FR2594294A1 - Enriched natural microbial complex for cultivated plants - Google Patents

Abstract

Combination of a pure nonpathogenic strain of the family "Pseudomonadaceae", consisting of a vegetable powder comprising very finely-ground stems, roots and leaves, composed of specific microelements and macroelements, vitamins, a growth factor and amino acids, using bacterised soil extracts. Application in agriculture, arboriculture, viticulture and horticulture.

Description

 The present invention is the result of a combination of 3 microorganisms selected to obtain an additional balanced rhizosphere effect allowing, with special culture media, a more balanced vegetative action, yields greater than those currently obtained in the following fields of agriculture, arboriculture, viticulture , horticulture, market gardening.

 Contribution to the soil both for the distant or near rhizosphere of pure bacterial strains and selected in association without antagonism.

Provides elements in the soil against drought allowing a rate of humidity necessary for the interactions of the rhizosphere, hence protective effect
Activation of the distant rhizosphere thus allowing synergy and rapprochement with the nearby rhizosphere.

 Perfect balance of microbial flora without dominance.

 Growth tripled by balanced growth factors.

 Solubility of mineral compounds for complete use of phosphorus without energy loss.

 No nitrogen immobilization, it is entirely used by the plant.

 Association of strains to avoid microbial interaction.

 Denitrification fully used.

 Superior resistance to parasitism.

 Decrease in fertilizer intake by 35%.

By this balance in the contributions, the regulation, the complete rhizosphere effect, possibility for several varieties of 2 harvests per year, without taking into account the normal dates of sowing or planting, finally development of the ears, various organs, flowers, fruits, very greatly increased or improved in their own values, ie color, volume, energy efficiency.

These microorganisms are part of the sub-branch of the Eubacteria, family of Pseudomonadaceae.

The cells of these bacteria are elongated in straight or slightly curved sticks; asporulated; gram-negative; pigmented or not; movable by polar eyelashes or immobile and aciliated. Optional aerobes growing well on the surface of usual organic media; heterotrophic in general with some chemoheterotrophic species; free, aquatic, or telluric forms saprophytic forms and parasitic forms of plants and animals.

Genus Pseudomonas not pathogenic for plants.

Pseudomonas Insolita Strain reference (STAFF 1940) Prévost 1960
Habitat: soil
Morphology: stick from 1.2 to 1.8 by 0.6, coccoid shape, movable by the 3 polar lashes, gram negative.

Physiology: Aerobic, optimum temperature 25 to 30 C, extreme temperatures 7 C and 48 C, killed at 60 C. PH 3.3 to 9.5.

Cultures Inclined celosis, flat, shiny, whitish colonies with sharp edges, slightly fluorescent. Unliquefied gelatin.

 Carbohydrates glucose, galactose, lebrulose, maltose, mannose, sucrose, raffinose, rhamnose, lactose, arabinose, xylose, adonital, glycerol, enositol, mannitol, sorbitol, dextrin, starch, salicin, fermented with gas, starch is hydrolized.

Biochemistry S H2; nitrates reduced to nitrites, ammonium salts are used; atmospheric nitrogen is fixed.

Non-pathogenic.

 Pseudomonas Proteus strain reference (HUMM 1946) Brisou 1954.

Isolated from marine sediments and Baronnet seawater 1.4 by 0.3
Mobile, gram negative.

Aerobic; positive catalase; optimum temperature 30 C.

On inclined agar, grayish, smooth colonies, the gelatin is quickly liquefied.

 I1 ferments: glucose, levrulose, galactose, mannose, maltose, sucrose, raffinose, arabinose, rhamnose, cellobiose, salicin, glycerol, inositol, mannitol, methanol, propylene glycol, it hydrolizes fats, it uses hippuric acid and glucosamine , as a source of C and N, as well as alanine, arginine, aspartic acid, creatine, cystine, glycine, glutamic acid, leucene, glucomic, lactic, malic, malonic, mucic, oxalic, succinic and tartaric.

Alginic acid is slowly hydrolyzed.

It produces NH3, SH2, urea and reduces nitrates to nitrites.

Not pathogenic.

Achromobacter Thalassius (reference Z obell and upham 1944)
Isolated from marine sediments.

Sticks from 0.8 to 2.3 out of 0.6 to 0.7
Isolated in pairs and short chains.

Mobile by peritrich eyelashes.

 Gram-negative.

Optional aerobic; optimum temperature 20 to 25 C grows better on salty environments.

Cultures: on inclined agar: punetiform, rough, translucent, raised colonies.

The gelatin is liquefied.

Carbohydrates are not fermented.

N H4 C L and 19 amino acids can serve as a source of nitrogen and carbon.

