TWI280852B - Bacillus subtile fungicide or soil improver with method - Google Patents

Abstract

This invention is related to the culture of Bacillus subtilis (CCR910203), and the usage of the culture as fungicide or soil amendment; this fungicide or soil amendment can be formulated as liquid or wettable powder, applied on the plant, soil and seed. The field trial of this inventive products show efficacy in controlling tomato bacterial wilt.

Description

1280852 IX. Description of the Invention: [Technical Field] The present invention relates to a method for preparing a Bacillus subtilis fungicide (CCRC91〇2〇3) or soil, and more particularly to the cultivation of Bacillus subtilis and subsequent preparation of a culture solution The fungicides or soil amendments of the dosage form or powder type or other dosage forms are derived from organisms and may also be referred to as biocides. [Prior Art] Strains 生ί spores and multi-function strains, different soil === for plant disease control (such as bowl bean powdery mildew) and improvement [invention content] The remaining conditions for cultivating nutrient base f The concentrate is concentrated under vacuum to remove water. Spray-dried; "body clothes, such as a good agent. Concentrate or soil according to Γ from the second Γ This unzirded body can be made into a powder type bactericide = good agent. Other dosage forms (such as tablets, emulsions, etc.) can be Liquid agent or = [Embodiment] Department::: ΪΪ草='s fungicide or soil modification preparation method, main h_〇, (胍) orphan, MPG4, _ = (_s_

MnS〇4 · h2〇^ ^ MgS〇4 · 7H2〇, CaCh · 2H2O and u are used as the medium, and the following steps are made according to the following steps: j 1280852; see Figure 1; 1. Species A: suitable for production sterilization An isolate of Bacillus subtilis or a soil amendment, such as strain CCRC910203. The following isolates did not find any function to antagonize plant diseases in the initial test. · ATCC6051, ATCC6633, ATCC7003, ATTCC7058, ATCC7059, ATCC9466, ATCC11774, ATCC15841, ATCC21228, ATCC21332, ATCC21336, ATCC21394, ATCC21556, ATCC21770, ATCC21777, ATCC21778, ATCC23059 , ATCC23856, ATCC23857, ATCC23858, ATCC23859, ATCC27505, ATCC27689, ATCC29056, ATCC33608, ATCC33677, ATCC39085, ATCC39093, ATCC39096, CCRC10257, CCRC10258, CCRC10259, CCRC10267, CCRC10872, CCRC11199, CCRC11357, CCRC12141, CCRC12142, CCRC12143, CCRC12144, CCRC12145, CCRC13836 , CCRC14192, CCRC14193, CCRC14199, CCRC14635, CCRC14639, CCRC14640, CCRC14641, CCRC14643, CCRC14645, CCRC14646, CCRC14716, CCRC14718, CCRC14719. 2. Fermentation tank B:

