CN100390272C - Fluorescent pseudomonads and its fermenting culture process and application - Google Patents

Fluorescent pseudomonads and its fermenting culture process and application Download PDF

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CN100390272C
CN100390272C CNB2005101125429A CN200510112542A CN100390272C CN 100390272 C CN100390272 C CN 100390272C CN B2005101125429 A CNB2005101125429 A CN B2005101125429A CN 200510112542 A CN200510112542 A CN 200510112542A CN 100390272 C CN100390272 C CN 100390272C
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pseudomonas fluorescens
plant
strain
culture
cgmcc
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CNB2005101125429A
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CN1772881A (en
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张力群
席先梅
潘林芳
张清霞
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中国农业大学
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Abstract

The present invention discloses a pseudomonas fluorescent strain MX01 CGMCC No. 1469, a fermentation culture method thereof and an application thereof. The strain effectively promotes the plant growth and has the conspicuous prevention and treatment effect on various plant soil-borne diseases such as tomato bacterial wilt, wheat take-all and rhizoctonia seedling blight of vegetables through being verified by greenhouse pot culture and field experiments. Through being indicated by molecular genetic study, the strain has a main mechanism for promoting the plant growth that the strain comprises 1-aminocyclopropane-1-carboxylic acid salt (1-aminocyclopropane-1-carboxylate, for short) aminase genes, the ACC aminase reduces the vinylic content of the plant root during the seedling period and the vinylic content around the plant roots, so the plant growth is promoted; the strain has a main mechanism for preventing and treating the plant diseases of strong plant rhizosphere colonization capability and capability of producing antibiotic materials for inhibiting the pathogenic bacterium growth. The pseudomonas fluorescent strain MX01 CGMCC No. 1469 has the advantages of short fermentation period and simple preparation process of microbial preparations thereof, and has the commercial process possibility. The strain of the present invention has wide application prospects on plant growth promotion and plant soil-borne disease prevention and treatment.

Description

One fluorescent pseudomonads and fermentation culture method thereof and application

Technical field

The present invention relates to a fluorescent pseudomonads and fermentation culture method thereof and application.

Background technology

Exist some beneficial bacterias in the plant rhizosphere, they can promote plant-growth by direct or indirect mode and to playing preventive and therapeutic effect by the microbial Plant diseases of cause of disease, the microorganism that these can promote plant-growth, prevent and treat disease, increase crop yield is referred to as the short border bacterium that takes root (plant growth promoting rhizobacteria is called for short PGPR).PGPR has the biological control effect to harmful pathogenic micro-organism in the soil and non-parasitics rhizosphere harmful microorganism (deleterious rhizosphere microorganisms is called for short DRMO).In addition, utilize mineral matter nutritional also to have promoter action, and can produce the meta-bolites of useful plants growth, thereby promote growth and development of plant plant absorbing.

Studies show that root can synthesize a certain amount of ethene during plant seedling growth, it is to plant-growth, and especially the growth of root is inhibited.1-amino-cyclopropane-1-carboxylate salt (1-aminocyclopropane-1-carboxylate is called for short ACC) deaminase can decompose the direct precursor substance ACC of ethene synthetic, thereby ethene synthesizes the reduction ethylene levels in the supression plant materials.

Summary of the invention

The purpose of this invention is to provide a strain and have Pseudomonas fluorescens and the fermentation culture method thereof that promotes plant-growth and prevent and treat the disease effect.

Pseudomonas fluorescens strain provided by the present invention is (Pseudomonas fluorescens) MX01, this bacterial strain is preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center on September 30th, 2005, and deposit number is CGMCC No.1469.

Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 belongs to gram negative bacillus, and cell is single, and the tool flagellum can move.This bacterium obligate is aerobic, cultivates 2-3 days for 28 ℃ on the LB solid medium, can grow single bacterium colony, and bacterium colony is white in color, circle, neat in edge, smooth.Its physio-biochemical characteristics are: can produce fluorochrome 4-37 ℃ of growth down, oxydase reaction, the two hydrolysis reaction of arginine, catalase reaction and gelatine liquefication reaction all are positive, and the hydrolyzed starch reaction is negative; Can utilize several kinds of carbon source to grow, as glucose, sucrose, trehalose, D-wood sugar, D-fructose, maltose, N.F,USP MANNITOL, inositol, L-proline(Pro), L-arginine, L-Xie Ansuan or Beta-alanine etc.

