CN105274020A - Ginger stem rot antagonistic bacterium and applications - Google Patents
Ginger stem rot antagonistic bacterium and applications Download PDFInfo
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Abstract
The invention discloses a ginger stem rot antagonistic bacterium. The ginger stem rot antagonistic bacterium is named after Burkholderia cenocepacia C3, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on October 17th, 2014. The address is Institute of Microbiology Chinese Academy of Sciences NO.1 Beichen West Road, Chaoyang District,Beijing. The preservation number is CGMCC No. 9791. The ginger stem rot antagonistic bacterium C3 has a control effect of 41.18% on the ginger stem rot, and the control effect is raised substantially compared with control effects of a control group. When C3 compared with CK2, the incidence and the disease index after processing are both lowered, which shows that diseases prevention effects of C3 are ideal.
Description
Technical field
The present invention relates to a kind of curing ginger stalk rot bacterium antagonistic bacterium and application.
Background technology
Curing ginger stalk rot is also known as ginger soft rot, ginger stem rot etc., infected by pythium spp and cause, within 1907, reported first finds in Surat of India, now generally betide the areas such as India, Australia, Japan, Nigeria, Hawaii, Fiji, Sri Lanka, Korea S and China Taiwan, this evil of being critically ill is serious.It is reported, the underproduction that part grave illness growing area causes because of curing ginger stalk rot every year, up to 80% ~ 90%, is even had no harvest.In the land for growing field crops loss 25% that Nepal causes because of this disease, shelf time loss 24%.In India's grave illness field loss up to more than 90%.Curing ginger stalk rot pathogenic bacteria is oomycetes door rotten mold genus, and in mycota classification, curing ginger stalk rot pathogenic bacteria is under the jurisdiction of Oomycete, pythiaceae, pythium.This pathogenic bacteria is a kind of lower fungi, in saprophytic mode at soil long-term survival, parasitizes higher plant, and cause harm root and basal part of stem cause rotten, show as damping off, root-rot and stem rot.
For aspects such as the factors that the research of curing ginger stalk rot cause of disease, occurrence regularity, Infection cycie rule and impact are fallen ill, the prophylactico-therapeutic measures on producing at present mainly contains cultural control, Agro-chemicals control and biological control.The plurality of advantages of chemical agent makes it be widely used in agriculture production, but along with the enhancing gradually of human health and environmental consciousness, long-term a large amount of uses lack of standardization of agricultural chemicals, adjoint problems also display gradually.Relatively outstanding as environmental pollution, pesticide residue, drug-fast generation and the direct murder by poisoning etc. to non-target organism.And biological control compensate for the defect of chemical pesticide to a certain extent, significant role will be played in the future of agriculture Sustainable development.But biological control at present is only confined to indoor pot experiment, and biological control is applied to field still needs long time.But from environmental safety and human health angle, finally will find out biological control method and approach safely and effectively, progressively replace existing chemical prevention.
Biocontrol fungicide has been widely studied and applied because of its environment friendly, advantage such as performance persistence, preventive effect specific aim etc., has a high potential.Report a large amount of biological prevention and control agent developments both at home and abroad and successful Application.As production applications of having become commercialized such as common Bacillus thuringiensis, muscardines, and achieve good preventive effect.
Summary of the invention
For above-mentioned prior art, the invention provides a kind of curing ginger stalk rot bacterium antagonistic bacterium and application.
The present invention is achieved by the following technical solutions:
Curing ginger stalk rot bacterium antagonistic bacterium provided by the invention, this Strain Designation is Burkholderia cepacia (Burkholderiacenocepacia) C3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 17th, 2014, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number is CGMCCNo.9791.
The main biological property of above-mentioned bacterial strains is: Burkholderia cepacia (Burkholderiacenocepacia), Gram-negative bacteria, bacterium colony yellow-white, smooth surface corrugationless, and single bacterium colony is circular, and on PDA substratum, colony growth rate is fast.
