CN102578152A - Bacillus cereus AR156 wettable powders as well as preparation and application thereof - Google Patents
Bacillus cereus AR156 wettable powders as well as preparation and application thereof Download PDFInfo
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Abstract
The invention discloses bacillus cereus AR156 (Analytical Reagent156) wettable powders as well as the preparation and application thereof. The bacillus cereus AR156 wettable powders comprise lactose, white carbon black, arabic gum, monopotassium phosphate and bacillus cereus AR156 spores. Bacillus cereus AR156 fermented concentrated solution is taken and mixed with lactose; mixed liquor is added in white carbon black with 325 meshes, and the mixture is stirred fully and is then put on a sterile tray; the mixture is dried for 11 to 13 hours under the temperature ranging from 60 to 70 DEG C, and the final moisture content can reach 20 to 30 percent; dried products are crushed, arabic gum is added during the crushing process, and then crushing is carried out again; and monosodium phosphate is added so as to adjust the pH value until the pH value reaches 7.0, so that the AR156 wettable powders are obtained. The AR156 wettable powders can successfully solve the negative influence that is caused when bacillus fermentation liquor is taken as preparation for using directly, and can achieve stable and reliable greenhouse control to tomato bacterial wilt.
Description
Technical field
The invention belongs to the pesticidal preparations field, relate to a kind of waxy Bacillus AR156 wetting powder.
Background technology
Bacillus (Bacillus spp.) is the dominant population of soil and plant microecology, as the plant rhizosphere beneficial microbe, and through secretion antibiotics and growth competition, performance multiple beneficial effect aspect controlling plant diseases.With it is that the biogenic pesticide of active ingredient exploitation is because of meeting the needs of environmental protection, food security and human health; Become the ideal substitute of chemical pesticide instantly, and acquired character is stable and bacillus preparation that be easy to transport and store is the basis of its smooth industrialization.
Bacillus can produce heat-resisting degeneration-resistant gemma in the later stage of breeding, and these characteristics make it in process, can reduce individual death to greatest extent, thereby avoid the forfeiture of active component, are beneficial to the development of microbial inoculum and the processing of formulation.And wetting powder is the formulation of technological comparative maturity in the pesticide processing; Mainly obtain through adsorbing with carrier to add auxiliary agent behind the former medicine and pulverize, with low cost, technology is simple; Convenient transportation; Because of it does not contain organic solvent as auxiliary agent, can not damage again, can not pollute yet environment to the former medicine of live body.Both are combined, can well remedy fermentation of bacillus liquid is directly used the defective that the cost of transportation that is brought is high, use inconvenience, pollute easily as preparation, so oneself becomes the main formulation of microbial manure and microbial pesticide production instantly.At present, multiple bacillus wettable powders such as existing hundred anti-, inferior treasured are successfully tested commercialization, are used for the control of crop field disease.But because the different bacteriums and the compatibility of auxiliary agent; Main frame has nothing in common with each other to the adsorptivity of bacterium; The prescription or the preparation technology of existing bacillus wettable powder are used to prepare waxy Bacillus AR156 wetting powder, and effectively the adsorbing capacity of bacterium, survival rate, preparation are all very undesirable to control efficiency and the stability of formulation of harmful bacterium in the preparation.And the selection of various auxiliary agents in the prescription; Neither those skilled in the art are just getable through the suitable adjustment of limited number of time test; Must take into account and consider that each carrier adsorption capacity, each carrier absorption oven dry back active constituent content, each carrier form the protectant effect of influence, heated drying, dispersant of the ability many factors such as influence to the preparation suspensibility to the AR156 biomembrane, can obtain just that the preparation performance is outstanding, preventive effect good, stable in storage wetting powder.
Summary of the invention
The objective of the invention is provides a kind of waxy Bacillus AR156 wetting powder to the AR156 zymotic fluid is directly used the defective that the cost of transportation that is brought is high, use inconvenience, pollute easily as preparation.
Another object of the present invention provides the preparation method of this wetting powder.
Another purpose of the present invention provides the application of this wetting powder.
The object of the invention can be realized through following technical scheme:
A kind of waxy Bacillus (Bacillus cereus) AR156 wetting powder; Said wetting powder comprises lactose, white carbon, gum Arabic, potassium dihydrogen phosphate and waxy Bacillus AR156 gemma and forms; Described waxy Bacillus AR156 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and culture presevation number is CGMCC NO.1929.
