30 days once, and during turning, moisturizing is to water content 40-70%, composting time remaining 120 days; Directly soil can be applied to after airing to drying after composting completes.

The subtilis of XHS0035Kc is numbered selected by the present invention, the streptomyces microflavus of the geotrichum candidum and XHS0032Xh that are numbered XHS0030B screens to obtain from warmhouse booth soil, simultaneously, although red good pseudomonas is common bacterial classification in marsh, but, the specificity of microbial strains and complicacy, various different bacterial classification can be used by compound compatibility, by the blending of various bacterial classification, compatibleness is combined with each bacterial classification attribute, the security of the composite bacteria considered, particularly be applied in warmhouse booth soil remediation and all need a large amount of infrastest checkings, the present invention is based on fundamental research accumulation in early stage, by adopting the composite test of bacterial classifications different in a large number, prove that the present invention adopts composite bacteria to prepare the agent of composite soil reparation group by adding auxiliary material, the agent of composite soil reparation group according to weight ratio proportioning by subtilis two 20%-25% being numbered XHS0035Kc, be numbered the geotrichum candidum 15%-20% of XHS0030B, the streptomyces microflavus 30%-35% and the Rhodopseudomonas palustris 20%-25% bacterial classification mixed solution that are numbered XHS0032Xh are fully adsorbed in carrier plastic shed soil, and mix mutually with selecting suitable auxiliary material, the agent of composite soil reparation group is prepared by technique, and the warmhouse booth soil-repairing agent obtained is applied by booth production practice, for effectively improveing and activating soil, objectionable impurities in degraded soil, solve the salinification problem of soil, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, sauce burns the aspects such as pesticide residue and all has great importance and act on.

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

(1) subtilis being numbered XHS0035Kc of the present invention's screening, the bacterial strain that is numbered the geotrichum candidum of XHS0030B and the streptomyces microflavus of XHS0032Xh all have stronger growth and breeding ability, growth velocity is fast, hereditary property is stablized, and has specific effect to soil remediation.

(2) screening and separating domestication is voluntarily utilized to obtain subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434, geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435 and prepare streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432 and Rhodopseudomonas palustris phase test for fusion, facts have proved that the composite soil reparation group agent of preparation is applied to the soil of improvement warmhouse booth, the quality of warmhouse booth soil can be improved, improvement and activating soil, objectionable impurities in degraded soil, solve the salinification problem of soil, solve soil nutrient in actual facility booth unbalance, salinification, desertify or the serious present situation that hardens, improve the growing environment of farm crop in booth, improve crop quality, comprehensively the disadvantageous effect for Vegetable produce is reduced to bottom line, be conducive to the yield and quality improving vegetables, obtain good technique effect, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, reduce the aspects such as pesticide residue and there is vital role, there is the extensively using value of reality simultaneously in warmhouse booth soil remediation.

Accompanying drawing explanation

Fig. 1 is shown as the 16SrDNA phylogeny tree graph of subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434.

Fig. 2 is shown as the ITSrDNA sequential system evo-devo tree graph of geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435.

Fig. 3 is shown as the 16SrDNA phylogeny tree graph of streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432.

Embodiment

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

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: the molecular level of subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434

1, the 16SrDNA sequence of pcr amplification subtilis and order-checking thereof

Single bacterium colony of picking a small amount of XHS0035Kc bacterial strain, put into the EP pipe filling 25 μ L sterilized waters, 100 DEG C are boiled 8-10min, put into rapidly mixture of ice and water 5min afterwards.Centrifugal 10000r/min, 5min, 4 DEG C of preservations, the used time gets supernatant.

The structure of 16SrDNA gene sequencing and systematic evolution tree thereof: the STb gene extracting bacterial strain according to a conventional method, will dilute universal primer with deionized water, and performing PCR of going forward side by side increases, and design of primers is as follows:

27f:AGAGTTTGATCCTGGCTCAG

1492r:TACGGCTACCTTGTTACGACTT

The structure of 16SrRNA gene sequencing and systematic evolution tree thereof: the STb gene extracting bacterial isolates according to a conventional method, with the pcr amplification of deionized water by dilution universal primer 16srDNA section.Primer is synthesized by Shanghai biotechnology company limited.Electrophoresis detection, delivers to the order-checking of Shanghai Sheng Gong bio-engineering corporation.