Curing ginger stalk rot bacterium antagonistic bacterium C3 for the preparation of the method for biocontrol fungicide is: cultivate curing ginger stalk rot bacterium antagonistic bacterium with Optimal Medium, when 600nmOD value is 1, by centrifugal for bacterial suspension 6000rpm 10min, thalline equal-volume sterilized water or phosphoric acid buffer Eddy diffusion, being diluted to bacterial concentration is 5 × 10
8cfu/ml, after packing, 4 DEG C of placements are for subsequent use.
Beneficial effect of the present invention,
1. indoor protection effect measures and shows, curing ginger stalk rot bacterium antagonistic bacterium C3 reaches 41.18% to curing ginger stalk rot preventive effect, is significantly increased compared with the control, and process sequela rate, disease index all decrease compared with CK2, illustrate that C3 protection effect is desirable;
2. pair curing ginger stalk rot bacterium antagonistic bacterium C3 fermentation condition is optimized, basic medium, nutrient media components single factor experiment are studied, result show glycerine and yeast powder best respectively as effect during substratum carbon nitrogen source, the orthogonal test of nutrient media components shows glycerine 2%, yeast powder 1%, K
2hPO
40.2%, MgSO
4the proportioning of 0.1% is optimal proportion, by incubation time, inoculum size, Medium bottling volume and pH etc., the influence research of thalli growth amount is shown, during with inoculum size 3%, the bottled amount of 150ml/250ml, incubation time 48h, pH for 8-9, this bacterial strain cell concentration is maximum;
3. pair Activities of Fermentation Broth material carries out the stability experiment of temperature, proteolytic enzyme and ultraviolet three aspects.Change not obvious to the suppression of pathogenic bacteria after fermentation liquor trypsinase and pepsin; After treatment of different temperature 30min compared with the control comparatively, pathogenic bacteria colony diameter is without considerable change; Fungistatic effect after ultraviolet Continuous irradiation 12h no significant difference compared with the control.Therefore this bacterium active substance has stronger stability to heat, proteolytic enzyme and ultraviolet.
Accompanying drawing explanation
Fig. 1 is curing ginger stalk rot bacterium antagonistic bacterium C3 colonial morphology;
Fig. 2 is the restraining effect of antagonistic bacterium C3 to curing ginger stalk rot bacterium;
Fig. 3 is that under opticmicroscope, antagonistic bacterium C3 is to the mycelial effect of curing ginger stalk rot bacterium, and wherein A is contrast, and B is for by C3 process mycelial form of cause of disease after 2 days;
Fig. 4 is antagonistic bacterium C3 to the inhibition zone size of curing ginger stalk rot bacterium and other pathogenic bacteria;
Fig. 5 is antagonistic bacterium C3 to the bacteriostatic action figure of curing ginger stalk rot bacterium and other pathogenic bacteria;
Fig. 6 is the impact of different culture media on thalline fermentation concentration;
Fig. 7 is the impact of different carbon source on thalline fermentation concentration;
Fig. 8 is the impact of different nitrogen sources on thalline fermentation concentration;
Fig. 9 is the impact of different bottling amount on thalline fermentation concentration;
Figure 10 is the impact of different vaccination amount on thalline fermentation concentration;
Figure 11 is the impact of different fermentations time on thalline fermentation concentration;
Figure 12 is the impact of different pH on thalline fermentation concentration;
Figure 13 is the impact for the treatment of of different temperature on antimicrobial substance activity in fermented liquid;
Figure 14 is the impact of UV treatment on antimicrobial substance activity in fermented liquid;
Figure 15 is the impact of protease treatment on antimicrobial substance activity in fermented liquid.
Embodiment
In conjunction with embodiment, the present invention is further illustrated, should be noted that following explanation is only to explain the present invention, not limiting its content.
The reagent of not detailed description in the present invention, method are conventional reagent, the method in affiliated field.
Embodiment 1: the acquisition of curing ginger stalk rot bacterium antagonistic bacterium C3 and qualification
From shallot rhizosphere screening antagonistic bacterium, morphology and molecular biology method is adopted to identify screening the antagonistic bacterium obtained.