Said wetting powder prepares through following method: get waxy Bacillus AR156 fermentation concentrate, mix with lactose, add in the 325 purpose white carbons; Fully stir, place on the aseptic pallet, under 60～70 ℃ of conditions, dry 11～13h; Make final water content reach 20～30%, the oven dry product is pulverized, add gum Arabic in the crushing process; Pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein said waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 180～220: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.0135～0.0165: 1, and the mass ratio of gum Arabic and white carbon is 0.07～0.09: 1.
Get waxy Bacillus AR156 fermentation concentrate, mix, add in the 325 purpose white carbons with lactose; Fully stir, place on the aseptic pallet, under 65 ℃ of conditions, dry 12h; Make final water content reach 24%～26%, the oven dry product is pulverized, add gum Arabic in the crushing process; Pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein, described waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 200: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.015: 1, and the mass ratio of gum Arabic and white carbon is 0.08: 1.
Described white carbon is the white loose powder, and 325 mesh sieve percent of pass are 99%, and water content is less than 3%, and purity is greater than 95%; Described gum Arabic is a pale yellow powder, and purity is greater than 98%; Said sodium dihydrogen phosphate is the colourless crystallization sprills, and purity is greater than 97%.
The preparation method of described waxy Bacillus AR156 wetting powder: get waxy Bacillus AR156 fermentation concentrate, mix, add in the 325 purpose white carbons with lactose; Fully stir, place on the aseptic pallet, under 60～70 ℃ of conditions, dry 11～13h; Make final water content reach 20～30%, the oven dry product is pulverized, add gum Arabic in the crushing process; Pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein said waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 180～220: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.0135～0.0165: 1, and the mass ratio of gum Arabic and white carbon is 0.07～0.09: 1.
The preparation method of described waxy Bacillus AR156 wetting powder is preferred: get waxy Bacillus AR156 fermentation concentrate, mix with lactose, add in the 325 purpose white carbons; Fully stir, place on the aseptic pallet, under 65 ℃ of conditions, dry 12h; Make final water content reach 24%～26%, the oven dry product is pulverized, add gum Arabic in the crushing process; Pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein, described waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 200: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.015: 1, and the mass ratio of gum Arabic and white carbon is 0.08: 1.
Described white carbon is the white loose powder, and 325 mesh sieve percent of pass are 99%, and water content is less than 3%, and purity is greater than 95%; Described gum Arabic is a pale yellow powder, and purity is greater than 98%; Said sodium dihydrogen phosphate is the colourless crystallization sprills, and purity is greater than 97%.
The application of described waxy Bacillus AR156 wetting powder in the medicine of preparation control of plant bacterial wilt.
The best preservation condition of described waxy Bacillus AR156 wetting powder is 4 ℃ and keeps in Dark Place.
Beneficial effect:
The present invention is directed to waxy Bacillus AR156 and can produce degeneration-resistant heat-resisting gemma in its growth later stage; The carrier white carbon that utilization has preferable adsorption capacity adsorbs the mixture of AR156 zymotic fluid with an amount of protectant lactose; The oven dry certain hour is removed wherein moisture, improves the ratio of gemma; The oven dry product is pulverized, obtained head product (former powder).In former powder, add auxiliary agents such as suspending agent, wetting agent according to a certain percentage and pulverize again and obtain the AR156 wetting powder.Demonstration test shows: waxy Bacillus wetting powder active constituent content of the present invention can reach 1.05 * 10
10CFU/g, shelf life reaches 1 year under 4 ℃ of conditions that keep in Dark Place, and its product can reach 85.76% to the greenhouse preventive effect of bacterial wilt of tomato.
7 kinds of carriers such as white carbon are all more common in wetting powder manufacturing process; The present invention takes all factors into consideration their adsorption capacity, the content influence of absorption AR156 zymotic fluid oven dry back gemma; And carrier self flowability and the wetability that have, the white carbon performance is more excellent.The present invention has studied the relation between wetting powder kind of carrier and the bacillus AR156 biomembrane formation ability first; Method through violet staining; Find that white carbon can significantly promote the biomembranous formation of waxy Bacillus, use the defence capability of back pathogen thereby improve it.In addition, white carbon self also has the certain protection effect to the AR156 gemma, has reduced the damage of heated drying process to it, so white carbon is the most suitable as the carrier of AR156 wetting powder.
