CN103045515A - Biological agent of bacillus methylotrophicus as well as preparation method and application thereof - Google Patents

Biological agent of bacillus methylotrophicus as well as preparation method and application thereof Download PDF

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CN103045515A
CN103045515A CN2012105798998A CN201210579899A CN103045515A CN 103045515 A CN103045515 A CN 103045515A CN 2012105798998 A CN2012105798998 A CN 2012105798998A CN 201210579899 A CN201210579899 A CN 201210579899A CN 103045515 A CN103045515 A CN 103045515A
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culture
genus bacillus
methylotrophy type
bacterial
type genus
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CN103045515B (en
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唐满仓
翟枫
马玉明
安德荣
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Shaanxi Hengtian Biological Agriculture Co. Ltd.
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SHAANXI HENTIAN CHEM-TECH Co Ltd
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Abstract

The invention discloses a novel bacillus methylotrophicus, which has the preservation register number of CGMCC No.6793, wherein the strain is separated from tobacco rhizosphere soil and is obtained by separating and purifying and screening strains. The invention further provides a fermentation method of the bacillus methylotrophicus, a fermentation product prepared by the method, as well as a biological agent based on the fermentation product and a preparation method thereof; the invention further provides a use of the biological agent in controlling a variety of plant diseases. According to the invention, the biological agent is good in environmental compatibility so that the number of effective living spores is more than 5,000,000,000/g, has high biological activity, and in particular has good control effect on tobacco bacterial wilt, wherein the control effect on tobacco bacterial wilt, soft rot of cabbage, rice bacterial leaf blight, rice bacterial stripe and cotton angular leaf spot is up to 80% or more.

Description

Bacteria agent of a kind of methylotrophy type genus bacillus and its preparation method and application
Technical field
The invention belongs to the biocontrol of plant disease field, particularly utilize beneficial microorganism that Plant diseases is carried out biological control, be specifically related to methylotrophy type genus bacillus (Bacillus methylotrophicus), and utilize this bacterial strain to prepare the application of bacteria agent and described bacteria agent controlling plant diseases.
Background technology
The popular crop failure that not only causes of Plant diseases 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 the 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, still, the general non-degradable of chemical pesticide, a large amount of uses along with chemical pesticide have caused the events such as environmental pollution, ecological damage, food contamination.This makes research and development utilize the biological pesticide person's that becomes the plant protection service important subject.Biological pesticide refers to the preparation that utilizes living organisms or its meta-bolites to kill or suppress for agricultural pest; have the characteristics such as safe, efficient, pollution-free; matching with the requirement of preserving the ecological environment and society develops in harmony, is the inexorable trend of pesticide in future development.
Microbial pesticide is a class that is most widely used in the biological pesticide, also is the important materials of biological control.The main component of microbial pesticide is microorganism live body and secondary metabolite thereof, 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 epiphyte venereal disease evil owing to environmentally safe, and can slow down pathogenic bacteria and develop immunity to drugs, become the main stream approach of current controlling plant diseases.
Methylotrophy type genus bacillus is a kind of have a liking for temperature, aerobic, sporiferous rod-shaped bacterium, its physiological characteristic is various, this bacterium is widespread in nature, has unique one carbon metabolism approach, can change into thalline self required nutritive substance such as the inexhaustible one-carbon compound of occurring in nature such as methyl alcohol, formaldehyde etc., can produce again simultaneously the metabolic by-prods L. Serine, vitamin B12, poly-p hydroxybutyric acid salt of multiple beneficial etc.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.Methylotrophy type genus bacillus not only can extensively exist in the external environments such as soil, plant rhizosphere body surface, and is common endogenetic bacteria in the plant materials, and is especially inner at root, the stem of plant.Methylotrophy type genus bacillus is by successfully surely growing in the plant rhizospheres such as tobacco, paddy rice, body surface or body, change its on every side flora environment and kind, with the site around the pathogenic bacteria competitive plant, and secrete multiple antimicrobial substance to suppress the pathogenic bacteria growth, the inducing plant system of defense is resisted the pathogenic bacteria invasion simultaneously, thereby reach the purpose of improvement soil and biological and ecological methods to prevent plant disease, pests, and erosion, have diseases prevention and growth-promoting functions.
Tobacco bacterial wilt be a kind of by the withered Raul Salmonella of green grass or young crops (Ralatonia solanacearum) cause, take the soil propagation as main, destructive bacterial disease.This disease is one of Major Diseases of tropical and subtropical region tobacco, China Yangtze valley and on the south the cigarette district generally occur, wherein heavier with Guangdong, Fujian, Hunan, Sichuan and Tobacco Planting Areas in Guizhou harm.At present, mainly adopt aborning chemical pesticide control, but can't prevent effectively that this disease from occuring, and use in a large number for a long time chemical pesticide germ was developed immunity to drugs, easily cause again serious environmental pollution.
