WO2023111330A1 - Beneficial acids for enhancing biocontrol efficacy of fungal spores - Google Patents

Beneficial acids for enhancing biocontrol efficacy of fungal spores Download PDF

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Publication number
WO2023111330A1
WO2023111330A1 PCT/EP2022/086495 EP2022086495W WO2023111330A1 WO 2023111330 A1 WO2023111330 A1 WO 2023111330A1 EP 2022086495 W EP2022086495 W EP 2022086495W WO 2023111330 A1 WO2023111330 A1 WO 2023111330A1
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Prior art keywords
acid
strain
composition
spores
formulation
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PCT/EP2022/086495
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French (fr)
Inventor
Daniel LANVER
Ulrike HILSCHER
Julia ULRICH
Angelika RAVE
Matthew TARVER
Oliver Gaertzen
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Danstar Ferment Ag
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Publication of WO2023111330A1 publication Critical patent/WO2023111330A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom

Definitions

  • dipicolinic acid has toxic activity against insects (Asaff et al., 2005, "Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus” Appl Microbiol Biotechnol DOI:10.1007/s00253-005-1909-2; Weng et al., 2019, “Secondary Metabolites and the Risks of Isaria fumosorosea and Isaria farinosa” molecules D01:10.3390/molecules24040664).
  • insects Adyceps fumosorosea, and it was further shown that dipicolinic acid has toxic activity against insects (Asaff et al., 2005, "Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus” Appl Microbiol Biotechnol DOI:10.1007/s00253-005-1909-2; Weng et al., 2019, “Secon
  • a first aspect of the present invention refers to a preparation comprising an acid and spores of an entomopathogenic fungus.
  • a second aspect of the present invention refers to a composition
  • a composition comprising water, an acid, and spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid is at least 1 g/1, and wherein the pH of the composition is between 2,0 and 7,0, and wherein the pH of said composition is lower than the pH of a comparative composition without said acid.
  • the invention refers to use of an acid as described herein for increasing the efficacy of entomopathogenic fungal spores as described herein to control insects and/or nematodes.
  • the invention refers to a method for enhancing the efficacy of fungal spores comprising contacting spores of an entomopathogenic fungus as described herein with an acid as described herein.
  • the invention refers to a method for controlling insects and/or nematodes, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of a preparation or composition of the invention to said plant or to a locus where plants are growing or intended to be grown.
  • the invention refers to a kit comprising entomopathogenic fungi as described herein and an acid as described herein, wherein the entomopathogenic fungi and the acid are spatially separated, and wherein said kit is optionally suitable for preparation of a composition of the invention.
  • a preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p -tol
  • a further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the acid is an organic acid selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, and glycine.
  • the acid is an organic acid selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, and glycine.
  • a further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the acid is an organic acid selected from the group consisting of malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, glycine.
  • the acid is an organic acid selected from the group consisting of malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, glycine.
  • a further preferred embodiment of Aspect 2 and any of its embodiments refers to the composition, wherein the decrease in pH is at least 0,1 pH units.
  • a further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the entomopathogenic fungus is selected from the group consisting of Beauveria bassiana (preferably strain ATCC 74040 (e.g. Naturalis® from CBC Europe, Italy; Contego BB from Biological Solutions Ltd.; Racer from AgriLife), strain GHA (Accession No. ATCC74250; e.g. BotaniGuard Es and Mycontrol-0 from Laverlam International Corporation), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339 (e.g. BroadBandTM from BASE), strain PPRI 7315, strain R444 (e.g.
  • Beauveria bassiana preferably strain ATCC 74040 (e.g. Naturalis® from CBC Europe, Italy; Contego BB from Biological Solutions Ltd.; Racer from AgriLife), strain GHA (Accession No. ATCC74250; e.g. BotaniGuard Es and My
  • strains IL197, IL12, IL236, IL10, IL131, IL116 all referenced in Jaronski, 2007, Use of Entomopathogenic Fungi in Biological Pest Management, 2007: ISBN: 978-81-308-0192-6
  • strain Bv025 strain BaGPK
  • strain ICPE 279 strain CG 716 (e.g. BoveMax® from Novozymes)
  • strain HF23 strain ANT-03 (from Bioceres)
  • strain NPP111B00 e.g.
  • strain Bbl 0 from Biagro Bb
  • strain ESALQ PL63 from Boveril, Koppert
  • strain IMI389521 strain IBCB 66
  • strain K4B1 Beaugenic from Ecolibrium Biologicals
  • strain K4B3 Beaublast from Ecolibrium Biologicals
  • strain CBMAI 1306 from Auin CE Agrivalle
  • Metarhizium brunneum formely known as Metarhizium anisopliae
  • strain Cbl5 e.g. ATTRACAP® from Biocare
  • strain ESALQ 1037 e.g. from Metarril® SP Organic
  • strain E-9 e.g.
  • strain M206077 from Metarril® SP Organic
  • strain C4-B NRRL 30905
  • strain ESC1 strain 15013- 1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448 (e.g. BIO 1020 by Bayer CropScience and also e.g. Met52 by Novozymes), strain ICIPE 78, strain ESF1 (from BIO-BLAST), strain IBCB 425 (from Metarhizium Oligos, Oligos Biotecnologia), strain LRC112 (from SpudSmart, AAFC)).
  • a further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the entomopathogenic fungus is selected from the group consisting of Cor dy ceps fumosorosea strain Apopka 97, strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367; Cordyceps javanica strain wf GA 17, strain GZQ-1, strain IjH6102, strain pfl85, strain pf212, strain JZ-7, or strain IJNL-N8 / CCTCC M-2017709; and Isaria farinosa strain ESALQ1205.
  • a further preferred embodiment of Aspect 2 and any of its embodiments refers to the composition, wherein the concentration of the fungi is between l,0*10 4 spores per ml composition and l,0*10 9 spores per ml composition.
  • a further preferred embodiment of Aspect 2 and any of its embodiments refers to the composition, wherein the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
  • a further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, further comprising at least one further component selected from the group consisting of liquids for spore formulations oil, antioxidant, emulsifier, rheology modifier, other components being present in a fungal spore formulation, impurities which can be found in water.
  • Another aspect refers to the use of an acid as defined in Aspect 1 , 2 or 3 and any of its embodiments for increasing the efficacy of fungal spores of the invention to control insects and/or nematodes.
  • Another aspect refers to a method for controlling insects and/or nematodes, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the preparation according to Aspect 1 , the composition according to Aspect 2 or any of its embodiments to said plant or to a locus where plants are growing or intended to be grown.
  • the amount of preparation or composition is at least 0,05 1/ha.
  • Kit-of-parts comprising a biological control agent selected from the group consisting of at least one spatially separated entomopathogenic fungi as defined in aspect 2 and any of its embodiments and a description how to prepare a composition according to aspect 2 and any of its embodiments.
  • Kit-in-parts further comprises at least one spatially separated acid as defined in aspect 2 or any of its preferred embodiments.
  • the term “acid” as used herein refers to a chemical compound that is able to transfer protons (H + ) to water. As a result of the proton transfer, oxonium ions (I I T) ) are formed and the pH value of an aqueous solution is lowered.
  • the term encompasses organic and inorganic acids.
  • the term “at least” indicates that in any case one agent as described herein (e.g. one acid or one antioxidant, etc.) is present in a composition according to the invention. However, more than one such as (at least) two, (at least) three, (at least) four, (at least) five or even more such agents (e.g. acids, antioxidants, etc.) may be present in a composition according to the invention.
  • one agent as described herein e.g. one acid or one antioxidant, etc.
  • more than one such as (at least) two, (at least) three, (at least) four, (at least) five or even more such agents (e.g. acids, antioxidants, etc.) may be present in a composition according to the invention.
  • antioxidants refers to compounds which inhibit oxidation of other molecules. Whereas Applicant does not wish to be bound by any scientific theory, it is believed that a certain concentration of antioxidant in a composition contributes to a better storage stability of the formulation, in particular the long stability of the fungal spores comprised therein.
  • An antioxidant may be any suitable antioxidant. Non-limiting examples are butylhydroxytoluol (BHT), butylhydroxyanisole (BHA), ascorbyl palmitate, tocopheryl acetate, ascorbyl stearate or the group of carotinoids (e.g. beta-carotin) or gallates (e.g. ethyl gallate, propyl gallate, octyl gallate, dodecyl gallate).
  • BHT butylhydroxytoluol
  • BHA butylhydroxyanisole
  • ascorbyl palmitate tocopheryl acetate
  • ascorbyl stearate or the group of carotinoids (e.
  • composition refers to a composition with essentially the same components as a composition according to the invention with the difference that the amount (% (w/w) of acid as described herein and which is an essential part of a composition according to the invention is replaced by water.
  • the missing amount of the acid i.e.
  • the ratios of all other components e.g., the entomopathogenic fungus and, e.g., optionally present additives of said fungus such as oils, emulsifiers, antioxidants, rheology modifiers etc.
  • the ratios of all other components e.g., the entomopathogenic fungus and, e.g., optionally present additives of said fungus such as oils, emulsifiers, antioxidants, rheology modifiers etc.
  • “Increase of efficacy” in the context of the present invention means the increase of the Abbott efficacy of a composition according to the invention compared to the Abbott efficacy of a comparative composition and the Abbott efficacy of the acid in the same concentration as in the formulation according to the invention.
  • inorganic acid refers to all acids that do not contain carbon, e.g. sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, silicic acid, boric acid.
  • organic acid refers to carbon-containing molecules that carry functional groups that are able to transfer protons to the aqueous medium and thus lower the pH.
  • H2CO3 carbonic acid
  • organic acids Non-limiting examples for organic acids are listed in Table A:
  • entomopathogenic fungus refers to a fungus that can act as a parasite of insects and kills or seriously disables them.
  • the entomopathogenic fungi include taxa from several of the main fungal groups.
  • Non-limiting examples of entomopathogenic fungi are in the order Hypocreales of the Ascomycota: the asexual (anamorph) phases Beauveria, Metarhizium, Cordyceps (Isaria, Paecilomyces), Hirsutella, Lecanicillium, Nomuraea and their sexual (teleomorph) states; or are in the order Entomophthorales of the Zygomycota: Entomophthora, Zoophthora, Pandora, Entomophaga.
  • “Fungal spores” include sexually (e. g. oospores, zygospores or ascospores) and asexually (e. g. conidia and chlamydospores, but also uredospores, teleutospores, ustospores and blastospores) formed spores.
  • the spores are conidia.
  • Integral values are usually given in a* 10 b format, wherein a and b can represent any number. The same information can be given by the aEb format. For example, l,0*10 2 is equal to l,0E+02, Both refer to the value 100.
  • Numbers as used in the present application are usually written with a e.g. 2,5 refers to 5/2.
  • 2,5 refers to 5/2.
  • a number in the present application is written with a instead of a”, such a number is regarded as being written with a
  • 2.5 is equal to 2,5 is equal to 5/2;
  • 1,000 does not mean 1000 (one thousand) but 1,000 (one);
  • l,0E+02 is equal to 1.0E+02 is equal to 100.
  • Plant oils or vegetable oils are oils derived from plant sources, as opposed to animal fats or petroleum.
  • the ones preferably used in the present invention are triglyceride -based vegetable oils which are liquid at least at room temperature, preferably also at temperatures below room temperature, such as at 15°C, at 10°C or even at 5°C or 4°C.
  • % refers to % (w/w) (weight percent) of not mentioned otherwise.
  • % (w/w) and “wt.-%.” can be used interchangeably, herein.
  • a and b as used herein (wherein a and b are numbers) means the threshold values a and b as well as all values between these the threshold values a and b.
  • the terms “spores/ml” and “s/ml” are used interchangeably, herein.
  • acids with specific characteristics can enhance the efficacy of entomopathogenic fungi in an aqueous composition, more preferably the efficacy of spores of an entomopathogenic fungi, even more preferred the efficacy of conidia. Even a synergistic effect can be observed.
  • the present disclosure provides a method for increasing the efficacy of fungal spores comprising contacting spores of an entomopathogenic fungus with an acid wherein upon contacting the fungal spores and the acid, the fungal spores exhibit an improved efficacy in inhibiting pests or exhibiting an increased pest control compared to contacting the fungal spores to the host without the acid.
  • the present invention refers inter alia to a composition
  • a composition comprising water, an acid, and an entomopathogenic fungus, preferably spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid is at least 1 g/1, and wherein the pH of the composition is between 2,0 and 7,0, and wherein the pH of said composition is lower than the pH of a comparative composition without said acid.
  • the present invention further refers to a composition
  • a composition comprising an acid and an entomopathogenic fungus, preferably spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid once the composition is diluted in water is at least 1 g/1, and wherein the pH of the composition diluted in water is between 2,0 and 7,0, and wherein the pH of said composition diluted in water is lower than the pH of a comparative composition without said acid.
  • the decrease of the pH value of a composition with acid (composition according to the invention) compared to a comparative composition is at least 0,1 pH units, more preferably at least 0,2 pH units, even more preferred at least 0,5 pH units.
  • a preparation of the invention may comprise any physical combination of entomopathogenic fungal spores and acid described herein, in any form.
  • the entomopathogenic fungal spores may be in a powder, for example a dry powder and/or a wettable powder.
  • the entomopathogenic fungal spores may be granulated (in granule form), for example a wettable granule form.
  • the entomopathogenic fungal spores may be in liquid form, for example dispersed in a solution, for example an aqueous solution, or dispersed in a non-aqueous liquid such as oil. Accordingly, the entomopathogenic fungal spores may be dispersed in an aqueous solution, or the entomopathogenic fungal spores may be oil- dispersed.
  • composition according to the invention is an aqueous formulation.
  • an aqueous formulation is a suspension (due to the presence of fungal spores).
  • the content of water in the composition according to the invention is at least 50%, more preferably at least 60% such as at least 70%, at least 80% or at least 95%, even more preferably at least 98% or even at least 99% or even at least 99,5%. pH of the final formulation
  • the pH range of composition according to the invention is adjusted by the presence of an acid so that the pH of the final composition is between pH 2 and 7 as described herein.
  • the pH depends inter alia on the amount of an acid used in a composition according to the invention.
  • the skilled person can, without undue burden, define amounts of acids to adjust the pH of a composition to be a composition in accordance with the present invention, wherein the pH is between pH 2 and pH 7 or any of the more preferred ranges, when the amount of an acid is between 0,0001% (w/w) and 2% (w/w).
  • a composition according to the invention may comprise on or more further additives (e.g., as comprised in a fungal formulation) or impurities such as e.g., a solubilizing agent (e.g. polyethylenglycol) or impurities as can be found in ground-water or rain-water.
  • a solubilizing agent e.g. polyethylenglycol
  • impurities as can be found in ground-water or rain-water.
  • Such additives may also influence the pH of a composition. However, as long as the pH is lowered by the presence of an acid (wherein the amount of acid is between 0,0001% (w/w) and 2% (w/w)) compared to such a composition without said acid and the pH is in the pH range of a composition as described herein, the beneficial effect of a composition according to the invention can be observed.
  • the phytotoxic effects (and/or also toxic effects for the spores) of low pH values compensate the beneficial effect of the increase of efficacy of spores of an entomopathogenic fungi.
  • increased pH values do not enhance efficacy of spores of an entomopathogenic fungi.
  • the pH of the final composition is between pH 2,0 and pH 6,5, more preferably between 2,5 and 6,5, even more preferably between 3,0 and 6,0.
  • such an acid may comprise more than one pKa values.
  • an acid has at least one, preferably one or two or three -COOH group(s), at least one of such an -COOH group has a pka between -3 and 5 such as between 1 and 5 or any of the other preferred pk a ranges.
  • said acid is an organic acid having the formula
  • said acid is an organic acid having the formula
  • said acid is an organic acid having the formula
  • said acid is an organic acid having the formula
  • an acid preferably an organic acid
  • has a -O-P( O)(OH) 2 group with a pka between -1 and 5 or the other preferred pka ranges.
  • said acid is an organic acid having the formula
  • One parameter that influences the pH value which is adjusted by the respective acid is the concentration of said acid in the final composition.
  • the concentration range of an acid in a composition according to the invention is between 0,0001 % (w/w) and 2 % (w/w), preferably between 0,00025 % (w/w) and 0,5 % (w/w) such as between 0,005 % (w/w) and 0,5 % (w/w), preferably between 0,001 % (w/w) and 0,25 % (w/w) such as between 0,01 % (w/w) and 0,25 % (w/w), more preferably between 0,0025% and 0,20% such as between 0,05 % (w/w) and 0,2 % (w/w). pH range
  • an acid suitable for the various aspects of the present invention results in a pH of the composition between 2,5 and 6,5 at 25 °C and 1013 hPa, more preferably between 3,0 and 6,5 at 25 °C and 1013 hPa, even more preferably between 3,0 and 6,0 at 25 °C and 1013 hPa.
  • an acid suitable for the various aspects of the present invention is present in a composition according to the invention in an amount between 0,005 % (w/w) and 0,5 % (w/w) and the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
  • an acid suitable for the various aspects of the present invention is present in a composition according to the invention in an amount between 0,01 % (w/w) and 0,25 % (w/w) and the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
  • an acid suitable for the various aspects of the present invention is present in a composition according to the invention in an amount between 0,05 % (w/w) and 0,2 % (w/w) and the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa
  • the pH can be measured using a laboratory pH meter (e.g. a Knick 766 Laboratory pH Meter). Water solubility
  • the acid suitable for the various aspects of the invention is sufficiently water soluble. More specific for the context of this invention, sufficient solubility in water at 25 °C and 1013 hPa is 1 g/1 (gram/liter) or more, preferably 2 g/1 or more, and more preferably 5 g/1 or more, e.g. 50 g/1 or more, 100 g/1 or more or even 250 g/1 or more. In one embodiment, the acid is completely miscible with water.
  • an acid is an organic acid having a water solubility of at least 1 g/1.
  • an acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p-toluenesulfonic acid, succinic acid, acrylic acid, aspartic acid,
  • an acid is an acid selected from the acids listed in Table A having a -COOH group with a pk a value between 1 and 5,
  • an acid is selected from the group consisting of oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, aspartic acid, chinolinic acid, c
  • an acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pk a value between 1 and 3,
  • an organic acid is selected from the group consisting of oxalic acid, dipicolinic acid, citric acid, malonic acid, tartaric acid, alpha-ketoglutaric acid, aconitic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, Isocinchomeronic acid, lutidinic acid, maleic acid, p
  • an acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pk a value between 2 and 3,
  • an organic acid is selected from the group consisting of dipicolinic acid, citric acid, malonic acid, tartaric acid, alpha-ketoglutaric acid, aconitic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, tyrosine, threonine, methionine, tryptophan, selenocysteine, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, Isocinchomeronic acid, lutidinic acid, maleic acid, phtalic acid, and salicylic acid.
  • an organic acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pk a value between above 3 and 5,
  • an organic acid is selected from the group consisting of malic acid, fumaric acid, adipic acid, pimelic acid, itaconic acid, trimesic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, succinic acid, acrylic acid, glutaric acid, glycolic acid, isocitric acid, isonicotinic acid, mandelic acid, nicotinic acid, picolinic acid, propionic acid, and tetronic acid.
  • an acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pk a value between above 4 and 5,
  • an organic acid is selected from the group consisting of adipic acid, pimelic acid, benzoic acid, sorbic acid, acetic acid, ascorbic acid, succinic acid, acrylic acid, glutaric acid, isonicotinic acid, nicotinic acid, and propionic acid.
  • an acid is an acid selected from the acids listed in Table A having a pka value between -3 and -1.
  • an organic acid is selected from the group consisting of p-toluenesulfonic acid, and methane sulfonic acid.
  • the acid is sulfuric acid. In another preferred embodiment, the acid is nitric acid. In another preferred embodiment, the acid is phosphoric acid. In another preferred embodiment, the acid is hydrochloric acid. In another preferred embodiment, the acid is boric acid.
  • the acid is oxalic acid. In another preferred embodiment, the acid is dipicolinic acid. In another preferred embodiment, the acid is citric acid. In another preferred embodiment, the acid is malonic acid. In another preferred embodiment, the acid is malic acid. In another preferred embodiment, the acid is fumaric acid. In another preferred embodiment, the acid is adipic acid. In another preferred embodiment, the acid is pimelic acid. In another preferred embodiment, the acid is tartaric acid. In another preferred embodiment, the acid is alpha-ketoglutaric acid. In another preferred embodiment, the acid is itaconic acid. In another preferred embodiment, the acid is trimesic acid. In another preferred embodiment, the acid is aconitic acid. In another preferred embodiment, the acid is benzoic acid.
  • the acid is sorbic acid. In another preferred embodiment, the acid is acetic acid. In another preferred embodiment, the acid is lactic acid. In another preferred embodiment, the acid is ascorbic acid. In another preferred embodiment, the acid is glutamic acid. In another preferred embodiment, the acid is glycine. In another preferred embodiment, the acid is serine. In another preferred embodiment, the acid is asparagine. In another preferred embodiment, the acid is alanine. In another preferred embodiment, the acid is phenylalanine. In another preferred embodiment, the acid is valine. In another preferred embodiment, the acid is leucine. In another preferred embodiment, the acid is isoleucine. In another preferred embodiment, the acid is histidine. In another preferred embodiment, the acid is arginine.
  • the acid is tyrosine. In another preferred embodiment, the acid is threonine. In another preferred embodiment, the acid is cysteine. In another preferred embodiment, the acid is methionine. In another preferred embodiment, the acid is tryptophan. In another preferred embodiment, the acid is proline. In another preferred embodiment, the acid is selenocysteine. In another preferred embodiment, the acid is p-toluenesulfonic acid. In another preferred embodiment, the acid is succinic acid. In another preferred embodiment, the acid is acrylic acid. In another preferred embodiment, the acid is aspartic acid. In another preferred embodiment, the acid is chinolinic acid. In another preferred embodiment, the acid is cinchomeronic acid.
  • the acid is dinicotinic acid. In another preferred embodiment, the acid is formic acid. In another preferred embodiment, the acid is glutaric acid. In another preferred embodiment, the acid is glycolic acid. In another preferred embodiment, the acid is isocinchomeronic acid. In another preferred embodiment, the acid is isocitric acid. In another preferred embodiment, the acid is isonicotinic acid. In another preferred embodiment, the acid is lutidinic acid. In another preferred embodiment, the acid is maleic acid. In another preferred embodiment, the acid is mandelic acid. In another preferred embodiment, the acid is nicotinic acid. In another preferred embodiment, the acid is phtalic acid. In another preferred embodiment, the acid is picolinic acid. In another preferred embodiment, the acid is propionic acid. In another preferred embodiment, the acid is salicylic acid. In another preferred embodiment, the acid is tetronic acid. In another preferred embodiment, the acid is methane sulfonic acid.
