CN102086444A - Paenibacillus elgii strain and application thereof - Google Patents
Paenibacillus elgii strain and application thereof Download PDFInfo
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- CN102086444A CN102086444A CN2010105791862A CN201010579186A CN102086444A CN 102086444 A CN102086444 A CN 102086444A CN 2010105791862 A CN2010105791862 A CN 2010105791862A CN 201010579186 A CN201010579186 A CN 201010579186A CN 102086444 A CN102086444 A CN 102086444A
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- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The invention discloses a paenibacillus elgii strain CGMCC No.4231. The microbial inoculant or moisture-retention organic fertilizer prepared from the strain has the functions of preventing the plant diseases, reducing the water loss of plant rhizosphere, strengthening the soil aggregate, improving the soil and promoting the plant growth. The bacteriostatic drought-resisting organic fertilizer is suitable for various crops and cash crops cultivated in arid areas and in dry seasons.
Description
Technical field
The present invention relates to agricultural biological technical field, specifically relate to the application of microorganism in agriculture production, mainly is the technology and the application of the antibacterial water conservation fertilizer of strain series bacillus production.
Technical background
Genus bacillus (
BacillusSpp.) be the aerobic or amphimicrobian of a group, sporiferous G
+The general name of bacillus.The genus bacillus kind is many, is widespread in nature, and can be distributed in the various ecotopes, and they are dominant populations of soil and plant rhizosphere microbe.Because genus bacillus has endogenous spore, strong stress resistance, reproduction speed is fast, nutritional requirement is simple and be easy to grow surely at plant rhizosphere, make it in biocontrol of plant disease, be studied widely and utilize.The biological and ecological methods to prevent plant disease, pests, and erosion genus bacillus of having reported at present has: subtilis (
B.subtilis), bacillus polymyxa (
B.polymyxa), bacillus cereus
(B.cereus), bacillus megaterium (
B.megaterium), bacillus pumilus (
B.pumilis), Bacillus coagulans (
B.coagulans), bacillus amyloliquefaciens (
B.amyloquefaciens), Bacillus circulans (
B.circulans) and Bacillus licheniformis (
B.licheniformis) etc.
The application of genus bacillus in the gardening plant disease control starts from the 1950's, and obtained swift and violent development in nearest 20 years.Up to now, the U.S. has had 4 bacillus biocontrol strains to obtain Environmental Protection Agency's (EPA) commercialization or limited commercialization production application permission, and they are QST713, GBO3, MBI600 and FZB24.The QST713(commodity are called serenade) be AgraQuest company product, foliage applying can be prevented and treated various bacteria and the fungal disease of crops such as vegetables, cherry, grape and English walnut.The GBO3(commodity are called Kodiak) by the exploitation of U.S. Gustafson company, can prevent and treat the Plant diseases that Fusarium and Rhizoctonia cause effectively, have the effect of diseases prevention and promotion plant-growth simultaneously, obtain large scale application the America and Europe.The MBI600(commodity are called Subtilex) by Microbio Ltd company exploitation, root is used or is dressed seed and can prevent and treat the root disease of the various crops that Fusarium, Aspergillus, Alternaria and Rhizoctonia cause.The FZB24(commodity are called TaegroTM) be the product of Taensa company, be applied to roots such as the sapling of greenhouse or indoor cultivation and flowers and trees, can prevent and treat by
FusariumWith
RhizoctoniaThe root disease that causes.In addition, the subtilis RB14 of Tokyo technical institute and NB22 respectively to dry thread Pyrenomycetes (
Rhizoctonia solani), the fusarium oxysporum bacterium (
Fusarium oxysporum) and blue or green withered false pseudomonas bacillus (
Psedomonas solanacearum) tomato disease that causes has good preventive effect.
China is a lot of about the research report that utilizes the genus bacillus controlling plant diseases, and filters out the biological control that a collection of good Bacillus strain is applied to Plant diseases, but the commodity preparation of registering and few.Early production has living grade of the Wang Jin of Agricultural University Of Nanjing to utilize subtilis B1(dish Fengning) the control soft rot of Chinese cabbage, its effect makes Chinese cabbage volume increase 5% ~ 40% on average more than 50%, has obtained large-area promotion and application in China." yield increasing fungus " that also has China Agricultural University to utilize multiple Bacillus strain to make in addition, field use has tangible disease-preventing and yield-increasing effect.Many bacillus category disease-preventing and yield-increasing commodity preparation registrations are arranged again later on, as: inferior precious (subtilis), Bacillus licheniformis, bacillus cereus, bacillus pumilus (A3), hundred anti-(B908), the withered peaceful B916 of line and wheat Fengning (B3) etc., played great role on the control of plant disease of field.