2,16SrDNA sequence alignment and Phylogenetic Analysis

Nucleotide sequence in the 16SrDNA sequence and GenBank database that obtain checking order carries out BLAST analysis, therefrom obtain close 16SrDNA sequence, after the 16SrDNA sequence of XHS0035Kc is carried out BLAST analysis in ncbi database, constructing system evolutionary tree.Shown in accompanying drawing 1, between strain X HS0035Kc and 30334-31756BacillusSubtilisKCTC1028, evolutionary distance is the shortest, is the allied species of 30334-31756BacillusSubtilisKCTC.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of XHS0035Kc, determine that it is subtilis (BacillusSubtilis) and belong to.By measured 16SrDNA sequence inputting Genbank, tetraploid rice is carried out with Blast program, the similarity maximum comparability finding the 16SrDNA sequence of it and 30334-31756BacillusSubtilisKCTC1028 is 84%, thus determines that it is BacillusSubtilis further.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of XHS0035Kc, to determine that it is bacterium numbering be XHS0035Kc comprehensive identification is subtilis (BacillusSubtilis).

Embodiment two: the molecular level of geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435

1, the ITSrDNA sequence of pcr amplification geotrichum candidum and order-checking thereof

Single bacterium colony of picking a small amount of XHS0030B bacterial strain, put into the EP pipe filling 25 μ L sterilized waters, 100 DEG C are boiled 8-10min, put into rapidly mixture of ice and water 5min afterwards.Centrifugal 10000r/min, 5min, 4 DEG C of preservations, the used time gets supernatant.

The structure of ITSrDNA gene sequencing and systematic evolution tree thereof: the STb gene extracting bacterial isolates according to a conventional method, with deionized water, dilution universal primer ITSl and ITS4 is carried out the pcr amplification of ITSrDNA section, design of primers is as follows:

ITS1(F)：5'-TCCGTAGGTGAACCTGCGG-3'

ITS4(R)：5'-TCCTCCGCTTATTGATATGC-3'

The reaction system of 50 μ l contains: 10 × PCR damping fluid 5 μ l, each 20pmol of primer, template DNA (1000ng/ μ l) 1 μ l, TaqTM (TaKaRa company) 0.5U, dNTP8 μ l.Pcr amplification condition: first at 94 DEG C of denaturation 2min, then 98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 1.5min, circulate 30 times, finally extends 10min at 72 DEG C.The purifying of PCR primer: the PCR primer of getting 8 μ l, electrophoresis in 1% sepharose, reclaims object fragment with TaKaRaPCRFragmentRecoveryKit from glue, is dissolved in the high purity water of 20 μ l.Make sequencing primer to PCR primer PA (+) and PB (-), measure ITSrDNA sequence, the gene order of bacterial classification XHS0030B is see attached gene order table.Need according to the single-row gene order table of the gene order provided.

2, the comparison of ITSrDNA gene order and Phylogenetic Analysis

Nucleotide sequence in the ITSrDNA sequence and GenBank database that obtain checking order carries out BLAST analysis, therefrom obtain close ITSrDNA sequence, after the ITSrDNA sequence of XHS0030B is carried out BLAST analysis in ncbi database, constructing system evolutionary tree.Shown in accompanying drawing 2, between strain X HS0030B and GeotrichumCandidumisolateL11B, evolutionary distance is the shortest, is the allied species of GeotrichumCandidumisolate.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of XHS0030B, determine that it is geotrichum candidum and belong to.By measured ITSrDNA sequence inputting Genbank, tetraploid rice is carried out with Blast program, find that the similarity of the ITSrDNA sequence of it and GeotrichumCandidumisolateL11B is maximum, be 94%, thus be defined as GeotrichumCandidum further.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of XHS0030B, to determine that it is bacterium numbering be XHS0030B comprehensive identification is geotrichum candidum (GeotrichumCandidum).

Embodiment three: the molecular level of streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432

1, the 16SrDNA sequence of pcr amplification streptomyces microflavus and order-checking thereof

Single bacterium colony of picking a small amount of XHS0032Xh bacterial strain, put into the EP pipe filling 25 μ L sterilized waters, 100 DEG C are boiled 8-10min, put into rapidly mixture of ice and water 5min afterwards.Centrifugal 10000r/min, 5min, 4 DEG C of preservations, the used time gets supernatant.