Second purpose of the present invention provides the fermentation culture method of a kind of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469.

The fermentation culture method of Pseudomonas fluorescens provided by the present invention (Pseudomonas fluorescens) MX01CGMCC No.1469, may further comprise the steps: 1) activated Pseudomonas fluorescens (Pseudomonasfluorescens) MX01CGMCC No.1469 is inoculated in this bacterium fermenting substratum, under 27-30 ℃, pH6.5-8.0, cultivated 36-48 hour, obtain seed liquor; 2) seed liquor with step 1) preparation is inoculated in this bacterium fermenting substratum, aerated culture under 26-32 ℃, pH 6.5-8.0; Described step 1) and step 2) in Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 fermenting culture medium prescription be: contain Semen Maydis powder 25-35g in every 1000mL water, soybean cake powder 25-35g, bran powder 16-24g, K 2HPO 416-24g, KH 2PO 46-10g, MgSO 40.5-1.0g, initial pH 7.2-8.0.

In above-mentioned fermentation culture method, Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCCNo.1469 bacterial strain is carried out the activatory method be: this inoculation in Jin Shi B substratum, was cultivated 24-48 hour down at 27-30 ℃; The prescription of described Jin Shi B substratum is: contain peptone 20g in every 1000mL water, glycerine 10mL, MgSO 47H 2O 1.5g, K 2HPO 4, 1.5g, agar 18g, pH 7.0-7.4.

Preferred Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 fermenting culture medium prescription is: contain Semen Maydis powder 30g in every 1000mL water, soybean cake powder 30g, bran powder 20g, K 2HPO 420g, KH 2PO 48g, MgSO 40.75g, pH 7.2-8.0.

For obtaining better ferment effect, also be added with certain density defoamer in above-mentioned Pseudomonas fluorescens (Pseudomonas fluorescens) the MX01CGMCC No.1469 fermenting substratum, the additive capacity of defoamer can be decided according to concrete used defoamer kind, described defoamer can be any one commercially available defoamer, preferred defoamer is the bubble enemy, and it adds concentration and is preferably 0.05%; Described percentage concentration is a mass percent concentration.

Inoculative proportion in the step 1) is 1-2%, and culture condition is preferably: shaking culture under 28 ℃, pH 7.0,200rpm.

Step 2) inoculative proportion in is 2-4%, and fermentating liquid volume and per minute air flow volume ratio are 1: 0.5-0.8, tank pressure are 1.5-2.0F/cm 2, stirring velocity is 150-250rpm, is preferably 200rpm, cultivates can arrive the cultivation terminal point in 24-48 hour; Wherein, under 28 ℃, pH 7.0 and above-mentioned condition aerated culture 36-48 hour be preferred fermentation culture conditions.

Another object of the present invention provides a kind of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 microbial inoculum.

Pseudomonas fluorescens provided by the present invention (Pseudomonas fluorescens) MX01CGMCC No.1469 microbial inoculum, its activeconstituents are Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469.

For obtaining better result of use, also contain stopping composition in the described microbial inoculum, as the attapulgite or the peat composed of rotten mosses etc., the fineness of stopping composition is the 150-250 order, is preferably 200 orders.

The production method of a kind of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 microbial inoculum, be will be through the bacterium liquid of Pseudomonas fluorescens (Pseudomonas fluorescens) the MX01 CGMCCNo.1469 of aforesaid method fermentation and drying, aseptic stopping composition 8-10 by ratio of weight and the number of copies: 1 mix after, press filtration, it is air-dry to shade after the press filtration, finally obtains Pseudomonas fluorescens (Pseudomonasfluorescens) the MX01 CGMCC No.1469 microbial inoculum that the viable bacteria amount is not less than 2,000,000,000/gram; Described stopping composition can be the attapulgite or the peat composed of rotten mosses, and fineness is the 150-250 order, is preferably 200 orders.

