WO2014163473A1 - Liquid formulation comprising two phosphorus-solubilizing pseudomonas fluorescens lr1 for use in agricultural fertilization - Google Patents

Abstract

The bacteria PGPR play a very important role in the solubilization of phosphorus which is insoluble in soil. However, they are too few to compete in the rhizosphere with other bacteria present in the soil. Thus the amount of phosphorus released by these small communities is generally insufficient to increase in-situ plant growth substantially. Consequently, it is necessary to inoculate the plants with a high concentration of a target microorganism which is higher than that normally found in the soil in order to benefit from the phosphate-solubilizing property in order to increase plant yields. The invention concerns the use of a biofertilizer product based on a phosphorus-solubilizing strain, known as Pseudomonas fluorescens. The invention also concerns the formulation method which was included from the outset in the mass production of the bacterial strain. Tests have been performed both on the stability of the strain in the product and on the efficiency thereof in improving crop yields.

Description

 title

 LIQUID FORMULATION OF PSEUDOMONAS FLUORESCENCE SOLUBILIZING PHOSPHORUS APPLICABLE FOR AGRICULTURAL FERTILIZATION

descriptive

Phosphorus (P) is one of the major macronutrients essential for growth and biological development. It is present at levels of 400-1200 mg / kg soil. Its cycle in the biosphere can be described as sedimentary because there is no exchange with the atmosphere. Microorganisms play a central role in the natural phosphorus cycle. This cycle occurs through cyclic oxidation and reduction of phosphorus compounds, where electron transfer reactions between the oxidation steps range from phosphine to phosphate. The genetic and biochemical mechanisms of these transformations are not yet fully understood. Soluble phosphorus concentration in the soil is generally very low, normally at 1 ppm or less. The cell can take up to several forms of phosphorus, but most of it is absorbed in the forms of HP0 ₄ ^{2 "} or H ₂ PO ₄ ^" . The largest phosphorus reserves are rocks and other deposits, such as primary apatites and other primary minerals formed during the geological age. In spite of this, they constitute the largest reservoirs of this element in the soil because, under appropriate conditions, they can be solubilized and become available to plants and microorganisms.

In vitro studies of the phosphate solubilization dynamics of bacterial strains were performed based on the measurement of P release in the culture broth, from cultures grown using an insoluble compound such as rock P. The solubilization rate P is often estimated by subtracting the final P concentration (minus that of an inoculated control) from the initial theoretical P provided by the P substrate.

Among the bacteria best known for their ability to solubilize P are strains of the species Pseudomonas fluorescens.

On the other hand, the spectacular increase in agricultural production in the 20th century was made possible thanks to several remarkable evolutions in cultivation practices - intensification and mechanization - development of agrochemicals - genetic selection. This evolution of productivist agricultural practices, however, generates many harmful effects for the environment (nitrates, phosphates, pesticides ...), and the development in the coming decades of practices more respectful of the environment has become an obligation.

The agriculture of tomorrow will be both intensive and ecological. This "eco-intensification" requires the use of new agronomic practices that are organic and respectful of the environment. Our project is fully in line with this strategic perspective and plans to develop products and services adapted to this new situation. The search for new strategies, aimed at maintaining or increasing the productivity of farming systems while reducing chemical inputs, can in particular be based on the results of recent research, which has highlighted the crucial importance of microorganisms in the functioning of agricultural systems. biological soil. This research has led to the emergence of the concept of "biofertilizers", defined as products containing different types of microorganisms with the ability to promote plant growth by various mechanisms still partially elucidated. Among these microorganisms there are bacteria, grouped under the term Plant Growth Promoting Rhizobacteria (PGPR), as well as fungi, in particular mycorrhizal fungi.

 Biofertilizers are defined as products containing live microorganisms that, when applied to the soil or plants, occupy the rhizosphere, or even colonize plant tissues, and stimulate plant growth, for example by increasing the uptake of nutrients and nutrients. the production of phytohormones.

The present invention is directed to the formulation and application of a product based on a strain of Pseudomonas fluorescens as a biofertilizer for phosphorus solubilization

The formulation of a product based on microorganisms remains the most important point. As such, the microorganism must remain viable and virulent for improved efficacy once re-enforced. In this work the inventors have found a technique for preserving solubilizing phosphorus bacteria.

 The present invention shows a formulation process that begins with the production of the bacterial strain. Indeed, the formulation is included in the manufacturing process.

The bacterium presented in this invention has been isolated from Moroccan soil by the following method:

 - Placing a biological sample likely to contain the Phosphorus solubilizing strain, with a suitable medium for the selection of strains with this function,

 - Treatment of said sample to isolate the strains,

 The isolation of the strain according to the invention can be carried out from samples of the soil rhizosphere of Morocco.

 The protocol described-by following:

 Samples of the Middle Atlas rhizosphere are obtained sterile after removing 15 cm from the root of the soil.

 Samples taken are placed in sterile closed polyethylene bags and stored at 4 ° C.

 Solubilizing strains of P are subsequently isolated on Rock P Agar medium after four types of successive treatments: treatment by grinding, heat, stirring and centrifugation.

After treatment of the samples, the solubilizing strains of P which have shown a light halot are isolated (see FIG. 1). The strains are transferred to the rich medium. Pure isolates are kept at 4 ° C for 2 months. Alternatively, the cultures are resuspended and stored in glycerol at -20 ° C.

Subsequent efficacy trials were conducted to show the role of the strain in solubilizing phosphorus and to promote plant growth. Other tests have also been carried out, namely the production of phytohormones.

Test 1: phytohormone production test

The following table shows the ability of both strains to produce auxin

Trial 1: Tests on life-size corn

The strains were applied in seed treatment on corn seeds. These last sown on the corn field. After harvesting the crops. It turned out that corn treated with solubilizing phosphorus strains had a choke effect and increased yield (see Figure 2A).

Also after physicochemical analysis of maize plants. The results show that the treatment of corn with the strain increased its phosphorus, potassium, boron and above all iron content (Figure 2B).

 Test 2: Stability of the product in the formulation

Several formulations have been tested to have a long stability of the strains during the storage period. The way to preserve the strains was ultimately to include the formulation process in the strains production process.

Indeed, production of strains in their adapted industrial culture medium in the stationary phase of production and before reaching the end of the exponential phase is added a stabilizing agent and conservation of the two strains.

The legend of the figures

Figure 1: Solubilization of insoluble P in a culture medium, appearance of a clear transparent halot.

 Figure 2: Life size corn tests

Claims

The revendications  1 - Biofertilizing product for phosphorus solubilization characterized by the liquid formulation consisting of:  Pseuodomonas fluorescens  CMC with a quantity that ranges from 0.01% to 1%  Preservative and stabilizer with a quantity that ranges from 5% to 10%  - Sterile water.  2- A method of manufacturing the product of claim 1 characterized by a step including the formulation process by adding the stabilizing agent before the stationary phase and shortly before the end of the exponential phase of the growth of the two bacterial strains .  3- The product of claim 1 and 2 is applied for the solubilization of phosphorus insoluble in the soil and make it available to the plants for the stimulation of their growth.  4- Application of the product, according to claims 1, 2 and 3, for agricultural fertilization characterized by the different types of application namely seed treatment, spot treatment, spray treatment on the ground.