27f:AGAGTTTGATCCTGGCTCAG

1492r:TACGGCTACCTTGTTACGACTT

The structure of 16SrRNA gene sequencing and systematic evolution tree thereof: the STb gene extracting bacterial strain according to a conventional method, with the pcr amplification of deionized water by dilution universal primer 16srDNA section.Primer is synthesized by Shanghai biotechnology company limited.Electrophoresis detection, delivers to the order-checking of Shanghai Sheng Gong bio-engineering corporation.

2,16SrDNA sequence alignment and Phylogenetic Analysis

Nucleotide sequence in the 16SrDNA sequence and GenBank database that obtain checking order carries out BLAST analysis, therefrom obtain close 16SrDNA sequence, after the 16SrDNA sequence of XHS0032Xh is carried out BLAST analysis in ncbi database, constructing system evolutionary tree.Shown in accompanying drawing 3, between strain X HS0032Xh and StreptomycessilaceusCB5G6, evolutionary distance is the shortest, is the allied species of Streptomyces.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of XHS0032Xh, determine that it is streptomyces microflavus (Streptomycesmicroflavus) and belong to.By measured 16SrDNA sequence inputting Genbank, tetraploid rice is carried out with Blast program, the similarity maximum comparability finding the 16SrDNA sequence of it and StreptomycessilaceusCB5G6 is 95%, thus is defined as Streptomyces further.In conjunction with Morphologic Characteristics and the physio-biochemical characteristics of XHS0032Xh, to determine that it is bacterium numbering be XHS0032Xh comprehensive identification is that streptomyces microflavus (Streptomycesmicroflavus) belongs to.

Embodiment four: the preparation of composite soil reparation group agent

(1) inoculate: preparation subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434, the solid medium of geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435 and streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432 and Rhodopseudomonas palustris four kinds of bacterial classifications, strictly aseptic technique is carried out after sterilizing, flat board is forwarded to from inclined-plane, the subtilis being numbered XHS0035Kc is cultivated 3 days at temperature 28 DEG C, the geotrichum candidum being numbered XHS0030B is cultivated 3 days at temperature 25 DEG C, the streptomyces microflavus of XHS0032Xh is cultivated 3 days at temperature 30 DEG C, Rhodopseudomonas palustris is temperature 32 DEG C-36 DEG C, intensity of illumination is cultivate 3 days under 3500-4500 lux.

The medium preparation method of described Rhodopseudomonas palustris is: Sodium Propionate 3.0g, ammonium sulfate 2.0g, potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.2g, sodium-chlor 0.5g, calcium chloride 0.1g, yeast extract paste 1.0g, moisturizing is natural to 1000mL, pH, packing, sterilizing, can prepare.

(5) auxiliary material is added: be fully adsorbed in carrier by composite bacteria mixed solution prepared by step (4), carrier selects warmhouse booth soil; Auxiliary material adopts urea and calcium superphosphate, and wherein the weight ratio of urea and calcium superphosphate is 1:2; The consumption of carrier and auxiliary material is the mixing of 1:1.5 proportioning according to weight ratio, add water to water content 40-70%, then every cubic metre of carrier and auxiliary material mixing materials add composite bacteria mixed solution 100-300g prepared by above-mentioned steps (4) by volume, and mix, prepare the agent of composite soil reparation group.

Meanwhile, the invention provides the application method of composite soil reparation group agent: composting at air themperature 20-30 DEG C; Composting carries out under covering straw screen or mat, and composting starts turning in latter every 30 days once, and during turning, moisturizing is to water content 40-70%, composting time remaining 120 days; Directly soil can be applied to after airing to drying after composting completes.

Further, the invention provides the application of the above-mentioned composite soil reparation group agent of application in warmhouse booth.Subtilis (BacillusSubtilis) XHS0035KcCGMCCNO.9434 is obtained by utilizing screening and separating domestication voluntarily, geotrichum candidum (GeotrichumCandidum) XHS0030BCGMCCNO.9435 and prepare streptomyces microflavus (Streptomycesmicroflavus) XHS0032XhCGMCCNO.9432, by practice and Rhodopseudomonas palustris phase test for fusion, facts have proved that the composite soil reparation group agent of preparation is applied to the soil of improvement warmhouse booth, for improvement and activating soil, objectionable impurities in degraded soil, solve the salinification problem of soil, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, reduce the aspects such as pesticide residue and obtain significantly outstanding technique effect.

