CN104164383A - Bacillus amyloliquefaciens used for antagonizing fusarium oxysporum f. sp. heterophylla - Google Patents
Bacillus amyloliquefaciens used for antagonizing fusarium oxysporum f. sp. heterophylla Download PDFInfo
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Abstract
The invention relates to bacillus amyloliquefaciens used for antagonizing fusarium oxysporum f. sp. heterophylla. A bacterial strain of bacillus amyloliquefaciens (BD-N) is registered and preserved in China General Microbiological Culture Collection Center, CGMCC on May 12th, 2014, with a preservation number of CGMCCNo.9114. The bacterial strain can efficiently suppress the fusarium oxysporum f. sp. heterophylla in rhizosphere soil, and has wide application prospects in the field.
Description
Technical field
The present invention relates to the bacillus amyloliquefaciens of a strain for antagonism Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum.
Background technology
Root of Heterophylly Faalsestarwort (
pseudstellariae heterophylla) be a kind of famousr and precious Chinese medicinal materials, containing amino acid, carbohydrate, glycoside, phospholipid, cyclic peptide and trace element etc.Have invigorating qi and benefiting blood, the effect of the tonifying the spleen and stomach that promotes the production of body fluid, be suitable for children's's summer heat of a specified duration and do not move back, cough, diet is depressed, the disease of weakness such as palpitaition and children's be weak unable, night sweat, mouth equality disease after being ill.Day by day deep along with Root of Heterophylly Faalsestarwort pharmacological effect research, Root of Heterophylly Faalsestarwort, as help medicine, becomes one of conventional bulk medicinal materials of China gradually.
Root of Heterophylly Faalsestarwort is of many uses, and the market requirement is very large.But its continuous cropping obstacle is extremely serious, the in the situation that of planting Root of Heterophylly Faalsestarwort continuously in same piece of land, even in the situation that enhancing field management, its upgrowth situation variation, even total crop failure, its product qualitative change is bad, Zong polysaccharide and content of ginsenoside significantly decline.Applicant passes through and studies confirm that, after Root of Heterophylly Faalsestarwort continuous cropping, Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum in rhizosphere soil (
fusarium oxysporumf. sp.
heterophylla) quantity extremely significantly increases, continuous cropping Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum (
fusarium oxysporumf. sp.
heterophylla) quantity be contrast nearly 5 times; The Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum being separated to is inoculated in the healthy soil of plantation Root of Heterophylly Faalsestarwort, Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum breeds in a large number in healthy soil, and infects Root of Heterophylly Faalsestarwort root, causes Root of Heterophylly Faalsestarwort disease.
In recent years, along with the research of the particular mechanism of action of green agriculture, Organic farming and microbial control is just flourish, obtaining efficient Antagonistic Fungi and making its effect perform to maximum was the basis of biological control.Many to the biological control of Fusarium oxysporum research in other crops (as banana, watermelon, cucumber, strawberry etc.), the Antagonistic Fungi kind that separates report is also more comprehensive.As mould in wood, bush mycorrhizal fungi (AM), Nonpathogenic Fusarium oxysporum, Paecilomyces lilacinus, false single-cell bacteria, genus bacillus, actinomycetes etc., wear brave grade of monarch and separated bacillus amyloliquefaciens KL-1 from Dianthus caryophyllus L., vegetable lamb rhizosphere soil, and for the control of Fusarium oxysporum f.sp. diathi (F. oxysporum f.sp.Diathi), cotton-wilt fusarium (F. oxysporum f.sp.vasinfectum).But the bacterial strain that this research separates is compared source with them different, for also difference of object, because Fusarium oxysporum exists the cause of genetic polymorphism.As the universal soil-borne disease fungal pathogens of one, the pathogenic strains in Fusarium oxysporum kind has the host preference of height, and the bacillus amyloliquefaciens KL-1 separating from cotton field might not have good effect to Root of Heterophylly Faalsestarwort specialized form sickle-like bacteria.The separated Antagonistic Fungi kind for Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum only has " for the Paenibacillus polymyxa of antagonism Root of Heterophylly Faalsestarwort rhizosphere soil Fusarium oxysporum " (patent No.: CN 102676435 B) at present, yet there are no the relevant report for the bacillus amyloliquefaciens of Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum.This seminar in early-stage Study, by dilution be coated with flat band method and dull and stereotyped dual test gather from experimental plot, Root of Heterophylly Faalsestarwort GAP base, Xi Yuan village, Ningde City Zherong County, Fujian Province the bacillus amyloliquefaciens that has been separated to a strain capable of high-efficiency continuous cropping Root of Heterophylly Faalsestarwort Rhizosphere sampling and suppressed Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum (
bacillus amyloliquefaciens), called after BN-D, the biological control that the separation of this Antagonistic Fungi can be Root of Heterophylly Faalsestarwort root rot provides new biocontrol fungicide.
