CN100445370C - Bacillus subtilis, bacterium agent and application thereof - Google Patents
Bacillus subtilis, bacterium agent and application thereof Download PDFInfo
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Abstract
The invention discloses a Bacillius subtilis Corn-1, which is reserved in the Chinese microbe bacterial committee center with reserving number at CGMCC No.1778.
Description
Technical field
The present invention relates to a kind of subtilis, also relate to the microbiobacterial agent, preparation method and the application that utilize this subtilis to produce.
Background technology
Corn leaf spot comprises that corn bends spore leaf spot, the leaf blight of corn and corn southern leaf blight.
The curved spore leaf spot of corn claims again to intend eye spot, black points, is the bigger maize diseases of a kind of danger that is caused by crescent Curvularia lunata (Curvularia lunata).This disease main harm blade and leaf sheath, scab gathers in flakes on the grave illness ground blade, causes blade dried-up.20th century the mid-80, China Henan Province finds this disease in Xinxiang.The disease developmenting spread is very fast, in Liaoning, northern corn producing region such as Beijing, Hebei, Henan, Shandong, Jilin, Tianjin generally takes place, and become the another important disease after leaf blight, helminthosporium maydis, head smut on the northern China corn, Maize Production has been constituted great threat.Four is that this disease seriously takes place for parent's seed farm as Hebei province's Longhua in 1994, Pingquan early on yellow 417 with Huang, the general underproduction 20%, and serious reaches 60%.Area takes place Hebei province in 1996 is more than 200,000 hm, and the underproduction 20%～60% does not wait.Because the maize curvularia leaf spot is sudden disease, spread rapidly, leaf portion scab is intensive in flakes when serious, and grave illness plot diseased plant rate and sick leaf rate have become one of the important disease in China corn producing region up to 100%.
The leaf blight of corn (Exserohilum turcicum) is one of important disease on the Maize Production, extensively betides all over the world, and should disease can cause serious production loss every year.This disease is since the seventies, northeastward, North of North China and south cool the mountain area once the several years popular, the general time underproduction about 20%, the serious popular time underproduction can reach more than 50%, sometimes also can increase the weight of the generation and the hazard rating of diseases such as corn root-rot, stem rot, have a strong impact on maize yield and quality.In recent years, because the establishing in large scale of susceptible variety and the appearance of new microspecies, disease is rise trend again.
Corn southern leaf blight (Bipolaria maydis) claim the corn spot disease again, is the important leaf diseases in China warm moist corn producing region.The sixties in 20th century, because big area is promoted susceptible cross-fertilize seed, causing harm of corn southern leaf blight is on the rise.After the seventies, along with the popularization of disease-resistant variety, the generation of corn southern leaf blight and harm are under control substantially, but because the big area simplification plantation and the Global Climate of disease resistance kind warm, corn southern leaf blight still happens occasionally in China, and has and increase the weight of trend gradually, and loss is serious.
At present, the method for preventing and treating to these three kinds of leaf spots mainly contains cultivation disease-resistant variety and chemical agent control.But because the big area of single disease-resistant variety is promoted, normal giving in the production brings the time of certain leaf spot popular, and loss is serious; And corn leaf spot pathogenic bacteria physiological strain is changeable, usually causes the forfeiture of disease-resistant variety resistance.
Chemical prevention occupies an important position on world's agriculture development history.For centuries, chemical pesticide is being brought into play positive effect in the control of phytopathy worm, grass, the plague of rats, for the stable yields of agricultural, increase production, retrieve a loss and improve quality and made significant contribution.But, influence non-target organism and cause the resistance generation of harmful organism and serious problems such as develop rapidly simultaneously also because of excessive using with pesticide abuse causes soil fertility decline and environmental pollution.Especially in recent years because pesticide residue exceed standard in the agricultural-food, cause person poultry poisoning's incident constantly to take place, the people's quality of life in serious threat, has also limited the foreign export of agricultural products in China.The potentially contaminated of chemical bactericide has become primary key issue in the agricultural safety in production.Along with the development of social civilization, the agricultural sustainable development problem makes human understanding to resource and environment that new leap arranged, and agricultural chemicals has been proposed efficient, low toxicity, environmentally friendly requirement.Countries in the world are also in the use of minimizing chemical synthetic pesticide that takes practical steps, just announced to cancel the registration of 91 kinds of chemical pesticides 90 years 20th century as U.S. EPA, Holland, Denmark have just formulated 5 years and the ten year plan that reduces half agricultural chemicals usage quantity in nineteen ninety, and European Union has also formulated similar plan.China has also cancelled the registration of tens kinds of chemical pesticides.For solve agricultural-food improve output and ensure the quality of products between contradiction, reach the great disease and pest of integrated control vegetables, reduce the purpose of chemical pesticide usage quantity, development biological control technology more and more receives national governments, scientific worker and the common people's concern.The chemical agent that is used for the control of maize leaf spot on producing at present mainly is the sterilant of using always, and the curved mould leaf spot of spore is doubly sprayed with 600-800 such as 50% Tuzet, 80% anthrax good fortune U.S., 50% thiram; Northern and southern leaf blight can doubly carry out the foliar spray control with medicament 500-800 such as 40% Hinosan, 50% derosal, 75% zinc manganese ethylenebisdithiocarbamates, but effect is not very desirable, and chemical pesticide is easily given human and environment brings a lot of negative impacts, and people are seeking effectively, the prophylactico-therapeutic measures of environmental protection.
