Field of invention The present invention relates to a microemulsion containing a phytosanitary agent and a humic substance for use in agriculture.

Background of the invention

Phytosanitary agents are often prepared in the form of concentrated formulations suitable to be diluted with water to obtain ready-to-use formulations. In the state of the art, different types of concentrated formulations are known (e.g., solutions, suspensions, emulsions, emulsifying concentrates, etc.), which consist of a phytosanitary agent and a carrier which can include various components. Phytosanitary agents are organic compounds, inorganic compounds, or microorganisms (bacterial or fungal inoculum), able to protect a plant or crop from harmful organisms or to stimulate their growth.

One of the main issues of the state-of-the-art concentrated formulations is the tendency to decrease in efficacy over time, for example during storage prior to their use in the field, due to various phenomena that can affect the formulation (e.g., flocculation, sedimentation, and precipitation) . This problem is particularly relevant when the phytosanitary agent is a microorganism, which, as known, is characterized by a limited cell viability over time. Even after application on plants or agricultural crops, phytosanitary agents can undergo degradation or inactivation phenomena due to the action of environmental factors, such as the action of microorganisms or the interaction with minerals and the organic substance present in the soils.

The use of growth promoting microorganisms in combination with humic substances as functional biostimulants to increase crop productivity is reported in the literature for different types of species of agricultural interest, such as, for example, corn, beans, tomato, pineapple, sugarcane. The responses activated by the inocula affect a wide range of biochemical reactions and physiological functions, comprising, among others, the activation of biochemical mechanisms related to the promotion of the H + -ATPase enzyme activity of the plasma membrane, the pseudo- hormonal action on the tissues and plant organs, the increase in the absorption efficiency of nutrients and limiting factors of the production (e.g. nitrogen and phosphorus), the induction of resistance and/or greater resilience capacity to biotic and abiotic stresses. Some examples of the use of bacterial inocula on non- leguminous crops described in the literature refer to the use of aqueous formulations containing rhizo- bacteria capable of promoting the development of root systems, increasing the biomass and the crop productivity.

One way of administration of said bacterial inocula is based on the application of a mixture of peat and inocula to the seeds of the crop, before sowing. It is also known to administrate bacterial inocula by direct foliar application of aqueous formulations wherein said inocula are dispersed. Direct foliar application, although a more efficient technique, has not yet found the desired practical application mainly due to the limited shelf life of the formulations containing the inocula of rhizo- bacteria. In fact, after just three months of storage, a drastic reduction in cell viability of rhizo-bacteria takes place, therefore making the formulation substantially ineffective.

Summary of the invention

The Applicant has faced the problem of overcoming the drawbacks highlighted above which afflict the phytosanitary formulations of the state of the art.

In particular, a specific purpose of the present invention is to provide a phytosanitary formulation that has a longer shelf life, i.e., that can be used effectively even after a longer shelf life as compared to the state of the art formulations.

A second purpose of the present invention is to provide a phytosanitary formulation that has a greater phytosanitary efficacy after the application to a crop of interest.

The Applicant has found that the above and other purposes, which will be better illustrated in the following description, can be achieved by using a phytosanitary formulation wherein the carrier of the phytosanitary agent is a microemulsion comprising an oil phase, water and at least one surfactant, and wherein at least one humic substance is present. In a preferred embodiment, the humic substance is a substance having a supramolecular conformation (hereinafter also referred to as "supramolecular humic substance").

It has in fact been found that the presence of humic substances in a microemulsion, in particular of supramolecular humic substances, is able to slow down the degradation processes of the phytosanitary agent by the aforementioned factors, thus prolonging the effectiveness of the phytosanitary formulation, both in storage conditions (i.e., longer shelf life of the formulation) and after its application to agricultural crops (i.e., lower degradability of the phytosanitary agent in the field). In particular, it was observed that the presence of humic substance in a microemulsion containing a microbial inoculum (e.g., bacterial or fungal) as a phytosanitary agent is particularly effective in preserving the cell viability of the inoculum microorganisms for a longer period of time.