Summary of the invention
For this reason, the present invention screens the bacillus amyloliquefaciens of a strain for antagonism Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum from Root of Heterophylly Faalsestarwort continuous cropping rhizosphere soil.
A strain provided by the invention is for the bacillus amyloliquefaciens of antagonism Root of Heterophylly Faalsestarwort rhizosphere soil Root of Heterophylly Faalsestarwort specialized form sickle-like bacteria, for bacillus amyloliquefaciens (
bacillus amyloliquefaciens) BN-D, registered preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 12nd, 2014, deposit number is CGMCCNo.9144.
This bacterium can be in genus bacillus collective media cultivation and fermentation.Medium component: Tryptones (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium-chlor (NaCl) 5g/L, pH 7.0.
(eyepiece 10X, object lens 100X) lower this bacterium gramstaining result of observing under oily mirror, this bacterium is bacillus, gramstaining is negative.This bacterial strain is through morphology and 16s RNA molecular biology identification, be defined as bacillus amyloliquefaciens (
bacillus amyloliquefaciens).
This bacterial strain can more extensively utilize carbon source, comprise: L-rhamnosyl, sucrose, D-Glucose and PEARLITOL 25C, but part carbon source is not utilized, comprise: D-Fructose, D-wood sugar, L-arabinose, L-inositol, maltose and cellobiose, also can more extensively utilize nitrogenous source, can make Starch Hydrolysis, not utilize Mierocrystalline cellulose.
Bacillus amyloliquefaciens of the present invention (
bacillus amyloliquefaciens) BN-D, can efficiently suppress the specialized form Fusarium oxysporum of Root of Heterophylly Faalsestarwort rhizosphere soil, having a extensive future of this field.This bacterial strain has been protected finish in the registration of China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 12nd, 2014, it is called for short CGMCC, and address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCCNo.9144.
Brief description of the drawings
Fig. 1 be bacillus amyloliquefaciens (
bacillus amyloliquefaciens) BN-D is at NA substratum growth figure.
Fig. 2 be bacillus amyloliquefaciens (
bacillus amyloliquefaciens) BN-D specially changes the fungistatic effect of Fusarium oxysporum to Root of Heterophylly Faalsestarwort.Left figure is the contrast of Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum; Right figure is the fungistatic effect of bacillus amyloliquefaciens BN-D antagonism Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum.
Fig. 3 is bacillus amyloliquefaciens (Bacil
lus amyloliquefaciens) BN-D is to Root of Heterophylly Faalsestarwort toxicity test; In picture, first row is the potted plant seedling of contrast Root of Heterophylly Faalsestarwort that does not add bacillus amyloliquefaciens BN-D, and second row is the potted plant seedling of Root of Heterophylly Faalsestarwort after treatment that adds bacillus amyloliquefaciens BN-D.
Fig. 4 be bacillus amyloliquefaciens (
bacillus amyloliquefaciens) BN-D Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum Root of Heterophylly Faalsestarwort tissue cultured seedling trains test altogether.Picture is from left to right followed successively by: the Root of Heterophylly Faalsestarwort tissue cultured seedling that does not add any bacterium; Only add the Root of Heterophylly Faalsestarwort tissue cultured seedling of Root of Heterophylly Faalsestarwort specialized form sickle-like bacteria; Add the Root of Heterophylly Faalsestarwort tissue cultured seedling of Root of Heterophylly Faalsestarwort specialized form sickle-like bacteria, bacillus amyloliquefaciens BN-D simultaneously.
Embodiment
1, the isolation and purification of bacillus amyloliquefaciens BN-D
The collection of soil sample: gather continuous cropping Root of Heterophylly Faalsestarwort Rhizosphere sampling for 2012 from experimental plot, Root of Heterophylly Faalsestarwort GAP base, Xi Yuan village, Ningde City Zherong County, Fujian Province.Adopt 5 point sampling methods, mix as sample.