Bacteria agent is good with its low toxicity, environment compatibility, have the ability of building the group at the crop seedling rhizosphere, lasting period is long, harmful organism is difficult for characteristics such as it develops immunity to drugs are paid close attention to widely and paid attention to, advanced in the world at present farming company plays an active part in the research and the marketization of bacteria agent mostly, particularly beneficial bacteria has been obtained certain progress in the control of soil biography, seed-borne disease, and the application that has series product to succeed, obtained remarkable economical and ecological benefits.Biological control is being brought into play more and more important effect as important measures of integrated control in the plant pest comprehensive regulation.
Bacillus subtilis Pseudomonas (Bacillius subtilis) is a class biocontrol bacteria that relatively is subjected to extensive concern at present.It is wide to distribute with it, easily separated cultivation, can produce the stronger gemma of resistance, storage period is long, characteristics such as easy to use, become a kind of ideal biological and ecological methods to prevent plant disease, pests, and erosion microorganism, after Johnson report producing bacillus subtilis in 1945 is given birth to antimicrobial substance, the research worker of various countries is expected to become a kind of biological control factor and expresses very big concern it since over half a century, and on various crop, carried out controlling widely disease and can test, many successful Application are arranged, in production practice, show tangible disease-preventing and yield-increasing effect, also further proved the stability of genus bacillus biocontrol fungicide in product, with the consistency of chemical pesticide and different plant not the same year preventive effect the consistence aspect obviously be better than non-genus bacillus and fungi biocontrol microorganisms.Chinese scholars successively reported respectively have the biological control effect each not the bacillus subtilis of homophyletic system biological property, grow correlative study such as condition, antimicrobial substance physicochemical property and surely in the research that the situation of growing, potential inducing plant produce disease resistance and promote aspects such as growth of seedling of deciding of Different Crop different sites.There are some researches show, embrace because of genus bacillus sprouts in producing that extremely strong anti-adversity ability is arranged, compare the biological and ecological methods to prevent plant disease, pests, and erosion factor of other types, more help the production of microbial inoculum, formulation processing is survived in environment, grows surely and breeding.Therefore, screening is one of most effectual way of the relevant Plant diseases of control to the inhibited subtilis of pathogenic bacteria.Through retrieval, also do not find to be used for the subtilis of control of maize leaf spot.
Summary of the invention
One of the object of the invention is at the single easy forfeiture disease resistance of disease-resistant variety, and cost height and problem of environment pollution caused that chemical pesticide control brought, a kind of efficient, active bacillus subtilis strain of broad-spectrum sterilization of having that can be used for biological control is provided.
Second purpose of the present invention provides a kind of microbiobacterial agent that utilizes subtilis production and preparation method thereof.
The 3rd purpose of the present invention provided the purposes of subtilis of the present invention on the control of maize leaf spot.
The present invention is achieved through the following technical solutions:
A kind of bacillus subtilis strain Corn-1 (Bacillius sub, lis Corn-1) is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 14th, 2006, and deposit number is CGMCC No.1778.
The microbiobacterial agent that utilizes above-mentioned subtilis Corn-1 to produce, its activeconstituents are subtilis Corn-1 thalline and its born of the same parents' extra-metabolite.
The mentioned microorganism microbial inoculum can be liquid formulation.
The preparation method of mentioned microorganism microbial inoculum comprises the steps: actication of culture, seed liquor preparation and fermentation culture.
