RU2851302C1 - Antimicrobial and agrochemical compositions - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: agrochemical composition that inhibits the formation of sclerotia by fungi contains the following starting components: copper oxychloride in the form of a wettable powder containing bentonite, or copper oxychloride in the form of a technical product, phosphoric acid, ammonium hydroxide, zinc salt and water, wherein phosphoric acid is added to a suspension of copper oxychloride in water at a concentration of 40 to 800 g/l in combination with ammonium hydroxide and zinc salt, wherein the total content of elemental copper in the composition is from 1 to 50 g/l, wherein the ratio of copper to zinc in the composition is from 10:1 to 1:10 and the ammonium hydroxide content is from 1 to 80 ml/l.

EFFECT: ensuring effective and long-term inhibition of fungal sclerosis formation in the field to prevent the continuation and development of pathogenic fungi.

5 cl, 3 dwg, 11 tbl, 5 ex

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to United States Patent Application No. 62/172,501, filed June 8, 2015, entitled "Antimicrobial and Agrochemical Compositions" by Dr. Tony John Hall, the entire disclosure of which is incorporated herein by reference as permitted by national or regional laws.

AREA OF TECHNOLOGY

The present invention generally relates to antimicrobial compositions that also inhibit the formation of sclerotia by fungi and, more specifically, to aqueous compositions containing copper ions, with or without zinc ions, a basic salt, and phosphorous acid as active ingredients. The compositions are additionally sporicidal and sclerotia-killing when combined with nitrous acid salts, such as sodium nitrite. The compositions inhibit the formation of sclerotia by fungi due to their content of phosphorous acid with a phosphite or phosphonate (PO ₃ ^3- ) group.

LEVEL OF TECHNOLOGY

The globalization of agriculture has led to crops being grown in areas where they may be exposed to new pathogens or new strains of existing pathogens, such as fungi and bacteria, to which they are susceptible. An estimated 70% of all major crop diseases are caused by phytopathogenic fungi, and plant diseases are now recognized as a threat to the global food supply.

The development of commercial fungicides in agriculture began with the copper-based Bordeaux mixture in the 19th century. In the 20th century, many new classes of synthetic organic fungicides with specific modes of action were created, but the development of fungal resistance to many of these substances is a growing problem. Furthermore, many of these fungicides have toxic effects on the environment or other species, and they can persist in crops and enter the food chain. Consequently, there is a growing need for new fungicides that are safer for the environment and consumers.

Many fungi disperse as spores, and wind-dispersed phytopathogenic fungi are responsible for some of the most damaging crop diseases, such as coffee rust (Hemileia vastairix), rice blast (Magnaporthe oryzae), and black sigatoka (Mycosphaerella fijiensis) on banana plants. Fungal (and bacterial) spores are highly resistant to cold, heat, ultraviolet light, and most fungicides. In fact, most spores are so resistant that chemicals such as hypochlorous acid (bleach) and hydrogen peroxide, which are sporicidal at high concentrations (e.g., 5-10% and >10%, respectively), also cause significant damage to plants, animals, and even the human environment at these sporicidal concentrations.

In addition to spores as a means of dispersal and survival, many fungi can form survival structures called sclerotia, which are an important source of phytopathogenic diseases associated with species of Rhizoctonia, Verticiliium, Sclerotinia, and Macrophormina. Sclerotia are asexual, multicellular, dormant, and highly chemically resistant structures. Sclerotia are typically melanized, making them resistant to ultraviolet radiation and therefore often identified as black spots or bodies in soil or on plants, fruits, and vegetables. If present in the soil alone, sclerotia can survive for many years, infecting newly planted crops through contact with plant roots. This leads to germination of the sclerotia and the growth of fungal hyphae, which penetrate the plant through the roots. Therefore, there is a need for an environmentally friendly and economically feasible product that can inhibit sclerotia formation in agriculture.

Copper-based fungicides are still widely used in agriculture, including organic farming, because they are widely available, inexpensive, and relatively safe to use. Furthermore, fungal resistance to copper-based products is low because copper has complex toxic effects, including damage to cell membranes and inactivation of iron-sulfur cluster dehydratase enzymes. However, currently available copper-based products include suspensions of copper compounds, such as copper hydroxide and copper oxychloride, which are applied preventatively by adhering to plant leaves to prevent fungal development. These copper-based fungicides require frequent applications and contain relatively high amounts of copper—Bordeaux mixture foliar sprays and copper oxychloride products typically contain 2.5 grams/liter of elemental copper—because they provide low amounts of ionic copper, which is the fungicidal/bactericidal form of copper.

Phosphorous acid in the form of salts, such as potassium phosphite, is classified by the U.S. Environmental Protection Agency as a biopesticide. Phosphites have both direct and indirect modes of action against oomycetes and fungi. Direct effects include inhibition of mycelial growth and suppression of spore formation and germination. Indirect effects of phosphites include activation of plant defense responses through mechanisms that are not yet fully elucidated. Phosphites have low toxicity and, like copper-based fungicides, have the advantage of being inexpensive, relatively safe to use, and acting through multiple sites of action, preventing the development of resistance.

Global health and environmental regulations are becoming increasingly stringent regarding pesticide residues. Farmers worldwide therefore face the dilemma of combating destructive pathogens, necessitating increased use of fungicides/bactericides, while government regulations require lower chemical residues on crops and soil. Plant pathogenic fungi that spread by spores are particularly difficult targets, and there are currently no commercially available, proven sporicidal products. Clearly, if spores could be destroyed/inactivated on infected plants before they are released into the air, this would prevent their dissemination and lead to effective disease control.

Therefore, there is a need for effective and safe sporicidal agricultural compositions that are not phytotoxic. It is known that the reaction between acids and sodium nitrite results in the formation of highly reactive nitrogen oxides, which exhibit antimicrobial and sporicidal activity. Experiments conducted by the inventor using compositions containing copper and/or zinc ions and phosphorous acid in combination with sodium nitrite unexpectedly revealed synergistic fungicidal, sporicidal, and sclerotia-killing activity compared to copper and/or zinc ions alone or phosphorous acid alone in combination with sodium nitrite. Importantly, these compositions in combination with sodium nitrite were not phytotoxic at sporicidal/sclerotia-killing concentrations. Furthermore, these compositions, when combined with sodium nitrite, advantageously exhibit both short-term (up to approximately 6 hours) sporicidal/sclerotia-killing activity and long-term (days to weeks) fungicidal and plant defense-inducing effects. Furthermore, these compositions can be prepared and applied in a safe, cost-effective, and environmentally friendly manner. The present invention describes such compositions.

Rhizoctonia solani (R. solani) is a widespread, soil-borne plant pathogen that causes root rot and blackleg in many plant species, and rhizoctonia in potato tubers. R. solani forms melanin-rich sclerotia that can survive in soil for long periods and act as a source of primary infection in newly planted crops. When determining the effects of compositions containing copper, zinc, and phosphorous acid in fungal growth inhibition assays with R. solani, which were typically tested after 1 to 2 days of cultivation, the inventor noted that in plates left for more than a week, sclerotia formation and/or development was inhibited at concentrations of some compositions that only partially inhibited R. solani growth after 1 to 2 days of cultivation. Such partially inhibited cultures typically confluent after 2 to 4 days of culture, at which point, when nutrients become limiting, the fungus begins to form sclerotia, which first appear as white spots or bodies and then become melanized, mature black structures that remain viable for many months, even in culture plates that have dried out.

This unexpected observation was further investigated, and the results presented here demonstrate that the presence of phosphorous acid in the formulations inhibited sclerotia formation. Further experiments unexpectedly revealed that widely used agrochemicals such as phosphorous acid, its salts, such as potassium phosphite, and the herbicide glyphosate (2-[(phosphonomethyl)amino]acetic acid), all of which contain a phosphite or phosphonate (PO ₃ ^3- ) group, are potent inhibitors of sclerotia formation.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be described with reference to the following drawings, in which like numerals refer to like elements and in which:

Fig. 1 shows the structures of selected chemical reagents: phosphites and phosphonates (PO ₃ ^3- ).

Fig. 2 is a graph showing the sclerotia-killing effect of Cu-Zn#31 alone and in combination with sodium nitrite (NaNO ₂ ).

CuZn№31 was used at a 1% stock solution with 1 to 100 millimolar sodium nitrite solutions. Immature (white) and mature (Mack) R. solani sclerotia were treated for 10 minutes, and subsequent fungal growth on PDA was assessed after 2 days of cultivation. NG = no growth.

Fig. 3 is a graph showing the effects of phosphorous acid (PA), glyphosate (GP) and aminomethylphosphonic acid (AMPA) on the formation of black sclerotia of Rhizoctonia solani.

These results represent the mean ± S.D. of three separate experiments (PK and GF) and two separate experiments (AMPK).