  • an acid is an inorganic acid, preferably, the inorganic acid is selected from the group consisting of phosphoric acid or boric acid. More preferably, the inorganic acid is phosphoric acid.
  • an acid is selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, glycine.
  • an acid is selected from the group consisting of malonic acid, malic acid, tartaric acid, acetic acid, lactic acid, ascorbic acid, glycine, and citric acid.
  • the acid is citric acid.
  • the acid is selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, glycine.
  • an acid is selected from the group consisting of malonic acid, malic acid, tartaric acid, acetic acid lactic acid, ascorbic acid, and glycine.
  • an acid is selected from the group consisting of malic acid, tartaric acid, and ascorbic acid.
  • an entomopathogenic fungi is selected from the group consisting of
  • Cordyceps fumosorosea (formely known as Isaria fumosorosea or Paecilomyces fumosoroseus) (preferably strain Apopka 97 which is now known as Cordyceps javanica (e.g. PFR97 or PreFeRal® WG from Biobest and Certis), strain IF-BDC01, strain FE 9901 / ARSEF 4490 (e.g. NoFly® from Natural Industries Inc., a Novozymes company), strain ESALQ 1296 (e.g. from Octane, Koppert) or strain CCM 8367 (e.g. WO 2010 006563));
  • Cordyceps fumosorosea (formely known as Isaria fumosorosea or Paecilomyces fumosoroseus) (preferably strain Apopka 97 which is now known as Cordyceps javanica (e.g. PFR97 or PreFeRal® WG from
  • Cordyceps javanica (formerly known as Isaria javanica) (preferably strain wf GAI 7 (referenced in Wu et al., 2020, Environmental Tolerance of Entomopathogenic Fungi: A New Strain of Cordyceps javanica Isolated from a Whitefly Epizootic Versus Commercial Fungal Strains, doi: 10,3390/insectsl 1100711), strain GZQ-1 (referenced in Ou et al., 2019 Identification of a new Cordyceps javanica fungus isolate and its toxicity evaluation against Asian citrus psyllid doi: 10,1002/mbo3,760), strain IjH6102 (e.g.
  • strain IJNL-N8 / CCTCC M-2017709 (Zhao et al., 2020 Sustainable control of the rice pest, Nilaparvata lugens, using the entomopathogenic fungus Isaria javanica. doi.org/10,1002/ps.6164); or strain GF 511 (which has been deposited by Lallemand Inc.
  • Isaria farinosa preferably strain ESALQ1205;
  • Beauveria bassiana preferably strain ATCC 74040 (e.g. Naturalis® from CBC Europe, Italy; Contego BB from Biological Solutions Ltd.; Racer from AgriLife), strain GHA (Accession No. ATCC74250; e.g. BotaniGuard Es and Mycontrol-0 from Laverlam International Corporation), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339 (e.g. BroadBandTM from BASF), strain PPRI 7315, strain R444 (e.g.
  • strains IL197, IL12, IL236, IL10, IL131, IL116 all referenced in Jaronski, 2007, Use of Entomopathogenic Fungi in Biological Pest Management, 2007: ISBN: 978-81-308-0192-6
  • strain Bv025 strain BaGPK
  • strain ICPE 279 strain CG 716 (e.g. BoveMax® from Novozymes)
  • strain HF23 strain ANT-03 (from Bioceres)
  • strain NPP 111 BOO e.g.
  • Beauveria brongniartii e.g. from Beaupro from Andermatt Biocontrol AG (preferably strain BIPESCO 2 (from Melocont, Agrifutur);
  • Metarhizium brunneum (formely known as Metarhizium anisopliae) (preferably strain Cbl5 (e.g. ATTRACAP® from Biocare), strain ESALQ 1037 (e.g. from Metarril® SP Organic), strain E-9 (e.g.
  • strain ICIPE 78 Met52 by Novozymes or LALGUARD M52 from Lallemand
  • strain ICIPE 78 strain ESF1 (from BIO-BLAST)
  • strain IBCB 425 from Metarhizium Oligos, Oligos Biotecnologia
  • strain LRC112 from SpudSmart, AAFC
  • Metarhizium acridum preferably strain ARSEF324 from GreenGuard by BASF or isolate IMI 330189, or ARSEF7486; e.g. Green Muscle by Biological Control Products;
  • Metarhizium robertsii preferably strain 23013-3 (NRRL 67075)
  • Metarhizium rileyi (formerly known as Nomuraea rileyi);
  • Myrothecium verrucaria preferably strain AARC-0255 (e.g. from DiTera DF, Biagro));
  • Hirsutella thompsonii (preferably Mycohit and ABTEC from Agro Bio-tech Research Centre, IN);
  • Lecanicillium lecanii (formerly known as Verticillium lecanii) (preferably conidia of strain KV01 (e.g. Mycotal® and Vertalec® from Koppert/Arysta));
  • Lecanicillium lecanii (formerly known as Verticillium lecanii), strain DAOM198499; strain DAOM216596; strain KV01 (e.g. from Vertalec®, Koppert/Arysta)
  • Entomophthora virulenta e.g. Vektor from Ecomic
  • Sporothrix insectorum e.g. Sporothrix Es from Biocerto, BR
  • Sporothrix insectorum e.g. Sporothrix Es from Biocerto, BR
  • Mucor haemelis e.g. BioAvard from Indore Biotech Inputs & Research
  • Purpureocillium lilacinum (forme ly also known as Paecilomyces lilacinus) (preferably strain 251 (AGAL 89/030550; e.g. BioAct from Bayer CropScience Biologies GmbH), strain PL11 (from BioStat, Certis), or strain UEL Pae 10 (from Unique, Ballagro Agro Tecnologia Ltda.));
  • strain Q-09 preferably Nemaquim® from Quimia, MX
  • Muscodor albus preferably strain QST 20799 (Accession No. NRRL 30547));
  • Muscodor roseus preferably strain A3-5 (Accession No. NRRL 30548));
  • Myrothecium verrucaria preferably strain AARC-0255 (e.g. DiTeraTM by Valent Biosciences);
  • Stagonospora phaseoli e.g. from Syngenta
  • Paraglomus sp in particular Paraglomus brasilianum
  • Pochonia chlamydosporia also known as Vercillium chlamydosporiuni
  • IMI SD 187 var. catenulata
  • KlamiC National Center of Animal and Plant Health
  • an entomopathogenic fungi is selected from the group consisting of Beauveria bassiana (preferably strain ATCC 74040 (e.g. Naturalis® from CBC Europe, Italy; Contego BB from Biological Solutions Ltd.; Racer from AgriLife), strain GHA (Accession No. ATCC74250; e.g. BotaniGuard Es and Mycontrol-0 from Laverlam International Corporation), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339 (e.g. BroadBandTM from BASF), strain PPRI 7315, strain R444 (e.g.
  • strains IL197, IL12, IL236, IL10, IL131, IL116 all referenced in Jaronski, 2007, Use of Entomopathogenic Fungi in Biological Pest Management, 2007: ISBN: 978-81-308-0192-6
  • strain Bv025 strain BaGPK
  • strain ICPE 279 strain CG 716 (e.g. BoveMax® from Novozymes)
  • strain HF23 strain ANT-03 (from Bioceres)
  • strain NPP 111 BOO e.g.
  • strain Bbl 0 from Biagro Bb
  • strain ESALQ PL63 from Boveril, Koppert
  • strain IMI389521 from strain IBCB 66 (e.g. from Atrevido, Koppert)
  • strain K4B1 Beaugenic from Ecolibrium Biologicals
  • strain K4B3 Beaublast from Ecolibrium Biologicals
  • strain CBMAI 1306 from Auin CE Agrivalle
  • Metarhizium brunneum (formely known as Metarhizium anisopliae) (preferably strain Cbl5 (e.g. ATTRACAP® from Biocare), strain ESALQ 1037 (e.g. from Metarril® SP Organic), strain E-9 (e.g.
  • strain ICIPE 78 Met52 by Novozymes or LALGUARD M52 from Lallemand
  • strain ICIPE 78 strain ESF1 (from BIO-BLAST)
  • strain IBCB 425 from Metarhizium Oligos, Oligos Biotecnologia
  • strain LRC112 from SpudSmart, AAFC
  • an entomopathogenic fungi is selected from the group consisting of Cordyceps fumosorosea strain Apopka 97 (which has now been reclassified as C. javanica strain Apopka 97), strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ 1296 or strain CCM 8367;
  • Lecanicillium muscarium strain VE 6 / CABI( IMI) 268317/ CBS102071/ ARSEF5128, strain K4V 1 , or strain K4V2;
  • Purpureocillium lilacinum strain 251 AGAL 89/030550, strain PL11, or strain UEL Pae 10;
  • Muscodor albus strain QST 20799 (Accession No. NRRL 30547;
  • Muscodor roseus strain A3-5 (Accession No. NRRL 30548;
  • an entomopathogenic fungi is selected from the group consisting of
  • Cordyceps fumosorosea (or C. javanica) strain Apopka 97, Cordyceps fumosorosea strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367;
  • Isaria farinosa strain ESALQ1205 even more preferably
  • Cordyceps fumosorosea (or C. javanica) strain Apopka 97, Cordyceps fumosorosea strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367;
  • Metarhizium brunneum (formely known as Metarhizium anisopliae) strain F52 (DSM3884/ ATCC 90448, IMI 507278 which has been deposited on November 18 th , 2022 according to the Budapest Treaty with CABI, Bakeham Lane, Egham, Surrey, TW209TY, UK); most preferably
  • the Apopka 97 strain was previously classified as Cordyceps fumosorosea (and, even earlier, as Isaria fumosorosea) but in 2017 was reclassified as Cordyceps javanica (Avery et al., (2021), Field Efficacy of Cordyceps javanica, White Oil and Spinetoram for the Management of the Asian Citrus Psyllid, Diaphorina citri Insects 2021, 12, 824).
  • the Apopka 97 strain is referred to using both or either classifications interchangeably.
  • the entomopathogenic fungus is a Cordyceps javanica strain. In a preferred embodiment the entomopathogenic fungus is a Cordyceps javanica Apopka 97 strain or a Cordyceps javanica GF 511 strain. In another preferred embodiment the entomopathogenic fungus is a Metarhizium brunneum strain. In a preferred embodiment the entomopathogenic fungus is a Metarhizium brunneum F52 strain (IMI 507278).
  • the number of fungal spores in a composition according to the invention is between l,0*10 4 spores per ml and l,0*10 9 spores per ml, such as between l,0*10 5 spores per ml and l,0*10 8 spores per ml. More preferably, the number of fungal spores is between 5,0* 10 5 spores per ml and 5,0* 10 7 spores per ml. Most preferably the number of spores is between 5,5* 10 5 spores per ml and 5,0* 10 7 spores per ml.
  • Liquids for spore formulations Preferred liquids for spore formulations suitable for the present invention are selected from the group consisting of polyethylene glycols, such as PEG-300; ethoxylated alcohols (such as Atplus 245, Berol 050, Berol 260, Fabricamul CO08, Fabricamul L03, Ziamul L05); mono-/polyethylene oxide diethers, such as Tetraglyme; mono-/polyethylene oxide ether-ester (such as n-Butyldiglycolacetat); ethoxylated carboxylic acids (such as Radiasurf 7403, PEG-400-monooleate, Radiasurf 7423); mono-/polyethylene oxide di-esters (such as Radiasurf 7442); polypropylene glycols(such as Dipropylene glycol); propoxylated alcohols (such as Dowanol DPM); mono-/polypropylene oxide diethers (such as Dipropylene glycol dimethyl
  • a composition according to the invention may comprise an oil, more preferably a mineral oil, plant oil or vegetable oil.
  • plant oils the ones preferably used in the present invention are triglyceride -based vegetable oils which are liquid at least at room temperature, preferably also at temperatures below room temperature, such as at 15°C, at 10°C or even at 5°C or 4°C.
  • Concentrations of oils, preferably plant oil, in a composition according to the invention may range between 0,005 % (w/w) and 0,9 % (w/w) and any other value in between this range, such as between 0,01 % (w/w) and 0,85 % (w/w) and any other value in between this range.
  • Preferred plant oils are wheat germ oil, soybean oil, peanut oil, rice bran oil, safflor oil, rapeseed oil, sunflower oil, com oil, walnut oil, hazelnut oil, almond oil and olive oil.
  • a composition according to the invention may comprise an antioxidant.
  • an antioxidant it is believed that a certain concentration of antioxidant in the formulation of the invention contributes to the superior storage stability of the formulation, in particular the long stability of the fungal spores comprised therein.
  • the concentration of antioxidants in the formulation according to the present invention is at least 0,00002 % (w/w) and may be increased to up to 0,05 % (w/w).
  • a preferred range is between 0,00003 % (w/w) and 0,007 % (w/w) and any other value in between this range.
  • the antioxidant may be any suitable antioxidant, but is preferably selected from the group consisting of butylhydroxytoluol (BHT), butylhydroxyanisole (BHA), ascorbyl palmitate, tocopheryl acetate, ascorbyl stearate or the group of carotinoids (e.g. beta-carotin) or gallates (e.g. ethyl gallate, propyl gallate, octyl gallate, dodecyl gallate).
  • BHT butylhydroxytoluol
  • BHA butylhydroxyanisole
  • ascorbyl palmitate tocopheryl acetate
  • ascorbyl stearate or the group of carotinoids (e.g. beta-carotin) or gallates (e.g. ethyl gallate, propyl gallate, octyl gallate, dodecyl gallate).
  • the antioxidant is butylhydroxytoluol which has been shown in the examples to contribute to the very good stability of the fungal spores in the formulation of the present invention. Further preferred, said butylhydroxytoluol is present in a concentration of between 0,00002 % (w/w) and 0,01 % (w/w) and any other value in between this range, preferably between 0,00004 % (w/w) and 0,006 % (w/w) and any other value in between this range.
  • Some plant oils naturally have a high content of antioxidants, e.g. wheat germ oil.
  • the further addition of an antioxidant may not be necessary, or the amount may be reduced in order to arrive at the concentrations described herein which are believed to be one factor responsible for the enhanced storage stability of the present formulation.
  • the required percentage of antioxidant according to the invention is comprised in the minimum concentration of plant oil according to the invention.
  • a composition according to the invention may further comprise an emulsifier.
  • the concentration of emulsifier should be at least 0,0002 % (w/w).
  • the maximum concentration of said at least one emulsifier should not exceed 0,3 % (w/w). Accordingly, useful ranges for emulsifiers range between 0,0002 % (w/w) and 0,2% (w/w), and any value in between this range.
  • Suitable emulsifiers include ethoxylated sorbitan esters, e.g. ethoxylated sorbitan trioleate 20EO, (e.g. Tween 85); ethoxylated sorbitan monooleate (e.g. Tween 80); ethoxylated sorbitan monolaurate (e.g. Emulsogen 4156, Tween 20 or Polysorbate 20); or ethoxylated sorbitol esters, e.g. ethoxylated sorbitol hexaoleate 40EO (e.g. Ariatone TV); ethoxylated sorbitol tetraoleate-laurate 40EO (e.g.
  • ethoxylated sorbitan esters e.g. ethoxylated sorbitan trioleate 20EO, (e.g. Tween 85); ethoxylated sorbitan monooleate (e.
  • emulsifiers are preferably present in a range of between about 0,0015 % (w/w) and 0,125 % (w/w) and any other value in between this range.
  • said emulsifier is an ethoxylated sorbitol ester, such as ethoxylated sorbitol hexaoleate 40EO.
  • said emulsifiers are combined with other emulsifiers such as ethoxylated alcohols or propoxylated-ethoxylated alcohols.
  • emulsifiers such as ethoxylated alcohols or propoxylated-ethoxylated alcohols.
  • emulsifiers such as ethoxylated alcohols or propoxylated-ethoxylated alcohols.
  • X is a branched or linear alcohol, saturated or partially unsaturated, with 1-24 carbon atoms, preferably 2-18, more preferably 3-14, most preferably 4-10, wherein m is an average number between 0 and 20, preferably 0-15; more preferably 0-10, and wherein n is an average number between 1 and 20, preferably 2-15, more preferably 3-10,
  • emulsifiers are preferably present in a range of between about 0% (w/w) to 0,1% (w/w) and any other value in between this range.
  • a composition according to the invention may further comprise a rheology modifier.
  • Rheology modifiers are preferably derived from minerals. These rheology modifiers provide long term stability when the formulation is at rest or in storage. Furthermore, it has been found in the course of the present invention that such rheology modifiers seem to contribute to the increased storage stability of the present formulation.
  • Suitable compounds are rheological modifiers selected from the group consisting of hydrophobic and hydrophilic fumed and precipitated silica particles, gelling clays including bentonite, hectorite, laponite, attapulgite, sepiolite, smectite, or hydrophobically/organophilic modified bentonite.
  • Rheology modifiers are preferably derived from minerals. These rheology modifiers provide long term stability when the composition is at rest or in storage. Furthermore, it has been found in the course of the present invention that such rheology modifiers seem to contribute to the increased storage stability of the present formulation.
  • Suitable compounds are rheological modifiers selected from the group consisting of hydrophobic and hydrophilic fumed and precipitated silica particles, gelling clays including bentonite, hectorite, laponite, attapulgite, sepiolite, smectite, or hydrophobically/organophilic modified bentonite.
  • fumed or precipitated silica is preferred as rheology modifier.
  • Fumed silica also known as pyrogenic silica, either hydrophilic or hydrophobic, usually is composed of amorphous silica fused into branched, chainlike, three-dimensional secondary particles which then agglomerate into tertiary particles. The resulting powder has an extremely low bulk density and high surface area. Both hydrophilic and hydrophobic fumed silica can be used in the present invention
  • Fumed silica usually has a very strong thickening effect.
  • the primary particle size is ca. 5-50 nm.
  • the particles are non-porous and have a surface area of ca. 50-600 m 2 /g.
  • Hydrophilic fumed silica is made from flame pyrolysis of silicon tetrachloride or from quartz sand vaporized in a 3000°C electric arc.
  • Major global producers are Evonik Industries, tradename AEROSIL®); Cabot Corporation, tradename Cab-O-Sil®; Wacker Chemie, HDK product range; and OCI, tradename Konasil®.
  • Hydrophilic fumed silica can be hydrophobized by further treatment with reactive silicium-containing agents in order to modify the physicochemical properties of the silica.
  • hydrophobisation takes place by treatment of a hydrophilic fumed silica with agents like hexaalkyldisilanes (e.g. ((CHajaSijz), trialkylsilylchlorides (e.g. (CHajaSiCl) or dialkyldichlorsilanes (e.g. (CHajzSiCE).
  • Hydrophobized fumed silica is available e.g. from Evonik Industries (AEROSIL R-types), and Cabot (Cab-O-Sil).
  • Precipitated silica is produced by acidifying aqueous alkaline silicate solutions with mineral acids. Variations of the precipitation process lead to different precipitated silica qualities namely with different specific surface areas. The precipitates are washed and dried. Precipitated silica having a particle size of below 10 pm are most effective for the present invention.
  • the specific surface area is typically from ca. 50-500 m 2 /g.
  • Global producers are for example Evonik Industries, tradename SIPERNAT® or Wessalon®; Rhodia, tradename Tixosil®; and PPG Industries, tradename Hi-SilTM.
  • fumed silica having a BET surface area of about 200 m 2 /g obtainable e.g. as Aerosil® 200,
  • Clay thickeners are generally micronized layered silicates that can be effective thickeners for a wide range of applications. They are typically employed either in their non-hydrophobized or hydrophobized form. In order to make them dispersible in non-aqueous solvents, the clay surface is usually treated with quaternary ammonium salts. These modified clays are known as organo-modified clay thickeners. Optionally, small amounts of alcohols of low molecular weight or water may be employed as activators.
  • rheological modifiers examples include smectite, bentonite, hectorite, attapulgite, seipiolite or montmorillonite clays.
  • Preferred rheological modifiers (b) are for example organically modified hectorite clays such as Bentone® 38 and SD3, organically modified bentonite clays, such as Bentone® 34, SD1 and SD2, organically modified sepiolite such as Pangel® B20, hydrophilic silica such as Aerosil® 200, hydrophobic silica such as Aerosil® R972, R974 and R812S, attapulgite such as Attagel® 50,
  • Another class of suitable examples for rheology modifiers are organic rheological modifiers based on modified hydrogentated castor oil (trihydroxystearin) or castor oil organic derivatives such as Thixcin® R and Thixatrol® ST. Table B: Examples of rheology modifiers
  • Said rheology-modifying agent may be present in the formulation of the invention in a concentration of up to 0,07 % (w/w), preferably between 0,00003 % (w/w) and 0,04 % (w/w) and any other value in between this range
  • the formulation may in addition comprise a polyether-modified trisiloxane which is preferably of formula I
  • R 1 represents independent from each other identical or different hydrocarbyl radicals having 1 -8 carbon atoms, preferred methyl-, ethyl-, propyl- and phenyl radicals, particularly preferred are methyl radicals.
  • R 2 represents independent from each other identical or different polyether radicals of general formula (II)
  • R 4 independent from each other identical or different hydrocarbyl radicals having 1-12 carbon atoms or hydrogen radical, preferably a methyl-, ethyl-, phenyl- or a hydrogen radical.
  • R 5 independent from each other identical or different hydrocarbyl radicals having 1 -16 carbon atoms, which are optionally contain urethane functions, carbonyl functions or carboxylic acid ester functions, or hydrogen radical, preferred methyl or H, particularly preferred H.
  • polyether-modified trisiloxanes described above can be prepared by methods well known to the practioner by hydrosilylation reaction of a Si-H containing siloxane and unsaturated polyoxyalkylene derivatives, such as an allyl derivative, in the presence of a platinum catalyst.
  • the reaction and the catalysts employed have been described for example, by W. Noll in “Chemie und Technologic der Silicone”, 2 nd ed., Verlag Chemie, Weinheim (1968), by B.