The bacterium that series bacillus belongs to equally with genus bacillus is not only common plant biocontrol bacteria, some also be the short endophytic bacteria of plant rhizosphere (Plant Growth-Promoting Rhizobacteria, PGPR).Because a lot of series bacillus have non-virulent to plant, have prophylaxis effect and growth-promoting functions simultaneously, therefore in agriculture production, has good application potential.Relevant research is deepening continuously, and Zhang Zhen etc. (2004) are separated to a strain Paenibacillus polymyxa A2 from ground, vegetable garden, studies show that this bacterium can promote cucumber growth, increases dry matter weight.Song Yongyan etc. (2006) have studied the growth-promoting functions of Paenibacillus polymyxa LM-3 to paddy rice, are provided with the bacteria suspension seed soaking, soak bud and water 3 kinds of processing of seedling, and the result shows, with 2 * 10
6The cell concn pouring seedling of cfu/ml is best to the growth-promoting effect of paddy rice to paddy growth, and every physical signs such as height of seedling is compared with contrast and other processing and all reached conspicuous level.The Paenibacillus polymyxa E681 that (2006) such as Choong-Min Ryu screen handles the sesame seed can increase the sesame volume, gains in weight.Wang Huiping etc. (2006) separate a strain series bacillus F53 from the eggplant rhizosphere, have studied the liquid fermentation condition that produces antimycotic product; Wen Xiaojuan etc. (2007) have carried out the solid fermentation Study on Conditions to this bacterial strain; Cui Xiaocan etc. (2009) have studied the inducing culture of the antimycotic material of product of this bacterial strain.Health ground flower bud must be 0.1 hundred million cfu/g Paenibacillus polymyxa granula subtilises of China's registration, by East China University of Science's research and development, is a kind of microbial bactericide of preventing and treating bacterial wilt.
The publication data of relevant water keeping organic fertilizer has: publication number is CN101585737, CN101054317, CN1530354, CN1310154, CN101654388, CN1411717, CN1426685, CN101215208, CN1580010, CN1498941, CN1853475, the drought resisting of adopting in 12 patents of CN101514290 and the method for water conservation are to add water-retaining agent that chemical process produces or water-holding agent etc., as materials such as polyacrylamide, the modified chitosan product with water retaining function that various nutritive ingredients or microbial cells etc. make that is mixed; The publication number of Patent data is CN1322699, and CN1663936 selection viscosity peat etc. is water-keeping material; Patent CN1587041 selects the effect of acid silicon dioxide as water conservation and slowly-releasing.But consult research and application that all disclosed documents are not seen the characteristic production water conservation fertilizer that utilizes the Production by Bacteria exocellular polysaccharide.Particularly the data of utilizing the class gemma bacterial strain that produces a large amount of exocellular polysaccharides and antimycotic material to produce antibacterial water conservation fertilizer is not seen open and report.In the current agriculture production particularly the agriculture production of arid area be badly in need of the strong fertilizer of water retaining function, the fertilizer that itself utilizes feces of livestock and poultry and agricultural waste fermentation to produce has certain water retaining function, but water retaining function can not satisfy the demand of arid area and agriculture production in dry season far away; In order to increase the water-retentivity of soil, many technology have adopted the method for adding chemical water-holding agent, and this method is an efficient ways, has also obtained a large amount of promotion and application, for the effect that ensures has been played in the increasing both production and income of agricultural.But material costs such as the polyacrylamide that adds, modified chitosan are higher, limited application, simultaneously these chemical materials have changed micro-flora in the original structure of soil and the soil in plant rhizosphere soil, produced the microflora of a large amount of these materials of degraded, equilibrium principle according to plant microecology: " procaryotic gained; be exactly Eukaryotic the mistake ", this phenomenon causes lot of energy to redirect to microbiological deterioration chemical substance aspect, must weaken the energy derive of plant, and influence the growth of plant.And these water-holding agents are disposable applying in the soil, have not just existed after being fallen by microbiological degradation, also will apply again in order to increase water retaining function.And the fertilizer that has added chemical substance is not a biological organic fertilizer truly.But series bacillus (
Paenibacillus elgii) CGMCC No.4231 be can long-term surviving in soil, as long as have water nutritious it just can grow, constantly secrete exocellular polysaccharide in the process of growth, the excretory exocellular polysaccharide can keep moisture, kept the growth of this thalline, in plant rhizosphere, just can keep such balance, there has been the existence of this bacterium just can constantly strengthen the crumb structure of soil and improved the soil, this bacterial strain can be secreted material that promotes plant-growth and the stable material that suppresses fungi simultaneously, has more strengthened its suitability and functional.