Bacterial blight of rice is by Gram-negative bacteria Xanthomonas campestris paddy rice mutation (Xanthomonas oryzaepv.oryzae, Xoo) cause, one of paddy rice three large diseases, nearly all paddy fields all has generation 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 long-grained nonglutinous rice producing region, Yangze river and Huai river Plain is Chang Faqu, have a strong impact on rice yield, mainly rely at present chemical pesticide control, 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 methylotrophy type genus bacillus.
Another object of the present invention is to provide the fermentation process of described methylotrophy type genus bacillus, and the tunning that is prepared gained by the method.
Bacteria agent that provides methylotrophy type genus bacillus and preparation method thereof is provided.
Another object of the present invention is to provide the purposes of the bacteria agent controlling plant diseases of utilizing described methylotrophy type genus bacillus.
The objective of the invention is to be achieved through the following technical solutions:
A kind of methylotrophy type genus bacillus, it belongs to bacillus, its called after Bacillus methylotrophicus; Preservation registration number is CGMCCNo.6793, and preservation mechanism is: China Committee for Culture Collection of Microorganisms common micro-organisms center; Depositary institution address: Datun Road, Chaoyang District, Beijing City; The preservation time is on November 07th, 2012.
Above-mentioned methylotrophy type genus bacillus of the present invention separates from tobacco rhizosphere soil and obtains, and separation, purifying and the screening step by bacterial strain obtains.
Identification of strains: adopt morphological specificity, Physiology and biochemistry experiment, molecular biology identification method, and 16SrDNA homologous sequence compare of analysis determines that finally the bacterial strain of above-mentioned deposit number is methylotrophy type genus bacillus (Bacillusmethylotrophilus).
The fermentation process of methylotrophy type genus bacillus of the present invention, described method comprises the steps:
Step 1: actication of culture: be that the methylotrophy type genus bacillus bacterial classification inoculation of CGMCCNo.6793 is above the LB solid medium with preservation registration number, in 30 ℃ of activation culture 48h, described LB solid culture based formulas is yeast extract 3-5g, peptone 8-10g, NaCl 5g, agar 15~20g, water 1000ml, pH 7.4~7.6, transfer continuously for 3 generations, after guaranteeing bacterial strain purity, inoculate in the seed liquor and use;
Step 2: the preparation of seed liquor: the picking bacterial classification inoculation is to the triangular flask that the LB liquid nutrient medium is housed from the LB solid medium, shaking culture in constant-temperature shaking incubator, namely obtain seed liquor, described LB liquid culture based formulas is: yeast extract 3-5g, peptone 8-10g, NaCl 5g, water 1000ml, pH 7.4~7.6; Culture condition: 30 ℃, 48h;
Step 3: fermentation culture: seed liquor is successively carried out seeding tank fermentation culture and large-scale industry fermentation culture, obtain fermented liquid; Wherein seeding tank fermentation culture and large-scale industry fermentation culture step are as follows respectively:
The seeding tank fermentation culture: the FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 ℃, 42h, inoculum size: 6%, air flow: 2.5L/min, mixing speed: 200r/min;
The large-scale industry fermentation culture: the FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 ℃, 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, listed per-cent is weight percentage.
Preferably, if in the fermentation culture process of step 3, there is foam to produce, can drip enemy's froth breaking.
Further, the invention provides the tunning that is prepared gained by the fermentation process of the described methylotrophy type of technique scheme genus bacillus.
Further, according to above-mentioned tunning, the present invention also provides the bacteria agent of a kind of methylotrophy type genus bacillus, and described bacteria agent makes as follows:
Step 1: filter and enriched product: will adopt tunning that the fermentation process of above-mentioned methylotrophy type genus bacillus prepares gained with careless acid for adjusting pH to 6.5-7.0, with the inorganic ceramic ultra-filtering film filtering separation of tunning by molecular weight cut-off 40000-100000k, service temperature is 35-37 ℃, membrane flux is 50-60LMH, the solution that is trapped within the liquid feeding side of film is gemma solution, gemma solution cycles of concentration is 6-7 times, make through concentrated gemma solution, described concentration method is for using the molecular screen membrane of 500,000 Da, the peristaltic pump flow velocity is 4 grades, flux is 420ml/min, and liquid outlet pressure is 0.08MPa;
Step 2: spraying drying: in the concentrated gemma solution of the warp of step 1, add 1.0% filler, subsequently gemma solution is made powder through spraying, spray-dired inlet temperature is 170-190 ℃, temperature out is 65-85 ℃, input speed 60mL/min, sprinkler pressure 0.10MPa namely obtains the bacteria agent of methylotrophy type 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 purposes with the bacteria agent controlling plant diseases of above-mentioned methylotrophy type genus bacillus; Especially for the disease in control tobacco, Chinese cabbage, paddy rice, the 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 the 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 (Xanthomonas oryzae pv.Oryzae); Described cotton disease is the cotton angular leaf spot, and described disease is that angular leaf spot of cotton Xanthomonas campestris (Xanthomonas campestrispv.malvacearum (E.F.Smith) Dowson) causes.
