CN103045515B - Bacteria agent of a kind of Methylotrophic genus bacillus and its preparation method and application - Google Patents
Bacteria agent of a kind of Methylotrophic genus bacillus and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to the Methylotrophic genus bacillus that a kind of new preservation registration number is CGMCCNo.6793, this bacterial strain is separated from tobacco rhizosphere soil and obtains, and is obtained by the separation of bacterial strain, purifying and screening step.The present invention further provides the fermentation process of described Methylotrophic genus bacillus, prepared the tunning of gained by the method, and based on the bacteria agent and preparation method thereof of this tunning, also provide described bacteria agent to prevent and treat the purposes of plurality of plant diseases further.This bacteria agent Environmental compatibility is good, effective spore count alive reaches 5,000,000,000/gram more than, there is very high biological activity, particularly to by tobacco bacterial wilt, there is good preventive effect, its to the preventive effect of tobacco bacterial wilt, soft rot of cabbage, bacterial blight of rice, bacterial leaf streak of rice and cotton angular leaf spot up to more than 80%.
Description
Technical field
The invention belongs to biocontrol of plant disease field, beneficial microorganism is particularly utilized to carry out biological control to Plant diseases, be specifically related to Methylotrophic genus bacillus (Bacillusmethylotrophicus), and utilize this bacterial strain to prepare the application of bacteria agent and described bacteria agent controlling plant diseases.
Background technology
The popular of Plant diseases not only causes crop failure, causes heavy economic losses to the mankind, even bringing on a disaster property consequence.The harm of Plant diseases also can cause quality of agricultural product inferior, can't bear to use or processing, even causes person poultry poisoning.In the control of Plant diseases, chemical pesticide because have that cost is low, first-selection that instant effect, the advantage such as time saving and energy saving were once once becoming people, but, the general non-degradable of chemical pesticide, along with a large amount of uses of chemical pesticide, the events such as environmental pollution, ecological damage, food contamination are caused.This makes research and development utilize the important subject of the biological pesticide person that becomes plant protection service.Biological pesticide refers to and utilizes living organisms or its meta-bolites to carry out the preparation killed or suppress for agricultural pest; there is the features such as safe, efficient, pollution-free; matching with the requirement of preserving the ecological environment and socio-intangible asset develops, is the inexorable trend of pesticide in future development.
Microbial pesticide is the class be most widely used in biological pesticide, is also the important materials of biological control.The main component of microbial pesticide is living microorganisms and secondary metabolite thereof, and because microbe species is various, its activity is also very extensive, comprises desinsection, sterilization, weeding and plant growth regulation etc.Occurring in nature is used for biocontrol bacteria, mainly contains bacillus, Rhodopseudomonas.Particularly in recent years, utilize microbial control plant fungal disease due to environmentally safe, pathogenic bacteria can be slowed down and develop immunity to drugs, become the main stream approach of current controlling plant diseases.
Methylotrophic genus bacillus is a kind of addicted to warm, aerobic, sporiferous rod-shaped bacterium, its physiological characteristic is various, this bacterium is widespread in nature, there is unique one carbon metabolism approach, by inexhaustible for occurring in nature one-carbon compound as methyl alcohol, formaldehyde etc. change nutritive substance needed for thalline self into, can produce again the metabolic by-prods L. Serine of multiple beneficial, vitamin B12, poly-p hydroxybutyric acid salt etc. simultaneously.Nontoxic to people and animals, free from environmental pollution, possess vitality strong, breeding can produce multiple antibiotic and enzyme fast, has anti-microbial activity and extremely strong high temperature resistance, anti-strong acid and strong base ability.Methylotrophic genus bacillus not only extensively can exist in the external environments such as soil, plant rhizosphere body surface, and is common endogenetic bacteria in plant materials, especially in root, the stem inside of plant.Methylotrophic genus bacillus passes through successfully surely to grow in the plant rhizosphere such as tobacco, paddy rice, body surface or body, change flora environment and kind around it, with the site around pathogenic bacteria competitive plant, and secrete multiple antimicrobial substance to suppress growth of pathogenic bacteria, inducing plant system of defense resists pathogenic bacteria invasion simultaneously, thus reach the object of improvement soil and biological and ecological methods to prevent plant disease, pests, and erosion, there is diseases prevention and growth-promoting functions.
Tobacco bacterial wilt is that one is caused by green grass or young crops withered Raul Salmonella (Ralatoniasolanacearum), based on soil-borne, destructive bacterial disease.This disease is one of Major Diseases of tropical and subtropical region tobacco, China Yangtze valley and on the south cigarette district generally occur, wherein with Guangdong, Fujian, Hunan, Sichuan and Tobacco Planting Areas in Guizhou harm heavier.At present, mainly adopt chemical pesticide control aborning, but cannot effectively prevent this disease from occurring, and for a long time use chemical pesticide in a large number and both easily made germ develop immunity to drugs, easily cause serious environmental pollution again.
