CN103756931A - Paenibacillus kribbensis and its application - Google Patents
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Abstract
The invention discloses a Paenibacillus kribbensis, and a preparation method and an application thereof. The Paenibacillus kribbensis has a bacterial code name of TRCC82001, is preserved in China General Microbiological Culture Collection Center, and has a preservation number of CGMCCNo.7996. The above bacterial strain has the advantages of good production property, good metabolite antimicrobial effect, and strong activity against rice blast and sheath blight, and a solution obtained after diluting a broth subjected to antibiotic treatment to 100 times has an inhibition effect of above 80% on the rice blast and sheath blight. The bacterial strain and its metabolites have a broad antimicrobial spectrum, can be used for preparing biocontrol preparations for the broad spectrum activity inhibition of pathogenic fungi to control the fungal diseases of plants.
Description
Technical field
The invention belongs to biotechnology prevention and control field, be specifically related to the preparation of a strain Ke Liben series bacillus (Paenibacillus kribbensis) and microbial inoculum thereof and suppressing pathogenic fungi and the application preventing and treating in fungal diseases of plants.
Background technology
Plant diseases is in modern agricultural production, the factor that plant biomass is exerted an influence, according to FAO data, the whole world is caused by disease invasion and attack every year loss because of plant accounts for the 10%-15% of crop production total amount, along with the adjustment of the agricultural planting industrial structure, the control of corps diseases is more and more paid attention to by people with control.The Plant diseases that plant pathogenic fungi causes is first of the large disease of plant three, accounts for 70% of all Plant diseasess.The disease that some farm crop mainly occur is as rice blast, rice sheath blight disease; Wheat rust, wheat powdery mildew; Corn northern leaf blight and southern leaf blight, banded sclerotial blight; Vegetables oidium etc., and farm crop gather after and the disease that occurs storage period mostly be fungal disease.For a long time, the agricultural chemicals that controlling plant diseases is used is chemical bactericide mostly, and sterilant kind is single, and phytopathogen has produced serious resistance to many kinds.In addition, due to long-term a large amount of a few sterilant that use, cause Pesticide Residue in Soil residue accumulation, cause environmental pollution, destroyed the microbial balance in soil and plant body, Plant diseases is occurred more frequent, even affect the healthy of the mankind.Therefore in the urgent need to exploitation, there is the biogenic sterilant that sterilant, especially " pollution-free food " the different mechanisms of action, novel structure, safe and effective environmental protection are produced.
Ke Liben series bacillus (Paenibacillus kribbensis) belongs to Prokaryota, Firmicutes, and bacillus guiding principle, genus bacillus order, Bacillaceae, series bacillus belongs to.By research, find that this bacterial strain can produce bacteriostatics outside born of the same parents in culturing process, the 13 kind of plant pathogenic fungies for examination are had to the growth activity of pressing down, has a broad antifungal spectrum, comparatively responsive to rice blast, rice sheath blight disease pathogenic bacteria, be the biocontrol strain that a strain has research and development application prospect.Research report about the antimycotic biologically active substance of Ke Liben series bacillus is less.The present invention has reported that a strain has the Ke Liben series bacillus of agricultural biological activity.
Summary of the invention
Technical problem to be solved by this invention is to provide the new Ke Liben series bacillus of a strain, this bacterial strain produces the active metabolite consumingly, suppress wide spectrum with pathogenic fungi by fermentation afterwards, this bacterial strain and active metabolite thereof can be used for preparing the biological prevention and control agent that wide spectrum suppresses pathogenic fungi, for preventing and treating fungal diseases of plants.
Ke Liben series bacillus bacterial strain provided by the present invention is (Paenibacillus kribbensis) TRCC82001, bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 13rd, 2013, and its preserving number is: CGMCC No.7996.
The present invention separately provides a kind of biological prevention and control agent, the activeconstituents of this biological prevention and control agent is the meta-bolites of described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996, or/and described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996.Described meta-bolites is to obtain from the thalline fermented liquid of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996.