(1) Morphological Identification:
Ne ar is relatively stable in suitable situation, the antagonistic bacterium filtered out is lined NA substratum, cultivate after 24h and observe single colony characteristics, comprise colonial morphology, size, the homogeneity of color, thalline smell, substratum carry out preliminary evaluation with or without variable color etc., result as shown in Figure 1;
(2) molecular biology identification:
By 16SrDNA order-checking and RecA gene sequencing, will in sequencing result sequence and GenBank, submit to sequence to compare, bacterial strain C3 has been accredited as Burkholderia cepacia (Burkholderiacenocepacia).
By above qualification, confirm that bacterial strain C3 is Burkholderia cepacia (Burkholderiacenocepacia), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number is CGMCCNo.9791, and the preservation time is on October 17th, 2014.
Embodiment 2: fungistatic effect
(1) curing ginger stalk rot bacterium antagonistic bacterium C3 is to the inhibition of pathogenic bacteria
C3 is to the restraining effect of ginger brown foot rot germ, and as shown in Figure 2, be C3 in the middle of culture dish, be around ginger brown foot rot germ, therefore, C3 obviously has restraining effect to ginger brown foot rot germ.As shown in Figure 3, wherein, A figure is contrast, and mycelium shows as surface smoothing, even thickness, in a tubular form; B figure is C3 and the mycelium of ginger brown foot rot germ opposite culture after 2 days, shows as the hyperplasia such as mycelium local bulkiness, hooks.
(2) curing ginger stalk rot bacterium antagonistic bacterium C3 is to the inhibition of instruction pathogenic bacteria
As shown in Figure 4,5, C3 shows obvious restraining effect to brown foot rot germ, tobacco ralstonia solanacearum and dry thread Pyrenomycetes; Geotrichum candidum is screening indicator.
Embodiment 3: curing ginger stalk rot bacterium antagonistic bacterium C3 is to the control efficacy of base rot disease
(1) preparation of biocontrol fungicide and cause of disease inoculum
Bacterial suspension prepares: cultivate curing ginger stalk rot bacterium antagonistic bacterium C3 with Optimal Medium, when 600nmOD value is 1, by centrifugal for bacterial suspension 6000rpm 10min, thalline equal-volume sterilized water or phosphoric acid buffer Eddy diffusion, being diluted to bacterial concentration is 5 × 10
8cfu/ml, after packing, 4 DEG C of placements are for subsequent use.
Assistant carrier: according to proportioning three kinds of material mixings of maltodextrin 60%, yeast powder 10%, wheat bran 30%.
Pathogenic bacteria inoculum: be inoculated in the wheat groat of sterilizing by brown foot rot pathogenic bacteria, cultivate 7d for 28 DEG C, for subsequent use, experimental design is specifically in table 1.
Table 1 experimental design and different treatment
First soil and matrix 3:1 are mixed rear 160 DEG C of sterilizing 2h, be dispensed in flowerpot.Assistant carrier a few days ago, mixes with every basin 10g and upper strata 1/3 soil by plantation.According to above-mentioned different treatment, the curing ginger stalk rot bacterium antagonistic bacterium C3 bacteria suspension prepared is mixed by every basin 10ml and 1000ml water, pours into soil.Two days later, plant anosis kind of ginger, 5, every basin, often processes 3 basins.Two days later, access base rot disease inoculum, inoculates the wheat that carries disease germs near each ginger bud in growth.
5d after inoculation, hangs liquid irrigating root with same concentrations biocontrol microorganisms, observes and measure and manage onset grade everywhere after 12d, according to disease scale criterion calculation disease index and protection effect, be treated to contrast with clear water.