Bacterial blight of rice is by Gram-negative bacteria Xanthomonas campestris rice varieties (Xanthomonasoryzaepv.oryzae, Xoo) cause, it is one of large disease of paddy rice three, all there is generation paddy fields nearly all in the world, all there is generation in various degree in China except the north in Xinjiang, Tibet and northeast, on the south the Changjiang river and Yangtze-Huai Plain long-grained nonglutinous rice producing region is Chang Faqu, have a strong impact on rice yield, mainly rely on chemical pesticide control at present, but life-time service, germ is easily developed immunity to drugs, and serious environment pollution.
Summary of the invention
The object of the present invention is to provide a kind of new Methylotrophic genus bacillus.
Another object of the present invention is to the fermentation process that described Methylotrophic genus bacillus is provided, and prepared the tunning of gained by the method.
Another object of the present invention is to bacteria agent that Methylotrophic genus bacillus is provided and preparation method thereof.
Another object of the present invention is to provide the purposes of the bacteria agent controlling plant diseases utilizing described Methylotrophic genus bacillus.
The object of the invention is to be achieved through the following technical solutions:
A kind of Methylotrophic genus bacillus, it belongs to bacillus, its called after Bacillusmethylotrophicus; Preservation registration number is CGMCCNo.6793, and preservation mechanism is: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Depositary institution address: Datun Road, Chaoyang District, Beijing City; The preservation time is on November 07th, 2012.
Above-mentioned Methylotrophic genus bacillus of the present invention is separated from tobacco rhizosphere soil and obtains, and is obtained by the separation of bacterial strain, purifying and screening step.
Identification of strains: adopt morphological specificity, bio-chemical characteristics, molecular biology identification method, and 16SrDNA homologous sequence compare of analysis finally determines that the bacterial strain of above-mentioned deposit number is Methylotrophic genus bacillus (Bacillusmethylotrophilus).
The fermentation process of Methylotrophic genus bacillus of the present invention, described method comprises the steps:
Step one: actication of culture: be that the Methylotrophic Bacillus of CGMCCNo.6793 is inoculated into above LB solid medium by preservation registration number, in 30 DEG C of activation culture 48h, described LB solid culture based formulas is yeast extract 3-5g, peptone 8-10g, NaCl5g, agar 15 ~ 20g, water 1000ml, pH7.4 ~ 7.6, continuously 3 generations of switching, after guaranteeing bacterial strain purity, inoculate in seed liquor and use;
Step 2: the preparation of seed liquor: from picking strain inoculation LB solid medium in the triangular flask that LB liquid nutrient medium is housed, shaking culture in constant-temperature shaking incubator, namely seed liquor is obtained, described LB liquid culture based formulas is: yeast extract 3-5g, peptone 8-10g, NaCl5g, water 1000ml, pH7.4 ~ 7.6; Culture condition: 30 DEG C, 48h;
Step 3: fermentation culture: seed liquor is successively carried out seeding tank fermentation culture and large-scale industry fermentation culture, obtains fermented liquid; Wherein seeding tank fermentation culture and large-scale industry fermentation culture step are distinguished as follows:
Seeding tank fermentation culture: FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 DEG C, 42h, inoculum size: 6%, air flow: 2.5L/min, mixing speed: 200r/min;
Large-scale industry fermentation culture: FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 DEG C, 60-70h, inoculum size: 6%, air flow: 2.0L/min, mixing speed: 220r/min;
Wherein in seeding tank fermentation culture and large-scale industry fermentation culture, the inoculum size of seed liquor all accounts for the 6-8% of liquid nutrient medium total amount;
Described FJ liquid culture based formulas is: soybean cake powder 1.5%, sucrose 1%, KH
2pO
40.05%, MgSO
40.02%, NaCl0.1%, all the other are water, and listed per-cent is weight percentage.
Preferably, if having foam to produce in the fermentation culture process of step 3, enemy's froth breaking can be dripped.
Further, the invention provides the tunning being prepared gained by the fermentation process of the Methylotrophic genus bacillus described in technique scheme.
Further, according to above-mentioned tunning, the present invention also provides the bacteria agent of a kind of Methylotrophic genus bacillus, and described bacteria agent obtains as follows:
Step one: filter and enriched product: prepare the tunning of gained with after careless acid for adjusting pH to 6.5-7.0 by adopting the fermentation process of above-mentioned Methylotrophic genus bacillus, by the inorganic ceramic ultra-filtering film filtering separation of tunning by molecular weight cut-off 40000-100000k, service temperature is 35-37 DEG C, membrane flux is 50-60LMH, the solution being trapped within the liquid feeding side of film is gemma solution, gemma solution cycles of concentration is 6-7 times, obtained through concentrated gemma solution, described concentration method is the molecular screen membrane of use 500,000 Da, peristaltic pump flow velocity is 4 grades, flux is 420ml/min, liquid outlet pressure is 0.08MPa,
Step 2: spraying dry: to the filler of the interpolation 1.0% in concentrated gemma solution of step one, subsequently gemma solution is made powder through spraying, spray-dired inlet temperature is 170-190 DEG C, temperature out is 65-85 DEG C, input speed 60mL/min, sprinkler pressure 0.10MPa, namely obtains the bacteria agent of Methylotrophic genus bacillus of the present invention; Described filler is white carbon black, micronized talc powder, light calcium carbonate or 2500 order ultrafine heavy calciums.