Without wishing to refer to any particular theory, it is theorized that the aforementioned effects of the humic substance are related, in general, to its surfactant characteristics and, therefore, to the ability of the humic substances to form stable colloidal aggregates (pseudo-micellar aggregates) in a microemeulsion . Colloidal aggregates constitute an optimal environment for receiving microbial inocula, thus protecting them from degradation phenomena. This effect is observable in the case of supramolecular humic substances, as these substances have a flexible molecular structure that favors their interaction with other molecules or microorganisms, thus becoming more effective in extending the shelf life of microemulsions.

Therefore, in accordance with the first aspect, the present invention relates to a microemulsion comprising: a. at least one phytosanitary agent; b. at least one humic substance; c. at least one oil phase; d. at least one surfactant; e. water .

In accordance with a second aspect, the present invention relates to a process for preparing a ready to use phytosanitary formulation comprising:

- providing at least the aforementioned microemulsion,

- diluting said microemulsion with water to obtain the aforementioned ready to use phytosanitary formulation.

In accordance with a third aspect, the present invention relates to a method for protecting an agricultural plant or crop from harmful organisms and/or stimulating the growth of said agricultural plant or crop which comprises applying at least one effective dose of the aforementioned microemulsion to a plant or agricultural crop in undiluted form or in diluted form with water.

In accordance with a further aspect, the present invention concerns the use of a humic substance in a microemulsion containing at least one viable microorganism to improve the maintenance of the cell viability of said microorganism.

The compositions according to the present invention can "comprise", "consist of" or "essentially consist of" the essential and optional components described in the present description and in the attached claims.

For the purposes of the present description and the appended claims, the expression "consist essentially of" means that the composition or components may include additional ingredients, but only to the extent that the additional ingredients do not alter the essential characteristics of the composition or of the component. The numerical limits and ranges expressed in the present description and in the appended claims also include the numerical value or numerical values mentioned. Furthermore, all values and sub-ranges of a numerical limit or range are to be considered as specifically included as if they were explicitly mentioned.

Description of the figures

The characteristics and advantages of the present invention will become evident from the following description in which reference will also be made to the following figures:

- Figure 1, which shows images obtained by optical microscopy of some microemulsions according to the invention.

- Figure 2, which shows results of the determination of bacterial population growth, on different substrates, for the microemulsions according to the invention and a control sample.

Detailed description of the invention

A microemulsion according to the present invention is a multiphase liquid system, comprising a dispersed phase and a continuous phase, thermodynamically stable, optically transparent and fluid. The average size (Z) of the particles (droplets) of the dispersed phase is less than 800 nm, preferably less than 600 nm, more preferably less than 500 nm. For the purposes of the present invention, the average size of the particles Z can be measured by Dynamic Light Scattering according to ISO 22412: 2017 (using for example a Zetasizer Nano ZS instrument produced by Malvern Panalytical). In consideration of the above average dimensions of the dispersed phase, the microemulsions according to the present invention can be defined as nanostructured microemulsions .

As known, microemulsions are formed when a surfactant, more frequently a mixture of at least one surfactant and at least one co-surfactant (or co solvent), reduces the oil/water interface tension to extremely low values, generally in the range ICh ³ N/m to 10 ⁹ N/m, preferably in the range from 10 ⁴ N/m to 10 ⁶ N/m, so that the two insoluble phases remain homogeneously dispersed due to thermal agitation.

The microemulsions according to the present invention can have an oil-in-water (0/W), water-in-oil (W/0) or bi-continuous (BC) structure.

The microemulsions according to the present invention comprise at least one phytosanitary agent.

The term "phytosanitary agent" means an organic compound, an inorganic compound or a microorganism that produces a growth promoting protection from harmful organisms, conservation and/or control effect on a plant or crop.

In one embodiment, the phytosanitary agent is a viable microorganism (bioeffector). Preferably, such microorganisms are bacteria or fungi. As for bacteria, plant growth promoting bacteria are particularly preferred, such as species capable of fixing atmospheric nitrogen, solubilizing insoluble forms of phosphorus and micronutrients, producing plant hormones and attenuating the effect of different types of stress (thermal, saline etc.). Examples of bacteria that can be used for the purposes of the present invention are: Herbaspirillum seropedicae; Gluconacetobacter diazotrophicus; Serratia marcensces; Azzospirum spp; Paraburkholderia spp; Bacillus spp; Paenebaccilus spp; Pseudomonas fluorescens; Enterobacter spp.