The nitrates are reduced to nitrites.

 I1 produces N H3 from the peptone.

Mineral sulfur can be used as a source of sulfur.

Not pathogenic.

The interest of this association of non-pathogenic, heterotrophic and chemotrophic bacteria requires complex metabolites and growth factors.

Heterotrophic bacteria synthesize all their cellular constituents from mineral carbon elements, which provide essential metabolites, it is necessary to respect all the characteristics drawn from their ecology and to know their nutritional and metabolic physiology.

Carbohydrates, for these selected bacteria therefore from pure strains provide energy sources, they are therefore demanding for organic compounds.

These bacteria can use a wide variety of compounds, for example sugars, which is our case, as also free nitrogen or better the nitrogen of the mineral compounds indicated in this file and thus reduce the nitrates to nitrites which are then reduced to ammonium.

The bacteria in this product assimilate nitrogen as a result of the ammoniacal transformation produced by the substrates on which they are grown.

An additional part of nitrogen is provided by the presence of amino acids in the culture media.

The latter also provide the phosphorus necessary for the constitution of nucleic acids, phospholipids, energetic substances of the ATP series, which allows the accumulation and distribution of energy in the cell.

This contribution of phosphorus can be organic compared to our environments.

The media include mineral elements - see the details of these in the file - which will constitute the enzymes and this creates the activation of enzymatic reactions, the constitution of growth factors.

 The bacteria in this product grow at the temperatures indicated, in this file which are strictly observed and all put in a ventilated oven so that they can take oxygen from the air, all performed in a room called sterile renewal of air controlled by laminar flow and controlled air conditioning.

In general, bacteria of the selected genus can undergo variations in pH from 6 to 9.

In our case the pH of 7 - 7.5 must be respected absolutely, hence strict control, and this in order to avoid sensitivity too often due to variations.

No specific contribution of 2% NACL to inhibit growth.

Our bacteria are strict aerobic, they need air and oxygen during their development and their metabolic activity, hence the requirement of cultures in a sterile room, flat bottom flask cultures and wide neck, boxes of kneaded should be unclogged for the first for the second without the cover but turned over on a support, let the air pass from below.

 Elements or foods are large molecules which will initially be fragmented into smaller molecules by enzymes, enzymes that secrete our bacterial cells (exocellular enzymes).

They will then be transformed inside the cell by intracellular enzymes.

Any substrate cannot cross the cytoplasmic membrane, hence the interest of our media selections.

 Our first seeding (see manufacturing technique) is carried out on a solid medium to facilitate the multiplication of bacteria.

All the others are made in nutritive broth therefore in liquid medium, to which are added all the solutions specified and specified in the list of media and manufacturing technique.

This condition, because, the plastic membrane permeable to water and certain organic media. It is moreover at this stage that the plant organ comprising rod, leaves, finely ground roots will be incorporated.

 This plasma membrane prevents the uncontrolled penetration of the constituents found in the exocellular medium, hence the selection of useful substances.

The media that are used for the product take these factors into account.

This membrane therefore plays a crucial physiological role in balancing the exchange between the endocellular and exocellular environment.

These substances will enter the cell at a certain speed and in a manner proportional to the extracellular concentration when the intracellular concentration is low, and this penetration will stop when the equilibrium is equal on both sides of the membrane. cellular.

In summary, the endocellular concentration cannot therefore exceed the exocellular concentration.

 The bacterial cell can accumulate a certain amount of substrates, it is by taking into account this possibility that our concentrations by our media or substrates are very important, in our case under control and dosage (technique indicated in the file) we arrive 9 stabilize or increase the concentration of enzymes in our product.

For external amino acids for example, we concentrate them at 500 times, which is a concentrate of high value.

The culture media or substrates that we use are synthetic, they better meet the nutritional requirements as a result of the contribution of elements necessary for the growth, the multiplication of our selected bacteria and this under defined physicochemical conditions.

We use (see list in the file) complex media of natural products with, as mentioned above, extracts from tissues or other organs of plants and animals.

These bacteria used having originated from pure strains, we do not need an isolation medium.

 The carbohydrates present in the intracellular content, therefore in the substrate are degraded by bacteria.

Certain molecules before entering the cell must first be broken down into fragments of low molecular weight by hydrolytic enzymes excreted by the bacteria in the medium.

 These are exoenzymes, the endoenzymes remaining inside the cell.

The products, thus produced can then diffuse slowly by osmosis through the semi-permeable membrane.

However, there is a certain activation of penetration by the action of enzymes also called permeases which have the role of accumulating in the cell and ensuring entry before.

 Cellular catabolism is then created.