Except as otherwise noted, " % " in the present specification all is weight percentage.
The present invention has following beneficial effect:
1, by the prepared bacteria agent of methylotrophy type genus bacillus of the present invention, its Environmental compatibility is good, has very high biological activity, particularly to had preferably preventive effect by tobacco bacterial wilt, proof by experiment, the bacteria agent of this methylotrophy type 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 that is made by methylotrophy type genus bacillus of the present invention is the novel high-activity high-concentration biological microbial inoculum of adopting new techniques fermentation, the research and development of advanced ultrafiltration and concentration isolation technique, and the spore count of effectively living reaches more than 5,000,000,000/gram.
Description of drawings:
Fig. 1 is the 16SrDNA homologous sequence compare of analysis dendrogram of methylotrophy type genus bacillus of the present invention.
Embodiment:
For making technical scheme of the present invention be convenient to understand, be further described below in conjunction with concrete test example.
Embodiment 1:The isolation and screening of the application's methylotrophy type genus bacillus (Bacillus methylotrophilus):
(1), the separation of genus bacillus:
Take by weighing tobacco rhizosphere soil 10g, pour in the Erlenmeyer flask that the 90ml aqua sterilisa is housed, the 200r/min 20min that vibrates, 80 ℃ of water-bath 30min, 10 doubling dilutions, coating separates.
(2), the purifying of genus bacillus: drop down onto on the LB solid medium method purifying that adopts line to separate according to the single bacterium of the timely picking of the feature of bacterium; The bacterium of purifying shows that through gramstaining and spore staining thalline is shaft-like, produces gemma, G +Isolate be genus bacillus; Described LB solid culture based formulas is: yeast extract 5g, and peptone 8g, NaCl 5g, agar 15g, water 1000ml, pH 7.4.
(3), the screening of antagonistic Bacillus:
Select the activated tobacco ralstonia solanacearum of tool as indicator with the TTC substratum, tobacco ralstonia solanacearum is made into 2 * 10 8The bacteria suspension of cfu/ml concentration is transferred on the BPA substratum, makes ralstonia solanacearum and contains bacterium culture medium.After drying up, connect the Bacillus strain of above-mentioned own separation and purification in dull and stereotyped central authorities, cultivate 2-3d for 30 ℃, measure the size at antibacterial circle diameter and antibacterial band (from the edge of active bacterium bacterium colony to the outer rim of inhibition zone).The prescription 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 prescription of described BPA substratum is: beef extract 3-5g, and peptone 5-10g, sucrose 10-12g, yeast extract 1-2g, agar 17-20g adds water to 1L.
Separation screening obtains the application's methylotrophy type genus bacillus, and the laboratory is numbered LW-6.
Embodiment 2:The evaluation of methylotrophy type genus bacillus (Bacillus methylotrophilus):
Qualification process to the methylotrophy type genus bacillus of embodiment 1 gained is:
(1), morphological specificity: the LW-6 bacterium colony presents creamy white on the NA solid medium, the bacterium colony surface irregularity, the edge presents spination, see through Electronic Speculum and show that LW-6 is shaft-like, Gram-positive, peritrichous, the individual cells size is 0.5-0.6um * 2.0-2.5um.The prescription of described NA solid medium is: beef extract 2-3g, and peptone 5g, glucose 2.5-3g, agar 16-18g adds water to 1000ml.
(2), Physiology and biochemistry experiment: by to the physio-biochemical characteristics tests (as shown in table 1) of methylotrophy type 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) is retrieved, and identifies that this bacterial strain is bacillus.
The physiological and biochemical property of table 1 the application methylotrophy type genus bacillus
Annotate: "+" expression positive reaction, "-" expression negative reaction.