The subtilis of XHS0035Kc is numbered selected by the present invention, the streptomyces microflavus of the geotrichum candidum and XHS0032Xh that are numbered XHS0030B screens to obtain from warmhouse booth soil, simultaneously, although red good pseudomonas is common bacterial classification in marsh, but, the specificity of microbial strains and complicacy, various different bacterial classification can be used by compound compatibility, by the blending of various bacterial classification, compatibleness is combined with each bacterial classification attribute, the security of the composite bacteria considered, particularly be applied in warmhouse booth soil remediation and all need a large amount of infrastest checkings, the present invention is based on fundamental research accumulation in early stage, by adopting the composite test of bacterial classifications different in a large number, prove that the present invention adopts composite bacteria to prepare the agent of composite soil reparation group by adding auxiliary material, the agent of composite soil reparation group is according to weight ratio proportioning, by subtilis two 20%-25% being numbered XHS0035Kc, be numbered the geotrichum candidum 15%-20% of XHS0030B, the streptomyces microflavus 30%-35% and the Rhodopseudomonas palustris 20%-25% bacterial classification mixed solution that are numbered XHS0032Xh are fully adsorbed in carrier plastic shed soil, and mix mutually with selecting suitable auxiliary material, the agent of composite soil reparation group is prepared by technique, and the composite soil reparation group agent obtained is applied by booth production practice, for effectively improveing and activating soil, objectionable impurities in degraded soil, solve the salinification problem of soil, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, reduce the aspects such as pesticide residue all have great importance and act on.

Embodiment five: the agent of composite soil reparation group is to the repair of warmhouse booth soil

1, test design and process

This test is carried out (30m × 70m) in the pattern plastic warmhouse booth of Korla City.The character of topsoil (0 ~ 15cm) is as table 1.Before this research, greenhouse soil planted continuously 2 season cucumber.

Test has 2 process and 1 contrast, that is: control group, commercially available soil-repairing agent group, soil-repairing agent group prepared by single geotrichum candidum, subtilis and streptomyces microflavus, composite soil reparation group agent prepared by the present invention.Repeat for 4 times, Gong12Ge community, each community 45m ², the group arrangement of completely random district is taked in test.In order to reduce edge effect, between community, stay the buffer zone of 1.2m.

Table 1: the physico-chemical property of greenhouse soil

The grains of sand (%)	Powder (%)	Clay (%)	Ph	Specific conductivity	Total carbon (%)	Total nitrogen (%)	Carbon-nitrogen ratio
								22.5	36.3	41.2	7.88	0.87	1.57	0.41	3.87

2, process of the test

Each community is transplanted into by 45000 strain every mu by cultivating 15d and growing homogeneous cucumber seedling.During cucumber growth, adopt furrow irrigation to keep field water holding 70%-80%.At the cucumber result initial stage, cucumber is carried out that sampling and measuring total biomass, climing length, stem are thick, leaf area index and the number of blade.The output of cucumber adopts cumulative method to measure.

3, test-results

(1) warmhouse booth soil-repairing agent is on the impact of greenhouse cucumber growth, yield and quality

As shown in Table 2, compare with contrast, composite soil reparation group agent prepared by the soil-repairing agent of single bacterial classification prepared by commercially available normal soil renovation agent, the present invention and the present invention all can significantly improve the output of cucumber.But, it is 58.7% that the composite soil reparation group agent that commercially available normal soil renovation agent improves output 10.1%, prepared by the present invention improves output, composite soil reparation group agent prepared by visible the present invention is obviously better than commercially available normal soil renovation agent, is better than single bacterial classification geotrichum candidum warmhouse booth soil-repairing agent, subtilis soil-repairing agent and streptomyces microflavus soil-repairing agent simultaneously.The process of warmhouse booth soil-repairing agent all can significantly reduce nitrate in cucumber.

Table 2: warmhouse booth soil-repairing agent is on the impact of greenhouse cucumber growth, yield and quality

(2) warmhouse booth soil-repairing agent affects result to cucumber nutritive ingredient

As can be seen from Table 3, warmhouse booth soil-repairing agent does not all have a significant impact nitrogen in cucumber fruits and Fe content.Compare with contrast, the copper content in cucumber fruits can be reduced.Visible, soil-repairing agent prepared by the present invention can improve the soil quality of warmhouse booth, repairs the soil of warmhouse booth.