As for fungi, the ones contributing to the biological control of phosphorus absorption and the increased bioavailability of the organic forms of phosphorus are preferred. Examples of fungi that can be used for the purposes of the present invention are: Thrichoderma longibracthium, Thricoderma hazarium, Trichoderma spp, mycorrhiza (glomus clarum, gigaspora margharita).

In one embodiment, the phytosanitary agent is an organic or inorganic compound having at least one function selected from: insecticide, fungicide, nematicide, herbicide, plant protector and growth regulator.

Examples of compounds having the above activities are gibberellin, auxin, brasinosteroid, abscisic acid, jasmonic acid, tryptophan, phenolic compounds.

The overall amount of phytosanitary agent in the microemulsion is generally in the range from 0.001% to 10% by weight, preferably from 0.01% to 1% by weight, more preferably from 0.05% to 1% by weight, the above percentages being referred to the weight of the microemulsion .

In the event that the phytosanitary agent is a microorganism, the total amount of microorganism in the microemulsion is generally in the range from 2 x 10 ⁸ to 2 x 10 ¹¹ CFU per mL of aqueous phase present in the microemulsion, preferably from 2 x 10 ⁹ to 2 x 10 ¹¹ CFU/mL, more preferably 2 x 10 ¹⁰ to 2 x 10 ¹¹ CFU/mL, wherein CFU means "colony forming units".

The microemulsions according to the present invention comprise at least one humic substance.

In a preferred embodiment, the humic substance is a humic substance having a supramolecular conformation. The humic substance is the product of the natural process of chemical and microbiological transformation of the residues of plants, fungi, microorganisms, and animals. On an industrial scale, preparations based on humic substances are mainly obtained starting from peat, compost, lignite, and humus-like materials obtainable, for example, by extraction from waste lignocellulosic materials from biorefineries, for example after a depolymerization treatment. In particular, the most widely used natural source of humic substance is Leonardite, a natural carbonaceous geochemical deposit based on lignite.

The humic substance has a non-stoichiometric elemental composition and an irregular and heterogeneous structure. Based on a first methodology, the humic substance can be operationally classified into the following fractions on the basis of its solubility in aqueous solutions at different pH: fulvic acids, which represent the soluble fraction at all pH values; humic acids, which represent the insoluble fraction at pH less than 2.0; humin, which represent the insoluble fraction at all pH values. The humic substance usable for the purposes of the present invention can be obtained, for example, by the extraction method described in Swift, R.S. 1996, Organic matter characterization (chap 35), pp. 1018-1020, in: D.L. Sparks et al. (eds) Methods of soil analysis. Part 3. Chemical methods, Soil Sci. Soc. Am. Book, Series: 5. Soil Sci. Soc. Am. Madison, WI.

Humic substances are supramolecular associations of low molecular weight organic components stabilized by weak interactions such as hydrophobic bonds, hydrogen bonds, van der Waals forces, n-n bonds. The humic substance has hydrophobic domains and contiguous hydrophilic domains, which give it surface-active properties. It is therefore also capable of forming stable micellar aggregates in aqueous solutions at different pH values, saline concentrations, and ionic strength values.

Preferably, the humic substance used for the purposes of the present invention is a humic substance having a supramolecular structure.

For the purposes of the present invention, a humic substance has a supramolecular structure if, following a treatment with acetic acid, it is fractionated into smaller aggregates with respect to the starting humic substance. For the purposes of the present invention, the supramolecular character of a humic substance can be experimentally determined according to the method described in the publication of Piccolo A. et al., 2002, "Reduced heterogeneity of a lignite humic acid by preparative HPSEC following interaction with an organic acid. Characterization of size-separates by PYR-GC-MS and 1H-NMR spectroscopy", Environmental Science and Technology, 36: 76-84, Materials and Methods chapter.

According to this method, the humic substance having a supramolecular structure can be detected by high pressure molecular exclusion chromatographic analysis, after treatment with glacial acetic acid. Glacial acetic acid, in fact, is able to break the bonds that keep the humic substance in aggregate form (supramolecular humic substance) forming aggregates of reduced size and molecular weight. The breaking of these bonds gives rise to a variation of the chromatographic profile as shown in figure 1 of the above publication.