The bacterial strain physiological and biochemical property shows: the application's bacterial strain LW-6 can make gelatine liquefication; Energy hydrolyzed starch and reduction nitrate; Glucose, pectinose, wood sugar, seminose, catalase, lipase, urase and oxydase all are positive; The V-P test is positive; Do not utilize Citrate trianion.Maximum growth temperature is 60 ℃, and minimum growth temperature is 10 ℃, and optimum growth temperature is 30 ℃; The highest pH value is 11.0, and minimum pH value is 3.0, and optimum pH is 7.0; Have salt tolerance, can in 15% NaCl substratum, grow.
(3), molecular biology identification: take the LW-6 genomic dna as template, carry out pcr amplification with primer 7f and 1540r, the 16S rDNA nucleotide sequence length that records this bacterial strain is 1454bp, and the GenBank accession number is JX220980.By with NCBI in the 16SrDNA sequence alignment of type strain analyze and the structure (Fig. 1) of phylogenetic tree, learn that LW-6 bacterial strain and methylotrophy type bacillus methylotrophicus CBMB205 (T) (the GenBank accession number is EU194897) homology is nearest, similarity is 100%.
The form of comprehensive LW-6 bacterium, cultural characteristic, Physiology and biochemistry are measured and 16SrDNA homologous sequence compare of analysis, and this bacterial strain of preliminary evaluation is methylotrophy type genus bacillus
Embodiment 3:The cultivation of methylotrophy type genus bacillus (Bacillus methylotrophilus)
(1), bacterial strain activation:
Substratum: the LB solid medium, described LB solid culture based formulas is: yeast extract 5g, peptone 8g, NaCl 5g, agar 20g, water 1000ml, pH 7.4;
Culture condition: 30 ℃, 48h;
Continuous 3 generations of switching, guarantee bacterial strain purity after, inoculate in the fermented liquid and use.
(2), the preparation of seed liquor (shake flask fermentation or the fermentation of eggplant bottle):
Substratum: the LB liquid nutrient medium, described LB liquid culture based formulas is: yeast extract 5g, peptone 8, NaCl 5g, water 1000ml, pH 7.4 (flask volume 250ml, liquid amount 50ml);
Culture condition: 30 ℃, 48h;
Inoculum size: 6-8%.
(3), seeding tank fermentation:
Substratum: the 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, listed per-cent is weight percentage.
Coefficient: 0.7;
Culture condition: 35-37 ℃, 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, listed per-cent is massfraction;
Coefficient: 0.7;
Culture condition: 35-37 ℃, 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 methylotrophy type genus bacillus
(1), fermented liquid is concentrated: after the tunning that will adopt embodiment 3 to make is adjusted to 6.5-7.0 with oxalic acid, with the inorganic ceramic ultra-filtering film filtering separation of tunning by molecular weight cut-off 40000-100000k, service temperature is 35-37 ℃, membrane flux is 50-60LMH, the solution that is trapped within the liquid feeding side of film is gemma solution, gemma solution cycles of concentration is 6 times, make through concentrated gemma solution, described concentration method is for using the molecular screen membrane of 500,000 Da, the peristaltic pump flow velocity is 4 grades, flux is 420ml/min, and liquid outlet pressure is 0.08MPa.
(2), spraying drying:
In the concentrated gemma solution of above-mentioned warp, add 1.0% white carbon black, gemma solution is made powder through spraying, namely obtain the bacteria agent of methylotrophy type genus bacillus of the present invention.
Embodiment 5:The application's methylotrophy type genus bacillus is to antagonistic experiment in the ware of tobacco ralstonia solanacearum, rice leaf spot bacteria, soft rot of cabbage bacterium:
Respectively take tobacco ralstonia solanacearum Ralstonia solanacearum, rice leaf spot bacteria Xanthomonasoryzae pv.Oryzae and soft rot of cabbage bacterium Erwinia carotovora as for the examination pathogenic bacteria, select the activated indicator of tool with the LB substratum, germ is made into 2 * 10 8The bacteria suspension of cfu/ml concentration is transferred on the LB substratum, makes to contain bacterium culture medium.After drying up, connect the Bacillus strain of above-mentioned own separation and purification in dull and stereotyped central authorities, cultivate 2-3d for 30 ℃, measure the size at antibacterial circle diameter and antibacterial band (from the edge of active bacterium bacterium colony to the outer rim of inhibition zone).The application's methylotrophy type genus bacillus is as shown in table 2 to the antibacterial situation of Different Kinds of Pathogens bacterium
Table 2 the application's methylotrophy type genus bacillus is to the antibacterial situation of various pathogenic bacteria
Pathogenic bacteria Antibacterial circle diameter/mm
Tobacco ralstonia solanacearum 21.05
The soft rot of cabbage bacterium 15.05
Rice leaf spot bacteria 18.90
[0084]Use inhibition zone size measurement methylotrophy type genus bacillus to the inhibition situation of tobacco ralstonia solanacearum Ralstoniasolanacearum, rice leaf spot bacteria Xanthomonas oryzae pv.Oryzae and soft rot of cabbage bacterium Erwinia carotovora by table 2, find that this bacterial strain suppresses situation to these pathogenic bacterias remarkable, especially more outstanding to tobacco bacterial wilt and bacterial blight of rice inhibition.