Lactose can be combined on the polar group of bacterial cell memebrane protein through hydrogen bond in the heated drying process as the heated drying protectant of said preparation, stops the gathering and the stretching, extension of protein, prevents that cell structure from being destroyed, and therefore has splendid protective effect.
Dispersant can improve the suspending power of active ingredient in the preparation, makes the aqueous solution of preparation keep favorable uniformity, and especially the AR156 gemma has hydrophobicity, and is the microorganism live body, and is very necessary to the appropriate selection of dispersant.Gum Arabic is as water-soluble high-molecular substance; Be the natural plant that condenses and form by myron, can significantly improve the suspending power of gemma in the AR156 wetting powder aqueous solution, have nontoxic characteristics again; It is individual big to solve gemma, the drawback that is difficult to suspend.
AR156 wetting powder of the present invention; Successful solution directly use the negative effect brought (cost of transportation is high, use inconvenience, pollute easily etc.) to fermentation of bacillus liquid as preparation; Raw material is easy to get, with low cost; And the organic additive that does not contain contaminated environment, this is consistent with biogenic pesticide green health, eco-friendly aim again.Through to the groping of preservation condition, can significantly improve the shelf life of AR156 preparation.Its product is reliable and stable to the greenhouse preventive effect of bacterial wilt of tomato.
In addition, because bacillus has similar characteristic mostly,, be used for the exploitation of many bacterium of bacillus mixture wetting powder so patent of the present invention also can be carried out somewhat modified.
Description of drawings
Each carrier of Fig. 1 and auxiliary agent are to the influence of waxy Bacillus AR156 survival
Active constituent content in each carrier adsorption capacity of Fig. 2 and the absorption oven dry stepmother powder
Form the contrast of biomembrane situation before and after the different female powder carrier wash-outs of Fig. 3
1 corn flour, 2 attapulgites, 3 talcum powder, 4 analysis for soybean powder, 5 bentonites, 6 kaolin, 7 white carbons, 8 zymotic fluids, 9 blanks.
The different protectants of Fig. 4 are to the protection usefulness of active ingredient in female powder
The different dispersants of Fig. 5 are to the influence of female powder suspensibility
Fig. 6 temperature (A), initial pH (B) and illumination condition (C) are to the influence of AR156 wetting powder shelf life
Fig. 7 AR156 wetting powder is to the control efficiency of bacterial wilt of tomato
Biomaterial preservation information
Waxy Bacillus AR156; Classification called after bacillus sp.; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preserving number is CGMCC NO.1929, and preservation date is on 01 26th, 2007.
Embodiment:
According to " State Standard of the People's Republic of China " waxy Bacillus AR156 wetting powder is measured in following examples.Wetting time is measured GB/T 5451-2001, and suspensibility is measured GB/T 14825-2006, and fineness is measured GB/T16150-1995, determination of moisture GB/T 1600-2001, pH pH-value determination pH GB/T1601-1993, heat endurance GB/T19136-2003.
1.1 each carrier and dispersant and waxy Bacillus AR156 compatibility detect
With carrier (white carbon, corn flour, bentonite, kaolin, attapulgite, analysis for soybean powder, talcum powder, potter's clay; Fineness is all through 325 mesh sieves), the ratio of dispersant (starch, xanthans, gum Arabic, sodium lignin sulfonate, carboxymethyl cellulose, DBSA amine, polyethylene glycol) according to 5% add (250ml triangular flask in the LB culture fluid respectively; Liquid amount 100ml) mixing; 1ml AR156 seed liquor is inserted in sterilization back, places shaking table, and 28 ℃, 180rpm are cultivated 24h.As contrast, the method for plate culture count is adopted in every processing repetition 3 times, measures thalline and gemma content in the zymotic fluid with the culture fluid of no carrier added and dispersant.Eliminate the carrier and the auxiliary agent that remove gemma growing amount influence big (compare growing amount descends greater than 40% with contrast), use all the other materials to carry out follow-up test.
After each carrier and auxiliary agent and waxy Bacillus AR156 cultivate altogether; To its survival all have in various degree influence (Fig. 1) wherein, it is bigger that talcum powder, sodium carboxymethylcellulose and dodecyl sodium sulfate generate influence to the gemma of AR156, compares with contrast; After adding above three kinds of materials; The gemma production rate descends all greater than 40%, and corn flour can improve the production rate of gemma as long-acting carbon source.