Further, the invention provides the purposes of the bacteria agent controlling plant diseases of above-mentioned Methylotrophic genus bacillus; Especially for the disease in control tobacco, Chinese cabbage, paddy rice, cotton crop;
Preferably, described tobacco diseases is tobacco bacterial wilt, and this disease is caused by tobacco ralstonia solanacearum (Ralstoniasolanacearum); Described Chinese cabbage disease is Chinese cabbage canker, and this disease is caused by soft rotten bacterium (Erwiniacarotovora); Described rice disease is bacterial blight of rice and bacterial leaf streak of rice, and described disease is all caused by rice Xanthomonas (Xanthomonasoryzaepv.Oryzae); Described cotton disease is cotton angular leaf spot, and described disease is that angular leaf spot of cotton Xanthomonas campestris (Xanthomonascampestrispv.malvacearum (E.F.Smith) Dowson) causes.
Except as otherwise noted, " % " in present specification is all weight percentage.
The present invention has following beneficial effect:
1, the bacteria agent obtained by Methylotrophic genus bacillus of the present invention, its Environmental compatibility is good, there is very high biological activity, particularly to by tobacco bacterial wilt, there is good preventive effect, prove by experiment, the bacteria agent of this Methylotrophic genus bacillus to the preventive effect of tobacco bacterial wilt, soft rot of cabbage, bacterial blight of rice, bacterial leaf streak of rice and cotton angular leaf spot up to more than 80%.
2, the bacteria agent obtained by Methylotrophic genus bacillus of the present invention is the novel high-activity high-concentration biological microbial inoculum adopting novel process fermentation, the research and development of advanced ultrafiltration and concentration isolation technique, spore count of effectively living reaches 5,000,000,000/gram more than.
Accompanying drawing illustrates:
Fig. 1 is the 16SrDNA homologous sequence compare of analysis dendrogram of Methylotrophic genus bacillus of the present invention.
Embodiment:
Being convenient to for making technical scheme of the present invention understand, being further described below in conjunction with concrete test example.
embodiment 1:the isolation and screening of the application's Methylotrophic genus bacillus (Bacillusmethylotrophilus):
(1), the separation of genus bacillus:
Take tobacco rhizosphere soil 10g, pour in the Erlenmeyer flask that 90ml aqua sterilisa is housed, 200r/min vibrates 20min, 80 DEG C of water-bath 30min, 10 doubling dilutions, and coating is separated.
(2), the purifying of genus bacillus: drop down onto on LB solid medium according to the single bacterium of the timely picking of the feature of bacterium, adopt the method purifying that line is separated; The bacterium of purifying is through gramstaining and spore staining, and display thalline is shaft-like, product gemma, G
+isolate be genus bacillus; Described LB solid culture based formulas is: yeast extract 5g, peptone 8g, NaCl5g, agar 15g, water 1000ml, pH7.4.
(3), the screening of antagonistic Bacillus:
Select the activated tobacco ralstonia solanacearum of tool as indicator with TTC substratum, tobacco ralstonia solanacearum is made into 2 × 10
8the bacteria suspension of cfu/ml concentration, is transferred on BPA substratum, makes ralstonia solanacearum containing bacterium culture medium.After drying up, connect the Bacillus strain of above-mentioned separation and purification in dull and stereotyped central authorities, cultivate 2-3d for 30 DEG C, measure the size of antibacterial circle diameter and antibacterial band (outer rim from the edge of active bacteria bacterium colony to inhibition zone).The formula of described TTC substratum is: Tryptones 17.0g, soya peptone 3.0g, sodium-chlor 2.5g, glucose 6.0g, sodium thioglycollate 0.5g, CYSTINE 0.25g S-WAT 0.1g agar 16.0g; The formula of described BPA substratum is: beef extract 3-5g, peptone 5-10g, sucrose 10-12g, yeast extract 1-2g, and agar 17-20g, adds water to 1L.
Separation screening obtains the Methylotrophic genus bacillus of the application, and laboratory is numbered LW-6.
embodiment 2:the qualification of Methylotrophic genus bacillus (Bacillusmethylotrophilus):
To the qualification process of the Methylotrophic genus bacillus of embodiment 1 gained be:
(1), morphological specificity: LW-6 bacterium colony presents creamy white on NA solid medium, bacterium colony surface irregularity, and edge presents spination, it is shaft-like for showing LW-6 through Electronic Speculum, Gram-positive, peritrichous, individual cells size is 0.5-0.6um × 2.0-2.5um.The formula of described NA solid medium is: beef extract 2-3g, peptone 5g, glucose 2.5-3g, and agar 16-18g, adds water to 1000ml.
