CN102250815B - Endogenous paenibacillus polymyxa - Google Patents
Endogenous paenibacillus polymyxa Download PDFInfo
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- CN102250815B CN102250815B CN 201110205215 CN201110205215A CN102250815B CN 102250815 B CN102250815 B CN 102250815B CN 201110205215 CN201110205215 CN 201110205215 CN 201110205215 A CN201110205215 A CN 201110205215A CN 102250815 B CN102250815 B CN 102250815B
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- paenibacillus polymyxa
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Abstract
The invention relates to the field of microorganisms and specifically discloses an endogenous paenibacillus polymyxa. The endogenous paenibacillus polymyxa provided by the invention is collected in the Center for General Microorganism of China Committee for Culture Collection of Microorganisms and the collection number is CGMCC No.4843. After the endogenous paenibacillus polymyxa provided by the invention is reproduced to the 40th generation, the paenibacillus polymyxa also has higher activity of inhibiting spores of a phgtophora palmivora butler bacterial strain and has higher genetic stability; meanwhile, a chemically preventing and treating effect is achieved for preventing and treating phgtophora palmivora butler; and the endogenous paenibacillus polymyxa provided by the invention does not pollute the environment, is ecological and safe and can be widely applied to prevention and treatment of the phgtophora palmivora butler and preparation of a preparation for preventing and treating the phgtophora palmivora butler.
Description
Technical field
The present invention relates to microorganism field, relate in particular a kind of endogenetic polymexa bacillus.
Background technology
Pepper (Piper nigrum L.) is piperaceae Piper perennial evergreen climbing vine plant, is the important tropical feature spice crop in the world, and one of, favorite seasonings the most frequently used for people is of many uses in fields such as food, health cares.
The pepper seasonal febrile diseases are a kind of weather dependency diseases, occur when being everlasting rainy season, and it is the very strong soil-borne disease of a kind of infectivity that is caused by the phytophthora infection.Since introducing a fine variety pepper, the pepper seasonal febrile diseases just are the primary diseases that harm China pepper produces always, the loss of pepper garden reaches more than 90% when being in a bad way, even full garden is destroyed, bring immeasurable loss for China's pepper plant husbandry, China has carried out comparatively deep research to the Prevention Technique of pepper seasonal febrile diseases for this reason.
At present, the control of pepper seasonal febrile diseases is mainly relied on Techniques For Chemical Control, as spray metaxanin Propamocarb WP.Although chemical prevention can reach preferably prevention effect, but the life-time service chemical pesticide not only can produce pesticide residue, reduce the pepper quality, but also can cause the resistance of pepper seasonal febrile diseases germ along with long-term frequent use of chemical pesticide, cause the pepper seasonal febrile diseases that the gesture that increases the weight of is year by year arranged in recent years.Therefore, effectively, safety, ecological prevent and treat the primary goal that the pepper seasonal febrile diseases just become pepper plant husbandry.
Biological control is a kind of technique means of preventing and treating the pepper seasonal febrile diseases that proposes present stage, it is take the strain of sudden death disease germ as indicator, filter out the microorganism that can suppress the strain of sudden death disease germ in from soil and carry out biological control, this method has effectively been avoided the resistance and the problem of environmental pollution that utilize Techniques For Chemical Control to bring.Yet the composition monoid of sudden death disease germ strain is very complicated, and during existing screening take typical case or single sudden death disease germ strain as indicator, so the microorganism that filters out has certain limitation, its prevention effect can not show a candle to the chemical prevention effect.In addition, the existing microorganism that filters out is along with the increase of passage number, and its inhibition activity to pepper seasonal febrile diseases germ spore reduces gradually, and genetic stability is relatively poor.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of endogenetic polymexa bacillus, so that the effect of the effect of this bacterial strain control pepper warm disease and chemical prevention pepper seasonal febrile diseases is without significant difference, and have higher genetic stability.