  • Examples of water soluble or self-emulsifyable polyether-modified (PE/PP or block-CoPo PEPP) trisiloxanes include but are not limited to those described by CAS-No 27306-78-1 (e.g. Silwet L77 from MOMENTIVE), CAS-No 134180-76-0 (e.g. BreakThru S233 or BreakThru S240 from Evonik), CAS-No 67674-67-3 (e.g Silwet 408 from WACKER), other BreakThru-types, and other Silwet-types.
  • CAS-No 27306-78-1 e.g. Silwet L77 from MOMENTIVE
  • CAS-No 134180-76-0 e.g. BreakThru S233 or BreakThru S240 from Evonik
  • CAS-No 67674-67-3 e.g Silwet 408 from WACKER
  • other BreakThru-types e.g. Silwet 408 from WACKER
  • Preferred polyether-modified trisiloxanes include those described by CAS-No 134180-76-0, in particular Break-Thru S240.
  • the polyether-modified trisiloxane has the chemical denomination oxirane, mono(3-(l,3,3,3-tetramethyl-l-((trimethylsilyl)oxy)disiloxanyl) propyl)ether. It is most preferred that the polyether-modified trisiloxane is Breakthru S240.
  • the amount of polyether-modified trisiloxane, if present in the formulation, is at least 0,001% (w/w), such as at least 0,002 % (w/w) or at least 0,004 % (w/w).
  • the amount of polyether modified trisiloxane ranges between 0,001 % (w/w) and 0,4 % (w/w).
  • impurities in the water may be present in a composition according to the invention.
  • the nature of such impurities depends on the water source, e.g. rain-water or ground-water.
  • impurities can be (metal) ions and their counter ions, small particles of dirt (soil or sand) or organic impurities.
  • the amount of such additional components (impurities) is 1 % (w/w) or less, more preferably 0,5 % (w/w) or less, even more preferably 0,05 % (w/w) or less.
  • water contains impurities of 0,005 % (w/w) or less.
  • impurities are encompassed by the weight values of the water in a composition according to the invention, i.e. it is not required to separately measure the amounts of impurities for evaluating if a composition is a composition according to the invention.
  • composition according to the invention the sum of all components in said composition always sum up to 100, If not mentioned otherwise, the % (w/w) values given herein refer to the total weight amount of a composition according to the invention.
  • the present invention relates to a method for controlling insects and/or nematodes, preferably insects, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the composition according to the invention as described above to said plant or to a locus where plants are growing or intended to be grown.
  • the method is a method wherein a synergistic composition according to the invention is applied.
  • the amount of the composition according to the invention when brought to the field in accordance with any of the methods described herein, is at least 0,05 1/ha (hectare), such as between 1 to 10000 1/ha, 1 to 7500 1/ha, such as 801/ha to 50001/h, e.g., 100 1/ha, 200 1/ha, 300 1/ha, 400 1/ha, 500 1/ha, 600 1/ha, 700 1/ha, 800 1/ha, 900 1/ha, 1000 1/ha, 1100 1/ha, 1200 1/ha, 1300 1/ha, 1400 1/ha, 1500 1/ha,
  • the amount to the field preferably ranges between 1 and 10000 1/ha, such as between 2 1/ha and 40 1/ha, e.g.
  • the amount to the field preferably ranges between 1 and 10000 1/ha, such as between 2 1/ha and 40 1/ha, e.g.
  • the composition comprises C.javanica spores, e.g., for insect control
  • the amount to the field preferably ranges between 1 and 10000 1/ha, such as between 2 1/ha and 40 1/ha, e.g.
  • the skilled person will understand that the exact rate being dependent on the spore concentration in the composition according to the invention as well as on the kind of spores.
  • the number of spores in a composition according to the invention is usually between 1 x ] 0 4 and I xlO 9 spores/g composition, e.g. for soil or foliar application. Further preferred amounts are mentioned above.
  • the increase of efficacy of controlling insects and/or nematodes, preferably insects, of a composition according to the invention vs the same composition without the acid (wherein the amount of (missing) acid is substituted by water) is at least 5%, more preferably at least 10%, even more preferably at least 15%, such as 20% or more, 25% or more or even 30% or more.
  • the present invention relates to a method for an increased control of insects and/or nematodes, preferably insects, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the composition according to the invention as described above to said plant or to a locus where plants are growing or intended to be grown.
  • the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared with the effect of a composition without the acid as described herein but otherwise with all components as a composition according to the invention.
  • the method is a method wherein a synergistic composition according to the invention is applied.
  • Another aspect refers to the use of an acid as defined herein for increasing the efficacy of fungal spores as defined herein to control insects and/or nematodes, preferably insects.
  • the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared to the efficacy of said fungal spores in the absence if said acid.
  • the use refers to an increase which is caused by the presence of said acid in an amount of between 0,0001% (w/w) and 2% (w/w) in a spore formulation.
  • the present invention also relates to the use of an acid as defined herein for enhancing the efficacy of fungal spores in a composition according to the invention in agriculture.
  • the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared to the efficacy of said fungal spores in the absence if said acid.
  • the use refers to an increase which is caused by the presence of said acid in an amount of between 0,0001% (w/w) and 2% (w/w) in a spore formulation.
  • the present invention relates to the use of a composition as disclosed herein for controlling insects and/or nematodes, preferably insects, in, on and/or around a plant, for enhancing growth of a plant or for increasing plant yield or root health.
  • the use is a use of a synergistic composition according to the invention.
  • the present invention relates to the use of a composition as disclosed herein for an enhances control of insects and/or nematodes, preferably insects, in, on and/or around a plant, for enhancing growth of a plant or for increasing plant yield or root health, wherein the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared with the effect of a composition without the acid as described herein but otherwise with all components as a composition according to the invention, wherein the weight of (missing) acid is replaced by water to ensure the % (w/w) values of the other components are essentially the same.
  • the use is a use of a synergistic composition according to the invention.
  • Plants which can be treated in accordance with the invention include the following main crop plants: maize, soya bean, alfalfa, cotton, sunflower, Brassica oil seeds such as Brassica napus (e.g. canola, rapeseed), Brassica rapa, B. juncea (e.g. (field) mustard) and Brassica carinata, Arecaceae sp. (e.g. oilpalm, coconut), rice, wheat, sugar beet, sugarcane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vine and various fruit and vegetables from various botanic taxa, e.g. Rosaceae sp. (e.g.
  • pome fruits such as apples and pears, but also stone fruits such as apricots, cherries, almonds, plums and peaches, and berry fruits such as strawberries, raspberries, red and black currant and gooseberry), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (e.g. olive tree), Actinidaceae sp., Lauraceae sp. (e.g. avocado, cinnamon, camphor), Musaceae sp. (e.g.
  • Rubiaceae sp. e.g. coffee
  • Theaceae sp. e.g. tea
  • Sterculiceae sp. e.g. lemons, oranges, mandarins and grapefruit
  • Solanaceae sp. e.g. tomatoes, potatoes, peppers, capsicum, aubergines, tobacco
  • Liliaceae sp. Compositae sp. (e.g. lettuce, artichokes and chicory — including root chicory, endive or common chicory), Umbelliferae sp. (e.g.
  • Cucurbitaceae sp. e.g. cucumbers — including gherkins, pumpkins, watermelons, calabashes and melons
  • Alliaceae sp. e.g. leeks and onions
  • Cruciferae sp. e.g. white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, pak choi, kohlrabi, radishes, horseradish, cress and Chinese cabbage
  • Leguminosae sp. e.g. peanuts, peas, lentils and beans — e.g. common beans and broad beans
  • Chenopodiaceae sp. e.g.
  • formulations according to the invention are used by spray application against insect pests from the following insect families:
  • Bemisia and Trialeurodes specifically to Bemesia tabaci (silverleaf whitefly) and Trialeurodes vaporariorum, (greenhouse whitefly) in crops such as, for example, soybean, melon, tabacco, tomatoes, squash, poinsettia, cucumber, eggplants, okra, beans and cotton.
  • Ceroplastes spp. Ceroplastes spp., Drosicha spp., Pulvinaria spp., Protopulminaria spp., Saissetia spp., Coccus spp., in perennial crops such as, for example, citrus fruit, pomaceous fruit, stone fruit, olives, grapevines, coffee, tea, tropical crops, ornamental plants, vegetables.
  • Diaspididae the family of the armoured scale insects
  • Quadraspidiotus spp. Aonidiella spp., Lepidosaphes spp., Aspidiotus spp., Aspis spp., Diaspis spp., Parlatoria spp., Pseudaulacaspis spp., Unaspis spp., Pinnaspis spp., Selenaspidus spp., in crops such as, for example, citrus fruit, pomaceous fruit, stone fruit, almonds, pistachios, nuts, olives, tea, ornamental plants, grapevines, tropical crops.
  • Thripidae preference is given from the family of the thrips (Thripidae) to: Anaphothrips spp., Basothrips spp., Caliothrips spp., Frankliniella spp., Heliothrips spp., Hercinothrips spp., Rhipiphorothrips spp., Scirtothrips spp., Kakothrips spp., Selenothrips spp. and Thrips spp., in crops such as, for example, fruit, cotton, grapevines, tea, nuts, tropical crops, ornamental plants, conifers, tobacco, spices, vegetables, soft fruit, melons, citrus fruit and potatoes.
  • crops such as, for example, fruit, cotton, grapevines, tea, nuts, tropical crops, ornamental plants, conifers, tobacco, spices, vegetables, soft fruit, melons, citrus fruit and potatoes.
  • Agromyza spp. Agromyza spp., Amauromyza spp., Atherigona spp., Chlorops spp., Liriomyza spp., Oscinella spp., Pegomyia spp. in crops such as, for example, vegetables, melons, potatoes, nuts, ornamental plants.
  • Caloptilia spp. Gracillaria spp., Lithocolletis spp., Leucoptera spp., Phtorimaea spp., Phyllocnistis spp. in crops such as pomaceous fruit, stone fruit, grapevines, nuts, citrus fruit, conifers, potatoes, coffee.
  • Anastrepha spp. Ceratitis spp., Dacus spp., Rhagoletis spp. in crops such as vegetables, soft fruit, melons, pomaceous and stone fruit, ornamental plants, potatoes, grapevines, tropical crops, citrus fruit, olives.
  • mites from the families of the spider mites (Tetranychidae) and the gall mites (Eriophydae):
  • Tetranychus spp. Tetranychus spp., Panonychus spp., Aculops spp. in crops such as vegetables, potatoes, ornamental plants, citrus fruit, grapevines, conifers.
  • the inventive treatment of the plants and parts of plants with the compositions according to the invention is effected directly or by allowing the compositions to act on their surroundings, environment or storage space by the customary treatment methods, for example by drenching, immersion, spraying, evaporation, fogging, scattering, painting on and, in the case of propagation material, in particular in the case of seeds, also by applying one or more coats.
  • the plant to be treated is selected from the group consisting of cotton, soya beans, tobacco, vegetables, spices, ornamental plants, conifers, citrus plants, fruit, tropical crops, nuts and grapevines.
  • the composition according to the invention acts against pests from the families of the woolly aphids, grape lice, jumping plant lice, soft scales, armored scale insects, ensign scales, mealy bugs, whiteflies, aphids, thrips, leafhoppers, planthoppers, leaf-miner flies, gall midges, fruit flies, leaf- miner moths, spider mites, gall mites.
  • the composition according to the invention acts against Diptera, including species from the family Culicidae such as Anopheles spp., Aedes spp., and Culex spp.
  • further families are Anthomyiidae, Tephritidae, Muscidae, Agromyzidae, Drosophilidae, Tabanidae, Calliphoridae, Cecidomyiidae, Tachinidae, Mycetophilidae, Simuliidae, Stratiomyidae or Psychodidae.
  • the composition may comprise spores of C. javanica Apopka 97 and citric acid.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of C. javanica Apopka 97 and one or more of the acids provided in Table 4.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of C.
  • javanica Apopka 97 and one or more of oxalic acid, dipicolinic acid, citric acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, phosphoric acid, aconitic acid, tartaric acid, succinic acid, malonic acid and/or malic acid.
  • the composition may comprise spores of C.
  • javanica Apopka 97 and one or more of 0,05% oxalic acid, 0,05% dipicolinic acid, 0,05% citric acid, 0,1% citric acid, 0,15% citric acid, 0,2% citric acid, 0,05% trimesic acid, 0,05% aconitic acid, 0,05% phosphoric acid, 0,1% benzoic acid, 0,1% sorbic acid, 0,1% acetic acid, 0,1% lactic acid, 0,1% ascorbic acid, 0,08% glutamic acid, 0,04% glycine, 0,05% serine, 0,025% phosphoric acid, 0,09% aconitic acid, 0,08% tartaric acid, 0,06% succinic acid, 0,05% malonic acid and/or 0,07% malic acid.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of C.javanica GF 511 and citric acid.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of C. javanica GF 511 and one or more of the acids provided in Table 4.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of C.
  • the composition may comprise spores of C. javanica GF 511 and 0,05% citric acid.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of M. brunneun F52 and citric acid.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of M. brunneun F52 and one or more of the acids provided in Table 4.
  • the composition may increase white fly mortality.
  • the composition may comprise spores of M.
  • the composition may increase white fly mortality.
  • the entomopathogenic fungi and the acid may be comprised within a kit.
  • the entomopathogenic fungi and the acid may be spatially separated within the kit, for example the kit may comprise at least two housings or containers, one comprising the entomopathogenic fungi and one comprising acid.
  • the entomopathogenic fungi and acid may be comprised in separate housings or containers.
  • the entomopathogenic fungi may be present as spores.
  • the entomopathogenic fungal spores may be in powder form, optionally a wettable powder form.
  • the entomopathogenic fungal spores may be dispersed in an aqueous solution or dispersed in a non-aqueous solution.
  • the entomopathogenic fungal spores may be spores of any species described herein, prefereably of Cordyceps fumosorosea, Cordyceps javanica or Metarhizium brunneum.
  • the entomopathogenic fungal spores may be spores of a Cordyceps javanica Apopka 97 strain or Cordyceps javanica GF 511 strain.
  • the entomopathogenic fungal spores may be spores of a Metarhizium brunneum F52 strain.
  • the kit may additionally comprise instructions or a description of how to prepare a composition of the invention or apply a method of the invention.
  • the present invention relates to a kit-of-parts comprising an entomopathogenic fungi to prepare a composition according to the invention and a description how to prepare a composition according to the invention.
  • kit-of-part further comprises one or more acids to prepare a composition according to the invention.
  • the entomopathogenic fungi being spatially separated in a container. More preferably, the entomopathogenic fungi and the acid (if present in a kits-of-parts) are separated from each other in two spatially separated containers, thus resulting in the kit-of-parts comprising two separated components and a description how to prepare a composition according to the invention.
  • the kit of parts according to the present invention can additionally comprise at least one auxiliary selected from the group consisting of extenders, solvents, spontaneity promoters, carriers, emulsifiers, dispersants, frost protectants, thickeners and adjuvants as mentioned herein.
  • This at least one auxiliary can be present in one or more than one component of the kit of parts, e.g. in the component comprising an entomopathogenic fungi or in the component comprising an acid, being spatially separated from each other, or in both of these components or in the form of a separate component thus resulting in the kit-of-parts comprising at least three components spatially separated from each other.
  • the description how to prepare a composition according to the invention gives the skilled person the information regarding mixing ratios between an acid, preferably being already part of the kits-in-parts, water and an entomopathogenic fungi formulation to arrive at a composition according to the invention.
  • an acid preferably being already part of the kits-in-parts, water and an entomopathogenic fungi formulation to arrive at a composition according to the invention.
  • the description will provide information how much solid form (powder) has to be combined with how much water and how much acid to arrive at a composition according to the invention.
  • the preparation of a composition according to the invention can include further steps, e.g., of adding further components or presolubilizing or pre -emulsifying the acid or the fungal spores, such as pre -emulsifying a solid entomopathogenic fungi-preparation, before combining the pre -emulsified or pre-solubilized component with a further component, such as (e.g., a pre-solubilized) acid.
  • the description preferably comprises time limits between the preparation of a composition according to the invention and its use (e.g. spraying the composition on a plant, plant part or habitat of a plant).
  • the preparation of a composition according to the invention takes place not longer than 48 hours before the use of the composition according to the invention. More preferably, the preparation of a composition according to the invention takes place not longer than 24 hours before the use of the composition according to the invention, even more preferably not longer than 12 or even 8 hours before the use of the composition according to the invention.
  • a composition comprising water, an acid, and an entomopathogenic fungus, preferably spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid is at least 1 g/1, and wherein the pH of the composition is between 2,0 and 7,0, and wherein the pH of said composition is lower than the pH of a comparative composition without said acid.
  • composition of embodiment 1, wherein the acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alphaketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p-toluenesulfonic acid, succinic acid, acrylic acid, aspartic
  • composition of embodiment 1 or embodiment 2, wherein the acid is an organic acid selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, and glycine.
  • the acid is an organic acid selected from the group consisting of malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid and glycine.
  • composition according to any one of the preceding embodiments wherein the decrease in pH is at least 0,1 pH units.
  • the entomopathogenic fungus is selected from the group consisting of Beauveria bassiana (preferably strain ATCC 74040, strain GHA (Accession No. ATCC74250), strain ATP02 (Accession No.
  • DSM 24665) strain PPRI 5339, strain PPRI 7315, strain R444, strains IL197, IL12, IL236, IL10, IL131, IL116, strain Bv025, strain BaGPK, strain ICPE 279, strain CG 716, strain HF23, strain ANT-03, strain NPP111B00, strain 203, strain BblO, strain ESALQ PL63, strain IMI389521, strain IBCB 66, strain K4B1, strain K4B3, strain CBMAI 1306; and Metarhizium brunneum (formely known as Metarhizium anisopliae) (preferably strain Cbl5, strain ESALQ 1037, strain E-9, strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013-1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448), strain ICIPE
  • composition according to any one of the preceding embodiments wherein the entomopathogenic fungus is selected from the group consisting of Cordyceps fumosorosea strain Apopka 97, strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367; Cordyceps javanica strain wf GA 17, strain GZQ-1, strain IjH6102, strain pfl85, strain pf212, strain JZ-7, or strain IJNL-N8 / CCTCC M-2017709; and Isaria farinosa strain ESALQ1205.
  • composition according to any one of the preceding embodiments, wherein the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
  • composition according to any one of the preceding embodiments further comprising at least one further component selected from the group consisting of liquids for spore formulations, oil, antioxidant, emulsifier, rheology modifier, other components being present in a fungal spore formulation, impurities which can be found in water.
  • a Method for controlling insects and/or nematodes, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the composition according to any one of embodiment 1 to 10 to said plant or to a locus where plants are growing or intended to be grown. 13. The method according to embodiment 12, wherein the amount of composition, is at least 0,05 1/ha.
  • Kit-of-parts comprising a biological control agent selected from the group consisting of at least one spatially separated entomopathogenic fungi as defined in embodiment 1 , 6 or 7 and a description how to prepare a composition according to any one of embodiments 1 to 10.
  • Kit-in-parts further comprising at least one spatially separated acid as defined in any one of embodiments 1 to 4.
  • C. fumosorosea strain Apopka 97 solid state fermentation can be used as described in Mascarin et al. (2013, “The virulence of entomopathogenic fungi against Bemisia tabaci biotype B (Hemiptera: Aleyrodidae) and their conidial production using solid substrate fermentation”, Biological Control https://doi.Org/10.1016/j.biocontrol.2013.05.001 ).
  • C. fumosorosea can be grown on Agar-based medium SDAY (sabouraud dextrose agar plus yeast extract) for 14 days at 26°C.
  • a conidial suspension was then obtained by washing off the conidia with water with surfactant (0.1 % of Tween80) and using a cell scraper.
  • surfactant 0.1 % of Tween80
  • spores were harvested by vacuuming and collecting them in a cyclone (Jaronski et al., 2007, “Chapter 11 - Mass Production of Entomopathogenic Fungi: State of the Art”, Mass Production of Beneficial Organisms https://doi.org/10.1016/B978-0-12-391453-8.00011-X). Harvested spores were dried resulting in a concentration of 2E+11 spores per g technical spore concentrate.
  • the ingredients were 20% (w/w) spores, 77,5 % (w/w) Polysorbate 20 and 2,5% (w/w) Aerosil R202. Blending was done using an Ultra-Turrax dispersing instrument (IKA, Germany). The formulations had spore concentrations of 4E+10 spores per g spore formulation. Corresponding blank type 1 and blank type 2 formulations were prepared according to the respective instructions given above, with the exception that the addition of fungal spores was omitted (i.e. they contain only formulants but no spores).
  • single-leaf cotton plants (Gossypium herbaceum) were infected with Bemisia tabaci by exposing the plants to respective white fly populations for 24h which results in an extensive deposition of eggs on the cotton leaves. Afterwards the plants were incubated for 8-10 days at 25°C until hatching and development of immobile N1 nymphal stages. Per experiment, five single-leaf cotton plants were used and treated as five biological replicates. For the treatments, C. fumosorosea conidia were suspended in tap water. If pure spores were used, the spore powder was put in suspension by using a Potter-Elvehjem homogenizer.
  • Formulated spores were added to water and stirred to gain homogenous suspensions. Likewise, blank formulations were dispersed and stirred in water to generate control treatments containing an equal concentrations of formulation ingredients. Spore concentrations for each treatment were determined using a haemocytometer and if not otherwise indicated spore concentrations were adjusted to a final concentration of l,0E+07 conidia/ml. E.g., to prepare a treatment with l,0E+07 spores/ml, 50 mg of the pure spore powder or 250 mg of the formulated spores were suspended in 100 ml water followed by a 1/10 dilution step with water.
  • stock solutions of the respective compound in water were made first, before adding an appropriate volume of the stock solutions to the treatment.
  • dipicolinic acid and glutamic acid a 0,5% stock solution was made.
  • oxalic acid citric acid, malonic acid, malic acid, fumaric acid adipic acid, pimelic acid, tartaric acid, itaconic acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, lysine, glycine, serine, p- Toluenesulfonic acid, succinic acid and propionic acid a 1% stock solution was made.
  • X represents the percentage of control (Abbott efficacy) of treatment 1
  • Y represents the percentage of control of treatment 2
  • E equals the expected percentage of control of a combined treatment 1 and 2
  • the observed values for the combined treatments were compared with the expected values: when E was less than the observed value, the composition was considered synergistic, when E was greater than the observed value, the composition was considered antagonistic, and when E equaled the observed value, the composition was identified as additive.