 The pseudomonas selected in the product have a very important action, that of being proteolytic and this action is related to the level of aeration and the concentration of our substrates.

As a reminder - Proteins, organic compounds of high molecular weight, are made up of amino acids linked together by peptide bonds.

Definition of the associative action of product microorganisms
To combat physiological disorders,
Stimulate growth in a ratio multiplied by 2,
-Increase the energy value without requiring contributions in amendments and treatments, on the contrary reduce them, of cultivated plants.

 The action of this association has no effect on lightning, frost, hail, pruning, poor execution of soil work by working implements, are all accidents due to physical and meteorological causes.

 Knowing that nutritional diseases are complex, that they can result from an effective absence of one or more elements in 801, that more often the deficiency is caused by the antagonistic action of another element which prevents the former from penetrating the roots or the block in the soil in an unassimilable form, finally that the physical and chemical conditions of the soil and the subsoil can also cause insolubilization or fixation of the elements.

 Our association of microorganisms of pure strains cultivated on special media, perfectly adapted to these nutritional diseases, will compensate by an additional supply of amino acids, vitamins, microelements produced by microorganisms, to these major drawbacks, but still by spraying at the level of the leaves and the soil, will immediately restore the biological cycle and further stimulate it by the growth factors present in the intra-cellular contents.

Our association of microorganisms will form a whole that can be assimilated directly.

 From where a revival of the biological activity and the rhyzophère effect by this supplementation.

 It should also be noted that nutritional disorders adversely predispose plants to attack by various parasites.

The action of this supplementation with the product which is the subject of the present invention will counter the attack of these parasites by giving the cultivated plants a marked and marked resistance.

 The present invention does not aim to describe the symptoms of deficiency, of deficiency encountered in cultures, but to explain the action of the product at the level of a normal plant, of a deficient or deficient plant, for restore balance on the one hand in these two cases and stimulate growth, on the other hand for the normal plant the action provided by the invention.

 Action of associated microorganisms, of pure strains, cultivated on specific culture media for cultivated plants.

 Nitrogen supply.

At the root level, from where aerial parts develop better, hence stimulation of vitality, flowering, fruiting, even if there has been leaching of nitrates from the soil and destruction of organic matter, or even an excess humidity lowering the activity of bacteria which can no longer ensure the decomposition of organic matter and the nitrification of nitrogen.

 The association in addition to this supply of assimilable nitrogen will also, by its bacterial concentration, reseed the rhizosphere, ensure nitrification and decomposition of the organic matter present by the specialized bacteria of the product whose physicochemical characteristics meet the demand of these needs, including urea.

 Phosphorus supply.

 There is a certain analogy with nitrogen, the product has been studied in relation to this, it will favor the opening of the buds more quickly and therefore a more advanced flowering.

The product will also act at the root level.

Particular advantage of the combination in the product, disregard the reaction of the soil.

 Intake of potassium.

Plant metabolism needs potassium, when the plant has reached a certain level of development, it is the only nutrient that does not enter into the composition of the main constituents of plants (proteins, lipids, carbohydrates, chlorophylls) but on the other hand, the sensitivity is accentuated to diseases, as well as the quality of the products.

Given these conditions so that potassium can be assimilated directly by the plant, the specific culture media transformed by our association of microorganisms contain potassium; the plant can therefore assimilate it on two levels, that of the leaf. either straight as a result of spraying, because this element is very mobile, it easily migrates from one point to another in the plant.

 Magnesium intake.

The dosage of potassium by the activity of the product's microorganisms avoids the relative excess of potassium. There is often an antagonism between magnesium and potassium, the product has taken account of this state, this risk does not therefore exist, moreover the nitrogen provided by the association of microorganisms cannot cause the imbalance of magnesium.

In addition, the PH is neutral.

The intake of magnesium by our product is calculated under conditions analogous to calcium.

Leaching is also fulfilled.

On the other hand, the spraying of the product is perfectly indicated at the level of the leaves, which allows a very clear assimilation of this intake of magnesium, hence a balance which avoids antagonism.

 Calcium intake.

Excess calcium where the deficiency creates significant disorders at the cellular level and meristematic for the formation of tissues.

Our product brings by spraying and also at ground level a restoration of functions in the case of disorders, but also by the supplementation developed by the microorganisms of our association, the stimulating quantity necessary and useful to strengthen the cell walls including the lamella medium is mainly formed of calcium pectate, but still the faster development of meristematic tissues, and also a total solubilization for provision of the plant.

Our product fills the leaching.

 Boron supply.

This micro-element is present in our product developed by our association of micro-organisms.

Spraying at the leaf level gives very satisfactory results, as well as at the scl level.