The preparation method of mentioned microorganism microbial inoculum specifically comprises the steps:
(1) actication of culture: the Corn-1 bacterial strain of cryopreservation is activated on the LB plate culture medium, and picking list bacterium colony expands numerous standby on the LB slant medium;
(2) preparation of seed liquor: make the LB liquid nutrient medium according to a conventional method, behind the high pressure moist heat sterilization, insert step (1) activatory bacterial strain Corn-1, shake down at 26～34 ℃ and cultivated 22～25 hours, cultivate obtained strains Corn-1 as seed liquor;
(3) fermentation culture: add sucrose soybean cake powder substratum and the water that mixes to fermentor tank, initial pH value is adjusted between 6.7～7.2 with NaOH, treating after the sterilization that temperature drops to below 30 ℃ the seed liquor that adopts negative pressure to insert step (2), is that 26～34 ℃, air flow are 1: 0.3～1.0, stirring velocity is that 150～230rpm condition bottom fermentation is cultivated 22～36h in temperature;
(4) detect thalline quantity in the fermented liquid, treat that the gemma rate reaches 90% in the fermented liquid and can stop fermentation culture; Gained is the liquid preparation of Corn-1 bacterial strain.
Culture temperature described in the above-mentioned preparation method is preferably 30～32 ℃, and air flow is preferably 0～5h ventilation than 1: 0.3～0.5; 5-10h 9.5～11.5m
3/ h (ventilation was than 1: 0.5); 20～22m behind the 10h
3/ h (ventilation was than 1: 1), stirring velocity is preferably 170～220rpm, and incubation time is preferably 22～25h.
The described sucrose soybean cake powder of above-mentioned preparation method substratum can add vegetables oil or organic oil as foam killer.
The weight percent of vegetables oil can be 1～2% described in the above-mentioned preparation method; The weight percent of described organic silicone oil can be 0.4～1.0%.
Sucrose soybean cake powder substratum described in the above-mentioned preparation method, its moiety and weight percent thereof are: sucrose 2.5～3.5%, soybean cake powder 1.5～2.5%, NaCl 0.1～0.3%, CaCO
30.1～0.3%, KH
2PO
4
0.01～0.04% and MgSO
47H
2O 0.02～0.04%, and all the other are water.
The preparation method of above-mentioned sucrose soya-bean cake substratum takes by weighing sucrose, soybean cake powder, NaCl, CaCO according to weight percent
3, KH
2PO
4And MgSO
4, pour in the fermentor tank after then they being mixed, add water to volume required getting final product again.
Air flow described in the above-mentioned preparation method is that per minute feeds the volume of air of fermentor tank and the volume ratio of fermentation cylinder for fermentation liquid.
The mentioned microorganism microbial inoculum, the viable count of its subtilis Corn-1 is greater than 5 * 10
9Cfug.
The application of mentioned microorganism microbial inoculum on controlling plant diseases.
The application of mentioned microorganism microbial inoculum on the control of maize leaf spot, described corn leaf spot comprise that corn bends spore leaf spot, the leaf blight of corn and corn southern leaf blight.
Bacillus subtilis strain Corn-1 between 62.50%～94.22%, has shown wider antimicrobial spectrum to the bacteriostasis rate of 11 kinds of pathogenic fungies such as cotton rhizoctonia solani, cotton-wilt fusarium, rhizoctonia cerealis, gaeumannomyces graminis, root rotof flax bacterium, fusarium graminearum, corn sheath blight fungus, stenocarpella maydis, botrytis cinerea, tomato early blight bacterium and cucumber fusarium axysporum.
Bacillus subtilis strain Corn-1 does not injure phenomenon to 7 kinds of staple crops (comprising unifacial leaf and dicotyledons) corn, wheat, cotton, soybean, cucumber, tomato, eggplant, and performance is to crop safety.
The using method of microbial inoculum of the present invention: with above-mentioned gained microbiobacterial agent dilute with water, liquid bacterial agent after will diluting then carries out foliar spray before the corn leaf spot onset peak, also can be sprayed on the face of land of corn field simultaneously, can reach the purpose of control control of maize leaf spot.
The screening and separating process of Corn-1 bacterial strain
To be Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie separate from the soil of the sick garden of its cotton verticillium wilt the Corn-1 bacterial strain obtains.Soil sample was gathered from 5 in field, the sick garden of its cotton verticillium wilt by Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie in 2004; taking by weighing 10g behind the mixing is put in the sterilization triangular flask of 250mL; add the 100mL sterilized water; be put on the shaking table; the 170r/min 30min that vibrates; leave standstill 2h, get supernatant liquor 1mL and add sterilized water 9mL, 10mL10
-2Times soil microorganisms suspension is diluted to 10 with soil supension then
-3, 10
-4, 10
-5, 10
-6Times diluent, each concentration are got 200 μ L microorganism suspensions and are applied on PDA, improvement PDA, LB and the KB culture medium flat plate, and each concentration repeats for 3 times, cultivates 1d, 3d and 5d respectively at 28 ℃ of constant temperature, carries out bacterium respectively, and fungi separates and purifying with actinomycetic.And be the target bacterium with the curved spore leaf spot pathogenic bacteria (Curvularia lunata) of corn, leaf blight of corn pathogenic bacteria (Exserohilum turcicum) and corn southern leaf blight pathogenic bacteria (Bipolaria maydis) respectively, carry out antimicrobial screening short of money.The result therefrom filters out one each is had the bacterial strain of obvious fungistatic effect for examination bacterium, names to be Corn-1.