(2), bio-chemical characteristics: by the biophysical and biochemical tests (as shown in table 1) to Methylotrophic genus bacillus and in conjunction with the morphological specificity of this bacterial strain, contrast " uncle Jie Shi bacterium handbook " (Buchanan & Gibbons, 1984) and " common bacteria system identification handbook " (eastern elegant pearl and Cai Miaoying, 2001) retrieve, identify that this bacterial strain is bacillus.
The physiological and biochemical property of table 1 the application Methylotrophic genus bacillus
Note: "+" represents positive reaction, " " represents negative reaction.
Bacterial strain physiological and biochemical property shows: the bacterial strain LW-6 of the application can make gelatine liquefication; Energy hydrolyzed starch and reduction nitrate; Glucose, pectinose, wood sugar, seminose, catalase, lipase, urase and oxydase are all positive; V-P test is for positive; Do not utilize Citrate trianion.Maximum growth temperature is 60 DEG C, and minimum growth temperature is 10 DEG C, and optimum growth temperature is 30 DEG C; Most high ph-values is 11.0, and minimum pH value is 3.0, and optimum pH is 7.0; There is salt tolerance, can grow in the NaCl culture medium of 15%.
(3), molecular biology identification: with LW-6 genomic dna for template, carry out pcr amplification with primer 7f and 1540r, the 16SrDNA nucleotide sequence length recording this bacterial strain is 1454bp, GenBank accession number is JX220980.By the structure (Fig. 1) of the 16SrDNA sequence alignment analysis and phylogenetic tree with type strain in NCBI, learn LW-6 bacterial strain and Methylotrophic bacillus methylotrophicusCBMB205 (T) (GenBank accession number is EU194897) homology recently, similarity is 100%.
The form of comprehensive LW-6 bacterium, cultural characteristic, physiological and biochemical test and 16SrDNA homologous sequence compare of analysis, this bacterial strain of preliminary evaluation is Methylotrophic genus bacillus
embodiment 3:the cultivation of Methylotrophic genus bacillus (Bacillusmethylotrophilus)
(1), bacterial strain activation:
Substratum: LB solid medium, described LB solid culture based formulas is: yeast extract 5g, peptone 8g, NaCl5g, agar 20g, water 1000ml, pH7.4;
Culture condition: 30 DEG C, 48h;
In continuous switching 3 generation, after guaranteeing bacterial strain purity, inoculate in fermented liquid and use.
(2), the preparation (shake flask fermentation or the fermentation of eggplant bottle) of seed liquor:
Substratum: LB liquid nutrient medium, described LB liquid culture based formulas is: yeast extract 5g, peptone 8, NaCl5g, water 1000ml, pH7.4 (flask volume 250ml, liquid amount 50ml);
Culture condition: 30 DEG C, 48h;
Inoculum size: 6-8%.
(3), seeding tank fermentation:
Substratum: FJ liquid nutrient medium, described FJ liquid culture based formulas is: soybean cake powder 1.5%, sucrose 1%, KH
2pO
40.05%, MgSO
40.02%, NaCl0.1%, all the other are water, and listed per-cent is weight percentage.
Coefficient: 0.7;
Culture condition: 35-37 DEG C, 48h;
Inoculum size: 6%;
Air flow: 2.5L/min;
Mixing speed: 200r/min;
(4), industrial fermentation:
Liquid nutrient medium: FJ liquid nutrient medium (20-36T), described FJ liquid culture based formulas is: soybean cake powder 1.5%, sucrose 1%, KH
2pO
40.05%, MgSO
40.02%, NaCl0.1%, all the other are water, and listed per-cent is massfraction;
Coefficient: 0.7;
Culture condition: 35-37 DEG C, 48h;
Inoculum size: 6%;
Air flow: 2.0L/min;
Mixing speed: 220r/min;
embodiment 4:the preparation of the bacteria agent of the application's Methylotrophic genus bacillus
(1), fermented liquid concentrates: after being adjusted to 6.5-7.0 by adopting the obtained tunning oxalic acid of embodiment 3, by the inorganic ceramic ultra-filtering film filtering separation of tunning by molecular weight cut-off 40000-100000k, service temperature is 35-37 DEG C, membrane flux is 50-60LMH, the solution being trapped within the liquid feeding side of film is gemma solution, gemma solution cycles of concentration is 6 times, obtained through concentrated gemma solution, described concentration method is the molecular screen membrane of use 500,000 Da, peristaltic pump flow velocity is 4 grades, flux is 420ml/min, and liquid outlet pressure is 0.08MPa.