  • Example 2 Synergistic action of the entomopathogenic fungus C. fumosorosea and citric acid for insect control
  • Figure 1 shows the mortality of white flies after treatment with C. fumosorosea and citric acid.
  • the mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 20 leaves were examined. To test for significant differences, the treatment of l,0E+07 spores/ml was set as a reference group (reference) for multiple pairwise t- tests. *** and **** denote p-values of ⁇ 0,001 and ⁇ 0,0001, respectively.
  • the error bars represent standard error and the pH value of each spray liquid is indicated on the right of the bars.
  • Example 3 Synergistic action of C. fumosorosea formulated in PEG400 mono oleate and citric acid for insect control
  • Figure 2 shows the mortality of white flies after treatment with type 1 formulated C. fumosorosea and citric acid.
  • the mortality of white flies was calculated for the indicated treatments as described in the examples of the main text.
  • a total of 45 leaves were examined (44 leaves for the water control).
  • the treatment of l,0E+07 spores/ml was set as reference group for multiple pairwise t-tests.
  • the error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
  • Table 2 Synergistic action of type 1 formulated C. fumosorosea and citric acid for the control of white flies
  • Example 4 Synergistic action of C. fumosorosea formulated in Polysorbate 20 and citric acid for insect control
  • Figure 3 shows the mortality of white flies after treatment with type 2 formulated C. fumosorosea and citric acid.
  • the mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 5 leaves were examined. To test for significant differences, the treatment of 1 ,0E+07 spores/ml was set as reference group for multiple pairwise t- tests. * and ** denote p-values of ⁇ 0,05 and ⁇ 0,01, respectively, ns denote p-values of >0,05.
  • the error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
  • Table 3 Synergistic action of type 2 formulated C. fumosorosea and citric acid for the control of white flies
  • Example 5 Impact of the concentration of citric acid to increase the efficacy of C. fumosorosea.
  • Figure 4 shows the mortality of white flies after treatment with formulated C. fumosorosea and citric acid concentrations of 0,005% - 0,05%.
  • the mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 15 leaves were examined. To test for significant differences, the treatment of l,0E+07 spores/ml was set as reference group for multiple pairwise t-tests. **** denotes p-values of ⁇ 0,0001. ns denote p-values of >0,05.
  • the error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
  • the insect mortalities of the treatments revealed that citric acid concentrations of 0,005% and 0,01% increase the efficacy of C. fumosorosea ( Figure 4), despite that the observed increase was not statistically significant.
  • Figure 5 shows the mortality of white flies after treatment with formulated C. fumosorosea and citric acid concentrations of 0,05% - 0,25%.
  • the mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 5 leaves were examined. To test for significant differences, the treatment of l,0E+07 spores/ml was set as reference group for multiple pairwise t-tests. *, ** and *** denote p-values of ⁇ 0,05, ⁇ 0,01 and ⁇ 0,001, respectively, ns denote p-values of >0,05.
  • the error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
  • Citric acid increases the efficacy of various spore concentrations of C. fumosorosea.
  • Figure 6 shows the mortality of white flies after treatment with various concentrations of formulated C. fumosorosea and citric acid concentrations of 0,05%.
  • the mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 5 leaves were examined. To test for significant differences, multiple pairwise t-tests were performed. ** and **** denote p-values of ⁇ 0,01 and ⁇ 0,0001, respectively.
  • the error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
  • Example 7 Mortality of white flies after combined treatments with C. fumosorosea and various acids
  • Blank type 1 formulation + 0,05% Aconitic acid 14 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Aconitic acid 62,2 (47,9) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,4
  • Blank type 1 formulation + 0,1% Benzoic acid 0,2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,1% Benzoic acid 55,3 (22,3) l,0E+7 s/ml + 0,02% Blank type 1 formulation 22,1
  • Blank type 1 formulation + 0,05% Serine 7,7 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Serine 53,2 (42,1) l,0E+7 s/ml + 0,02% Blank type 1 formulation 37,3
  • Blank type 1 formulation + 0,04% p-Toluenesulfonic acid -11.2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,04% p-Toluenesulfonic acid 29,2 (30,3) l,0E+7 s/ml + 0,02% Blank type 1 formulation 27
  • Figure 7 shows the correlation between pH of the spray liquid containing acids and C. fumosorosea spores and mortality of white flies. In total, 359 leaves which were all treated with l,0E+07 spores/ml with or without addition of various acids, were examined.
  • Example 8 Synergistic action of an enthomopathogenic fungus C. javanica strain GF 511 and citric acid, for insect control
  • the amount of this suspension needed to reach l,0E+07 spores per ml in 20 ml was calculated.
  • the initial spore suspension contained l,0E+08 spores/ml then 2 ml of this spore suspension was added to 17 ml of water followed by 1 ml of the 1% stock solution of citric acid.
  • Nymphal mortality was assessed under a dissecting stereomicroscope (Leica) at x40 magnification.
  • Leica dissecting stereomicroscope
  • the leaves were then incubated inside Gerbox box with a wet cotton added to the leaf petiole for four days at room temperature.
  • the dead marked nymphs presenting sporulation i.e., mycosed nymphs
  • Tween 80 (0.05% v/v) was set as control treatment and Abbott’s formula (Abbott, 1925) was used to calculate the efficacy of the treatment with pure spores and the efficacy of the treatment with citric acid and the efficacy of the compositions of both treatments (Table 6).

Abstract

The present invention refers to the use of acids to enhance efficacy of fungal spores and formulations comprising the same.

Description

Beneficial acids for enhancing biocontrol efficacy of fungal spores
Background
The application of entomopathogenic fungi against insect pests is an effective and sustainable approach in plant protection. However, full control with such treatments has been proven difficult to achieve, and the reached efficacy is usually lower than the efficacy of current chemical crop protection agents. It is desired to increase the efficacy of entomopathogenic fungi in order to provide more flexibility for growers to control important insect pests.
Bidochka and Khachatourians (1991, “The implication of metabolic acids produced by Beauveria bassiana in pathogenesis of the migratory grasshopper, Melanplus sanguinipes.”, Journal of Invertebrate Pathology DOI: 10.1016/0022-2011 (91)90168-P) demonstrated that the entomopathogenic fungus Beauveria bassiana produces oxalic acid and citric acid. In addition, it was shown that the external addition of oxalic acid and citric acid to B. bassiana increased the virulence of this fungus towards grasshoppers. Sabbour et al. (2002, “The role of chemcial additives in enhancing the efficacy of Beauveria bassiana and Metarhizium anisopliae against the potato tuber moth Phthorimaea operculella.”, Pakistan Journal of Biological Control DOI: 10.3923/PJBS.2002.1155.1159) showed that several additives including oxalic acid and citric acid, seem to have a positive effect on virulence of B. bassiana and M. anisopliae against potato tuber moths.
Based on literature, it was only known for two species of entomopathogenic fungi, i.e. for B. bassiana and M. anisopliae that external addition of organic acids such as oxalic acid and citric acid can increase their virulence. Such a virulence increasing effect was however not described for species of the fungal genus Cordyceps (formely known as Isaria or Paecilomyces), nor was it found for any other entomopathogenic fungi. Cordyceps spp. are in particular known for their high virulence against lepidopteran and hemipteran pests (Zimmermann, 2008, “The entomopathogenic fungi Isaria farinosa (formerly Paecilomyces farinosus) and the Isaria fumosorosea species complex (formerly Paecilomyces fumosoroseus): biology, ecology and use in biological control” Biocontrol Science and Technology DOI:10.1080/09583150802471812). Interestingly, Cordyceps fumosorosea abundantly produces dipicolinic acid, and it was further shown that dipicolinic acid has toxic activity against insects (Asaff et al., 2005, "Isolation of dipicolinic acid as an insecticidal toxin from Paecilomyces fumosoroseus" Appl Microbiol Biotechnol DOI:10.1007/s00253-005-1909-2; Weng et al., 2019, "Secondary Metabolites and the Risks of Isaria fumosorosea and Isaria farinosa" molecules D01:10.3390/molecules24040664). Thus, it could well be that the external addition of dipicolinic acid to C. fumosorosea spores increases the fungal virulence. This hypothesis was however not tested. To which extend Cordyceps spp. (formely known as Isaria spp.) actively secrete organic acids including citric acid is not disclosed and largely unknown. By supplementing the spores of C. fumosorosea with low concentrations of acids in the spray liquid, it was surprisingly found that the addition of various acids including citric acid and dipicolinic acid increases the efficacy of this entomopathogenic fungus against white flies after spray application.
Summary
A first aspect of the present invention refers to a preparation comprising an acid and spores of an entomopathogenic fungus.
A second aspect of the present invention refers to a composition comprising water, an acid, and spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid is at least 1 g/1, and wherein the pH of the composition is between 2,0 and 7,0, and wherein the pH of said composition is lower than the pH of a comparative composition without said acid.
In a third aspect the invention refers to use of an acid as described herein for increasing the efficacy of entomopathogenic fungal spores as described herein to control insects and/or nematodes.
In a fourth aspect the invention refers to a method for enhancing the efficacy of fungal spores comprising contacting spores of an entomopathogenic fungus as described herein with an acid as described herein.
In a fifth aspect the invention refers to a method for controlling insects and/or nematodes, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of a preparation or composition of the invention to said plant or to a locus where plants are growing or intended to be grown.
In a sixth aspect the invention refers to a kit comprising entomopathogenic fungi as described herein and an acid as described herein, wherein the entomopathogenic fungi and the acid are spatially separated, and wherein said kit is optionally suitable for preparation of a composition of the invention.
A preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p -toluenesulfonic acid, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, isocinchomeronic acid, isocitric acid, isonicotinic acid, lutidinic acid, maleic acid, mandelic acid, nicotinic acid, phtalic acid, picolinic acid, propionic acid, salicylic acid, tetronic acid, methane sulfonic acid. A further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the acid is an organic acid selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, and glycine.
A further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the acid is an organic acid selected from the group consisting of malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, glycine.
A further preferred embodiment of Aspect 2 and any of its embodiments refers to the composition, wherein the decrease in pH is at least 0,1 pH units.
A further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the entomopathogenic fungus is selected from the group consisting of Beauveria bassiana (preferably strain ATCC 74040 (e.g. Naturalis® from CBC Europe, Italy; Contego BB from Biological Solutions Ltd.; Racer from AgriLife), strain GHA (Accession No. ATCC74250; e.g. BotaniGuard Es and Mycontrol-0 from Laverlam International Corporation), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339 (e.g. BroadBand™ from BASE), strain PPRI 7315, strain R444 (e.g. Bb-Protec from Andermatt Biocontrol), strains IL197, IL12, IL236, IL10, IL131, IL116 (all referenced in Jaronski, 2007, Use of Entomopathogenic Fungi in Biological Pest Management, 2007: ISBN: 978-81-308-0192-6), strain Bv025, strain BaGPK, strain ICPE 279, strain CG 716 (e.g. BoveMax® from Novozymes), strain HF23, strain ANT-03 (from Bioceres), strain NPP111B00, strain 203 (e.g. Phoemyc from Glen Biotech), strain Bbl 0 (from Biagro Bb), strain ESALQ PL63 (from Boveril, Koppert), strain IMI389521, strain IBCB 66 (e.g. from Atrevido, Koppert), strain K4B1 (Beaugenic from Ecolibrium Biologicals), strain K4B3 (Beaublast from Ecolibrium Biologicals), strain CBMAI 1306 (from Auin CE Agrivalle); and Metarhizium brunneum (formely known as Metarhizium anisopliae) (preferably strain Cbl5 (e.g. ATTRACAP® from Biocare), strain ESALQ 1037 (e.g. from Metarril® SP Organic), strain E-9 (e.g. from Metarril® SP Organic), strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013- 1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448 (e.g. BIO 1020 by Bayer CropScience and also e.g. Met52 by Novozymes), strain ICIPE 78, strain ESF1 (from BIO-BLAST), strain IBCB 425 (from Metarhizium Oligos, Oligos Biotecnologia), strain LRC112 (from SpudSmart, AAFC)).
A further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, wherein the entomopathogenic fungus is selected from the group consisting of Cor dy ceps fumosorosea strain Apopka 97, strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367; Cordyceps javanica strain wf GA 17, strain GZQ-1, strain IjH6102, strain pfl85, strain pf212, strain JZ-7, or strain IJNL-N8 / CCTCC M-2017709; and Isaria farinosa strain ESALQ1205.
A further preferred embodiment of Aspect 2 and any of its embodiments refers to the composition, wherein the concentration of the fungi is between l,0*104 spores per ml composition and l,0*109 spores per ml composition.
A further preferred embodiment of Aspect 2 and any of its embodiments refers to the composition, wherein the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
A further preferred embodiment of Aspect 1 or 2 and any of its embodiments refers to the preparation or composition, further comprising at least one further component selected from the group consisting of liquids for spore formulations oil, antioxidant, emulsifier, rheology modifier, other components being present in a fungal spore formulation, impurities which can be found in water.
Another aspect refers to the use of an acid as defined in Aspect 1 , 2 or 3 and any of its embodiments for increasing the efficacy of fungal spores of the invention to control insects and/or nematodes.
Another aspect refers to a method for controlling insects and/or nematodes, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the preparation according to Aspect 1 , the composition according to Aspect 2 or any of its embodiments to said plant or to a locus where plants are growing or intended to be grown.
In some embodiments the amount of preparation or composition, is at least 0,05 1/ha.
Another aspect refers to a Kit-of-parts comprising a biological control agent selected from the group consisting of at least one spatially separated entomopathogenic fungi as defined in aspect 2 and any of its embodiments and a description how to prepare a composition according to aspect 2 and any of its embodiments.
In some embodiments the Kit-in-parts further comprises at least one spatially separated acid as defined in aspect 2 or any of its preferred embodiments.
Definition
The term “about”, whenever used in connection with the present invention, relates to the mentioned sumerical value +/- 10%.
The term “acid” as used herein refers to a chemical compound that is able to transfer protons (H+) to water. As a result of the proton transfer, oxonium ions (I I T) ) are formed and the pH value of an aqueous solution is lowered. The term encompasses organic and inorganic acids.
The term “at least” indicates that in any case one agent as described herein (e.g. one acid or one antioxidant, etc.) is present in a composition according to the invention. However, more than one such as (at least) two, (at least) three, (at least) four, (at least) five or even more such agents (e.g. acids, antioxidants, etc.) may be present in a composition according to the invention.
The term “antioxidants” refers to compounds which inhibit oxidation of other molecules. Whereas Applicant does not wish to be bound by any scientific theory, it is believed that a certain concentration of antioxidant in a composition contributes to a better storage stability of the formulation, in particular the long stability of the fungal spores comprised therein. An antioxidant may be any suitable antioxidant. Non-limiting examples are butylhydroxytoluol (BHT), butylhydroxyanisole (BHA), ascorbyl palmitate, tocopheryl acetate, ascorbyl stearate or the group of carotinoids (e.g. beta-carotin) or gallates (e.g. ethyl gallate, propyl gallate, octyl gallate, dodecyl gallate).
The term “comparative composition” as used herein refers to a composition with essentially the same components as a composition according to the invention with the difference that the amount (% (w/w) of acid as described herein and which is an essential part of a composition according to the invention is replaced by water. In other words, in a comparative composition, the missing amount of the acid (i.e. a value between 0,0001% (w/w) and 2% (w/w)) is replaced by water in said comparative composition, the ratios of all other components (e.g., the entomopathogenic fungus and, e.g., optionally present additives of said fungus such as oils, emulsifiers, antioxidants, rheology modifiers etc.) based on the total weight of the composition according to the invention, stay the same as in the composition according to the invention which is compared to a comparative composition.
“Increase of efficacy” in the context of the present invention means the increase of the Abbott efficacy of a composition according to the invention compared to the Abbott efficacy of a comparative composition and the Abbott efficacy of the acid in the same concentration as in the formulation according to the invention. For example, when the Abbott efficacy of a composition of the invention (AE1) is 57,0 and the Abbott efficacy of the comparative composition (AE2) is 34,3 and the Abbott efficacy of the acid (AE3) is 7,6, then the increase of efficacy is calculated as follows (AEl/(AE2+AE3)*100)-100 = increase of efficacy: (57,0/(34,3+7,6)* 100)-l 00=36%.
The term “inorganic acid” as used herein refers to all acids that do not contain carbon, e.g. sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, silicic acid, boric acid.
The term "organic acid" as used herein refers to carbon-containing molecules that carry functional groups that are able to transfer protons to the aqueous medium and thus lower the pH. Thus, H2CO3 (carbonic acid) is for the purpose of this invention an organic acid. Non-limiting examples for organic acids are listed in Table A:
Table A: Examples of organic acids
Figure imgf000006_0001
Figure imgf000007_0001
Figure imgf000008_0001
The term “entomopathogenic fungus” as used herein refers to a fungus that can act as a parasite of insects and kills or seriously disables them. The entomopathogenic fungi include taxa from several of the main fungal groups. Non-limiting examples of entomopathogenic fungi are in the order Hypocreales of the Ascomycota: the asexual (anamorph) phases Beauveria, Metarhizium, Cordyceps (Isaria, Paecilomyces), Hirsutella, Lecanicillium, Nomuraea and their sexual (teleomorph) states; or are in the order Entomophthorales of the Zygomycota: Entomophthora, Zoophthora, Pandora, Entomophaga.
“Fungal spores” include sexually (e. g. oospores, zygospores or ascospores) and asexually (e. g. conidia and chlamydospores, but also uredospores, teleutospores, ustospores and blastospores) formed spores. Preferably the spores are conidia.
Integral values are usually given in a* 10b format, wherein a and b can represent any number. The same information can be given by the aEb format. For example, l,0*102 is equal to l,0E+02, Both refer to the value 100.
Numbers as used in the present application, if applicable, are usually written with a e.g. 2,5 refers to 5/2. In case a number in the present application is written with a instead of a”,”, such a number is regarded as being written with a In other words, 2.5 is equal to 2,5 is equal to 5/2; “1,000” does not mean 1000 (one thousand) but 1,000 (one); l,0E+02 is equal to 1.0E+02 is equal to 100.
Plant oils or vegetable oils are oils derived from plant sources, as opposed to animal fats or petroleum. Among plant oils, the ones preferably used in the present invention are triglyceride -based vegetable oils which are liquid at least at room temperature, preferably also at temperatures below room temperature, such as at 15°C, at 10°C or even at 5°C or 4°C.
When referring to amounts of a component or ranges of amounts of a component, the term “%” refers to % (w/w) (weight percent) of not mentioned otherwise. The terms “% (w/w)” and “wt.-%.” can be used interchangeably, herein.
The term “between a and b” as used herein (wherein a and b are numbers) means the threshold values a and b as well as all values between these the threshold values a and b. The terms “spores/ml” and “s/ml” are used interchangeably, herein.
Detailed description
It was surprisingly found that acids with specific characteristics can enhance the efficacy of entomopathogenic fungi in an aqueous composition, more preferably the efficacy of spores of an entomopathogenic fungi, even more preferred the efficacy of conidia. Even a synergistic effect can be observed.
The present disclosure provides a method for increasing the efficacy of fungal spores comprising contacting spores of an entomopathogenic fungus with an acid wherein upon contacting the fungal spores and the acid, the fungal spores exhibit an improved efficacy in inhibiting pests or exhibiting an increased pest control compared to contacting the fungal spores to the host without the acid.
Thus, the present invention refers inter alia to a composition comprising water, an acid, and an entomopathogenic fungus, preferably spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid is at least 1 g/1, and wherein the pH of the composition is between 2,0 and 7,0, and wherein the pH of said composition is lower than the pH of a comparative composition without said acid.
The present invention further refers to a composition comprising an acid and an entomopathogenic fungus, preferably spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid once the composition is diluted in water is at least 1 g/1, and wherein the pH of the composition diluted in water is between 2,0 and 7,0, and wherein the pH of said composition diluted in water is lower than the pH of a comparative composition without said acid.
In one preferred embodiment, the decrease of the pH value of a composition with acid (composition according to the invention) compared to a comparative composition is at least 0,1 pH units, more preferably at least 0,2 pH units, even more preferred at least 0,5 pH units.
Preparation
A preparation of the invention may comprise any physical combination of entomopathogenic fungal spores and acid described herein, in any form. The entomopathogenic fungal spores may be in a powder, for example a dry powder and/or a wettable powder. The entomopathogenic fungal spores may be granulated (in granule form), for example a wettable granule form. The entomopathogenic fungal spores may be in liquid form, for example dispersed in a solution, for example an aqueous solution, or dispersed in a non-aqueous liquid such as oil. Accordingly, the entomopathogenic fungal spores may be dispersed in an aqueous solution, or the entomopathogenic fungal spores may be oil- dispersed.
Composition In one preferred embodiment, a composition according to the invention is an aqueous formulation. For example, an aqueous formulation is a suspension (due to the presence of fungal spores).
In another preferred embodiment, the content of water in the composition according to the invention is at least 50%, more preferably at least 60% such as at least 70%, at least 80% or at least 95%, even more preferably at least 98% or even at least 99% or even at least 99,5%. pH of the final formulation
The pH range of composition according to the invention is adjusted by the presence of an acid so that the pH of the final composition is between pH 2 and 7 as described herein. As can be seen from, e.g., the table 4 (see, e.g., the different concentration dependent pH values of citric acid or dipicolinic acid), the pH depends inter alia on the amount of an acid used in a composition according to the invention. The skilled person can, without undue burden, define amounts of acids to adjust the pH of a composition to be a composition in accordance with the present invention, wherein the pH is between pH 2 and pH 7 or any of the more preferred ranges, when the amount of an acid is between 0,0001% (w/w) and 2% (w/w).
Notably, a composition according to the invention may comprise on or more further additives (e.g., as comprised in a fungal formulation) or impurities such as e.g., a solubilizing agent (e.g. polyethylenglycol) or impurities as can be found in ground-water or rain-water. Such additives may also influence the pH of a composition. However, as long as the pH is lowered by the presence of an acid (wherein the amount of acid is between 0,0001% (w/w) and 2% (w/w)) compared to such a composition without said acid and the pH is in the pH range of a composition as described herein, the beneficial effect of a composition according to the invention can be observed.
Without being bound to the following explanation, the phytotoxic effects (and/or also toxic effects for the spores) of low pH values compensate the beneficial effect of the increase of efficacy of spores of an entomopathogenic fungi. On the other hand, increased pH values do not enhance efficacy of spores of an entomopathogenic fungi.
