CN1053923C

CN1053923C - Cultivation of vegetable active fungus extract of short bacillus family

Info

Publication number: CN1053923C
Application number: CN 90100470
Authority: CN
Inventors: 陈希萍; 邵宝富
Original assignee: People's Government Of Fengshan Town Hangzhou; ZHEJIANG SEED CORP
Current assignee: People's Government Of Fengshan Town Hangzhou; ZHEJIANG SEED CORP
Priority date: 1990-01-25
Filing date: 1990-01-25
Publication date: 2000-06-28
Anticipated expiration: 2005-01-25
Also published as: CN1053640A

Abstract

The present invention provides a method for producing a plant active bacterial agent of brevibacterium, which relates to a method for producing products for promoting plant growth by using a microbial nutrient material as an effective component. A strain is obtained from soil or plant stems via separation, belongs to the brevibacterium, is a beneficial strain newly discovered and is used as an effective component of the bacterial agent to produce the plant active bacterial agent. After an original bacterial agent is diluted with water or is mixed with auxiliary agents, such as a binding agent, a protective agent, a buffering agent, a nutritional agent, etc., the obtained mixture can be applied to crops by various methods, such as bud agitation, sprout root dipping, seed coating, etc.; the bacterial agent has favorable effects on the promotion, the yield increase and the resistance of crops, and the performance is superior to that of similar products at present.

Description

A kind of production method of vegetable active microbial inoculum of brevibacterium sp

The present invention relates to a kind of microorganisms cultures that utilizes and be effective ingredient, produce the former agent and process for producing same of product that plant-growth is had promoter action, i.e. the production method of vegetable active microbial inoculum.

The general agricultural bacterium of domestic and international application (as bacterial manure, bacterium medicine, antibiotic etc.) in the past, belonging to soil ecology is category, and employed microorganism is fungi or actinomyces, and these microorganisms are from the bacterium of meeting by chance of soil, rhizosphere or root face.This century the fifties some scholars are to the root face in the world, the research on blade face has disclosed the root face, there is microbial population in the blade face, with plant materials confidential relation is arranged.Particularly " plant natural ecosystem " notion that domestic expert proposes over past ten years and " plant materials microbial ecological engineering " theory of proposition on this basis, and be developed into then to plant have the health care and the volume increase effectiveness " volume increase microbial inoculum ", the effective ingredient of " volume increase microbial inoculum " is a combination colony of containing multiple bacterial strain, belongs to bacillus.

The objective of the invention is to learn principle, obtain vegetable active bacterium (Brevibacterium SP), and a kind of production method that plant is had the vegetable active microbial inoculum of promotion and antagonistic action is provided from soil or axis separation based on agricultural ecological.

The contriver finds that through the long fruit of jute being planted investigation, the practice of product examination same kind is widely different in the morbidity of each pilot, falls ill hardly in some place, and the pilot that has then sickness rate can be up to 50-70%.This species diversity can't be explained from aspects such as soil, moisture content, fertilising and management by analysis.According to the principle that agricultural ecological is learned, infer this phenomenon, be likely by due to " the microcosmic eubiosis " of crop growth.I.e. morbidity gently or is not fallen ill and is had a large amount of beneficial microorganisms in the soil in field or the antagonism bacterium exists, and the heavy field of morbidity may not have or seldom has tangerine antibiotic.According to above-mentioned principle, the contriver takes up to separate the content of different soils bacterial classification class, in the hope of selecting a kind of antagonism bacterium of the best, carry out artificial culture after, use plant again and get on.Through repetition test, confirmed above-mentioned supposition, and finished technical scheme of the present invention that particular content is summarized following step:

1, the separation contriver of bacterial classification takes the soil of different pilots, adopts dish dilution method to separate, and gets 10 in sterilization culture dish, and every culture dish adds 10 milliliters of aqua sterilisas.Take by weighing soil sample 1 and restrain the aqua sterilisa that places first culture dish, soil is ground, left standstill 30 minutes, the various microorganisms in the soil are flowed in the water (being made into suspension) with the sterilization glass rod; From first culture dish, get one with the dropper of sterilization again and drip in second culture dish, get one behind the thorough mixing again and drip in the 3rd culture dish, be diluted to successively till the tenth culture dish; Then, other gets three of sterilization plates, from last three dilution plates, get a bacterium liquid in ware respectively, the agar medium of fusing is cooled to pour mixing into about 45 ℃, after the cooled and solidified, with the culture dish upset, cultivate under 28 ℃ of temperature: last, the various microorganisms that these separation are obtained compare.The three kinds of grave illness districts of selecting and remain out do not exist, and do not have more bacterial strain and there is the lesion.

On this basis, the dull and stereotyped antagonism mensuration that further exsomatizes and soil plate biology are measured relatively, big and best vegetable active (Brevibacterium SP) bacterial strain of protection effect of the inhibition zone of selecting and remain out.Bacterial strain of the present invention has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, and CGMCC, deposit number: NO:0150 are called for short in this center; Preservation date is on December 25th, 1989.

For proving this bacterium, whether also be present in and do on the object, the contriver is many with some bar leafiness shows and scab, is tending towards withered plant, has carried out sampling and has separated, and separation method adopts dish dilution method, has checked the microbe species similarities and differences on this two classes plant materials.The result is separated to above-mentioned vegetable active bacterium (Brcvibacterium SP) equally on the plant of bar leafiness show, then be not separated on the ill plant.