(2), spraying dry:
In the gemma solution that above-mentioned warp concentrates, add the white carbon black of 1.0%, gemma solution is made powder through spraying, namely obtains the bacteria agent of Methylotrophic genus bacillus of the present invention.
embodiment 5:the application's Methylotrophic genus bacillus is to antagonistic experiment in the ware of tobacco ralstonia solanacearum, rice leaf spot bacteria, Erwinia carotorora:
Pathogenic bacteria is tried for supplying respectively with tobacco ralstonia solanacearum Ralstoniasolanacearum, rice leaf spot bacteria Xanthomonasoryzaepv.Oryzae and Erwinia carotorora Erwiniacarotovora, select the activated indicator of tool with LB substratum, germ is made into 2 × 10
8the bacteria suspension of cfu/ml concentration, is transferred on LB substratum, makes containing bacterium culture medium.After drying up, connect the Bacillus strain of above-mentioned separation and purification in dull and stereotyped central authorities, cultivate 2-3d for 30 DEG C, measure the size of antibacterial circle diameter and antibacterial band (outer rim from the edge of active bacteria bacterium colony to inhibition zone).The antibacterial situation of Methylotrophic genus bacillus to Different Kinds of Pathogens bacterium of the application is as shown in table 2
The Methylotrophic genus bacillus of table 2 the application is to the antibacterial situation of various pathogenic bacteria
| Pathogenic bacteria | Antibacterial circle diameter/mm |
| Tobacco ralstonia solanacearum | 21.05 |
| Erwinia carotorora | 15.05 |
| Rice leaf spot bacteria | 18.90 |
Use inhibition zone size measurement Methylotrophic genus bacillus to the suppression situation of tobacco ralstonia solanacearum Ralstoniasolanacearum, rice leaf spot bacteria Xanthomonasoryzaepv.Oryzae and Erwinia carotorora Erwiniacarotovora by table 2, find that this bacterial strain suppresses situation remarkable to these pathogenic bacterias, especially to tobacco bacterial wilt and bacterial blight of rice inhibition more outstanding.
embodiment 6:the determination of activity of the biocontrol fungicide of the application's Methylotrophic genus bacillus
For examination pathogenic bacteria: tobacco ralstonia solanacearum Ralstoniasolanacearum, TTC substratum is cultivated, makes 1 × 10
6cfu/ml bacteria suspension, stand-by.
For examination chemical agent: 72% agricultural Vetstrep wettable powder.
Method: test soil is mixture Nutrition Soil V (soil): V (Nutrition Soil)=1: l of sterilizing.Sterilized soil is cultivated cigarette seedling to the 4 ~ 5 leaf phase, seedling is taken out.
Process 1: blank, root 30min is soaked in cigarette shoot root portion in clear water.
Process 2: chemical agent contrasts, and root 30min soaks in 72% agricultural Vetstrep wettable powder, 2000 times of liquid in cigarette shoot root portion, after inoculation 1d, root-pouring method inoculation tobacco Ralstonia solanacearum, inoculum size is 5ml/ strain.
Process 3: soak root 30min in biocontrol fungicide 200 times of diluents of the Methylotrophic genus bacillus of embodiment 4, after inoculation 1d, root-pouring method inoculation tobacco Ralstonia solanacearum, inoculum size is 5ml/ strain.
Often 3 repetitions are established in process, often repeat 10 strain cigarette seedlings, and process is transplanted in the seedling alms bowl that sterilized soil is housed, under process is positioned over 28 ~ 36 DEG C, greenhouse condition, and 15 days " Invest, Then Investigate " tobacco plant incidences, statistics sickness rate and disease index.
Grade scale:
0 grade: complete stool is anosis
1 grade: stem is even to be had chlorisis scab or have minority blade wilting in streak side
3 grades: stem's black scab.But do not reach top, or sick side 5% blade is wilting
5 grades: stem's black scab reaches plant top.Or sick side 2/3 blade is wilting
7 grades: diseased plant stem is withered
The biocontrol fungicide of the application's Methylotrophic genus bacillus to the greenhouse preventive effect result of study of tobacco bacterial wilt as table 3:
The biocontrol fungicide of table 3 the application Methylotrophic genus bacillus is to the greenhouse prevention effect of tobacco bacterial wilt
embodiment 7:the biocontrol fungicide of the application's Methylotrophic genus bacillus is tested bacterial blight of rice preventive effect:
For examination pathogenic bacteria: rice leaf spot bacteria (Xanthomonasoryzaepv.Oryzae), LB substratum is cultivated, makes 1 × 10
6cfu/ml bacteria suspension, stand-by.
For examination chemical agent: kasugamycin aqua.
Method: test soil is mixture Nutrition Soil V (soil): V (Nutrition Soil)=1: 1 of sterilizing.Sterilized soil is cultivated rice seedling to the 4 ~ 5 leaf phase, seedling is taken out.
Process 1: blank, rice seedlings root soaks root 30min in clear water.