The feature of Corn-1 bacterial strain:
(1) morphological specificity
It is shaft-like cultivating thalline on the LB substratum, produces gemma behind the cultivation 8h, give birth in the gemma, and ellipse, sporangiocyst does not expand, the acid-fast stain feminine gender, no parasporal crystal can move flagellum Zhousheng.On nutrient agar plate, cultivate initial stage bacterium colony light oyster white, the purulence shape, circle, neat in edge, the bacterium colony protuberance is steamed bun shape, surface wettability; The late stage of culture bacterium colony is faint yellow, and the edge is irregular, and surface drying has fold; Streak culture on the nutrient agar medium inclined-plane, shape linearly; Static cultivation in the liquid medium within, the surface forms white mycoderm.
(2) 16SrDNA sequencing
The primer of bacterial 16 S rDNA of being used to increase is: 530F (5 '-GTGCCAGCMGCCGCGG-3 ') and 1494R (5 '-GGYTACCTTGTTACGACTT-3 ').Genomic dna with Corn-1 is template amplification 16S rDNA, and checks order, and the 16SrDNA sequence of Corn-1 is seen sequence table as a result.The 16SrDNA sequence of gained Corn-1 is carried out homology relatively in Genbank, the 16SrDNA homology of results strain Corn-1 and Bacillus subtilis reaches 98% (among Fig. 2, "-" expression is identical with the corresponding base of Corn-1 in the Bacillus subtilis isolate BS-2 sequence, " g, a, c, t " expression is different with the base in the corresponding site of Corn-1), illustrate that the Corn-1 bacterial strain belongs to the subtilis of bacillus (Bacillus) (Bacillus subtilis).
(3) physiological and biochemical property
Corn-1 bacterial strain and subtilis reference culture 1504 (Chinese culture presevation management committee) are carried out the Physiology and biochemistry proterties detect (the results are shown in Table 1) under identical experiment condition.As can be seen from Table 1, the physiological and biochemical property of Corn-1 bacterial strain is identical with subtilis reference culture 1504, illustrates that the Corn-1 bacterial strain belongs to a kind of subtilis.
Table 1 bacterial strain Corn-1 qualification result
Annotate: a represents that Corn-1 also can grow in 10%NaCI."+" expression positive findings, "-" expression negative findings.
The conventional identification handbook of bacterium (" general bacterium authentication method commonly used " etc.) show that subtilis is movable, Gram-positive, gemma ellipse or column, middle life or the middle partially catalase test positive, V-P tests positive, grow in the 7%-10% sodium-chlor, produce acid from glucose, pectinose, wood sugar and N.F,USP MANNITOL, hydrolyzed starch, utilize Citrate trianion as carbon source, reduction nitrate becomes nitrite, separates casein, the egg yolk reaction feminine gender, on glucose and tyrosine agar, do not form melanochrome, do not utilize propionic salt, do not decompose characteristics such as tyrosine.The part Physiology and biochemistry proterties detected result of above Corn-1 bacterial strain is compared with the subtilis reference culture, (Chinese Academy of Sciences's microbial bacterial research division bacteria group is compiled according to " general bacterium authentication method commonly used ", Science Press, 1978), " bacillus ") (Cai Miaoying, war Garrick etc. is translated, industry press, 1988) and " microbial taxonomy " (Zhang Jizhong, Fudan University in Shanghai press, 1995) key is retrieved, and determines that the corresponding proterties with subtilis of bacterial strain Corn-1 Physiology and biochemistry proterties is identical.Simultaneously 16SrDNA sequence and analyze the evolutionary tree that obtains according to the 16SrDNA sequence with DNAMAN Version 4.0 and tie (Fig. 1) and further confirm the biochemical identification result identifies that the Corn-1 bacterial strain is subtilis (Bacillus subtilis).
Advantage that the present invention had and beneficial effect: (1) the invention provides the bacillus subtilis strain of Plant diseasess such as a kind of energy broad-spectrum high efficacy control of maize leaf spot, and a kind of new way of biological control is provided for Plant diseasess such as control of maize leaf spots; (2) fungistatic effect of the present invention is remarkable, and the fungistatic effect of pathogenic bacteria is reached 60.14%～94.22%; (3) antimicrobial spectrum of the present invention is wide, and it is not only to corn leaf spot, and many pathogenic fungies such as cotton rhizoctonia solani, cotton-wilt fusarium, rhizoctonia cerealis are all had good inhibitory effect; (4) preparation method of the present invention is simple, cost is low, be easy to apply; (5) the present invention is pollution-free, and is nuisanceless, and low residue is environmentally friendly, has vital role to improving agricultural product quality and output, can improve the world of agricultural products in China simultaneously and strive power unexpectedly.