 Boron will be assimilated directly by the leaves and roots by whatever quality of the soil, unless there is too much liming, which is not the responsibility of the product, but of the accidents mentioned at the beginning of the development of the soil. activity of our product, accidents due to physical or meteorological causes.

 Copper supply.

This element is found in the product, its action in this composition is to strengthen the activity of potassium to fight against parasitic diseases, thus providing the plant with better resistance to parasitism.

 Iron supply.

Element essential for the formation of pigment, chlorophyll.

The contribution of the product at the level of the roots, will solve the serious problem of the non assimilation of iron in certain grounds, also of its insolubilization.

Advantage also in the case of too much liming.

The balance of product development by microorganisms also takes into account the potassium deficiency which worsens levric chlorosis, which does not manifest itself as a result of the product's PH and calcium intake such as iron dissolve quickly, like the iron naturally present in the soil, the spraying of the product is better indicated at the level of the leaves, than of the roots, without however abandoning the treatment on the ground.

Manganese intake
Interest of the product by spraying on the leaves.

As this contribution is developed by vectorized microorganisms, there is a dosage studied for the various cultures of microorganisms of the association providing ammonia at the root level, advantage in the effect which is very often limited by the soils and fertilizers.

 Supply of molybdenum.

Molybdenum acts as a co-factor in the reduction process
tion of nitrates to ammonia.

The association of microorganisms by its properties and the quality of these responds perfectly in the reduction of nitrates, the addition of molybdenum increases the potential of co-factor, which allows to reinforce the nitrogen produced
by our product and fix it by the association of microorganisms present.

 There is no risk of insolubilization, because the product does not undergo the reaction of the soil.

Zinc intake
In the combination of the product, the zine plays a role of growth factor, stimulator.

 The culture media used and their properties. Their provenance.

Before situating in detail the places of culture for the microbial multiplication of the species that we have chosen, it is necessary to point out that
all these culture media include
An inert support of silica gel impregnated with a nutritive liquid
of determined composition, by adding a food component to this liquid
preferably consumable by the bacteria in presence which allows in terms of control to determine the physiological group that consumes it. We thus arrive at a natural condition of existence of the bacteria. There is thus obtained, having a pure microbial strain at the start, with specific culture media, a dominant microbial flora.

to return to silica gel, the one used must be taken immediately.

We will therefore create cultures of bacteria with "ammonifying", nitric, nitrous, cellulolytic, glucidolytic, fixative, nitrogen, demitrificator etc ...

We will also use media with soil dilution suspension.

 Technique for preparing the silico-gel plates, in the event of difficulty finding them commercially.

Mix in equal parts
A solution of sodium silicate diluted to 7 Baumé and a solution of hydrochloric acid diluted to 13 Baumé, pouring the acid into the silicate and stirring.

 It does not appear lumps or the acid is not diluted enough.

 Then pour 30 ml into the 10 cm diameter petri dishes. Leave the boxes open on the bench until the gel sets (around 30 hours).

To note that the gel is very vibrating, for that it is necessary to strike on the edge of the box.

Then put these boxes in a very large crystallizer and proceed to wash the boxes under running water (by tap) until the excess acid is eliminated. The pH checked on paper must be neutral.

Before using these boxes it is necessary to immerse them in boiling water. Then boil in a test tube the next solution from which 1 ml was taken to which 1 ml of the energetic substance was added which will give a production.

 Winogradsky's solution formula.

Bi potassium phosphate 5 gr
Magnesia sulfate 2 gr 5
Sodium chloride 2 gr 5
Ferric sulfate 0 gr 05
Manganese sulfate 0 gr 05
Dissolve these salts in 1,000 ml of normal water but not containing antiseptics (watch out for tap water), then take Vittel-type water.

Check the PH which must be 7 - 7.5.

Indicator: use Bromothymol blue in 0.02% solution. Blue tint at PH 7 - 7.5.

If the pH of the solution is acidic (blue Bromothymol turns green or yellow). Then add the PH with a KOH solution. Distribute this solution in a tube or bottle of 100 to 1,000 ml. Cotton butcher. Sterilize at 110 C for 20 minutes. The solution presents a precipitate surmounted by a clear liquid. Before use, shake to homogenize the solution. Pour the contents of the tules on the plates (1 tube per plate). Bring everything to the oven at 45 - 50 for 2 hours until the water has completely evaporated, of course avoiding drying of the gel. Then deposit on the surface of
of soil the box thus impregnated with the seeds / debacterized using a stretched and buttoned agitator whose end is moistened. Arrange the seeded plates, the culture medium on the ceiling, (which means the culture at the top)
culture centre
lid
In a humid room and bring everything to the oven.