The present invention will be described in the context of a preferred embodiment, but it should be understood that there is no intention to limit the invention to the embodiment described. On the contrary, there is an intention to cover all alternatives, modifications and equivalents that may be included within the spirit and scope of the present invention as defined in the said description and accompanying drawings.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided an antimicrobial, sporicidal and sclerotia-killing composition comprising an aqueous solution containing copper and/or zinc ions, optionally a basic salt and phosphorous acid in combination with a nitrous acid salt; wherein in a preferred embodiment of the invention, the nitrous acid salt is sodium nitrite. The present invention includes, in some embodiments of the invention, chemical reagents containing a phosphite or phosphonate group (PO ₃ ^3- ), which can inhibit the formation of sclerotia at concentrations that are easily achievable for agricultural applications. The compositions of the present invention inhibit the formation of sclerotia by fungi and can therefore be used in field practice to prevent the regeneration and development of pathogenic fungi.

The above paragraph has been provided as an introduction and is not intended to limit the scope of the present invention as described in the present description and accompanying drawings and claims.

THE BEST OPTION FOR IMPLEMENTING THE INVENTION

The present invention and various embodiments of the invention described and presented herein include compositions, formulations, methods and uses of compositions having previously unknown synergistic antimicrobial activity, containing copper ions and/or zinc ions with phosphorous acid in combination with salts of nitrous acid, such as sodium nitrite. The present invention will be described by way of example and not by way of limitation. Modifications, improvements and additions to the invention described in this application can be determined upon reading the present description and viewing the accompanying drawings; and such modifications, improvements and additions are considered to be included within the spirit and broad scope of the claims according to the present invention and in its various embodiments described or provided in this application.

The term antimicrobial as used in this application includes antibacterial, fungicidal, anti-oomycetous and anti-pathogenic, sporicidal and sclerotiacidal.

The antimicrobial, sporicidal, and sclerotia-killing activity of the described compositions is unexpectedly more potent than that of copper ions with or without zinc ions alone, or than that of phosphorous acid alone in combination with sodium nitrite. Importantly, these compositions are also non-phytotoxic at effective antimicrobial, sporicidal, and sclerotia-killing concentrations. Because the compositions contain copper and/or zinc ions and phosphorous acid, they also remain effective as antimicrobial and plant defense-promoting chemicals after the sporicidal/sclerotia-killing reaction with sodium nitrite is complete (approximately 6 hours).

One embodiment of the present invention relates to unexpectedly synergistic inhibition of plant pathogenic fungi growth and sporicidal and sclerotium killing activity of compositions containing copper ions with or without zinc ions and phosphorous acid in combination with a nitrous acid salt, compared to the fungicidal and sporicidal activity of copper ion solutions with or without zinc ions taken alone, or with phosphorous acid taken alone in combination with a nitrous acid salt, preferably sodium nitrite.

In another embodiment of the present invention, it is shown that the aforementioned compositions containing copper salts, with or without zinc salts, and phosphorous acid are unexpectedly potent inhibitors of sclerotia formation by fungi. In fact, it has been additionally and unexpectedly shown that various chemical reagents containing a phosphite or phosphonate group (PO ₃ ^3- ) can inhibit sclerotia formation at concentrations that are readily achievable for agricultural applications. Chemical reagents in particular include the fungicides potassium phosphite, phosphorous acid, and Aliette, and the herbicide glyphosate, all of which are widely used agrochemicals.

Compositions containing copper ions, with or without zinc ions, and phosphorous acid are conveniently prepared according to the general method described below. The disclosed embodiments of the present invention serve as examples of certain preferred compositions; however, such examples are not intended to limit the scope of the present invention. As will be apparent to those skilled in the art, numerous changes and modifications can be made without departing from the spirit and broad scope of the present invention.

The antimicrobial compositions of the present invention comprise aqueous solutions of copper ions that are obtained from copper oxychloride, two forms of which are: (1) Technical copper oxychloride product (CuOCl-TP, CuOCl-TP) contains approximately 57% elemental copper and 1% suspension in water that has a pH of 6.2 (2) Wettable copper oxychloride powder (CuOCl-WP, CuOCl-SP) contains approximately 50% elemental copper and 1% suspension in water that has a pH of 8.3. CuOCl-SP (CuOCl-WP) contains approximately 10% bentonite and a surfactant to increase the "wettability" of the water-insoluble copper oxychloride so that it remains in suspension longer than CuOCl-TP (CuOCl-TP). Copper ions in the compositions may also be obtained from copper sulfate pentahydrate either mixed with CuOCl during the preparation of the composition or prepared separately as a composition and later mixed together with the CuOCl-based composition.

Copper ions in the compositions can be with or without zinc ions and with phosphorous acid, with the addition of a base if necessary to adjust the acidity of the compositions. In this regard, the use of sodium hydroxide or potassium hydroxide sometimes led to instability (turbidity, crystallization) of compositions obtained from CuOCl-TP or copper sulfate, but not compositions obtained from CuOCl-WP. Surprisingly, it was found that ammonium hydroxide can be used with all three copper compounds to adjust the acidity of the compositions without problems with turbidity or crystallization, and therefore it is the preferred hydroxide for use in the compositions. Some observations in a series of such copper-based compositions and copper-zinc-based compositions are presented in Table 1. The results show, for example, that copper sulfate is more preferable than copper chloride for obtaining stable compositions. The results also unexpectedly show that CuOCl-WP is preferable to CuOCl-TP for the preparation of copper-only composites due to the latter's instability (crystallization). However, it was unexpectedly discovered that the presence of zinc ions prevents instability (crystallization) in composites obtained from CuOCl-TP.

Table 1. Stability observations of various copper and copper-zinc compositions with phosphorous acid (H ₃ PO ₃ ) with or without the addition of ammonium hydroxide (NH ₄ OH), prepared and stored at room temperature (22°C). In the table, gram or grams are abbreviated as g, milliliter or milliliters are abbreviated as ml, and deciliter is abbreviated as dl.

Composition H ₃ PO ₃ at 17.5 g/dl
NH ₄ OH (56.6% solution) at 2 ml/dl Copper Elemental copper at a concentration of 2.5 g/dL Copper and Zinc Elemental copper and elemental zinc, both at 1.25 g/dL Conclusions CuSO ₄ ⋅5H ₂ O ± ZnSO ₄ ⋅7H ₂ O Stable blue solution ± addition of ammonium hydroxide. Stable blue solution ± ammonium hydroxide. Copper sulfate based compositions are preferred over copper chloride based compositions. CuCl ₂ ⋅2H ₂ O ± ZnSO ₄ ⋅7H ₂ O Crystallization begins within a few hours ± addition of ammonium hydroxide. Signs of crystallization after a few days ±
addition of ammonium hydroxide. CuOCl-TP*
CuOCl-TP* (CuOCl-TP*) ± ZnSO ₄ ⋅7H ₂ O
*Technical product Turbid blue solution with crystallization after several days ± addition of ammonium hydroxide. A slightly cloudy blue solution without the addition of ammonium hydroxide, but without crystallization in
Anyway. CuOCl-WP, but not CuOCl-TP, can be used to obtain stable copper-only composites.
Stable CuZn compositions can be obtained from any product
CuOCl. CuOCl-WP*
CuOCl-SP* (CuOCl-WP*) ± ZnSO ₄ ⋅7H ₂ O *Wettable powder Clear green solution. Bentonite precipitates more effectively when ammonium hydroxide is added at low temperatures, such as 37°C. Clear green solution.
Bentonite does not precipitate with or without the addition of ammonium hydroxide.

The antimicrobial compositions of the present invention, and in particular the sporicidal and sclerotia-killing compositions of the present invention, comprise (i) aqueous solutions of copper ions with or without zinc ions and phosphorous acid with the addition, if necessary, of a base such as sodium hydroxide, potassium hydroxide and preferably ammonium hydroxide, and (ii) in combination with a salt of nitrous acid, preferably sodium nitrite.

In one embodiment of the present invention, the antimicrobial composition comprises a solution containing copper oxychloride or copper sulfate pentahydrate with or without a zinc salt (preferably zinc sulfate), dissolved in water, followed by the addition of phosphorous acid to the solution, with the addition, if necessary, of a base (preferably ammonium hydroxide) to adjust the pH of the acidic copper or copper-zinc compositions thus formed. Such solutions are designated, for example, Cu-31 or CuZn-43-N and Cu-32-N (N = with the addition of NH ₄ OH, see Table 2).

In one embodiment of the present invention, the antimicrobial composition comprises a suspension containing copper oxychloride-WP mixed in water with the addition of ammonium hydroxide before the addition of phosphorous acid, which promotes the precipitation of bentonite at low temperatures (20°C to 50°C (see EXAMPLE below for more information)).