Adopt isolation by dilution method to the separation and purification of soil sample microorganism, take 10g soil and add in the triangular flask that 90mL sterilized water is housed, 120r/min, vibration 30min, makes 10 by the soil supension having shaken
-1, 10
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7etc. the different dilution soil solution.The suspension of above-mentioned dilution is applied in and LB substratum is housed (fills a prescription as follows: Tryptones (Tryptone) 10g/L, yeast extract (Yeast extract) 5g/L, sodium-chlor (NaCl) 10g/L, regulate pH to 7.0 ~ 7.4 of this substratum with NaOH, every liter adds 10g ~ 15g agar powder) flat band method on, each concentration repeats 3 times, be placed in the incubator overnight incubation of 30 DEG C, choose the suitable flat board of colony number, picking single bacterium colony wherein, adopt the method for parallel scribing to carry out purifying, continuous 3 switchings, confirm as single bacterium.The soil microorganisms of getting respectively after separation and purification is inoculated on LB substratum, 30 DEG C activate 2 days, then receive on PDA substratum, beat and get Root of Heterophylly Faalsestarwort Fusarium oxysporum bacterium cake and receive PDA (potato 200g/L with punch tool, glucose 20g/L, every liter, agar adds 15 ~ 20 grams, natural PH) carry out flat board face-off experiment on flat board, the bacterial strain of choosing obvious antagonistic action carries out preserving after purifying, for subsequent use.
2, the screening of bacillus amyloliquefaciens and fungistatic effect are measured
Adopt face-off culture method further to screen the each bacterial strain that has certain antagonistic action of above-mentioned purifying and Root of Heterophylly Faalsestarwort Fusarium oxysporum: the bacterium liquid 100 μ L that take out above-mentioned purifying pour on LB plate culture medium, be coated with rod with the glass of sterilizing diluent is coated with uniformly, cultivate 7 days for 30 DEG C.Beat and get Antagonistic Fungi bacterium piece access PDA culture medium flat plate on one side with diameter 5mm punch tool, and beat and get the onesize Fusarium oxysporum bacterium piece access the other end, carry out flat board face-off and cultivate.Screening antagonistic effect good bacterial strain identifies it, through morphology and 16s rRNA molecular biology identification, be defined as bacillus amyloliquefaciens (
bacillus amyloliquefaciens), called after BN-D.
Bacillus amyloliquefaciens BN-D bacterial strain ferments through seeding tank, further amplifies after fermentation to pilot scale fermentation tank, and after Root of Heterophylly Faalsestarwort plantation, spray application, in soil, or after the fermentation of bacterium liquid, is mixed base manure and is applied to the soil that is about to plantation Root of Heterophylly Faalsestarwort immediately.Bacterium liquid spray application or mix base manure and use, its application concentration is 10
6-10
8cFUs/mL.After using, will significantly improve Root of Heterophylly Faalsestarwort yield and quality.
3, the fungistatic effect of bacillus amyloliquefaciens BN-D is measured
In laboratory, with punch tool, Fusarium oxysporum and bacillus amyloliquefaciens BN-D respectively being broken into diameter is the small bacteria block of 5mm the two ends that moved into respectively PDA plate culture medium.The bacterium piece of not inoculating the PDA plate culture medium central position access formed objects of this bacterium shines for empty.28 DEG C of constant temperature culture 5 days, carry out that colony diameter size is measured and inhibiting rate calculates, and determine the antagonism performance of bacterial strain.
Bacteriostasis rate calculation formula: Fusarium oxysporum colony diameter × 100% of bacteriostasis rate (%)=(the Fusarium oxysporum colony diameter of contrast-inoculate the Fusarium oxysporum colony diameter of this bacterium)/contrast.
As a result, this bacterial strain reaches 93.2% to 5 days bacteriostasis rates of Fusarium oxysporum, as shown in Figure 2.
4, bacillus amyloliquefaciens BN-D bacterial strain toxicity test
By in this bacillus amyloliquefaciens BN-D access 100mL LB substratum, cultivate after 3 days, get apply after 5 times of this bacterium bacterium liquid 100 mL sterilized water dilutions of cultivating 3 days Root of Heterophylly Faalsestarwort potted plant in, observe the growing state of Root of Heterophylly Faalsestarwort every day, observe this bacillus amyloliquefaciens to the whether toxic effect of Root of Heterophylly Faalsestarwort.As shown in Figure 3, compared with the control, the potted plant seedling well-grown of Root of Heterophylly Faalsestarwort group, shows that this bacillus amyloliquefaciens BN-D, to not murder by poisoning of Root of Heterophylly Faalsestarwort growth itself, can not make Root of Heterophylly Faalsestarwort cause a disease.
5, bacillus amyloliquefaciens BN-D, Root of Heterophylly Faalsestarwort Fusarium oxysporum and Root of Heterophylly Faalsestarwort tissue cultured seedling are trained test altogether.