The supramolecular humic substance, if dispersed in water, organizes itself in aggregates having a conformation capable of adapting to the shape of other molecules and surfaces with which it comes into contact (this conformation is also referred to here as a "metastable conformation").

The overall amount of humic substance in the microemulsion according to the invention can vary in a relatively wide range of values. The mass of humic substance in the microemulsion is generally in the range 0.2 - 50 mmol of carbon of the humic substance per liter of aqueous phase, preferably in the range 2 - 20 mmol C/L. The use of humic substance concentrations higher than 20 mmol C/L of carbon is not recommended in the event that the phytosanitary agent is a microorganism, as an excessive concentration of humic substance can have toxic effects on microorganisms.

The microemulsions according to the present invention comprise at least one oil phase.

The oil phase, in general, is an organic liquid phase, substantially immiscible with water. Components of the oil phase suitable for the purposes of the present invention include, but are not limited to, hydrocarbons and esters of fatty acids, liquid at room temperature. Examples of such hydrocarbons include mineral oils, vegetable oils, silicone oils, paraffin oils and liquid polyolefins. Fatty acid esters include, for example, methyl and isopropyl esters of fatty acids containing 12 to 22 carbon atoms, such as: methyl laurate, methyl stearate, methyl oleate, methyl erucate, isopropyl palmitate, isopropyl stearate, isopropyl oiled.

Other components of the oil phase can be n-butyl stearate, n-hexyl laurate, n-decyl oleate, isooctyl stearate, isononyl palmitate, isononyl isononanoate, 2- ethylhexyl palmitate, 2-ethylhexyl laurate, 2-oxydecyl stearate, oleyl oleate, oleyl erucate, erucyl oleate and esters obtainable from aliphatic alcohol mixtures and aliphatic carboxylic acids, for example esters of saturated and unsaturated fatty alcohols containing 12 to 22 carbon atoms and saturated and unsaturated fatty acids containing 12 to 22 carbon atoms which are obtainable from animal and vegetable fats.

Other components of the oil phase include: esters of dicarboxylic acids such as, for example, di-n-butyl adipate, di-n-butyl sebacate, di- (2-ethylhexyl) - adipate, di- (2-ethylhexyl) -succinate, diisotridecyl esters of azelate and diol such as, for example, ethylene glycol dioleate, ethylene glycol diisotridecanoate, di- (2-ethylhexanoate of propylene glycol), butanediol diisostearate or dicaprylate of neopentyl glycol.

Liquid triglycerides can also be used as components of the oil phase. These triglycerides comprise, for example, olive oil, corn oil, sunflower oil, soybean oil, peanut oil, rapeseed oil, almond oil, palm oil or the liquid fractions of coconut or palm oil. These liquid triglycerides also comprise synthetic triglyceride oils obtainable, for example, by esterification of glycerol with caprylic acid/capric acid mixtures, or palmitic acid/oleic acid mixtures. Preferably, the components of the oil phase of the invention are mineral oils, vegetable oils (in particular, olive oil, corn oil, sunflower oil, rapeseed oil), paraffin oils, silicone oils or esters, including fatty acid esters and methyl esters, more preferably isopropyl myristate.

The total amount of oil phase in the microemulsion according to the invention generally depends on the amount of water used to disperse the phytosanitary agent. The weight ratio between the oil phase and the aqueous phase wherein the phytosanitary agent is dispersed is generally in the range from 1: 4 to 4: 1, preferably in the range from 1: 3 to 3: 1, more preferably in the range from 1: 2 to 2: 1.

Preferably, for the BC structure microemulsions the above ratio is about 1: 1. Preferably, for microemulsions with an 0: W structure the above ratio is about 0.5: 1. Preferably, for the W: 0 structure microemulsions the above ratio is about 2: 1.

The total amount of oil phase in the microemulsion is generally in the range of 1% to 25% by weight with respect to the total weight of the microemulsion, more preferably in the range of 5% to 25% by weight, even more preferably in the range of 10 % to 20% by weight. Preferably, in the case of the 0: W and bicontinuous microemulsions, the oil phase is in the range from 5% to 15% by weight with respect to the total weight of the microemulsion. Preferably, in the case of the W: 0 microemulsions, the oil phase is in the range from 15% to 25% by weight with respect to the total weight of the microemulsion . The microemulsions according to the present invention comprise at least one surfactant. The surfactant serves to reduce the surface tension between the continuous and the dispersed phase, thus stabilizing the droplets of the dispersed phase. The surfactant also helps the solubilization of the phytosanitary agent, particularly when this is an organic compound.