2, fungus characteristic is cultivated proterties: on the solid inclined-plane of Yeast protein peptone substratum, lawn is irregular corrugation decorative pattern, the look Huang, the edge dentation: in identical liquid nutrient medium, mycoderm corrugation, bacterium liquid muddiness, throw out is arranged: on flat board, the bacterium colony circle, middle slightly projection belongs to pure bacterial classification.

Identification feature; This bacterial strain is the shaft-like organism of unicellular or paired, colourless, no gemma; Size 0.8-0.83 * 1.7-2.2 micron; Motion; Flagellum Dan Sheng; Slightly partially: Gram-positive; Good gas; Heterotrophism; Produce not aerogenesis of acid with carbohydrate oxidation: 30-35 ℃ of growth optimum temperuture, growth scope 15-45 ℃; Growth optimum pH 6.8-7.0, growth scope 4.8-8.5: other proterties is as follows: agricultural anti-mycotic agent reaction (derosal, jingganmycin)-catalase+litmus milk decomposing casein+reduction reindeer moss+starch hydrolysis+reduction KNO ₃Be NO ₂+ gelatin liquefaction+methyl red test-VP test+indole test-H2S generation-7%NaCl+from glucose produce acid+sucrose+galactolipin+lactose+mannose+sweet mellow wine+sorbose-

According to The above results, the brevibacterium sp (Brevibacterium SP) of this Pseudomonas the 17th part in the 8th edition " uncle Jie Shi determinative bacteriology handbook ".

3, the cultivation of former microbial inoculum

The vegetalitas bacterium (Brevibacterium SP) that above-mentioned separation obtains is cultivated by following technology:

The liquid nutrient medium preparation

↓

Autoclaving

↓

Inoculation

↓

Liquid culture

↓

Former microbial inoculum

Liquid nutrient medium is prepared as following weight percent: peptone: 0.4-0.6%; Glucose: 1.0-1.5%; Yeast extract paste: 0.2-0.4%; Buffer reagent 0.1-0.3% transfers to pH value in the 6.8-7.0 scope then.

Sterilization and inoculation: the liquid nutrient medium that is mixed with was sterilized 20-40 minute under 15-20 pound high pressure, connect after cooling, filled in tampon with vegetable active bacterial classification (Brevibacterium SP).Used inoculation method is conventional microbial inoculant method.

Liquid culture: under 30-35 ℃ of constant temperature, cultivated through 3-5 days, treat that the liquid culture primary surface grows the corrugation mycoderm, promptly become for producing the former microbial inoculum that the vegetable active microbial inoculum is used, former bacteria suspension concentration is about every milliliter 10 ⁹Individual.

4, the preparation of vegetable active microbial inoculum

The vegetable active microbial inoculum is to be effective ingredient with vegetable active bacterium (Brevibacterium SP), former microbial inoculum with the vegetable active bacterium is made main raw material, dilute with water can be applied to farm crop, can use by chemical substance application process or microbial inoculant method, also available agricultural tackiness agent etc. fits in glue microbial inoculum, Cotton seeds seed.Its weight percent consists of: former microbial inoculum: 15-25%; Tackiness agent: 75-85% is equipped with auxiliary agents such as protective material, buffer reagent, nutrition agent again according to different applications, optional wherein one or more, and the weight percent during preparation is: protective material: 1-3%; Buffer reagent: 0.1-0.3%; Nutrition agent: 0.3-0.5%.And stir at normal temperatures.Described various auxiliary agent is an agricultural agent, allly mixes with vegetable active bacterium (Brevibacterium SP), and the various auxiliary agents (or weighting agent) that the phase mutual energy produces positive-effect or do not influence this bacterium all can use.

Technology of the present invention is simple, and effective strain is simple, harmless, and is easy to use with the vegetable active microbial inoculum that described method is produced, and plant is had good diseases prevention, production-increasing function, and result of use is better than present like product.Below further be illustrated with embodiment:

Provided several specific embodiments of the present invention and result of use thereof in the subordinate list, the separation of bacterial classification and the culturing process of former microbial inoculum are as previously mentioned.In the table:

B ₁: be former microbial inoculum:

A ₀Be tackiness agent, embodiment the tackiness agent that uses as the production of China brass hill insecticide factory;

C ₁: be protective material, what use among the embodiment is agricultural antiseptic-germicide--derosal;

C ₂Be buffer reagent, what use among the embodiment is dipotassium hydrogen phosphate;

M ₁, M ₂: be nutrition agent, use calcic, boron ionic compound among the embodiment respectively.

Claims

1, a kind of production method of vegetable active microbial inoculum of brevibacterium sp is characterized in that: will separate the tyrothricin Brevibacterium SP CGMCC No0150 that obtains and cultivate from soil, and obtain former microbial inoculum.

2, production method according to claim 1 is characterized in that: the cultural method of the former microbial inoculum of this bacterium is:

A. substratum preparation: by following component and weight percent obtaining liq substratum, peptone 0.4-0.6%, glucose 1.0-1.5%, yeast extract paste 0.2-0.4%, buffer reagent 0.1-0.3%, PH6.8-7.0:

B. sterilize and inoculation: liquid nutrient medium under 15-20 pound high pressure, through 20-40 minute autoclaving, is used the conventional inoculation method inoculation of microorganism after the cooling;

C. liquid culture: press the microorganism fermentation process ordinary method, under 30-35 ℃ constant temperature,, promptly obtain former microbial inoculum through 3-5 days fermentation culture.