Description of drawings
Fig. 1 analyzes the evolutionary tree result who obtains according to the 16SrDNA sequence with DNAMAN 5.2.2.
Fig. 2 is the 16SrDNA gene order homology comparative result of subtilis Corn-1 bacterial strain and subtilis BS-2 bacterial strain (Bacillius subtilisisolate BS-2).
Embodiment
Come further clearly to explain the present invention with specific embodiment below, but be construed as limiting the invention never in any form.Experimental technique among the following embodiment if no special instructions, is ordinary method; Percentage composition among the following embodiment if no special instructions, is weight percentage.
The preparation of Corn-1 microbiobacterial agent, carry out according to following steps:
(1) actication of culture: the bacterial strain Corn-1 that will be stored in-40 ℃ activates (30 ℃) on the LB plate culture medium, and picking list bacterium colony expands numerous standby on the LB slant medium;
(2) preparation of seed liquor: make the LB liquid nutrient medium according to a conventional method, LB nutrient solution 200mL packs in the 500mL triangular flask, the high pressure moist heat sterilization, after treating that temperature drops to room temperature, insert the good Corn-1 bacterial strain of the above-mentioned activation of step (1) one transfering loop in every bottle, on shaking table, carry out shaking culture, rotating speed 170rpm, and under 30 ℃, cultivated 22h hour, gained can be used as seed liquor;
(3) preparation of sucrose soya-bean cake substratum: in the 500L fermentor tank, add sucrose 10.5kg, soybean cake powder 7kg, NaCl 0.35kg, CaCO
30.35kg, KH
2PO
40.07kg and MgSO
47H
2O 0.105kg adds water 350L then, mix sucrose soya-bean cake substratum; At 121 ℃ sucrose soya-bean cake substratum was sterilized 30 minutes, cool to again 30 ℃ standby;
(4) fermentation culture: to step (3) gained sucrose soya-bean cake inoculation of medium step (2) gained seed liquor 1.75L; 30 ℃ of constant temperature culture 17 hours, became 35 ℃ of constant temperature culture later in 17 hours, be aided with rotating speed 180rpm simultaneously, tank pressure 0.05-0.06Mpa, aeration condition is respectively: 0-5h 6.3m
3/ h; 5-10h 10.5m
3/ h; 21m behind the 10h
3/ h carries out fermentation culture;
(5) after the 17h, every 30 minutes from the fermentor tank of step (4) sampling carry out microscopy, the gemma in the visual field and total thalline number are counted, and are calculated the gemma rate; The gemma rate reaches 90% can stop fermentation culture; Fermentation culture 22～25h gets subtilis Corn-1 liquid preparation.
This test is carried out in the greenhouse of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, selecting for use corn to bend spore disease pathogen bacterium the Liao Dynasty 9605, No. 1 microspecies of leaf blight of corn pathogenic bacteria and corn southern leaf blight pathogenic bacteria 2010-1 respectively is the infective pathogen bacterium, three kinds of pathogenic bacterias are the Yellow River and Huai He River Haiti district corn leaf spot dominant races, are provided by plant protection institute of Hebei Prov. Academy of Agricultural ﹠. Forest Sciences integrated control laboratory; Corn southern leaf blight and curved spore leaf spot are selected susceptible variety 9831 for use, and susceptible variety 8112 is selected in leaf blight of corn experiment for use, by being provided by plant protection institute of Hebei Prov. Academy of Agricultural ﹠. Forest Sciences integrated control laboratory.In flowerpot, grow seedlings, 10 seedlings of every basin, every processing repeats for four times.The subtilis Corn-1 microbial inoculum water of embodiment 1 gained is diluted (mycetome 10
8Cfu/mL), evenly be sprayed on the maize seedling blade face with throat spray, inoculate pathogenic bacteria (10 spore/visuals field of big spot bacterium under the low power lens behind the 24h respectively with the breeding of sorghum grain substratum, stigma bacterium 20-30/visual field, Curvularia lunata 100-200/visual field), the cultivation (big spot bacterium 20 ℃, about 30 ℃ of stigma bacterium and Curvularia lunatas) of preserving moisture.Treat the blank sick level of " Invest, Then Investigate " of fully falling ill, and calculate disease index and preventive effect.The corn leaf spot diseased plant rank criteria for classifying and preventive effect method of calculation reference " State Standard of the People's Republic of China's pesticide field efficacy medicine test criterion " (two) the 107th parts: bactericidal agent for preventing and treating corn northern and southern leaf blight (the results are shown in Table 2).