Here you have a pure and selected culture medium that will allow the first multiplication of the bacteria selected for our products.

Formula "soil extract" necessary as a component in culture media in silico-gel plates.

Choose a ground of neutral pH or very little alkaline 7.5 for example.

Take this soil 10 or 25 cm deep (10 kg of soil gives 7 liters of extracts). Mix cold equal earth and neutral PH spring water, or Vittelourobinet if it does not contain antiseptics.

Autoclave for 1 hour at 130 C. As soon as the temperature of the autoclave (after 1 hour at 130 ° C) drops to 105 -100 C, let the steam escape gently to avoid browning of the soil extract.

Filter the decanted mixture on a filter, for example Laurent n 7 hot, to eliminate in a certain way the possible appearance of a colloidal disorder, filter on a filtering washer (asbestos-cellulose), under vacuum.

Check the PH, it must be close to neutral.

Distribute in tubes of 22 for immediate use or in vials. Cotton butcher. Sterilize in an autoclave for 30 minutes at 115 C.

Culture medium for ammomifying bacteria.

Take 2 silico-gel plates, impregnate them with 2 ml of Whinogradsky medium and 0.5 gr of Peptonebacteriological (organic nitrogen source). Inoculate the plates with the bacterial strain coming from the silico-gel plates, add the technique of earth seeds. Bring to the oven at 30 C.

After 24 hours: invasive development of the colonies.

 Examining under the microscope, you must have a pure culture.

 Look for the gram next to the bacteria that compose it that you know (means of control).

 You detect ammonia by the Nessler reagent (this reagent is commercially available (means of control).

Demonstration of nitrifying bacteria
Nitrous ferments. (means of control)
Take two plates (Petri dishes) impregnate them with 2 ml of Winogradsky medium + 0.1 gr of ammonium chloride (source of ammoniacal nitrogen).

Inoculate the plates by the technique of earth seeds. Bring to the oven at 30 ° C.

After 24 hours: examine the plates: Around the seeds of the earth we can see a halo more cu less brown.

Search for nitrites by the griess reagent using this technique (cut using a small tube used as a cookie cutter, a silico-gel washer around a grain of earth, push the washer to the bottom of the tube and look for nitrites). Pink coloring characterizes the presence of nitrites. This reagent is commercially available in 2 parts which must be mixed, for this search for nitrites, 1 ml of the mixture must be taken. On a second washer, look for ammonia by Nessler's reagent. This reagent is commercially available. Due to the function of the incubation time, that of ammonia is still positive or negative.

Nitric ferments. (means of control)
Take 2 plates (petri dishes) impregnate with 2 ml of medium.
Winogradsky and 0.1 gr of potassium nitrite (source of nitrous nitrogen).

Inoculate the plates using the grain of earth technique. Bring to an oven at 30 ° C. After 24 Hours examine the plates. Around the grains of earth there is a more or less brown halo.

Search for nitrates and note the more or less extensive disappearance of nitrites.

To do this: operate using the technique applied to nitrous enzymes. Look for nitrates by diphenylanine and nitrites by grievance reagent.

The reaction of nitrates must be positive, while depending on the duration of incubation that of nitrites is still positive or negative.

 Demonstration of nitrogen-fixing bacteria in the air. (Means of control).

Impregnate two plates each with 2 ml of Winogradsky medium + 0.1 of glucose and 0.3 of calcium carbonate (which enamels the surface). This medium does not contain nitrogen.

 Impregnate two other plates, but replace the glucose with 0.1 gr of sodium benzoate. This medium is also deficient in nitrogen. Inoculate the plates prepared by the technique of earth seeds and bring them to the oven at 30 ° C.

After 24 hours, note the color of the colonies.

In the middle with glucose: white, rounded, creamy colonies, which later turn brown.

In the middle with benzoate: flatter, black colonies, with metallic reflection.

In both cases, it is nitrogen fixing Azotobacter. Of course you must seed with bacteria from the purchased strain, this procedure gives you the opportunity to control. The value of your strain whose function is to fix the nitrogen in the air. It is the same what I forgot to report (previously) for nitrites, nitrates ammonia, ammonifying bacteria. These are technical means of control to assure you of the quality of your strains and their possibilities of multiplication of enrichment.

Medium of trace elements. (enrichment)
Potassium molybdate 0.10 gr
Sodium borate 0.10 gr
Cobalt nitrate 0.10 gr
Cadmium sulfate 0.10 gr
Copper sulphate 0.10 gr
Zinc sulfate 0.10 gr
Manganese sulfate 0.10 gr
Iron perchloride 3 drops
1.000 ml distilled water through which a current of carbon dioxide is passed.