Table 3: warmhouse booth soil-repairing agent is on the impact of cucumber nutritive ingredient

Utilize the composite soil reparation group agent of preparation can improve the quality of warmhouse booth soil, improvement and activating soil, objectionable impurities in degraded soil, solve the salinification problem of soil, solve soil nutrient in actual facility booth unbalance, salinification, desertify or the serious present situation that hardens, improve the growing environment of farm crop in booth, improve crop quality, comprehensively the disadvantageous effect for Vegetable produce is reduced to bottom line, be conducive to the yield and quality improving vegetables, obtain good technique effect, at the anti-continuous cropping of farm crop, prevention rotten of greenhouse crops, blight, reduce the aspects such as pesticide residue and there is vital role, there is the extensively using value of reality simultaneously in warmhouse booth soil remediation.

Above-described embodiment is only for example of the present invention is clearly described, and the restriction not to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And apparent change extended thus or variation are still among protection scope of the present invention.

SEQUENCELISTING1

<110> Huisen Biotech Co., Ltd., Xinjiang

<120> subtilis and the application in warmhouse booth soil remediation thereof

<130> subtilis

<160>1

<170>PatentInversion3.3

<210>1

<211>1420

<212>DNA

<213> subtilis

<220>

<221>16SrDNA

<222>(1)..(1420)

<400>1

tgcaagtcgagcggacagatgggagcttgctccctgatgttagcggcggacgggtgagta60

acacgtgggtaacctgcctgtaagactgggataactccgggaaaccggggctaataccgg120

atggttgtttgaaccgcatggttcaaacataaaaggtggcttcggctaccacttacagat180

ggacccgcggcgcattagctagttggtgaggtaacggctcaccaaggcaacgatgcgtag240

ccgacctgagagggtgatcggccacactgggactgagacacggcccagactcctacggga300

ggcagcagtagggaatcttccgcaatggacgaaagtctgacggagcaacgccgcgtgagt360

gatgaaggttttcggatcgtaaagctctgttgttagggaagaacaagtaccgttcgaata420

gggcggtaccttgacggtacctaaccagaaagccacggctaactacgtgccagcagccgc480

ggtaatacgtaggtggcaagcgttgtccggaattattgggcgtaaagggctcgcaggcgg540

tttcttaagtctgatgtgaaagcccccggctcaaccggggagggtcattggaaactgact600

tgagtgcagaagaggagagtggaattccacgtgtagcggtgaaatgcgtagagatgtgga660

ggaacaccagtggcgaaggcgactctctggtctgtaactgacgctgaggagcgaaagcgt720

ggggagcgaacaggattagataccctggtagtccacgccgtaaacgatgagtgctaagtg780

ttagggggtttccgccccttagtgctgcagctaacgcattaagcactccgcctggggagt840

acggtcgcaagactgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatg900

tggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctctgacaatcct960

agagataggacgtccccttcgggggcagagtgacaggtggtgcatggttgtcgtcagctc1020

gtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttgatcttagttgccag1080

cattcagttgggcactctaaggtgactgccggtgacaaaccggaggaaggtggggatgac1140

gtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggacagaacaaa1200

gggcagcgaaaccgcgaggttaagccaatcccacaaatctgttctcagttcggatcgcag1260

tctgcaactcgactgcgtgaagctggaatcgctagtaatcgcggatcagcatgccgcggt1320

gaatacgttcccgggccttgtacacaccgcccgtcacaccacgagagtttgtaacacccg1380

aagtcggtgaggtaacctttttaggagccagccgccgaag1420

SEQUENCELISTING2

<110> Huisen Biotech Co., Ltd., Xinjiang

<120> streptomyces microflavus and the application in warmhouse booth soil remediation thereof

<130> streptomyces microflavus

<160>1

<170>PatentInversion3.3

<210>1

<211>1302

<212>DNA

<213> streptomyces microflavus

<220>

<221>16SrDNA

<222>(1)..(1302)