Embodiment 6:The determination of activity of the biocontrol fungicide of the application's methylotrophy type genus bacillus
For trying pathogenic bacteria: tobacco ralstonia solanacearum Ralstonia solanacearum, cultivate at the TTC substratum, make 1 * 10 6The cfu/ml bacteria suspension, stand-by.
For trying chemical agent: 72% agricultural Vetstrep wettable powder.
Method: test is Nutrition Soil V (soil): the V (Nutrition Soil) that is mixed that sterilizes=1: 1 with soil.The earth culture of will sterilizing is educated cigarette seedling to 4~5 leaf phases, and seedling is taken out.
Process 1: blank, root 30min soaks in cigarette shoot root section in clear water.
Process 2: the chemical agent contrast, root 30min soaks in cigarette shoot root section in 2000 times of liquid of 72% agricultural Vetstrep wettable powder, behind the inoculation 1d, root-pouring method inoculation tobacco Ralstonia solanacearum, inoculum size is the 5ml/ strain.
Process 3: in 200 times of diluents of biocontrol fungicide of the methylotrophy type genus bacillus of embodiment 4, soak root 30min, behind the inoculation 1d, root-pouring method inoculation tobacco Ralstonia solanacearum, inoculum size is the 5ml/ strain.
3 repetitions are established in every processing, and every repetition 10 strain cigarette seedlings are processed and transplanted to the seedling alms bowl that sterilization soil is housed, and process being positioned under 28~36 ℃ of conditions in greenhouse, 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's idol has the chlorisis scab or has the minority blade wilting in streak one side
3 grades: stem's black scab.But do not reach the top, or sick side 5% blade is wilting
5 grades: stem's black scab reaches the plant top.Or sick side 2/3 blade is wilting
7 grades: diseased plant stem is withered
The biocontrol fungicide of the application's methylotrophy type genus bacillus is to greenhouse preventive effect result of study such as the table 3 of tobacco bacterial wilt:
The biocontrol fungicide of table 3 the application methylotrophy type genus bacillus is to the greenhouse prevention effect of tobacco bacterial wilt
Embodiment 7:The biocontrol fungicide of the application's methylotrophy type genus bacillus is tested the bacterial blight of rice preventive effect:
For trying pathogenic bacteria: rice leaf spot bacteria (Xanthomonas oryzae pv.Oryzae), cultivate at the LB substratum, make 1 * 10 6The cfu/ml bacteria suspension, stand-by.
For trying chemical agent: the kasugamycin aqua.
Method: test is Nutrition Soil V (soil): the V (Nutrition Soil) that is mixed that sterilizes=1: 1 with soil.The earth culture of will sterilizing is educated rice seedling to 4~5 leaf phases, and seedling is taken out.
Process 1: blank, the rice seedlings root soaks root 30min in clear water.
Process 2: the chemical agent contrast, the rice seedlings root soaks root 30min in 600 times of liquid of kasugamycin, behind the inoculation 1d, root-pouring method Inoculated Rice bacterial leaf spot cause of disease bacterium bacteria suspension, inoculum size is the 5ml/ strain.
Process 3: in 200 times of diluents of biocontrol fungicide of the methylotrophy type genus bacillus of embodiment 4, soak root 30min, behind the inoculation 1d, root-pouring method Inoculated Rice bacterial leaf spot bacterium, inoculum size is the 5ml/ strain.
3 repetitions are established in every processing, and every repetition 10 strain rice seedlings are processed and transplanted to the seedling alms bowl that sterilization soil is housed, and process being positioned over 28~36 ℃ in greenhouse
Under the 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 below 1/5 of leaf area;
2 grades: lesion area be leaf area 1/3 below;
3 grades: lesion area is below 1/2 of leaf area;
4 grades: lesion area is below 3/5 of leaf area;
5 grades: lesion area is more than 3/5 of leaf area
The biocontrol fungicide of the application's methylotrophy type genus bacillus is to greenhouse preventive effect result of study such as the table 4 of bacterial blight of rice:
The biocontrol fungicide of table 4 the application methylotrophy type genus bacillus is to the greenhouse prevention effect of bacterial blight of rice
Embodiment 8:The biocontrol fungicide of the application's methylotrophy type genus bacillus is tested the former bacterium preventive effect of soft rot of cabbage:
For trying pathogenic bacteria: soft rot of cabbage bacterium (Erwinia carotovora), cultivate at the LB substratum, make 1 * 10 6The cfu/ml bacteria suspension, stand-by.