Biological prevention and control agent described in the present invention is divided into three kinds: biological prevention and control agent 1, the mixed preparation of thalline and metabolite, be the fermented liquid of described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996, contain and state Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 thalline and metabolite thereof; Biological prevention and control agent 2, thalline, is the thalline of described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996, comprises nourishing body and gemma; Biological prevention and control agent 3, meta-bolites, be described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 without fermented liquid.
Described biological prevention and control agent of the present invention is specifically according to following A) or the acquisition of method B):
A) described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 is inoculated in the liquid czapek's solution II of improvement that loading amount is shaking flask 20%-30% volume with picking list bacterium colony or lawn after the solid czapek's solution II activation of improvement.30 degrees Celsius, 180r/min shaking culture 84 hours, the fermented liquid obtaining is described biological prevention and control agent 1.
B) described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 is inoculated in the liquid czapek's solution II of improvement that loading amount is shaking flask 20%-30% volume with picking list bacterium colony or lawn after the solid czapek's solution II activation of improvement.30 degrees Celsius, 180r/min shaking culture 84 hours, carries out centrifugation by obtained fermented liquid, obtains supernatant liquor and solid precipitation.The solid obtaining is described biological prevention and control agent 2; Supernatant liquor is described biological prevention and control agent 3 through sterilizing.
The czapek's solution II formula of described improvement is as follows:
Add quantitatively to 100% of sterilized water, if add the agar of 1.5-2%, pH7-8 as solid medium.
Described biological prevention and control agent is following 1)-2) in arbitrary biological prevention and control agent:
1) for preventing and treating the biological prevention and control agent of fungal diseases of plants;
2) for suppressing the biological prevention and control agent of pathogenic fungi.
Described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 is in preparation following 1)-2) in application in arbitrary biological prevention and control agent also belong to protection scope of the present invention:
1) for preventing and treating the biological prevention and control agent of fungal diseases of plants;
2) for suppressing the biological prevention and control agent of pathogenic fungi.
In the application of above-mentioned biological prevention and control agent and biological prevention and control agent, described pathogenic fungi be following at least one: rice blast (Phyricularia grisea (Cooke) Sacc.), rice sheath blight disease (Thanatephorus cucumeris (Frank) Donk), root rotof flax (Drechslera SoFokiniana (Sacc.) Subram.Gain), lichee anthrax (Colletotrichum litchi), Exserohilum turcicum (Exerohilum turcicum), southern corn leaf blight (Helminthosporium maydis), Maize yellow spot bacterium (Curvularia lunata), corn-based Pathogen (Fusarium graminearum), verticillium dahliae (Verticillium dahliae), withered germ of water-melon (Fusarium oxysporum f.sp.niveum), Colletotrichum capsici (Colletotrichum capsici), Alternaria brassicae (Alternaria brassicae (Berk.) Sace.), purple blotch of onion bacterium (Alternaria porri (Ell.) Cifferri).
In one embodiment of the invention, described fungal diseases of plants is rice blast, and pathogenic bacteria is grey pears spore (Phyricularia grisea (Cooke) Sacc.); Described plant is paddy rice, and described rice varieties is glad Rong You 08.
The preparation method of described biological prevention and control agent also belongs to protection scope of the present invention.Preparation method is as follows: by the meta-bolites of described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996, or/and described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996, as active ingredient, obtains described biological prevention and control agent.Described meta-bolites is to obtain from the thalline fermented liquid of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996.
Described biological prevention and control agent of the present invention is specifically according to following A) or the acquisition of method B):
A) described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 is inoculated in the liquid czapek's solution II of improvement that loading amount is shaking flask 20%-30% volume with picking list bacterium colony or lawn after the solid czapek's solution II activation of improvement, 30 degrees Celsius, 180r/min shaking culture 84 hours, the fermented liquid obtaining is described biological prevention and control agent 1.
B) described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 is inoculated in the liquid czapek's solution II of improvement that loading amount is shaking flask 20%-30% volume with picking list bacterium colony or lawn after the solid czapek's solution II activation of improvement.30 degrees Celsius, 180r/min shaking culture 84 hours, carries out centrifugation by obtained fermented liquid, obtains supernatant liquor and solid precipitation.The solid obtaining is described biological prevention and control agent 2; Supernatant liquor is described biological prevention and control agent 3 through sterilizing.