Sterile calcium carbonate medium (enrichment)
Distribute in tubes of 17 powdered calcium carbonate S at the rate of 0.2 gr per tube. Butcher with carded cotton. Sterilize 20 minutes at 120 ° C. You can put in a bottle or in a tube.

 Medium for ammonifiers. (means of control) finished products.

0.2 gr Asparagine
Winogradsky standard saline solution 50 ml.

1 ml trace element solution
Distilled water 950 ml
Distribute in a tube of 22 (10 ml per tube) or in a bottle, butcher with cotton,
Sterilize at 110 C for 20 minutes.

 Environment for the search for nitrous enzymes. (means of control) finished products.

Ammonium sulphate 0.5 gr
Calcium carbonate 1 gr
Winogradsky Standard Saline Solution 50 ml
Distilled water 950 ml
Dispense into hemolysis tubes at a rate of 1 ml per tube. Butcher with carded cotton. Sterilize 20 minutes at 110 C.

 Medium for the search for nitric ferments. (means of control) finished products.

Sodium nitrite 1 gr
Calcium carbonate 1 gr
Winogradsky Standard Saline Solution 50 ml
Distilled water 950 ml
Divide into hemolysis tubes at a rate of 1 ml per tube or use a bottle.

Butcher with carded cotton. Sterilize 20 minutes at 110 C.

 Environment for the search for denitrification. (means of control) finished product.

Potassium nitrate 2 gr
Calcium carbonate 5 gr
Glucose 10 gr
Standard Winogradsky Saline Solution 50 ml
1 ml trace element solution
Distilled water 950 ml
Distribute in 22 i tubes at a rate of 10 ml per tube or put in a bottle. Sterilize 20 minutes at 110 C.

Purified mineral medium (enrichment)
Purified glucose 20 gr
Washed agar 20 gr
Monopotassium phosphate 1.5 gr
0.25 gr magnesia sulfate
Biotin 10 - 9 gr
Thiamine 10 - 6 gr
Nicotinic acid 10 - 6 gr
Distilled water 1,000 ml
Medium for growth (vitamins)
Miso Ynositol 300 mg liter
Thiamine 1 mg liter
Ascorbic acid 5 mg liter
Citric acid 50 mg liter
Growth medium (amino acids)
Glutamine 250 mg / liter
Glycine 100 mg / liter
Asparagine 100 mg / liter
Proline 100 mg / liter
Tyrosine 100 mg / liter
Phenylalanine 100 mg / liter
Methionine 100 mg / liter
Cysteine 100 mg / liter
Mix all the solutions.

Growth stimulating medium yeast extract 1 gr
Growth stimulating medium
Casein hydrolyzate 3 gr / liter
Sugar enrichment medium
Sucrose 5%
Glucose $ 8
Enriching mineral composition of micro-elements
Manganese sulfate 10 mg
Zinc sulfate 10 mg
Copper sulfate 0.03 mg
Aluminum chloride 0.03 mg
Cobalt chloride 5 mg
Nikel chloride 0.03 mg
Potassium chloride 0.01 mg EDTA 0.03 mg
Iron sulfate 0.03 mg
Distilled water q SP 1.000 ml
Origin of these environments
All chemicals can be purchased from the following companies
Merk Paris
Rhône-Poulenc Paris
Institut Pasteur for certain environments prepared in advance - Paris
Pointet Girard - Paris
These products must be vigorously pure, not technical products.

You will notice that some environments only contain minerals and organic substances. They must be considered as contributions in the development of microbial strains with enriching properties, the solution of vitamins, amino acids, sugars of yeast extract, are to be added to the contributions of minerals.

Origin
The same companies as those mentioned above, with the addition of vitamins, the Hoffman La Roche company in Paris.

 Mineral composition of some macro-element media.

Yeast water
Yeast autolysate 50 ml
Distilled water 1,000 ml
Divide into 17 cm tubes. Stopper with carded cotton. Sterilize at 120 for 20 minutes.

Preparation of the yeast autolysate 1000 g of baker's yeast are dissolved in 1000 ml in distilled water. Bring the suspension to the husband's bath at 50 C for 24 hours. Boil then. Neutrallscr and filter. Autoclave the filtrate for 20 minutes at 120 ° C. Concentrated yeast autolysate is sometimes found commercially, of which 1 gr corresponds to approximately 10 ml of the above solution.

Yeast water gelose agar 20 gr
Yeast water 1000 ml
Dissolve the agar in an autoclave for 20 minutes at 120 C. Filter while hot.

Divide into tubes at the rate of 10 ml per tube of 17. Close the tubes with carded cotton. Sterilize 20 minutes at C to avoid possible precipitation.

This preparation is very rich and can replace the "yeast water" medium.