Surfactants suitable for formulating the microemulsions of the present invention are known in the state of the art. In principle, it is possible to use any surfactant of any of the known surfactant groups, such as nonionic surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants and mixtures thereof. Particularly preferred are the anionic surfactants, the nonionic surfactants, and mixtures thereof.

Examples of non-ionic surfactants suitable for the purposes of the present invention are:

- sugars esterified with fatty acids (sorbitans), such as sorbitan monooleate, sorbitan mono-stearate, sorbitan monopalmitate, sorbitan dioleate, etc. (eg. SPAN® 20, 40, 60, 80))

- etherified-esterified sugars with fatty acids (polysorbates), such as polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate (eg TWEEN® 20, 40, 60, 80)

- polyoxyethylene alkyl ethers of formula CnEm, wherein n is the number of methylene groups, which is in the range from 10 to 18, and m the number of oxyethylene chains, in the range from 1 to 20. The anionic surfactants suitable for the purposes of the present invention are, for example, sulphates of fatty alcohols having from 8 to 22, preferably from 10 to 18, carbon atoms, such as for example cetyl sulfate, myristyl sulphate, palmite sulphate, sulphate of stearyl.

Other suitable anionic surfactants are: ethoxylated sulfated alcohols having from 8 to 22 carbon atoms; the alkan sulfonates, having from 8 to 24, preferably from 10 to 18; soaps, such as the Na or K salts of carboxylic acids having from 8 to 24 carbon atoms; N-acylsarcosin with aliphatic radicals, saturated or unsaturated, having from 8 to 25 carbon atoms, for example N- oleoylsarcosinate; linear or branched alkylbenzenesulphonates having 10 to 13 carbon atoms (LAS).

The cationic surfactants suitable for the purposes of the present invention are, for example, hexadecyltrimethyl ammonium bromide (CTAB) diodecyltrimethyl ammonium bromide (DDAB).

The zwitterionic surfactants suitable for the purposes of the present invention are, for example, esterified phospholipids, c.d. lecithins, such as soy lecithin (e.g., ALCOLEC®).

In one embodiment, the at least one surfactant comprises or consists of a mixture of two or more surfactants, more preferably a mixture of at least one anionic surfactant and at least one non-ionic surfactant.

The total amount of surfactant in the microemulsion is generally in the range from 5% to 45% by weight, preferably from 25% to 45% by weight, preferably from 35% to 40% by weight, with respect to the total weight of the microemulsion.

The microemulsions according to the present invention optionally comprise at least one co-surfactant (or co-solvent). The co-surfactant favors the formation of the microemulsion, helping to reduce the surface tension between the continuous phase and the dispersed phase. It can also promote the dissolution of the phytosanitary agent in the microemulsion.

The at least one co-surfactant can be chosen for example from: aliphatic alcohol having from 1 to 8 carbon atoms and a number of OH groups equal to 1, 2, 3 or 4; carboxylic acid having from 4 to 10 carbon atoms, monosubstituted amine with a Cl - C6 alkyl group, polyethylene glycol (PEG), propylene glycol (PG) and mixtures thereof.

Suitable co-surfactants are for example: glycerol, ethanol, propanol, isopropanol, butanol, pentanol, hexanol, 1,2-butanediol, pentanoic acid and butylamine.

The total amount of co-surfactant in the microemulsion is generally in the range from 0% to 45% by weight, preferably from 25% to 45% by weight, even more preferably from 35% to 40% by weight, with respect to the total weight of the microemulsion.

In microemulsions wherein at least one co surfactant is present, the ratio between the total weight of the surfactant and the total weight of the co surfactant is preferably comprised in the range 1:1 to 3:1.

The microemulsion of the invention also contains an aqueous phase. The aqueous phase comprises at least water. The amount of water is generally in the range from 1% to 80% by weight, for example in the range from 5% to 50% by weight, in particular from 10% to 40% by weight and more preferably from 15% to 30 % by weight, with respect to the total weight of the microemulsion. It is evident that in the microemulsion the amount of water together with the amounts of the other ingredients amount to 100% by weight of the weight of the microemulsion .