The czapek's solution II formula of described improvement is as follows:
Add quantitatively to 100% of sterilized water, if add the agar of 1.5-2%, pH7-8 as solid medium.
Ke Liben series bacillus provided by the present invention (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 tool has the following advantages:
1, Ke Liben series bacillus provided by the present invention (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 and have strong broad spectrum antibiotic activity without fermented liquid, to rice blast (Phyricularia grisea (Cooke) Sacc.), rice sheath blight disease (Thanatephorus cucumeris (Frank) Donk), root rotof flax (Drechslera SoFokiniana (Sacc.) Subram.Gain), lichee anthrax (Colletotrichum litchi), Exserohilum turcicum (Exerohilum turcicum), southern corn leaf blight (Helninthosporium maydis), Maize yellow spot bacterium (Curvularia lunata), corn-based Pathogen (Fusarium graminearum), verticillium dahliae (Verticillium dahliae), withered germ of water-melon (Fusarium oxysporumf.sp.niveum), Colletotrichum capsici (Colletotrichum capsici), Alternaria brassicae (Alternaria brassicae (Berk.) Sace.), (Alternaria porri (Ell.) Cifferri) is remarkably productive for purple blotch of onion bacterium.
2, the biological prevention and control agent 2 of Ke Liben series bacillus provided by the present invention (Paenidacillus kribbensis) TRCC82001CGMCC No.7996, biological prevention and control agent 3 are in the field test of rice blast, and prevention effect is better than contrasting 30 milliliters/mu of medicament 1,000 hundred million living spores/20 grams/acre of gram subtilis wettable powders, 250 grams per liter Azoxystrobin suspension agents.Biological prevention and control agent 1 prevention effect in the field test of rice blast of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC8200l CGMCC No.7996 reaches 84.92%.
3, the aseptic fermentation energy of Ke Liben series bacillus provided by the present invention (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 stands 121 degrees Celsius of pyroprocessing of 20 minutes, still can keep strong bacteriostatic activity.
4, three kinds of biological prevention and control agent storage periods that prepared by the active ingredient of Ke Liben series bacillus provided by the present invention (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 are long, under room temperature, preserve and still can keep strong bacteriostatic activity in 2 years, this Application and Development that is biotechnological formulation provides good foundation.
Preservation explanation
Strain name: Ke Liben series bacillus
Latin name: (Paenibacillus kribbensis)
Strain number: TRCC82001
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on August 13rd, 2013
The preservation center numbering of registering on the books: CGMCC No.7996
Embodiment
If the experimental technique using in following embodiment, without specified otherwise, is ordinary method.Material used, reagent etc., if without specified otherwise, all can obtain from commercial channels.
Strains tested:
Rice blast (Phyricularia grisea (Cooke) Sacc.): China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterial strain stock CGMCC3.3283.
Rice sheath blight disease (Thanatephorus cucumeris (Frank) Donk): Chinese industrial microbial strains preservation center, bacterial strain stock CCAM041076.
Root rotof flax (Helminthosporium sativum): China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterial strain stock CGMCC3.2881.
Lichee anthrax (Colletotrichum litchi): microbial strains preservation center, Guangdong Province, bacterial strain stock GIM1.751
Exserohilum turcicum (Exerohilum turcicum): China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterial strain stock CGMCC3.3114.
Southern corn leaf blight (Helminthosporium maydis): microbial strains preservation center, Liaoning, bacterial strain stock LICC30166.
Maize yellow spot bacterium (Curvularia lunata): microbial strains preservation center, Liaoning, bacterial strain stock LICC30317.
Corn-based Pathogen (Fusarium graminearum): China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterial strain stock ACCC36258
Verticillium dahliae (Verticillium dahliae): China Committee for Culture Collection of Microorganisms's common micro-organisms center, bacterial strain stock CGMCC3.2869.