Copper oxychloride-based compositions are conveniently prepared using CuOCl-WP, which contains copper oxychloride, bentonite, and surfactant(s). It was found that the addition of phosphorous acid (i) to solubilized CuOCl-WP to form a stable solution and (ii) to a suspension of CuOCl-WP followed by incubation at 70°C to 90°C for 15 to 30 minutes promoted rapid precipitation of the bentonite component, leaving a clear dark green solution with or without the addition of ammonium hydroxide.

The following example describes a general protocol for the preparation of a 2X acid-solubilized copper oxychloride stock composition (Cu-32-2X), which can be used in turn to prepare compositions such as Cu-32 and Cu-Zn-32. Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich Company Ltd., The old Brickyard, New Road, Gillingham, Dorset SP8 4XT, UK. Unless otherwise stated, Hortiphyte was purchased from Hortifeeds, Park Farm, Park Farm Road, Kettlethorpe, Lincoln LN1 2LD, UK. Aliette (Fosethyl-aluminum) and glyphosate (2-[(phosphonomethyl)amino]acetic acid) were purchased from Bayer CropScience, 230 Cambridge Science Park Milton Road, Cambridge CB4 0WB, UK. Aminomethylphosphonic acid was purchased from Fisher Scientific UK, Bishop Meadows Road, LE115RG, Loughborough, Leicestershire, UK. Potassium phosphite was purchased from Wuhan Rison Trading Ltd., 498 Jianshe Ave, Wuhan, China. Copper oxychloride 50-wettable powder (CuOCl-WP) and copper oxychloride-technical product (CuOCl-TP) were purchased from Manica S.P.A., 4 Via all'Adige, 38068 Rovereto, Italy.

10.0 grams of CuOCl-WP (containing 50% elemental copper) are added to 70 milliliters of distilled water in a glass beaker; a magnetic stirrer and stir bar are used to mix and form a greenish-blue suspension. If bentonite precipitates at low temperatures (20°C to 50°C), adding 8.0 milliliters of ammonium hydroxide (56.6% solution) at this point to the CuOCl-WP suspension will unexpectedly accelerate the precipitation of bentonite. 35.0 grams of phosphorous acid is added gradually, which solubilizes CuOCl-WP to form a dark green solution, which is stirred for another 5 minutes, and then the solution is made up to 90 milliliters with distilled water. The solution is then heated, for example, to 37°C for 24 hours or until bentonite of the CuOCl-WP composition precipitates. The bentonite is removed by centrifugation, followed by decanting the soluble composition. If bentonite requires precipitation at high temperatures (e.g., from 60°C to 90°C), the pH of the acidic solution can be adjusted at this point by adding ammonium hydroxide (from 1 to 80 milliliters/liter), and the total volume can then be finally brought to 100 milliliters with distilled water.

This Cu-32-2X concentrate can be mixed with other compounds to conveniently produce a range of other products. Thus, to obtain Cu-32, 2X concentrate is mixed 1:1 with distilled water. To obtain CuZn-32, equal volumes of Cu-32 and Zn-32 (see Table 2) are combined with stirring.

It should be noted that CuOCl-TP cannot be used to prepare stable copper-only compositions by adding phosphorous acid and, if necessary, neutralizing with ammonium base, since such compositions prepared from CuOCl-TP (with or without the addition of ammonium hydroxide) crystallize within a few days (see Table 1). However, copper-zinc-based compositions prepared by suspending CuOCl-TP and a zinc salt (preferably zinc sulfate) in water followed by the addition of phosphorous acid (with or without the addition of ammonium hydroxide) are unexpectedly stable compositions that show essentially no signs of crystallization even after several weeks and do not require time and/or heating for bentonite precipitation, as is the case with the CuOCl-WP-based compositions described above (see Table 1).

Compositions containing copper sulfate are preferably prepared with the addition of ammonium hydroxide.

In addition, copper-based compositions and copper-zinc-based compositions prepared from copper oxychloride (-WP or -TP) can be combined with copper-based compositions and copper-zinc-based compositions prepared from copper sulfate in ratios ranging from 1:1 to 1:100 without any noticeable change in antimicrobial activity, as illustrated by the various compositions prepared by combining Cu-31 and Cu-32 in different ratios, as shown in Table 2.

For copper-based compositions and copper-zinc-based compositions, it is preferred that the total concentration of elemental copper and zinc in the compositions be in the order of 1 to 50 grams/liter.

In copper and zinc based compositions, the concentration ratio of elemental copper to elemental zinc may be in the order of 100:1 to 1:100, preferably 10:1 to 1:10, and even more preferably 1:1.

If the concentration of the base solution (preferably ammonium hydroxide) is 14.5 mol/liter, it is added to the compositions in the order of 1 to 80 milliliters/liter.

The acid selected for the compositions is phosphorous acid. Preferably, the phosphorous acid concentration in the compositions should be between 40 and 800 grams per liter.

Distilled or deionized water is the preferred solvent phase for the formulations.

Examples of other copper oxychloride-based compositions according to the protocols described above are shown in Table 2. In some embodiments of the present invention, copper and copper-zinc compositions obtained from copper oxychloride and copper sulfate may be combined in ratios ranging from 1:100 to 100:1. Because copper oxychloride is typically significantly more expensive than copper sulfate and the protocol for preparing the corresponding composition is more complex, such combinations may be beneficial not only for their practical applications but also for their cost effectiveness.

For examples of other phosphorous acid solubilized CuNi, Cu-ZnNi, and ZnNi compositions according to the specified protocol, see Table 2. In the Table, grams or grams are abbreviated as g, milliliter or milliliters are abbreviated as ml.

Table 2. Examples of compositions and their components, and their 50% inhibitory concentration (IC ₅₀ ) on the growth of Rhizoctonia solani during cultivation.

Ingredients added per deciliter of distilled water

Composition CuSO4⋅5H ₂ O (CS), CuOCl-SP (SP), CuOCl-TP (TP) ZnSO ₄ ⋅7H ₂ O H3PO3 NH ₄ OH 56.6% solution (if necessary) IC50 (% of initial solution) on the growth of R. solani*** Cu-31 CS 10.0 g - 17.5 g - 0.15 ^& Cu-32-2X SP 10.0 g 35.0 g (8.0 ml) - Cu-32* SP 5.0 g - 17.5 g - 0.19 ^& Cu-32-N* SP 5.0 g - 17.5 g 2.0 ml 0.17 Cu-32-PO4 SP 5.0 g - 20.6 ml H ₃ PO ₄ (85% solution) 2.0 ml 0.15 Cu-31:Cu-32 1:1 mixture ^& Cu-31:Cu-32 10:1 mixture Cu-31:Cu-32 100:1 mixture Cu-31:CuZn-32-T mixture Cu-31:CuZn-32-TA mixture - - - 0.17 ^& 0.16 0.15 0.17 0.15 Zn-32 - 11.0 g 17.5 g (2.0 ml) 0.19 CuZn-32 SP 5.0 g 5.5 g 17.5 g (2.0 ml) 0.17 Cu-32-T TP 4.4 g - 17.5 g 2.0 ml 0.16 CuZn-32-T TP 2.2 g 2.8 g 17.5 g (2.0 ml) 0.17 CuZn-32- TA TP 2.2 g Zinc acetate dihydrate 3.6 g 17.5 g (2.0 ml) 0.17 CuZn-32-TN TP 2.2 g Zinc nitrate hexahydrate 2.8 g 17.5 g (2.0 ml) 0.15 Cu-36 SP 5.0 g - 5.0 ml
H ₂ SO ₄
(18.7M) 2.0 ml 0.26 CuZn-46-N CS 2.5 g 2.75 g 17.5 g 4.0 ml 0.28 CuZn-47-N CS 10.0 g 11.0 g 80.0 g 8.0 ml 0.14

Composition CuSO ₄ ⋅5H ₂ O ZnSO⋅7H ₂ O H ₃ PO ₃ NH ₄ OH (19% or 56.6% solution) Cu№28** CS 16.0 g - 4.0 g 4.0 ml (19%) 0.18 Cu№31 CS 10.0 g - 17.5 g 2.0 ml (56.6%) 0.15 CuZn№12 CS 8.0 g 8.8 g 4.0 g 4.0 ml (19%) 0.14 ± 0.02 ^$
Mean ± SD, n=6 Zn№4 - 17.6 g 4.0 g 4.0 ml (19%) 0.17 CuZn№31 CS 5.0 g 5.5 g 17.5 g 2.0 ml (56.6%) 0.20 CuOCl-SP SP 5.0 g - - - 2.0 CuOCl-TP TP 4.4 g - - - 1.8 _CuSO4 CS 16.0 g - - - 0.39 ZnSO ₄ - 17.6 g - - 0.33 H ₃ PO ₃ - - 4.0 g - 0.64 ⁺

* In compositions that did not contain a basic ammonium salt (e.g., Cu-32) or ammonium hydroxide, they were added, if necessary, after phosphorous acid to increase the pH of the composition (e.g., Cu-32-N). **Compositions designated with No. were obtained as insoluble metal-ammonium complexes that were solubilized with phosphorous acid. ***Concentration of compositions required to inhibit the growth of R. solani by 50% (IC ₅₀ ) on potato dextrose agar after 24 hours of cultivation at 22°C. ^& The 1:1 mixture contains a total of 2.5 g/dL elemental copper, 1.25 g/dL from CuSO4∙5H2O and CuOCl-WP composites, and has the expected IC ₅₀ against R. solani based on the Cu-31 and Cu-32 results, as do the 10:1 and 100:1 Cu-31:Cu-32 mixtures. ^s CuZnNo. 12 was used as the internal standard in most experiments. + Equivalent concentrations in CuNo. 28, ZnNo. 4, and CuZnNo. 12.

