Use of a composition for plant protection

Abstract

The present disclosure provides for an aqueous composition comprising 1 - 5 mM HNO2, and having a pH between 1 – 4, or between 3 - 4. The aqueous composition may comprise a base which may be NH₄OH. The aqueous composition may be plasma-activated water. It is preferred that the aqueous composition comprises a wetting agent, preferably a silicone based wetting agent. Preferably, the amount of total dissolved solids (TDS) in the aqueous composition, is less than 1 g / L, preferably less than 0.1 g / L. The present disclosure also provides for a method for preparing the aqueous composition, the method comprising combining a composition comprising 1-5 mM HNO2 and a base, wherein the base is preferably NH₄OH. The aqueous composition may be used for protecting plants, by applying the composition to a plant or plant seed and / or to soil supporting the plant or plant seed.

Description

[0001] P36882PCOO / MJO

[0002] Compositions for plant protection

[0003] Technical field

[0004] The present disclosure relates to aqueous formulations comprising HNO2, and particularly plasma activated water formulations. The aqueous formulations find use in protecting plants, by applying the formulations to a plant or plant seed and / or to soil supporting the plant or plant seed.

[0005] Background

[0006] During the cultivation of plants there is a need to protect the plants against pests, diseases and fungi. Within the agriculture the dependency on plant protection products (PPP) is significant. Without PPP it is estimated that 30 to 50% of crop production is lost.

[0007] The cultivation of plants, particularly crops, is fundamental to global agriculture and food security. Crops such as wheat, maize, rice, and vegetables are essential for human nutrition and economic stability. However, plant cultivation faces challenges, primarily due to pests, diseases, and fungi that can severely reduce yields. Farmers heavily rely on plant protection products (PPPs) to safeguard crops. These products are crucial for controlling and mitigating the impact of various biotic stresses, ensuring sustainable agricultural productivity.

[0008] It is estimated that without the use of PPPs, crop production could face a significant yield reduction of 30% to 50%. This dependency underscores the critical need for effective pest and disease management strategies. Conventional PPPs, including fungicides, insecticides, and herbicides, have been instrumental in achieving high yields. However, their extensive use has raised concerns regarding environmental impact, human health, and the development of resistance among pests and pathogens.

[0009] Among the various PPPs, formulations comprising nitrous acid (HNO2) have gained attention for their potential in plant protection. Nitrous acid is known for its antimicrobial properties, making it a promising candidate for developing effective plant protection agents. Plasma- activated water (PAW) is an example of a formulation comprising HNO2. PAW has shown efficacy in controlling a range of pathogens, including bacteria, fungi, and viruses. PAW is generated by exposing water to a plasma discharge, creating a reactive mixture of species such as nitrogen oxides, hydroxyl radicals, and reactive oxygen species, which are detrimental to pathogens. Despite its potential, the use of PAW, particularly those formulations containing HNO2, presents a significant challenge: phytotoxicity. Plasma activated water, while effective against pathogens, can also be harmful to plants and seeds, limiting its practical application in agriculture. Phytotoxicity refers to the toxic effects that certain substances can have on plant growth and development, leading to reduced germination rates, stunted growth, or even plant death. This presents a critical limitation, as the goal of any PPP is not only to protect crops against pathogens but also to ensure their health and viability. Another important challenge is that PAW, i.e. formulations with HNO2 without salts, are unstable and cannot be stored for a prolonged period of time.

[0010] It is an objective of the present disclosure to overcome one or more of the drawbacks associated with current formulations of HNO2, particularly plasma activated water. The aim is to provide improved plant protection formulations that maintain or enhance effectiveness against a broad spectrum of pathogens while significantly reducing phytotoxicity. By doing so, the present disclosure seeks to provide for safer, more reliable tools for crop protection, ultimately contributing to higher yields, reduced environmental impact, and sustainable agricultural practices. The present disclosure outlines novel methodologies and compositions that achieve these objectives, promising advancements in the field of plant protection.

[0011] Summary

[0012] The present inventors considered that pH could be an important parameter determining the level of phytotoxicity of HNO2 formulations, in particular plasma activated water formulations. It was surprisingly found that there is a sweet spot formulation which has a HNO2 concentration of 1-20, 1-15, 1-8, 1-5 mM, particularly 3-4 mM, in combination with a pH of 1- 8, 1-4, more particularly pH 3-4 and most preferably pH3.5. It was found that a lower pH may lead to plant death, while a higher pH turns the chemical balance towards higher concentrations of nitrite (NO2-) which also leads to more phytotoxicity. In addition, it was found that HNO2 is the only or primary active ingredient in plasma activated water, and that a HNO2 concentration of 1-5 mM, preferably 3-4 mM is sufficient to counter pathogens.