Withered germ of water-melon (Fusarium oxysporum f.sp.niveum): Chinese Academy of Agricultural Sciences's agricultural microbial strains preservation center, bacterial strain stock ACCC36367.
Colletotrichum capsici (Colletotrichum capsici): Chinese Academy of Agricultural Sciences's agricultural microbial strains preservation center, bacterial strain stock ACCC36182.
Alternaria brassicae (Alternaria brassicae (Berk.) Sace.): microbial strains preservation center, Liaoning, bacterial strain stock LICC30382.
Purple blotch of onion bacterium (Alternaria porri (Ell.) Cifferri): microbial strains preservation center, Guangdong Province, bacterial strain stock GIM1.643.
The czapek's solution II of improvement, fills a prescription as follows:
Add quantitatively to 100% of water, if add the agar of 1.5-2% as solid medium, pH7-8,121 degrees Celsius of sterilizings 20 minutes.
Beef extract-peptone nutrient agar, fill a prescription as follows:
Add quantitatively to 100% of water, pH7.0-7.2,121 degrees Celsius of sterilizings 20 minutes.
PDA substratum, potato 200g is cut into small pieces, and adds poach rotten (boiling 20～30 minutes), with after filtered through gauze, adds glucose 20g, agar 15g, then supply water to 1000 milliliter, pH nature, 121 degrees Celsius of sterilizings 20 minutes.
Rice varieties is glad Rong You 08: Jiangxi Province seeds company, the hybrid early combination of the first extensive 9898 hybridization apolegamys of glad flourish Ax.
Separation, the evaluation of embodiment 1, Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996.
One, the separation of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996: Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996, in the soil in Shi Cong Agricultural University Of South China herbarium garden, separation obtains.Method is as follows: from Agricultural University Of South China's herbarium garden, gather pedotheque, take soil 1.0g, put into the Erlenmeyer flask that is added with 99mL sterilized water, vibration evenly, is dilution 10
-2soil supension.Then use ten times of gradient dilutions, prepare successively 10
-3, 10
-4, 10
-5, 10
-6, 10
- 7dilution soil diluent.With aseptic title device, draw each concentration soil diluent of 0.1mL, with aseptic technique, be seeded on beef extract-peptone nutrient agar flat board, coating is even, 5 repetitions of each concentration.The flat board of inoculation is after 30 degrees Celsius of constant temperature culture 2～5d, and it is dull and stereotyped that picking list bacterium colony continues to be inoculated into beef extract-peptone nutrient agar, and culture condition is the same, and culture purified obtains pure culture.Using the pathogenic fungies such as rice blast, banded sclerotial blight as target, utilize face-off method on beef extract-peptone nutrient agar, PDA culture medium flat plate, to carry out respectively the primary dcreening operation of Antagonistic Fungi.Bacterial strain primary dcreening operation to antagonistic activity carries out liquid shaking bottle fermentation, and fermented liquid filters and obtains without fermented liquid through aseptic bacterium filtering membrane.Utilize Oxford agar diffusion method further to measure the impact that aseptic fermentating liquid filtrate grows on pathogenic bacteria, utilize greenhouse pot culture inoculation test to verify without fermented liquid the greenhouse prevention effect to rice blast, banded sclerotial blight, finishing screen is selected the biocontrol strain that preventive effect is good, and the strain bacterium that screening is obtained is numbered TRCC82001.
Two, the evaluation of bacterial strain TRCC82001:
From morphology, Physiology and biochemistry and three aspects of 16S rRNA sequence homology, obtained bacterial strain TRCC82001 having been carried out to classification respectively identifies.
(1) Morphological Identification:
Bacterial strain TRCC82001 gradient dilution method coating beef extract-peptone nutrient agar is dull and stereotyped, cultivate 18-24h for 30 degrees Celsius, observe form, size, edge, surface, camber, the transparency of bacterium colony; Then carry out gramstaining and spore staining, examine under a microscope its thalli morphology.