For trying chemical agent: the agricultural streptomycin wettable powder.
Method: test is Nutrition Soil V (soil): the V (Nutrition Soil) that is mixed that sterilizes=1: 1 with soil.The earth culture of will sterilizing is educated Chinese cabbage to 4~5 leaf phases, and shoot is taken out.
Process 1: blank, the Chinese cabbage root soaks root 30min in clear water.
Process 2: the chemical agent contrast, the Chinese cabbage root soaks root 30min in 4000 times of liquid of agricultural streptomycin, behind the inoculation 1d, the former bacterium bacteria suspension of root-pouring method inoculation soft rot of cabbage, inoculum size is the 5ml/ strain.
Process 3: in 200 times of diluents of biocontrol fungicide of the methylotrophy type genus bacillus of embodiment 4, soak root 30min, behind the inoculation 1d, the soft rotten bacterium of root-pouring method inoculation Chinese cabbage, inoculum size is the 5ml/ strain.
3 repetitions are established in every processing, and every repetition 10 strain Chinese cabbages are processed and transplant to the seedling alms bowl that sterilization soil is housed, and process being positioned under 28~36 ℃ of conditions in greenhouse, 15 days " Invest, Then Investigate " tobacco plant incidences, statistics sickness rate and prevention effect.
The biocontrol fungicide of the application's methylotrophy type genus bacillus is as shown in table 5 to the greenhouse preventive effect result of study of soft rot of cabbage:
The biocontrol fungicide of table 5 the application methylotrophy type genus bacillus is to the greenhouse prevention effect of soft rot of cabbage
Embodiment 9:Residential quarter, the field experiment of the biocontrol fungicide control tobacco bacterial wilt of the application's methylotrophy type genus bacillus, bacterial blight of rice, soft rot of cabbage:
Be the biocontrol fungicide of the checking the application methylotrophy type genus bacillus preventive effect to tobacco bacterial wilt, bacterial blight of rice, soft rot of cabbage, we carry out the 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 the Baoji, testing ground area 900m 2(1.36 mu), soil property are light loam, and organic content is 1.01%, pH value 7.7, and all experimental plot cultivation conditions and control measures are consistent.200 times of liquid of biocontrol fungicide, 72% agricultural Vetstrep wettable powder and totally 3 processing of not dispenser clear water of methylotrophy type genus bacillus are established in this experiment, and each processes 4 repetitions, totally 12 residential quarters, each residential quarter random alignment, each residential quarter 40m 2The minizone arranges the wide isolation strip of 0.6m around reaching experimental field.Sow in April, 2010, average spacing in the rows 8cm, and after planting respectively on May 5th, 2010,15 days and 25 days root filling once, it is workers and peasants that mu is used liquid 500kg, medicinal sprayer tool---16 type atomizers, take off sprinkling irrigation with shower nozzle.
Meteorological conditions
For the first time dispenser (May 5) was partly cloudy the same day, 3 grades of wind-force, and 25 ℃ of the highest temperatures, 15 ℃ of the lowest temperatures, relative humidity is 80%.For the second time dispenser (May 15) was cloudy the same day, 3 grades of wind-force, and 23 ℃ of the highest temperatures, 15 ℃ of the lowest temperatures, relative humidity is 80%.For the first time dispenser (May 25) was partly cloudy the same day, 5 grades of wind-force, and 25 ℃ of the highest temperatures, 14 ℃ of the lowest temperatures, relative humidity is 80%.
Investigated in 7 days after the last dispenser, 4 point samplings are adopted in every residential quarter, look into 20 strains at every, record total strain number and diseased plant number at different levels.
Stage division:
0 grade: complete stool is anosis
1 grade: stem's idol has the chlorisis scab or has the minority blade wilting in streak one side
3 grades: stem's black scab.But do not reach the top, or sick side 5% blade is wilting
5 grades: stem's black scab reaches the 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 biocontrol fungicide of the application's methylotrophy type genus bacillus reaches the average preventive effect of tobacco bacterial wilt.