In a preferred embodiment, the pH of the final composition is between pH 2,0 and pH 6,5, more preferably between 2,5 and 6,5, even more preferably between 3,0 and 6,0.
Acids pKa
Depending on the structure of an acid suitable for the various aspects of the invention (e.g., uses, methods and compositions), such an acid may comprise more than one pKa values. For example, lysine has three pKa values: p Ta, coon = 2,20 p Ta, a.NH3+ = 8,90 and p Ta, E-NH3+ = 10,28, However, due to the pH of the compositions according to the invention, the relevant pKa is a pKa between -3 and 5, i.e. the p Ta, coon (= 2,20).
Organic acids
In another preferred embodiment, an acid has at least one, preferably one or two or three -COOH group(s), at least one of such an -COOH group has a pka between -3 and 5 such as between 1 and 5 or any of the other preferred pka ranges.
More preferably, said acid is an organic acid having the formula
Ri-C(=O)-OH wherein Ri represents phenyl, optionally substituted with one or more substituents selected from the group consisting of (Ci- C4)alkyl, -(Co-C4)alkyl-OH, -O-R2, -(C0-C4)alkyl-(NR3R4); pyridyl, optionally substituted with one or more substituents selected from the group consisting of Ci- C4 alkyl, -OH, -O-R2, -(NR3R4), -COOH, and -C(=O)-(NR3R4); or a linear or branched C1-C12 alkyl group which optionally can be substituted with one or more additional substituents selected from the group consisting of phenyl, 2-pyridyl, -OH, -O-R2, -(NR3R4), -C(O)OH, -O-S(=O)2OH, -S(=O)2OH, and -C(=O)(NR3R4); wherein R2 represents C1-C4 alkyl; and wherein R3 and R4 independently from each other represent -H or C1-C4 alkyl), -(Co-C4)alkyl-COOH, and -(C0-C4)alkyl-C(=O)-(NR3R4).
In a preferred embodiment, said acid is an organic acid having the formula
Ri-C(=O)-OH wherein Ri represents a linear or branched C1-C12 alkyl group which optionally can be substituted with one or more additional substituents selected from the group consisting of phenyl, 2-pyridyl, -OH, -O- R2, -(NR3R4), -C(O)OH, -O-S(=O)2OH, -S(=O)2OH, and -C(=O)(NR3R4).
Organic sulfuric acids
In another preferred embodiment, an acid, has at least one, preferably one, -O-S(=O)2OH group with a pka between -3 and 5 or the other preferred pka ranges.
More preferably, said acid is an organic acid having the formula R5-O-S(=O)2OH wherein Rs represents phenyl, optionally substituted with one or more substituents selected from the group consisting of (Ci- C4)alkyl, -(Co-C4)alkyl-OH, -O-R2, -(NR3R4), -(C0-C4)alkyl-COOH, and -(C0-C4)alkyl-C(=O)(NR3R4); a linear or branched C1-C12 alkyl group which optionally can be substituted with one or more additional groups selected from the group consisting of -OH, -O-R2, -C(O)OH, and -C(=O)(NR3R4); wherein R3 and R4 independently from each other represent -H or C1-C4 alkyl), -(Co-C4)alkyl-COOH, and -(C0-C4)alkyl-C(=O)-(NR3R4); or
Rs represents a residue of formula
R6-O-[CH2-CH(CH3)O-]x-[CH2-CH2-O-]y- wherein x and y are independently from each other 1 (one) or greater than 1 (one), e.g. between 1 and 8 or between 1 and 5, and wherein Re is a linear or branched (Ci-C8)Alkyl.
In a more preferred embodiment, said acid is an organic acid having the formula
RS-O-S(=O)2OH wherein Rs represents a linear or branched C1-C12 alkyl group which optionally can be substituted with one or more additional groups selected from the group consisting of -OH, -O-R2, -C(O)OH, and - C(=O)(NR3R4).
Organic sulfonic acid
In another preferred embodiment, an acid, has a -S(=O)2OH group with a pka between -3 and 5 or the other preferred pka ranges.
More preferably, said acid is an organic acid having the formula
RS-S(=O)2OH wherein Rs is as defined above.
Organic phosphates
In another preferred embodiment, an acid, preferably an organic acid, has a -O-P(=O)(OH)2 group with a pka between -1 and 5 or the other preferred pka ranges.
More preferably, said acid is an organic acid having the formula R5-O-P(=O)(OH)2 wherein Rs is as defined above.
Organic phosphonates
In another preferred embodiment, an acid, preferably an organic acid, has a -P(=O)(OH)2 group with a pka between 1 and 5 or the other preferred pka ranges.
More preferably, said acid is an organic acid having the formula
RS-P(=O)(OH)2 wherein Rs is independently from each other as defined above.
Concentration range
One parameter that influences the pH value which is adjusted by the respective acid is the concentration of said acid in the final composition.
In a preferred embodiment, the concentration range of an acid in a composition according to the invention is between 0,0001 % (w/w) and 2 % (w/w), preferably between 0,00025 % (w/w) and 0,5 % (w/w) such as between 0,005 % (w/w) and 0,5 % (w/w), preferably between 0,001 % (w/w) and 0,25 % (w/w) such as between 0,01 % (w/w) and 0,25 % (w/w), more preferably between 0,0025% and 0,20% such as between 0,05 % (w/w) and 0,2 % (w/w). pH range
In a preferred embodiment, an acid suitable for the various aspects of the present invention results in a pH of the composition between 2,5 and 6,5 at 25 °C and 1013 hPa, more preferably between 3,0 and 6,5 at 25 °C and 1013 hPa, even more preferably between 3,0 and 6,0 at 25 °C and 1013 hPa.
In yet another preferred embodiment, an acid suitable for the various aspects of the present invention is present in a composition according to the invention in an amount between 0,005 % (w/w) and 0,5 % (w/w) and the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
In yet another preferred embodiment, an acid suitable for the various aspects of the present invention is present in a composition according to the invention in an amount between 0,01 % (w/w) and 0,25 % (w/w) and the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
In yet another preferred embodiment, an acid suitable for the various aspects of the present invention is present in a composition according to the invention in an amount between 0,05 % (w/w) and 0,2 % (w/w) and the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa
The pH can be measured using a laboratory pH meter (e.g. a Knick 766 Laboratory pH Meter). Water solubility
In one embodiment, the acid suitable for the various aspects of the invention is sufficiently water soluble. More specific for the context of this invention, sufficient solubility in water at 25 °C and 1013 hPa is 1 g/1 (gram/liter) or more, preferably 2 g/1 or more, and more preferably 5 g/1 or more, e.g. 50 g/1 or more, 100 g/1 or more or even 250 g/1 or more. In one embodiment, the acid is completely miscible with water.
Preferred acids
In one preferred embodiment, an acid is an organic acid having a water solubility of at least 1 g/1.
In one preferred embodiment, an acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p-toluenesulfonic acid, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, isocinchomeronic acid, isocitric acid, isonicotinic acid, lutidinic acid, maleic acid, mandelic acid, nicotinic acid, phtalic acid, picolinic acid, propionic acid, salicylic acid, tetronic acid, methane sulfonic acid.
In another preferred embodiment, an acid is an acid selected from the acids listed in Table A having a -COOH group with a pka value between 1 and 5, In one preferred embodiment, an acid is selected from the group consisting of oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, isocinchomeronic acid, isocitric acid, isonicotinic acid, lutidinic acid, maleic acid, mandelic acid, nicotinic acid, phtalic acid, propionic acid, salicylic acid, and tetronic acid.
In another preferred embodiment, an acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pka value between 1 and 3, In one preferred embodiment, an organic acid is selected from the group consisting of oxalic acid, dipicolinic acid, citric acid, malonic acid, tartaric acid, alpha-ketoglutaric acid, aconitic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, Isocinchomeronic acid, lutidinic acid, maleic acid, phtalic acid, and salicylic acid.
In another preferred embodiment, an acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pka value between 2 and 3, In one preferred embodiment, an organic acid is selected from the group consisting of dipicolinic acid, citric acid, malonic acid, tartaric acid, alpha-ketoglutaric acid, aconitic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, tyrosine, threonine, methionine, tryptophan, selenocysteine, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, Isocinchomeronic acid, lutidinic acid, maleic acid, phtalic acid, and salicylic acid.
In another preferred embodiment, an organic acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pka value between above 3 and 5, In one preferred embodiment, an organic acid is selected from the group consisting of malic acid, fumaric acid, adipic acid, pimelic acid, itaconic acid, trimesic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, succinic acid, acrylic acid, glutaric acid, glycolic acid, isocitric acid, isonicotinic acid, mandelic acid, nicotinic acid, picolinic acid, propionic acid, and tetronic acid.
In another preferred embodiment, an acid is an organic acid selected from the acids listed in Table A having a -COOH group with a pka value between above 4 and 5, In one preferred embodiment, an organic acid is selected from the group consisting of adipic acid, pimelic acid, benzoic acid, sorbic acid, acetic acid, ascorbic acid, succinic acid, acrylic acid, glutaric acid, isonicotinic acid, nicotinic acid, and propionic acid.
In another preferred embodiment, an acid is an acid selected from the acids listed in Table A having a pka value between -3 and -1. In one preferred embodiment, an organic acid is selected from the group consisting of p-toluenesulfonic acid, and methane sulfonic acid.
In one preferred embodiment, the acid is sulfuric acid. In another preferred embodiment, the acid is nitric acid. In another preferred embodiment, the acid is phosphoric acid. In another preferred embodiment, the acid is hydrochloric acid. In another preferred embodiment, the acid is boric acid.
In another preferred embodiment, the acid is oxalic acid. In another preferred embodiment, the acid is dipicolinic acid. In another preferred embodiment, the acid is citric acid. In another preferred embodiment, the acid is malonic acid. In another preferred embodiment, the acid is malic acid. In another preferred embodiment, the acid is fumaric acid. In another preferred embodiment, the acid is adipic acid. In another preferred embodiment, the acid is pimelic acid. In another preferred embodiment, the acid is tartaric acid. In another preferred embodiment, the acid is alpha-ketoglutaric acid. In another preferred embodiment, the acid is itaconic acid. In another preferred embodiment, the acid is trimesic acid. In another preferred embodiment, the acid is aconitic acid. In another preferred embodiment, the acid is benzoic acid. In another preferred embodiment, the acid is sorbic acid. In another preferred embodiment, the acid is acetic acid. In another preferred embodiment, the acid is lactic acid. In another preferred embodiment, the acid is ascorbic acid. In another preferred embodiment, the acid is glutamic acid. In another preferred embodiment, the acid is glycine. In another preferred embodiment, the acid is serine. In another preferred embodiment, the acid is asparagine. In another preferred embodiment, the acid is alanine. In another preferred embodiment, the acid is phenylalanine. In another preferred embodiment, the acid is valine. In another preferred embodiment, the acid is leucine. In another preferred embodiment, the acid is isoleucine. In another preferred embodiment, the acid is histidine. In another preferred embodiment, the acid is arginine. In another preferred embodiment, the acid is tyrosine. In another preferred embodiment, the acid is threonine. In another preferred embodiment, the acid is cysteine. In another preferred embodiment, the acid is methionine. In another preferred embodiment, the acid is tryptophan. In another preferred embodiment, the acid is proline. In another preferred embodiment, the acid is selenocysteine. In another preferred embodiment, the acid is p-toluenesulfonic acid. In another preferred embodiment, the acid is succinic acid. In another preferred embodiment, the acid is acrylic acid. In another preferred embodiment, the acid is aspartic acid. In another preferred embodiment, the acid is chinolinic acid. In another preferred embodiment, the acid is cinchomeronic acid. In another preferred embodiment, the acid is dinicotinic acid. In another preferred embodiment, the acid is formic acid. In another preferred embodiment, the acid is glutaric acid. In another preferred embodiment, the acid is glycolic acid. In another preferred embodiment, the acid is isocinchomeronic acid. In another preferred embodiment, the acid is isocitric acid. In another preferred embodiment, the acid is isonicotinic acid. In another preferred embodiment, the acid is lutidinic acid. In another preferred embodiment, the acid is maleic acid. In another preferred embodiment, the acid is mandelic acid. In another preferred embodiment, the acid is nicotinic acid. In another preferred embodiment, the acid is phtalic acid. In another preferred embodiment, the acid is picolinic acid. In another preferred embodiment, the acid is propionic acid. In another preferred embodiment, the acid is salicylic acid. In another preferred embodiment, the acid is tetronic acid. In another preferred embodiment, the acid is methane sulfonic acid.
In yet another preferred embodiment, an acid is an inorganic acid, preferably, the inorganic acid is selected from the group consisting of phosphoric acid or boric acid. More preferably, the inorganic acid is phosphoric acid.
In a more preferred embodiment, an acid is selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, glycine. In an even more preferred embodiment, an acid is selected from the group consisting of malonic acid, malic acid, tartaric acid, acetic acid, lactic acid, ascorbic acid, glycine, and citric acid. In an especially preferred embodiment, the acid is citric acid.
In another preferred embodiment, the acid is selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, glycine. In another even more preferred embodiment, an acid is selected from the group consisting of malonic acid, malic acid, tartaric acid, acetic acid lactic acid, ascorbic acid, and glycine. In another especially preferred embodiment, an acid is selected from the group consisting of malic acid, tartaric acid, and ascorbic acid.
Entomopathogenic fungi
In a preferred embodiment, an entomopathogenic fungi is selected from the group consisting of
Cordyceps fumosorosea (formely known as Isaria fumosorosea or Paecilomyces fumosoroseus) (preferably strain Apopka 97 which is now known as Cordyceps javanica (e.g. PFR97 or PreFeRal® WG from Biobest and Certis), strain IF-BDC01, strain FE 9901 / ARSEF 4490 (e.g. NoFly® from Natural Industries Inc., a Novozymes company), strain ESALQ 1296 (e.g. from Octane, Koppert) or strain CCM 8367 (e.g. WO 2010 006563));
Cordyceps javanica (formerly known as Isaria javanica) (preferably strain wf GAI 7 (referenced in Wu et al., 2020, Environmental Tolerance of Entomopathogenic Fungi: A New Strain of Cordyceps javanica Isolated from a Whitefly Epizootic Versus Commercial Fungal Strains, doi: 10,3390/insectsl 1100711), strain GZQ-1 (referenced in Ou et al., 2019 Identification of a new Cordyceps javanica fungus isolate and its toxicity evaluation against Asian citrus psyllid doi: 10,1002/mbo3,760), strain IjH6102 (e.g. CN 111961598 A), strain pfl 85 and pf212 (Bocco et al., 2021 Endophytic Isaria javanica pfl 85 Persists after Spraying and Controls Myzus persicae (Hemiptera: Aphididae) and Colletotrichum acutatum (Glomerellales: Glomerellaceae) in Pepper. doi.org/10,3390/insects!2070631), strain JZ-7 (Sun et al., 2021 The roles of entomopathogenic fungal infection of viruliferous whiteflies in controlling tomato yellow leaf curl virus. doi.org/10,1016/j.biocontrol.2021.104552), strain IJNL-N8 / CCTCC M-2017709 (Zhao et al., 2020 Sustainable control of the rice pest, Nilaparvata lugens, using the entomopathogenic fungus Isaria javanica. doi.org/10,1002/ps.6164); or strain GF 511 (which has been deposited by Lallemand Inc. of 1620 rue Prefontaine, Montreal, QC, H1W 2N8, CANADA on 8th June 2021 according to the Budapest Treaty under accession number 080621-01 with the International Depository Authority of Canada (IDAC), 1015 Arlington Street, Winnipeg, Manitoba, R3E 3R2, Canada) (e.g. LALGUARD JAVA, Lallemand);
Isaria farinosa (preferably strain ESALQ1205);
Beauveria bassiana (preferably strain ATCC 74040 (e.g. Naturalis® from CBC Europe, Italy; Contego BB from Biological Solutions Ltd.; Racer from AgriLife), strain GHA (Accession No. ATCC74250; e.g. BotaniGuard Es and Mycontrol-0 from Laverlam International Corporation), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339 (e.g. BroadBand™ from BASF), strain PPRI 7315, strain R444 (e.g. Bb-Protec from Andermatt Biocontrol), strains IL197, IL12, IL236, IL10, IL131, IL116 (all referenced in Jaronski, 2007, Use of Entomopathogenic Fungi in Biological Pest Management, 2007: ISBN: 978-81-308-0192-6), strain Bv025, strain BaGPK, strain ICPE 279, strain CG 716 (e.g. BoveMax® from Novozymes), strain HF23, strain ANT-03 (from Bioceres), strain NPP 111 BOO, strain 203 (e.g. Phoemyc from Glen Biotech), strain Bbl 0 (from Biagro Bb), strain ESALQ PL63 (from Boveril, Koppert), strain IMI389521, strain IBCB 66 (e.g. from Atrevido, Koppert), strain K4B1 (Beaugenic from Ecolibrium Biologicals), strain K4B3 (Beaublast from Ecolibrium Biologicals), strain CBMAI 1306 (from Anin CE Agrivalle);
Beauveria brongniartii (e.g. from Beaupro from Andermatt Biocontrol AG (preferably strain BIPESCO 2 (from Melocont, Agrifutur);
Metarhizium brunneum (formely known as Metarhizium anisopliae) (preferably strain Cbl5 (e.g. ATTRACAP® from Biocare), strain ESALQ 1037 (e.g. from Metarril® SP Organic), strain E-9 (e.g. from Metarril® SP Organic), strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013- 1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448; IMI 507278 which has been deposited on November 18th, 2022 according to the Budapest Treaty with CABI, Bakeham Lane, Egham, Surrey, TW209TY, UK) (e.g. BIO 1020 by Bayer CropScience and also e.g. Met52 by Novozymes or LALGUARD M52 from Lallemand), strain ICIPE 78, strain ESF1 (from BIO-BLAST), strain IBCB 425 (from Metarhizium Oligos, Oligos Biotecnologia), strain LRC112(from SpudSmart, AAFC));
Metarhizium acridum (preferably strain ARSEF324 from GreenGuard by BASF or isolate IMI 330189, or ARSEF7486; e.g. Green Muscle by Biological Control Products);
Metarhizium robertsii (preferably strain 23013-3 (NRRL 67075));
Metarhizium flavoviride;
Metarhizium rileyi (formerly known as Nomuraea rileyi);
Myrothecium verrucaria (preferably strain AARC-0255 (e.g. from DiTera DF, Biagro));
Hirsutella citriformis;
Hirsutella minnesotensis;
Hirsutella thompsonii (preferably Mycohit and ABTEC from Agro Bio-tech Research Centre, IN);
Lecanicillium lecanii (formerly known as Verticillium lecanii) (preferably conidia of strain KV01 (e.g. Mycotal® and Vertalec® from Koppert/Arysta));
Lecanicillium lecanii (formerly known as Verticillium lecanii), strain DAOM198499; strain DAOM216596; strain KV01 (e.g. from Vertalec®, Koppert/Arysta)
Lecanicillium muscarium (formerly known as Verticillium lecanii), in particular strain VE 6 / CABI(=IMI) 268317/ CBS 102071/ ARSEF5128 (e.g. Mycotal from Koppert); strain K4V 1 (from eNtokill, Ecolibrium Biologicals); strain K4V2 (from eNtoblast, Ecolibrium Biologicals)
Aschersonia aleyrodis;
Conidiobolus obscurus;
Lagenidium giganteum;
Entomophthora virulenta (e.g. Vektor from Ecomic);
Zoophthora sp.;
Entomophaga sp.;
Pandora delphacis;
Sporothrix insectorum (e.g. Sporothrix Es from Biocerto, BR);
Zoophtora radicans;
Mucor haemelis (e.g. BioAvard from Indore Biotech Inputs & Research);
Purpureocillium lilacinum (forme ly also known as Paecilomyces lilacinus) (preferably strain 251 (AGAL 89/030550; e.g. BioAct from Bayer CropScience Biologies GmbH), strain PL11 (from BioStat, Certis), or strain UEL Pae 10 (from Unique, Ballagro Agro Tecnologia Ltda.));
Paecilomyces variotii, strain Q-09 (preferably Nemaquim® from Quimia, MX));
Muscodor albus (preferably strain QST 20799 (Accession No. NRRL 30547));
Muscodor roseus (preferably strain A3-5 (Accession No. NRRL 30548));
Monacrosporium cionopagum;
Monacrosporium psychrophilum;
Myrothecium verrucaria (preferably strain AARC-0255 (e.g. DiTeraTM by Valent Biosciences);
Stagonospora phaseoli (e.g. from Syngenta);
Monacrosporium drechsleri;
Monacrosporium gephyropagum;
Nematoctonus geogenius;
Nematoctonus leiosporus; Neocosmospora vasinfecta; and
Paraglomus sp, in particular Paraglomus brasilianum;
Pochonia chlamydosporia (also known as Vercillium chlamydosporiuni) (preferably var. catenulata (IMI SD 187; e.g. KlamiC from The National Center of Animal and Plant Health (CENSA), CU);
Stagonospora heteroderae;
Meristacrum asterospermum;
Arthrobotrys dactyloides;
Arthrobotrys oligospora; and
Arthrobotrys superba.
In another preferred embodiment, an entomopathogenic fungi is selected from the group consisting of Beauveria bassiana (preferably strain ATCC 74040 (e.g. Naturalis® from CBC Europe, Italy; Contego BB from Biological Solutions Ltd.; Racer from AgriLife), strain GHA (Accession No. ATCC74250; e.g. BotaniGuard Es and Mycontrol-0 from Laverlam International Corporation), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339 (e.g. BroadBand™ from BASF), strain PPRI 7315, strain R444 (e.g. Bb-Protec from Andermatt Biocontrol), strains IL197, IL12, IL236, IL10, IL131, IL116 (all referenced in Jaronski, 2007, Use of Entomopathogenic Fungi in Biological Pest Management, 2007: ISBN: 978-81-308-0192-6), strain Bv025, strain BaGPK, strain ICPE 279, strain CG 716 (e.g. BoveMax® from Novozymes), strain HF23, strain ANT-03 (from Bioceres), strain NPP 111 BOO, strain 203 (e.g. Phoemyc from Glen Biotech), strain Bbl 0 (from Biagro Bb), strain ESALQ PL63 (from Boveril, Koppert), strain IMI389521, strain IBCB 66 (e.g. from Atrevido, Koppert), strain K4B1 (Beaugenic from Ecolibrium Biologicals), strain K4B3 (Beaublast from Ecolibrium Biologicals), strain CBMAI 1306 (from Auin CE Agrivalle); and
Metarhizium brunneum (formely known as Metarhizium anisopliae) (preferably strain Cbl5 (e.g. ATTRACAP® from Biocare), strain ESALQ 1037 (e.g. from Metarril® SP Organic), strain E-9 (e.g. from Metarril® SP Organic), strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013- 1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448 / IMI 507278 which has been deposited on November 18th, 2022 according to the Budapest Treaty with CABI, Bakeham Lane, Egham, Surrey, TW209TY, UK) (e.g. BIO 1020 by Bayer CropScience and also e.g. Met52 by Novozymes or LALGUARD M52 from Lallemand), strain ICIPE 78, strain ESF1 (from BIO-BLAST), strain IBCB 425 (from Metarhizium Oligos, Oligos Biotecnologia), strain LRC112(from SpudSmart, AAFC)).