Under microscope, bacterial strain TRCC82001 thalli morphology is shown in accompanying drawing 1; Result shows, cultivates 30 degrees Celsius of constant temperature culture 48h on beef extract-peptone nutrient agar, bacterium colony oyster white, purulence shape, subcircular, neat in edge, bacterium colony protuberance, surperficial thickness.Under microscope, the thalli morphology of TRCC82001 bacterial strain is shaft-like, is uniformly distributed, and can form gemma, and gemma is circular, and gramstaining is positive.
(2) Analysis of The Physiological And Biochemical Properties:
According to < < uncle Jie Shi Bacteria Identification handbook > >, TRCC82001 bacterial strain is carried out to Physiology and biochemistry mirror, qualification result is as following table 1.
(3) 16S rRNA sequence homology analysis:
The extraction of the total DNA of TRCC82001 bacterial strain:
1. TRCC82001 bacterial strain is after PDA activation, get in the czapek's solution II liquid nutrient medium that a ring is inoculated into 100mL improvement the fermented liquid 1.5mL of take the logarithm vegetative period (14 hours-18 hours), the centrifugal 5min of 8000r/min, collect thalline, repeat 2-3 time.
2. bacterial sediment is suspended to 400 μ LTE damping fluids, adds the lysozyme soln 20 μ L of 50mg/mL, 37 degrees Celsius, 4-24h incubation.
3. add 400 μ L " SDS " Buffer Extraction buffers, 65 degrees Celsius of incubation 45min, put upside down vibration once every 15min, are cooled to room temperature.
4. the saturated phenol of Tris that adds 400 μ L: chloroform: primary isoamyl alcohol (25:24:1), mixes latter 4 degrees Celsius, the centrifugal 10min of 12000r/min.Isopyknic chloroform extracting 1 time for supernatant liquor.
5. the ice-cold isopropanol precipitating DNA of 1 times of volume for supernatant then, adds 3mol/L KAc2-3 and drips, the washing with alcohol with 70%.DNA crude extract is dissolved in 400 μ LTE damping fluids, adds 20 μ LRNAase (1mg/mL), 37 degrees Celsius of incubation 30min.Repeating step 4., 5. respectively once.
6. get supernatant liquor in new centrifuge tube, add 2 times of volume dehydrated alcohols and 3mol/LKAc2-3 to drip-20 degrees Celsius and place 60min, take out the centrifugal 20min of 11000r/min, then use 70% washing with alcohol once.
7. dry, add in 15-20 μ L TE, 1% agarose electrophoresis detects the separation case of (2-3 μ L) DNA.DNA concentration is adjusted to 100mg/uL, the standby pcr template of doing.
The pcr amplification of TRCC82001 bacterial strain 16S rDNA:
Using the total DNA of bacterial strain that extracts as gene amplification masterplate, adopt bacterial 16 S rDNA universal primer, 27f:5`-AGAGTTTGATCCTGGCTCAG-3`, 1492r:5`-GGTTACCTTGTTACGACTT-3`, carries out PCR reaction system: DNA (70ng/ μ L) template 2 μ L; DNTP Mixture (2.5mmol/L) 2.5 μ L; 1495F (20 μ mol/L) 1.5 μ L; 27F (20 μ mol/L) 1.5 μ L; E xTaqDNA polysaccharase 0.2 μ L; 10 * ExTaq Buffer (Mg2+pluse), 5 μ L; Supply ddH2O to 50 μ L.PCR condition is: 94 degrees Celsius of denaturation 3min; Then 94 degrees Celsius of sex change 1min, 55 degrees Celsius of annealing 1min, 72 degrees Celsius of extensions 3min, totally 30 circulations; Last 72 degrees Celsius are extended 5min.With 1.0% sepharose, detect PCR product, reach the order-checking of purity Hou Song biotechnology Services Co., Ltd.