It is contemplated that for practical application in agricultural applications as a foliar spray, for example, the stock solution of the composition would be added to water with stirring or agitation until dilution is achieved, for example, by 100-1000 times. An appropriate amount of sodium nitrite stock solution, for example, 690 grams/liter (10 M), would then be added with continuous stirring or agitation until dilution is achieved, for example, by 200 times, to obtain a final concentration of 50 mM in the combined product. An appropriate auxiliary, adhesive, adjuvant, carrier, surfactant, or filler can then be added at an appropriate concentration to the effective amount of the combined product.

In order that the present invention may be illustrated, more readily appreciated and more readily carried out by those skilled in the art, embodiments of the invention will now be presented by way of non-limiting examples only and described with reference to the accompanying drawings.

EXAMPLE 1. Analysis of fungal growth inhibition using compositions.

Fungal growth inhibition assay. Rhizoctonia solani (R. solani) was isolated from potato peel sclerotia and maintained on potato dextrose agar (PDA) at room temperature (22°C). To determine the effect of the compounds on fungal growth, 10 microliters of the compounds diluted in sterile distilled water were placed in the wells of a 12-well tissue culture plate, and 1 milliliter of PDA was pipetted into each well. The plate was vortexed to evenly distribute the compound throughout the agar (distilled water alone served as a control), and the agar was then allowed to solidify. Agar inserts containing fungal hyphae (3 × 3 mm) from the established fungal culture were excised with a scalpel and inserted into holes cut in the center of the agar in each well of a 12-well plate, which were then cultured at room temperature. To determine the effect of the compositions on fungal radial growth, the fungal hyphal diameter was measured twice at a 90° angle using a ruler, and the average diameter in millimeters was calculated. The concentrations of the compositions required to inhibit R. solani radial growth by 50% (IC ₅₀ ) were determined graphically.

Results:

As shown in Table 2, the various Cu, CuZn, CuNa, and Cu-ZnNa compositions all had _IC50 values ranging from 0.14% to 0.28% of the stock solution (approximately 700 to 350 times the dilution of the stock solutions, respectively), indicating that the method by which the compositions were prepared and the copper source did not significantly affect the ability of the compositions to inhibit the growth of the plant pathogenic fungus R. solani in culture. Furthermore, copper (and CuZn) compositions prepared from copper oxychloride and copper sulfate can be mixed together over a wide range of concentrations and still maintain full fungicidal activity.

It should be noted that the compositions were more active than the components from which they were prepared. CuOCl-WP, CuOCl-TP, copper sulfate, zinc sulfate and phosphorous acid had IC ₅₀ values of 2.0%, 1.8%, 0.39%, 0.33% and 0.64% respectively against R. solani, so it is a fact that all compositions had significantly lower IC ₅₀ values (approximately 0.15%) than would be expected from combining the copper composition and phosphorous acid; this indicates the synergism of the components when combined in the described compositions. Such synergism is particularly noticeable with the copper oxychloride-derived compositions, as CuOCl-WP and -TP had particularly low IC ₅₀ values of 2.0 and 1.8%, respectively, and phosphorous acid had an IC ₅₀ of 0.64%, yet Cu-32 and Cu-32-T had IC ₅₀ values of 0.19% and 0.17%, respectively. Taken together, these results demonstrating the synergistic activity of the constituents when combined in compositions demonstrated unexpectedly enhanced antimicrobial activity of such compositions prepared using the protocols described above in the "Best Mode for Carrying Out the Invention" section.

It should also be noted that copper-zinc (CuZn) formulations have specific advantages: (i) Commercially available copper-based fungicides (e.g., Bordeaux mixture, copper oxychloride, copper hydroxide, and copper oxide) typically contain approximately 2.5 grams/liter of elemental copper when diluted, for example, for foliar sprays. The copper salt in such products is largely insoluble and exists in suspension, causing the products to adhere to plant leaves; when it rains and the product on the leaves becomes wet, a small amount of active ionic copper is released, which has antimicrobial activity. A negative side effect of rain is that it washes the copper suspension from the leaves to the ground, so frequent applications of these products are required.

Most of the copper-only formulations described in Table 2 contained 25 grams/liter of elemental copper (4.4, 5, and 10 grams/deciliter of CuOCl-TP, CuOCl-WP, and copper sulfate, respectively) in 100X stock solutions, so a 1:100 dilution of foliar spray containing the formulation would contain 0.25 grams/liter of elemental copper, which is 10 times less than typical commercial products. The presence of ionic copper (and zinc) in the formulations (in addition to the synergistic antimicrobial action with phosphorous acid) partly explains why they were so much more active (10- to 15-fold) in inhibiting the growth of R. solani compared to CuOCl-WP and -TP suspension products, even in PDA culture media in the presence of water (see Table 2). Furthermore, suspensions of existing copper-based products tend to precipitate and can block spray equipment, whereas the described formulations are ionic solutions that are more suitable for use with spray equipment.

Agricultural crops such as grapevines and potatoes are typically sprayed 10 to 15 times per season, whereas the Cu or CuZn composition described in this application, applied as a spray treatment, would contain approximately the same amount of copper per treatment over the entire season as a single application of a commercially available copper-based product. CuZn compositions are typically formulated with only 50% of the amount of copper contained in comparable copper-only compositions (e.g., see Cu#28 and CuZn#12 in Table 2), yet they are comparable in activity as fungicidal compositions; this means that the copper concentration is reduced to 1/20th that of commercial copper-based products. It is well known that many commercial copper-based fungicides (insoluble, non-ionic copper) must be applied frequently to be effective, and this results in rain or irrigation washing large amounts of potentially toxic copper into the soil.

Cultivated agricultural soils (where copper-based fungicides are not applied) are often deficient in micronutrients such as copper and especially zinc (Sillinpaa. M. (1982) Micronutrients and the nutrient status of soils: A global study. FAO Soils Bulletin, No. 48. FAO, Rome, Italy), and therefore the CuZn compositions described in this application could provide effective nutritional amounts of such micronutrients when applied to plants.

EXAMPLE 2. Fungicidal tests with compositions and sodium nitrite.

Fungicidal assay. Agar inserts containing fungal hyphae (3 x 3 mm) were excised from Magnaporthe oryzae (M oryzae) cultured on potato dextrose agar (FDA) and placed into wells of a 96-well plate containing 75 microliters of varying concentrations of sodium nitrite or sterile distilled water (DW). To initiate the reaction, 75 microliters of the compositions (diluted in DW) were added to the wells. The plate was gently shaken and incubated at room temperature for 30 minutes, after which the liquid was removed, and 150 microliters of CM-1 medium were added for 5 minutes to stop the reaction. The CM-1 medium was removed, and agar inserts were inserted into holes cut in the PDA in 9-centimeter-diameter Petri dishes (6 to 7 agar inserts per Petri dish). The dishes were then cultured at room temperature for 4 days. Fungal growth was then measured (twice at a 90° angle using a ruler), and the average diameter in millimeters was calculated. If fungal growth was not detectable by eye, the cultures were examined using phase microscopy (40X) to confirm no growth (NG), as determined by visual inspection.

Results:

The results in Table 3 show that in the absence of sodium nitrite (NaNO ₂ ), CuZnNo. 12 and its components, copper sulfate-zinc sulfate (CuZn), and phosphorous acid (H ₃ PO ₃ ) at the highest concentration tested (1% of the stock solution) were not fungicidal for M. oryzae. However, in the presence of 10 mM sodium nitrite, all three formulations showed a concentration-dependent increase in fungicidal activity after only 30 minutes of exposure. Clearly, CuZn was slightly less fungicidal than phosphorous acid, and that both were significantly less active than CuZnNo. 12. The result with phosphorous acid was unexpected, since the formation of nitrogen oxides from NaNO ₂ is pH-dependent, and phosphorous acid has a lower pH than CuZnNo. 12 (Table 3). However, this unexpected result was explained when the synergism between CuZn and phosphorous acid was calculated.