1.2 the screening of carrier
1.2.1 each carrier adsorption capacity is measured and each carrier absorption oven dry back active constituent content compares
The single bacterium colony of picking AR156 (CGMCC NO.1929) places the test tube that the LB culture fluid is housed, and 28 ℃, 200rpm cultivated 24 hours, as seed liquor.Inoculum concentration according to 5% is inoculated in optimization medium (maltose 0.25%, corn flour 0.5%, analysis for soybean powder 0.5%, tryptone 0.5%, CaCl with seed liquor
22H
2O 0.05%, MnSO
4H
2O 0.05%, K
2HPO
40.1%, boil the back and filter, transfer pH to 7.0) in, 28 ℃, 200rpm cultivates 48h, is concentrated into 25% (content that is gemma has improved 4 times) of original volumes again, obtains the concentrate that ferments, and is subsequent use.
Accurately above-mentioned each the carrier 5.0g of weighing puts into the dry beaker of 50ml, with the slow dropping AR156 zymotic fluid in the carrier of 1ml syringe, constantly stirs with glass rod simultaneously, begins to bunch up and stops dropping when not disperseing to sample.Record is added drop-wise to the volume of bacterium liquid in the carrier.Test repetition 3 times for every group, calculate the average saturated extent of adsorption of each carrier of 5.0g bacterium liquid.
The fermentation concentrate according to the average saturated extent of adsorption of above-mentioned definite each carrier to bacterium liquid, is mixed with each carrier of equivalent respectively in beaker, sample is placed constant incubator, dry under 65 ℃ of conditions, sampling detects thalline and gemma content behind the 12h.
In the wetting powder, carrier is very big to the physicochemical property influence of preparation.Former medicine is liquid preparation, selects the strong carrier of absorption property very important to preparation processing.Can know that by Fig. 2 in 8 kinds of alternative carriers, white carbon is the strongest to the adsorption capacity of fermentation concentrate, reaches 294.17ml/100g, and mobile splendid, dissolve each other with zymotic fluid and potter's clay is almost difficult.With drying after each carrier absorption zymotic fluid, obtain female powder.With the female powder of white carbon as carrier, because of the zymotic fluid of himself absorption is many, its structure itself has the certain protection effect to gemma in addition, causes that gemma content can reach 7.56 * 10 in this mother's powder
9CFU/g, active constituent content all significantly is lower than white carbon in female powder that all the other carriers are made.
1.2.2 each carrier forms the influence of ability to the AR156 biomembrane
In 96 orifice plates, add the LB liquid nutrient medium by 180 μ L/ holes, the wetting powder that different carriers is processed inserts wherein according to the principle that active constituent content equates, is contrast with the LB medium that only adds the fermentation concentrate and do not add preparation; Mixing; Add a cover, in 25 ℃ of greenhouses, leave standstill cultivation, wait to produce naked eyes it is thus clear that behind the biomembrane; Utilize crystal violet staining assay to detect, every processing repeats for five times.Thalline separates in the wetting powder that different carriers is processed, and obtains repeating above-mentioned experiment after the pure culture.
Can be known by Fig. 3, be that female powder of carrier can form fine and close biomembrane on LB culture fluid surface with the white carbon, and its dyeing liquor light absorption value under 570nm is the highest, and talcum powder, attapulgite and corn flour then can weaken AR156 and form biomembranous ability.After each carrier wash-out, utilize the thalline pure culture to repeat above-mentioned experiment, find that the light absorption value of biomembrane formation compactness extent and biomembrane dyeing liquor does not all have significant difference.
Comprehensive above experimental result, selected white carbon is as the carrier of waxy Bacillus AR156 wetting powder.
1.3 the screening of auxiliary agent
1.3.1 heated drying protectant
In the fermentation concentrate, add 1.5% glucose, 1.5% sucrose, 1.5% lactose, 1.5% cellobiose, 1.5% trehalose, 2% peptone, 2mol/L glycerine, 0.25mol/L sodium glutamate respectively; With 1.2 in the carrier confirmed mix; Make various female powder after the oven dry; Detect wherein gemma content, not compare with protectant, each handles repetition 3 times.Experimental result is seen Fig. 4, and carbohydrate has the better protect effect to the gemma of AR156.Preparation adds 0.5% lactose as the heated drying protectant, and behind the 12h heated drying, active constituent content is the highest in female powder, reaches 1.05 * 10
10CFU/g.