[0013] To increase the pH of the HNO2 formulations, in particular plasma activated water, a few methods can be used: adding a base such as NaOH, KOH or other *OH molecules. However, it was found particularly advantageous to use ammonia as a pH regulation method. The ammonia can react into NH4NO2 which is an unstable compound resulting in a non-salted residue which reduces the risk of stable toxic residues on plant, when compared to other bases. Additionally, it was found that by adding a wetting agent to the formulation, phytotoxicity can be dramatically further reduced, wherein a silicone based wetting agent may provide the best results.

[0014] The HNO2 in the PAW is produced by an air plasma source resulting in dissolved HNO2 that does not originate from salts. This results in limited to no residues on the plants.

[0015] The present inventors have prepared HNO2 formulations by using an air plasma source resulting in dissolved HNO2 that does not originate from salts. This gives the opportunity to locally produce HNO2 containing plasma activated water without needing nitrite salts. This reduces the risk of nitrate salt residues on the plants after treatment.

[0016] Detailed description

[0017] The present disclosure provides for an aqueous composition comprising (between) 0.01 - 100 mM HNO2, preferably (between) 0.15-25 or 2-20 mM HNO2 , more preferably (between) 1-8, (between) 1 - 5 mM HNO2, preferably (between) 2 - 5, or (between) 2-4 mM HNO2, most preferably (between) 3 - 4 mM HNO2 and / or having a pH between 1-8, 1 - 7, preferably between 2-6, between 2-5, between 1-4, or between 2-4, most preferably between 3-4.

[0018] The aqueous composition particularly finds use as plant protection product (which can for example be a biocide).

[0019] In addition, or alternatively, the aqueous composition may comprise

[0020] (between) 0.01-100 mM, preferably (between) 2-60 mM or (between) 0.1 - 25 mM or (between) 5-20 mM nitrite, more preferably (between) 0.1-5 mM nitrite, most preferably (between) 2-3 mM nitrite (e.g. at pH (between) 1-5, or pH (between) 3-4) or (between) 2-10 or (between) 4-6 mM nitrite; and / or

[0021] (between) 0-300 mM, preferably (between) 0-100 mM nitrate.

[0022] In the present disclosure, reference to HNO2 concentration does not include the combined concentration of NO2- and H+.

[0023] HNO2 (nitrous acid) is a weak acid in equilibrium with its’ ion form when in liquid:

[0024] Ka HNO2H++ NO2

[0025] (Total) nitrite refers to the sum of the nitrous acid and nitrite ion. The ratio of these is determined by the pH of the solution and the acid dissociation constant (pKa) of nitrous acid. If the pH of the solution changes, this ratio changes instantly too.

[0026] [Brackets] suggest concentration in [mol / L] = M(olar) mM stands for milliMolar

[0027] HNO3 (nitrate or nitric acid) is a strong acid dissociating into ion form when in liquid:

[0028] HNO3 -> H++ NO3 .

[0029] This is what makes can make the pH of the solution so acidic as the pH is dependent on the H+present: pH = - log10[H+]

[0030] The equilibrium between nitrous acid and nitrite in ionic form in the aqueous composition typically is pH dependent and the pH typically determines the ratio between nitrous acid and nitrite ions. The pKa value of the equilibrium is 3.15 and it can be seen as the 50 / 50 mark in the equilibrium. The fraction nitrous acid to total nitrite can be calculated based on the pH of the solution and is given in Figure 1.

[0031] After understanding the effect of pH on the nitrite equilibrium, the concentration of total nitrite is introduced to illustrate the effect of the combination of both. Figure 2 shows the effect of the pH on the nitrite equilibrium with variable total nitrite concentration. As can be seen, increasing the total nitrite concentration can be a method to increase the concentration of nitrous acid while keeping pH constant.

[0032] The aqueous composition according to the present disclosure preferably has an amount of total dissolved solids (TDS), preferably not including any wetting agent (i.e. preferably not taking any possibly present wetting agent into account), which is less than 1 g / L, preferably less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 , 0.05, 0.01, 0.001 g / L.

[0033] The amount of total dissolved solids may be measured by evaporation of the aqueous composition followed by weighing of the residue.

[0034] Similarly, the present aqueous composition preferably comprises less than 10, 5, 1 , 0.5, 0.1 M cation(s) other than H+. This may avoid formation of nitrite salts, e.g. after repetitive application of the aqueous composition. Nitrite salts can harm the plant or consumer of the plant or plant part.