Nutrient agar medium. (nutritive agar) agar 20 gr
Nutritional broth 1000 ml
Melt the agar in the autoclave for 20 minutes at 120 ° C. Filter hot. Divide into "base" tubes and "inclined" tube. Butcher with carded cotton.

Sterilize 20 minutes at 115 ° C.

Whirlpool water
Bacterological Peptone 2 gr
Biammonium phosphate monopotassium phosphate
Tartaric acid
Ammonium sulfate
Sucrose
Whirlpool water q S
After dissolution, filter, divide into tubes, stopper with cotton, sterilize 20 minutes at 120 ° C.

Preparation of churning water
In a canvas bag introduce 15 g of churn. Immerse the bag in water and boil for 30 minutes. Filter. Reduce the final volume to 1 liter. Sterilize 20 minutes at 120 ° C. The medium should not be used immediately. Touraillon: barley germ which is dried in an oven called TOURAILLE.

Winogradsky saline
Phosphate 5 gr
Sulfate of magnesia 2.5 gr
Sodium chloride 2.5 gr
Ferric sulfate 0.05 gr
Manganese sulfate 0.05 gr
Dissolve these salts in 1000 ml of water, but not containing an antiseptic like chlorine in tap water. The PH should be 7 - 7.5. Divide into vials or tubes. Cotton butcher. Sterilize for 20 minutes.

Trace element solution
Potassium molybdate 0.05 gr
Sodium borate 0.05 gr
Cobalt nitrate 0.05 gr
Cadmium sulfate 0.05 gr
Copper sulphate 0.05 gr
Zinc sulfate 0.05 gr
Manganese sulfate 0.05 gr
Iron perchloride 1 drop
1000 ml distilled water through which a stream of carbon dioxide C02 is passed.

Sterile sugar solution
Prepare 30% solutions in distilled water neutralized at pH 7.2 the following sugars glucose - sucrose - maltose - lactose - galactose.

Distribute in tubes of 17 at a rate of 10 ml per tube. Cotton butcher. Sterilize for 15 minutes at 115-107 ° C. The solution should not be yellow on leaving the autoclave.

 Mobility medium for bacteria to control whether they live well.

Yeast extract 3 gr
Peptone 10 gr
Sodium chloride 5 gr
Agar 20 gr
Distilled water q SP l 000 ml
QSP means sufficient quantity to spread in tubes of 17. Sterilize 20 minutes at 120 C. Cotton stopper.

Purified mineral medium
Glucose purifies 20 gr
Washed agar 20 gr
Monopotassium phosphate 1.5 gr
Sulfate of magnesia 0.25 gr Biotin 10 9 o gr
Thiamine 10 - 6 gr
Nicotinic acid 10 - 6 gr
Glucose purification
Boil a 20% pure glucose solution with activated charcoal for at least 10 minutes. Filter. Reduce the volume to 1 liter after dissolving the various ingredients in the medium.

The agar cut into small pieces is washed with running water for 48 hours in distilled water and dried with alcohol.

Preparation of suspensions - dilution of earth material necessary (which must be sterilized)
500 ml mortar and pestle
Sterile 500 ml vial graduated to 100 ml and marked n 1 15 test tubes of 22 5 sterile graduated pipettes
Sterile distilled water.

Procedure 9 tubes of 22 are placed on a rack and marked with 2 to 10.

Add 18 ml of sterile distilled water to each.

Grind to dry in the mortar 10 grs of sieved and homogeneous earth.

Grind again after adding approximately 20 ml of distilled water.

Grind again by adding a little distilled water.

Transfer to the sterile graduated flask and make up to 100 ml of the volume of the suspension.

Shake the flask containing the soil suspension at dilution 10
Take out using a sterile 5 ml graduated pipette, 2 ml and place them in a marked tube.

Shake by rolling it between. hands, tube closed without wetting the cotton.

We obtain a sus pension at dilution 10
Do the same, but changing the sterile pipette between each dilution, up to 10-10
NB: The addition of sodium pyrophosphate helps the dissociation of soil aggregates and the dispersion of germs in water. In some cases, for the first suspension-dilution, a 1 to 1000 aqueous solution of sodium pyrophosphate may be used.

Immediately use the suspensions-dilutions after preparation by seeding the different media, starting with the most diluted suspensions.

Operate under strict aseptic conditions.

Particular culture process (plant powder)
It is absolutely necessary to cultivate each plant under specific cultivation conditions to obtain a pure culture whose destination is to be finely ground (stems, roots, leaves) and this up to the 3rd leaf development stage.

The seeds or seeds must be placed on a very fine trellis above a water tank containing lithothane, the water must be flush with the base of the trellis, sprayed once or twice a day with the plants to activate development and maintain thus a certain amount of humidity.