Process 2: chemical agent contrasts, and rice seedlings root soaks root 30min in kasugamycin 600 times of liquid, after inoculation 1d, root-pouring method Inoculated Rice bacterial leaf spot pathogenic bacteria bacteria suspension, inoculum size is 5ml/ strain.
Process 3: soak root 30min in biocontrol fungicide 200 times of diluents of the Methylotrophic genus bacillus of embodiment 4, after inoculation 1d, root-pouring method Inoculated Rice bacterial leaf spot bacterium, inoculum size is 5ml/ strain.
Often 3 repetitions are established in process, often repeat 10 strain rice seedlings, and process is transplanted in the seedling alms bowl that sterilized soil is housed, and process is positioned over 28 ~ 36 DEG C, greenhouse
Under condition, 15 days " Invest, Then Investigate " tobacco plant incidences, statistics sickness rate and disease index.
Grade scale:
0 grade: anosis;
1 grade: lesion area is less than 1/5 of leaf area;
2 grades: lesion area is less than 1/3 of leaf area;
3 grades: lesion area is less than 1/2 of leaf area;
4 grades: lesion area is less than 3/5 of leaf area;
5 grades: lesion area is more than 3/5 of leaf area
The biocontrol fungicide of the application's Methylotrophic genus bacillus to the greenhouse preventive effect result of study of bacterial blight of rice as table 4:
The biocontrol fungicide of table 4 the application Methylotrophic genus bacillus is to the greenhouse prevention effect of bacterial blight of rice
embodiment 8:the biocontrol fungicide of the application's Methylotrophic genus bacillus is to the former bacterium preventive effect experiment of soft rot of cabbage:
For examination pathogenic bacteria: Erwinia carotorora (Erwiniacarotovora), LB substratum is cultivated, makes 1 × 10
6cfu/ml bacteria suspension, stand-by.
For examination chemical agent: agricultural streptomycin wettable powder.
Method: test soil is mixture Nutrition Soil V (soil): V (Nutrition Soil)=1: 1 of sterilizing.Sterilized soil is cultivated Chinese cabbage to the 4 ~ 5 leaf phase, shoot is taken out.
Process 1: blank, Chinese cabbage root soaks root 30min in clear water.
Process 2: chemical agent contrasts, and Chinese cabbage root soaks root 30min in agricultural streptomycin 4000 times of liquid, after inoculation 1d, the former bacterium bacteria suspension of root-pouring method inoculation soft rot of cabbage, inoculum size is 5ml/ strain.
Process 3: soak root 30min in biocontrol fungicide 200 times of diluents of the Methylotrophic genus bacillus of embodiment 4, after inoculation 1d, the soft rotten bacterium of root-pouring method inoculation Chinese cabbage, inoculum size is 5ml/ strain.
Often 3 repetitions are established in process, often repeat 10 strain Chinese cabbages, and process is transplanted in the seedling alms bowl that sterilized soil is housed, under process is positioned over 28 ~ 36 DEG C, greenhouse condition, and 15 days " Invest, Then Investigate " tobacco plant incidences, statistics sickness rate and prevention effect.
The greenhouse preventive effect result of study of biocontrol fungicide to soft rot of cabbage of the application's Methylotrophic genus bacillus is as shown in table 5:
The biocontrol fungicide of table 5 the application Methylotrophic genus bacillus is to the greenhouse prevention effect of soft rot of cabbage
embodiment 9:the biocontrol fungicide of the application's Methylotrophic genus bacillus prevents and treats the field plot experiment of tobacco bacterial wilt, bacterial blight of rice, soft rot of cabbage:
For the biocontrol fungicide of checking the application Methylotrophic genus bacillus is to the preventive effect of tobacco bacterial wilt, bacterial blight of rice, soft rot of cabbage, we carry out land for growing field crops extend trial in June, 2012 to August, and experiment condition is as follows:
(1), tobacco: practice ground is selected in Baoji, testing ground area 900m
2(1.36 mu), soil property is light loam, and organic content is 1.01%, pH value 7.7, all experimental plots cultivation condition and control measures consistent.Biocontrol fungicide 200 times of liquid of Methylotrophic genus bacillus, 72% agricultural Vetstrep wettable powder and totally 3 process of not dispenser clear water are established in this experiment, each process 4 repetition, Gong12Ge community, each community random alignment, each community 40m
2minizone and experimental field surrounding arrange the wide isolation strip of 0.6m.In April, 2010 is sowed, average spacing in the rows 8cm, and after planting fill with once respectively on May 5th, 2010,15 days and 25 days root, mu liquid 500kg, medicinal sprayer tool is workers and peasants---and 16 type atomizers, take off sprinkling irrigation by shower nozzle.