Bacillus subtilis is aerobic and requires a large amount of oxygen to maintain its growth. Therefore, in the case of liquid acid fermentation, it is cultured in a ventilated or relatively high oxygen supply. Ben, the large-scale industrial liquid stirred ventilated acid fermentation tank B with a capacity of 50 liters to 50,000 liters (50 tons) is used as an example to cultivate the dry grass rod g, and the fermentation tank B outside this capacity range can also be used. 3. The culture medium c: _ The substance of the culture medium C has been optimized to determine the appropriate ratio of carbon/nitrogen t ra ίο) to other micronutrients to promote the balanced production of B. subtilis and spores and metabolites. generate. The optimum culture" Bay 77 ^ example is starch 5 grams · protein 3 grams · molasses 1 gram: @ 1280852 mother powder 1 · 5 grams: corn steep immersion liquid (corn steep iiqu〇r) 〇 · 5 grams: ( NH4)2S〇4 〇· 3 g: ΜΡ〇4〇·4 g: MgS〇4 · 7H2〇0· 2 g: CaCl2 · 2M 10 mg·· MnS〇4 · Μ 6 mg. The composition of the culture medium C suitable for the growth of the cells is 5-40 g/L, powder 3-24 g/L, molasses 1-8 g/L, yeast powder 1·5-12. 0 g/L, Corn soaking liquid 〇·5-4·0g/L, (nh4)2S〇4 0· 3-2· 4 g/L, K2HPO4O· 4-3· 2 g/L, MgS〇4 · 7H2O 0· 2 -1· 6 g/L two CaCL· · 2 out 0 10-8〇 mg/L, MnS〇4 · Μ 6-48 mg, male=. B. subtilis growth retardation in a culture medium having a concentration lower than this composition: growth was inhibited in a medium having a concentration higher than this composition. The preferred medium for obtaining faster growth is composed of starch 15-30 liters, protein 9-18 gram/liter, molasses 3-6 gram/liter, yeast L"5-Q9·G gram/liter , corn soaking liquid 丨 · 5 " · 3 · G gram / liter, (10) 4) orphan η · 9 t / liter, MP 〇 4 L 2 - 2. 4 grams / liter, MgS 〇 4 · 】 8 grams, liters, CaCh·2H2. 3°-6° mg/L, MnS〇4 ·H2〇18-36耄/L. = The range of the hard-to-turn silk area is: 丨6丨15 158 eggs 15 liters, molasses 4-1 gram/liter, yeast powder ιί. m corn soaking solution 2. °-2.5 gram/liter, (10)(10) 〇· - · 269-6-2 〇 gram / liter, _ · TM 2H2 〇 40-50 mg / liter, Mn muscle · disassociation of the difference in the effect of the powder or protein ° use of the mineral salt c is not added 1280852 u The class complements it. 4. Fermentation operation D: The culture medium C of the sputum, under the following adjusted operating conditions, the production of the product, the test, the hay (4) the spore and the metabolite screen, the medium f ^ lin ship volume S, Reserve the age of 1曰21^11 pain level and Wei·___. The culture substrate is sterilized by ==:ΐ: gas. Culture medium C was maintained at 121. The time of ° depends on the acid fermentation tank. Long time ί* ‘but the shortest possible time is ok’ to avoid the heat of the culture medium, Ai. The present invention shows that the 5 liter liter fermentation tank β is maintained at 12 〇 ^, , , 5 〇〇 25 ^ mile, which is more than j soil. Vapor sterilization in the culture medium C = electricity _ steam, temperature rise, _ warm, cooling enzyme = water, ventilation, valve switch and other procedures. The sentence (4) from the 4 ° C refrigerated culture dish without the need for thermal shock (heat shock) activated bacteria in the fermenter B and other ways to expand the culture into the inoculum after inoculation to the big, leave # D temperature is 2 (M (TC, In this range, the growth of the cells is slow. The temperature of the fast-growing chicks is 24 - voyage. The faster growth is preferably c = 2: C. The spore production period (printing _, broth The pH of the acid and the alkali solution (such as sulfuric acid and hydroxide surface solution) which have been separately sterilized are maintained at 5·5-8·5, and the growth of the cells is inhibited outside the pH range. The preferred pH for rapid growth is preferred. It is 6. 0-8. 0. It is better to obtain faster growth = 6.5-7.5. During this growth process, the pH is decreased from the lag phase to the middle of the vegetative growth phase. It must be added with lye to maintain it. From the vegetative growth _ towel segment to the fresh production period, the pH rises, and the acid solution must be added: The dissolved oxygen concentration (DO) in the acid fermentation broth is maintained above 30% saturation. 8 1280852 and the amount (bacterial decomposition) phenomenon. A faster segment can be obtained, D0 decreases; the change from the mid-image and PH of the camping period from the lag phase to the vegetative growth period = two segments To: Buns production period, D0 is broken. This current fermentation tank B scramble rate is detected by the secrets / 広 ,, and vice versa (the disturbance rate can be changed) or the fermentation tank Β original factory setting ^ i; Rr5 500 When operating at the stirring rate of the old dead, the space after filtration and sterilizing: month 1 = ; ϊ; the lower limit of D0 saturation of the liquid is controlled by the acid fermentation tank. In the growth process, * Hysteresis period to #养生长ϊίϊίίη, the lower limit of iDG saturation; from the middle of the vegetative growth period to the time, the ventilation may increase to the setting · ^^^巾&® body 4 takes the big, the side contains protein The acid fermentation process can produce spores in 80-95% of cells, and the economic efficiency of the operation of the vine fermentation tank is changed. The size of the whole fermentation tank B of the germination culture changes. The present invention shows that 5 liters of liter acid is 2M, hour (10) 'called _ (four) 23 butterfly ^ 1280852 within 25 hours, 50 tons of fermentation tank within 26 hours. 5 · vacuum concentration E: After the acid fermentation is completed, the culture solution is concentrated in vacuo to remove water. Vacuum concentrator Operating conditions, the degree of vacuum is 〇·5 atm (atm), and the preferred vacuum is below 〇·1 atm. The best vacuum is 〇·〇 Below atmospheric pressure; the boiling point of the culture solution is below 6 (rc, ^good boiling point is 4 〇. (: below, the best boiling point is below 35. ,, the removal of water can be traced to the south and the product is saturated and extended Preservation period 6. Liquid preparation f: You can add a suitable and appropriate amount of additives after concentration, and adjust the water to achieve the specified product specifications, then you can make a liquid product. Add 2 ΐ 3 t Preservative, pH-adjusting acid or test solution. This liquid product has sterilization

