Process for obtaining a formulation with fertilizing and phytoprotective capability, a formulation with fertilizing and phytoprotective capability, use of a formulation with fertilizing and phytoprotective capability

Abstract

The present invention falls into the context of green chemistry and generically relates to a fertilizing and phytoprotective formulation and, in particular embodiment, to a film forming formulation that induces resistance to plants. The respective formulation, when applied to plants and/or fruits, results in the formation of a film on the surface of the material, which has a characteristic of photoprotection against UV-B and UV-C radiations, resistance kept in water, even after high hygroscopicity, greater stability at high ambient temperatures, formation of desired porosity and surface homogeneity.

Claims

1. A process for obtaining a thermally stable formulation with fertilizing and phytoprotective capability, comprising the following steps: A) Obtaining a distilled pyroligneous extract (DPE) by vacuum distillation of a crude pyroligneous extract (CPE) at a temperature of 60 C. to 75 C.; B) Obtaining a composition comprising the DPE and chitosan; C) Obtaining a fertilizing mineral solution; D) Mixing the composition obtained in step B with the solution obtained in step C.

2. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 1, wherein the composition of step B of the process is obtained by mixing the chitosan and the DPE.

3. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 2, wherein the chitosan has a minimum distillation degree of 97%.

4. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 2, wherein the concentration of the chitosan in the composition of step B ranges from 0.05 g/L to 30 g/L.

5. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 2, wherein conductivity of the composition obtained in step B ranges from 1038 S cm.sup.1 to 4970 S cm.sup.1.

6. The process for obtaining a thermally stable formulation with fertilizing and phytoprotective capability according to claim 2, wherein the composition obtained in step B has a concentration of the chitosan of 1 g/L.

7. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 2, wherein the composition obtained in step B has a conductivity ranging from 1938 S cm.sup.1 to 2190 S cm.sup.1.

8. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 1, wherein the solution in step C is obtained by adding minerals to water.

9. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 8, wherein the minerals are selected from silicon and/or boron and/or molybdenum and/or manganese and/or zinc and/or calcium and/or copper.

10. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 9, wherein concentrations of minerals in an aqueous solution are: silicon: 0.07 g/L to 0.5 g/L; boron: 0.04 g/L to 0.08 g/L; molybdenum: 0.02 g/L to 0.09 g/L; manganese: 0.04 g/L to 0.13 g/L; zinc: 0.02 g/L to 0.1 g/L; calcium; 0.03 g/L to 0.3 g/L; copper 0.065 g/L to 0.2 g/L.

11. The process for obtaining the thermally stable formulation with fertilizing and phytoprotective capability according to claim 1, wherein the mixing ratio between the solutions B:C ranges from 0.05:99.95 to 30:70.

12. A thermally stable formulation with fertilizing and phytoprotective capability, comprising distilled pyroligneous extract (DPE), chitosan and mineral solution prepared by a process of A) Obtaining a distilled pyroligneous extract (DPE) by vacuum distillation of a crude pyroligneous extract (CPE) at a temperature of 60 C. to 75 C.; B) Obtaining a composition comprising the DPE and chitosan; C) Obtaining a fertilizing mineral solution; D) Mixing the composition obtained in step B with the solution obtained in step C.

13. The thermally stable formulation with fertilizing and phytoprotective capability according to claim 12, wherein concentration of chitosan in the formulation ranges from 2.510.sup.5 g/L to 9 g/L.

14. The thermally stable formulation with fertilizing and phytoprotective capability according to claim 12, wherein the minerals in the formulation are selected from silicon and/or boron and/or molybdenum and/or manganese and/or zinc and/or calcium and/or copper.

15. The thermally stable formulation with fertilizing and phytoprotective capability according to claim 14, wherein concentrations of the minerals are: silicon: 0.049 g/L to 0.5 g/L; boron: 0.028 g/L to 0.08 g/L; molybdenum: 0.014 g/L to 0.09 g/L; manganese: 0.028 g/L to 0.13 g/L; zinc: 0.014 g/L to 0.1 g/L; calcium; 0.021 g/L to 0.3 g/L; copper 0.046 g/L to 0.2 g/L.

16. A process comprising applying the thermally stable formulation with fertilizing and phytoprotective capability according to claim 12 to plants.

17. A process comprising forming a film of the thermally stable formulation with fertilizing and phytoprotective capability according to claim 12 on a plant.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1Thermograms obtained through differential scanning calorimetry of chitosan on distilled pyroligneous acid, with heating rate of 10 C. min-1;

(2) FIG. 2A) Transmittance as a function of the wavelength of chitosan/distilled pyroligneous acid films with thickness of 50 m; B) Molar absorptivity of chitosan/distilled pyroligneous films as a function of the wavelength;

(3) FIG. 3X-ray diffraction spectrum of the chitosan/distilled pyroligneous acid film, =0.1542 m.

(4) FIG. 4Thermogravimetric analysis profile, and first derivative of the chitosan/distilled pyroligneous acid films;

(5) FIG. 5Relative mass variation of the chitosan/distilled pyroligneous acid films after different times of immersion in distilled water at 25 C.;

(6) FIG. 6Electronic micrography of the chitosan/distilled pyroligneous acid film after spraying on a smooth surface at a temperature ranging from 18 to 25 C.;

(7) FIG. 7Electronic micrography of the chitosan/distilled pyroligneous acid film after spraying on a smooth surface at a temperature ranging from 18 to 25 C.;

(8) FIG. 8A partial view of the experiments for evaluation of the efficiency of the formulates in reducing the incidence of anthracnose on beans plant, (A) plants in wet chambers after inoculation of Colletotrichum lindemuthianum spores, (B) plants before inoculation of the fungus;

(9) FIG. 9Evaluation of the effect of the phytoprotector of chitosan/pyroligneous acid film (F1), chitosan/pyroligneous/minerals (F2) after spraying, for evaluation of vigor and development on beans (C) and potato (D);

(10) FIG. 10Hybrid pepper plants cv. Mitla inoculated with nematodes and treated with the formulations pyroligneous extract/chitosan (T3) and pyroligneous extract/chitosan/minerals (T4), positive witness (T1), witness with nematodes (T2), showing the presence of necrosis on the leaves;

(11) FIG. 11Difference in the vigor of hybrid pepper cv. Mitla plants inoculated with nematode and treated with the formulations pyroligneous extract/chitosan (T3) and pyroligneous extract/chitosan/minerals (T4), positive witness (T1), witness with nematode (T2);

(12) FIG. 12Inhibition of the micellar growth of the isolate 5.7 Colletotrichum gloeosporioides, caused by the fertilizing phytoprotective formulation pyroligneous acid/chitosan, reference in the photo for 1.1% and 2.3%, (2) the standard fungicide used for controlling the fungus, (3) witness and 10 diluted standard fungicide;

(13) FIG. 13Inhibition of the micellar growth of the isolate 02/08 of Monilinia fruticola by the phytoprotective formulation pyroligneous acid/chitosan, reference in the photo for 1.1 and 2.3% (1) and (2) the standard fungicidal used for controlling the fungus, (3) witness and 10 diluted standard fungicide.

DETAILED DESCRIPTION OF THE INVENTION

(14) The present invention relates to a process for obtaining a formulation, as well as to a formulation with phytoprotective and fertilizing characteristic and which represents a feasible alternative composition for application to plants and fruits, while keeping it characteristics by forming a stable film, with greater durability, thermal resistance and keeping the characteristics upon absorption of water, being ideal for field application.

(15) Said invention uses a by-product of the process for obtaining charcoal from the burning of wood for, after a specific treatment, using it in combination with chitosan and specific minerals, thus obtaining a formulation that, after application to plants, has desirable characteristics and so far not fully achieved by similar products, such as induction of the systemic resistance, proven fungitoxic and nematicidal action, formation of a film on the plant surface after being sprayed, photoprotection against UV-B and UV-C radiations, resistance of the film being kept in water even after high absorption, greater stability of the film at high ambient temperature, formation of desired porosity and homogeneity of photoprotective surface.

(16) After application to plants and fruits, the film formed efficiently blocks the radiation in the UV-B and UV-C regions. The high molar absorptivity decreases with the increase in wavelength. The formulation is thermally stable up to 60 C. and the resulting film loses a small amount of water under heating, but is thermally stable at a wide temperature range, undergoing decomposition only at 300 C. The film exhibits semi-crystalline structure, which imparts to it flexibility and porosity, which are desirable characteristics in the water-penetration and gas-exchange processes.

(17) The film maintains its integrity under immersion in water for up to 7 days and exhibits excellent hygroscopicity, and can absorb water up to 300% of its mass with little loss of the initial characteristics, which enables the use thereof as coverings for plants in ambient conditions.

(18) The invention relates to a process for obtaining a formulation with fertilizing and phytoprotective capability. The invention also deals with the formulation with fertilizing and phytoprotective capability. The respective formulation promotes the formation of a film capable of coating the surface where it is applied, be it a plant or a fruit. The film produced from this formulation keeps stability in water for up to one week, efficiently blocks UV-B and UV-C radiation, is thermally stable up to 60 C. and has semi-crystalline structure, which imparts to it flexibility and porosity, which are desirable characteristics in the water-penetration and gas-exchange processes carried out by the plants. The formulation of the present invention exhibits fungitoxic action in vitro for Monilinia fructicola and Colletotrichum, and nematicidal action on second-stage juveniles of M. graninicola and M. javanica, with in vitro mortality. The formulation also stimulates the enzymes related to the environmental defense and stress mechanisms of the plants (peroxidase (PO), phenylalaninammonia-liase (FAL), 1,3 glucanase ( 1,3)). Said formulation partially inhibits the natural senescence process of the fruit coming from plants treated with promotion of total or partial healing of the wounds. It further acts by decelerating the hydrolysis process of pectin in stored apples, keeping the pectin contents for a longer period of time, and natural juiciness in apples, imparting greater quality to the fruits in pre-harvest applications.

(19) The process for obtaining the formulation with fertilizing and phytoprotective capability of the present invention comprises the following steps: A) Obtaining distilled pyroligneous extract (DPE); B) Obtaining a composition comprising DPE and chitosan; C) Obtaining a fertilizing mineral solution; D) Mixing the composition obtained in step B with the solution obtained in step C.

(20) In the present invention, the distilled pyroligneous extract (DPE) is obtained from crude pyroligneous extract (CPE). By crude pyroligneous extract one understands the liquid phase obtained upon condensation of smoke during the burning of the wood for the production of charcoal. The CPE is also called pyroligneous liquid or pyroligneous acid or wood vinegar or pyroligneous liquor or liquid smoke or bio-oil. In the case of the CPE of the present invention, it should be produced by using control parameters that enable one to obtain a product with the smallest amount of tar possible. The presence of tar in the CPE makes it toxic and unfeasible for use in agriculture. In the case of the present invention, the CPE is obtained according to the obtainment guidelines described in Campos, A. D. (Tcnicas de produo de extrato pirolenhoso para use agricola. Embrapa Clime Temperado, Circular Tcnica no. 65, 2007. ISSN 1981-5999). As part of the process of obtaining/separating, the CPE is kept at rest for 3 to 6 months and separated by decantation from the other components resulting from the condensation of smoke. Alternatively, after separation thereof from the other components resulting from the condensation of smoke, the CPE obtained may further be subjected to a filtration process with a view to eliminate remaining impurities. In the present invention, the EPD is obtained from vacuum distillation of the CPE. More specifically, the EPD is obtained from vacuum distillation at minimum and maximum temperatures of 60 and 75 C., respectively.

(21) Step B of the process for obtaining a fertilizing and phytoprotective formulation of the present invention comprises obtaining a precursor composition containing DPE and chitosan. In order to obtain the respective precursor composition, chitosan is mixed with the DPE. Preferable, for use thereof in the present invention, chitosan should have a minimum degree of deacetylation, of 97%. Further preferably, the concentration of chitosan ion DPE in the composition obtained in step B of the invention should range from 0.05 g/L to 30 g/L, resulting in a conductivity of the composition obtained in B that should range from 1038 S cm.sup.1 to 4970 S cm.sup.1. In a preferred embodiment, the concentration of chitosan in DPE in the composition obtained ion step B of the process is of 10 g/L, resulting in a conductivity of 1938 S cm.sup.1 to 2190 cm.sup.1.