For achieving the above object, the invention provides a kind of endophyte that obtains that from the pepper blade, separates, called after bacterial strain S, bacterial strain S is circular at the bacterium colony of PDA planar surface growth, surperficial center protrusion is glossy, and the grower thickness; Gramstaining is negative; Spore staining is positive, and gemma is generally in the middle of thalline; Capsule stain positive (having pod membrane to form); Flagella staining is positive, and flagellum is that give birth at two ends, and motor capacity is not strong; Thalli morphology is shaft-like, Dan Sheng.Catalase, V.P. reaction, Starch Hydrolysis, nitrate reduction react, gelatine liquefication is all positive.Citrate trianion utilization, urease test, clark and Lubsreaction are all negative.Thalline is well-grown on the substratum of pH5.7, can not grow at the substratum that contains 7%NaCl.In addition, analyze through the 16S DNA detection, 50 bacterial strains high with bacterial strain S homology all are series bacillus, and homology in conjunction with physiological and biochemical property, identifies that it is endogenetic polymexa bacillus up to 99%.
Endogenetic polymexa bacillus of the present invention has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 12nd, 2011, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.4843.
In endogenetic polymexa bacillus of the present invention go down to posterity 1st generation, the 10th generation, the 20th generation, the 30th generation, the 40th generation of cultivating, show the inhibition test of pepper seasonal febrile diseases germ spore, and described endogenetic polymexa bacillus suppresses the EC of the pepper seasonal febrile diseases germ spore germination from 40 generations of 1st generation to the
50Without significant difference, and all below 5.13%, show that endogenetic polymexa bacillus of the present invention has preferably inhibition, and genetic stability is higher, can reduce with the increase of passage number the inhibition of pepper seasonal febrile diseases germ spore active.
Endogenetic polymexa bacillus of the present invention and metaxanin Propamocarb WP show that to pepper seasonal febrile diseases efficiency test result both preventive effects are suitable, no significant difference.Show that endogenetic polymexa bacillus preventive effect of the present invention is higher, reach the effect of chemical prevention, can be applied in control pepper seasonal febrile diseases and the preparation control pepper seasonal febrile diseases preparation.
The present invention also provides a kind of biological prevention and control agent, comprises that deposit number is the endogenetic polymexa bacillus of CGMCC No.4843.Biological prevention and control agent of the present invention can use before pepper is transplanted in spraying, also can make compost and use, and perhaps fills with root after the dilution and use when transplanting.
The preparation method of described biological prevention and control agent of the present invention is as follows:
The endogenetic polymexa bacillus that with deposit number is CGMCC No.4843 is cultivated in the PDA nutrient solution, and then filtering and regulate bacterium liquid spore count is 10
8-10
10Individual/mL, namely get the biological prevention and control agent finished product.
Wherein, the temperature of described cultivation is preferably 26-30 ℃, and more preferably 28 ℃, the fate of described cultivation is preferably 4-6d, more preferably 5d.
By above technical scheme as can be known, endogenetic polymexa bacillus of the present invention is passaged to the activity that 40 generations still had higher inhibition sudden death disease germ strain spore, has higher genetic stability, the effect of its control pepper seasonal febrile diseases reaches the effect of chemical prevention simultaneously, and free from environmental pollution, ecological, safety can be widely used in the control and preparation control pepper seasonal febrile diseases preparation of pepper seasonal febrile diseases.
Biological preservation explanation
Classification And Nomenclature: Paenibacillus polymyxa, Paenibacillus polymyxa. is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on May 12nd, 2011, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.4843.
Embodiment
The invention discloses a kind of endogenetic polymexa bacillus, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are deemed to be included in the present invention.Endogenetic polymexa bacillus of the present invention is described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described within not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: the separation method of endogenetic polymexa bacillus of the present invention
Heavily adopt the long healthy blade of bringing back to life in the district from the morbidity of pepper seasonal febrile diseases, be cut into the tissue block of about 2cm * 2cm specification with the aseptic operation cutter, aseptic water washing for several times, in 75% alcohol, soak 1min, aqua sterilisa flushing 3 times places aseptic mortar with tissue block, adds sterilized water after shredding with scissors and grinds, leave standstill 30min, be diluted to 10
-1, 10
-2, 10
-33 gradients pipette respectively last 1 washing fluid and each 0.3mL of lapping liquid, are applied on the PDA culture medium flat plate, and last 1 washing fluid, is observed in 28 ℃ of lower cultivations day by day as CK.Select among the CK without bacterium colony, and grow the bacterial strain of bacterium colony in the lapping liquid, and according to feature picking list bacterium colonies such as colonial morphology, colors, after the purifying numbering, face-off is cultivated and is detected its restraining effect to pepper seasonal febrile diseases germ in plate, selects to form the bacterial strain of antibacterial band, called after bacterial strain S.