<400>1

gggtgagtaacacgtgggcaatctgcccttcactctgggacaagccctggaaacggggtc60

taataccggataacactctctctcgcatgggagagggttgaaagctccggcggtgaagga120

tgagcccgcggcctatcagctagttggtgaggtagaagctcaccaaggcgacgacgggta180

gccggcctgagagggcgaccggccacactgggactgagacacggcccagactcctacggg240

aggcagcagtggggaatattgcacaatgggcgaaagcctgatgcagcgacgccgcgtgag300

ggatgacggccttcgggttgtaaacctctttcagcagggaagaagcgaaagtgacggtac360

ctgcagaagaagcgccggctaactacgtgccagcagccgcggtaatacgtagggcgcaag420

cgttgtccggaattattgggcgtaaagagctcgtaggcggcttgtcacgtcggttgtgaa480

agcccggggcttaaccccgggtctgcagtcgatacgggcaggctagagttcggtagggga540

gatcggaattcctggtgtagcggtgaaatgcgcagatatcaggaggaacaccggtggcga600

aggcggatctctgggccgatactgacgctgaggagcgaaagcgtggggagcgaacaggat660

tagataccctggtagtccacgccgtaaacggtgggaactaggtgtgggcgacattccacg720

tcgtccgtgccgcagctaacgcatccccgcctggggagtacggccgcaaggctaaaactc780

aaaggaattgacgggggcccgcacaagcggcggagcatgtggcttaattcgacgcaacgc840

gaagaaccttaccaaggcttgacatacaccggaaagcatcagagatggtgccccccttgt900

ggtcggtgtacaggtggtgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaag960

tcccgcaacgagcgcaacccttgtcccgtgttgccagcaagccccttcgggggtgttggg1020

gactcacgggagaccgccggggtcaactcggaggaaggtggggacgacgtcaagtcatca1080

tgccccttatgtcttgggctgcacacgtgctacaatggccggtacaatgagctgcgatac1140

cgcgaggtggagcgaatctcaaaaagccggtctcagttcggattggggtctgcaactcga1200

ccccatgaagtcggagtcgctagtaatcgcagatcagcattgctgcggtgaatacgttcc1260

cgggccttgtacacaccgcccgtcacgtcacgaaagtcggta1302

SEQUENCELISTING3

<110> Huisen Biotech Co., Ltd., Xinjiang

<120> geotrichum candidum and the application in warmhouse booth soil remediation thereof

<130> geotrichum candidum

<160>1

<170>PatentInversion3.3

<210>1

<211>371

<212>DNA

<213> geotrichum candidum

<220>

<221>ITSrDNA

<222>(1)..(371)

<400>1

tccgtaggtgaacctgcggaaggatcattatgaattaataatatttgtgaaatttcaaca60

aacaacatcaattttatagtctattatttttaattaaaacttttaacaatggatctcttg120

gttctcgtatcgatgaagaacgcagcgaaacgcgatatttcttgtgaattgcagaagtga180

atcatcagtttttgaacgcacattgcactttggggtatcccccaaagtatacttgtttga240

gcgttgtttctctcttggaattgctttgctcttctaaaatttcgaatcaaattcgtttga300

aaaacaacactattcaacctcagatcaagtaggattacccgctgaacttaagcatatcaa360

taagcggagga

Claims

1. the subtilis being applicable to warmhouse booth soil remediation ( bacillusSubtilis) XHS0035Kc, it is characterized in that, described subtilis ( bacillusSubtilis) XHS0035Kc deposit number is CGMCCNO.9434, gene order is as SEQUENCELISTING1.

2. the streptomyces microflavus being applicable to warmhouse booth soil remediation ( streptomycesmicroflavus) XHS0032Xh, it is characterized in that, described streptomyces microflavus ( streptomycesmicroflavus) XHS0032Xh deposit number is CGMCCNO.9432, gene order is as SEQUENCELISTING2.

3. the geotrichum candidum being applicable to warmhouse booth soil remediation ( geotrichumCandidum) XHS0030B, it is characterized in that, described geotrichum candidum ( geotrichumCandidum) XHS0030B deposit number is CGMCCNO.9435, gene order is as SEQUENCELISTING3.