(22) The obtention of the fertilizing mineral solution described in step C of the process for obtaining the fertilizing and phytoprotective formulation of the present invention is carried out by adding minerals to the water. Various minerals with fertilizing capability may be used in obtaining the mineral solution (step C) of the present invention. Preferably, the minerals are selected from silicon and/or boron and/or molybdenum and/or manganese and/or zinc and/or calcium and/or copper. Further preferably, the concentrations of the respective minerals used are: silicon: 0.07 g/L to 0.50 g/L; boron: 0.04 g/L to 0.08 g/L; molybdenum: 0.02 g/L to 0.09 g/L; manganese: 0.04 g/L to 0.13 g/L; zinc: 0.02 g/L to 0.10 g/L; calcium; 0.03 g/L to 0.30 g/L; copper 0.065 g/L to 0.2 g/L.

(23) Step D of the process for obtaining the fertilizing and phytoprotective formulation of the present invention comprises mixing the composition obtained in step B with the solution obtained in step C of the process. Preferably, the mixture ratio between the solutions B:C ranges from 0.05:99.95 to 30:70. The mixture of the solutions B and C at the ratios described before results then in the fertilizing and phytoprotective formulation of the invention.

(24) The present invention also relates to a formulation with fertilizing and phytoprotective capability, comprising such formulation, DPE, chitosan and minerals. More specifically, the invention relates to a fertilizing and phytoprotective formulation comprising DPE, chitosan and minerals, where preferably the concentration of chitosan in the formulation ranges from 2.510.sup.5 g/L to 9 g/L. Various minerals having fertilizing function may be present in the formulation of the invention. Preferably, the minerals present in the fertilizing and phytoprotective formulation of the invention are selected from silicon and/or boron and/or molybdenum and/or manganese and/or zinc and/or calcium and/or copper, which, when present, exhibit the following concentrations: silicon: 0.049 g/L to 0.5 g/L; boron: 0.028 g/L to 0.08 g/L; molybdenum: 0.014 g/L to 0.09 g/L; manganese: 0.028 g/L to 0.13 g/L; zinc: 0.014 g/L to 0.1 g/L; calcium: 0.021 g/L to 0.3 g/L; copper 0.046 g/L to 0.2 g/L.

(25) The present invention further relates to a formulation with fertilizing and phytoprotective capability comprising distilled pyroligneous extract (DPE), chitosan and minerals and obtained according to the formulation obtainment process described in this document. More specifically, the invention relates to a fertilizing and phytoprotective formulation comprising DPE, chitosan and minerals, obtained according to the formulation obtainment process described in this document, where preferably the concentration of chitosan in the formulation ranges from 2.510.sup.5 g/L to 9 g/L. Various minerals with fertilizing function may be present in the formulation obtained according to the process described in this document. Preferably, the minerals present in the formulation are selected from silicon and/or boron and/or molybdenum and/or manganese and/or zinc and/or calcium and/or copper, which, when present, exhibit the following concentrations: silicon: 0.049 g/L to 0.5 g/L; boron: 0.28 g/L to 0.08 g/L; molybdenum: 0.014 g/L to 0.09 g/L; manganese: 0.028 g/L to 0.13 g/L; zinc: 0.014 g/L to 0.1 g/L; calcium; 0.021 g/L to 0.3 g/L; copper 0.046 g/L to 0.2 g/L.

(26) The present invention further relates to the use of a formulation with fertilizing and phytoprotective capability as described before, for application to plants, parts of plants, including fruits. More specifically, the invention relates to the use of the respective formulation described in the invention for obtaining a film on plants and/or fruits, which has phytoprotective and fertilizing characteristic.

EXPERIMENTAL RESULTS

(27) Physicochemical Characterization of the Composition Obtained in Step B of the Process for Obtaining a Fertilizing and Phytoprotective Formulation

(28) The compositions of chitosan in distilled pyroligneous acid were characterized as to the presence of electrolytes in solution through measurements of pH and conductivity, which were carried out on Digimed equipment, models DM-20 and DM-31, respectively.

(29) The conductivity and the pH of the solutions of chitosan in distilled pyroligneous acid, at different concentrations, are shown in Table 1. The determination of the conductivity is important to characterize the presence of electrolytes in solution, since it has direct influence on the formation of gel and on the polymer hydration radius. The pH is important, since studies suggest that chitosan has greater antifungal potential at acidic pH in the range 3 to 4.