Embodiment 2: the mensuration of endogenetic polymexa bacillus Physiology and biochemistry of the present invention and 16S dna sequence dna
1, Physiology and biochemistry detects
Detect growing state, colonial morphology and color, gramstaining, spore staining and the flagella staining etc. of bacterial strain S on the PDA flat board, the method that " the planting the disease research method " that method is edited with reference to Fang Zhongda is 182 pages is carried out.
Detect bacterial strain S physio-biochemical characteristics, comprise: the utilization of litmus milk, catalase, V.P. reaction, Starch Hydrolysis, nitrate reduction reaction, gelatine liquefication reaction, oxydase, M.R, phenylalanine deaminase, Citrate trianion utilization, urease test, propionic salt, whether can be in the substratum that contains 7%NaCl and the pH5.7 test such as grow, method is with reference to the elegant pearl in east, the Cai Miaoying chief editor's " method of common bacteria system identification handbook 353-385 page or leaf.
Detected result is as follows:
Bacterium colony in the growth of PDA planar surface is circular, and surperficial center protrusion is glossy, and the grower thickness; Gramstaining is negative; Spore staining is positive, and gemma is generally in the middle of thalline; Capsule stain positive (having pod membrane to form); Flagella staining is positive, and flagellum is that give birth at two ends, and motor capacity is not strong; Thalli morphology is shaft-like, Dan Sheng.Catalase, V.P. reaction, Starch Hydrolysis, nitrate reduction react, gelatine liquefication is all positive.Citrate trianion utilization, urease test, clark and Lubsreaction are all negative.Thalline is well-grown on the substratum of pH5.7, can not grow at the substratum that contains 7%NaCl.
2, the mensuration of 16S dna sequence dna
With reference to the bacterial genomes DNA preparation method of Ausubel, carry out pcr amplification with bacterium universal primer 27F and 1492R.
The PCR reaction system is 25 μ l, 10 * PCR Buffer, 2.5 μ l wherein, each 1 μ l of Primer27F and Primer1492R (25pmoL), dNTP Mixture 0.5 μ l, Taq DNA Polymerase (5U/ μ l) 0.5 μ l, ddH
2O 17.5 μ l, dna profiling 2 μ l.
Pcr amplification reaction condition: 95 ℃ of 4min; 95 ℃ of 1min, 55 ℃ of 40s, 72 ℃ of 2min, totally 30 circulations; Then 72 ℃ keep 7min.The PCR product reclaims test kit with EZ-10 SpinColum DNA Gel Extraction Kit and reclaims, and entrusts precious biotechnology (Dalian) company limited to check order.The gene order that records is carried out the sequence alignment analysis with BLAST software, and with GenBank in known 16S rDNA carry out homology relatively.
Detected result is as follows:
With this pcr amplification product (accession number: JF747221) carry out the BLAST comparison in GenBank.The result shows, 50 bacterial strains high with S bacterial strain homology all are that series bacillus belongs to (Paenibacillus sp.), its homology reaches 99%, wherein table 1 is depicted as 10 bacterial strains the highest with the 16S rDNA homology of bacterial strain S, and deducibility bacterial strain S belongs to series bacillus and belongs to (Paenibacillus sp.) thus.
Table 1 16S rDNA homology relatively
| The sequence accession number | Bacterial strain | Homology |
| GQ849013.1 | Paenibacillus polymyxa strain y-10 | 99％ |
| GU328684.1 | Paenibacillus sp.Nz29 | 99％ |
| EU362601.1 | Paenibacillus polymyxa isolate TN61 | 99％ |
| GU328691.1 | Paenibacillus sp.Sx52 | 99％ |
| AB480885.1 | Paenibacillus sp.Mis2 | 99％ |
| AB247329.2 | Paenibacillus sp.Mis2 | 99％ |
| GU328683.1 | Paenibacillus sp.Nz28 | 99％ |
| EU430119.1 | Paenibacillus polymyxa strain YC0136 | 99％ |
| EF634024.1 | Paenibacillus polymyxa strain ISSDS-851 | 99％ |
| EF620468.1 | Paenibacillus polymyxa strain ISSDS-792 | 99％ |
In conjunction with Physiology and biochemistry and 16S rDNA detected result, bacterial strain S is accredited as Paenibacillus polymyxa (Paenibacillus polymyxa.).