TRCC82001 bacterial strain 16S rDNA sequential analysis and phylogenetic tree construction:
PCR product reclaims purifying and after row DNA sequencing, compares homology with all procaryotic 16S rDNA sequences of having measured in GenBank, builds TRCC82001 bacterial strain phylogenetic tree (accompanying drawing 2, accompanying drawing 3).Result shows that TRCC82001 bacterial strain and AF391123 (Paenibacillus kribbensis) similarity are 98.90%, and its thalli morphology and physiological and biochemical index meet the feature of (Paenibacillus kribbensis), so can identify that TRCC82001 bacterial strain is for (Paenibacillus kribbensis).By institute of microbiology of the Chinese Academy of Sciences by (Paenibacillus kribbensis) called after Ke Liben series bacillus.TRCC82001 bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 13rd, 2013, and deposit number is: CGMCC No.7996.
The Antibacterial Activity of embodiment 2, Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 and meta-bolites thereof.
One, Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 thalline and
The thermostability of its antibacterial meta-bolites.
Get Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996,30 degrees Celsius, 6 parts of the fermented liquids that the dilution of 84 hours of 180r/min shaking culture is 100 times are wherein processed 30min for 5 parts under 40 degrees Celsius, 60 degrees Celsius, 80 degrees Celsius, 100 degrees Celsius, 120 degrees Celsius; Another part do not process, and as a control group, each processes 5 repetitions.Connect respectively that flat board is done thalline survival and to rice blast bacteriostatic test, result is as following table 2.Result shows, the antibacterial metabolite that ferments is stable under the temperature environment 100 degrees Celsius below, and to the bacteriostasis rate of rice blast, 79.71%～89.00%, and over after 100 degrees Celsius, bacteriostasis rate only has 55.15%.Further evidence, 95 degrees Celsius of 30min can kill live body in fermented liquid and not affect the fungistatic effect of meta-bolites.
Two, Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 and without the preparation of fermented liquid.
Described Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 is inoculated in the liquid czapek's solution II of improvement that loading amount is shaking flask 20%-30% volume with picking list bacterium colony or lawn after the solid czapek's solution II activation of improvement.30 degrees Celsius, 180r/min shaking culture 84 hours, through 95 degrees Celsius, after 35min sterilizing obtains obtained fermented liquid without viable bacteria fermentation liquid.
Three, the mensuration of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 Antifungal activity.
Pathogenic fungi for examination is: rice blast (Phyricularia grisea (Cooke) Sacc.), rice sheath blight disease (Thanatephorus cucumeris (Frank) Donk), root rotof flax (Drechslera SoFokiniana (Sacc.) Subram.Gain), lichee anthrax (Colletotrichum litchi), Exserohilum turcicum (Exerohilum turcicum), southern corn leaf blight (Helminthosporium maydis), Maize yellow spot bacterium (Curvularia lunata), corn-based Pathogen (Fusarium graminearum), verticillium dahliae (Verticillium dahliae), withered germ of water-melon (Fusarium oxysporum f.sp.niveum), Colletotrichum capsici (Colletotrichum capsici), Alternaria brassicae (Alternaria brassicae (Berk.) Sace.), purple blotch of onion bacterium (Alternaria porri (Ell.) Cifferri).
Get acquisition and be added in the liquid czapek's solution II of sterilized improvement in the ratio of 1:99 without viable bacteria fermentation liquid, be down flat plate, after flat board is cooling, each pathogenic bacterium bacterium cake that is 0.5cm at dull and stereotyped central authorities access diameter, 5 flat boards of every strain pathogenic bacterium repetition.Another each pathogenic bacterium are cooked 5 blanks that do not conform to without fermented liquid.After inoculation, flat board is placed in 30 degrees Celsius of constant incubators, cultivates 3 days.Observe and measure each flat-plate bacterial colony diameter, measure calculation result as following table 3.
Table 3 result shows, the antimicrobial substance that Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 produces all has bacteriostatic activity to pathogenic fungi used, and the bacteriostatic activity of the pathogenic fungies such as rice sheath blight disease, root rotof flax, rice blast, lichee anthrax is surpassed to 80%.To Exserohilum turcicum, 9 kinds of bacteriostatic activities for examination pathogenic fungi such as southern corn leaf blight are higher than 60%.
Embodiment 3, Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 without fermented liquid greenhouse and field application test.