In actual production, requirement according to the microbial inoculum water content, the bacterium liquid of Pseudomonas fluorescens (Pseudomonasfluorescens) MX01 CGMCC No.1469 is mixed with stopping composition, after the press filtration, can add a certain amount of auxiliary agent again, as the peat composed of rotten mosses, attapulgite or diatomite, be preferably the peat composed of rotten mosses, fineness is the 150-250 order, be preferably 200 orders, the additive capacity of auxiliary agent can be decided according to the practical situation of bacterium amount and water content, under the prerequisite that does not influence the product effect, make the water content of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 microbial inoculum reach 30-40% and get final product.

Use Pseudomonas fluorescens of the present invention (Pseudomonas fluorescens) MX01 CGMCC No.1469 or be that the microbial inoculum of activeconstituents promotes the method for plant-growth can be: before the sowing, be 10 plant seed concentration with it 7-10 9The Pseudomonas fluorescens of cfu/mL (Pseudomonas fluorescens) MX01 CGMCC No.1469 bacterium liquid seed soaking 0.5-2 hour, or be the microbial inoculum seed dressing of activeconstituents in order to Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCCNo.1469, the ratio of weight and number of microbial inoculum and seed is 1: 0.5-2, through sowing, cultivation, obtain growth and obtain promoted plant again.

In the method for above-mentioned promotion plant-growth, Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 bacterial concentration is preferably 10 8Cfu/mL, seed soaking time are preferably 1 hour; With Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 is that the microbial inoculum of activeconstituents is 1: 1 with seed blended weight fraction ratio.

Use Pseudomonas fluorescens of the present invention (Pseudomonas fluorescens) MX01 CGMCC No.1469 or be that the method for the microbial inoculum controlling plant diseases of activeconstituents can be with it: treating that plant-growth to 3-4 leaf during the phase, takes out seedling, is 10 with concentration 7-10 9The Pseudomonas fluorescens of cfu/mL (Pseudomonas fluorescens) MX01CGMCC No.1469 bacterium liquid soaked root 20-40 minute, be that the microbial inoculum of activeconstituents is converted water and irritated root with Pseudomonas fluorescens (Pseudomonasfluorescens) MX01CGMCC No.1469 maybe with the seedling of taking out, the ratio of weight and number of microbial inoculum and water is 1: 400-600, through transplanting, cultivating, obtain the plant of disease resistance again.

In the pest control method of above-mentioned plant, Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 bacterial concentration is preferably 10 8Cfu/mL soaks the root time to be preferably 30 minutes; With Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 is that the microbial inoculum and the water blended ratio of weight and number of activeconstituents is 1: 500.

The invention provides a fluorescent pseudomonads (Pseudomonas fluorescens) MX01 CGMCC No.1469 and be the microbial inoculum of activeconstituents with it, greenhouse pot culture and field test prove that this bacterial strain has short giving birth to and the diseases prevention dual function, not only can effectively promote plant-growth, and, has remarkable prevention effect as the rhizoctonia damping-off of bacterial wilt of tomato, take-all, vegetable crop to the various plants soil-borne disease.Molecule genetics research shows that this bacterial strain promotes that the main mechanism of plant-growth is that it has 1-amino-cyclopropane-1-carboxylate salt (1-aminocyclopropane-1-carboxylate, be called for short ACC) the deaminase gene, the ACC deaminase can reduce the plant root in seedling stage and the content of ethene on every side thereof, thus the stimulating plant growth.The main mechanism of controlling plant diseases is its good plant rhizosphere colonization ability and produces the antibiotics material that suppresses the pathogenic bacteria growth.

Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 fermentation period is short, and its fungicide preparation technology is simple, has the possibility of suitability for industrialized production.The present invention has broad application prospects in the control that promotes plant growth and plant soil-borne diseases.

The present invention will be further described below in conjunction with specific embodiment.

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and all percentage concentrations are mass percent concentration, and the solvent in all substratum is water.

The prescription of Jin Shi B substratum is: contain peptone 20g in every 1000mL water, glycerine 10mL, MgSO 47H 2O1.5g, K 2HPO 4, 1.5g, agar 18g, pH 7.0-7.4.

Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 fermenting culture medium prescription is: contain Semen Maydis powder 30g in every 1000mL water, soybean cake powder 30g, bran powder 20g, K 2HPO 420g, KH 2PO 48g, MgSO 40.75g, 0.05% bubble enemy, pH 7.2-8.0.