Embodiment 3: endogenetic polymexa bacillus of the present invention is to the inhibition test of pepper seasonal febrile diseases germ spore germination
With the activation endogenetic polymexa bacillus streak inoculation of the present invention in the PDA culture dish, 28 ℃ of cultivations turn ware 1 time every 24h, corotation 40 times.Measure the endogenetic polymexa bacillus bacterial strain in 1st generation, 10 generations, 20 generations, 30 generations and 40 generations with sessile drop method to the inhibition of pepper seasonal febrile diseases germ spore germination, the results are shown in Table 2.
Table 2 endogenetic polymexa bacillus of the present invention is to the inhibition test of pepper seasonal febrile diseases germ spore germination
By table 2 result as can be known, endogenetic polymexa bacillus of the present invention suppresses the EC of pepper seasonal febrile diseases germ spore germination from the bacterium liquid in 40 generations of 1st generation to the
50Difference is very little, and it is active to show that endogenetic polymexa bacillus of the present invention can not reduce with the increase of passage number the inhibition of pepper seasonal febrile diseases germ spore, has higher genetic stability.Simultaneously, endogenetic polymexa bacillus bacterium liquid of the present invention suppresses the EC of pepper seasonal febrile diseases germ spore germination
50Below 5.13%, show that it has good inhibition spore germination effect.
Embodiment 4: endogenetic polymexa bacillus of the present invention is to the efficiency test of pepper seasonal febrile diseases
3 processing are established in test: (spore count is 10 to endogenetic polymexa bacillus bacterium liquid of the present invention
8Individual/mL) 500 times of diluents; 1000 times of diluents of commercially available 25% metaxanin Propamocarb WP; Clear water.
The mensuration of provide protection: spray the liquid of 3 processing at the potted plant seedling of pepper first, whole strain all squirts, take the liquid that drips as degree.Under the moisturizing condition, inoculate sudden death disease disease pathogen bacterium behind the 24h.Each is processed 3 times and repeats, 10 seedlings of every repetition, 3 leaves of every seedling inoculation.Carry out state of an illness investigation by table 3 grade scale behind the 5d.
The mensuration of therapeutic action: inoculate sudden death disease disease pathogen bacterium under the moisturizing condition, spray the liquid of 3 processing behind the 24h at potted plant seedling, whole strain all squirts, take the liquid that drips as degree.Each is processed 3 times and repeats, 10 seedlings of every repetition, 3 leaves of every seedling inoculation.Carry out state of an illness investigation by table 3 grade scale behind the 5d.
Table 3 severity grade scale
| 0 grade | Without scab |
| 1 grade | Lesion area accounts for below 1/8 of one-piece blade area |
| 3 grades | Lesion area accounts for the 1/8-1/4 of one-piece blade area |
| 5 grades | Lesion area accounts for the 1/4-1/2 of one-piece blade area |
| 7 grades | Lesion area accounts for more than 1/2 of one-piece blade area |
Wherein, disease index=∑ (the sick numbers of sheets at different levels * this disease level level value)/(investigation leaf sum * the highest sick level value) * 100; prevention effect (%)=(contrast disease index-processing disease index)/contrast disease index * 100%; variance analysis is carried out in the provide protection of statistics and the anti-effectiveness SAS software of therapeutic action; the preventive effect of clear water contrast is with 0 note, and the result sees Table respectively 4 and table 5.
The preventive effect of table 4 endogenetic polymexa bacillus provide protection of the present invention
The preventive effect of table 5 endogenetic polymexa bacillus therapeutic action of the present invention
By table 4 and table 5 result as can be known; endogenetic polymexa bacillus of the present invention reaches 80.64% to the preventive effect of pepper seasonal febrile diseases provide protection; the preventive effect of therapeutic action is 78.54%, and is suitable with the chemical prevention effect of carrying out with 25% metaxanin Propamocarb WP, no significant difference.
Embodiment 5: prepare biological prevention and control agent of the present invention
The endogenetic polymexa bacillus that with deposit number is CGMCC No.4843 is cultivated in the PDA nutrient solution, and culture temperature is 28 ℃, and fate is 5d, and rotating speed is 200rpm, and then filtering and regulate bacterium liquid spore count is 10
9Individual/mL, namely get the biological prevention and control agent finished product.