[0035] By using plasma as a source for the HNO2 comprising aqueous composition according to the present disclosure, said composition can be made without the formation of salts. The salts may end up as undesired residues, e.g. on treated plants.

[0036] Accordingly, the aqueous composition according to the present disclosure preferably is plasma-activated water. Plasma activated water (PAW) typically refers to water that has been exposed to a plasma discharge, which may impart it with a variety of reactive species. These species can include free radicals, ions, and other reactive molecules, such as ozone (O3), hydrogen peroxide (H2O2), nitrogen oxides (NOx), and hydroxyl radicals (•OH).

[0037] In other words, plasma activated water can be produced by making use of water, air and electricity. Ambient air is brought into the plasma phase with electrical energy, the activated air is then brought into contact with water. Reactive oxygen and nitrogen dissolve into the water creating plasma activated water (PAW).

[0038] The process of producing plasma activated water typically involves the following steps: generation of plasma: plasma is generated by applying a high voltage to a gas (usually air or oxygen) in the presence of water. This can be achieved using various plasma sources, such as dielectric barrier discharge (DBD), corona discharge, or microwave plasma. The electrical discharge ionizes the gas, creating a plasma containing a mixture of ions, electrons, and excited molecules. interaction with water: the plasma interacts with the surrounding water, leading to the formation of reactive species. This interaction can be direct, where the plasma species directly react with the water molecules, or indirect, where the plasma-generated species react with the dissolved gases in the water, such as oxygen and nitrogen.

[0039] In other words, water can be "activated" by applying plasma in contact with the water, for instance by creating plasma inside (bubbles in) the water, or along a water surface. Plasma activated water (PAW) typically contains hydrogen peroxide, nitrates, nitrites, where peroxynitrite is formed due to a reaction with nitrite and hydrogen peroxide in an acidic environment, and is present in PAW for period of approximately 15 minutes after activation. Alternatively, water can be activated by bringing gasses of plasma in contact with water. For the process of producing plasma-activated water, a plasma activated fluid processing system can be used, which may comprise:

[0040] - an alternating current, AC, source (10),

[0041] - one reaction chamber (14), wherein the one reaction chamber comprises a plurality of reaction electrodes (11) and a corresponding ground electrode (12), each reaction electrode and corresponding ground electrode separated by a gap; or

[0042] - a plurality of reaction chambers (14), wherein each reaction chamber comprises at least one reaction electrode (1 1) and a corresponding ground electrode, the reaction electrode and corresponding ground electrode separated by a gap; wherein the AC source is electrically connected on one side to each ground electrode in parallel and on another side to each reaction electrode in parallel, wherein each reaction electrode is provided with a capacitor (21) connected to the ground (13), and wherein an inductor (20) is provided between each reaction electrode and the AC source, preferably wherein an AC operating frequency is between 10 kHz and 50 MHz.

[0043] Alternatively, a thermal and non-thermal plasma activated water reactor system may be used, which may comprise: a) a reaction chamber, wherein said reaction chamber comprises

[0044] - a gas inlet,

[0045] - a water inlet,

[0046] - a combined gas and water outlet,

[0047] - a ground electrode and reaction electrodes configured to generate, in operation, reactive oxygen species, ROS, and reactive nitrogen species, RNS, in gas form, wherein said gas inlet and said water inlet are disposed off-center from said reaction chamber and said combined gas and water outlet is disposed at a center of a bottom surface of said reaction chamber or at a height thereof, such that in operation, when water flows there through a vortex movement of water and air is generated so as to mix the water with said ROS and RNS, wherein said reaction electrodes i) comprise a thermal plasma electrode and a non-thermal plasma electrode, wherein said thermal plasma electrode is configured to generate a thermal plasma and wherein said non- thermal plasma electrode is configured to generate a non-thermal plasma, or ii) are configured to be switchable connectable to a power supply so as to generate in operation both the thermal and the non-thermal plasma with predefined duty cycles, wherein the thermal and non-thermal plasma activated water reactor is configured to generate plasma activated water having a pH ranging from 0 to 7, or an oxidation reduction potential, ORP, value ranging from 200 mV to 800 mV, and b) a plasma activated water reservoir, wherein said water reservoir is disposed to receive said plasma activated water from said reaction chamber and disposed to return said plasma activated water to said reaction chamber.

[0048] In the present disclosure, the HNO2 in the aqueous composition, e.g. plasma-activated water, is preferably produced by an air plasma source resulting in dissolved HNO2 that does not originate from salts. This results in limited to no residues on the plants. To increase the pH of the HNO2 formulations, in particular plasma activated water, a few methods can be used: adding a base such as NaOH, KOH or other *OH molecules.