Or the function of a soil amendment. The country \ spray drying G work> □, 彳 § E after the condensate to spray to remove water. The spores of the spores can be maintained below the mouth temperature [Donyun Baqie, the 8th powder preparation Η: and the appropriate and appropriate amount of carrier, adjuvant or diluent, carrier and bismuth product specifications can be added. Made into a powder product. Available 9. Other preparations I: Implementation (eg (4), emulsions, etc.) can be derived from the processing of liquid or powder. Method for cultivating Bacillus subtilis············································································································ Short for standard medium) consists of corn granule powder 12 g / liter, molasses 4 g / liter, yeast liter, MP0] Ar Yh rice sap liquid 2 g / liter, (10) 4) 2S 〇 4 丨 2 gram / male 40 mg Λ l μ / η liter, % S 〇 4 · 〇. 8 gram / liter, CaCl2 · _ t / cut milliliter shake flask containing (10) ml of medium. Shake flasks were cultured in an incubator at 31 ± 1 C and 2 rpm. The optical density of the cells (10), or the absorbance - coffee.) The right q 酦 in the yeast sample was transferred to a 5 liter fermentation tank to expand the culture. 5 liters ^ 酦 ^ to make _ = f quasi-medium ° in a sufficient amount of shake flask culture inoculation 5 gong and ώ 7 〇 ^ mouth ^ Lida 〇% above t liters of fermentation tank in 31 soil lt:, pH 7. 1 i# 操作 Operate under the above conditions. Under this D0 condition, the stirring rate of the fermentation tank is above __, and the aeration rate is above 1 liter/min. Reduce the amount of bacteria in the culture medium in the acid fermentation tank 1)0 to 0.5 or more, then culture the 椤 典 典 典 X X X X X X X X X X X X X X 5 () liters of the groove when 35 liters of acid fermentation tank 31 soil rc, Qiu 7. _· 1 and DO 70% saturated two, made. Under this condition, the fermentation rate of the fermentation tank is above liters per minute in 300-20 hours. The acid fermentation can be carried out until the end (after the inoculation, the bacteria body is exhausted, the bacteria have produced mature spores and released, and the second nutrient solution can be concentrated in vacuum for subsequent treatment. The yeast can also be used for bacteria. σ 〇之,囷脰0D reaches 0.5 or more as 500 liters of fermented culture, and the culture medium of the cultured liquid in the vinegar fermentation tank reaches 〇. 5 or more can be cultured r °500 'so Fermentation tank 31 rc, PH 7. 1 and DO 60% = mouth and above conditions. Under this DQ condition, ride above pm, the ventilation is above 15 liters / minute. Fermentation can be seen to see = 11 1280852 The sub-injection is exhausted, the bacteria have been matured, and the fermentation can be carried out until the culture of sn can be concentrated in vacuum for subsequent treatment. #体(10)Achieves G. 5 or more as 5000 liters (5 nutrient solution ^D〇 reaches 〇·5 or more can be used to raise the standard medium of Peisheng. Under the condition of more than the saturation saturation in the tank, the fermentation tank 31± 1 C, Qiu 7· _. 1 and DO 50% 50 rpm or more, the aeration rate is %n /. Under this D〇 condition, the stirring rate of the fermentation tank is within 25 hours after the inoculation. The acid fermentation can be carried out until the end (the bacterium is released and the bacteria are released in vitro. The mature spore fermentation can be carried out until the culture is carried out, and the cold liquid can be concentrated in a vacuum for subsequent treatment. Tons) , the solid limb D reaches 0.5 or more as 50,000 liters (50 when the 5,000 liter fermentation tank is cultured, the culture medium is transferred to 5_male mD〇 to reach 〇. 5 or more can be used to raise the _ liter of fresh medium and DO Conditions above 50% saturation rise f 31 ° 1 C, pH 7. 