To determine whether the combined activity of CuZn and phosphorous acid in the form of CuZnNo. 12 in the presence or absence of sodium nitrite was synergistic, the following formula was used:

Sf=M / [A+0.01 x B (100 – A)]

Where: Sf is the synergism coefficient, A is the percentage influence of CuZn; B is the percentage influence of phosphorous acid; M is the percentage influence of the mixture, CuZn№12. If Sf>1, then synergism existed. (Ref: Samoucha Y and Cohen Y (1984). Synergy between Metalaxyl and Mancozeb in controlling downy mildew in cucumbers. Phytopathology 74:1434-1437).

When the synergism coefficient values were calculated (Table 3), clear synergism was observed at concentrations of the composition of 0.3% (Sf=1.37) and 1% (Sf=1.57) in combination with 10 mM sodium nitrite.

Therefore, these results indicate that the combination of CuZn and phosphorous acid in the form of CuZn#12 composition acts synergistically in combination with sodium nitrite to produce unexpectedly greater than expected antimicrobial/fungicidal activity.

Table 3. Fungicidal effects of _the CuZn№12 composition and its components: copper-zinc (CuZn) component and phosphorous acid ( _H3PO3 ) at equivalent concentrations against M. oryzae in the presence and absence of 10 mMolar sodium nitrite. NG indicates no growth. The control growth of fungi with distilled water (DW) was 20 mm. The percentage of control values in parentheses was used to calculate the synergism coefficient (Sf; a value >1.0 means synergism), as described in the text.

M. oryzae mushroom growth (mm) with composition NaNO ₂ (mM) Concentration of the composition CuZn H ₃ PO ₃ CuZn№12 Sf 0 1% 20 21 20 1 10 0.1% 20 20 20 1 10 0.3% 17 (A=15%) 13 (B=38%) 7 (M=65%) 1.37 10 1% 14 (A=30%) 11 (B=48%) NG (M=100%) 1.57 pH of 1% solution in active ingredient 4.91 2.12 2.48

EXAMPLE 3. Sporicidal tests with compositions and sodium nitrite.

Sporicidal assays: (1) Spores of Bolrylis cinerea (B. cinerea, also known as gray mold) and Fusarium oxysporum f. sp. cubense (F. oxysporum, causes "Panama disease" of banana) were isolated from mature cultures grown on potato dextrose agar (PDA) in 9-centimeter Petri dishes by gentle washing with sterile distilled water, DW. The spore-containing solution was passed through a 40-micron filter to remove debris, and the filtrate was adjusted to 2 x 10 ⁶ (B. cinerea) or 5 x 10 ⁶ (F. oxysporum) spores/milliliter DW. Ten microliters of sodium nitrite samples (dissolved in sterile DW) or DW were added to the wells of a 48-well tissue culture plate containing 10 microliters of spore solutions. To initiate the reaction, the compounds were added (time = 0), and the reagents were mixed thoroughly by gently shaking the plate. After 30 minutes of incubation at room temperature, the reaction was stopped by adding 1 milliliter of PDA at 60°C. The plates were then cultured at room temperature for 3 to 4 days, at which time fungal growth was assessed visually. Fungal growth was scored as follows: fungal growth equivalent to the control cultures (with DW) was scored as 2. If fungal growth was clearly less than the control, it was scored as 1. No fungal growth was scored as 0. When PDA was added immediately after the formulation (within 2 seconds), no fungal growth was observed regardless of the concentrations of sodium nitrite (up to 100 millimoles/liter) or the formulation (up to 1% dilution of the stock solution), indicating that PDA effectively inhibits the reaction even at the highest concentrations of _NaNO2 (100 mM) and CuZnNo2 (1%) used in the experiments.

(2) To determine the sporicidal activity of the individual formulations, B. cinerea spores (1 x 10 ⁴ ) were exposed to various concentrations of the formulations or DW at 22°C in microcentrifuge tubes. After 1 hour of exposure, the spores were washed twice with 1 ml of DW, then suspended in PDA and incubated for 6 days, and fungal growth was assessed as described in Table 4.

Results:

The results in Table 4 show that the CuZn#12, Cu#28, and Zn#4 compositions were slightly sporicidal at 10% stock solution, but not at lower concentrations, while the Cu-32 composition showed slight sporicidal activity at 10% and 5% stock solution, but not at lower concentrations. The greater sporicidal activity of Cu#32 can be explained by the higher concentration of phosphorous acid (17.5 grams/deciliter) compared to the other 3 compositions (4 grams/deciliter, see Table 2).

Table 4. Sporicidal activity of compositions against B. cinerea spores after 1 hour of incubation at 22°C in the absence of sodium nitrite. Spores exposed to distilled water for 1 hour served as controls*. Fungal growth was assessed 6 days after the treated spores were suspended in potato dextrose agar.

Mushroom growth
2*=Control growth; 1=Reduced growth; 0=No growth % of the original solution Cu№28 Zn№4 CuZn№12 Cu-32 10 1 1 1 0 5 2 2 2 1 2 2 2 2 2 1 2 2 2 2

2. The sporicidal activity of the four formulations tested in the experiment, shown in Table 4, was relatively weak given the high concentrations applied (the formulations are designed for application at <1% of the initial solution). However, since the formulations are acidic due to the presence of phosphorous acid, it was expected that they might exhibit enhanced sporicidal activity when combined with sodium nitrite, as the subsequent reaction would form nitrous acid, which releases highly reactive nitrogen oxides, which are known to be sporicidal.

The results in Table 5 show that sodium nitrite alone (NaNO ₂ + DW) and all formulations alone (NaNO ₂ = 0) had no sporicidal activity against B. cinerea spores at any concentration tested (all received a 2 = control growth). Copper sulfate (Cu), zinc sulfate (Zn), and copper sulfate with zinc sulfate (CuZn), taken alone, showed sporicidal activity in the presence of 100 mM sodium nitrite, while phosphorous acid was active at 1% (equivalent to its concentration in the 3 formulations at 1%) but showed little activity at lower concentrations. All 3 formulations showed a concentration-dependent increase in sporicidal activity with sodium nitrite. The most active formulation was Cu#28, followed by CuZn#12 and Zn#4. The sporicidal activity of CuZn#12 was greater than expected from its Cu-Zn components and phosphorous acid (as discussed in the fungicidal experiment - see Table 3), despite the latter having a lower pH than CuZn#12, both in the presence and absence of 100 mM sodium nitrite. Since Cu#28 was significantly more sporicidal than CuZn#12 and Zn#4, despite the pH values of these three compositions being the same, it turned out that the presence of copper in the Cu#28 and CuZn#12 compositions (the latter was less active than Cu#28, apparently due to the presence of only half the concentration of copper ions - see Table 2) together with phosphorous acid resulted in an unexpected enhancement of sporicidal activity in the presence of sodium nitrite, which is unrelated to simple pH aspects.

Time-dependent experiments showed (i) that effective sporicidal activity was obtained within 1 minute after mixing 100 millimolar sodium nitrite and CuZnNo.12 (1% stock solution) and (ii) that sporicidal activity was exhausted approximately 6 hours after mixing the reactants (data not shown).

Experiments with various formulations and sodium nitrate or urea showed no evidence of sporicidal activity against B. cinerea spores (data not shown), so sodium nitrite is the preferred compound for obtaining sporicidal activity in combination with the formulations.

Table 5. Sporicidal activity of three compositions and their components: copper sulfate (Cu), zinc sulfate (Zn), copper sulfate and zinc sulfate (CuZn), and phosphorous acid ( _H3PO3 ) with and without sodium nitrite ( _NaNO2 ) against B. cinerea spores. The pH _values of selected 1% solutions with and without 100 millimolar sodium nitrite are also shown.

The results in Table 6 show that copper sulfate and copper oxychloride-SP(-WP) had no sporicidal activity when combined with sodium nitrite at concentrations up to 30 millimolar/liter (only 100 millimolar sodium nitrite was sporicidal against F. oxysporum spores, data not shown). The two formulations tested, Cu-31 and Cu-32, contain copper sulfate and CuOCl-SP (CuOCl-WP), respectively, as their copper salt with 17.5 grams/deciliter phosphorous acid (Table 2), and both formulations showed synergistic sporicidal activity when combined with sodium nitrite compared to their components taken separately in combination with sodium nitrite at equivalent concentrations.

These results confirm and extend the results presented in Table 5, showing that the two different copper salts, even in simple formulations with phosphorous acid, show synergistically enhanced sporicidal activity compared to their components taken separately in combination with sodium nitrite.