The biocontrol fungicide of table 6 the application methylotrophy type genus bacillus is prevented and treated situation to the tobacco bacterial wilt field test
(2), paddy rice: practice ground is selected in the Yang County, Shaanxi, practice ground 800m 2(1.21 mu), soil property are light loam, and organic content is 1.2%, pH value 7.5, and all experimental plot cultivation conditions and control measures are consistent; 200 times of liquid of biocontrol fungicide, 600 times of liquid of kasugamycin and totally 3 processing of not dispenser clear water of the application's methylotrophy type genus bacillus are established in this experiment, and each processes 4 repetitions, totally 12 residential quarters, each residential quarter random alignment, each residential quarter 60m 2The minizone arranges the wide isolation strip of 0.5m around reaching experimental field.Sow in March, 2010, average spacing in the rows 7cm, and respectively on April 15th, 2010, May 5 and 25 days, root was filled with once respectively, it is workers and peasants that mu is used liquid 500kg, medicinal sprayer tool behind the insemination and emergence---16 type atomizers, take off sprinkling irrigation with shower nozzle.
Meteorological conditions
For the first time dispenser (April 15) was partly cloudy the same day, 3 grades of wind-force, and 14 ℃ of the highest temperatures, 1 ℃ of the lowest temperature, relative humidity is 30%.For the second time dispenser (May 15) was cloudy the same day, 3 grades of wind-force, and 19 ℃ of the highest temperatures, 3 ℃ of the lowest temperatures, relative humidity is 30%.For the first time dispenser (May 25) was partly cloudy the same day, 5 grades of wind-force, and 23 ℃ of the highest temperatures, 10 ℃ of the lowest temperatures, relative humidity is 20%.
Investigated in 7 days after the last dispenser, 4 point samplings are adopted in every residential quarter, look into 20 strains at every, record total strain number and diseased plant number at different levels.
Stage division:
0 grade: anosis;
1 grade: lesion area is below 1/5 of leaf area;
2 grades: lesion area be leaf area 1/3 below;
3 grades: lesion area is below 1/2 of leaf area;
4 grades: lesion area is below 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 biocontrol fungicide of the application's methylotrophy type genus bacillus reaches 70% to the average preventive effect of bacterial blight of rice, is higher than the preventive effect of kasugamycin aqua.
The biocontrol fungicide of table 7 the application methylotrophy type genus bacillus is prevented and treated 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 are light loam, and organic content is 1.07%, pH value 7.2, and all experimental plot cultivation conditions and control measures are consistent.200 times of liquid of biocontrol fungicide, 4000 times of liquid of agricultural streptomycin and totally 3 processing of not dispenser clear water of the application's methylotrophy type genus bacillus are established in this experiment, and each processes 4 repetitions, totally 12 residential quarters, each residential quarter random alignment, each residential quarter 40m 2The minizone arranges the wide isolation strip of 0.6m around reaching experimental field.Sow in July, 2010, average spacing in the rows 8cm, and after planting respectively on August 5th, 2010,15 days and 25 days root filling once, it is workers and peasants that mu is used liquid 500kg, medicinal sprayer tool---16 type atomizers, take off sprinkling irrigation with shower nozzle.
Meteorological conditions
For the first time dispenser (August 5) was partly cloudy the same day, 3 grades of wind-force, and 25 ℃ of the highest temperatures, 15 ℃ of the lowest temperatures, relative humidity is 80%.For the second time dispenser (August 15) was cloudy the same day, 3 grades of wind-force, and 23 ℃ of the highest temperatures, 15 ℃ of the lowest temperatures, relative humidity is 80%.For the first time dispenser (August 25) was partly cloudy the same day, 5 grades of wind-force, and 25 ℃ of the highest temperatures, 14 ℃ of the lowest temperatures, relative humidity is 80%.
Last dispenser was investigated after 10 days, and 4 point samplings are adopted in every residential quarter, look into 20 strains at every, record total strain number and diseased plant number at different levels.
Stage division:
0 grade: complete stool is anosis
1 grade: stem's idol has the chlorisis scab or has the minority blade wilting in streak one side
3 grades: stem's black scab.But do not reach the top, or sick side 5% blade is wilting
5 grades: stem's black scab reaches the 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 biocontrol fungicide of the application's methylotrophy type genus bacillus reaches 74.1% to the average preventive effect of soft rot of cabbage, is higher than 4000 times of liquid of agricultural streptomycin.
Table 8 methylotrophy type genus bacillus LW-6 microbial inoculum is prevented and treated situation to the field test of soft rot of cabbage
Bacteria agent of a kind of methylotrophy type 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 corresponding other purpose, its relevant change does not all break away from content of the present invention, all similar replacements and change will become apparent to those skilled in the art that and all be deemed to be included within the scope of the present invention.

Claims (8)

1. methylotrophy type 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 common micro-organisms center; The preservation time is on November 07th, 2012.