0 ± 0.1 rate above 2 rpm, aeration rate; 乍 2 second = fermentation tank stirring (about 26 hours after inoculation) Cultivate its heart. The acid yeast is carried out to the most mature spores and the bacteria are released in vitro. The _, _, and _ have been produced. Bu, Pei, can be vacuum-concentrated, follow-up Example 2 The method of sterilizing or soil improvement is condensed with the cultivating solution: 5:2 fertilizer, this method can also eliminate the problem of the residual nutrient 12 1280852 in the broth. Add the appropriate amount of water and acid or alkali. Liquid to the above concentrated liquid, so that the spores can be produced in a concentration of 1〇9/m^ or more and the pH is 7·0±0·5. In addition, the above-mentioned liquid shrinkage machine can completely remove water after the spray outlet temperature is below 70 ° C. The powder can be obtained by adding an appropriate amount of clay powder to the powder so that the number of spores reaches 1 〇 9 / gram or more. The powder product can be prepared. Example 3. The field test sample of the dosage form Bacillus subtilis fungicide or soil improver · The liquid product in the example! diluted with water 400 or 800 times plus the appropriate amount of interface. Exhibitor, if added with fertilizer grade amino acid solution diluted 1〇〇〇 times mixed and allowed to sit for more than γ hours will help the activation of B. subtilis spores. Apply this dilution mixture to more than 200 liters per metric f The rate is evenly sprayed on the stems and leaves of the newly-occurring pea seedlings, and the daily application - times 'four times in a row, can prevent the white bean white reduction and the plant growth potential is strong, silk recorded, fruit is large and beautiful. The test details are as follows : Baitai Biotechnology Co., Ltd., Field Test Report, June, 84 = Test Purpose: To investigate the effect of Bacillus subtilis or soil amendment 4% solution (s) on the control of pea powdery mildew. · Bacillus subtilis 4% s _ three. Test design: Pan 1 · Test unit: Technology Promotion Department of Baitai Biotechnology Co., Ltd. _ 2·Test time: April, 1994~May, May 3·Test location: Taichung Xinshe 4·Test crop: wrist Bean 5·Test method: Apply at the beginning of the disease, and apply once every 7 days, ping-pong four times, spray the whole agent evenly when spraying. /, 6·Field design: Full area of the machine Set design, 4 plants per plot, three replicates. 7.·Pharmaceutical treatment: 13 1280852 枯·Bacillus subtilis 4%S 400X Β·Bacillus subtilis 4%S 800X C·Pingke seat 10.5% 1500X D·Control area no application 8. Date of application: A. 84/4/16 Β· 84/4/23 C· 84/4/30 D. 84/5/7 9·Investigation date·· 84/5/14 'Statistical disease ΐ 〇·Efficacy investigation: After the last application, the questionnaire is listed every other week, and the results are observed. ~ Test agent Bacillus subtilis 4% S Bacillus subtilis 4〇 / 〇S Pingke seat 10. 5% Control group dilution factor 400 times 4 · 9 800 times 6. 8 1500 times 3.2 25.6 IV. Test results: Note: Spraying the dry grass g _ domain plants grow vigorously, the knot is HiHi month to 7 ribs), the fruit is large and beautiful (color green light, bright). There was no phytotoxicity during the month of the test. The control effect of 4% s of bacillus on cowpea powdery mildew is 仫 and the plant grows vigorously, the result period is long, and the fruit is large and beautiful. Example 4 Powder type dry turn a miscellaneous _ or earth view good _ interlaboratory example 1280852 The powder product in example 2 is diluted 1000 times with the secondary solution, diluted 6 times () () times, if added with fertilizer grade amino acid activation . This dilution mixture above H will help the planting of the spores of the 8th spores. (4) Seedling roots = ^ 500 ml or more application rate perfusion for more than 4 times, can be controlled in the near soil, every 7 days, Continuous fruit is big and Wu Li. The details of the interest are as follows: Qianbaitai Biotechnology Co., Ltd., Field Test Report, October 1990, 瞧Milk Powder II. Test Agent: Bacillus subtilis 5〇〇/JP II. Test Design: 1. Test Unit: 100 Tai Biotech Co., Ltd. Technology Promotion Department 2·Test time: July 1990~90 August 3. Test location: Puli, Nantou 4·Test crop: Tomato 5. Test method: 10 days after crop transplant, bandit Perfusion, four times in a row for seven times. 6. Field design: The design of the complete area of the harvesting machine is 50 plants per plot and three repetitions. 7.·Pharmaceutical treatment: A·Bacillus subtilis 5〇%WP 600X Β·CuO hydroxide 77% WP 800X C. Control area is not applied 8. Application date: A. 90/7/10 B. 90/7/17 C. 90/7/24 D· 90/7/31 9. Date of investigation: 1280852 A· 90/7/17 B· 90/7/24 C· 90/7/31 D· 90/8/6 E. 90/8/13 F· 90/8/20 10·Efficacy investigation: investigate after each application and check the results. The statistics of the rickets are listed, and the results are as follows: 1. Since the previous two investigations have not occurred, they are not listed. The area of the plant grows vigorously. Bacillus subtilis