4. a composite soil reparation group agent, is characterized in that, described a kind of composite soil reparation group agent is obtained by following preparation method:

(1) inoculate: preparation subtilis ( bacillusSubtilis) XHS0035KcCGMCCNO.9434, geotrichum candidum ( geotrichumCandidum) XHS0030BCGMCCNO.9435 and streptomyces microflavus ( streptomycesmicroflavus) solid medium of XHS0032XhCGMCCNO.9432 and Rhodopseudomonas palustris four kinds of bacterial classifications, strictly aseptic technique is carried out after sterilizing, flat board is forwarded to from inclined-plane, the subtilis being numbered XHS0035Kc is cultivated 3 days at temperature 28 DEG C, the geotrichum candidum being numbered XHS0030B is cultivated 3 days at temperature 25 DEG C, the streptomyces microflavus of XHSOO32Xh is cultivated 3 days at temperature 30 DEG C, and Rhodopseudomonas palustris is cultivated 3 days under temperature 32 DEG C-36 DEG C, intensity of illumination are 3500-4500 lux;

Described subtilis ( bacillusSubtilis) substratum of XHS0035KcCGMCCNO.9434 is tryptose soya agar (TSA) substratum, preparation method is: Tryptones 15g, soy peptone 5g, NaCI5g, agar 20g, moisturizing is to 1000mL, pH7.2, packing, sterilizing, can prepare tryptose soya agar (TSA) substratum;

Described geotrichum candidum ( geotrichumCandidum) substratum of XHS0030BCGMCCNO.9435 is PDA substratum, preparation method is: potato is cleaned peeling, gets 200 grams and be cut into small pieces, add water 1000 milliliters, after boiling half an hour, supply moisture; In filtrate, add 10 grams of agar, to boil after dissolving with sucrose 20 grams, supply moisture, packing, sterilizing, PDA solid medium can be prepared;

Described streptomyces microflavus ( streptomycesmicroflavus) substratum of XHS0032XhCGMCCNO.9432 is ISP2 solid medium, preparation method is: yeast leaching powder 4.0g, Fructus Hordei Germinatus leaching powder 10.0g, glucose 4.0g, agar 20.0g, moisturizing is to 1000mL, pH7.2, packing, sterilizing, can prepare ISP2 solid medium;

The medium preparation method of described Rhodopseudomonas palustris is: Sodium Propionate 3.0g, ammonium sulfate 2.0g, potassium primary phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.2g, sodium-chlor 0.5g, calcium chloride 0.1g, yeast extract paste 1.0g, moisturizing is natural to 1000mL, pH, packing, sterilizing, can prepare;

(2) one-level is cultivated: from the solid medium step (1), picking list bacterium colony is forwarded to and is equipped with in the 50mL Erlenmeyer flask of liquid nutrient medium, aseptic technique, the subtilis being numbered XHS0035Kc cultivates 24h-48h at temperature 28 DEG C, 120r/min, the geotrichum candidum being numbered XHS0030B cultivates 24h-48h at temperature 25 DEG C, 120r/min, the streptomyces microflavus of XHS0032Xh cultivates 24h-48h at temperature 30 DEG C, 120r/min, Rhodopseudomonas palustris temperature 32 DEG C-36 DEG C, intensity of illumination be 3500-4500 lux, 120r/min cultivates 24h-48h;

(3) second order fermentation: above-mentioned steps one-level is cultivated strain inoculation in the 500mL Erlenmeyer flask filling liquid nutrient medium, aseptic technique, the subtilis being numbered XHS0035Kc cultivates 24h-48h at temperature 28 DEG C, 120r/min, the geotrichum candidum being numbered XHS0030B cultivates 24h-48h at temperature 25 DEG C, 120r/min, the streptomyces microflavus of XHS0032Xh cultivates 24h-48h at temperature 30 DEG C, 120r/min, Rhodopseudomonas palustris temperature 32 DEG C-36 DEG C, intensity of illumination be 3500-4500 lux, 120r/min cultivates 24h-48h;

(4) compatibility of strain fermentating liquid: assess according to weight part ratio, the subtilis second order fermentation liquid 20%-25% of prepared by optional step (3) be numbered XHS0035Kc, the geotrichum candidum second order fermentation liquid 15%-20% being numbered XHS0030B, the streptomyces microflavus second order fermentation liquid 30%-35% being numbered XHS0032Xh and Rhodopseudomonas palustris 20%-25% second order fermentation liquid mix, and prepare the mixed solution of composite bacteria;

5. the using method of composite soil reparation group agent as claimed in claim 4, is characterized in that, described using method step is as follows:

Composite soil reparation group agent composting at air themperature 20-30 DEG C prepared by claim 4; Composting carries out under covering straw screen or mat, and composting starts turning in latter every 30 days once, and during turning, moisturizing is to water content 40-70%, composting time remaining 120 days; Directly soil can be applied to after airing to drying after composting completes.

6. the application of composite soil reparation group agent in warmhouse booth soil remediation as claimed in claim 4.