Onset grade is with reference to sprout term disease grade scales such as Li Changsong:
0 grade: anosis
1 grade: the slightly aobvious scab of basal part of stem or root or slightly variable color
There is scab in 2 grades: 1/3 ~ 1/2 basal part of stem or root, variable color or rotten
3 grades: scab is around whole basal part of stem or root, and variable color is rotted
4 grades: withered death
(2) control efficacy result
Result is as shown in table 2, and curing ginger stalk rot bacterium antagonistic bacterium C3 reaches 41.18% to curing ginger stalk rot preventive effect, is significantly increased compared with the control, and process sequela rate, disease index all decrease compared with CK2, illustrate that protection effect is desirable.
Table 2 different treatment is to the prevention effect of curing ginger stalk rot
Embodiment 4: curing ginger stalk rot bacterium antagonistic bacterium C3 cultivation and fermentation condition optimizing
(1) screening of basal fermentation medium
With KB1, KB2, NYBD, YPG, LB, Ppm substratum is alternative basic medium, nutrient media components and proportioning are as table 3, seed liquor being inoculated into 4% inoculum size is equipped with in the 100mL triangular flask of 50mL substratum, 28 DEG C, 180rpm cultivates 72h and obtains bacteria suspension, by bacteria suspension 4 DEG C, 10000rpm frozen centrifugation 20min, precipitation is microorganism, by OD value under ultraviolet spectrophotometer mensuration 600nm after dilution, 0 is adjusted with sterilized water, measure the impact of different culture media type on thalline fermentation concentration, result as shown in Figure 6, as seen from the figure, maximum with KB1 substratum thalline fermentation concentration, so choose substratum based on KB1 substratum.
Table 3 basic media components and proportioning
(2) nutrient media components experiment of single factor
Basic medium KB1 is carried out to the screening of Carbon and nitrogen sources.According to identical proportioning using glycerine, sucrose, glucose, lactose, maltose, Zulkovsky starch as the carbon source in basic medium, other compositions and proportioning constant, measure heterogeneity carbon source to the impact of cell concentration.According to identical proportioning by extractum carnis, yeast powder, Tryptones, (NH
4)
2sO
4, NH
4cl, peptone are respectively as nitrogenous source in basic medium.As can be seen from Fig. 7-8, glycerine is as culture medium carbon source, and when yeast powder is as culture media nitrogen source, OD value is maximum, illustrates that curing ginger stalk rot bacterium antagonistic bacterium C3 cell concentration is maximum, therefore using Carbon and nitrogen sources that glycerine and yeast powder are optimized as nutrient media components.
After determining basic medium, on the basis of single factor experiment with inorganic salt in the Carbon and nitrogen sources filtered out and basic medium for constant, select upper, middle and lower three levels according to former basic medium proportioning, medium component proportioning is carried out to the L of 4 factor 3 levels
9(3
4) orthogonal test, the design consideration DPS of orthogonal table generates table 4 automatically.By bacteria suspension 4 DEG C, 10000rpm frozen centrifugation 20min, precipitation is microorganism, by OD value under ultraviolet spectrophotometer mensuration 600nm after dilution, adjusts 0 with sterilized water, measures the impact of different components proportioning on thalline fermentation concentration.
Table 4 Optimal Medium proportioning orthogonal test factor and level
By orthogonal trial, each level of factor, experimental result and range analysis are in table 5, and four kinds of component impacts on thalline fermentation concentration are followed successively by glycerine > MgSO
4> K
2hPO
4> yeast powder, namely in fermented liquid, the percentage composition of yeast powder has the greatest impact to thalline fermentation, and optimum combination is A
1b
2c
3d
1, i.e. glycerine 2%, yeast powder 1%, K
2hPO
40.2%, MgSO
40.1%.
Table 5 fermention medium Orthogonal experiment results and analysis
(3) fermentation condition optimization
Based on the Medium Proportion optimized, measure the impact on thalline fermentation concentration of substratum different vaccination amount, bottling amount, fermentation time and medium pH respectively.