Preservation and the fermentation culture thereof of embodiment 1, Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469

1, the preservation of bacterial classification

Pseudomonas fluorescens strain (Pseudomonas fluorescens) MX01 separates from Shandong Province's wheat rhizosphere soil, be preserved in Chinese common micro-organisms culture presevation management committee common micro-organisms center on September 30th, 2005, deposit number is CGMCC No.1469.

2, the fermentation of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469

The fermentation culture method of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 may further comprise the steps:

1) activation of bacterial classification

Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 inoculation in Jin Shi B liquid nutrient medium, was cultivated 36 hours down at 28 ℃.

2) seed tank culture

The Pseudomonas fluorescens that step 1) is activated (Pseudomonas fluorescens) MX01 CGMCC No.1469 is inoculated in this bacterium fermenting substratum by 2% inoculative proportion, shaking culture is 40 hours under 28 ℃, pH 7.0,200rpm, obtains seed liquor.

3) fermentor cultivation

With step 2) seed liquor of preparation is inoculated in this bacterium fermenting substratum by 3% inoculative proportion, per minute air flow volume), tank pressure 1.5-2.0F/cm at 28 ℃, pH 7.0, stirring velocity 200rpm, ventilation 1: 0.5-0.8 (fermentating liquid volume: 2Following aerated culture 40 hours obtains Pseudomonas fluorescens (Pseudomonasfluorescens) MX01 CGMCC No.1469 bacterium liquid.

The preparation of embodiment 2, Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 microbial inoculum

The preparation method of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 microbial inoculum is: the bacterium liquid of Pseudomonas fluorescens (Pseudomonas fluorescens) the MX01 CGMCCNo.1469 that embodiment 1 fermentation is made is after the 200 purpose peats composed of rotten mosses mixed in 10: 1 by ratio of weight and the number of copies with dry, aseptic, fineness, press filtration, add the 200 an amount of purpose peats composed of rotten mosses again and make bacterium liquid and the abundant mixing of the peat composed of rotten mosses, it is air-dry to shade, and obtains Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 microbial inoculum.Finished product is qualified after testing, and water content is 35%, and viable bacteria content is greater than 2,000,000,000/gram.

Embodiment 3, detection Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 are to the test of plant growth-promoting effect

With the rape is example, detects short the come into force fruit of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 to plant, and concrete grammar is: after the Semen Brassicae campestris surface sterilization, place vernalization in 12-24 hour down at 28 ℃; Be 10 with Semen Brassicae campestris with the concentration that embodiment 1 fermentation obtains then 8The Pseudomonas fluorescens of cfu/mL (Pseudomonas fluorescens) MX01 CGMCC No.1469 bacterium liquid seed soaking 1 hour, or be the microbial inoculum seed dressing of activeconstituents in order to Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469, microbial inoculum is 1: 1 with the weight fraction ratio of seed, with undressed Semen Brassicae campestris is contrast, again through sowing, cultivation, observe the plant strain growth situation after 15 days, weighing root fresh weight is measured the overground part plant height.The result is that the root fresh weight of the microbial inoculum of the activeconstituents plant of handling on average increases by 32.8% than adjoining tree through Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 bacterium liquid or with it, the over-ground part plant height also increases by 29.1%, has the short preferably fruit of coming into force.With carrying out field test with quadrat method, the short fruit of coming into force of result also can reach 10-20%.

Embodiment 4, detection Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469 are to the test of control of plant disease effect

With the bacterial wilt of tomato is example, detect the prevention effect of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCCNo.1469 to Plant diseases, concrete grammar is: the tomato seedling that stand-by sterilization earth culture is educated grows to the 3-4 leaf during phase, seedling is taken out, and be 10 with concentration 8The Pseudomonas fluorescens of cfu/mL (Pseudomonas fluorescens) MX01 CGMCC No.1469 bacterium liquid soaked root 30 minutes, be that the microbial inoculum of activeconstituents is converted water and irritated root with Pseudomonas fluorescens (Pseudomonasfluorescens) MX01 CGMCC No.1469 maybe with the seedling of taking out, the ratio of weight and number of microbial inoculum and water is 1: 500, with unprocessed and compare with the plant that aqua sterilisa is irritated root, again with seedling replanting to the seedling alms bowl of 8cm bore, slow seedling is irritated the root inoculum density after 3 days be 10 7The bacterial wilt of tomato bacteria culture fluid 50mL/ alms bowl of cfu/mL, regularly by disease stage division investigation disease index, the result is that the prevention effect of the microbial inoculum of the activeconstituents plant of handling reaches 86.3% through Pseudomonas fluorescens (Pseudomonasfluorescens) MX01 CGMCC No.1469 bacterium liquid or with it behind the inoculation pathogenic bacteria.Potted plant and the field resistance test-results of the take-all of carrying out with same procedure, the rhizoctonia damping-off of vegetable crop shows that bacterial strain of the present invention also is higher than 60% to the prevention effect of above-mentioned disease.Above-mentioned test-results shows that all Pseudomonas fluorescens of the present invention (Pseudomonas fluorescens) MX01 CGMCC No.1469 can significantly improve the disease resistance of plant.