Agriculture and market gardening: (same technique)
Wine growing: take roots and branches of the year.

Fruit crops: same
Horticultural crops: seeds or seeds. For the cuttings (grind these).

The plant powder thus obtained is added to the medium extracted from the earth in the manufacture of this microbial complex.

This delivery technique is a manufacturing secret.

Manufacturing technique
In a sterile room and under laminar flow, all the material used for handling must be sterile.

1) Put in the bottles or tubes strains of microorganisms received, a few mls or drops to put them in suspension.

2) Distribute the suspensions obtained in equal parts in the large size petri dishes (diameter 18 cm) or in large capacity red roux bottles which is preferable. Compare the number of bottles of roux required with the number of drops obtained in a suspended strain.

3) Pour an inclined agar with yeast extract into the red falcons, or buy bottles prepared with this medium.

4) Locate in parallel the medium with small gaps between each parallel with platinum wire previously flamed with the bunsen burner and this each time that this work is carried out in each bottle that box, in order to avoid the transport of a contamination of an urban bottle or box to another therefore always possible despite the laminar flow and the sterile room of a microorganism present in the ambient air having withstood sterility.

5) When this streaking work is carried out, it is understood that the platinum wire has been previously soaked in the medium of the microorganisms in suspension. It is therefore very difficult despite as indicated in N'2 to count the number of drops contained in the suspension, because each time there is a sample with the platinum wire in it, it is not always said that this one will take a drop. It is therefore advantageous to provide a large number of bottles of roux. Another observation, it is easier to streak in a petri dish, than in a bottle of roux. Both cases are however reported with preference to petri dishes.

6) After incubation in an oven at 25 C bacteria for 4 days, collect all the developed colonies and seed them in new dishes or flasks.

The importance of the development will force to double the number of boxes.

7) Prepare the broth media for each type of strain because each strain put in suspension must be cultivated separately and not associated.

8) Collect all the developed colonies, add to soiled red boxes an extract of soil or dilution in suspension (for soil extract 10 grs, for dilution in suspension 10 ml) seeded all the developed colonies.

Add to this medium 1 gr of the plant (per box) which is going to be treated (for example powder of malus plants for corn treatment).

Incubate for 5 days in a humid chamber oven at a temperature of 25 C.

9) Empty all of the boxes aseptically into a glass container.

Add to this mixture a solution of trace elements, a vitamin solution which is made to develop in a crystallizer in an oven S at a temperature of 25 C in a sterile humid room for 5 days.

10) Resume everything, add 500 gr of earth extract or 500 ml of diluted suspension, add the micro-element medium + calcium carbonate medium. Use a larger container, incubate at 25 ° C for 5 days in a humid chamber oven.

 11) Resume everything, add 500 grs of soil extract or dilution suspension. Add growth medium (amino acids) incubate at 25 ° C in a humid chamber oven for 5 days.

12) Resume everything add 500 grs of soil extract or dilution suspension in a larger container, add the growth stimulating medium to the yeast extract. Incubate at 25 ° C for 5 days (humid chamber incubator).

13) Resume everything, add 500 grs of soil extract or dilution suspension in a container suitable for the amount, add the composition of micro-enriching elements + the sugar enrichment medium. Incubate at 25 C in the humid chamber for 5 days.

This product is then lyophilized (only in this form).

Concentration in gr 50 billion bacteria.

Origin of strains (microorganisms)
Some shopping addresses
Institut Pasteur, 28 rue du Docteur Roux, Paris 15th
Centraalbureau, Voor schimmelcutures, 5 Javalaan Baarn, (Holland)
American type culture collection, 2029 M. Street, Washington (USA)
Sté Biòpox, BP 177, 8 rue Barhes, 92305 Levallois-Perret.

Claims (5)

 1) Association of bacteria of pure, non-pathogenic strain of the Pseudomonadaceae family, order of Bacterials constituted by a vegetable powder in the substrates resulting from a particular culture under sterile conditions, vegetable powder comprising very finely ground stems, roots and leaves ,  2) Product according to claim (1) characterized by a substrate composed of micro-elements, specific macro-elements, vitamin solution, growth factor, amino acid complexes, with regard to the nutritional physiology of bacteria.  3) Freeze-dried product according to claims (1 and 2) characterized by the concentration in grime of 50 billion active series beXS, having to be re-dissolved in normal water.  4) Product according to claims (1 to 3) characterized by a particular manufacturing technique using debacterized earth extracts.  5) Product according to all of claims (1 to 4) characterized by the use in the formula of yeast extract and yeast water as a nutritional source.