[0049] To neutralize the acidic pH, the H+of both species is to be considered, even though the H+concentration of the nitrous acid is dependent on the pH value. To calculate the ratio in nitrous acid or the concentration of the species there are three formula’s that can be used. As long as 2 out of the 3 parameters (concentration H+, NCh' or HNO2) are known you can calculate the ratio. If the concentration of NC>2' or HNO2 is unknown, but the total nitrite concentration is known, you can rewrite the equation and solve the system.

[0050] Thus the concentration of NOa' is not a requirement to calculate the ratio, however, the nitric acid is usually the main supply for the concentration of H+.

[0051] A way to neutralize the acidic pH is to add alkali to the solution, for example:

[0052] 1. With metal ion:

[0053] Can be added via compound which can contribute to base ions in the solution. This includes, but not limited to, potassium hydroxide, potassium bicarbonate, sodium hydroxide.

[0054] 2. With ammonium ion

[0055] NH4+: can be added via sources containing ammonium ion or which are able to produce ammonium in on making the solution. This includes, but not limited to, like ammonium hydroxide, ammonium bicarbonate, ammonia gas.

[0056] Reaction routes:

[0057] Reaction routes for adjusting pH of plasma activated water with KOH and NH4OH involved acid-base neutralization reactions. :

[0058] 1. With potassium hydroxide The overall reaction amongst nitric acid, nitrous acid and potassium hydroxide can be written as follows. This includes the complete neutralization reaction between potassium base and acids from PAW and leftover acids from the PAW:

[0059] H++ NO2+ NO2+ HN02+ KOH K++ H++ N02+ N02+ HN02+ H20

[0060] Here, the amount of KOH added could be adjusted to control the desired pH and hence the composition of HNO2 and NO2'.

[0061] 2. With ammonium hydroxide

[0062] The reaction pathway is similar to that of with KOH. Hence, similarly the overall reaction can be written as follows. This includes the complete neutralization reaction between ammonium base and acid from PAW and leftover acids from the PAW:

[0063] H++ NO2+ NO2+ HN02+ NH40H NH4++ H++ NO2+ NO2+ HN02+ H2O

[0064] Similar to KOH, the amount of ammonium hydroxide can be adjusted to control the desired pH and hence the composition of HNO2 and NO2'.

[0065] It was found particularly advantageous to use ammonia as a pH regulation method, e.g. ammonium hydroxide, ammonium bicarbonate, ammonia gas. The ammonia can react into NH4NO2 which is an unstable compound resulting in a non-salted residue which reduces the risk of stable toxic residues on plant, when compared to other bases.

[0066] NH4N02-> N21 + 2H2O

[0067] Accordingly, the aqueous composition, e.g. plasma-activated water, preferably comprises a base. The concentration of the base is preferably chosen such that the desired pH is achieved.

[0068] The base is preferably selected from NH40H, KOH, NaOH, KHCO3 and / or NH3. A particularly preferred base is ammonium hydroxide, ammonium bicarbonate, ammonia gas, preferably NH4OH. Adding NH40H may allow for similar stability as when using KOH but does not result in / reduces toxic residues.

[0069] The present disclosure also provides for a method for preparing an aqueous composition according to the disclosure, the method comprising combining an aqueous composition as disclosed herein (e.g. comprising (between)1-5 mM HNO2) and a base, wherein the base is preferably selected from NH40H or KOH, most preferably NH40H. Optionally, a wetting agent (e.g. as taught herein) is added. It is preferred that, during the method, the molar ratio of oxygen-containing radicals to nitrogen-containing radicals remains below 2, preferably below 1. For example, the molar ratio of oxygen-centered radicals (e.g., hydroxyl radicals, superoxide radicals) to nitrogen-centered radicals (e.g., nitric oxide, nitrogen dioxide) remains below 2, preferably below 1. Optionally, the method is performed in the absence of oxygen, or in the presence of less then 5, 4, 3, 2, 1 vol.% oxygen.

[0070] Preferably the method does not comprise the use of an alkali or alkaline earth metal nitrite or nitrate.

[0071] The aqueous composition (e.g. comprising 1-5 mM HNO2) is preferably provided by using an air plasma reactor and subsequent dissolving NOx gases from the reactor in water in the form of HNO2, NO and NO3’.

[0072] It is preferred that the aqueous composition of the present disclosure, particularly the plasma- activated water, is not in contact with oxygen or hydrogen peroxide; and / or not in contact with gas comprising more than 1 , 2, 3, 4, 5 vol.% oxygen and / or more than 1, 2, 3, 4, 5 vol.% hydrogen peroxide.

[0073] This is because there is a oxidation reaction of nitrous acid to nitric acid (HNO2 to HNO3)With oxygen sources. This can happen with oxygen or hydrogen peroxide for example. This is one of the reasons to prevent oxygen with the aqueous composition according to the disclosure.

[0074] HNO2 may slowly (minutes / hours / days) degrade to NO gas. The reaction rate constant increases largely with temperature, thus the higher the temperature, the quicker the degradation. The degradation does not happen to the nitrite form, only to the nitrous acid. This is another reason to prevent air being stored with the PAW. Accordingly, the aqueous composition, e.g. the plasma-activated water according to the present disclosure preferably has a temperature of below 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 degrees Celsius.

[0075] The aqueous composition, e.g. plasma activated water, according to the present disclosure can advantageously be combined with one or more adjuvants, such as (super)surfactants, spreaders, stickers, emulsifiers, or wetting agents, rop oils, crop oil concentrates, and vegetable oil concentrates, stickers (deposition agent), penetration improvers, driftreductors / drift control agents, fertilizers, compatibility agents, and / or defoaming agents.

[0076] It is particularly advantageous if the aqueous composition, e.g. plasma-activated water according to the present disclosure comprises a wetting agent (or any kind of surfactant), because it can dramatically reduce phytotoxicity effects. Suitable wetting agents are for example a silicone based wetting agent or a polyglycerol based wetting agent, wherein a silicone based wetting agent is particularly preferred because it has markedly better reduced phytotoxicity effects as compared to other types of wetting agents.

[0077] The wetting agent may be present in the aqueous composition, e.g. plasma activated water, in a concentration of (between) 0.001-100 g / L, preferably (between) 0.01-10 g / L, more preferably (between) 0.1-1 g / L.

[0078] The present disclosure provides for a method for protecting plants (e.g. against plant pathogen(s)), wherein the method comprises applying the aqueous composition according to the present disclosure to a plant or plant seed and / or to soil (or other material) supporting the plant or plant seed and / or soil, water and / or air surrounding a plant or plant seed. Preferably, the aqueous composition is in situ produced, wherein in situ means within 10, or within 5 preferably within 1 km of the plant(s).

[0079] The application is preferably by submerging, spraying, soaking, (low volume) misting, vapouring, drip irrigating and / or injecting the applying the composition in, on or near (e.g. within 50, 25, 10 cm) the leaves of the plant, base of the plant and / or soil supporting the plant and / or applying the composition in, on or near (e.g. within 50, 25, 10 cm) plant sees or soil (or other material) supporting the plant seed.

[0080] In other words, the application of the aqueous composition e.g. plasma-activated water may be done either by submerging, spraying, soaking, (low volume) mist, vapour, drip irrigation, injection ETC... Foliar, basal, soil, space application.

[0081] In addition or alternative, the application may be performed at least once every 3-14 days. In addition or alternatively, the aqueous composition e.g. plasma-activated water, may be applied in amount of (between) 50-6000L / ha, or (between) 200-4000L / ha, or (between) 300- 3500L / ha. As will be clear, the aqueous composition, e.g. the plasma-activated water, according to the present disclosure may find use as a plant protection product.

[0082] The components of the aqueous composition, particularly the plasma activated water have synergistic antimicrobial effects against bacteria, biofilms, yeasts and other microorganisms, and also work as a natural fertilizer, as seed germination and plant growth is stimulated.

[0083] The present disclosure particularly provides for the control of microorganisms, fungi or oomycetes e.g. in agriculture, for example in a method comprising applying to plants or to the environment thereof, the aqueous composition, e.g. plasma-activated water, according to the present disclosure.

[0084] In an embodiment of the present disclosure, the aqueous composition, e.g. plasma-activated water, according to the present disclosure is applied to a plant or plant seed, and in a subsequent step (e.g. between 1-3 days or between 1-72 hours of the first step), water is applied to the plant or plant seed so as to wash the surface thereof.

[0085] The present disclosure may aim for prophylaxis or treatment of plant fungal or oomycete diseases, for example mildew. It may be used to control fungi selected from Magnaporthe grisea, Blumeria gramina Mycosphaerella graininicola Fusarium spp., Rhizoctonia solani Gaewnannomyces graminis, Botrytis cinerea or the oomycete Phytophthora infestans.