1.3.2 the screening of dispersant
In female powder, add certain amount of dispersant respectively, measure the suspensibility of each preparation according to the method for GB/T 14825-2006, each handles repetition 3 times.The result sees Fig. 5, and is visible in five kinds of alternative dispersants, and except that polyethylene glycol did not significantly improve the suspending power of AR156 wetting powder, all the other dispersants all presented increase in various degree to the raising of preparation suspensibility along with the increase of addition.Gum Arabic is as dispersant, and when addition reached 8%, suspensibility can reach 77.14%.
2.1AR156 the preparation of fermentation concentrate
The single bacterium colony of picking AR156 (CGMCC NO.1929) places the test tube that the LB culture fluid is housed, and 28 ℃, 200rpm cultivated 24 hours, as seed liquor.Inoculum concentration according to 5% is inoculated in optimization medium (maltose 0.25%, corn flour 0.5%, analysis for soybean powder 0.5%, tryptone 0.5%, CaCl with seed liquor
22H
2O 0.05%, MnSO
4H
2O 0.05%, K
2HPO
40.1%, boil the back and filter, transfer pH to 7.0) in, 28 ℃, 200rpm cultivates 48h, obtains gemma content and is about 1 * 10
9The zymotic fluid of CFU/mL is concentrated into 25% (content that is gemma has improved 4 times) of original volumes, obtains the concentrate that ferments, and is subsequent use.
Gemma content detecting method: adopt the method for plate culture count: get 100 μ L AR156 zymotic fluids, join in the 900 μ L aqua sterilisas, behind the concussion mixing, therefrom draw 100 μ L and join in the 900 μ L aqua sterilisas, shake mixing again.So repeatedly, until the AR156 zymotic fluid is diluted to 10 of original concentration
-7Choosing concentration is original 10
-7～10
-4The dilution of four gradients, each gradient are drawn 10 μ L, put on the LB flat board, count after placing 28 ℃ of incubator dark culturing 12h, obtain number of viable (x); Take out after placing 80 ℃ of water-baths to handle 10min this 100 μ L AR156 zymotic fluid, according to the quadrat method gradient dilution, the some plate is cultivated, and obtains gemma quantity (y).Gemma content (%)=x/y * 100.
Gemma content is greater than 97% in the fermentation concentrate of above-mentioned preparation.
2.2AR156 the preparation of wetting powder (is example with 10g)
(active constituent content is about 4 * 10 to measure the fresh AR156 fermentation concentrate for preparing among the 30ml 1.1
9CFU/ml), fully mix, add in the 10g 325 purpose white carbons, fully stir, place on the aseptic pallet, under 65 ℃ of conditions, dry 12h, final water content is reached about 25% with the 0.15g lactose.The oven dry product is pulverized, during add the 0.8g gum Arabic, pulverize again, add the 0.7g sodium dihydrogen phosphate, pH is adjusted to 7.0, obtain finished product.
The AR156 wetting powder of above-mentioned preparation is dissolved in the sterile water, carries out gradient dilution, utilize drip method to detect active constituent content in the AR156 wetting powder.Each index to finished product is measured; Wetting time is measured with reference to GB/T 5451-2001, and suspensibility is measured with reference to GB/T 14825-2006, and fineness is measured with reference to GB/T16150-1995; Determination of moisture is with reference to GB/T1600-2001, and the pH pH-value determination pH is with reference to GB/T1601-1993.
The result shows that waxy Bacillus AR156 wetting powder gemma content is 1.05 * 10
10CFU/g, pH value 7.0, moisture 25%, suspensibility 77.14%, wetting time 41s, fineness percent of pass 91.74%.
Embodiment 3AR156 wetting powder condition of storage and shelf life detect
3.1 temperature is to the influence of preparation shelf life
Make microbial inoculum as stated above, it is placed respectively under 4 ℃, 25 ℃ and 37 ℃ of conditions preserve.In the 0th, 3,7,15, the 30d sampling detects active constituent content.Each handles repetition 3 times.