(1) bottling amount is on the impact of thalline fermentation concentration
Based on proportion optimizing substratum, in 250ml triangular flask, regulate Medium bottling volume to be 50ml, 10ml, 150ml, 200ml under 4% seed liquor inoculum size, pH natural condition, after 28 DEG C of 180rpm cultivate 72h, by OD value under ultraviolet spectrophotometer mensuration 600nm, adjust 0 with sterilized water, determine bottled amount during maximum cell concentration.Can find out in Fig. 9 that cell concentration increases with bottled amount and increases, reach maximum during the bottled amount of 150ml/200ml, subsequent concn declines, and reason may be because with the increase of bottled amount, the oxygen that can utilize for bacterium in bottle reduces, and metabolism reduces.So determine that bottled amount is 150ml/250ml, namely in 250mL triangular flask, 150mL substratum is housed.
(2) inoculum size is on the impact of thalline fermentation concentration
With Optimal Medium component and proportioning, seed liquor inoculum size is regulated to be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% under the bottled amount of 150ml/250ml, pH natural condition, on the impact of thalline fermentation concentration as shown in Figure 10, during 3% inoculum size, cell concentration is maximum, therefore determines that inoculum size is 3%.
(3) fermentation time is on the impact of thalline fermentation concentration
With Optimal Medium component and proportioning, under the bottled amount of 150ml/250ml, pH nature, 3% inoculum size condition, different incubation time 12h, 24h, 36h, 48h, 60h, 72h, 96h, 108h are set, OD value under ultraviolet spectrophotometer mensuration 600nm, 0 is adjusted with sterilized water, analyze different incubation time section cell concentration, result as shown in figure 11, as can be seen from the figure cell concentration increases with fermentation time and increases, during 48h, cell concentration is maximum, subsequently in steady state, therefore determine that fermentation time is 48h.
(4) pH is on the impact of thalline fermentation concentration
With Optimal Medium component and proportioning, the substratum of different pH is set under 150ml (250ml) bottling amount, 3% inoculum size condition, pH is respectively 4,5,6,7,8,9,10 and shakes bacterium 48h, by bacteria suspension 4 DEG C, 10000rpm frozen centrifugation 20min, precipitation is microorganism, by OD value under ultraviolet spectrophotometer mensuration 600nm after dilution, adjust 0 with sterilized water, measure the impact of different pH on thalline fermentation concentration.The results are shown in Figure 12, as can be seen from the figure pH8-9 cell concentration is maximum, shows that the growth of this condition hypothallus is the fastest.
Embodiment 5: fermented liquid physical and chemical property determining
With the substratum optimized and fermentation condition culturing bacterium, fermentation liquor is centrifugal, obtain bacteria-free filtrate, adopts mixing to fall flat band method and measures the temperature of fermented liquid, ultraviolet and protease stability.
(1) temperature stability:
By fermented liquid 10mL centrifuge tube packing, respectively at 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 100 DEG C process 30min, place filtrate for contrast with 4 DEG C, adopt and be mixed into flat band method with filtrate: ratio a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices of substratum=1:5, each process repetition 3 times, measures fermented liquid bacteriostatic activity difference.
Result is as Figure 13, and colony diameter is no significant difference compared with the control, and 100 DEG C of process 30min fungistatic effects are identical with contrast, illustrate that bacteriostatic active ingredients is insensitive to high temperature.
(2) UV stable:
Get appropriate fermented liquid and be placed in sterile petri dish, after sealing under 30W ultraviolet lamp distance 30cm Continuous irradiation 12h, with 4 DEG C place filtrates for contrast, with reference to aforesaid method measure bacteriostatic activity.
As shown in figure 14, fermentation liquor ultraviolet closely after Continuous irradiation 12h fungistatic effect with contrast no significant difference, illustrate that in fermented liquid, activeconstituents has good stability to uv irradiating.
(3) protease stability:
Get appropriate fermented liquid, add appropriate trypsinase and pepsin solution respectively, make the final concentration of enzyme be 0.5g/L, 37 DEG C of water-bath 3h make enzyme fully reflect.Place filtrate for contrast with 4 DEG C, measure bacteriostatic activity with reference to aforesaid method.