Meteorological conditions
Dispenser (May 5) same day first time is partly cloudy, and wind-force 3 grades, the highest temperature 25 DEG C, the lowest temperature 15 DEG C, relative humidity is 80%.Second time was cloudy for dispenser (May 15) same day, and wind-force 3 grades, the highest temperature 23 DEG C, the lowest temperature 15 DEG C, relative humidity is 80%.Dispenser (May 25) same day first time is partly cloudy, and wind-force 5 grades, the highest temperature 25 DEG C, the lowest temperature 14 DEG C, relative humidity is 80%.
Within after last dispenser 7 days, investigate, every community adopts 4 point samplings, looks into 20 strains at often, records total strain number and diseased plant number at different levels.
Stage division:
0 grade: complete stool is anosis
1 grade: stem is even to be had chlorisis scab or have minority blade wilting in streak side
3 grades: stem's black scab.But do not reach top, or sick side 5% blade is wilting
5 grades: stem's black scab reaches plant top.Or sick side 2/3 blade is wilting
7 grades: diseased plant stem is withered
Investigation result is as shown in table 6 below, and wherein, all sampling spots all do not have poisoning to occur, and show through field plot trial, and the average preventive effect of biocontrol fungicide to tobacco bacterial wilt of the Methylotrophic genus bacillus of the application reaches.
The biocontrol fungicide of table 6 the application Methylotrophic genus bacillus prevents and treats situation to tobacco bacterial wilt field test
(2), paddy rice: practice ground is selected in Yang County, Shaanxi, practice ground 800m
2(1.21 mu), soil property is light loam, and organic content is 1.2%, pH value 7.5, all experimental plots cultivation condition and control measures consistent; Biocontrol fungicide 200 times of liquid of the application's Methylotrophic genus bacillus, kasugamycin 600 times of liquid and totally 3 process of not dispenser clear water are established in this experiment, each process 4 repetition, Gong12Ge community, each community random alignment, each community 60m
2minizone and experimental field surrounding arrange the wide isolation strip of 0.5m.In March, 2010 is sowed, average spacing in the rows 7cm, respectively on April 15th, 2010, May 5 and 25 days, root was filled with once respectively after insemination and emergence, and mu liquid 500kg, medicinal sprayer tool is workers and peasants---16 type atomizers, take off sprinkling irrigation by shower nozzle.
Meteorological conditions
Dispenser (April 15) same day first time is partly cloudy, and wind-force 3 grades, the highest temperature 14 DEG C, the lowest temperature 1 DEG C, relative humidity is 30%.Second time was cloudy for dispenser (May 15) same day, and wind-force 3 grades, the highest temperature 19 DEG C, the lowest temperature 3 DEG C, relative humidity is 30%.Dispenser (May 25) same day first time is partly cloudy, and wind-force 5 grades, the highest temperature 23 DEG C, the lowest temperature 10 DEG C, relative humidity is 20%.
Within after last dispenser 7 days, investigate, every community adopts 4 point samplings, looks into 20 strains at often, records total strain number and diseased plant number at different levels.
Stage division:
0 grade: anosis;
1 grade: lesion area is less than 1/5 of leaf area;
2 grades: lesion area is less than 1/3 of leaf area;
3 grades: lesion area is less than 1/2 of leaf area;
4 grades: lesion area is less than 3/5 of leaf area;
5 grades: lesion area is more than 3/5 of leaf area
Investigation result is as shown in table 7 below, wherein, all sampling spots all do not have poisoning to occur, and show through field plot trial, the average preventive effect of biocontrol fungicide to bacterial blight of rice of the application's Methylotrophic genus bacillus reaches 70%, higher than the preventive effect of kasugamycin aqua.
The biocontrol fungicide of table 7 the application Methylotrophic genus bacillus prevents and treats situation to field trials of rice bacterial blight
(3), Chinese cabbage: practice ground is selected in Yangling Shaanxi, practice ground 800m
2(1.21 mu), soil property is light loam, and organic content is 1.07%, pH value 7.2, all experimental plots cultivation condition and control measures consistent.Biocontrol fungicide 200 times of liquid of the application's Methylotrophic genus bacillus, agricultural streptomycin 4000 times of liquid and totally 3 process of not dispenser clear water are established in this experiment, each process 4 repetition, Gong12Ge community, each community random alignment, each community 40m
2minizone and experimental field surrounding arrange the wide isolation strip of 0.6m.In July, 2010 is sowed, average spacing in the rows 8cm, and after planting fill with once respectively on August 5th, 2010,15 days and 25 days root, mu liquid 500kg, medicinal sprayer tool is workers and peasants---and 16 type atomizers, take off sprinkling irrigation by shower nozzle.
Meteorological conditions
Dispenser (August 5) same day first time is partly cloudy, and wind-force 3 grades, the highest temperature 25 DEG C, the lowest temperature 15 DEG C, relative humidity is 80%.Second time was cloudy for dispenser (August 15) same day, and wind-force 3 grades, the highest temperature 23 DEG C, the lowest temperature 15 DEG C, relative humidity is 80%.Dispenser (August 25) same day first time is partly cloudy, and wind-force 5 grades, the highest temperature 25 DEG C, the lowest temperature 14 DEG C, relative humidity is 80%.