[0086] For agricultural applications, protection, disinfection and germination of seeds, plants, flowers, vegetables and crops, and increasing plant growth are enabled by the disclosure, preferably wherein the aqueous composition / activated liquid / water is sprayed immediately, or after being stored (preferably for at most 3, 2, 1 days or at most 24, 16, 12, 8, 6, 4, 3, 2, 1 hours), or applied in any other form. This ensures the use in its most active form. The vase life of cut flowers can be significantly extended. Also buds of flowers (roses) infected with botrytis can be reduced by about 60 %.

[0087] Other applications can be waste / drink water cleaning, removal of fouling by biofilms of membranes, such as membranes for cleaning of drinking water. Specifically, the production of nitrogen components that can be used as fertilizer for crops and plants is a very interesting application.

[0088] For medical applications, the present disclosure allows disinfection of human skin, wounds, root channels in teeth, medical instruments, equipment and surfaces. In addition to disinfection, sterilization is enabled due to a synergetic effect when combining with a mild disinfectant. Where (concentration) ranges are disclosed herein, endpoint may be included in the range. Alternatively, i.e. when the term “ between” is used, endpoint may not be included. Description of Figures

[0089] Figure 1: Effect of pH on total nitrite equilibrium

[0090] Figure 2: Effect of pH on the nitrite equilibrium with different total nitrite concentrations Figure 3: Test strategy

[0091] Figure 4: In vitro tests - HNO2 is active compound

[0092] Figure 5: In vitro tests - No effectiveness of NO3.

[0093] Figure 6: Dose effectiveness in vitro on pathogen - in vitro

[0094] Figure 7: Dose effectiveness on pathogen on crop

[0095] Figure 8: Effect of adjuvants

[0096] Figure 9: The effectiveness of plasma activated water against bacteria in combination with wetting agent

[0097] Figure 10: The effectiveness of plasma activated water against bacteria in combination with wetting agent

[0098] Figure 11 : In lettuce: Added effectiveness by adding wetting agent.

[0099] Figure 12: In cucumber: Added effectiveness due to combination liquefaction. Figure 13: In rose - Decrease in effectiveness with surfactant removal.

[0100] Figure 14: System / process overview

[0101] EXAMPLES

[0102] 1. Production of plasma activated water

[0103] The production of Plasma activated water was performed in a high voltage plasma reactor. Said plasma reactor has an air input, liquid input and a combined gas and liquid output, a high voltage electrode and a ground electrode. The reactor is operated in a Continuous Stirred-Tank Reactor (CSTR) mode wherein both air input and water input are sensor regulated based on the desired concentration of the components in the liquid and gas resulting in a continuous output of PAW (1-8 mM HNO2and pH 1-4). The plasma is generated by a high voltage AC power source generating sufficient voltage and frequency to discharge from the high voltage electrode to the liquid. The liquid is in direct contact with a ground electrode. The combined gas and liquid output is intensely mixed to transfer all NOx components from the gas to the liquid resulting in a high concentration of HNO2 and NO2- whilst reducing the amount of NO3-. To maintain a pre determined pH the pH is measured and an additive (e.g. KOH, NH4OH, etc) can be added before or after the process.

[0104] The reactor is equipped with two closed-loop control system that regulates the PAW composition and the pH (see also Figure 14): Residence-time control forHNO2concentration - The system monitors the concentration of the HNO2. Based on the selected output concentration the output flow is controlled by a PI D controller to ensure accurate HNO2 concentration at the output. pH monitoring and correction - The water flow passes through temperature-compensated pH probes. The addition of base (NH40H or KOH) is controlled by a PI D controller adjusting the setpoint of the injection pump to ensure accurate pH.

[0105] Optimal plant protection composition - Components in plasma activated water responsible for phytotoxicity / methods to increase pH and reduce phytotoxicity

[0106] The composition of PAW can be chosen to fit the plant pathogen combination and the ability for the plant to withstand a low pH. Acidity can burn the leaves therefore adjusting the pH to a higher level will reduce phytotoxic effect, however it will reduce the HNO2 concentration due to the chemical balance of the HNO2 and NO2-.

[0107] 2. Optimal plant protection composition - Components in plasma activated water responsible for phytotoxicity / methods to increase pH and reduce phytotoxicity

[0108] Goal

[0109] • Determine components responsible for phytotoxicity

[0110] • Determine upper limit in plasma activated water concentration and pH

[0111] Methods

[0112] Young, three-leaf cucumber plants, grown in the winter, have been exposed to various plasma activated water formulations to subsequently assess crop damage. Each application was performed three times. The trial was conducted in December 2023.