The result shows that subtilis Corn-1 all has the better prevention effect to three kinds of leaf spots in the greenhouse, and the preventive effect of corn being bent spore leaf spot, leaf blight and helminthosporium maydis is respectively 100.00%, 83.30% and 100.00%.
Table 2 in the greenhouse subtilis Corn-1 to the preventive and therapeutic effect of corn leaf spot
Embodiment 3
This test is carried out in the experimental plot of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie, every sub-district two row, and four repetitions, district's group is arranged at random.(water dilutes, mycetome 10 to adopt knapsack hand sprayer inoculation pathogenic bacteria and embodiment 1 prepared subtilis Corn-1 microbial inoculum
8Cfu/mL); Other establishes contrast medicament (45% amobam SL, commercial, Hebei Shuan Ji Chinese workers company limited produces, 1500 times of water diluent spraying processing) and blank (clear water spraying); Treatment process is watered sufficient water with embodiment 2 behind the inoculation pathogenic bacteria.Treat the blank sick level of " Invest, Then Investigate " (with embodiment 2) of fully falling ill, and calculate disease index and preventive effect.Test-results shows (seeing Table 3), and the disease index that the microbial inoculum of subtilis Corn-1 is handled back three kinds of corn leaf spots significantly is lower than blank, and its preventive effect reaches 43.56%～52.04%.Bacillus subtilis strain Corn-1 has all showed the better prevention effect to three kinds of leaf spots, preventive effect has reached 52.04% respectively, and (corn is bent the spore leaf spot, be significantly higher than medicament contrast), 43.56% (Exserohilum turcicum, be equivalent to the medicament contrast) and 48.16% (southern corn leaf blight is significantly higher than the medicament contrast).
Table 3 is at the action effect of field subtilis Corn-1 to corn leaf spot
Embodiment 4
This test is carried out in the greenhouse of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie.Processing is divided into thallus suspension liquid and removes thalline born of the same parents extra-metabolite.Subtilis Corn-1 microbial inoculum dilution (mycetome 10 with embodiment 1 gained
8Cfu/mL), then Corn-1 microbial inoculum diluent is divided into two parts, portion is used for direct spraying (being treated to nutrient solution, mycetome and meta-bolites); A under 5000 rev/mins of conditions centrifugal 20 minutes, supernatant liquor was born of the same parents' extra-metabolite of subtilis Corn-1; Precipitation is thallus suspension liquid (mycetome 10 with 0.9% physiological saline suspension of equivalent before centrifugal
8Cfu/mL); Water is as blank.Result's (seeing Table 4) shows, the disease index difference that the nutrient solution of subtilis Corn-1 (mycetome and meta-bolites), born of the same parents' extra-metabolite, thallus suspension liquid are handled is not remarkable, but all significantly be lower than blank, illustrate that subtilis Corn-1 born of the same parents extra-metabolite and viable bacteria body all have preventive and therapeutic effect to corn leaf spot.Therefore the effective active composition of the curved spore leaf spot of subtilis Corn-1 control of maize comprises thalline and the meta-bolites thereof of subtilis Corn-1.
The outer metabolite of table 4 subtilis Corn-1 thalline and spore is to the prevention effect of the curved spore leaf spot of corn
Embodiment 5,
Adopt the face-off culture method to detect the restraining effect of subtilis Corn-1 bacterial strain, comprising: corn sheath blight fungus (Rhizoctonia solani) 11 kind of plant pathogenic fungies, cotton rhizoctonia solani (Rhizoctonia solani), cotton-wilt fusarium (F.oxysporium f.sp.vasinfectum), botrytis cinerea (Botrytis cinerea), tomato early blight bacterium (Alternaria solani), cucumber fusarium axysporum (Fusarium oxysporum f.sp.cucumerinum), stenocarpella maydis (F.graminearum), fusarium graminearum (F.graminearum), rhizoctonia cerealis (R.cerealis), gaeumannomyces graminis (Gaeumannomyces graminis), the antimicrobial spectrum of root rotof flax bacterium (Alternaria triticna).These 11 kinds of pathogenic fungies and subtilis Corn-1 bacterial strain are preserved by Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie biocontrol of plant disease laboratory.Concrete grammar is as follows:
Subtilis Corn-1 inhibition pathogenic fungi effect detection adopts dull and stereotyped face-off culture method, and subtilis Corn-1 inclined-plane activates 48h down for 28 ℃, and is to be measured.Get 11 kinds of pathogenic fungies that this testing laboratory preserves, on the PDA substratum, activate 5d, beat the bacterium sheet of getting the cell age unanimity with the punch tool of diameter 5mm at colony edge then, mycelia is transferred to the dull and stereotyped central authorities of another PDA down, be " product " font inoculation tested bacteria in three weeks of bacterium sheet apart from bacterium sheet 2cm place, not connect bacterium is contrast, every processing repeats for 4 times, be put in 25 ℃ of constant temperature culture 5d, the spacing (antibacterial band) to the ash arrhizus bacteria colony edge is put in measurement processing colony radius and microbionation, determine bacteriostatic level, calculate bacteriostasis rate.