Table 6. Sporicidal activity of two compositions and their components against F. oxysporum spores: copper sulfate (CM (CS), in Cu-31), copper oxychloride-WP (-WP) (CuOCl, in Cu-32) and phosphorous acid (H ₃ PO ₃ ) with sodium nitrite (NaNO ₂ ) and without it against B. cinerea spores.

Fungal growth: 2*=Control growth; 1=Reduced growth; 0=No growth NaNO ₂ mM DV CM CuOCl H ₃ PO ₃ (%) Cu-31 (%) Cu-32 (%) 1% 1% 0.03 0.1 0.3 0.03 0.1 0.3 0.03 0.1 0.3 0 2* 2 2 - - 2 - - 2 - - 2 1 2 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 2 2 1 2 1 0 2 1 0 10 2 2 2 2 2 0 2 0 0 2 0 0 30 2 2 2 2 1 0 2 0 0 1 0 0

Taken together, these results have important implications for the potential use of these sporicidal compositions in field applications. It is well known that acidified sodium nitrite forms nitrous acid, which in turn decomposes into sporicidal nitrogen oxides. However, the results presented in this application demonstrate that copper-based compositions (and copper-zinc-based compositions) containing phosphorous acid unexpectedly exhibit greater sporicidal activity than equivalent concentrations of phosphorous acid alone combined with sodium nitrite.

Furthermore, application of the present sodium nitrite compositions, for example as a foliar spray, would not only provide a sporicidal effect that would kill spores before they are released into the air and thereby reduce the spread of associated fungal infection, but after the sporicidal effect had been exhausted (after approximately 6 hours), there would be continued, persistent antimicrobial and plant defense promoting activity provided by the copper or copper and zinc composition and phosphorous acid remaining on the treated plants.

When a foliar spray mixture containing 1% CuZn#12 and 50 mM sodium nitrite was applied twice daily for 5 days to 10-day-old rice seedlings, there was no significant effect on seedling growth, and no chlorosis was observed compared to control rice seedlings sprayed with water alone. Interestingly, when the same foliar spray was applied to rose bushes whose rose buds were infested with aphids (both green and black), the insects were found dead within 5 minutes of spraying; observation of the sprayed rose bushes over the next several days revealed that neither the plants nor the rose buds were damaged by the spray.

Taken together, these results demonstrate that the composition combined with sodium nitrite was not phytotoxic to rice seedlings or rose bushes, while the spray was effective as an insecticide against aphids. Thus, various embodiments of the present invention include the compositions described herein used as insecticides.

EXAMPLE 4. Analysis of sclerotium-killing activity with compositions and sodium nitrite.

Sclerotium-killing activity assays: Agar inserts containing white immature sclerotia (3 × 3 mm) were excised from R. solani cultured for 6 to 8 days, while mature melanized (black) sclerotia (approximately 10 x 3 mm) were excised from R. solani cultured for 13 days, and the inserts were cut into 2 x 3 mm pieces using a scalpel. Sclerotia samples were then placed in wells of a 96-well plate containing 75 microliters of sodium nitrite at various concentrations or sterile distilled water (DW) as a control. To initiate the reaction, 75 microliters of CuZn#3l (diluted in DW) were added to the wells. The plate was gently shaken and incubated at room temperature for 10 minutes, after which the liquid was carefully removed, and 150 microliters of CM-1 medium was added for 5 minutes to stop the reaction. The CM-1 medium was removed, and agar plugs were carefully inserted into holes cut in the PDA in 9 cm Petri dishes (4 inserts per Petri dish), which were then cultured at room temperature until fungal growth was measured twice at a 90° angle using a ruler, and the average diameter in millimeters was calculated. If fungal growth could not be detected by eye, the cultures were examined using phase microscopy (40X) to confirm no growth (NG), as indicated by visual inspection.

Results:

The results in Fig. 2 show that the combination of CuZn™31 (1%) with sodium nitrite (1 to 100 mmol/L) had sclerotia-killing activity that was concentration-dependent against both immature (white) and mature (black, pigmented) R. solani sclerotia with only 10 minutes of exposure. White sclerotia were more sensitive to the reagent combination than black sclerotia. CuZn™31 (1%+0) and sodium nitrite (0+100), taken alone, had little or no significant sclerotia-killing effect on either white or black sclerotia, as fungal growth was similar to that observed in the controls (0), indicating that nitrogen oxide production was necessary for the observed sclerotia-killing activity.

It should be noted that naturally occurring microsclerotia of Verticillium dahliae (V. dahliae) in potato fields are mostly (~90%) less than 125 micrometres in diameter (Smith VL and Rowe RC. Phytopathology 74: 553-556, 1984), and it is likely that microsclerotia would be more susceptible to the sclerotia-killing action of Cu-ZnNo. 31 in combination with sodium nitrite than the 2 x 3 mm portions of mature sclerotia used in the patent application.

The results in Table 7 show the sclerotia-killing activity of four similar formulations (the components of which are shown in Table 7) against melanized (black) sclerotia of R. solani in the presence and absence of increasing concentrations of sodium nitrite. None of the formulations had sclerotia-killing activity without sodium nitrite (0+1%), and equally, the highest sodium nitrite concentration tested (30+0) had no sclerotia-killing activity. However, all four formulations showed a dose-dependent increase in sclerotia-killing activity with increasing sodium nitrite concentrations, indicating that nitrogen oxide formation was necessary for the observed sclerotia-killing activity. Overall, the four formulations had similar sclerotia-killing activity (measured by the ability of sclerotia to form hyphae when cultured on PDA after treatment), with the two formulations containing copper sulfate being slightly more active than those containing copper oxychloride. All four compositions had complete sclerotium killing activity with 30 mM sodium nitrite, as observed with the CuZn#31 composition (Fig. 2).

Table 7. Sclerotia-killing activity of four compositions with and without sodium nitrite ( _NaNO2 ) against black (melanized) sclerotia of R. solani. Treated sclerotia were cultured on PDA for 2 days, after which radial hyphal growth was measured (in millimeters). The composition components are shown at the bottom of the table.

Average mushroom growth (mm ± SD, n=2)
black sclerotia after treatment NaNO ₂ (mM) + 1% of the composition Cu-31 Cu№31 Cu-32 Cu-32-N Control 39 ± 4 42 ± 3 0+1% 39 ± 2 40 ± 2 30+0 41 ± 2 43 ± 2 1+1% 38 ± 2 37 ± 2 37 ± 4 39 ± 4 3+1% 22 ± 3 26 ± 2 29 ± 3 33 ± 3 10+1% 13 ± 2 16 ± 2 19 ± 3 22 ± 0 30+1% NG NG NG NG Amount of compound in the composition (g/dL or *mL/dL) CuSO ₄ ⋅5H ₂ O** 10 10 - - CuOCl-SP** - - 5 5 NH ₄ OH (30%)* - 2 - 2 Phosphorous acid 17.5 17.5 17.5 17.5

**Both compounds provide 2.5 g/dL elemental copper at the concentrations listed

EXAMPLE 5. Inhibition of sclerotia formation by fungi.

Fungal growth inhibition and sclerotia formation inhibition assay: The Rhizoctonia solani (R. solani) strain used in these experiments was isolated from sclerotia on potato and maintained in culture on potato dextrose agar (PDA) at room temperature (22°C). To determine the effect of the formulations on fungal growth, 10 microliters of the test formulations diluted with sterile distilled water were placed in the wells of a 12-well tissue culture plate, and 1 milliliter of PDA was pipetted into each well. The plate was shaken to evenly distribute the test formulation throughout the agar (distilled water alone was used as a control), and the agar was then allowed to solidify. Agar inserts containing fungal hyphae (3 x 3 mm) from the established fungal culture were excised with a scalpel and inserted into holes cut in the center of the agar in each well of a 12-well plate, which was then cultured at room temperature (22°C). To determine the effect of the compositions on the radial growth of fungi, the diameter of fungal hyphae was measured twice at a 90° angle using a ruler, and the average diameter in millimeters was calculated. If fungal growth could not be detected by eye, the cultures were examined using phase microscopy (40X) to confirm the absence of growth (NG), as indicated by visual inspection.

To determine the effects of the compounds and formulations on sclerotia formation, R. solani cultures were examined daily and the radial growth of the fungi was measured until the cultures confluent, after which the development of white (immature) sclerotia and mature (black pigmented) sclerotia was recorded and the number of mature sclerotia was counted on day 10 of culture.