The AR156 wetting powder is behind different preservation condition held certain hours, and decline in various degree can appear in gemma content.Temperature becomes positive correlation with this fall off rate, and cryogenic conditions can suppress the sprouting of gemma, makes gemma can continue to keep resting state, therefore, preserve under 4 ℃ of conditions of said preparation after 30 days, gemma content still can keep higher level (Fig. 6, A).
3.2 initial pH is to the influence of preparation shelf life
Making microbial inoculum as stated above, with sodium dihydrogen phosphate its pH value is transferred to acidity (6.0), neutral (7.0) and alkalescence (8.0), is contrast with natural pH.Place 25 ℃ of isoperibols to preserve, in the 0th, 3,7,15, the 30d sampling detects active constituent content.Each handles repetition 3 times.Under neutrallty condition, preserve after 30 days, gemma content is significantly higher than other processing (Fig. 6 B), this shows, environmental condition peracid or cross the survival that alkali all is unfavorable for gemma in the preparation.
3.3 illumination is to the influence of preparation shelf life
Make solid fungicide as stated above, be packaged in respectively in plated film packaging bag and the transparent wrapper bag, place 25 ℃ of isoperibols to preserve, in the 0th, 3,7,15, the 30d sampling detects active constituent content.Each handles repetition 3 times.Contain a certain amount of ultraviolet ray in the natural daylight, gemma had stronger lethal effect, handle, can exempt the injury of ultraviolet ray gemma through lucifuge, thus slow down gemma content fall off rate (Fig. 6, C).
3.4 shelf life detects
Get 10g bacillus subtilis AR156 wetting powder; Be adjusted to optimal pH; Place under Optimal Temperature and the illumination condition to store, active constituent content in the regular test sample, the mensuration duration is 360d; Thereby confirm that preparation stores the variation of active constituent content during the 360d, compare with the variation of this index under the normal temperature.
Comprehensive above optimum condition (4 ℃, pH7.0, lucifuge), the shelf life of detection AR156 wetting powder, when preserving 360 days, its active constituent content still can reach 6.21 * 10
9CFU/g, other conditions are constant, and when normal temperature was preserved, active constituent content was merely 2.13 * 10
9CFU/g.
The preventive effect checking of embodiment 4AR156 wetting powder greenhouse
Pathogenetic bacteria Solanaceae Raul Salmonella (Ralstonia solanacearum) is prior to cultivating 2-3 days on the YPGA flat board, and the collection thalline expands numerous, and compound concentration is OD600=2.0 (about 2.0 * 10
8CFU/ml) bacteria suspension is subsequent use.
Tomato (kind is Shanghai 903) seedling is educated in the dish of cave.After 30 days, be 1.0 * 10 with gemma content
8The aqueous solution of the waxy Bacillus AR156 wetting powder of CFU/ml and the dilution of AR156 zymotic fluid water tomato seedling respectively, every seedling 20ml.After 3 days, every seedling pouring concentration is 1.0 * 10
8The culture fluid 15ml of CFU/ml pathogenetic bacteria Solanaceae Raul Salmonella.Behind the tomato seedling pouring equivalent clear water of control group, with same method inoculation pathogen.In the test overall process, the tomato growth condition is 28 ℃, relative moisture 30%, photoperiod 12h/12h.3 repetitions are established in each processing, and each repetition is made up of 24 young plants.Each greenhouse test all repeats 3 times.
The sick grade standard that proposes in nineteen eighty-three according to Kempe writes down disease progression: 0 grade, do not have blue or green withered symptom; 1 grade, the wilting symptom appears in the blade of 1-25%; 2 grades, the wilting symptom appears in the blade of 26-50%; 3 grades, the wilting symptom appears in the blade of 51-75%; 4 grades, the wilting symptom appears in the blade of 76-100%.The computing formula of disease severity and preventive effect is following:
Waxy Bacillus AR156 wetting powder carries out bacterial wilt of tomato control greenhouse test, and the result sees table 1, Fig. 7.
Table 1AR156 wetting powder is to the control efficiency of bacterial wilt of tomato
Annotate: the different letter representations of * are handled under the significance level of P=0.05, and otherness is (LSD test) significantly, and data are 3 mean values that repeat.