As Figure 15, after fermentation liquor trypsinase and pepsin 3h, fungistatic effect is without considerable change, illustrates that in fermented liquid, bacteriostatic active ingredients is insensitive to proteolytic enzyme.
To sum up, after fermentation liquor treatment of different temperature 30min compared with the control pathogenic bacteria colony diameter without considerable change; After fermentation liquor ultraviolet Continuous irradiation 12h compared with the control pathogenic bacteria colony diameter without considerable change; After fermentation liquor trypsinase and pepsin 3h compared with the control pathogenic bacteria colony diameter without considerable change; Therefore the antimicrobial substance that this bacterium produces has stronger stability to heat, proteolytic enzyme and ultraviolet.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned; but not limiting the scope of the invention; one of ordinary skill in the art should be understood that; on the basis of technical scheme of the present invention, those skilled in the art do not need to pay various amendment or distortion that creative work can make still within protection scope of the present invention.
Claims (7)
1. curing ginger stalk rot bacterium antagonistic bacterium, it is characterized in that, called after Burkholderia cepacia (Burkholderiacenocepacia) C3, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 17th, 2014, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number is CGMCCNo.9791.
2. curing ginger stalk rot bacterium antagonistic bacterium as claimed in claim 1, is characterized in that, cultivates each component concentration in the substratum of curing ginger stalk rot bacterium antagonistic bacterium to be: lactose 3%, extractum carnis 2%, K
2hPO
40.2%, MgSO
40.15%.
3. curing ginger stalk rot bacterium antagonistic bacterium as claimed in claim 1, it is characterized in that, during fermented ginger brown foot rot germ antagonistic bacterium, bottled amount is 150ml/250ml, and inoculum size is 3%, and fermentation time is 48h.
4. a C3 microbial inoculum, is characterized in that, its activeconstituents is curing ginger stalk rot bacterium antagonistic bacterium according to claim 1.
5. microbial inoculum as claimed in claim 4, it is characterized in that, the packing of product formulation of described microbial inoculum is liquid bacterial agent.
6. the preparation method of microbial inoculum described in claim 4 or 5, it is characterized in that, adopt following steps: cultivate curing ginger stalk rot bacterium antagonistic bacterium with Optimal Medium, when 600nmOD value is 1, by centrifugal for bacterial suspension 6000rpm 10min, thalline equal-volume sterilized water or phosphoric acid buffer Eddy diffusion, being diluted to bacterial concentration is 5 × 10
8cfu/ml, after packing, 4 DEG C of placements are for subsequent use.
7. microbial inoculum described in bacterial strain described in claim 1 and/or claim 4 is in the application of preparation treatment curing ginger stalk rot.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739860A (en) * | 2018-05-02 | 2018-11-06 | 华南农业大学 | Bacterium and its application as biocontrol microorganisms is quenched in a kind of micropopulation inductive signal |
| CN116267997A (en) * | 2022-09-07 | 2023-06-23 | 湖北科技学院 | Application of paenibacillus polymyxa in preventing and treating ginger stem basal rot |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101999371A (en) * | 2010-10-09 | 2011-04-06 | 亓冬英 | Preparation for preventing and curing ginger stalk rot |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101999371A (en) * | 2010-10-09 | 2011-04-06 | 亓冬英 | Preparation for preventing and curing ginger stalk rot |
Non-Patent Citations (2)
| Title |
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| K. HEUNGENS等: "Zoospore Homing and Infection Events: Effects of the Biocontrol Bacterium Burkholderia cepacia AMMDR1 on Two Oomycete Pathogens of Pea (Pisum sativum L.)", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108739860A (en) * | 2018-05-02 | 2018-11-06 | 华南农业大学 | Bacterium and its application as biocontrol microorganisms is quenched in a kind of micropopulation inductive signal |
| CN116267997A (en) * | 2022-09-07 | 2023-06-23 | 湖北科技学院 | Application of paenibacillus polymyxa in preventing and treating ginger stem basal rot |
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