2. The third investigation of rickets (90/7/31) treatment of rickets rate 枯 · Bacillus subtilis 50% WP 600X Β · copper hydroxide 77% WP 800X C · control no application area Note: regional plants infused with Bacillus subtilis The growth is strong. 1 2 3 Average 0 0 0 0 0 2 0 0.66 3 5 3 3.66 3. The fourth investigation of rickets (9〇/8/6) treatment

1 2 3 average A · Bacillus subtilis 50 ° / 〇 WP 600X 1 0 0 0. 33 Β · copper hydroxide 77% WP 800X 4 4 5 4. 33 C · Control no application area 5 8 7 6. 67 Note: The plants infused with Bacillus subtilis have strong growth potential, and the fruit pieces are large and beautiful (high color and high brightness). 4. The fifth investigation of rickets (9〇/8/13) 16 1280852 罹 12 12 3 Average 12 0 l.oo 8 6 7 7· 00 7 12 12 10.33

Α·Bacillus subtilis 50°/〇WP 600X

Β·CuO hydroxide 77% WP 800X C·Control no application area Note: The plants in the area where B. subtilis is infused have a strong growth potential, and the fruit pieces are large and beautiful (high color and high brightness). 5. The sixth investigation of rickets (9〇/8/13) 罹 率 rate treatment 丄 ό average Α · Bacillus subtilis 50 ° / JP 600X 2 2 1 1 β γ Β copper hydroxide 77% WP 800X l 〇 8 9 9·· 00 Control no application area 15 16 15 14.00 Note: The area of plants inoculated with Bacillus subtilis grows vigorously, and the fruit of the plant is large and beautiful (high brightness of fresh color plate). V. Conclusions: 1. No phytotoxicity occurred during the test. 2. From the above data, it can be seen that Bacillus subtilis 5〇%wp is more suitable for Fanjia, and it can be harvested to more than η month at the beginning of August, and the average is large and beautiful (high color and high brightness). )fruit

Claims (1)

1280852 X. The scope of application for patents: fi type fif = 5 赖 or 土观良杂作绿, the main money using the sediment f〇i, %S〇r survey, (10) 2 · and secret. M for the von medium, the following production steps : Separation of Bacillus subtilis such as fungicide or soil amendment b · Fermentation tank · · _ grass turned into a good New Zealand, material = liquid fermentation should be ventilated or quite high;; C · support I quality: from the most appropriate women 5-40 grams / liter, protein 3-24 male corn soaked 5-4. G grams / liter, (10) said (10) / \ gram ^ liter, MP 〇 4 0. 4-3 2 . %s〇4. 7Μ 〇> ^ 2β ^ C^aCl, - 2H2〇10-80 . MnS〇4 . ^ 6_48 d' ^Ph6· °-· 〇^ m will be concentrated in vacuum to remove water Made of concentrated liquid ===== Air pressure (atm) or less 'The temperature is below the machine. Take the soil spoon [force in 〇.〇1 L liquid preparation: After the really concentrated atrophy, adjust the water The spore concentration is achieved by the additive product and the function of the sterilizing or soil improvement agent; the mouth gauge is σ, and the liquid can be prepared. The spray drying: the concentrated liquid after vacuum concentration is sprayed with a spray. The outlet temperature can be less than 7 generations. Spores maintain full removal of water, h powder formulation: Spray fiscal material of formula I prepared the end of the water was removed, and add appropriate 181280852 When and with the right amount of carrier, adjuvant or diluent ^ - product specifications, you can make a powder product; set the number of weeks to the established I. Other preparations: other dosage forms (such as lozenges, milky得 荨 荨 荨 荨 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 可由 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Draw at least a preservative, pH-adjusting acid or test solution. Nu 4·If you apply for the 帛 3 method of ageing, #巾含_ _ = The ship is made up of concentrated water and Wei Wei, 7·0±0. 5, so that the spore concentration reaches 1〇v ml or more. form. The production method according to the item 1, wherein the addition of the powder preparation, the adjuvant or the diluent comprises at least wood chips, clay powder, and an interface active agent. 6 ^ Patent application of the preparation method of the fifth item, the towel containing the Bacillus subtilis, the preparation of the culture solution by concentration, spray drying of the powder formed, adding an appropriate amount of clay 'to achieve a spore concentration of 1 〇 9 / gram or more formed. 19 1280852 VII. Designated representative map: (1) The representative representative of the case is: (1). (2) A brief description of the symbol of the representative figure: A strain B fermentation tank C culture medium D fermentation operation E vacuum concentration F liquid preparation G spray drying Η powder preparation I other preparations VIII, if there is a chemical formula in this case , please reveal the chemical formula that best shows the characteristics of the invention: