AQUEOUS FORMULATION THAT REDUCES DAMAGE CAUSED BY SPRING FROSTS IN PLANTS AND PRODUCTION METHOD THEREOF

Abstract

An aqueous formulation that reduces the damage caused by spring frosts in plants having: (a) 0.001-2% by weight PVA micro/nanoparticles having a molecular weight of 10,000-100,000 g/mol and 1-20% acetate groups; (b) 0.0001-0.4% by weight stabilising agents; (c) 0.0005-0.05% by weight category IV adjuvants; (d) 0.00025-4% by weight emulsifier; (e) 0.003-6% by weight sodium hydroxide; and (f) 0.0065-13% by weight hydrochloric acid at 37% weight/weight and 1.18 g/mL density; a process for producing the aqueous formulation, and to uses.

Claims

1. An aqueous formulation that reduces spring frost damage to plants comprising the following components: i. 0.001-2% by weight of PVA micro/nanoparticles of molecular weight between 10,000-100,000 g/mol and percentage of acetate groups between 1 and 20%; ii. 0.0001-0.4% by weight of stabilizing agents; iii. 0.0005-0.05% by weight of category IV adjuvants; iv. 0.00025-4% by weight of emulsifier; v. 0.003-6% by weight of sodium hydroxide; and vi. 0.0065-13% by weight of hydrochloric acid to 37% w/w and 1.18 density g/ml.

2. The aqueous formulation that reduces spring frost damage to plants according to claim 1 wherein the stabilizing agent is polyvinylpyrrolidone and/or a nonionic surfactant.

3. The aqueous formulation that reduces damage caused by spring frost on plants according to claim 1 wherein the category IV adjuvants are of the Induce pH® type.

4. The aqueous formulation that reduces spring frost damage in plants according to claim 1 wherein the emulsifier is soy lecithin.

5. A process for producing the aqueous formulation according to claim 1 comprising the following steps: a. dissolving PVA in water in at least a 1/50 ratio in a mineral bath and with temperatures fluctuating between 40-100° C., depending on the molecular weight and degree of hydrolysis of the PVA and for at least 2 hours, using agitation with speeds between 500-900 rpm; b. dissolving sodium hydroxide in water at room temperature (20° C.), using agitation with speeds between 500-900 rpm and concentrations fluctuating between 2-8% weight/volume; c. obtaining PVA micro/nanoparticles: the solution obtained in stage “a” is mixed in a reactor containing the solution of stage “b” in at least a 1/1 ratio, by dripping using a peristaltic pump with speeds varying between 10-50 ml/h and using agitation with speeds between 700-8000 rpm, to obtain PVA micro/nanoparticles whose size varies between 300-3000 nm; d. neutralizing the dispersion obtained in step “c” with hydrochloric acid up to pH 7, using agitation with speeds between 100-300 rpm; and e. mixing of stabilizers, emulsifiers and/or adjuvants: the neutralized dispersion is mixed in a reactor with the different stabilizers, emulsifiers and/or adjuvants in at least a 1/10 ratio at room temperature, using agitation with speeds between 4000-8000 rpm.

6. A method for inhibiting recrystallization of ice in a plant comprising applying the formulation of claim 1 to the plant.

7. A method for reducing damage to plants in frosts down to −5° C. and for 4 hours comprising applying the formulation of claim 1 to the plant.

8. The aqueous formulation that reduces spring frost damage to plants according to claim 2 wherein the nonionic surfactant is polysorbate.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0025] FIG. 1: corresponds to scanning electron microscopy images (a, d and g), histograms from scanning microscopy (b, e and h) and transmission electron microscopy images of PVA micro/nanoparticles obtained from PVA of different weight-average molecular weight (c, f and i); where: (a), (b) and (c) correspond to L-PVA micro/nanoparticles; (d), (e) and (f) m-PVA amicro/nanoparticles; and (g), (h) and (i) to h-PVA micro/nanoparticles.

[0026] FIG. 2: corresponds to FTIR spectra of PVA macromolecules of different weight-average molecular weight (a, c and e) and PVA micro/nanoparticles (b, d and f), where: (a) and (b) corresponds to L-PVA; (c) and (d) to m-PVA; and (e) and (f) to h-PVA.