The microemulsion of the invention may also contain conventional additives used for the preparation of liquid phytosanitary formulations, such as antifoams, preservatives, dyes, stabilizers and similar components. The total amount of these additives generally does not exceed 10% by weight, preferably 5% by weight, more preferably 3% by weight of the microemulsion.

In one embodiment, the microemulsion comprises:

- 1% - 25% of an oil phase

- 5% - 45% of at least one surfactant

- 0% - 45% of at least one co-surfactant

- complement to 100% of an aqueous phase comprising water, at least one humic substance and at least one phytosanitary agent, where:

- the humic substance is present in an amount equal to 0.2 - 50 mmol of carbon per liter of aqueous phase

- the at least one phytosanitary agent is present in a total amount from 0.001% to 10%, if the phytosanitary agent is an organic or inorganic compound, or in a total amount from 2-10 ⁸ to 2 -10 ¹¹ CFU per mL of aqueous phase if the phytosanitary agent is a viable microorganism; the aforementioned percentages being percentages by weight referred to the weight of the microemulsion. The microemulsions of the present invention can be prepared by simply mixing the components until a homogeneous liquid is formed. The sequence of adding the ingredients is not of particular importance. For example, the ingredients can be poured into a container and the mixture thus obtained is homogenized, e.g., stirring, until a homogeneous liquid is formed.

The mixing temperature and mixing conditions are not of particular importance. Generally, the ingredients are mixed at a temperature between 10 ⁰ C and 90 ⁰ C, preferably between 10 ⁰ C and 60 ⁰ C.

In one embodiment, when the phytosanitary agent is a microorganism, it is possible to prepare the microemulsion according to a process, which represents a further object of the present invention, comprising the following steps: i. providing an aqueous phase comprising water and at least one humic substance; ii. adding at least one phytosanitary agent to the aqueous phase leaving step i; iii. adding at least one surfactant, and optionally at least one co-surfactant, to the aqueous phase leaving step ii; iv. adding an oil phase to the aqueous phase leaving step iii to obtain the microemulsion.

Preferably, after step iii and before step iv, the pH of the dispersion of the humic substance can advantageously be brought into the range from 5 to 8, for example by adding a strong diluted base or a strong diluted acid. It has in fact been observed that a pH value in the above range favors the formation of pseudo micelles of humic substance in the microemulsion. When used to protect a plant or agricultural crop from harmful organisms and/or to stimulate the growth of a plant or agricultural crop, the microemulsions according to the invention can be applied as such or in diluted form with water, on any part of the plant or agricultural cultivation, for example on leaves, stems, branches, and roots, or on the seeds themselves before sowing, or on the soil where the plant grows.

Examples of crops of interest are fruit trees, horticultural and extensive crops such as rice, wheat, and soy.

The amount of microemulsion to be applied to an agricultural crop to obtain the desired phytosanitary effect may vary depending on various factors such as, for example, the crop, the climatic conditions, the characteristics of the soil, the method of application. The amount to be applied can be easily determined by the expert in the field based on his knowledge.

The following examples are provided for illustrative purposes only of the present invention and should not be construed as limiting the scope of protection defined by the attached claims.

EXAMPLES

1, Preparation of the microemulsions

The humic substances were extracted from different matrices: superficial horizons of an Oxisol (ox) soil and a Dark Earth (de) "terra preta do indio" (both Brazilian), vermicompost (v) and Leonardite (1) soil. The extraction of the humic substance from the soils was performed as described in Swift, R.S. 1996, Organic matter characterization (Chapter 35), pp. 1018-1020, in: D.L. Sparks et al. (eds) Methods of soil analysis. Part 3. Chemical methods, Soil Sci. Soc. Am. Book, Series: 5. Soil Sci. Soc. Am. Madison, WI, except for Leonardite, for which a humic substance from commercial preparations was used (Heringer Co, Sao Paulo, Brazil). Each extract was dialyzed against deionized water with 1000 Da membranes and subsequently lyophilized.

The choice of different matrices allows the use of humic substances with adequate variability in terms of origin and nature of the molecular composition and chemical-physical characteristics.