Summary of the invention
In order to reduce arid to the influence of agriculture production, increase the function of fertilizer, the invention provides a strain series bacillus bacterial strain, and with the antibacterial water conservation fertilizer of this bacterial strain production.
The said series bacillus of the present invention, its code name is FQ35, be series bacillus (
Paenibacillus elgii) CGMCC No.4231.
The present invention also provides a kind of microbial inoculant, and the content of series bacillus CGMCC No.4231 gemma is 10 in this Inoculant
8More than the cfu/g.
Above-mentioned said microbial inoculant can adopt the method production of liquid fermenting or solid fermentation:
Solution fermentation: adopt liquid fermentation and culture series bacillus CGMCC No.4231, fermented product by volume 1:1 is mixed in the peat composed of rotten mosses or the plant ash, stirs, and 60 ℃ of oven dry are pulverized, sampling Detection, and gemma content is 10
8Cfu/g is above for reaching the product standard of microbial inoculant.
Solid fermentation method: adopt solid fermentation to cultivate series bacillus CGMCC No.4231, fermentation ends admittedly expect 60 ℃ of oven dry, is pulverized, sampling Detection, and gemma content is 10
8Cfu/g is above for reaching the product standard of microbial inoculant.
Among the present invention on single-factor factor research basis, adopt orthogonal experiment to optimize liquid fermenting best medium prescription and the fermentation condition that the FQ35 bacterial strain produces antimycotic material to be: sucrose 0.3%, W-Gum 0.3%, peptone 1.0%, soybean cake powder 0.3%, NaCl 0.5%, and FeSO4 0.002%, and MgSO4 0.04%; The initial pH value 7.0 of fermented liquid, 35 ℃ of rotating speed 150r/min, inoculum size 4%, temperature, fermentation time 48h.
Bacterial strain solid fermentation optimization formula and fermentation condition are: the cow dung 70% of becoming thoroughly decomposed, thin field soil 20%, wheat bran 10%, pH7.0, material-water ratio 1:1.5,35 ℃ of leavening temperatures, inoculum size 10%.
The invention also discloses a kind of antibacterial water conservation fertilizer by series bacillus CGMCC No.4231 preparation, production method is: livestock and poultry waste discharge things such as cow dung are inoculated 20% microbial inoculant in digest process, and inoculation time is at ferment middle.The method operation of becoming thoroughly decomposed according to fertilizer is continued in the inoculation back.The fermentation ends sampling Detection, gemma content is 10
5Cfu/g is above for reaching the product standard of antibacterial water conservation fertilizer.
The invention also discloses the application of series bacillus CGMCC No.4231 aspect the antibacterial drought-resistant organic fertilizer of production.The application of series bacillus CGMCC No.4231 plant prevention soil-borne disease, promotion growth and water conservation aspect is disclosed.
Measure by the antibacterial and water conservation of carrying out Inoculant of the present invention and antibacterial water conservation fertilizer:
(1) Inoculant and the fertilizer of FQ35 bacterial strain preparation, the solid fermentation thing after oven dry is pulverized are with distilled water (solid-to-liquid ratio is 1:4) lixiviate 2h, and the centrifugal 20min of 5000rpm gets supernatant liquor through 0.22 μ m filtering with microporous membrane, promptly gets aseptic leach liquor.Be added drop-wise to and be mixed with in the intestinal bacteria and the saccharomycetic pair of dish (with reference to the method in the Chinese Pharmacopoeia), observing has inhibition zone.