Results:

The results in Table 8 show that the Cu-32 formulation (containing phosphorous acid) completely inhibited the formation of immature and mature sclerotia in R. solani cultures. Although Cu-32 partially inhibited the radial growth of R. solani on days 1 (and 2) of cultivation, growth was continuous by day 3 of cultivation, but sclerotia formation was completely absent at all three concentrations. The Cu-32-PO4 formulation (containing phosphorous acid) also partially inhibited the growth of R. solani, but once confluent white sclerotia were achieved, black sclerotia formed at the two lower concentrations tested, as was seen in the control cultures (with distilled water). At the highest concentration tested, Cu-32-PO4 was similar to Cu-32 in inhibiting growth and preventing the formation of white and black sclerotia. The Cu-36 formulation (containing sulfuric acid) was the least potent inhibitor of R. solani growth, and sclerotia formation was very similar in time course and quantity to control cultures at all three concentrations tested.

These results clearly demonstrate that Cu-32 was the most potent inhibitor of sclerotia formation, with Cu-32-PO4 having a similar effect at the highest concentration tested, and Cu-36 having little effect on sclerotia formation. Since the three formulations were identical except for the acid used and had similar pH values (Table 8 caption), the results unexpectedly revealed that the presence of phosphorous acid in the formulations was necessary for optimal inhibition of R. solani sclerotia formation.

Table 8. Effect of three compositions, differing only in the acid they contain, on radial growth and sclerotia formation of Rhizoctonia solani.

Control (n=3) Cu-32 (%)* Cu-32-PO ₄ (%)* Cu-36 (%)* 0.05 0.1 0.2 0.05 0.1 0.2 0.05 0.1 0.2 Day 1 growth in mm 19, 19, 19 16 13.5 9 18 14 11 19 19 14 Day of Solid Growth 2, 2, 2 3 3 3 2 3 3 2 2 3 White SCL Day 1 spotted** 3, 3, 4 - - - 4 4 - 4 4 5 Black SCL Day 1 spotted** 5, 5, 5 - - - 5 5 - 5 5 6 Number of black SCL per day 10 38, 48, 31 0 0 0 40 34 0 39 32 28

pH of the composition (diluted 1:1000 with distilled water, equivalent to 0.1% v/v of the original solution): Cu-32=2.69; Cu-32-P04=2.63; Cu-36=2.46.

*Formulations were identical except for the acid used - see Table 1; (%) = percentage of original solution. **White/black SCL = immature/mature (pigmented) sclerotia.

The results in Table 9 show that phosphorous acid and its alkali metal salt, potassium phosphite, both completely inhibited the formation of mature (black, pigmented) sclerotia of R. solani at concentrations of 40 milligrams/liter and higher. At these concentrations, both products were only moderate inhibitors of fungal growth, so the inhibitory effect of phosphite on sclerotia formation occurred despite fungal growth.

Commercial potassium phosphite products are typically applied at concentrations of approximately 4 grams/liter as a foliar spray to stimulate plant defenses against pathogen attack; this concentration is 100 times higher than the concentration required to completely prevent sclerotia formation (40 milligrams/liter) of R. solani in culture.

Table 9. Effect of phosphorous acid and potassium phosphite on radial growth and sclerotia formation of R. solani.

Control (n=2) Phosphorous acid(mg/l) Potassium phosphite (mg/l) 10 20 40 80 160 10 20 40 80 160 Day 1 growth in mm 19, 19 18 18 16 14 11 19 19 19 18 15 Day of Solid Growth 2, 2 2 2 2 3 3 2 2 2 2 3 White SCL Day 1 spotted* 3, 3 3 4 - - - 3 4 - - - Black SCL Day 1 spotted* 5, 5 5 - - - - 5 7 - - - Number of black SCL per day 10 34, 27 17 0 0 0 0 9 2 0 0 0

* White/black SCL = immature/mature (pigmented) sclerotia.

The results in Table 10 show that Hortiphyte and Aliette, commercial products that contain potassium phosphite and Phosethyl-aluminum (phosphonate), respectively, have nearly identical effects on the growth and sclerotium formation of R. solani when adjusted to equivalent concentrations of phosphite as phosphorous acid.

Hortiphyte is sold as a liquid fertilizer, providing nitrogen and potassium, as well as phosphorus in the form of potassium phosphite. Aliette is sold as a fungicide, containing a PO3 _3- ^group in the form of aluminum tris(O-ethylphosphonate).

These results indicate that it is the concentration of the phosphite/phosphonate group (PO ₃ ^3- ), rather than the form in which it is present, that is important for inhibiting R. solani sclerotia formation during culture.

Table 10. Effect of three different forms of phosphite (PO ₃ ^3- ) on radial growth and sclerotia formation of Rhizoctonia solani during cultivation.

Control
(n=2) Phosphorous acid (mg/l)* Hortiphyte (mg/L)* Aliette (mg/L)* 12 40 120 400 12 40 120 400 12 40 120 400 Day 1 growth in mm 19, 19 18 16 10 5 19 17 16 13 15 11 9 7 Day of Solid Growth 2, 2 2 2 4 - 2 2 2 3 2 2 3 4 White SCL Day 1 spotted* 3, 3 3 - - - 3 - - - 3 8 - - Black SCL Day 1 spotted* 6, 6 6 - - - 6 - - - 6 - - - Number of black SCL per day 10 >20,>20 >20 0 0 0 >20 0 0 0 >20 0 0 0

*All formulations were adjusted to contain equal amounts of the phosphite/phosphonate group (PO ₃ ^3- ). Hortiphyte contains potassium phosphite (25%). Aliette is aluminum tris(O-ethylphosphonate) or Fosethyl-aluminum (80% by weight). **White/black SCL = immature/mature (pigmented) sclerotia.

The results in Table 11 show that phosphorous acid was a more potent inhibitor of fungal growth (on day 1 of cultivation) and sclerotia formation (on day 10 of cultivation) than glyphosate or aminomethylphosphonic acid (AMPA), which is a product of microbial degradation of glyphosate in soil. Due to solubility issues, AMPA could only be tested at the maximum concentration of 1 gram/liter, where it was similar in activity against R. solani growth and sclerotia formation to glyphosate but significantly less active than phosphorous acid.

Figure 3 shows the number of black sclerotia formed with phosphorous acid, glyphosate, and AMPA from two to three replicate experiments. It is evident that phosphorous acid was approximately 20 times more active than glyphosate and approximately 30 times more active than AMPA in inhibiting the formation of black sclerotia in R. solani.

Phosphorous acid and phosphite salts are typically applied at 4 g/L as a foliar spray, but as shown in Tables 9 and 11, both products completely inhibited sclerotia formation at 1/100 of the stated concentration (40 milligrams/L). The glyphosate product tested in these experiments is recommended for application as a foliar spray at 8 grams/L (containing 6.8 grams/L active ingredient), but sclerotia formation occurred (albeit at lower levels than the controls) at only 1/3 to 1/10 of the stated concentration.

However, although glyphosate (a herbicide) and phosphite (a fungicide/plant protection promoter) have very different agricultural uses, it is of potential practical importance that either product, when applied alone, can significantly inhibit sclerotia formation at concentrations 10 to 100 times lower, respectively, than those at which they are applied as a foliar spray for the product's primary agricultural application.

In other experiments in which phosphorous acid and glyphosate were combined, there was no synergistic or inhibitory effect of either compound on the other (data not shown); the effects of the combined products were additive and dominant over those of phosphorous acid, as would be expected from the results shown in Table 11 and Fig. 3. Since glyphosate and phosphorous acid and its potassium salt were found to be compatible in solution, a combination of, for example, glyphosate and potassium phosphite could also be used as an effective herbicide/sclerotium formation inhibitor product in field practice, particularly since both products are highly effective when applied to plants as a foliar spray.

Table 11. Effect of phosphorous acid, glyphosate and aminomethylphosphonic acid (AMPA) on radial growth and sclerotia formation of Rhizoctonia solani during cultivation.

Control (n=2) Phosphorous acid (mg/l)* Glyphosate (g/L)* AMPK (g/l) ^$ 12 40 120 400 0.27 0.8 2.7 8 0.1 0.3 1 Day 1 growth in mm 20, 20 20 19 14 6 20 14 8 6 20 19 17 Day of Solid Growth 2, 2 2 2 3 - 2 2 6 - 2 2 2 White SCL Day 1 spotted* 3, 3 3 - - - 3 3 6 - 3 3 5 Black SCL Day 1 spotted* 5, 5 6 - - - 5 6 8 - 5 8 9 Number of black SCL per day 10 >20,>20 >20 0 0 0 >20 8 4 0 >20 >20 5

* Glyphosate is recommended for foliar spray application at 8 g/L, which translates to 6.8 g/L of active ingredient. ^AMPA stands for aminomethylphosphonic acid. **White/black SCL = immature/mature (pigmented) sclerotia.

It is well known that acidification of sodium nitrite results in the formation of reactive, biocidal nitrogen oxides, but the results presented herein clearly demonstrate that the novel copper-based and copper-zinc-based ionic compositions with phosphorous acid unexpectedly have synergistically greater antimicrobial (Table 3), sporicidal (Table 6), and sclerotium-killing (Table 7) activity than equivalent concentrations of phosphorous acid alone in combination with sodium nitrite, even though copper salt solutions or copper and zinc salt solutions alone in combination with sodium nitrite are only weakly antimicrobial (Table 3) or sporicidal (Table 5).