(30) TABLE-US-00001 TABLE 1 Physicochemical characteristics of the solutions of chitosan in distilled pyroligneous acid Concentration Conductivity (g L.sup.1) (S cm.sup.1) pH 0 1035 3.26 0.05 1038 2.95 0.1 1035 n.d. 0.5 967 2.91 1.0 991 2.95 2.0 n.d. 3.06 2.5 1101 n.d. 5.0 1410 n.d. 10.0 2180 3.23 15.0 n.d. 2600 30.0 3.43 4970

(31) The thermal behavior of gels was determined through differential scanning calorimetry (DSC). The DSC measurements were carried out on a DSC Q 20 from TA Instruments, at a temperature interval of 40 C. to 60 C., with heating rate of 10 C.min.sup.1 under nitrogen flow of 50 mL.Math.min.sup.1.

(32) FIG. 1 shows the thermal behavior of the gel formed by the chitosan/distilled pyroligneous acid system. The DSC analysis was carried out with two heating cycles and one cooling cycle. One carried out consecutive heating ramps.

(33) One observes in FIG. 1 a discontinuity at approximately 24 C. in the curve of the 1 heating, which does not repeat in the 2 heating, which suggests only a loss of water by the gel in the first heating. It was not possible to observe any phase transition showing that the gel remained stable in the temperature range studied.

(34) Characterization of the Films

(35) Capability of Blocking the UV/VIS Radiation

(36) The typical behavior of UV/VIS transmittance of chitosan/distilled pyroligneous acid films is shown in FIG. 2-A. The transmittance of the films was evaluated at a thickness interval that obeys the Lambert-Beer Law. The molar absorptivity was calculated for different wavelengths through the expression of Lambert-Beer (1) described below:
A()=()bc(1),
wherein A is the absorbance of the films, is the is the molar absorbance, b is the film thickness and c is the concentration.

(37) Considering the thickness of the films and the concentration, one calculated the partial molar absorptivities that were expressed as a function of the wavelength in FIG. 2-B. The results for the molar absorptivity as a function of the wavelength after 320 m were obtained through the equation (2) described below:
y=4.6.109e(x/100)+1.4.107(2).

(38) The spectral range covered showed that these films can be used as photoprotectors, blocking almost completely the UV-B (310-280 m) and UV-C (279-200 m) radiations.

(39) Structural Characteristics of the Films

(40) The X-ray diagram of the films (FIG. 3) showed the peaks at 2, 8.4-8.6 and 11.55, located over a huge halo characteristic of amorphous materials. The films then exhibited a semi-crystalline structure. This semi-crystalline characteristic is interesting, since it provides the film with flexibility and porosity, which are desirable characteristics in the water-penetration and gas-exchange processes.

(41) Thermal Stability

(42) The profiles of the thermogravimetric analysis and of the first derivative of the chitosan/distilled pyroligneous acid films are shown in FIG. 4. At 45 C., the films lost about 20% of mass, which is attributed to the release of water and acetic acid caught in their structure. At 300 C., chitosan began to degrade. The remaining material (about 40% by mass) exhibited characteristics of amorphous carbon.

(43) Behavior of the Films in Water

(44) The films proved to be stable in water, without undergoing disintegration for up to one weak of immersion. The hygroscopic characteristic of the film was determined by varying the mass of water absorbed by the films according to the equation (3):

(45) $\begin{array}{cc}m=\left(\frac{m_i-m_0}{m_0}\right)*100,& \left(3\right)\end{array}$
wherein m is the relative increase in mass, m.sub.0 is the initial mass of the film and m.sub.i is the mass of the film in the immersion time i.

(46) FIG. 5 shows the increase in water absorption of the films as a function of the time. The film came to the point of increasing by 300% its mass in water.

(47) FIGS. 6 and 7 show electronic micrographies of the chitosan/distilled pyroligneous acid film, after spraying on a smooth surface at a temperature ranging from 18 to 25 C.

(48) Behavior of the Plant after Treatment

(49) The phytoprotective and fertilizing formulation of the invention promotes an increase in the adhesion of the molecules to the plant cuticle, enabling better contact between the formulation of the invention, nutrient and the leaf surface. In FIGS. 8A and 8B, one shows partial views of the experiments for evaluation of the efficiency of the formulations in reducing the incidence of anthracnose on beans plant. In FIG. 8A, one shows plants in wet chambers after inoculation of Colletotrichum lindemuthianum spores, and in FIG. 8B one shows the plants before inoculation of the fungus.