(2) Inoculant and the fertilizer of FQ35 bacterial strain preparation, dry fully absorb water to the solid fermentation thing of constant weight after, claim weight in wet base, dry again to constant weight, claim dry weight, calculate water-intake rate, water-intake rate=weight in wet base/dry weight * 100%.Water-intake rate is qualified product more than 200%.
The result shows that Inoculant of the present invention and antibacterial water conservation fertilizer have bacteriostatic action and water retention preferably.Except producing the stable material that suppresses plant soil-borne disease fungal pathogens, produce a large amount of exocellular polysaccharides simultaneously in the fermented product of bacterial strain of the present invention, these exocellular polysaccharides are exactly natural water-absorbing water-retaining agent.Being administered to plant root with the solids of this bacterial strain preparation can suppress plant soil-borne disease fungal pathogens, promote plant-growth and water-absorbing-retaining.Antibacterial drought-resistant organic fertilizer with this bacterial strain production has prevention Plant diseases, the water loss of minimizing plant rhizosphere, enhancing soil aggregate, improves the soil, promotes the effect of plant-growth that this antibacterial drought-resistant organic fertilizer is applicable to the various farm crop and the cash crop of arid area and cultivation in dry season.
Embodiment
The per-cent of mentioning among the present invention if no special instructions, is mass ratio.
Embodiment 1: the acquisition of function stem
Gather the eggplant plant rhizosphere soil, utilize dilution-plate method the soil diluent to be coated on the potato glucose protein culture medium that is added with the eggplant verticillium wilt spore, obtain producing bacterial isolates 24 strains of inhibition zone, the sporiferous bacterial isolates of 8 strains is wherein arranged through microscopy.Be further purified bacterial strain and go out eggplant verticillium wilt bacterium inhibition zone greatly from 8 strain bacterial screenings, can produce the bacterial strain FQ35 of a large amount of exocellular polysaccharides again.This bacterial strain through form and Physiology and biochemistry be accredited as the kind that series bacillus belongs to (
PaenibacillusSp.); Again through 16S rDNA sequential analysis, under the prerequisite of extracting bacterium colony DNA, amplify the clear band of 1442bp with the bacterium universal primer, through order-checking and on Genbank comparison and
Paenibacillus elgiiSimilarity is more than 98%.
The FQ35 bacterial strain is submitted China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation on October 20th, 2010, and deposit number is CGMCC No.4231, the biology name
Paenibacillus elgii, depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.
Embodiment 2:FQ35 bacterial strain is to the mensuration of pathogenic micro-organism antimicrobial spectrums such as plant soil-borne pathogen
The bacterial classification that FQ35 bacterial strain inclined-plane is preserved is after the beef-protein medium activation, and point is planted and is inoculated on the potato glucose flat board, inoculates phytopathogen simultaneously, and face-off is cultivated, and observes the fungistatic effect of FQ35 bacterial strain.Bacterium (pathogenetic bacteria) is adopted and mixes the bacterium culture method, plants inoculation FQ35 bacterial strain again, observes fungistatic effect.
By the visible FQ35 bacterial strain of table 1 10 kind of plant pathogenic bacterias (the bacterial wilt of tomato bacterium is a bacteria pathogeny, and all the other are fungal pathogen) and the three strain environmental pollution indicator microoraganisms that detected all there is restraining effect.
The antimicrobial spectrum measurement result of table 1. FQ35 bacterial strain
| Detected bacterial strain | The Latin formal name used at school | Fungistatic effect |
| Intestinal bacteria | Escherichia coli | ++++ |
| Streptococcus aureus | Staphalococcus aureus | ++ |
| Yeast saccharomyces cerevisiae | Saccharomyces cerevisiae | ++ |
| The bacterial wilt of tomato bacterium | Ralstonia solanacearum | ++++ |
| Cotton-wilt fusarium | Fusarium oxysporumf.sp. vasinfectum | ++ |
| Withered germ of water-melon | Fusarium oxysporum f.sp .niveum | ++ |
| Cucumber fusarium axysporum | Fusariumoxysporum f.sp. cucumerinum | +++ |
| Verticillium dahliae | Verticilliumdahliae | ++ |
| Bean anthrax bacteria | Colletoteichum lindemuthianum | ++ |
| The Phytophthora capsici germ | Phytophthora capsici | ++ |
| Fusarium graminearum | Fusarium graminearum | ++ |
| Tobacco brown spot pathogen | Alternariaalternata | +++ |
| Sclerotinia sclerotiorum | Sclerotinia sclertiorum | +++ |
(annotate: the power of "+" " ++ " " +++" " ++ ++ " expression bacteriostatic action)
Embodiment 3:FQ35 strain liquid Optimizing Conditions of Fermentation:
(1) the FQ35 bacterial strain that the inclined-plane is preserved connects one and encircles in the seed culture fluid, 250mL triangular flask liquid amount 50mL, and 31 ℃, 180r/min shaking culture 36h(bacterium amount is 3.5 * 10
8Cfu/mL).