This Antagonistic Fungi bacterium liquid of 200 μ L is joined around Root of Heterophylly Faalsestarwort tissue cultured seedling rhizosphere, cultivate after 2 days in tissue culture bottle edge access sickle-like bacteria, observe every day and make mutually effect between pathogenic bacteria, Antagonistic Fungi, Root of Heterophylly Faalsestarwort tissue cultured seedling.
As shown in Figure 4, Root of Heterophylly Faalsestarwort tissue cultured seedling well-grown, the Root of Heterophylly Faalsestarwort tissue cultured seedling that only accesses sickle-like bacteria is withered and final death gradually by fusarium infection.And being connected in the Root of Heterophylly Faalsestarwort tissue culture bottle of Root of Heterophylly Faalsestarwort Fusarium oxysporum and bacillus amyloliquefaciens BN-D, bacillus amyloliquefaciens BN-D is centered around tissue cultured seedling and forms inhibitory area around, cause sickle-like bacteria cannot cross inhibitory area and Root of Heterophylly Faalsestarwort is caused a disease, and crown prince's seedling upgrowth situation is the same with blank grows fine.Finally in the tissue culture bottle of access Antagonistic Fungi, formed centered by crown prince's seedling, periphery is respectively Antagonistic Fungi, Antagonistic Fungi inhibitory area, the sickle-like bacteria three situation of work mutually.
5, the application prospect of bacillus amyloliquefaciens BN-D
Plant diseases more than 80% is by fungus-caused, controls fungal disease of crucial importance for improving crop yield.Fusarium oxysporum is the soil-borne disease fungi of a quasi-representative, in soil and organic matter, quantity, enlivening more, its scope of living away from home is very extensive, can cause the 100 various plants generation blights such as melon, Solanaceae, banana, cotton, pulse family and flowers, in agriculture production, cause serious crop failure, reduce quality, impact supply and export trade, even cause person poultry poisoning, cause serious financial loss.Applicant is through studies confirm that: after plantation Root of Heterophylly Faalsestarwort, in rhizosphere soil, Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum quantity extremely significantly increases; The Fusarium oxysporum being separated to is inoculated in the healthy soil of plantation Root of Heterophylly Faalsestarwort, Fusarium oxysporum breeds in a large number in healthy soil, and infects Root of Heterophylly Faalsestarwort root, causes Root of Heterophylly Faalsestarwort disease.Approximately more than 50,000 mu of the annual total cultivated area of Root of Heterophylly Faalsestarwort in the whole nation, by existing market price budget, approximately more than 5,000,000,000 yuan of Renminbi of the whole nation output value, but after continuous cropping, in Root of Heterophylly Faalsestarwort rhizosphere soil, the pathogenic bacterium such as specialized form Fusarium oxysporum and pathogenic bacteria are increased, make output sharply drop to below 1/3 of the first planting (contrast), produce serious financial loss, be one and be badly in need of the major issue solving.Bacillus amyloliquefaciens BN-D can efficiently suppress Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum, having a extensive future of this field.
Claims (1)
- One strain for the bacillus amyloliquefaciens of antagonism Root of Heterophylly Faalsestarwort specialized form Fusarium oxysporum ( bacillus amyloliquefaciens) BN-D, registered preservation at China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 12nd, 2014, deposit number is CGMCCNo.9144.
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| CN201410263153.5A CN104164383B (en) | 2014-06-14 | 2014-06-14 | One strain is for the bacillus amyloliquefaciens of antagonism radix pseudostellariae specialized form Fusarium oxysporum |
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Cited By (2)
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| CN105586303A (en) * | 2016-03-28 | 2016-05-18 | 福建农林大学 | Korean pseudomonas and application thereof |
| CN106399170A (en) * | 2016-09-18 | 2017-02-15 | 中南民族大学 | Bacillus amyloliquefaciens clx-60 bacterial strain producing polyphyllin VI and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586303A (en) * | 2016-03-28 | 2016-05-18 | 福建农林大学 | Korean pseudomonas and application thereof |
| CN105586303B (en) * | 2016-03-28 | 2019-03-12 | 福建农林大学 | One plant of South Korea pseudomonad and its application |
| CN106399170A (en) * | 2016-09-18 | 2017-02-15 | 中南民族大学 | Bacillus amyloliquefaciens clx-60 bacterial strain producing polyphyllin VI and application thereof |
| CN106399170B (en) * | 2016-09-18 | 2019-06-07 | 中南民族大学 | The bacillus amyloliquefaciens clx-60 bacterial strain and application thereof of one plant of production polyphyllin Ⅵ |
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