(50) Table 2 below shows the disease index according to McKINNEY for incidence of anthracnose after application of the phytoprotective and fertilizing formulation of the invention.

(51) TABLE-US-00002 TABLE 2 disease index according to McKINNEY for incidence of anthracnose Chocolate Cultivars Macanudo Treatments Exp. III* Exp. IV* Exp. III Exp. IV Formulation A 0.97 0.47 0.33 0.20 Pyroligneous 0.96 0.43 0.20 0.13 acid/chitozan/minerals Pyroligneous acid/ 0.99 0.33 0.29 0.17 chitosan T1a-Test. with 1 0.88 0.37 0.24 inoculum T1b-Test. with 0.20 0.11 0.11 0.11 inoculum and with fungicide *Experiment IIIone application *Experiment IVthree applications, except for witnesses

(52) One can observe in Table 2 that, after three applications of the formulations, the responses of the plants were significant as to the resistance to anthracnose (Colletotrichum lindemuthianum). The Mackinney index equal to 1 corresponds to the high incidence of disease and high susceptibility to anthracnose. After three applications of the formulation of the invention, one found that the Mckinney index came down to less than 0.50, which means that plants that were susceptible before, had now intermediate resistance. The cultivar Macanudo, considered susceptible to anthracnose, became resistant, exhibiting a Mckinney index of 0.33 (DPE+chitosan) and 0.43 (DPE+chitosan+minerals). The cultivar chocolate exhibited, after 3 applications, Mckinney indexes of 0.17 (DPE+chitosan) and 0.13 (DPE+chitosan+minerals), respectively.

(53) FIG. 9 shows the good development of the plants treated with formulations of the invention (DPE+chitosan) and (DPE+chitosan+minerals). The evaluation of the activity of the peroxidase (PO), phenylamineammonia-liase (FAL), 1,3 GLUCANASE ( 1,3) after application of the formulations pyroligneous acid/chitosan and pyroligneous acid/chitosan/minerals, in cultivation of hybrid pepper cv. Mitla, inoculated with nematodes Meloidogyne is shown in Table 3 below.

(54) TABLE-US-00003 TABLE 3 Activity of the peroxidase (PO), phenylamineammonia-liase (FAL), 1,3 glucanase ( 1,3) after application of the formulations DPE/chitosan and DPE/chitosan/minerals, in cultivation of hybrid pepper cv. Mitla, inoculated with nematodes Meloidogyne PO FAL 1,3 PFO ue/min/g ue/min/g ue/g ue/min/g DPE/chitosan of tissue of tissue of tissue of tissue DPE/chitosan 385.91 b 28.41 ab 39.68 b 438.90 b DPE/chitosan/ 320.00 c* 32.29 a 37.56 b 426.67 b nutrients Test. Inoculated with 472.58 a 27.45 b 62.86 a 498.77 a nematode Test. Positive 134.80 d 29.98 ab 29.26 c 354.43 c *Different letters differ from each other in the columns by the Duncan test (p < 0.05).

(55) In table 3, the activity of the proteins related to the pathogenesis ( 1,3 glucanase, PO, PFO and FAL) involved in defense responses and resistance to various types of environmental stress exhibited significant alterations, when the plants were treated with the formulation of the invention. This indicates that the formulation of the invention activated the defense metabolism at the moment when the plant was attacked in some way, promoting a rapid defense. In this case, one observed the presence of necrosis on the leaves of the witnesses (FIG. 10), indicating that the phytoprotection process did not take place on these untreated plants. This is confirmed by observing Table 4, where it is possible to observe that the phenolic compounds (polyphenols, monophenols, and ortho- and diphenols, etc.), which are substrates for the enzymes PO, PFO and FAL, were not synthetized by the plant in the absence of the formulation of the invention. One observed that, on the witness, there was a significant decrease in the concentration of these phenols. Thus, the plant, upon not managing to defend itself from the attacks, demanded a higher expenditure of energy by its organism and thereby caused the vigor and the production to be drastically reduced (FIG. 11 and Table 5). In this way, one concluded that the defense process was not activated and the plant became more susceptible. The phenolic compounds are tannins, which, when present in the leaves, participate in the lignification process and production of phytoalexines, and also make the plants more indigestible and/or less attractive to the phylophagous insects (insects that feed on leaves) and sucking insects, rendering these plants more resistant to these pests as well. From the results presented, one can conclude that the formulation of the invention exhibited the characteristic of the action of inducing systemic resistance of the plants.