Last dispenser was investigated after 10 days, and every community adopts 4 point samplings, looked into 20 strains at often, recorded total strain number and diseased plant number at different levels.
Stage division:
0 grade: complete stool is anosis
1 grade: stem is even to be had chlorisis scab or have minority blade wilting in streak side
3 grades: stem's black scab.But do not reach top, or sick side 5% blade is wilting
5 grades: stem's black scab reaches plant top.Or sick side 2/3 blade is wilting
7 grades: diseased plant stem is withered
Investigation result is as shown in table 8 below, wherein, all sampling spots all do not have poisoning to occur, and show through field plot trial, the average preventive effect of biocontrol fungicide to soft rot of cabbage of the application's Methylotrophic genus bacillus reaches 74.1%, higher than agricultural streptomycin 4000 times of liquid.
The field test of table 8 Methylotrophic genus bacillus LW-6 microbial inoculum to soft rot of cabbage prevents and treats situation
Bacteria agent of a kind of Methylotrophic genus bacillus of the present invention and its preparation method and application is described by concrete example, those skilled in the art can use for reference content of the present invention, the links such as appropriate change raw material, processing condition realize other object corresponding, its relevant change does not all depart from content of the present invention, all similar replacements and change will become apparent to those skilled in the art that and be all deemed to be included within scope of the present invention.
Claims (1)
1. a purposes for the bacteria agent control tobacco bacterial wilt of Methylotrophic genus bacillus, it is characterized in that: described Methylotrophic genus bacillus, it belongs to bacillus, its called after
bacillusmethylotrophicus; Preservation registration number is CGMCCNo.6793, and preservation mechanism is: China Committee for Culture Collection of Microorganisms's common micro-organisms center; The preservation time is on November 07th, 2012; Described bacterial strain is separated from tobacco rhizosphere soil and obtains;
Described bacteria agent obtains as follows:
Step one: actication of culture: above above-mentioned strain inoculation to LB solid medium, in 30 DEG C of activation culture 48h, described LB solid culture based formulas is yeast extract 3-5g, peptone 8-10g, NaCl5g, agar 15 ~ 20g, water 1000ml, pH7.4 ~ 7.6, continuously 3 generations of switching, after guaranteeing bacterial strain purity, inoculate in seed liquor and use;
Step 2: the preparation of seed liquor: from picking strain inoculation LB solid medium in the triangular flask that LB liquid nutrient medium is housed, shaking culture in constant-temperature shaking incubator, namely seed liquor is obtained, described LB liquid culture based formulas is: yeast extract 3-5g, peptone 8-10g, NaCl5g, water 1000ml, pH7.4 ~ 7.6; Culture condition: 30 DEG C, 48h;
Step 3: fermentation culture: seed liquor is successively carried out seeding tank fermentation culture and large-scale industry fermentation culture, obtains fermented liquid; Wherein seeding tank fermentation culture and large-scale industry fermentation culture step are distinguished as follows:
Seeding tank fermentation culture: FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 DEG C, 42h, inoculum size: 6%, air flow: 2.5L/min, mixing speed: 200r/min;
Large-scale industry fermentation culture: FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 DEG C, 60-70h, inoculum size: 6%, air flow: 2.0L/min, mixing speed: 220r/min;
Described FJ liquid culture based formulas is: soybean cake powder 1.5%, sucrose 1%, KH
2pO
40.05%, MgSO
40.02%, NaCl0.1%, all the other are water, and listed per-cent is weight percentage;
Above-mentioned tunning is prepared bacteria agent further:
(1): filter and enriched product: by above-mentioned tunning with after careless acid for adjusting pH to 6.5-7.0, by the inorganic ceramic ultra-filtering film filtering separation of tunning by molecular weight cut-off 40000-100000k, service temperature is 35-37 DEG C, membrane flux is 50-60LMH, the solution being trapped within the liquid feeding side of film is gemma solution, gemma solution cycles of concentration is 6-7 times, obtained through concentrated gemma solution, described concentration method is the molecular screen membrane of use 500,000 Da, peristaltic pump flow velocity is 4 grades, flux is 420ml/min, and liquid outlet pressure is 0.08MPa;
(2): spraying dry: to the filler of the interpolation 1.0% in concentrated gemma solution of (1), subsequently gemma solution is made powder through spraying, spray-dired inlet temperature is 170-190 DEG C, temperature out is 65-85 DEG C, input speed 60mL/min, sprinkler pressure 0.10MPa, namely obtains the bacteria agent of Methylotrophic genus bacillus; Described filler is white carbon black, micronized talc powder, light calcium carbonate or 2500 order ultrafine heavy calciums;
Described tobacco bacterial wilt be by tobacco ralstonia solanacearum (
ralstoniasolanacearum) cause.
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