One, testing without fermented liquid greenhouse production control of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996:
Rice varieties is glad Rong You 08, from Jiangxi Province seeds company, buy, after the seed soaking of paddy rice pot-culture method, vernalization, after radicle grows to 2cm left and right, select the consistent rice seedlings of growing way to transplant to earth culture in plastic square basin, grew to for 3 leaf phases, rhizome portion Inoculated Rice banded sclerotial blight germ, observe incidence simultaneously.The results are shown in accompanying drawing 4.
Test-results shows, the blade of blank group is paved with Rhizoctonia solani Kuhn mycelia after 3 days, and the withered flavescence of rear blade in 5 days, forms a large amount of sclerotium after 7 days; And there is not withered phenomenon through the blade of the aseptic fermentation liquor treatment of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996, it is green that blade still keeps, Rhizoctonia solani Kuhn mycelia is diffusion not, almost do not form sclerotium, show good bacteriostatic activity.Prevent and treat statistics and show, Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 has good preventive effect without 100 times of dilutions of fermented liquid to rice sheath blight disease, and preventive effect reaches more than 80%.
Two, Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 without fermented liquid control rice blast field controling test:
Test is located in rice field morning, Ji Nan village, Bai Tang street, Ji'an City Jizhou District, Jiangxi Province.Test arranges 6 processing, repeats 24Ge community, each community 67m for 4 times
2, adopting conventional spray method dispenser, every mu of liquid consumption 45kg, respectively at twice of paddy rice cut phase and full heading time dispenser.
The preventive effect of each processing of investigation in 15 days to tassel blast after medicine.Investigation method is pressed 5 samplings of diagonal lines, every some investigation 10 places, and totally 50 places, record respectively disease spike number, sick fringe morbidity severity.Drug effect is calculated as follows:
Ke Liben series bacillus (Paenibacillus kribbensis) TRCC8200l CGMCC No.7996 prevents and treats rice blast without fermented liquid, every mu of consumption 150-450 milliliter, in paddy rice cut phase, full heading time, each sprays medicine one time, after medicine, the preventive effect of 15 days three consumptions is respectively 69.99%, 75.21% and 84.92%, the preventive effect that its low dosage is 150 milliliters every mu is lower than contrast chemicals treatment B and process C, significant difference; The preventive effect that middle dosage is 225 milliliters every mu is with to contrast chemicals treatment C suitable, and difference is not remarkable, and higher than the preventive effect of contrast chemicals treatment B, significant difference; Preventive effect significant difference between each dosage processing of test medicine.Test of pesticide effectiveness typical result is shown in accompanying drawing 5.
Table 1TRCC82001 bacterial strain Physiology and biochemistry measurement result
The impact of table 2 different treatment temperature on thalline and metabolite bacteriostatic activity
The measurement result of table 3TRCC82001CGMCC No.7996 Antifungal activity
| Pathogenic fungi | Colony diameter/cm | CK／cm | Inhibiting rate/% |
| Rhizoctonia solani Kuhn | 1.50 | 6.98 | 85.67(±0.52) |
| Pyricularia oryzae | 1.60 | 7.13 | 84.57(±0.25) |
| Exserohilum turcicum | 2.24 | 7.12 | 75.56(±1.46) |
| Southern corn leaf blight | 3.12 | 7.43 | 64.74(±1.30) |
| Maize yellow spot bacterium | 2.35 | 7.52 | 75.40(±0.84) |
| Corn-based Pathogen | 3.24 | 7.12 | 61.52(±0.57) |
| Verticillium dahliae | 3.14 | 7.32 | 76.39(±0.93) |
| Cotton-wilt fusarium | 2.95 | 6.96 | 64.80(±2.12) |
| Withered germ of water-melon | 2.80 | 7.17 | 67.92(±0.82) |
| Root Rot of Wheat germ | 1.40 | 7.12 | 87.34(±0.64) |
| Glorosprium musarum Cookeet Mass | 2.76 | 7.10 | 68.17(±1.26) |
| Lichee anthrax bacteria | 1.64 | 7.22 | 84.21(±1.02) |
| Colletotrichum capsici | 1.94 | 7.04 | 79.55(±0.66) |
| Cucumber phytophthora root rot bacterium | 2.31 | 6.92 | 73.84(±0.39) |
| Alternaria brassicae | 2.01 | 7.12 | 78.79(±0.57) |
| Purple blotch of onion bacterium | 1.91 | 6.94 | 79.68(±1.39) |
The design of table 4TRCC82001CGMCC No.7996 field controling test medicament
Table 5TRCC82001CGMCC No.7996 field control rice blast test-results
* each processing data is the mean value repeating for three times.Significance of difference analytical procedure adopts " DMRT " method to carry out.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope microscopic morphology of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996.