(56) TABLE-US-00004 TABLE 4 Evaluation of the contents of phenolic compounds (mg/100 g) extracted in methanol, 50% methanol and water after application of the formulations pyroligneous acid/chitosan and pyroligneous acid/chitosan/minerals, in cultivation of hybrid cv. Mitla pepper Total 50% 100% phenolic methanol methanol water compounds Phytoprotectors mg/100 g mg/100 g mg/100 g mg/100 g Pyrolignoleus acid/ 2.21 a 3.69 a 1.18 a 7.08 a chitosan Pyroligneous/chitosan/ 2.29 a 3.72 a 1.20 a 7.21 a micronutrients Test. Inoculated with 1.86 b 2.19 b 1.18 a 5.23 b nematode Test. Positive 1.98 b 2.42 b 0.77 b 5.17 b * Different letters differ from each other in the column by the Duncan test (p < 0.05).

(57) TABLE-US-00005 TABLE 5 Evaluation of the vigor and production of hybrid cv. Mitla pepper after treatments with the formulations pyroligneous acid/chitosan and pyroligneous acid/chitosan/minerals in cultivation of hybrid cv. Mitla pepper. Number of Weight/plant fruits per Formulations g plant Pyroligneous acid/chitosan 131.00 b 25.00 b Pyroligneous 158.33 a 31.00 a acid/chitosan/nutrients Test. Inoculated with 126.00 b 17.00 c nematodes Test. Positive 120.00 b 12.00 c * Different letters differ from each other in the column by the Duncan test (p < 0.05).
FIGS. 12 and 13 show results of the inhibition of the micellar growth of isolates of Colletotrichum and Monilinia by using the formulation of the invention, proven the fungitoxic action of the formulation.

(58) Experiments for evaluation of the effect of the formulation of the invention were also carried out with a view to test the resistance of diseases and quality of fruit on apple trees cv Fuji. The interest of an alternative product in this crop is the adaptation to international rules for the integrated production, reduction of agricultural defensives and of environmental attacks. The experiments were carried out by using three plants by repetition and three repetitions by treatment. One bordure plant was left between the repetitions and 108 plants were used in the experiment.

(59) The first analysis was carried out in the experiment in conventional crops was for the contents of pectin in the fruits. The presence of pectin guarantees the juiciness of the fruit, when the pectinase acts by hydrolyzing the pectin. In a normal senescence process, apple exhibits flour-like texture, losing quality. So, it is ideal to keep the juiciness for as long as possible during storage. The results are shown in Table 6 below.

(60) TABLE-US-00006 TABLE 6 Contents of pectin (g/mg) in apple from an orchard treated with formulates pyroligneous extract/chitosan and pyroligneous extract/chitosan/minerals and witness with conventional treatment of the crop, during the period of storage of the fruits at average room temperature of 24 to 26 C. In order to accelerate the senescence process, the apples were stored at room temperature ranging from 24 to 26 C., for 120 days. Pectin Pectin Pectin Pectin (g/mg) (g/mg) (g/mg (g/mg Formulations 08/04 27/04 18/05 23/06 Formulation-T5 51.24 b 48.93 bc 41.72 a 34.89 bcd Pyroligneous 54.31 a 52.17 a 44.83 a 36.43 abc extract/chitosan Pyroligneous 58.99 a 56.06 a 39.69 a 37.09 ab extract/chitosan/ minerals Formulation 15-T9 50.60 b 48.98 bc 39.93 a 38.00 a Witness-T1 49.90 b 46.04 c 41.48 a 33.24 d Variation 5.4 5.7 6.2 3.9 Coefficient (%) * Different letters differ from each other in the column by the Duncan test (p < 0.05).

(61) The analysis of Table 6 shows that, even in conditions suitable for acceleration of senescence, the plants treated with the formulation of the invention exhibited, after 120 days' storage, higher contents of pectin in the fruit.