(2) adopt fermention medium, 250mL triangular flask liquid amount 50mL, shake 31 ℃ of bottle speed 180r/min, inoculum size 2%, temperature, incubation time 48h detects the influence to FQ35 bacterial strain fermentation liquor fungistatic effect of different carbon sources, nitrogenous source and inorganic salt respectively, repeats 3 times.
(3) to 9 kinds of common carbon sources: Semen Maydis powder, W-Gum, maltose, glucose, molasses, lactose, sucrose, wood sugar, Zulkovsky starch (concentration is 3g/L), make comparisons with CK (not adding carbon source).The result shows: sucrose or W-Gum are made carbon source, and the fermented liquid fungistatic effect is best, and antibacterial circle diameter reaches 1.97cm and 1.88cm; The no bacteriostatic activity of CK group that does not add carbon source.
(4) to 8 kinds of common nitrogenous sources: imported fish meal, dried silkworm chrysalis meal, soybean cake powder, peptone, yeast extract paste, ammonium nitrate, ammonium sulfate, ammonium chloride (organic nitrogen source concentration is 10g/L, and inorganic nitrogen-sourced concentration is 5g/L).The result shows: with peptone as nitrogenous source antibacterial circle diameter maximum.
(5) add different inorganic salt: 0.07%K in the substratum respectively
2HPO
4, 0.002%FeSO
4, 0.001%ZnSO
4, 0.02%MnSO
4And 0.04%MgSO
4, with broth culture as CK.The result shows, 0.002%FeSO
4And 0.04%MgSO
4Can improve FQ35 bacterial strain fermentation liquor fungistatic effect.
(6) respectively at inoculation back 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h sampling and measuring bacteriostatic activity.The fermented liquid bacteriostatic activity reaches the highest when being cultured to 48h, and being cultured to 96h fermented liquid bacteriostatic activity no longer increases.
(7) with NaOH and HCl the initial pH value of substratum is adjusted to 5.0,6.0,7.0,8.0,9.0,10.0 respectively, pH is that 7.0 ~ 8.0 o'clock FQ35 bacterial strain fermentation liquor bacteriostatic activities are higher.
(8) will shake that bottle speed is made as 120r/min, 150 r/min, 175 r/min, 200 r/min respectively, 230 r/min cultivate, FQ35 bacterial strain fermentation liquor fungistatic effect is best when shaking bottle speed 150r/min.
(9) under 25 ℃, 28 ℃, 31 ℃, 35 ℃, 37 ℃ conditions, cultivate the FQ35 bacterial strain respectively, in 25 ~ 34 ℃ of scopes, the fermented liquid bacteriostatic activity improves with the rising of culture temperature, it is the highest to cultivate F53 bacterial strain fermentation liquor bacteriostatic activity during with 35 ℃, when culture temperature at 37 ℃, the fermented liquid fungistatic effect begins to descend.
(10) it is as follows to be suitable for the culture medium prescription and the culture condition of FQ35 strain growth most: sucrose 0.3%, and W-Gum 0.3%, peptone 1.0%, soybean cake powder 0.3%, NaCl 0.5%, FeSO
40.002%, MgSO
40.04%; The initial pH value 7.0 ~ 8.0 of substratum, shake 35 ℃ of bottle speed 150 r/min, inoculum size 4%, temperature, incubation time 48h.
Embodiment 3:
(1) adopts the fermentation of 250ml triangular flask, every bottled 20 grams of expecting admittedly add suitable quantity of water, stir, substratum is through 121 ℃ of sterilization 60min, cooling back inoculum size is 10%(v/m, v-liquid spawn volume number, the quality of m-solid fermentation substratum, down together), put static cultivation in 35 ℃ of substratum then, patted once, make its even growth every 12 hours.Reach 80% when above to forming gemma, stop fermentation.In 60 ℃ of oven dry, pulverize then, detect.3 repetitions are established in 3 bottles of every processing.