The data of the elemental composition, determined by means of an elemental analyzer, are reported in Table 1. Table 1

Elemental composition of the humic substance. Origin of the humic substance: ox = oxisol, v = vermicompost; de = terra preta do indio, 1 = leonardite.

Microorganisms of the Herbaspirilium seropedicae Z67 species were used as a model of growth promoting microorganisms of agricultural crops. A bacterial colony was prepared in glass tubes on JNFb culture medium with the addition of NH4C1 (1 g/L), at 34 ⁰ C, kept under stirring (150 rpm) for 16 hours. The cells were then separated by centrifugation (4,000 g for 15 min) and dispersed in an aqueous solution of humic substance at a concentration of 48 mg C/L (equivalent to 4 mmol C/L) and pH equal to 5.8. The concentration of the cells in the dispersion was 109 CFU/mL.

The suspensions thus obtained were subsequently used as aqueous phase (W) in the preparation of the microemulsions .

The microemulsions were prepared by mixing together:

- oil phase (0) consisting of isopropyl myristate (Sigma Aldrich)

- aqueous phase (W) prepared as described above

- surfactant system (ST) consisting of a mixture of Tween 20 and glycerol (weight ratio 2:1).

The tested microemulsions have the following compositions ST:W:0, expressed as percentage ratios by weight

- 80:10:10 (bicontinuous microemulsion);

- 70:20:10 (O/W microemulsion);

- 70:10:20 (W/O microemulsion).

The microemulsions were prepared at room temperature and let stand for 48 hours to allow thermodynamic stabilization, before subjecting them to characterization .

2. Physico-chemical characterization

The average diameter of the dispersed phase particles and the polydispersion index were evaluated by Dynamic Light Scattering (DLS) using 1.5 mL aliquots of volume for each microemulsion sample (173 ⁰ angle, 120 s measurement time). The zeta (Z) potential, electrophoretic mobility and conductivity of the microemulsions were determined by LDV (laser doppler velocimetry) with 1 mL of sample in electrophoretic cells with potential equal to ± 150 mV, using the Zetasizer Nano ZS device (Malvern Instruments, UK). The values of the Zeta potential were derived from the average electrophoretic mobility using the Smoluchowski equation. All analyses were conducted at 25 ⁰ C, preliminary adjusting the pH value to 5.9 ± 0.1 (Cambo HI 98129, Hanna Instruments, UK) by placing the electrode in 5 ml of each microemulsion.

The shelf life of the Herbaspirilium seropedicae inoculum in the microemulsion in the presence of the humic substance was determined as follows. Aliquots of 20 pL of each microemulsion were transferred, in triplicate, into glass tubes containing the JNFb growth medium diluted 1:1000 in distilled water and sterilized in an autoclave. The tubes were incubated at 30 ⁰ C for seven days. The reference of the viability and growth of the bacterial population was represented by the formation of an extensive white patina on the culture medium. Viable cell count was performed after transfer to JNFb growth medium in the absence of N by turbidimeter at 460 nm with reference to a standard calibration curve.

3. Effectiveness of microemulsions

Corn hybrid seeds {Zea mays L., cultivar DEKALB 7815) were surface-sterilized with a 0.5% NaHClO (sodium hypochlorite) solution for three minutes and then washed with distilled water. The seeds were then transferred to distilled water for six hours to favor a sufficient and uniform imbibition, and then placed in a sowing substrate consisting of a mixture of sterilized sand and vermiculite (3:1 v:v) in 500 mL pots. The substrate was then added with a Clark nutrient solution (at 25% concentration corresponding to an ionic strength 1/41), adjusting the total N content with a 0.5 mmol/L solution of nitrate ions and 0.5 mmol/L of ammonium ions. Seven days after sowing, the foliar application of the microemulsions was performed after dilution in water (1:1000 v:v). A reference control consisting of foliar application of distilled water was used for each microemulsion .

The experiments were performed in triplicate with a randomized block scheme.

In a first experiment, after two weeks of growth, the maize seedlings were harvested for the evaluation of the development of the bacterial population on the plant tissues (leaves and root system).

A second growth experiment of potted corn plants was conducted in the same way, using the microemulsions according to the invention after 12 months shelf storage, at room temperatures and in the absence of lighting. The bacterial population was evaluated with the most probable number (MPN) technique, expressed as the log of the number of cells per gram fresh tissue (root and bud), based on the development of the patinas after transferring to JNFb growth medium in the absence of N.