[0027] FIG. 3: corresponds to optical microscopy images of a frozen drop of water containing: PVA solution of different weight average molecular weight at 1.0 wt % w/v (A, B, E, F, I and J); PVA micro/nanoparticles dispersion 1.0 wt % w/v (C, D, G, H, K and L) and NaCl solution at 0.137 mol/L (0 and P), and at 0.7 mol/L (Q and R) at zero time (A, E, I, O, C, G, K and Q) and at 60 min (B, F, J, P, D, H, L and R) of maintaining the samples at −6° C.; where: (A), (B), (C) and (D) correspond to L-PVA; E, F, G and H to m-PVA; and (I), (J), (K) and (L) to h-PVA.

[0028] FIG. 4: corresponds to 1 optical microscopy images of a frozen drop of water containing: PVA solution of different weight average molecular weight at 0.01% w/v (A, B, E, F, I and J) and PVA micro/nanoparticles dispersion 0.01% w/v (C, D, G, H, H, K and L) and NaCl solution at 0.07 mol/L (O and P), at zero time (A, E, I, O, C, G and K) and at 60 min (B, F, J, P, D, H and L) of keeping the samples at −6° C.; where: (A), (B), (C) and (D) correspond to L-PVA; (E), (F), (G) and (H) to m-PVA; and (I), (J), (K) and (L) to h-PVA.

[0029] FIG. 5: corresponds to the microchamber tests on the effect of the application of different products (water, PVA solution, micro/nanoparticle dispersion and commercial cryoprotectant) on the incidence of frost damage in flower primordia under simulated frost conditions (−5° C. for 4 h); the values represent the average of different phenological stages.

[0030] FIG. 6: corresponds to the results of the field trial expressed in flower primordium damage at the phenological stages of: receding bud, swollen bud, green button and flowering when applying the different products (water, PVA solution, micro/nanoparticle dispersion and commercial cryoprotectant); where (a) corresponds to PVA solution at 0.1% w/v; (b) to PVA micro/nanoparticle dispersion (0.01% w/v), (c) to commercial cryoprotectant agent and (d) to water.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present technology corresponds to an aqueous formulation whose active ingredient is a biomimetic solute that reduces plant damage caused by spring frosts. This formulation contains micro/nanoparticles of PVA, a biomimetic polymer with frost recrystallization inhibitory activity, which achieves plant protection, regardless of the phenological state, in conditions of up to −5° C. and for 4 hours.

[0032] Specifically, the aqueous formulation comprises at least the following components:

[0033] a. 0.001-2% by weight of PVA micro/nanoparticles, whose molecular weight fluctuates between 10,000-100,000 g/mol and the percentage of acetate groups between land 20%;

[0034] b. 0.0001-0.4% by weight of stabilizing agents such as polyvinylpyrrolidone and/or nonionic surfactants such as tween;

[0035] c. 0.0005-0.05% by weight of category IV commercial adjuvants such as Induce pH® which acts as a buffer or stopper when diluting the formulation concentrate;

[0036] d. 0.00025-4% by weight of emulsifier such as soy lecithin to enhance formulation adhesion;

[0037] e. 0.003-6% by weight of sodium hydroxide forming agent for PVA micro/nanoparticles;

[0038] f 0.0065-13% by weight of Hydrochloric acid (37% w/w and 1.18 density g/ml) as neutralizing agent.

[0039] The formulation based on PVA nanoparticles is obtained through a precipitation or alkaline treatment process, which comprises at least the following steps: [0040] a. dissolution of PVA: PVA must be dissolved in water in at least a 1/50 ratio in a mineral bath and with temperatures fluctuating between 40-100° C., depending on the molecular weight and degree of hydrolysis of the PVA and for at least 2 hours, using agitation with speeds between 500-900 rpm. [0041] b. dissolution: sodium hydroxide must be dissolved in water at room temperature (20° C.), using agitation with speeds between 500-900 rpm and concentrations fluctuating between 2-8% weight/volume;

[0042] c. mixing: the solution obtained in step (a) is mixed in a reactor containing a solution of step (b) in at least one ratio of 1/1, by dripping using a peristaltic pump with speeds fluctuating between 10-50 ml/hr. During mixing, agitation is used with speeds between 700-8000 rpm. At this stage micro/nanoparticles of PVA are obtained whose size fluctuates between 300-3000 nm;

[0043] d. neutralization: the dispersion obtained in step (c) is neutralized with hydrochloric acid (37% w/w and 1.18 density g/ml) up to pH=7, using agitation at speeds between 100-300 rpm; and

[0044] e. mixing of stabilizers, emulsifiers and/or coadjuvants: the dispersion obtained in step (d) is mixed in a reactor with the different stabilizers, emulsifiers and/or coadjuvants in at least one 1/10 ratio at room temperature, using stirring speeds between 4000-8000 rpm.

[0045] Advantageously, this formulation allows: [0046] inhibit ice recrystallization at concentrations greater than and equal to 0.01% w/v of PVA micro/nanoparticles; [0047] reduce frost damage to flower buds of stone fruit trees by 20% with respect to water; and [0048] do not cause damage to the harvested fruit, nor to the fruit during post-harvest.

APPLICATION EXAMPLES

Example 1. Preparation of an Aqueous Formulation of PVA Micro/Nanoparticles and Evaluation of their Properties

[0049] Fully hydrolyzed PVA of three different weight-average (Mw) molecular masses (M.sub.w=13000-23000 g/mol, M.sub.w=31000-50000 g/mol and M.sub.w=89000-98000 g/mol) were dissolved in nanopure water using a natural oil bath and under a magnetic stirrer at 100° C. for about 2 h. A 2% w/v solution was added dropwise to a 6% w/v sodium hydroxide solution at a rate of 30 ml/h using a syringe pump (TE 331, Terumo, Japan) and under constant stirring at 700 rpm. The ratio of PVA:NaOH solution was 1:1. The dispersion was then neutralized with hydrochloric acid (37% w/w and density of 1.18 g/mL). The samples were labeled according to the molecular mass of the PVA. Thus, the L-PVA label corresponded to the low Mw (13000-23000 g/mol) PVA micro/nanoparticle dispersion, the m-PVA label to the medium Mw (31000-50000 g/mol) PVA micro/nanoparticle dispersion and the h-PVA label to the medium Mw (31000-50000 g/mol) PVA micro/nanoparticle dispersion and the h-PVA label to the medium Mw (31000-50000 g/mol) PVA micro/nanoparticle dispersion. PVA micro/nanoparticles dispersion of high Mw. (89000-98000 g/mol).

[0050] The obtained PVA micro/nanoparticles were evaluated by the following assays:

[0051] a.—Scanning Electron Microscopy (SEM):

[0052] The morphology and size of PVA micro/nanoparticles before and after neutralization were analyzed using a JEOL-JSM 6380LV (Tokyo, Japan) scanning electron microscope operated at 20 kV. A droplet of PVA micro/nanoparticle dispersion was dried at room temperature and coated with a gold film (50 nm). The magnifications were 2000×, 5000× and 10000×. The microscopy images can be seen in the FIG. 1, where (A), (D) and (G) correspond to micro/nanoparticles of L-PVA; (B), (E) and (H) correspond to micro/nanoparticles of m-PVA; and (C), (F) and (I) correspond to h-PVA micro/nanoparticles.

[0053] Alkaline treatment to the aqueous PVA solution produces particles of spherical shape as can be seen in FIGS. 1A, B and C. The size of these PVA micro/nanoparticles was measured directly from SEM micrographs, using lmageJ software, and size distribution plots were constructed from at least 190 particles (see FIGS. B1, E and H). The average size of the PVA micro/nanoparticles was 900±300, 1300±400, 1500±700 nm for L-PVA, m-PVA and h-PVA samples, respectively. The mean particle size increased significantly (Tukey's test, α=0.05) with increasing PVA molecular weight. The m-PVA and h-PVAmicro/nanoparticles size distributions were broader than those corresponding to L-PVA nanoparticles. In general, the h-PVA particle samples showed higher amount of chain-like aggregates of the spherical micro/nanoparticles (see FIGS. 1C, F and I).

[0054] The effect of the molecular weight of PVA on the average size of the micro/nanoparticles can be explained by taking into account that: at the 1% w/v concentration, the ball was formed by a single chain (Budhlall et al., 2003), and that the distance between the chains of fully hydrolyzed PVA was relatively short, due to the competition between the polymer-solvent and polymer-polymer interactions (Mansur et al., 2008). The addition of the aqueous PVA solution to the NaOH solution decreased the solubility of PVA, as the hydration of Na.sup.+ and OH ions reduces the number of free water molecules available to interact with PVA. As a result, the polymer-polymer interaction was dominant, and the PVA single tangle chains tended to coalesce, forming a spherical-shaped micellar structure. Therefore, the size of this micellar structure depends on the degree of polymerization of the component PVA chains. It is proposed that the micro/nanoparticles were stabilized by electrostatic repulsion of their negative charges, probably due to the absorption of free OH groups on the PVA particles by hydrogen bridge interactions, as can be deduced from the FTIR infrared spectroscopy results. The change in the dielectric environment of the PVA micro/nanoparticles by the addition of HCl led to a pseudo-linear arrangement of micro/nanoparticles, as can be observed in the SEM images.

[0055] b.—Transmission Electron Microscopy (TEM):

[0056] This test was performed on a JEOL/JEM1200 EX transmission microscope II (USA) operated up to 120 kV. A drop of a dilute dispersion of PVA micronanoparticles before and after neutralization was placed on a carbon-coated Cu grid (G200 Hex) and then air-dried.

[0057] FIGS. 1 (C, F and H) shows the transmission electron microscopy images of the micro/nanoparticles obtained from PVA of different weight-average molecular weights, where it is observed that the neutralized particles were coated by sodium chloride crystals, suggesting the interaction of sodium hydroxide with the PVA chains.

[0058] c.—Fourier Transform Infrared Spectroscopy (FTIR):

[0059] The FTIR spectra of the PVA micro/nanoparticles and PVA macro molecules were recorded in triplicate in a Perkin Elmer Spectrum Two FTIR (USA) spectrophotometer, in the range of 600-4000 cm.sup.−1 to 4 cm.sup.−1 of resolution. The FTIR spectrum was taken with attenuated total internal reflectance (ATR). The spectrum is shown in transmittance mode. Baseline correction of the spectrum was performed using Spectral Manager software, version 2. The data were processed according to Pozo et al. (2018), using the Origin software, version 8.6.

[0060] The FTIR spectra of PVA mico/nanoparticles and PVA precursors are shown in FIG. 2. The spectra show peaks characteristic of PVA (Awada and Daneault, 2015). The broad band between 3100-3400 cm.sup.−1 is assigned to the stretching vibrations of OH groups. The symmetric vibration and asymmetry of the CH.sub.2 groups is observed at 2910 and 2942 cm.sup.−1, respectively. The peaks around 840 and 1420 cm.sup.−1 are associated with in-plane bending and stretching vibrations, respectively, of the CH.sub.2 group. The most intense peak at 1080 cm.sup.−1 corresponds to C—OH stretching in aliphatic alcohols. As expected, the vibrations associated with C═O of the acetate group around 1715 cm.sup.−1 is observed at lower intensity for PVA precursors with high hydrolyzed degree (98-99%). The band at 1142 cm.sup.−1 is attributed to the C—O—C stretching vibration and is related to the crystallinity of PVA. This band was similar for both the PVA macromolecule and the PVA micro/nanoparticles, suggesting that no increase in crystallinity increases in crystallinity upon formation of PVA micro/nanoparticles.

[0061] The main differences between the spectra of the PVA micro/nanoparticles and the PVA precursors are the absence of bands between 1735-1750 cm.sup.−1 and the presence of a new band at 1647 cm.sup.−1 in the spectra of the micro/nanoparticles of PVA. The absence of bands in the region between 1735-1750 cm.sup.−1 accounts for the fact that PVA was completely hydrolyzed during the alkaline treatment. The band at 1647 cm.sup.−1 was attributed to the out-of-plane bending vibration or the result of δ(HOH), which is influenced by bridging-type hydrogen bonds. The δ(OH) band at wave number higher at 1645 cm.sup.−1 indicates a high proportion of bridging hydrogen bonds in the PVA microparticles.

[0062] d.—Splash Cooling Test:

[0063] IRI activity was assessed using a method adapted from Knight et al (1988). A drop of 10 μL was dropped from a height of 1.85 m onto a microscope slide, previously cooled for 1 h with dry ice. Then, the slide was rapidly transferred to the Peltier LTS120 system (Linkam, UK) attached to the Olympus BX43 microscope (Japan) and kept at −6° C. for 1 h. The recrystallization process was followed during thawing at −6° C. using a polarizer for transmitted light (U-POT). Images were collected using an Olympus SC50 video camera at 10× magnification. A NaCl solution was used as a negative control at different concentrations and NaCl was also added to the precursor PVA solution to better distinguish the ice crystal outline. The NaCl concentration selected was equal to that present in the PVA micro/nanoparticles, except for the PVA macromolecules at 1% w/v, where a lower concentration of NaCl 0,137 mol/L was used (Biggs et al. 2019), due to the insolubility of the macromolecule in a 0.7 mol/L NaCl solution. The ice crystal size before (zero time) and after (1 hr) keeping the PVA samples at −6° C. was compared to determine the ice recrystallization inhibition activity of PVA micro/nanoparticles with respect to PVA precursors.

[0064] FIGS. 3 and 4 show the evolution of ice crystals for samples with PVA micro/nanoparticles and for the precursor PVA macromolecules at two concentrations: 1% w/v (FIG. 3) and 0.01% w/v (FIG. 4). The ice crystal sizes increased over time in NaCl solution of different concentrations. Precursor PVA solutions of different molecular weights and PVA micro/nanoparticle dispersions showed smaller crystal sizes at min 60 and at −6° C. compared to NaCl solution, despite the fact that the 1% w/v PVA solution was more stable than the NaCl solution had the same NaCl concentration as the negative control. The PVA micro/nanoparticle dispersions exhibited higher IRI activity than the precursor PVA solutions. This is explained by the hydrodynamic bulk presented by the PVA particles when adsorbed on the ice surface, which prevents adjacent ice crystals from bonding to form a larger one (Knight et al., 1995). Furthermore, it is observed that as the molecular weight of PVA increases, the degree of ice recrystallization inhibition was higher, which supports our theory.

[0065] e.—Evaluation of the Cryoprotectant Properties of PVA Micro/Nanoparticles on Fruit Trees.

[0066] The cryoprotective properties of PVA micro/nanoparticles on fruit trees were evaluated by microchamber test and field trial:

[0067] e.1.—Microchamber Test:

[0068] The microchamber trial consisted of placing naked cherry darts of different phenological stages in a cold chamber and applying different products to inhibit frost damage. The darts of 4-5 cherry buds of the Sweethearr variety were placed in a plastic tray with hydrated cotton. The bracts of each bud were then stripped to expose the inner tissue to low temperatures. The darts were sprayed with different products: water, 0.1% w/v PVA solution, 0.01% w/v h-PVA micro/nanoparticle dispersion or commercial cryoprotective agrochemical, and immediately placed in a micro-chamber at −5° C. and 70% relative humidity for a period of 4 hours.

[0069] The phenological stages tested were: bud recess, swollen bud and bud burst. The FIG. 5 shows the effect of the application of the different products on the incidence of frost damage in flower buds in the microchamber test, where the most effective product was the dispersion of micro/nanoparticles of PVA at 0.01% w/v. This formulation reduced the incidence of frost damage on flower primordia by about 10% with respect to the control (water).

[0070] e.2.—Field Test:

[0071] Field trials were conducted during the 2018-2019 season in a commercial cherry orchard of the Sweetheart cultivar. The Sweetheart cultivar orchard consisted of nine-year-old trees grafted on the Colt rootstock and planted at a distance of 4.5×3 m. As experimental unit, four branches per tree were selected to which hydrogenated cyanamide (Dormex®, Trostberg, Germany) was applied in the bud break period (July 2018) at a dose of 2% v/v together with a concentrated soluble adjuvant (Break®, Hopewell, USA) at a dose of 0.01% v/v. The experiment was planned in a completely randomized block design with four replications (trees).

[0072] The products applied were: water, 0.1% w/v PVA solution, 0.01% w/v h-PVA micro/nanoparticle dispersion and commercial cryoprotectant agrochemical. Applications were made at the phenological stages of: bud recess, swollen bud, green button and flowering; and before a frost event. Applications of hydrogenated cyanamide and the products were made through a manual motorized sprayer (SWISSMEX®, Jalisco, Mexico) with a 15 L capacity.

[0073] In the 2018-2019 season, the number of accumulated hours with temperatures below 0° C. exceeded 200 hours in the locality where the commercial orchard under study is located. In this case, the 0.01% w/v PVA micro/nanoparticle formulation reduced the amount of damaged flower primordia in buds by 85% (FIG. 6). For this product, the number of damaged flower primordia per dart was lower at all phenological stages, except at 54 and 41 days before flowering (recessing bud stage), at 54 and 41 days before flowering (recessing bud stage). The formulation with micro/nanoparticles was used in the formulation with micro/nanoparticles in the formulation with micro/nanoparticles in the formulation with micro/nanoparticles in the formulation with micro/nanoparticles. The formulation with dispersed PVA micro/nanoparticles was 45% more effective in reducing the incidence of damage compared to the commercial antifreeze.

[0074] Finally, these trials demonstrated that the PVA nanoparticle-based formulation can reduce frost damage to plants.