In a more preferred embodiment, an entomopathogenic fungi is selected from the group consisting of Cordyceps fumosorosea strain Apopka 97 (which has now been reclassified as C. javanica strain Apopka 97), strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ 1296 or strain CCM 8367;
Cordyceps javanica strain wf GA 17, strain GZQ-1, strain IjH6102, strain pfl 85, strain pf212, strain JZ-7, strain IJNL-N8 / CCTCC M-2017709, or strain GF 511 (e.g. LALGUARD JAVA, Lallemand);
Isaria farinosa strain ESALQ 1205;
Beauveria brongniartii strain BIPESCO 2;
Metarhizium acridum strain ARSEF324, isolate IMI 330189, or ARSEF7486;
Metarhizium robertsii strain 23013-3 (NRRL 67075);
Metarhizium rileyi;
Myrothecium verrucaria strain AARC-0255;
Lecanicillium lecanii strain KV01;
Lecanicillium lecanii strain DAOM198499, strain DAOM216596, or strain KV01;
Lecanicillium muscarium strain VE 6 / CABI(=IMI) 268317/ CBS102071/ ARSEF5128, strain K4V 1 , or strain K4V2;
Purpureocillium lilacinum strain 251 (AGAL 89/030550, strain PL11, or strain UEL Pae 10;
Paecilomyces variotii strain Q-09;
Muscodor albus strain QST 20799 (Accession No. NRRL 30547;
Muscodor roseus strain A3-5 (Accession No. NRRL 30548; and
Myrothecium verrucaria strain AARC-0255,
In yet another more preferred embodiment, an entomopathogenic fungi is selected from the group consisting of
Cordyceps fumosorosea (or C. javanica) strain Apopka 97, Cordyceps fumosorosea strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367;
Cordyceps javanica strain wf GA 17, strain GZQ-1, strain IjH6102, strain pfl 85, strain pf212, strain JZ-7, strain IJNL-N8 / CCTCC M-2017709, or strain GF 511; Metarhizium brunneum (formely known as Metarhizium anisopliae) strain F52 (DSM3884/ ATCC 90448, IMI 507278 which has been deposited on November 18th, 2022 according to the Budapest Treaty with CABI, Bakeham Lane, Egham, Surrey, TW209TY, UK), and
Isaria farinosa strain ESALQ1205; even more preferably
Cordyceps fumosorosea (or C. javanica) strain Apopka 97, Cordyceps fumosorosea strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367;
Cordyceps javanica strain wf GA 17, strain GZQ-1, strain IjH6102, strain pfl 85, strain pf212, strain JZ-7, strain IJNL-N8 / CCTCC M-2017709, or strain GF 511;
Metarhizium brunneum (formely known as Metarhizium anisopliae) strain F52 (DSM3884/ ATCC 90448, IMI 507278 which has been deposited on November 18th, 2022 according to the Budapest Treaty with CABI, Bakeham Lane, Egham, Surrey, TW209TY, UK); most preferably
Cordyceps strain Apopka 97 (ATCC 20874);
Cordyceps javanica strain GF 511 (ID AC 080621-01); and
Metarhizium brunneum strain F52 (IMI 507278).
The Apopka 97 strain was previously classified as Cordyceps fumosorosea (and, even earlier, as Isaria fumosorosea) but in 2017 was reclassified as Cordyceps javanica (Avery et al., (2021), Field Efficacy of Cordyceps javanica, White Oil and Spinetoram for the Management of the Asian Citrus Psyllid, Diaphorina citri Insects 2021, 12, 824). Herein the Apopka 97 strain is referred to using both or either classifications interchangeably.
In a preferred embodiment the entomopathogenic fungus is a Cordyceps javanica strain. In a preferred embodiment the entomopathogenic fungus is a Cordyceps javanica Apopka 97 strain or a Cordyceps javanica GF 511 strain. In another preferred embodiment the entomopathogenic fungus is a Metarhizium brunneum strain. In a preferred embodiment the entomopathogenic fungus is a Metarhizium brunneum F52 strain (IMI 507278).
Amount of fungus
Preferably, the number of fungal spores in a composition according to the invention is between l,0*104 spores per ml and l,0*109 spores per ml, such as between l,0*105 spores per ml and l,0*108 spores per ml. More preferably, the number of fungal spores is between 5,0* 105 spores per ml and 5,0* 107 spores per ml. Most preferably the number of spores is between 5,5* 105 spores per ml and 5,0* 107 spores per ml.
Further components
Liquids for spore formulations Preferred liquids for spore formulations suitable for the present invention are selected from the group consisting of polyethylene glycols, such as PEG-300; ethoxylated alcohols (such as Atplus 245, Berol 050, Berol 260, Lucramul CO08, Lucramul L03, Lucramul L05); mono-/polyethylene oxide diethers, such as Tetraglyme; mono-/polyethylene oxide ether-ester (such as n-Butyldiglycolacetat); ethoxylated carboxylic acids (such as Radiasurf 7403, PEG-400-monooleate, Radiasurf 7423); mono-/polyethylene oxide di-esters (such as Radiasurf 7442); polypropylene glycols(such as Dipropylene glycol); propoxylated alcohols (such as Dowanol DPM); mono-/polypropylene oxide diethers (such as Dipropylene glycol dimethyl ether); mono-/polypropylene oxide ether-ester (such as Dipropylene glycol methyl ether acetate); propoxylated carboxylic acids; mono-/polypropylene oxide di-esters (such as Propylenglycol diacetate); alcohol propoxylate-ethoxylates (such as Atlas G-5002L, Lucramul HOT 5902); carboxylic acid propoxylate -ethoxylate; carboxylic acid propoxylate -ethoxylate ether (such as Leofat OC0503M).
Overview of suitable liquids:
Figure imgf000023_0001
Figure imgf000024_0001
Oils
As at least one further component, a composition according to the invention may comprise an oil, more preferably a mineral oil, plant oil or vegetable oil. Among plant oils, the ones preferably used in the present invention are triglyceride -based vegetable oils which are liquid at least at room temperature, preferably also at temperatures below room temperature, such as at 15°C, at 10°C or even at 5°C or 4°C.
Concentrations of oils, preferably plant oil, in a composition according to the invention may range between 0,005 % (w/w) and 0,9 % (w/w) and any other value in between this range, such as between 0,01 % (w/w) and 0,85 % (w/w) and any other value in between this range. Preferred plant oils are wheat germ oil, soybean oil, peanut oil, rice bran oil, safflor oil, rapeseed oil, sunflower oil, com oil, walnut oil, hazelnut oil, almond oil and olive oil.
Antioxidants
As at least one further component, a composition according to the invention may comprise an antioxidant. Whereas Applicant does not wish to be bound by any scientific theory, it is believed that a certain concentration of antioxidant in the formulation of the invention contributes to the superior storage stability of the formulation, in particular the long stability of the fungal spores comprised therein.
The concentration of antioxidants in the formulation according to the present invention is at least 0,00002 % (w/w) and may be increased to up to 0,05 % (w/w). A preferred range is between 0,00003 % (w/w) and 0,007 % (w/w) and any other value in between this range.
The antioxidant may be any suitable antioxidant, but is preferably selected from the group consisting of butylhydroxytoluol (BHT), butylhydroxyanisole (BHA), ascorbyl palmitate, tocopheryl acetate, ascorbyl stearate or the group of carotinoids (e.g. beta-carotin) or gallates (e.g. ethyl gallate, propyl gallate, octyl gallate, dodecyl gallate).
In a more preferred embodiment, the antioxidant is butylhydroxytoluol which has been shown in the examples to contribute to the very good stability of the fungal spores in the formulation of the present invention. Further preferred, said butylhydroxytoluol is present in a concentration of between 0,00002 % (w/w) and 0,01 % (w/w) and any other value in between this range, preferably between 0,00004 % (w/w) and 0,006 % (w/w) and any other value in between this range.
Some plant oils naturally have a high content of antioxidants, e.g. wheat germ oil. In case such plant oil is used, the further addition of an antioxidant may not be necessary, or the amount may be reduced in order to arrive at the concentrations described herein which are believed to be one factor responsible for the enhanced storage stability of the present formulation. Accordingly, for such plant oils with a high content of antioxidants, such as at least 0,00008 % (w/w), no addition of further antioxidant may be necessary. In such cases, the required percentage of antioxidant according to the invention is comprised in the minimum concentration of plant oil according to the invention.
Emulsifier
As at least one further component, a composition according to the invention may further comprise an emulsifier. If used, the concentration of emulsifier should be at least 0,0002 % (w/w). The maximum concentration of said at least one emulsifier should not exceed 0,3 % (w/w). Accordingly, useful ranges for emulsifiers range between 0,0002 % (w/w) and 0,2% (w/w), and any value in between this range.
Suitable emulsifiers include ethoxylated sorbitan esters, e.g. ethoxylated sorbitan trioleate 20EO, (e.g. Tween 85); ethoxylated sorbitan monooleate (e.g. Tween 80); ethoxylated sorbitan monolaurate (e.g. Emulsogen 4156, Tween 20 or Polysorbate 20); or ethoxylated sorbitol esters, e.g. ethoxylated sorbitol hexaoleate 40EO (e.g. Ariatone TV); ethoxylated sorbitol tetraoleate-laurate 40EO (e.g. Atlox 1045- A); or ethoxylated castor oils, e.g. Emulsogen EL400, Emulsogen EL360, Emulsogen EL300, Lucramul CO30, Agnique CSO25 or Etocas 10; or polyethylenglycol (e.g. PEG 400). These emulsifiers are preferably present in a range of between about 0,0015 % (w/w) and 0,125 % (w/w) and any other value in between this range.
In a preferred embodiment, said emulsifier is an ethoxylated sorbitol ester, such as ethoxylated sorbitol hexaoleate 40EO.
In another preferred embodiment, said emulsifiers are combined with other emulsifiers such as ethoxylated alcohols or propoxylated-ethoxylated alcohols. These substances are best described by the general formula X — O — [CEE — CH(CH3) — O]m — [CEE — CEE — O — ]n — OH where X is a branched or linear alcohol, saturated or partially unsaturated, with 1-24 carbon atoms, preferably 2-18, more preferably 3-14, most preferably 4-10, wherein m is an average number between 0 and 20, preferably 0-15; more preferably 0-10, and wherein n is an average number between 1 and 20, preferably 2-15, more preferably 3-10, These emulsifiers are preferably present in a range of between about 0% (w/w) to 0,1% (w/w) and any other value in between this range.
Rheology modifier
As at least one further component, a composition according to the invention may further comprise a rheology modifier. Rheology modifiers are preferably derived from minerals. These rheology modifiers provide long term stability when the formulation is at rest or in storage. Furthermore, it has been found in the course of the present invention that such rheology modifiers seem to contribute to the increased storage stability of the present formulation.
Suitable compounds are rheological modifiers selected from the group consisting of hydrophobic and hydrophilic fumed and precipitated silica particles, gelling clays including bentonite, hectorite, laponite, attapulgite, sepiolite, smectite, or hydrophobically/organophilic modified bentonite.
Rheology modifiers are preferably derived from minerals. These rheology modifiers provide long term stability when the composition is at rest or in storage. Furthermore, it has been found in the course of the present invention that such rheology modifiers seem to contribute to the increased storage stability of the present formulation.
Suitable compounds are rheological modifiers selected from the group consisting of hydrophobic and hydrophilic fumed and precipitated silica particles, gelling clays including bentonite, hectorite, laponite, attapulgite, sepiolite, smectite, or hydrophobically/organophilic modified bentonite.
In connection with the present invention, fumed or precipitated silica is preferred as rheology modifier. Fumed silica, also known as pyrogenic silica, either hydrophilic or hydrophobic, usually is composed of amorphous silica fused into branched, chainlike, three-dimensional secondary particles which then agglomerate into tertiary particles. The resulting powder has an extremely low bulk density and high surface area. Both hydrophilic and hydrophobic fumed silica can be used in the present invention
Fumed silica usually has a very strong thickening effect. The primary particle size is ca. 5-50 nm. The particles are non-porous and have a surface area of ca. 50-600 m2/g.
Hydrophilic fumed silica is made from flame pyrolysis of silicon tetrachloride or from quartz sand vaporized in a 3000°C electric arc. Major global producers are Evonik Industries, tradename AEROSIL®); Cabot Corporation, tradename Cab-O-Sil®; Wacker Chemie, HDK product range; and OCI, tradename Konasil®.
Hydrophilic fumed silica can be hydrophobized by further treatment with reactive silicium-containing agents in order to modify the physicochemical properties of the silica. Typically hydrophobisation takes place by treatment of a hydrophilic fumed silica with agents like hexaalkyldisilanes (e.g. ((CHajaSijz), trialkylsilylchlorides (e.g. (CHajaSiCl) or dialkyldichlorsilanes (e.g. (CHajzSiCE). Hydrophobized fumed silica is available e.g. from Evonik Industries (AEROSIL R-types), and Cabot (Cab-O-Sil).
Best results are obtained using a hydrophilic fumed silica having a BET surface area of 150 to 350 m2/g, e. g. 150, 200, 250, 300 or 350,
Precipitated silica is produced by acidifying aqueous alkaline silicate solutions with mineral acids. Variations of the precipitation process lead to different precipitated silica qualities namely with different specific surface areas. The precipitates are washed and dried. Precipitated silica having a particle size of below 10 pm are most effective for the present invention. The specific surface area is typically from ca. 50-500 m2/g. Global producers are for example Evonik Industries, tradename SIPERNAT® or Wessalon®; Rhodia, tradename Tixosil®; and PPG Industries, tradename Hi-Sil™.
Major global producers for fumed (pyrogenic) hydrophilic or hydrophbized silicas are Evonik (tradename Aerosil®), Cabot Corporation (tradename Cab-O-Sil®), Wacker Chemie (HDK product range), Dow Coming, and OCI (Konasil®). Another class of suitable rheology modifiers are precipitated silicas, and major global producers are Evonik (tradenames Sipemat® or Wessalon®), Rhodia (Tixosil) and PPG Industries (Hi-Sil).
Particularly preferred in connection with the present composition is fumed silica having a BET surface area of about 200 m2/g obtainable e.g. as Aerosil® 200,
Another class of suitable examples for rheology modifiers are clay thickeners. Clay thickeners are generally micronized layered silicates that can be effective thickeners for a wide range of applications. They are typically employed either in their non-hydrophobized or hydrophobized form. In order to make them dispersible in non-aqueous solvents, the clay surface is usually treated with quaternary ammonium salts. These modified clays are known as organo-modified clay thickeners. Optionally, small amounts of alcohols of low molecular weight or water may be employed as activators. Examples for such clay-based rheology modifiers include smectite, bentonite, hectorite, attapulgite, seipiolite or montmorillonite clays. Preferred rheological modifiers (b) are for example organically modified hectorite clays such as Bentone® 38 and SD3, organically modified bentonite clays, such as Bentone® 34, SD1 and SD2, organically modified sepiolite such as Pangel® B20, hydrophilic silica such as Aerosil® 200, hydrophobic silica such as Aerosil® R972, R974 and R812S, attapulgite such as Attagel® 50, Another class of suitable examples for rheology modifiers are organic rheological modifiers based on modified hydrogentated castor oil (trihydroxystearin) or castor oil organic derivatives such as Thixcin® R and Thixatrol® ST. Table B: Examples of rheology modifiers
Figure imgf000028_0001
Figure imgf000029_0002
Said rheology-modifying agent may be present in the formulation of the invention in a concentration of up to 0,07 % (w/w), preferably between 0,00003 % (w/w) and 0,04 % (w/w) and any other value in between this range Polyether-modified trisiloxane
The formulation may in addition comprise a polyether-modified trisiloxane which is preferably of formula I
Figure imgf000029_0001
Formula (I) where
R1 represents independent from each other identical or different hydrocarbyl radicals having 1 -8 carbon atoms, preferred methyl-, ethyl-, propyl- and phenyl radicals, particularly preferred are methyl radicals. a = 0 to 1, preferred 0 to 0,5, particularly preferred 0, b = 0,8 to 2, preferred 1 to 1.2, particularly preferred 1, in which: a + b < 4 and b>a, preferred a + b <3 and particularly preferred a + b <2,
R2 represents independent from each other identical or different polyether radicals of general formula (II)
-R3O[CH2CH2O]c[CH2CH(CH3)O]d[CHR4CHR4O]eR5
Formula (II)
R3 = independent from each other identical or different, bivalent hydrocarbyl radicals having 2 - 8 carbon atoms, which are optionally interrupted by oxygen atoms, preferred rest is the general formula (III) where n = 2 - 8, particularly preferred -CH2-CH2-CH2-,
Figure imgf000030_0001
Formula (III)
R4 = independent from each other identical or different hydrocarbyl radicals having 1-12 carbon atoms or hydrogen radical, preferably a methyl-, ethyl-, phenyl- or a hydrogen radical.
R5 = independent from each other identical or different hydrocarbyl radicals having 1 -16 carbon atoms, which are optionally contain urethane functions, carbonyl functions or carboxylic acid ester functions, or hydrogen radical, preferred methyl or H, particularly preferred H.
C = 0 to 40, preferred 1 to 15, particularly preferred 2 to 10 d = 0 to 40, preferred 0 to 10, particularly preferred 1 to 5 e = 0 to 10, preferred 0 to 5, particularly preferred 0, in which c + d + e > 3 The polyether-modified trisiloxanes described above can be prepared by methods well known to the practioner by hydrosilylation reaction of a Si-H containing siloxane and unsaturated polyoxyalkylene derivatives, such as an allyl derivative, in the presence of a platinum catalyst. The reaction and the catalysts employed have been described for example, by W. Noll in “Chemie und Technologic der Silicone”, 2nd ed., Verlag Chemie, Weinheim (1968), by B. Marciniec in “AppL Homogeneous Catal. Organomet. Compd. 1996, 1, 487). It is common knowledge that the hydrosilylation products of SiH- containing siloxanes with unsaturated polyoxyalkylene derivatives can contain excess unsaturated polyoxyalkylene derivative.
Examples of water soluble or self-emulsifyable polyether-modified (PE/PP or block-CoPo PEPP) trisiloxanes include but are not limited to those described by CAS-No 27306-78-1 (e.g. Silwet L77 from MOMENTIVE), CAS-No 134180-76-0 (e.g. BreakThru S233 or BreakThru S240 from Evonik), CAS-No 67674-67-3 (e.g Silwet 408 from WACKER), other BreakThru-types, and other Silwet-types.
Preferred polyether-modified trisiloxanes include those described by CAS-No 134180-76-0, in particular Break-Thru S240. In one preferred embodiment, the polyether-modified trisiloxane has the chemical denomination oxirane, mono(3-(l,3,3,3-tetramethyl-l-((trimethylsilyl)oxy)disiloxanyl) propyl)ether. It is most preferred that the polyether-modified trisiloxane is Breakthru S240.
The amount of polyether-modified trisiloxane, if present in the formulation, is at least 0,001% (w/w), such as at least 0,002 % (w/w) or at least 0,004 % (w/w). Preferably, the amount of polyether modified trisiloxane ranges between 0,001 % (w/w) and 0,4 % (w/w).
Depending on the nature of the water used to prepare a composition according to the invention, the skilled person is aware that further components such as impurities in the water, may be present in a composition according to the invention. The nature of such impurities depends on the water source, e.g. rain-water or ground-water. For example, such impurities can be (metal) ions and their counter ions, small particles of dirt (soil or sand) or organic impurities. However, preferably, the amount of such additional components (impurities) is 1 % (w/w) or less, more preferably 0,5 % (w/w) or less, even more preferably 0,05 % (w/w) or less. Most preferably, water contains impurities of 0,005 % (w/w) or less. For the sake of this invention, such impurities are encompassed by the weight values of the water in a composition according to the invention, i.e. it is not required to separately measure the amounts of impurities for evaluating if a composition is a composition according to the invention.
The skilled person will understand that in a composition according to the invention, the sum of all components in said composition always sum up to 100, If not mentioned otherwise, the % (w/w) values given herein refer to the total weight amount of a composition according to the invention.
Method and uses
In a further aspect, the present invention relates to a method for controlling insects and/or nematodes, preferably insects, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the composition according to the invention as described above to said plant or to a locus where plants are growing or intended to be grown. In one preferred embodiment, the method is a method wherein a synergistic composition according to the invention is applied.
The amount of the composition according to the invention when brought to the field in accordance with any of the methods described herein, is at least 0,05 1/ha (hectare), such as between 1 to 10000 1/ha, 1 to 7500 1/ha, such as 801/ha to 50001/h, e.g., 100 1/ha, 200 1/ha, 300 1/ha, 400 1/ha, 500 1/ha, 600 1/ha, 700 1/ha, 800 1/ha, 900 1/ha, 1000 1/ha, 1100 1/ha, 1200 1/ha, 1300 1/ha, 1400 1/ha, 1500 1/ha,
1600 1/ha, 1700 1/ha, 1800 1/ha, 1900 1/ha, 2000 1/ha, 2200 1/ha, 2400 1/ha, 2600 1/ha, 2800 1/ha, 3000 1/ha, 3200 1/ha, 3400 1/ha, 3600 1/ha, 3800 1/ha, 4000 1/ha, 4200 1/ha, 4400 1/ha, 4600 1/ha, 4800 1/ha. In one embodiment where the composition comprises C. fumosorosea spores, e.g., for insect control, the amount to the field preferably ranges between 1 and 10000 1/ha, such as between 2 1/ha and 40 1/ha, e.g. between 5 1/ha and 30 1/ha, between 30 1/ha and 50 1/ha such as around 40 1/ha, between 200 1/ha to 1000 1/ha, such as between 200 1/ha to 500 1/ha, or up to 10000 1/ha such as between 5000 1/ha and 10000 1/ha, between 6000 1/ha and 10000 1/ha, between 7000 1/ha and 10000 1/ha, between 8000 1/ha and 10000 1/ha, between 9000 1/ha and 10000 1/ha. In one embodiment where the composition comprises M. brunneum spores, e.g., for insect control, the amount to the field preferably ranges between 1 and 10000 1/ha, such as between 2 1/ha and 40 1/ha, e.g. between 5 1/ha and 30 1/ha between 30 1/ha and 50 1/ha such as around 40 1/ha, between 100 to 2000 1/ha, between 500 to 1500 1/ha, or up to 10000 1/ha such as between 5000 1/ha and 10000 1/ha, between 6000 1/ha and 10000 1/ha, between 7000 1/ha and 10000 1/ha, between 8000 1/ha and 10000 1/ha, between 9000 1/ha and 10000 1/ha. In one embodiment where the composition comprises C.javanica spores, e.g., for insect control, the amount to the field preferably ranges between 1 and 10000 1/ha, such as between 2 1/ha and 40 1/ha, e.g. between 5 1/ha and 30 1/ha between 30 1/ha and 50 1/ha such as around 40 1/ha, between 100 to 2000 1/ha, between 500 to 1500 1/ha, or up to 10000 1/ha such as between 5000 1/ha and 10000 1/ha, between 6000 1/ha and 10000 1/ha, between 7000 1/ha and 10000 1/ha, between 8000 1/ha and 10000 1/ha, between 9000 1/ha and 10000 1/ha. The skilled person will understand that the exact rate being dependent on the spore concentration in the composition according to the invention as well as on the kind of spores. The number of spores in a composition according to the invention is usually between 1 x ] 04 and I xlO9 spores/g composition, e.g. for soil or foliar application. Further preferred amounts are mentioned above.
In a preferred embodiment, the increase of efficacy of controlling insects and/or nematodes, preferably insects, of a composition according to the invention vs the same composition without the acid (wherein the amount of (missing) acid is substituted by water) is at least 5%, more preferably at least 10%, even more preferably at least 15%, such as 20% or more, 25% or more or even 30% or more.
In yet a further aspect, the present invention relates to a method for an increased control of insects and/or nematodes, preferably insects, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the composition according to the invention as described above to said plant or to a locus where plants are growing or intended to be grown. Preferably, the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared with the effect of a composition without the acid as described herein but otherwise with all components as a composition according to the invention. In one preferred embodiment, the method is a method wherein a synergistic composition according to the invention is applied.
Another aspect refers to the use of an acid as defined herein for increasing the efficacy of fungal spores as defined herein to control insects and/or nematodes, preferably insects. Preferably, the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared to the efficacy of said fungal spores in the absence if said acid. Preferably, the use refers to an increase which is caused by the presence of said acid in an amount of between 0,0001% (w/w) and 2% (w/w) in a spore formulation.
The present invention also relates to the use of an acid as defined herein for enhancing the efficacy of fungal spores in a composition according to the invention in agriculture. Preferably, the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared to the efficacy of said fungal spores in the absence if said acid. Preferably, the use refers to an increase which is caused by the presence of said acid in an amount of between 0,0001% (w/w) and 2% (w/w) in a spore formulation.
In another aspect the present invention relates to the use of a composition as disclosed herein for controlling insects and/or nematodes, preferably insects, in, on and/or around a plant, for enhancing growth of a plant or for increasing plant yield or root health. In one preferred embodiment, the use is a use of a synergistic composition according to the invention.
In another aspect the present invention relates to the use of a composition as disclosed herein for an enhances control of insects and/or nematodes, preferably insects, in, on and/or around a plant, for enhancing growth of a plant or for increasing plant yield or root health, wherein the increase is at least 5%, more preferably at least 10%, more preferably at least 15%, even more preferably at least 20% compared with the effect of a composition without the acid as described herein but otherwise with all components as a composition according to the invention, wherein the weight of (missing) acid is replaced by water to ensure the % (w/w) values of the other components are essentially the same. In one preferred embodiment, the use is a use of a synergistic composition according to the invention.
Plants
Plants which can be treated in accordance with the invention include the following main crop plants: maize, soya bean, alfalfa, cotton, sunflower, Brassica oil seeds such as Brassica napus (e.g. canola, rapeseed), Brassica rapa, B. juncea (e.g. (field) mustard) and Brassica carinata, Arecaceae sp. (e.g. oilpalm, coconut), rice, wheat, sugar beet, sugarcane, oats, rye, barley, millet and sorghum, triticale, flax, nuts, grapes and vine and various fruit and vegetables from various botanic taxa, e.g. Rosaceae sp. (e.g. pome fruits such as apples and pears, but also stone fruits such as apricots, cherries, almonds, plums and peaches, and berry fruits such as strawberries, raspberries, red and black currant and gooseberry), Ribesioidae sp., Juglandaceae sp., Betulaceae sp., Anacardiaceae sp., Fagaceae sp., Moraceae sp., Oleaceae sp. (e.g. olive tree), Actinidaceae sp., Lauraceae sp. (e.g. avocado, cinnamon, camphor), Musaceae sp. (e.g. banana trees and plantations), Rubiaceae sp. (e.g. coffee), Theaceae sp. (e.g. tea), Sterculiceae sp., Rutaceae sp. (e.g. lemons, oranges, mandarins and grapefruit); Solanaceae sp. (e.g. tomatoes, potatoes, peppers, capsicum, aubergines, tobacco), Liliaceae sp., Compositae sp. (e.g. lettuce, artichokes and chicory — including root chicory, endive or common chicory), Umbelliferae sp. (e.g. carrots, parsley, celery and celeriac), Cucurbitaceae sp. (e.g. cucumbers — including gherkins, pumpkins, watermelons, calabashes and melons), Alliaceae sp. (e.g. leeks and onions), Cruciferae sp. (e.g. white cabbage, red cabbage, broccoli, cauliflower, Brussels sprouts, pak choi, kohlrabi, radishes, horseradish, cress and Chinese cabbage), Leguminosae sp. (e.g. peanuts, peas, lentils and beans — e.g. common beans and broad beans), Chenopodiaceae sp. (e.g. Swiss chard, fodder beet, spinach, beetroot), Linaceae sp. (e.g. hemp), Cannabeacea sp. (e.g. cannabis), Malvaceae sp. (e.g. okra, cocoa), Papaveraceae (e.g. poppy), Asparagaceae (e.g. asparagus); useful plants and ornamental plants in the garden and woods including turf, lawn, grass and Stevia rebaudiana; and in each case genetically modified types of these plants. Other examples plants of which can be treated in accordance with the invention could also include macadamia, pecan, cashew, walnut, coconut, guava, mango, papaya, acerola, passion fruit, pineapple or soursop.
Insects & Acari (mites and ticks)
Preferably, the formulations according to the invention are used by spray application against insect pests from the following insect families:
Preference is given to Coleopteran, Hemiptera, Diptera or Lepidopteran plant pest, including a Diabrotica, Leptinotarsa, Phyllotreta, Acyrthosiphan, Bemisia, Halyomorpha, Ostrinia, Lygus, Helicoverpa, Nezara, Spodoptera plant pest, Curculionidae, Chrysomelidae, of Scolytinae , Cerambycidae, Bruchinae, Buprestidae, Scarabaeidae, Dynastinae, Lyctinae, Anobiidae, Bostrichidae, Coccoidea (scale insects) Cercopoidea (spittlebugs, froghoppers), Membracoidea (Cicadelloidea), Fulgoroidea, Heteroptera, Reduviidae (assassin bugs), Cimicidae (bedbugs and flower bugs), Miridae (plant bugs) Coreidae (Leaf-footed bugs, squash bugs & sweet potato bugs), Lygaeidae (seed bugs) Rhyparochromidae (seed bugs), Pentatomidae (stink bugs or shield bugs), Stemorrhyncha Aphididae (aphids), Eriosomatinae (gall aphids), Pemphigidae (gall aphids), Adelgidae (conifer aphids), Chermesidae (conifer aphids), Phylloxeridae (phylloxerans), Aleyrodidae (whiteflies), Psyllidae (jumping plant lice), Noctuoidea, Anthomyiidae, Tephritidae, Muscidae, Agromyzidae, Drosophilidae, Tabanidae, Calliphoridae, Cecidomyiidae, Tachinidae, Mycetophilidae, Simuliidae, Stratiomyidae, Psychodidae, Opilioacarida, Holothyrida, Ixodida, Mesostigmata, Trigynaspida or Monogynaspida. Especially, preference is given to Bemisia and Trialeurodes, specifically to Bemesia tabaci (silverleaf whitefly) and Trialeurodes vaporariorum, (greenhouse whitefly) in crops such as, for example, soybean, melon, tabacco, tomatoes, squash, poinsettia, cucumber, eggplants, okra, beans and cotton.
Preference is further given from the family of the woolly aphids (Pemphigidae) to: Eriosoma spp., Pemphigus spp., in crops such as, for example, citrus fruit, pomaceous fruit, stone fruit, leaf vegetables, root and tuber vegetables and ornamental plants.
Preference is given from the family of the grape lice (Phylloxeridae) to: Phylloxera spp. in grapevines, nuts, citrus fruit.
Preference is given from the family of the coffee berry borer (Hypothenemus hampei) (Coleoptera: Curculionidae, Scolytinae in coffee.
Preference is given from the family of the com leafhopper, Dalbulus maidis (Hemiptera, Cicadellidae) in maize, apples, beans, carrots, celery, onions, potatoes, peppers, soybean, melon, tabacco, tomatoes, squash, poinsettia, cucumber, eggplants, okra, beans and cotton.
Preference is given from the family of the fall army worm and cotton bowl worm (Noctuidae) in cereals (maize, sorghum, millet, rice), soybean, melon, tabacco, tomatoes, squash, poinsettia, cucumber, eggplants, okra, beans and cotton.
Preference is further given from the family of the jumping plant lice (Psyllidae) to: Psylla spp., Paratrioza spp., Tenalaphara spp., Diaphorina spp., Trioza spp., in crops such as, for example, pomaceous fruit, stone fruit, citrus fruit, vegetables, potatoes, in tropical crops.
Preference is further given from the family of the soft scales (Coccidae) to: Ceroplastes spp., Drosicha spp., Pulvinaria spp., Protopulminaria spp., Saissetia spp., Coccus spp., in perennial crops such as, for example, citrus fruit, pomaceous fruit, stone fruit, olives, grapevines, coffee, tea, tropical crops, ornamental plants, vegetables.
Preference is further given from the family of the armoured scale insects (Diaspididae) to: Quadraspidiotus spp., Aonidiella spp., Lepidosaphes spp., Aspidiotus spp., Aspis spp., Diaspis spp., Parlatoria spp., Pseudaulacaspis spp., Unaspis spp., Pinnaspis spp., Selenaspidus spp., in crops such as, for example, citrus fruit, pomaceous fruit, stone fruit, almonds, pistachios, nuts, olives, tea, ornamental plants, grapevines, tropical crops.
Preference is further given from the family of the ensign scales (Ortheziidae) to: Orthezia spp. in citrus fruit, pomaceous fruit, stone fruit. Preference is given from the family of the mealy bugs (Pseudococcidae) to: Pericerga, Pseudococcus spp., Pianococcus spp., Dysmicoccus spp., in crops such as, for example, citrus fruit, stone fruit andpomaceous fruit, tea, grapevines, vegetables, ornamental plants and tropical crops. Preference is furthermore given from the family of the white flies (Aleyrodidae) to: Bemisia tabaci, Bemisia argentifolii, Trialeurodes vaporariorum, Aleurothrixus floccosus, Aleurodes spp., Dialeurodes spp., Parabemisia myricae in crops such as, for example, vegetables, melons, potatoes, tobacco, soft fruit, citrus fruit, ornamental plants, cotton, soya beans and tropical crops.
Moreover, preference is given from the family of the aphids (Aphidae) to:
Myzus spp. in tobacco, stone fruit, soft fruit, fruit vegetables, leafy vegetables, tuber and root vegetables, melons, potatoes, ornamental plants, spices, Acyrthosiphon onobrychis in vegetables, Aphis spp. in tobacco, citrus fruit, pomaceous fruit, stone fruit, melons, strawberries, soft fruit, fruit vegetables, leafy vegetables, tuber, stem and root vegetables, ornamental plants, potatoes, pumpkins, spices, Rhodobium porosum in strawberries, Nasonovia ribisnigri in leafy vegetables, Macrosiphum spp. in ornamental plants, potatoes, leafy vegetables and fruit vegetables, strawberries, Phorodon humuli in hops, Brevicoryne brassicae in leafy vegetables, Toxoptera spp. in citrus fruit, stone fruit, almonds, nuts, spices, Aulacorthum spp. in citrus fruit, potatoes, fruit vegetables and leafy vegetables, Anuraphis cardui in vegetables, Brachycaudus helycrisii in sunflowers, Acyrthosiphon onobrychis in vegetables.
Likewise, preference is given from the family of the thrips (Thripidae) to: Anaphothrips spp., Baliothrips spp., Caliothrips spp., Frankliniella spp., Heliothrips spp., Hercinothrips spp., Rhipiphorothrips spp., Scirtothrips spp., Kakothrips spp., Selenothrips spp. and Thrips spp., in crops such as, for example, fruit, cotton, grapevines, tea, nuts, tropical crops, ornamental plants, conifers, tobacco, spices, vegetables, soft fruit, melons, citrus fruit and potatoes.
Moreover, preference is given from the families of the leaf-miner flies (Agromyzidae) and root-maggot flies (Anthomyiidae) to: Agromyza spp., Amauromyza spp., Atherigona spp., Chlorops spp., Liriomyza spp., Oscinella spp., Pegomyia spp. in crops such as, for example, vegetables, melons, potatoes, nuts, ornamental plants.
Preference is given from the families of the leafhoppers (Cicadellidae) and planthoppers (Delphacidae) to: Circulifer spp., Dalbus spp., Empoasca spp., Erythroneura spp., Homalodisca spp., lodioscopus spp., Laodelphax spp., Nephotettix spp., Nilaparvata spp., Oncometopia spp., Sogatella spp., in crops such as, for example, citrus fruit, fruit, grapevines, potatoes, vegetables, ornamental plants, conifers, melons, soft fruit, tea, nuts, rice and tropical crops.
Preference is given from the family of the leaf-miner moths (Gracillariidae) to:
Caloptilia spp., Gracillaria spp., Lithocolletis spp., Leucoptera spp., Phtorimaea spp., Phyllocnistis spp. in crops such as pomaceous fruit, stone fruit, grapevines, nuts, citrus fruit, conifers, potatoes, coffee.
Preference is given from the family of the gall midges (Cecidomyiidae) to: Contarinia spp., Dasineura spp., Diplosis spp., Prodiplosis spp., Thecodiplosis spp., Sitodiplosis spp., Haplodiplosis spp. in crops such as citrus fruit, pomaceous fruit, stone fruit, vegetables, potatoes, spices, soft fruit, conifers, hops.
Likewise, preference is given from the family of the fruit flies (Tephritidae) to:
Anastrepha spp., Ceratitis spp., Dacus spp., Rhagoletis spp. in crops such as vegetables, soft fruit, melons, pomaceous and stone fruit, ornamental plants, potatoes, grapevines, tropical crops, citrus fruit, olives.
Moreover, preference is given to mites from the families of the spider mites (Tetranychidae) and the gall mites (Eriophydae):
Tetranychus spp., Panonychus spp., Aculops spp. in crops such as vegetables, potatoes, ornamental plants, citrus fruit, grapevines, conifers.
Preference is given from the family of the weevils, Sphenophorus levis in sugarcane and the banana weevil Cosmopolites sordidus in banana (Coleoptera: Curculionidae).
The inventive treatment of the plants and parts of plants with the compositions according to the invention is effected directly or by allowing the compositions to act on their surroundings, environment or storage space by the customary treatment methods, for example by drenching, immersion, spraying, evaporation, fogging, scattering, painting on and, in the case of propagation material, in particular in the case of seeds, also by applying one or more coats. Preferably, the plant to be treated is selected from the group consisting of cotton, soya beans, tobacco, vegetables, spices, ornamental plants, conifers, citrus plants, fruit, tropical crops, nuts and grapevines.
Preferably, the composition according to the invention acts against pests from the families of the woolly aphids, grape lice, jumping plant lice, soft scales, armored scale insects, ensign scales, mealy bugs, whiteflies, aphids, thrips, leafhoppers, planthoppers, leaf-miner flies, gall midges, fruit flies, leaf- miner moths, spider mites, gall mites.
In another preferred embodiment, the composition according to the invention acts against Diptera, including species from the family Culicidae such as Anopheles spp., Aedes spp., and Culex spp. In another preferred embodiment, further families are Anthomyiidae, Tephritidae, Muscidae, Agromyzidae, Drosophilidae, Tabanidae, Calliphoridae, Cecidomyiidae, Tachinidae, Mycetophilidae, Simuliidae, Stratiomyidae or Psychodidae.
In some embodiments the composition may comprise spores of C. javanica Apopka 97 and citric acid. The composition may increase white fly mortality. In some embodiments the composition may comprise spores of C. javanica Apopka 97 and one or more of the acids provided in Table 4. The composition may increase white fly mortality. In some embodiments the composition may comprise spores of C. javanica Apopka 97 and one or more of oxalic acid, dipicolinic acid, citric acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, phosphoric acid, aconitic acid, tartaric acid, succinic acid, malonic acid and/or malic acid. In some embodiments the composition may comprise spores of C. javanica Apopka 97 and one or more of 0,05% oxalic acid, 0,05% dipicolinic acid, 0,05% citric acid, 0,1% citric acid, 0,15% citric acid, 0,2% citric acid, 0,05% trimesic acid, 0,05% aconitic acid, 0,05% phosphoric acid, 0,1% benzoic acid, 0,1% sorbic acid, 0,1% acetic acid, 0,1% lactic acid, 0,1% ascorbic acid, 0,08% glutamic acid, 0,04% glycine, 0,05% serine, 0,025% phosphoric acid, 0,09% aconitic acid, 0,08% tartaric acid, 0,06% succinic acid, 0,05% malonic acid and/or 0,07% malic acid. The composition may increase white fly mortality.
In some embodiments the composition may comprise spores of C.javanica GF 511 and citric acid. The composition may increase white fly mortality. In some embodiments the composition may comprise spores of C. javanica GF 511 and one or more of the acids provided in Table 4. The composition may increase white fly mortality. In some embodiments the composition may comprise spores of C. javanica GF 511 and one or more of oxalic acid, dipicolinic acid, citric acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, phosphoric acid, aconitic acid, tartaric acid, succinic acid, malonic acid or malic acid. In some embodiments the composition may comprise spores of C. javanica GF 511 and 0,05% citric acid. The composition may increase white fly mortality.
In some embodiments the composition may comprise spores of M. brunneun F52 and citric acid. The composition may increase white fly mortality. In some embodiments the composition may comprise spores of M. brunneun F52 and one or more of the acids provided in Table 4. The composition may increase white fly mortality. In some embodiments the composition may comprise spores of M. brunneun F52 and one or more of oxalic acid, dipicolinic acid, citric acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, phosphoric acid, aconitic acid, tartaric acid, succinic acid, malonic acid or malic acid. The composition may increase white fly mortality.
Kit
In a further aspect, the entomopathogenic fungi and the acid may be comprised within a kit. The entomopathogenic fungi and the acid may be spatially separated within the kit, for example the kit may comprise at least two housings or containers, one comprising the entomopathogenic fungi and one comprising acid. The entomopathogenic fungi and acid may be comprised in separate housings or containers. The entomopathogenic fungi may be present as spores. The entomopathogenic fungal spores may be in powder form, optionally a wettable powder form. The entomopathogenic fungal spores may be dispersed in an aqueous solution or dispersed in a non-aqueous solution. The entomopathogenic fungal spores may be spores of any species described herein, prefereably of Cordyceps fumosorosea, Cordyceps javanica or Metarhizium brunneum. The entomopathogenic fungal spores may be spores of a Cordyceps javanica Apopka 97 strain or Cordyceps javanica GF 511 strain. The entomopathogenic fungal spores may be spores of a Metarhizium brunneum F52 strain. The kit may additionally comprise instructions or a description of how to prepare a composition of the invention or apply a method of the invention.
In a further aspect, the present invention relates to a kit-of-parts comprising an entomopathogenic fungi to prepare a composition according to the invention and a description how to prepare a composition according to the invention.
In a preferred embodiment, the kit-of-part further comprises one or more acids to prepare a composition according to the invention.
Preferably, the entomopathogenic fungi being spatially separated in a container. More preferably, the entomopathogenic fungi and the acid (if present in a kits-of-parts) are separated from each other in two spatially separated containers, thus resulting in the kit-of-parts comprising two separated components and a description how to prepare a composition according to the invention.
Moreover, the kit of parts according to the present invention can additionally comprise at least one auxiliary selected from the group consisting of extenders, solvents, spontaneity promoters, carriers, emulsifiers, dispersants, frost protectants, thickeners and adjuvants as mentioned herein. This at least one auxiliary can be present in one or more than one component of the kit of parts, e.g. in the component comprising an entomopathogenic fungi or in the component comprising an acid, being spatially separated from each other, or in both of these components or in the form of a separate component thus resulting in the kit-of-parts comprising at least three components spatially separated from each other.
The description how to prepare a composition according to the invention gives the skilled person the information regarding mixing ratios between an acid, preferably being already part of the kits-in-parts, water and an entomopathogenic fungi formulation to arrive at a composition according to the invention. For example, if the acid is present in a separated aqueous solution or as a solid in the kit-of-part and the fungi is present in form of a separated solid form (such as a powder) or a dispersion, the description will provide information how much solid form (powder) has to be combined with how much water and how much acid to arrive at a composition according to the invention. The preparation of a composition according to the invention can include further steps, e.g., of adding further components or presolubilizing or pre -emulsifying the acid or the fungal spores, such as pre -emulsifying a solid entomopathogenic fungi-preparation, before combining the pre -emulsified or pre-solubilized component with a further component, such as (e.g., a pre-solubilized) acid. Furthermore, the description preferably comprises time limits between the preparation of a composition according to the invention and its use (e.g. spraying the composition on a plant, plant part or habitat of a plant). Preferably, the preparation of a composition according to the invention takes place not longer than 48 hours before the use of the composition according to the invention. More preferably, the preparation of a composition according to the invention takes place not longer than 24 hours before the use of the composition according to the invention, even more preferably not longer than 12 or even 8 hours before the use of the composition according to the invention.