4._ Physico-chemical_ characteristics_ of microemulsions

The results of the zeta potential, electrophoretic mobility and electrical conductivity measurements are shown in Table 3.

The values of the zeta potentials (mV), all slightly higher than the value of -30 mV, and the values of the polydispersion index, comprised in the range from -10 to -30 mV, are generally considered as indicative of thermodynamically stable microemulsions with increasing stability towards lower mV values. The average diameter of the dispersed phase particles in the tested microemulsions falls within the field of nanostructures, since the measured average diameter values vary from 34 nm to 505 nm, according to the following order of magnitude: bicontinuous> oil: water> water: oil (Table 2).

The presence of humic substance and microorganisms in the aqueous phase produces an average decrease of 35% in the average diameter of the dispersed phase particles in the final microemulsions compared to the control represented by corresponding microemulsions obtained using an aqueous phase free of humic substance.

The largest decrease was observed in the oil: water system. The reduction of the average diameter is a further parameter indicative of the stability of the systems formed by the microemulsions in the presence of the aqueous suspensions of H. seropedicae containing the humic substances.

Table 2 - Physico-chemical characterization of microemulsions at pH = 5.9; HA _V = humic substance from vermicompost; HA _ox = humic substance from oxisol; HA _e = humic substance from dark earth; HAi = humic substance from leonardite.

Figure 1 shows the images obtained by optical microscopy of the microemulsions containing HA _V and microorganisms, used for the assays to evaluate the shelf life of the bacterial population (see below).

Light microscopy reveals that the microemulsions have a regular and reproducible shape and size, in line with the physico-chemical property data of Table 3.

Figure 1-A refers to the water: oil microemulsion (W/0) having the ratio ST:W:0 equal to 70:10:20. The phase contrast optical microscopy image highlights the presence of encapsulated bacteria indicated with the star symbol and aggregates of humic acids indicated with the arrow symbol. We note the high density of bacterial cells within the vesicle of the dispersed phase compared to the dispersing phase (bar = 5 pm).

Figure 1-B depicts the same microemulsion as Figure 1-A. The differential interference contrast microscopy (DIC) image shows the dimensions of the microemulsion with a different magnification (bar = 25 pm).

Figure 1-C refers to the oil: water microemulsion (0/W) having the ratio ST:W:0 equal to 70:20:10. The phase contrast optical microscopy image highlights the presence of encapsulated bacteria and particles of humic substances; the particles are surrounded by a translucent halo (bar = 20 pm). Figure 1-D refers to the same microemulsion as Figure 1-C. The differential interference contrast microscopy (DIC) image shows the dimensions of the microemulsion with a different magnification (bar = 30 pm).

5. Evaluation of the survival of microorganisms in microemulsions

The maintenance of bacterial cell viability was determined after 1 week and 50 weeks after their insertion into microemulsions containing humic vermicompost substances (HA _V ).

The presence of patinas on the surface of the growth substrate in the tubes was indicative of the survival and development of bacterial cells. The results of determining bacterial population growth by MPN are shown in Figure 2-A. The number of viable cells is drastically reduced (from 109.4 to 103.7 cells/mL) in the system consisting of the aqueous suspension of HA _V and H. seropedicae after 50 weeks of storage. Conversely, the reduction observed for bacterial cells included in the microemulsions was much less intense; the microemulsions in fact showed a high number of viable cells at the end of the conservation period. Consequently, the infection potential of the plants also remained at high levels (Figure 2-B). The microbial population present on the leaves and roots of maize seedlings after application of the microemulsions diluted in water (1:1000 v:v) is shown in Figures 2-B and 2-C. The almost constant maintenance of cell viability recorded for the population of H. seropedicae on the leaf surface following inoculation of the microemulsions after one year from their preparation is highlighted (Figure 2-B). The inoculation based on the aqueous suspension of HA _V and H. seropedicae showed a total loss of the potential for infection at 52 weeks.

The results obtained confirm that the present invention is an effective innovative technology to produce environmentally friendly substrates capable of preserving the functionality of phytosanitary agents, in particular the vitality of microorganisms, to be used in agronomy.