Can find out that by above result the AR156 zymotic fluid is processed after the wetting powder, and its control efficiency to bacterial wilt of tomato is 85.76%, compares no significant difference with zymotic fluid.It is thus clear that the process of wetting powder does not impact the biological and ecological methods to prevent plant disease, pests, and erosion potentiality of AR156 thalline and gemma, the AR156 zymotic fluid is processed into the performance that this kind formulation can not influence its biocontrol effect yet.
The white carbon that uses among the above embodiment is the white loose powder, and 325 mesh sieve percent of pass are 99%, and water content is less than 3%, and purity is greater than 95%; Gum Arabic is a pale yellow powder, and purity is greater than 98%; Sodium dihydrogen phosphate is the colourless crystallization sprills, and purity is greater than 97%.
Claims (8)
1. a waxy Bacillus (Bacillus cereus) AR156 wetting powder; It is characterized in that; Said wetting powder comprises lactose, white carbon, gum Arabic, potassium dihydrogen phosphate and waxy Bacillus AR156 gemma and forms; Described waxy Bacillus AR156 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and culture presevation number is CGMCC NO.1929.
2. wetting powder according to claim 1 is characterized in that, said wetting powder prepares through following method: get waxy Bacillus AR156 fermentation concentrate, mix with lactose; Add in the 325 purpose white carbons, fully stir, place on the aseptic pallet; Under 60～70 ℃ of conditions, dry 11～13h, make final water content reach 20～30%, the oven dry product is pulverized; Add gum Arabic in the crushing process, pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein said waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 180～220: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.0135～0.0165: 1, and the mass ratio of gum Arabic and white carbon is 0.07～0.09: 1.
3. wetting powder according to claim 2 is characterized in that, said wetting powder prepares through following method: get waxy Bacillus AR156 fermentation concentrate, mix with lactose; Add in the 325 purpose white carbons, fully stir, place on the aseptic pallet; Under 65 ℃ of conditions, dry 12h, make final water content reach 24%～26%, the oven dry product is pulverized; Add gum Arabic in the crushing process, pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein, described waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 200: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.015: 1, and the mass ratio of gum Arabic and white carbon is 0.08: 1.
4. wetting powder according to claim 2 is characterized in that, described white carbon is the white loose powder, and 325 mesh sieve percent of pass are 99%, and water content is less than 3%, and purity is greater than 95%; Described gum Arabic is a pale yellow powder, and purity is greater than 98%; Said sodium dihydrogen phosphate is the colourless crystallization sprills, and purity is greater than 97%.
5. the preparation method of the described waxy Bacillus AR156 of claim 1 wetting powder is characterized in that, gets waxy Bacillus AR156 fermentation concentrate, mixes with lactose; Add in the 325 purpose white carbons, fully stir, place on the aseptic pallet; Under 60～70 ℃ of conditions, dry 11～13h, make final water content reach 20～30%, the oven dry product is pulverized; Add gum Arabic in the crushing process, pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein said waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 180～220: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.0135～0.0165: 1, and the mass ratio of gum Arabic and white carbon is 0.07～0.09: 1.
6. the preparation method of waxy Bacillus AR156 wetting powder according to claim 5 is characterized in that, gets waxy Bacillus AR156 fermentation concentrate, mixes with lactose; Add in the 325 purpose white carbons, fully stir, place on the aseptic pallet; Under 65 ℃ of conditions, dry 12h, make final water content reach 24%～26%, the oven dry product is pulverized; Add gum Arabic in the crushing process, pulverize again, add sodium dihydrogen phosphate pH is adjusted to 7.0 and make; Wherein, described waxy Bacillus AR156 fermentation concentrate viable bacteria content is 1～6 * 10
9CFU/ml, gemma content is greater than 97%; Described waxy Bacillus AR156 fermentation concentrate is 200: 1 with the volume mass ratio of lactose, and the mass ratio of lactose and white carbon is 0.015: 1, and the mass ratio of gum Arabic and white carbon is 0.08: 1.
7. the preparation method of waxy Bacillus AR156 wetting powder according to claim 5 is characterized in that, described white carbon is the white loose powder, and 325 mesh sieve percent of pass are 99%, and water content is less than 3%, and purity is greater than 95%; Described gum Arabic is a pale yellow powder, and purity is greater than 98%; Said sodium dihydrogen phosphate is the colourless crystallization sprills, and purity is greater than 97%.
8. the application of each described waxy Bacillus AR156 wetting powder in the medicine of preparation control of plant bacterial wilt in the claim 1～4.
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