Subtilis Corn-1 is to for examination pathogenic fungi restraining effect result (as shown in table 5): subtilis Corn-1 to for the bacteriostasis rate of 11 kinds of pathogenic fungies of examination between 60.14%～94.22%, bacteriostasis rate to gaeumannomyces graminis has reached 94.22%, bacteriostasis rate to the root rotof flax bacterium is minimum, be 60.14%, illustrate that bacillus subtilis strain Corn-1 of the present invention has wider antimicrobial spectrum.
Table 5 bacillus subtilis strain Corn-1 is to the restraining effect of plant pathogenic fungi
Embodiment 6
Indoor seed germination security detects: with 5 times, 10 times of the subtilis Corn-1 bacterium liquid dilutions of embodiment 1 gained and stoste respectively as three processing, if clear water is a blank, wheat, corn, cotton, cucumber, eggplant, soybean, 7 kinds of staple crop seeds of potato (potato seed) are soaked seed handled 30 minutes, be sowed in the flowerpot then, 50 seeds of every processing or potato seed repeat 3 times.Each processing of observation is to the security of seed germination.The result shows (table 6), and 7 kinds of budding gesture such as the wheat after the bacteria suspension kind is handled, corn, cotton, cucumber, eggplant, soybean, potato and percentage of germination and contrast do not have significant difference.
Indoor foliar spray detects: respectively as three processing, establishing clear water is blank with 5 times, 10 times of the subtilis Corn-1 bacterium liquid of embodiment 1 gained dilutions and stoste, and 20 seedlings of every processing repeat 3 times; Adopt the foliar spray inoculation method to inoculate in seedling stage, the result shows (table 6), and compared with the control, subtilis Corn-1 all shows not have to the upgrowth situations of 7 kinds of crops and causes a disease and do not have injury effect, and is safe in utilization to crop.
Table 6 subtilis Corn-1 is to the influence of emerging and growing of 7 kinds of field crops
Sequence table
＜110〉Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie
＜120〉a kind of subtilis and microbial inoculum thereof and application
<160>1
<170>PatentIn?version?3.3
<210>1
<211>947
<212>DNA
<213>Bacillus?subtilis
<400>1
tatctgtcca?ccttcggcgg?ctggctcata?aaggttacct?caccgacttc?gggtgttaca 60
acttctcggg?gtgtgacggg?cggtgtgtac?aaggcccggg?aacgtattca?ccgcggcatg 120
ctgatccgcg?attactagcg?attccagctt?cacgcagtcg?agttgcaaac?tgcgatccga 180
actgaaaaca?aatttgtggg?attggcttaa?cctcgcggtt?tcgctgccct?ttgttctgtc 240
cattgtagca?cgtgtgtacc?ccaggtcata?aggggcatga?tgatttgacg?tcatccccac 300
cttcctccgg?tttgtcaccg?gcagtcacct?taaagtgccc?aactgaatgc?tggcaactaa 360
aatcaagggt?tgcgctcgtt?gcgggactta?acccaacatc?tcacgacacg?agctgacaac 420
aaccatgcac?cacctgtcac?tgggcccccg?aaggggacgt?cctatctcta?ggattgtcaa 480
aggatgtcaa?gacctggtaa?ggttcttcgc?gttgcttcaa?attaaaccac?atgctccacc 540
gcttgtgcgg?gcccccgtca?attcctttga?gtttcagtct?tgcgaccgta?ctccccaggc 600
ggagtgctta?atgcgttagc?tgcagcacta?aggggcggaa?accccctaac?acttagcact 660
catcgtttac?ggcgtgaact?accagggtat?ctaatcctgt?tcgctcccca?cgctttcgct 720
cctcagcgtc?agttacagac?cagagagtcg?ccttcgccac?tggtgttcct?ccacatctct 780
acgcatttca?ccgctacacg?tgaaattcca?ctctcctctt?ctgcactcaa?gttccccagt 840
ttccaatgac?cctccccggt?tgagccgggg?ctttcacatc?agacttaaga?aaccgcctgc 900
gagcccttta?cgcccaataa?ttccggacaa?cgctgccacc?tacgtat 947
Claims (10)
1, a kind of bacillus subtilis strain Corn-1 (Bacillius subtilis Corn-1), in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is: CGMCC No.1778.