(2) be 4 component factors with the cow dung of becoming thoroughly decomposed, thin field soil, wheat bran, material-water ratio, each component factor is established 3 levels by the different ratios quality respectively, and scheme sees Table 2.Be configured to 9 kinds of prescriptions by orthogonal design and be optimized combined sorting.
(3) contain the mensuration of gemma amount: take by weighing the 1g sample, add in the 9ml sterilized water, fully vibration, behind 80 ℃ of heating in water bath 15min, then per sample in the difference of bacteria containing amount, does 10 times of dilutions, the method for plate culture count detection living spores number is established 3 repetitions.
(4) the FQ35 bacterial strain solid fermentation optimization formula of You Huaing is: the cow dung 70% of becoming thoroughly decomposed, thin field soil 20%, wheat bran 10%, pH7.0, material-water ratio 1:1.5,35 ℃ of leavening temperatures, inoculum size 10%.
Embodiment 4:FQ35 bacterial strain produces the stability of antibacterial substance and measures
(1) with the FQ35 inoculation in broth culture, 35 ℃, 150r/min shaking culture 48h, the centrifugal 10min of 5000r/min, supernatant liquor must not have the thalline fermented liquid by 0.22 μ m filtering with microporous membrane.
(2) will not having fermented liquid and place 100 ℃ of water-baths, and handle 20min, 40 min, 60min, 80min, 100min respectively, and detect saccharomycetic bacteriostatic activity, is CK with the untreated samples, and fixed its bacteriostatic activity is 100%.
(3) (1) fermented liquid pH value being adjusted to 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0 respectively, detecting bacteriostatic activity after placing 48h under the room temperature, is that 3.0 ~ 10.0 aseptic broth culture is CK with the pH value.
(4) (1) fermented liquid pH value being adjusted to 3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0 respectively, detecting bacteriostatic activity after placing 48h under the room temperature, is that 3.0 ~ 10.0 aseptic broth culture is CK with the pH value.
(5) be 10ug/mL with (1) fermented liquid adding Proteinase K and trypsin concentration), 37 ℃ of water-bath 60min detect bacteriostatic activities, are CK with the sample that does not add proteolytic enzyme, and fixed its bacteriostatic activity is 100%.
(6) place 4 ℃ of refrigerators to preserve (1) fermented liquid, detect its bacteriostatic activity, detect altogether 6 times every 30d.
(7) The above results shows: the antibacterial substance that the FQ35 bacterial strain produces is stable to heat, soda acid and proteolytic enzyme, and 6 months activity of storage endurance remain unchanged.
Embodiment 6:FQ35 bacterial strain is tested short the giving birth to of plant
(1) the FQ35 bacterial strain is with optimum liquid fermentation medium prescription and fermentation condition fermentation, and fermented product is centrifugal, and throw out suspends into thalline content 10 with sterilized water
8The cfu/ml bacteria suspension; Supernatant also becomes aseptic fermented liquid supernatant with the bacterium membrane filtration.Cultivate and directly seed soaking cultivation employing seed soaking back respectively.The result shows: the bacteria suspension of FQ35 bacterial strain and fermented supernatant fluid all can improve the percentage of germination of eggplant seed, and eggplant birth seedling is had significant promotion growth effect (seeing Table 3).Cultivate after wherein seed soaking cultivation growth-promoting functions long to root and hypocotyl length is better than soaking bacterial classification.Bacterium liquid is better than fermented supernatant fluid to the promoter action of hypocotyl elongation, may can be colonizated in seed inside with bacterial strain, and it is relevant to play a role lastingly.
(2) the FQ35 bacterial strain is with optimum liquid fermentation medium prescription and fermentation condition fermentation, and fermented product is centrifugal, and throw out suspends into thalline content 108cfu/ml bacteria suspension with sterilized water; Supernatant also becomes aseptic fermented liquid supernatant with the bacterium membrane filtration.Adopt the aseptic fermented liquid supernatant of bacteria suspension bacterium is poured onto on the potted plant eggplant seedling.The average plant height of bacteria suspension treatment group, long, the total fresh weight of root and gross dry weight increase by 17.1%, 22.5%, 98.0%, 74.6% than control group respectively.Bacterium liquid is not remarkable with the short fruit difference that comes into force of the supernatant processing of fermenting, but on the whole, the short fruit of coming into force of bacteria suspension is better than fermented supernatant fluid (table 4), and it is relevant to think that thalline can be grown the interior continuous release active substance of plant materials surely.