2. according to claim 1 the fermentation process of methylotrophy type genus bacillus, described method comprises the steps:
Step 1: actication of culture: be that the methylotrophy type genus bacillus bacterial classification inoculation of CGMCCNo.6793 is above the LB solid medium with preservation registration number, in 30 ℃ of activation culture 48h, described LB solid culture based formulas is yeast extract 3-5g, peptone 8-10g, NaCl 5g, agar 15~20g, water 1000ml, pH 7.4~7.6, transfer continuously for 3 generations, after guaranteeing bacterial strain purity, inoculate in the seed liquor and use;
Step 2: the preparation of seed liquor: the picking bacterial classification inoculation is to the triangular flask that the LB liquid nutrient medium is housed from the LB solid medium, shaking culture in constant-temperature shaking incubator, namely obtain seed liquor, described LB liquid culture based formulas is: yeast extract 3-5g, peptone 8-10g, NaCl 5g, water 1000ml, pH 7.4~7.6; Culture condition: 30 ℃, 48h;
Step 3: fermentation culture: seed liquor is successively carried out seeding tank fermentation culture and large-scale industry fermentation culture, obtain fermented liquid; Wherein seeding tank fermentation culture and large-scale industry fermentation culture step are as follows respectively:
The seeding tank fermentation culture: the FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 ℃, 42h, inoculum size: 6%, air flow: 2.5L/min, mixing speed: 200r/min;
The large-scale industry fermentation culture: the FJ liquid nutrient medium, coefficient 0.7, culture condition: 35-37 ℃, 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, listed per-cent is weight percentage.
3. according to claim 2 fermentation process wherein drips enemy's froth breaking in the fermentation culture process of step 3.
4. the tunning that makes to the fermentation process of 3 arbitrary described methylotrophy type genus bacillus according to claim 2.
5. the bacteria agent of a methylotrophy type genus bacillus, described bacteria agent makes as follows:
Step 1: filter and enriched product: with the tunning of claim 4 with careless acid for adjusting pH to 6.5-7.0, with the inorganic ceramic ultra-filtering film filtering separation of tunning by molecular weight cut-off 40000-100000k, service temperature is 35-37 ℃, membrane flux is 50-60LMH, the solution that is trapped within the liquid feeding side of film is gemma solution, gemma solution cycles of concentration is 6-7 times, make through concentrated gemma solution, described concentration method is for using the molecular screen membrane of 500,000 Da, the peristaltic pump flow velocity is 4 grades, flux is 420ml/min, and liquid outlet pressure is 0.08MPa;
Step 2: spraying drying: in the concentrated gemma solution of the warp of step 1, add 1.0% filler, subsequently gemma solution is made powder through spraying, spray-dired inlet temperature is 170-190 ℃, temperature out is 65-85 ℃, input speed 60mL/min, sprinkler pressure 0.10MPa namely obtains the bacteria agent of methylotrophy type genus bacillus of the present invention; Described filler is white carbon black, micronized talc powder, light calcium carbonate or 2500 order ultrafine heavy calciums.
6. according to claim 5 the purposes of bacteria agent controlling plant diseases of methylotrophy type genus bacillus.
7. according to claim 6 purposes, described Plant diseases is the disease in tobacco, Chinese cabbage, paddy rice, the cotton crop.
8. according to claim 6 purposes, described Plant diseases is tobacco bacterial wilt, Chinese cabbage canker, bacterial blight of rice, bacterial leaf streak of rice or cotton angular leaf spot.
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CN105039220A (en) * 2015-07-30 2015-11-11 武汉华恩绿农生物科技有限公司 Bacillus methylotrophicus AR3, bacillus subtilis AR4 and bacillus amyloliquefaciens AR10 and application thereof
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CN110627566A (en) * 2019-09-29 2019-12-31 云南云之叶生物科技有限公司 Bio-organic fertilizer with clubroot disease prevention and treatment function and preparation method and application thereof
CN110934154A (en) * 2019-12-06 2020-03-31 西北农林科技大学 Nutrient composition for promoting growth of cucumber seedlings and application thereof
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CN104017750A (en) * 2014-04-14 2014-09-03 青岛海大生物集团有限公司 Salt-resistance Bacillusmethylotrophicus strain LJ and application thereof
CN106686983A (en) * 2014-07-31 2017-05-17 格拉纳达大学 Use of bacillus methylotrophicus as a stimulant of plant growth and biological control means, and isolates of said species
CN106686983B (en) * 2014-07-31 2020-05-12 格拉纳达大学 Use of bacillus methylotrophicus as plant growth stimulator and biological control element, and isolate of said species
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