2, the microbiobacterial agent that utilizes the described bacterial strain Corn-1 of claim 1 to produce is characterized in that its activeconstituents is subtilis Corn-1 thalline and its born of the same parents' extra-metabolite.
3, according to the described microbiobacterial agent of claim 2, it is characterized in that preparing as follows: fermentation subtilis Corn-1 in sucrose soybean cake powder substratum, then the fermented liquid that obtains is mixed with matrix, promptly obtain microbiobacterial agent.
4, the preparation method of claim 2 or 3 described microbiobacterial agents is characterized in that comprising the steps: actication of culture, seed liquor preparation and fermentation culture.
5, according to the described preparation method of claim 4, it is characterized in that comprising the steps:
(1) actication of culture: the Corn-1 bacterial strain of cryopreservation is activated on the LB plate culture medium, and picking list bacterium colony expands numerous standby on the LB slant medium;
(2) seed liquor preparation: make the LB liquid nutrient medium according to a conventional method, behind the high pressure moist heat sterilization, insert step (1) activatory Corn-1 bacterial strain, shake down at 26～34 ℃ and cultivated 22～25 hours, cultivate back gained Corn-1 bacterium liquid as seed liquor;
(3) fermentation culture: the sucrose soybean cake powder substratum that will proportionally mix is put into fermentor tank, and add entry, initial pH value is between 6.7～7.2, high-temperature sterilization, treat that then temperature drops to the seed liquor that adopts negative pressure access step (2) below 30 ℃, be 26～34 ℃ in temperature again, air flow is 1: 0.3～1.0, and stirring velocity is that the condition bottom fermentation of 150～230rpm is cultivated 22～36h;
(4) detect thalline quantity in the fermented liquid, treat that the gemma rate reaches 90% in the fermented liquid and stop fermentation culture, promptly get the liquid preparation of Corn-1 bacterial strain.
6,, it is characterized in that adding in the described sucrose soybean cake powder substratum vegetables oil or organic silicone oil according to the described preparation method of claim 5.
7,, it is characterized in that described fermentation culture temperature is 30～32 ℃ according to claim 5 or 6 described preparation methods.
8,, it is characterized in that described fermented incubation time is 22～25h according to claim 5 or 6 described preparation methods.
9,, it is characterized in that described fermentation stirring velocity is 170～220rpm according to claim 5 or 6 described preparation methods.
10, the application of the described bacillus subtilis strain Corn-1 of claim 1 on the control of maize leaf spot.
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| CN100540656C (en) * | 2007-09-03 | 2009-09-16 | 河北省农林科学院植物保护研究所 | A kind of subtilis and microbial inoculum thereof and application |
| CN101717723B (en) * | 2009-12-14 | 2013-04-03 | 北京理工大学 | Mixed cooperative fermentation process of bacillus subtilis |
| CN102732444B (en) * | 2012-02-09 | 2013-06-19 | 河北省农林科学院植物保护研究所 | Bacillus pumilus for controlling cottonbollblight and microorganism bacterium agent thereof |
| CN102628023B (en) * | 2012-03-12 | 2014-01-22 | 鹤山市新的生物制品有限公司 | Bacillus subtilis.XD08B having high ganoderma lucidum polysaccharide yield, feeding microbial preparation prepared from bacillus subtilis.XD08B and preparation method of the feeding microbial preparation |
| CN102604871A (en) * | 2012-03-27 | 2012-07-25 | 福建省农业科学院土壤肥料研究所 | Probiotics fungicide for fermenting bean pulp and preparation method thereof |
| CN103966977B (en) * | 2014-04-25 | 2016-03-02 | 王夕刚 | A kind of construction process of ecological water channel |
| CN104946553B (en) * | 2015-02-11 | 2018-06-26 | 江苏大学 | A kind of bacillus subtilis and its application in anti-radix pseudostellariae leaf spot |
| CN105859628B (en) * | 2016-04-19 | 2018-08-07 | 清远职业技术学院 | A kind of antimicrobial compound is to N imidazoles benzaldehyde contracting ortho-aminotoluene schiff bases and its preparation method and application |
| CN107043721B (en) * | 2017-03-16 | 2020-02-14 | 河北省农林科学院植物保护研究所 | Bacillus subtilis HMB28948 and application thereof |
| CN106939290B (en) * | 2017-03-16 | 2020-02-11 | 河北省农林科学院植物保护研究所 | Bacillus subtilis HMB26553 and application thereof |
| CA3145376A1 (en) * | 2019-06-27 | 2020-12-30 | Idemitsu Kosan Co., Ltd. | Plant disease control agent and plant disease control method |
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