Fig. 2 is the 16S rDNA sequence of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996.
Fig. 3 is the phylogenetic tree of Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 based on 16sRNA sequence construct.
Fig. 4 be Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 without fermented liquid to rice sheath blight disease pot experiment effect.
Fig. 5 be Ke Liben series bacillus (Paenibacillus kribbensis) TRCC82001CGMCC No.7996 without fermented liquid to rice blast field test effect.
Claims (6)
1. a strain Ke Liben series bacillus, its Latin is called Paenibacillus kribbensis, bacterial classification code name is TRCC82001, and this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 13rd, 2013, and its preserving number is: CGMCC No.7996.
2. a biological prevention and control agent, its activeconstituents is Ke Liben series bacillus claimed in claim 1, its Latin is called Paenibacillus kribbensis, bacterial classification code name is TRCC82001, its preserving number is meta-bolites and/or the Ke Liben series bacillus claimed in claim 1 of CGMCC No.7996, its Latin is called Paenibacillus kribbensis, and bacterial classification code name is TRCC82001, and preserving number is: CGMCC No.7996.
3. biological prevention and control agent according to claim 2, is characterized in that: described biological prevention and control agent is following 1)-2) in arbitrary biological prevention and control agent:
1) for preventing and treating the biological prevention and control agent of fungal diseases of plants;
2) for suppressing the biological prevention and control agent of pathogenic fungi.
4. Ke Liben series bacillus claimed in claim 1, its Latin is called Paenibacillus kribbensis, bacterial classification code name is TRCC82001, preserving number is: CGMCC No.7996, it is in preparation following 1)-2) in application in arbitrary biological prevention and control agent:
1) for preventing and treating the biological prevention and control agent of fungal diseases of plants;
2) for suppressing the biological prevention and control agent of pathogenic fungi.
5. the application described in biological prevention and control agent according to claim 3 or right 4, it is characterized in that: described pathogenic fungi be following at least one: rice sheath blight disease, its Latin is called Thanatephorus cucumeris (Frank) Donk, root rotof flax, its Latin is called Drechslera SoFokiniana (Sacc.) Subram.Gain, rice blast, its Latin is called Phyricularia grisea (Cooke) Sacc., lichee anthrax, its Latin is called Colletotrichum litchi, Exserohilum turcicum, its Latin is called Exerohilum turcicum, southern corn leaf blight, its Latin is called Helminthosporium maydis, Maize yellow spot bacterium, its Latin is called Curvularia lunata, corn-based Pathogen, its Latin is called Fusarium graminearum, verticillium dahliae, its Latin is called Verticillium dahliae, withered germ of water-melon, its Latin is called Fusarium oxysporum f.sp.niveum, Colletotrichum capsici, its Latin is called Colletotrichum capsici, Alternaria brassicae, its Latin is called Alternaria brassicae (Berk.) Sace., purple blotch of onion bacterium, its Latin is called Alternaria porri (Ell.) Cifferri.
6. the preparation method of biotechnological formulation described in claim 2,3 or 5, comprise the steps: described Ke Liben series bacillus, its Latin is called Paenibacillus kribbensis, bacterial classification code name is TRCC82001, preserving number is: the meta-bolites of CGMCC No.7996, or/and described Ke Liben series bacillus, bacterial classification code name is TRCC82001, preserving number is: CGMCC No.7996, as activeconstituents, obtains described biotechnological formulation.
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