[0113] Results

[0114] The hypothesis was that pH would be a damaging factor in plasma activated water.

[0115] Therefore, a pH series (nitrate water + KOH) was tested on the cucumber plants. The results showed that at a pH below 1 , the plants were completely dead. Plants treated with a pH of 3 looked significantly better. However, all tested pH's resulted in crop damage. pH is clearly a factor in phytotoxicity.

[0116] Subsequently, a matrix of plasma activated water compositions was tested in order to determine the role of HNO2(active ingredient), NO2 (HNO2 reservoir), NO3 (causative agent of pH) in crop damage (in addition to the pH itself).

[0117] See Figure 3.

[0118] It was surprisingly found that there is an optimum formulation which has a HNO2 concentration of 1-5 mM, particularly 3-4 mM, and a pH of 1-4, more particularly pH 3-4 and most preferred pH3.5. At a higher pH range, a higher concentration of nitrite (NO2-) is needed to achieve the same amount of active ingredient, which is why more phytotoxicity occurs. A too high concentration HNO2 was also found to be responsible for crop damage. The contribution of NO2- in crop damage is less, but still present (horizontal line 0 vs horizontal line 1). NO3- itself does not cause any damage, but the low pH it causes does (nitrate + KOH at high pH does not show any damage). To achieve a pH of 1-4, more particularly pH 3-4 or pH 3.4, a base typically need to be added to the plasma activated water.

[0119] The optimum at pH3.5 may somewhat differ per crop. This optimum does not depend on the nitrate concentration in the plasma activated water.

[0120] Conclusions

[0121] 1. pH, HNO2, NO2- are the harmful factors in plasma activated water, with pH and HNO2

[0122] (to which NO2- is linked) being the most important.

[0123] 2. There is a sweet spot at a HNO2 concentration of 1-5 mM, particularly 3-4 mM, in combination with a pH of 1-4, more particularly pH 3-4 and most preferably pH 3.5.

[0124] 3. Optimal composition - Components in PA l / V responsible for effectivity

[0125] It was found that HNO2 is the only or primary active ingredient in plasma activated water.

[0126] Not NO3 which has no effectiveness, and only leads to low pH, and not SLS (not present, still activity after an hour), and not NO2 (because at high pH no activity).

[0127] HNO2 is always or typically present in a mix with NO2 (ratio) and NO3 (decay). That is why this combination of components has been tested. Subsequently, NO2 and NO3 have been shown to be ineffective against pathogens.

[0128] In vitro tests HNO2 is active compound. See Figure 4.

[0129] Conclusion: HNO2 is the active substance, not NO2-.

[0130] No effectiveness of NO3. See Figure 5. Conclusion: No effectivity of NO3-.

[0131] Overall conclusion: HNO2 is the active substance. NO2- acts as a reservoir for HNO2.

[0132] NO3- contributes to the low pH.

[0133] 4. Optimal composition for plasma activated water - HNO2 concentration and effectivity

[0134] Dose effectiveness in vitro on pathogen

[0135] Figure 6 shows one application of in vitro.

[0136] Dose effectiveness on pathogen on crop

[0137] See Figure 7.

[0138] PAW A, B, C: dose range of HNO21.7mM + 3.3 mM NO2-, HNO23.4mM + 6.6mM NO2-, HNO2 4.7mM + 10.3mM NO2-. Application of PAW via spraying in a 4 / 5 day interval.

[0139] Conclusion: Higher dose of HNO2 results in a higher effectivity against pathogens. 1-5 mM, preferably 3-4, or particularly 3.4mM of HNO2 shows sufficient effectivity against pathogens.

[0140] 5. Optimal composition of PAW- Effect of an adjuvants on phytotoxicity and effectivity against pathogens.

[0141] Adjuvants - to reduce phytotoxicity

[0142] It was found that by adding a wetting agent, phytotoxicity can be reduced. Two wetting agents have been tested (Silwet Gold 20cc -and Elasto).

[0143] Phyto results C02 (and mildew)

[0144] Phyto C02 % mildew (indication) + = high, -= low

[0145] Elasto 0 Elasto +

[0146] Silwet 0 Silwet + water 0 water +++ untreated 0 untreated ♦+++

[0147] See also Figure 8. In general, the combination of plasma activated water with Silwet leads to a lower phytotoxicity on the crop. In addition, the combination with a wetting agent does indeed reduce crop damage, but crop damage does not go to zero. As a bonus, the effect of plasma activated water in combination with the wetting agent on mildew was also examined. Conclusion

[0148] Surfactant should be used to reduce crop damage wherein a silicone based wetting agent provides the best results. 6. Adjuvants - to improve PAW effectivity against pathogen

[0149] In vitro lab testing

[0150] The effectiveness of plasma activated water against bacteria in combination with Elasto G5 150cc and Silwet Gold 20cc has been determined in the microbiology lab of the Radboud university medical center. Both Silwet and Elasto had no effect on the effectiveness of PAW against Staphylococcus aureus. See Figure 9 and 10.