The FQ35 bacterial strain is with optimum liquid fermentation medium prescription and fermentation condition fermentation, and fermented product is centrifugal, and throw out suspends into thalline content 10 with sterilized water
8The cfu/ml bacteria suspension; Supernatant also becomes aseptic fermented liquid supernatant with the bacterium membrane filtration.Adopt the aseptic fermented liquid supernatant of bacteria suspension bacterium be poured onto inoculate with nonvaccinated potted plant eggplant seedling on (seeing Table 5).The bacteria suspension of FQ35 bacterial strain and fermented supernatant fluid all have certain prevention effect to eggplant verticillium wilt, the processing prevention effect of wherein inoculating inoculation verticillium wilt pathogen behind the FQ35 bacteria suspension 3d is best, preventive effect can reach 74.3%, and visible bacterial strain is better than prevention effect for the preventive effect of verticillium.The prevention effect of bacteria suspension is better than fermented supernatant fluid, may be that thalline can well be grown at plant root soil surely, has occupied favourable ecological site more fully, has reduced the chance that germ is invaded.The fermented supernatant fluid effect may be relevant with the amount that applies, and along with the prolongation of time, part degraded or lost efficacy in soil.
Adopt potted plant experiment, irritate the root inoculation method, experimental design sees Table 6.The result shows: antimicrobial content short of money is big in the soil, will get well the eggplant verticillium wilt prevention effect.
(1) the FQ35 bacterial strain fermentation liquor is removed thalline and impurity through the distilled water diluting of 2 times of volumes and after stirring 15min with the centrifugal 30min of 8000r/min.With supernatant concentration 0.5 times to original volume, add 2 times of volume precooled ethanol, place 24h, centrifugal, collecting precipitations down in 4 ℃.Throw out washs through ether, and vacuum-drying gets the exocellular polysaccharide crude product.Crude product is soluble in water again, the chloroform and propyl carbinol (4:1v/v) mixed solution of 0.5 times of volume of adding, vibration, extraction, layering, 3 times so repeatedly, to remove protein and small amount of coloring matter.The upper strata gets supernatant liquor with the centrifugal 10min of 4000 r/min, ethanol sedimentation, and vacuum-drying obtains the pure product of exocellular polysaccharide.
(2) choose 5 kinds of polysaccharide mark product: the D-semi-lactosi, D-ribose, D-fructose, lactose, the sample of glucose and (1) preparation, point sample carries out thin-layer chromatography.Developping agent is a propyl carbinol: acetic acid: water (3:3:1) (v/v).Launch once back oven dry, repeat 2 times again and launch, each the about 5-6 of developing time hour (, need carry out three times and launch) in order to reach good separating effect.Developer is: pentanoic 2g, aniline 2ml, 85% phosphoric acid (v/v), 10 ml are dissolved in the 100 ml acetone.Be placed in 85 ℃ of baking ovens after the thin layer plate spraying and dried by the fire 15 minutes.The result generates glucose after according to relative position and the acid hydrolysis of Rf value decidable sample.
(2) Infrared spectroscopy adopts the KBr pressed disc method, through infrared spectrometric analyzer analytical characteristic group.What results of IR showed is a typical polysaccharide spectrogram, and the characteristic group of polysaccharide all has charateristic avsorption band.
(3) solid fungicide of FQ35 bacterial strain is manured into soil according to 2% amount, mixing, and fully suction is filtered with quantitative paper, till filtration time does not drip to 1h, the title weight in wet base.Put in 60 ℃ of baking ovens and dry to constant weight title dry weight, calculating water-intake rate.Water-intake rate=weight in wet base/dry weight * 100%.Taking by weighing three sample water-intake rates is respectively: 155.5%, 186.2%, 180.0%.Average water-intake rate is 173.9%.In the sand, saturation moisture content is in 25～60% scopes in the ordinary course of things.The saturation moisture content of organophilic clay such as peat soil or saprolite can reach more than 100%.The solid fungicide that applies the FQ35 bacterial strain has improved about 70% the water-intake rate of farmland soil.