The various copper-based and copper-zinc-based ionic compositions described are effective antimicrobial products, as demonstrated by their ability to inhibit the growth of R. solani with IC ₅₀ values ranging from 0.14 to 0.28% of the original solutions (Table 2), indicating that the method by which the compositions are prepared does not significantly affect their potent antimicrobial activity. Furthermore, copper (and copper-zinc) compositions based on copper oxychloride and copper sulfate can be prepared or mixed together over a wide range of concentrations while maintaining full antimicrobial activity. Surprisingly, compositions containing copper ions or copper and zinc ions together with phosphorous acid are more antimicrobial than the components from which they are prepared (see Table 2). Taken together, these results demonstrate the synergistic activity of the compositions compared to their components and indicate unexpectedly enhanced antimicrobial activity of the compositions obtained as described in the Best Mode for Carrying Out the Invention section.

It is important to note that when combining the compositions with sodium nitrite, their antimicrobial and sporicidal activities are synergistically enhanced (Table 3 and Table 5, respectively). Such a combination, applied, for example, as a foliar spray, would not only provide sporicidal activity, which could destroy fungal spores before they are released into the air and thereby reduce the spread of associated fungal infections, but after the sporicidal effect has been exhausted (after approximately 6 hours), there would be continued, persistent antimicrobial and plant defense-promoting activity provided by the copper or copper-zinc components of the composition and the phosphorous acid component of the composition remaining on the treated plants. It should also be possible to extend the sporicidal action of the combination by including a slow-release form of sodium nitrite. This would be useful if the time required to prepare and apply the sporicidal combination is more than 6 hours.

Furthermore, combining the formulations with sodium nitrite (neither of which, taken individually, exhibited sclerotia-killing activity) also unexpectedly exhibited sclerotia-killing activity (Table 7). In fact, full sclerotia-killing activity was achieved by combining the formulations, diluted 1:100 (1%) of their stock solutions, with 30 millimolar sodium nitrite after only 10 minutes of exposure. These results indicate that such formulations can be effectively used in field practice not only to prevent sclerotia formation, primarily by inhibiting fungal growth, but also by killing sclerotia in dry plant material before application, which could otherwise lead to costly, long-term sclerotia infestation of the soil.

Sclerotia-forming fungi such as R. solani, V. dahliae, and M. phaseolina are a serious problem in soils of fields used to produce lettuce, potatoes, spinach, and strawberries. Destruction of sclerotia in soil by fumigation is now prohibited or being phased out, but no effective alternative strategies are currently available. Combining the formulations with sodium nitrite destroys sclerotia and strongly inhibits fungal growth, so sclerotia would not have formed in the first place; furthermore, phosphorous acid in the formulations has been shown to inhibit sclerotia formation at concentrations (>40 milligrams/liter) that are significantly lower than those present in the fungicidal concentrations of the formulations (at 1% stock solution, phosphorous acid concentrations typically range from 400 to 1750 milligrams/liter - see Table 2).

In addition to phosphorous acid, potassium phosphite, glyphosate, and Aliette, which are widely used agrochemicals containing a phosphite or ^phosphonate ( _PO33- ) group, have all been shown to inhibit sclerotia formation. However, phosphorous acid, potassium phosphite, and Aliette were the most potent inhibitors of sclerotia formation, and because they are inexpensive and widely used products, it might be reasonable to expect that daily application of these products to crops at low concentrations in water supplies, such as sprays, drip irrigation lines, or center pivots, could provide a convenient and cost-effective way to prevent sclerotia formation by fungi in field applications.

It has been unexpectedly shown that a sclerotia-killing composition comprising a compound containing a phosphite or phosphonate (PO ₃ ^3- ) prevents the formation of sclerotia by fungi. The compound can be, for example, phosphorous acid or a salt thereof, such as potassium, sodium, calcium, copper, aluminum, zinc or ammonium phosphites or combinations thereof. In some embodiments of the present invention, the compound can also be glyphosate (N-(phosphonomethyl)-glycine).

The composition may also be, in some embodiments of the present invention, a combination of glyphosate with phosphorous acid or a salt thereof.

In another embodiment of the present invention, a copper or copper-zinc composition with phosphorous acid in combination with sodium nitrite to produce nitrous acid and nitrogen oxides results in antimicrobial (bactericidal, fungicidal) and sporicidal, and sclerotium-killing compositions for agricultural applications.

In some embodiments of the present invention, the salt of nitrous acid is a sodium or potassium salt. In some embodiments of the present invention, the copper salt may be copper oxychloride or copper sulfate. In some embodiments of the present invention, the copper salt may be copper oxychloride in the form of a wettable product. In some embodiments of the present invention, the zinc salt may be zinc sulfate, zinc acetate or zinc nitrate. In some embodiments of the present invention, the base may be ammonium hydroxide, sodium hydroxide or potassium hydroxide, with ammonium hydroxide being preferred. In some embodiments of the present invention, water may be distilled water, deionized water, purified water, filtered water, pharmaceutical grade water, medical grade water and reverse osmosis water; in some embodiments of the present invention, the copper and/or zinc salt used to prepare the solution is hydrated. In some embodiments of the present invention, the ratio of copper to zinc in the composition is in the range of 100:1 to 1:100, more preferably from 10:1 to 1:10, and even more preferably 1:1. In some embodiments of the present invention, the composition further comprises excipients, adjuvants, carriers, surfactants, or fillers.

Unexpectedly, Cu/CuZn compositions with phosphorous acid (PA) exhibited stronger antimicrobial/fungicidal and sporicidal activity than compositions with phosphorous acid (PA) alone, regardless of whether they were applied alone or mixed with a nitrite salt. Phosphorous acid also stimulates plant defense mechanisms.

The unexpected enhancement of sporicidal activity due to the presence of copper ions when using phosphorous acid as a _NaNO2 activator for agricultural applications was unexpected. Furthermore, fungicidal experiments in this application demonstrate that the enhanced antimicrobial activity provided by the presence of copper with phosphorous acid was an unpredictable synergistic effect.

However, although phosphorous acid and potassium phosphite, taken alone, are very effective inhibitors of sclerotia formation by R. solani (at concentrations of 40 mg/L and above), they are relatively weak inhibitors of fungal growth (IC ₅₀ values with R. solani are approximately 250 and 400 mg/L, respectively), and neither product, taken alone, is sporicidal or sclerotia-killing at concentrations lower than the 4 grams/L typically applied as a foliar spray. Therefore, there are significant advantages to using one of the compositions described herein, with or without sodium nitrite, in field practice.

Interestingly, when applied as a foliar spray to rice seedlings and rose bushes, the formulations combined with sodium nitrite were not phytotoxic, and on aphid-infested rose bushes, the combination was found to have rapid insecticidal action.

Ideally, agrochemicals should be easy and safe to use, environmentally friendly, and inexpensive and effective. These formulations, combined with sodium nitrite, are preferred in this regard, as they are inexpensive and relatively safe to use, and environmentally friendly, as they contain significantly lower levels of copper (approximately 10 times less at a 1% dilution of the stock solution) than the most currently available copper-based agrochemicals.

Claims (11)

1. An agrochemical composition that inhibits the formation of sclerotia by fungi, containing as initial components: copper oxychloride in the form of a wettable powder containing bentonite, or copper oxychloride in the form of a technical product, phosphorous acid, ammonium hydroxide, zinc salt and water, wherein phosphorous acid is added to a suspension of copper oxychloride in water at a concentration of 40 to 800 g/l in combination with ammonium hydroxide and zinc salt, wherein the total content of elemental copper in the composition is from 1 to 50 g/l, wherein the ratio of copper to zinc in the composition is from 10:1 to 1:10 and the content of ammonium hydroxide is from 1 to 80 ml/l. 2. An agrochemical composition according to claim 1, additionally containing copper sulfate pentahydrate, wherein the total content of elemental copper in the composition is from 1 to 50 g/l. 3. The agrochemical composition of claim 1, wherein the zinc salt is selected from the group consisting of zinc sulfate heptahydrate, anhydrous zinc sulfate, zinc acetate, and zinc nitrate, providing a total elemental zinc in the composition in the range of 1 to 50 g/l. 4. An agrochemical composition according to claim 1, wherein the water is selected from the group consisting of distilled water, deionized water, purified water, filtered water, pharmaceutical grade water, water for medical use, and reverse osmosis water. 5. An agrochemical composition according to claim 1, having an effect of inhibiting sclerotia by fungi from 0 to 6 hours after production, and an antimicrobial effect for a period of at least two days after production.