[0151] Practical tests - wetting agent

[0152] In lettuce: Added effectiveness by adding wetting agent. See Figure 11.

[0153] In cucumber: Added effectiveness due to combination liquefaction. See Figure 12.

[0154] In rose - Decrease in effectiveness with surfactant removal. See figure 13.

[0155] Conclusions

[0156] Plasma activated water is compatible with multiple types of wetting agents.

[0157] 7. Optimal composition of PA W per crop / pathogen

[0158] Cucumber - powdery mildew:

[0159] Dosage: 10mM total nitrites (3.4mM HN02) pH: 3.5 method of application: spraying frequency of application: 4 / 5 days interval surfactant: Silwet Gold 20cc or Elasto G5250cc

[0160] Roses - powdery mildew

[0161] Dosage: 10mM total nitrites (3.4mM HN02) pH: 3.5 method of application: spraying frequency of application: 3 / 4 days interval surfactant: Silwet Gold 10cc or Elasto G5 100cc

[0162] Overall optimal plasma activated water composition (based on evidence)

[0163] Dosage: 1-5 mM HNO2 all applications, preferably >2.5mM HNO2 in greenhouse pH: 3.5 in greenhouse; 1-4 in other application fields method of application: spraying (foliar) in greenhouse, spraying (foliar), misting, submerging in other application fields frequency of application: twice a week to once every two weeks

[0164] Surfactant: Silwet Gold 10-20cc or Elasto G5 100-250cc

Claims

CLAIMS1. Use of an aqueous composition as a plant protection product, wherein the aqueous composition comprises 1 - 8 mM HNO2 and has a pH between 1 — 4, wherein the aqueous composition is plasma-activated water.

2. Use according to claim 1, wherein the aqueous composition comprises less than 1 mM cations other than H+.

3. Use according to any one of the preceding claims, wherein the amount of total dissolved solids (TDS) in the aqueous composition, is less than 1 g / L, preferably less than 0.1 g / L.

4. Use according to any one of the preceding claims, wherein the aqueous composition has a pH between 3 - 4.

5. Use according to any one of the preceding claims, the aqueous composition further comprising a wetting agent.

6. Use according to claim 5, wherein the wetting agent is a silicone based wetting agent or a polyglycerol based wetting agent, preferably a silicone based wetting agent.

7. Use according to claim 5 or 6, wherein the wetting agent is present in a concentration of 0.001-100 g / L, preferably 0.01-10 g / L, more preferably 0.1-1 g / L.

8. Use according to any one of the preceding claims, wherein the aqueous composition comprises a base.

9. Use according to claim 8, wherein the base is selected from NH40H, KOH, NaOH, KHCO3 and / or NH3.

10. Use according to claim 9, wherein the base is NH40H.

11. Use according to any one of the previous claims, wherein the use comprises applying the aqueous composition to- a plant or plant seed;- soil supporting a plant or plant seed; and / or- soil, water and / or air surrounding a plant or plant seed.

12. Use according to claim 11, wherein application is preferably by submerging, spraying, soaking, (low volume) misting, vapouring, drip irrigating and / or injecting the applying the composition in, on or near the leaves of the plant, base of the plant and / or soil supporting the plant.

13. Use according to any one of claims 11 or 12, wherein application is performed at least once every 3-14 days.

14. Use according to any one of the previous claims, wherein the aqueous composition is in situ produced, wherein in situ means within 10, preferably within 5 km of the plant(s).

15. Method for preparing an aqueous composition as defined in any one of the preceding claims, the method comprising combining a composition comprising HNO2 and a base, wherein the base is preferably selected from NH40H or KOH.

16. Method for preparing an aqueous composition according to claim 15, wherein the base is NH40H.

17. Method for preparing an aqueous composition according to any one of claims 15-16, wherein the aqueous composition comprising 1-8 mM HNO2 is provided by using an air plasma reactor and subsequent dissolving NOx gasses from the reactor in water in the form of HNO2, NO2- and NO3-.

18. Method according to any one of claims 15-17, wherein the method does not comprise the use of an alkali or alkaline earth metal nitrite or nitrate.

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