Embodiment 10:FQ35 bacterial strain is grown mensuration surely at plant rhizosphere
Employing contains Streptomycin sulphate meat soup gradient culture method.Measure earlier
FQ35To the Streptomycin sulphate sensitive concentration is 80 μ g/ml, disposes the gravy peptone nutrient solution that contains different concns Streptomycin sulphates such as 100,150,200,250,300,400,500,600 μ g/mL then, will
FQ35Begin to cultivate in being lower than the pastille nutrient solution of sensitive concentration, the method inducing culture that improves drug level is step by step adopted in the back, and adapts to for 3 generations in each drug level, until the bacterial strain that produces anti-Streptomycin sulphate 600 μ g/mL.With anti-Streptomycin sulphate 600 μ g/mL's
FQ35Respectively through pastille meat soup and dull and stereotyped alternately the continuation were not cultivated for 4 generations, tieback contains the substratum detection of 600 μ g/mL Streptomycin sulphates again, confirming chemical sproof genetic stability, and the mark bacterial strain is done bacteriostatic activity measure, to confirm bacterial strain not change of bacteriostatic activity through inducing after.Thereby obtain research
FQ35Necessary anti-Streptomycin sulphate 600 μ g/mL mark bacterial strains when growing surely.
Test establishes 10
8, 10
7, 10
6Three gradient inoculations
FQ35, respectively after inoculation 0,6,12,20,30 and 40d, taking by weighing the 5g soil 30min that in sterile saline, vibrates among the 45ml respectively, serial dilution is cultivated counting on the NA flat board that contains 600 μ g/mL Streptomycin sulphates, the meter colony number.The bacterium amount that is detected with the 1st sub-sampling is an inoculum size.The bacterium amount is grown quantity cfu/g with deciding in the soil and is represented.Every processing repeats 3 times.
The result shows
FQ35At the eggplant root very strong colonization ability is arranged.When inoculum density is 3.51 * 10
8During cfu/g, the bacterial content of inoculating back 40 days soil still can reach 1.33 * 10
8Cfu/g.In addition, in the soil
FQ35Final colonization ability with the initial concentration of introducing bigger relation is arranged.Usually concentration is high more, and colonization ability is strong more, and the time of growing surely is also long more.See Table 7.
Under the table 7 inoculation different concns
FQ35Decide the time of growing (* cfu/ g) at the eggplant root
| Handle | 0d | 6d | 12d | 20d | 30d | 40d |
| 10 8 | 3.51 | 1.96 | 1.68 | 1.45 | 1.34 | 1.33 |
| 10 7 | 2.40 | 0.60 | 0.56 | 0.40 | 0.35 | 0 |
| 10 6 | 4.78 | 1.79 | 1.20 | 1.07 | 0 | 0 |
Claims (7)
- One strain series bacillus ( Paenibacillus elgii), deposit number is CGMCC No.4231.
- 2. series bacillus CGMCC No.4231 as claimed in claim 1 is characterized in that and can exist at plant rhizosphere for a long time, produces the stable material of inhibition plant soil-borne disease fungal pathogens and a large amount of exocellular polysaccharides.
- 3. a microbial inoculant is characterized in that, the content of series bacillus CGMCC No.4231 gemma is 10 in the Inoculant 8More than the cfu/g.
- 4. an antibacterial water conservation fertilizer is characterized in that, be to make by following method: livestock and poultry waste discharge things such as cow dung are inoculated 20% series bacillus CGMCC No.4231 microbial inoculant in digest process, inoculation time is at ferment middle; Continue after the inoculation to become thoroughly decomposed; The fermentation ends sampling Detection makes gemma content 10 5More than the cfu/g.
- 5. the described series bacillus CGMCC of claim 1 No.4231 prevents soil-borne disease plant, promotes the application aspect growth and the water conservation.
- 6. the application of the described microbial inoculant of claim 3 aspect the antibacterial drought-resistant organic fertilizer of production.
- The described antibacterial water conservation fertilizer of claim 4 in the prevention Plant diseases, reduce the plant rhizosphere water loss, strengthen soil aggregate, improve the soil, promote the application aspect the plant-growth.
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