Embodiment 6: prepare biological prevention and control agent of the present invention
The endogenetic polymexa bacillus that with deposit number is CGMCC No.4843 is cultivated in the PDA nutrient solution, and culture temperature is 30 ℃, and fate is 4d, and rotating speed is 200rpm, and then filtering and regulate bacterium liquid spore count is 10
10Individual/mL, namely get the biological prevention and control agent finished product.
Embodiment 7: prepare biological prevention and control agent of the present invention
The endogenetic polymexa bacillus that with deposit number is CGMCC No.4843 is cultivated in the PDA nutrient solution, and culture temperature is 26 ℃, and fate is 6d, and rotating speed is 200rpm, and then filtering and regulate bacterium liquid spore count is 10
8Individual/mL, namely get the biological prevention and control agent finished product.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a Paenibacillus polymyxa (Paenibacillus polymyxa) is characterized in that, deposit number is CGMCC No.4843.
2. the application of the described Paenibacillus polymyxa of claim 1 in control pepper seasonal febrile diseases.
3. the application of the described Paenibacillus polymyxa of claim 1 in preparation control pepper seasonal febrile diseases preparation.
4. a biological prevention and control agent is characterized in that, comprises that deposit number is the Paenibacillus polymyxa of CGMCC No.4843.
5. the application of the described biological prevention and control agent of claim 4 in control pepper seasonal febrile diseases.
6. the preparation method of a biological prevention and control agent is characterized in that, is that the Paenibacillus polymyxa of CGMCCNo.4843 is cultivated in the PDA nutrient solution with deposit number, and then filtering and regulate bacterium liquid spore count is 10
8-10
10Individual/mL, namely get the biological prevention and control agent finished product.
7. described preparation method according to claim 6 is characterized in that the temperature of described cultivation is 26-30 ℃.
8. described preparation method according to claim 7 is characterized in that the temperature of described cultivation is 28 ℃.
9. described preparation method according to claim 6 is characterized in that the fate of described cultivation is 4-6d.
10. described preparation method according to claim 9 is characterized in that the fate of described cultivation is 5d.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103250741B (en) * | 2013-03-05 | 2015-02-18 | 吉林农业大学 | Preparation method of ginseng endophytic Paenibacillus polymyxa biological control microbial inoculum |
| CN103173386B (en) * | 2013-03-12 | 2015-01-28 | 山东农业大学 | Bio-control strain G1 for preventing and controlling pepper phytophthora blights and applications thereof |
| CN107711890A (en) * | 2017-07-27 | 2018-02-23 | 上海万力华生物科技有限公司 | A kind of Paenibacillus polymyxa sterilization aqua and preparation method thereof |
| CN113151079B (en) * | 2021-04-19 | 2022-05-17 | 河北省农林科学院谷子研究所 | Paenibacillus polymyxa KDB and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696395A (en) * | 2009-10-29 | 2010-04-21 | 南京农业大学 | Antagonistic bacterium for preventing and killing off continuous cropping tobacco black shank and microbial organic fertilizer thereof |
| WO2011074831A2 (en) * | 2009-12-17 | 2011-06-23 | 경기도 | Novel paenibacillus polymyxa and microbial agent for prevention of phytophthora rot comprising same |
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2011
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101696395A (en) * | 2009-10-29 | 2010-04-21 | 南京农业大学 | Antagonistic bacterium for preventing and killing off continuous cropping tobacco black shank and microbial organic fertilizer thereof |
| WO2011074831A2 (en) * | 2009-12-17 | 2011-06-23 | 경기도 | Novel paenibacillus polymyxa and microbial agent for prevention of phytophthora rot comprising same |
Non-Patent Citations (2)
| Title |
|---|
| Chen, XL."Antifungal peptide produced by Paenibacillus polymyxa BRF-1 isolated from soybean rhizosphere".《AFRICAN JOURNAL OF MICROBIOLOGY RESEARCH》.2010,第4卷(第24期), |
| 陈文俊."西瓜根际细菌JK-3的鉴定及其拮抗性的初步研究".《上海农业学报》.2011,第27卷(第1期),68-72. |
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