Claims (9)

1. Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469.
2. the fermentation culture method of Pseudomonas fluorescens (Pseudomonas fluorescens) MX01 CGMCC No.1469, may further comprise the steps: 1) activated Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469 is inoculated in this bacterium fermenting substratum, under 27-29 ℃, pH 6.5-8.0, cultivated 36-48 hour, obtain seed liquor; Describedly Pseudomonas fluorescens MX01 is carried out the activatory method be: this inoculation in Jin Shi B substratum, was cultivated 24-48 hour down at 27-30 ℃; The prescription of described Jin Shi B substratum is: contain peptone 20g in every 1000mL water, glycerine 10mL, MgSO 4.7H 2O 1.5g, K 2HPO 4, 1.5g, agar 18g, pH 7.0-7.4; 2) seed liquor with step 1) preparation is inoculated in this bacterium fermenting substratum, aerated culture under 26-32 ℃, pH 6.5-8.0; Described step 1) and step 2) in Pseudomonas fluorescens MX01 fermenting culture medium prescription be: contain Semen Maydis powder 25-35g in every 1000mL water, soybean cake powder 25-35g, bran powder 16-24g, K 2HPO 416-24g, KH 2PO 46-10g, MgSO 40.5-1.0g, initial pH 7.2-8.0.
3. fermentation culture method according to claim 2 is characterized in that: described Pseudomonas fluorescens MX01 fermenting culture medium prescription is: contain Semen Maydis powder 30g in every 1000mL water, soybean cake powder 30g, bran powder 20g, K 2HPO 420g, KH 2PO 48g, MgSO 40.75g, pH 7.2-8.0.
4. according to claim 2 or 3 described fermentation culture methods, it is characterized in that: also be added with mass percent concentration in the described Pseudomonas fluorescens MX01 fermenting substratum and be 0.05% bubble enemy.
5. according to claim 2 or 3 described fermentation culture methods, it is characterized in that: the inoculative proportion in the described step 1) is 1-2%, and culture condition is: shaking culture under 28 ℃, pH 7.0,200rpm; Step 2) inoculative proportion in is 2-4%, and fermented liquid and per minute air flow volume ratio are 1: 0.5-0.8, tank pressure are 1.5-2.0F/cm 2, stirring velocity is 150-250rpm, incubation time is 24-48 hour.
6. fermentation culture method according to claim 5 is characterized in that: fermentation culture described step 2) is carried out for 7.0 times at 28 ℃, pH, and incubation time is 36-48 hour.
7. be the microbial inoculum of activeconstituents with Pseudomonas fluorescens (Pseudomonas fluorescens) MX01CGMCC No.1469.
8. the production method of the described microbial inoculum of claim 7, be with the bacterium liquid of Pseudomonas fluorescens (Pseudomonasfluorescens) MX01CGMCC No.1469 and dry, aseptic stopping composition 8-10 by ratio of weight and the number of copies: 1 mix after, press filtration, after the press filtration, it is air-dry to shade, and obtains Pseudomonas fluorescens MX01 microbial inoculum; Described stopping composition is the attapulgite or the peat composed of rotten mosses, and fineness is the 150-250 order.
9. the production method of described microbial inoculum according to Claim 8 is characterized in that: the described microbial inoculum fineness that obtains is that to be adjusted to final water content be 30-40% for the 150-250 purpose peat composed of rotten mosses, attapulgite or diatomite.
CNB2005101125429A 2005-10-10 2005-10-10 Fluorescent pseudomonads and its fermenting culture process and application CN100390272C (en)

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