The invention may be described by the following numbered embodiments:
1. A composition comprising water, an acid, and an entomopathogenic fungus, preferably spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid is at least 1 g/1, and wherein the pH of the composition is between 2,0 and 7,0, and wherein the pH of said composition is lower than the pH of a comparative composition without said acid.
2. The composition of embodiment 1, wherein the acid is selected from the group consisting of sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alphaketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p-toluenesulfonic acid, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, isocinchomeronic acid, isocitric acid, isonicotinic acid, lutidinic acid, maleic acid, mandelic acid, nicotinic acid, phtalic acid, picolinic acid, propionic acid, salicylic acid, tetronic acid and methane sulfonic acid.
3. The composition of embodiment 1 or embodiment 2, wherein the acid is an organic acid selected from the group consisting of citric acid, malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid, and glycine.
4. The composition according to any one of the preceding embodiments, wherein the acid is an organic acid selected from the group consisting of malonic acid, malic acid, pimelic acid, tartaric acid, trimesic acid, aconitic acid, dipicolinic acid, acetic acid, lactic acid, ascorbic acid and glycine.
5. The composition according to any one of the preceding embodiments, wherein the decrease in pH is at least 0,1 pH units. 6. The composition according to any one of the preceding embodiments, wherein the entomopathogenic fungus is selected from the group consisting of Beauveria bassiana (preferably strain ATCC 74040, strain GHA (Accession No. ATCC74250), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339, strain PPRI 7315, strain R444, strains IL197, IL12, IL236, IL10, IL131, IL116, strain Bv025, strain BaGPK, strain ICPE 279, strain CG 716, strain HF23, strain ANT-03, strain NPP111B00, strain 203, strain BblO, strain ESALQ PL63, strain IMI389521, strain IBCB 66, strain K4B1, strain K4B3, strain CBMAI 1306; and Metarhizium brunneum (formely known as Metarhizium anisopliae) (preferably strain Cbl5, strain ESALQ 1037, strain E-9, strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013-1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448), strain ICIPE 78, strain ESF1, strain IBCB 425, strain LRC112.
7. The composition according to any one of the preceding embodiments wherein the entomopathogenic fungus is selected from the group consisting of Cordyceps fumosorosea strain Apopka 97, strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367; Cordyceps javanica strain wf GA 17, strain GZQ-1, strain IjH6102, strain pfl85, strain pf212, strain JZ-7, or strain IJNL-N8 / CCTCC M-2017709; and Isaria farinosa strain ESALQ1205.
8. The composition according to any one of the preceding embodiments, wherein the concentration of the fungi is between 1,0*104 spores per ml composition and 1,0*109 spores per ml composition.
9. The composition according to any one of the preceding embodiments, wherein the pH of the composition is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
10. The composition according to any one of the preceding embodiments, further comprising at least one further component selected from the group consisting of liquids for spore formulations, oil, antioxidant, emulsifier, rheology modifier, other components being present in a fungal spore formulation, impurities which can be found in water.
11. Use of an acid as defined in embodiments 1 to 4 for increasing the efficacy of fungal spores as defined in any one of embodiments 1, 6 or 7 to control insects and/or nematodes.
12. A Method for controlling insects and/or nematodes, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of the composition according to any one of embodiment 1 to 10 to said plant or to a locus where plants are growing or intended to be grown. 13. The method according to embodiment 12, wherein the amount of composition, is at least 0,05 1/ha.
14. Kit-of-parts comprising a biological control agent selected from the group consisting of at least one spatially separated entomopathogenic fungi as defined in embodiment 1 , 6 or 7 and a description how to prepare a composition according to any one of embodiments 1 to 10.
15. The Kit-in-parts further comprising at least one spatially separated acid as defined in any one of embodiments 1 to 4.
The following examples illustrate the invention in a non-limiting fashion.
Examples
Example 1: Materials and Methods
Production of spores and spore formulations of C. fumosorosea strain Apopka 97
To obtain conidia of C. fumosorosea strain Apopka 97, solid state fermentation can be used as described in Mascarin et al. (2013, “The virulence of entomopathogenic fungi against Bemisia tabaci biotype B (Hemiptera: Aleyrodidae) and their conidial production using solid substrate fermentation”, Biological Control https://doi.Org/10.1016/j.biocontrol.2013.05.001 ). To prepare the inoculum for the solid state fermentation, C. fumosorosea can be grown on Agar-based medium SDAY (sabouraud dextrose agar plus yeast extract) for 14 days at 26°C. A conidial suspension was then obtained by washing off the conidia with water with surfactant (0.1 % of Tween80) and using a cell scraper. For the fermentation, moistened and autoclaved rice (moisture content 40-50%) was placed in flasks, capped with a cotton ball and aluminum foil, and inoculated with a conidial suspension (l,0E+07 conidia per g moist rice). Spore concentrations were determined using a haemocytometer. The inoculated flasks were incubated for 11 days at 26°C. At the end of the fermentation process, spores were harvested by vacuuming and collecting them in a cyclone (Jaronski et al., 2007, “Chapter 11 - Mass Production of Entomopathogenic Fungi: State of the Art”, Mass Production of Beneficial Organisms https://doi.org/10.1016/B978-0-12-391453-8.00011-X). Harvested spores were dried resulting in a concentration of 2E+11 spores per g technical spore concentrate.
Two different formulations were prepared. For the type 1 formulation, technical spore concentrate was blended resulting in formulations consisting of 20% (w/w) spores, 77,5% (w/w) PEG400 mono oleate and 2,5% (w/w) Aerosil 200.
For the type 2 formulation, the ingredients were 20% (w/w) spores, 77,5 % (w/w) Polysorbate 20 and 2,5% (w/w) Aerosil R202. Blending was done using an Ultra-Turrax dispersing instrument (IKA, Germany). The formulations had spore concentrations of 4E+10 spores per g spore formulation. Corresponding blank type 1 and blank type 2 formulations were prepared according to the respective instructions given above, with the exception that the addition of fungal spores was omitted (i.e. they contain only formulants but no spores).
Testing of insecticidal activity
For testing insecticidal activity against white flies, single-leaf cotton plants (Gossypium herbaceum) were infected with Bemisia tabaci by exposing the plants to respective white fly populations for 24h which results in an extensive deposition of eggs on the cotton leaves. Afterwards the plants were incubated for 8-10 days at 25°C until hatching and development of immobile N1 nymphal stages. Per experiment, five single-leaf cotton plants were used and treated as five biological replicates. For the treatments, C. fumosorosea conidia were suspended in tap water. If pure spores were used, the spore powder was put in suspension by using a Potter-Elvehjem homogenizer. Formulated spores were added to water and stirred to gain homogenous suspensions. Likewise, blank formulations were dispersed and stirred in water to generate control treatments containing an equal concentrations of formulation ingredients. Spore concentrations for each treatment were determined using a haemocytometer and if not otherwise indicated spore concentrations were adjusted to a final concentration of l,0E+07 conidia/ml. E.g., to prepare a treatment with l,0E+07 spores/ml, 50 mg of the pure spore powder or 250 mg of the formulated spores were suspended in 100 ml water followed by a 1/10 dilution step with water. For additive testing, stock solutions of the respective compound in water were made first, before adding an appropriate volume of the stock solutions to the treatment. For dipicolinic acid and glutamic acid a 0,5% stock solution was made. For oxalic acid, citric acid, malonic acid, malic acid, fumaric acid adipic acid, pimelic acid, tartaric acid, itaconic acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, lysine, glycine, serine, p- Toluenesulfonic acid, succinic acid and propionic acid a 1% stock solution was made.
Before spraying, whitefly nymphs were checked for good coverage on the leaves and the infected plants were sprayed with the prepared solutions by using an automatic spraying system machine with integrated residue drying and afterwards incubated in climate chambers under controlled conditions (25 °C and 70 % RH). The spraying was done by using a two-nozzle system so that plants were sprayed from above and below (20 ml per plant and per treatment in total) to achieve the best coverage of the plants/insects. Twelve days after treatment, one representative leaf per plant and 30 to 60 nymphs belonging to various developmental stages (N1-N3) were examined to calculate insect mortality (percentage of dead insects). The efficacy of treatments was calculated using Abbott’s formula (Abbott, 1925, “A Method of Computing the Effectiveness of an Insecticide”, Journal of Economic Entomology https://doi.org/10.1093/jee/18-2.265a):
Abbott efficacy
Figure imgf000043_0001
100 Where X represents the percentage of living insects after control treatment and Y represents the percentage of living insects after treatment.
In order to determine the expected efficacy of the composition of two treatments the Colby formula was used (Colby, 1967, “Calculating Synergistic and Antagonistic Responses of Herbicide Combinations”, Weeds https://doi.org/10.2307/4041058): v. Y
E = X + Y - — 100
Where X represents the percentage of control (Abbott efficacy) of treatment 1, Y represents the percentage of control of treatment 2, and E equals the expected percentage of control of a combined treatment 1 and 2, The observed values for the combined treatments were compared with the expected values: when E was less than the observed value, the composition was considered synergistic, when E was greater than the observed value, the composition was considered antagonistic, and when E equaled the observed value, the composition was identified as additive.
Example 2: Synergistic action of the entomopathogenic fungus C. fumosorosea and citric acid for insect control
Spores of C. fumosorosea strain Apopka 97 were produced, dried and applied as described in Example 1. Pure spores in a concentration of l,0E+07 spores/ml were either treated alone, or in composition with 0,05% citric acid. To exemplify the preparation of 20 ml spray liquid containing l,0E+07 spores/ml and 0,05% citric acid, the following applies: 50 mg of pure spores were suspended in 100 ml water. After determining the number of spores per ml using a haemocytometer (e.g. a Thoma chamber), the amount of this suspensions needed to reach l,0E+07 spores per ml in 20 ml was calculated. E.g., if the initial spore suspension contained l,0E+08 spores/ml, then 2 ml of this spore suspension were added to 17 ml of water followed by 1 ml of the 1 % stock solution of citric acid. A solution of 1 ml of the 1 % stock solution of citric acid in 19 ml of water and pure water served as control experiments. Four independent experiments were conducted to calculate the insect mortality for each treatment. The composition of C. fumosorosea spores and citric acid reached a significantly higher mortality than C. fumosorosea spores alone (Figure 1).
Figure 1 shows the mortality of white flies after treatment with C. fumosorosea and citric acid. The mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 20 leaves were examined. To test for significant differences, the treatment of l,0E+07 spores/ml was set as a reference group (reference) for multiple pairwise t- tests. *** and **** denote p-values of <0,001 and <0,0001, respectively. The error bars represent standard error and the pH value of each spray liquid is indicated on the right of the bars. To calculate treatment efficacy, pure water was set as control treatment and Abbott’s formula (Abbott, 1925) was used to calculate the efficacy of the treatment with pure spores, the efficacy of the treatment with citric acid, and the efficacy of the composition of both treatments (Table 1).
Table 1 : Synergistic action of C. fumosorosea and citric acid for the control of white flies
Treatment Abbott efficacy (expected Colby efficacy) l,0E+07 s/ml 34,3
0,05% Citric acid 7,6 l,0E+7 s/ml + 0,05% Citric acid 57,0 (39,3)
The expected efficacy of the composition of both treatments was calculated with Colby’s formula (Colby, 1967) and this expected control was found to be 17,7% less than the observed level of control indicating that the treatment of spores and the treatment of citric acid are synergistic for insect control (Table 1).
Example 3: Synergistic action of C. fumosorosea formulated in PEG400 mono oleate and citric acid for insect control
Spores of C. fumosorosea strain Apopka 97 were formulated according to type 1 (vide infra) and applied to white flies as described in Example 1. 1 ,0E+07 spores/ml were either treated alone or in composition with 0,05% citric acid. To exemplify the preparation of 20 ml spray liquid containing l,0E+07 spores/ml and 0,05% citric acid, the following applies: 250 mg of formulated spores were suspended in 100 ml water. After determining the number of spores per ml using a haemocytometer (e.g. a Thoma chamber), the amount of this suspensions needed to reach l,0E+07 spores per ml in 20 ml was calculated. E.g., if the initial spore suspension contains l,0E+08 spores/ml, then 2 ml of this spore suspension were added to 17 ml of water followed by 1 ml of the 1 % stock solution of citric acid. As control experiments treatments with the blank formulation alone and the composition of the blank formulation and 0,05% citric acid were used. To exemplify the preparation of 20 ml of spray liquid containing 0,02% blank type 1 formulation and 0.05% citric acid the following procedure applies: 200 mg of blank type 1 formulation were dispersed in 100 ml water. Then, 2 ml of this premix were added to 17 ml of water followed by 1 ml of the 1% stock solution of citric acid. As additional control experiment pure water was employed. Nine independent experiments were conducted to calculate the insect mortality for each treatment. The supplementation of formulated C. fumosorosea spores with citric acid reached a significantly higher mortality than formulated C. fumosorosea spores alone (Figure 2).
Figure 2 shows the mortality of white flies after treatment with type 1 formulated C. fumosorosea and citric acid. The mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 45 leaves were examined (44 leaves for the water control). To test for significant differences, the treatment of l,0E+07 spores/ml was set as reference group for multiple pairwise t-tests. **** denotes p-values of <0,0001. The error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
To calculate treatment efficacy, blank formulation was set as control treatment and Abbott’s formula (Abbott, 1925) was used to calculate the efficacy of the treatment with formulated spores, the efficacy of the treatment with citric acid and blank formulation, and the efficacy of the composition of both treatments (Table 2).
Table 2: Synergistic action of type 1 formulated C. fumosorosea and citric acid for the control of white flies
Treatment Abbott efficacy
(expected Colby efficacy) l,0E+07 s/ml + 0,02% Blank type 1 formulation 35,8
0,05% Citric acid + 0,02% Blank type 1 formulation 9,5
1 ,0E+7 s/ml + 0,05% Citric acid + 0,02% Blank type 1 formulation 60,7 (42,0)
The expected efficacy of the composition of both treatments was calculated with Colby’s formula (Colby, 1967) and this expected control was found to be 18,6% less than the observed level of control indicating that the treatment of spores and the treatment of citric acid are synergistic for insect control (Table 2).
Example 4: Synergistic action of C. fumosorosea formulated in Polysorbate 20 and citric acid for insect control
Spores of C. fumosorosea strain Apopka 97 were type 2 formulated and applied to white flies as described in Example 1. l,0E+07 spores/ml were either treated alone or in composition with 0,05% citric acid. As controls served treatments with the blank formulation alone, the composition of the blank formulation and 0,05% citric acid and pure water. The insect mortality was counted for each treatment. The supplementation of formulated C. fumosorosea spores with citric acid reached a significantly higher mortality than formulated C. fumosorosea spores alone (Figure 3).
Figure 3 shows the mortality of white flies after treatment with type 2 formulated C. fumosorosea and citric acid. The mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 5 leaves were examined. To test for significant differences, the treatment of 1 ,0E+07 spores/ml was set as reference group for multiple pairwise t- tests. * and ** denote p-values of <0,05 and <0,01, respectively, ns denote p-values of >0,05. The error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
To calculate treatment efficacy, blank formulation was set as control treatment and Abbott’s formula (Abbott, 1925) was used to calculate the efficacy of the treatment with formulated spores, the efficacy of the treatment with citric acid and blank formulation, and the efficacy of the composition of both treatments (Table 3).
Table 3: Synergistic action of type 2 formulated C. fumosorosea and citric acid for the control of white flies
Treatment Abbott efficacy
(expected Colby efficacy) l,0E+07 s/ml + 0,02% Blank type 2 formulation 16,9
0,05% Citric acid + 0,02% Blank type 2 formulation 11,4
1 ,0E+7 s/ml + 0,05% Citric acid + 0,02% Blank type 2 formulation 50,3 (26,4)
The expected efficacy of the composition of both treatments was calculated with Colby’s formula (Colby, 1967) and this expected control was found to be 23,9% less than the observed level of control indicating that the treatment of spores and the treatment of citric acid are synergistic for insect control (Table 3).
Example 5: Impact of the concentration of citric acid to increase the efficacy of C. fumosorosea.
Spores of C. fumosorosea strain Apopka 97 were type 1 formulated and applied to white flies as described in Example 1. 1 ,0E+07 spores/ml were either treated alone or in composition with citric acid at the concentrations 0,005%, 0,01% (Figure 4), 0,05% (Figure 4 and 5), 0,1%, 0,15%, 0,2% and 0,25% (Figure 5). As controls served treatments with the blank formulation alone and the compositions of the blank formulation and various concentrations of citric acid (Figure 4 and 5).
Figure 4 shows the mortality of white flies after treatment with formulated C. fumosorosea and citric acid concentrations of 0,005% - 0,05%. The mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 15 leaves were examined. To test for significant differences, the treatment of l,0E+07 spores/ml was set as reference group for multiple pairwise t-tests. **** denotes p-values of <0,0001. ns denote p-values of >0,05. The error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars. The insect mortalities of the treatments revealed that citric acid concentrations of 0,005% and 0,01% increase the efficacy of C. fumosorosea (Figure 4), despite that the observed increase was not statistically significant.
Figure 5 shows the mortality of white flies after treatment with formulated C. fumosorosea and citric acid concentrations of 0,05% - 0,25%. The mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 5 leaves were examined. To test for significant differences, the treatment of l,0E+07 spores/ml was set as reference group for multiple pairwise t-tests. *, ** and *** denote p-values of <0,05, <0,01 and <0,001, respectively, ns denote p-values of >0,05. The error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
The mortality increasing effect of citric acid was even more pronounced at concentrations of 0,05% and 0,1% (Figure 4 and 5). While the insect mortality of combinatorial treatments of C. fumosorosea and citric acid at concentrations of greater than 0, 1 % was also high, citric acid concentrations of 0, 15% and above showed a strong intrinsic efficacy against white flies, which even exceeded the efficacy of formulated spores alone at a citric acid concentration of 0,25% (Figure 5). Liquids with such high concentrations of citric acids having a pH of less than 3 also showed phytotoxicity towards the plants.
Example 6: Citric acid increases the efficacy of various spore concentrations of C. fumosorosea.
Spores of C. fumosorosea strain Apopka 97 were type 1 formulated and applied to white flies as described in Example 1. Different spore concentrations of the spray liquid, i.e. 2,5E+06 spores/ml, 5,0E+06 spores/ml and l,0E+07 spores/ml, were either treated alone or in composition with citric acid at the concentrations 0,05% (Figure 6). For all tested spore concentrations, the insect mortality was significantly increased by the addition of citric acid.
Figure 6 shows the mortality of white flies after treatment with various concentrations of formulated C. fumosorosea and citric acid concentrations of 0,05%. The mortality of white flies was calculated for the indicated treatments as described in the examples of the main text. For each treatment, a total of 5 leaves were examined. To test for significant differences, multiple pairwise t-tests were performed. ** and **** denote p-values of <0,01 and <0,0001, respectively. The error bars represent standard error, and the pH value of each spray liquid is indicated on the right of the bars.
Example 7: Mortality of white flies after combined treatments with C. fumosorosea and various acids
Spores of C. fumosorosea strain Apopka 97 were type 1 formulated and applied to white flies as described in Example 1. 1 ,0E+07 spores/ml were either treated alone or in composition with various acids summarized in Table 4. Table 4: List of acids supplemented to spray liquid
Acid in spray liquid (f.c.) pH
0, 1 % Acetic acid 4,3
0,05% Aconitic acid 3,5
0,09% Aconitic acid 2,8
0,05% Adipic acid 4,7
0,1% Ascorbic acid 4, 1
0, 1 % Benzoic acid 4,5
0,005% Citric acid 6,8
0,01% Citric acid 6,4
0,05% Citric acid 3,9
0,1% Citric acid 3 ,2
0,15% Citric acid 3,0
0,2% Citric acid 2,9
0,25% Citric acid 2,8
0,005% Dipicolinic acid 7,0
0,01% Dipicolinic acid 6,6
0,05% Dipicolinic acid 3,0
0,05% Fumaric acid 3,4
0,08% Glutamic acid 4,4
0,04% Glycine 8,0
0,05% Itaconic acid 4,3
0, 1 % Lactic acid 6,7
0,08% Lysine 9,6
0,05% Malic acid 3,9
0,07% Malic acid 3,6
0,05% Malonic acid 3,3
0,005% Oxalic acid 6,7
0,01% Oxalic acid 6,0 0,05% Oxalic acid 2,4
0,04% p-Toluenesulfonic acid 6,0
0,005% Phosphoric acid 6,7
0,01% Phosphoric acid 6,1
0,025% Phosphoric acid 3,0
0,05% Phosphoric acid 2,5
0,05% Pimelic acid 4,8
0,05% Propionic acid 4,6
0,05% Serine 7,8
0, 1 % Sorbic acid 4,3
0,06% Succinic acid 4,3
0,05% Tartaric acid 3,4
0,08% Tartaric acid 3,2
0,05% Trimesic acid 4,0
To calculate treatment efficacy, the respective blank formulation treatments were set as control and Abbott’s formula (Abbott, 1925) was used to calculate the efficacy of each of the acid treatments, the respective treatment with formulated spores, and the efficacy of the composition of both treatments (Table 5). The expected efficacy of the combined treatments was calculated with Colby’s formula (Colby, 1967) and this expected control was found to be less than the observed level of control for the organic acid treatments 0,05% oxalic acid, 0,05% dipicolinic acid, 0,05% citric acid, 0,1% citric acid, 0,15% citric acid, 0,2% citric acid, 0,05% trimesic acid, 0,05% aconitic acid, 0,05% phosphoric acid, 0,1% benzoic acid, 0,1% sorbic acid, 0,1% acetic acid, 0,1% lactic acid, 0,1% ascorbic acid, 0,08% glutamic acid, 0,04% glycine, 0,05% serine, 0,025% phosphoric acid, 0,09% aconitic acid, 0,08% tartaric acid, 0,06% succinic acid, 0,05% malonic acid and 0,07% malic acid, i.e. those acids acted synergistically with the Cordyceps spores for insect control. By contrast, the acid treatments 0,25% citric acid, 0,05% itaconic acid, 0,05% propionic acid, 0,005% phosphoric acid, 0,01% phosphoric acid, 0,08% lysine and 0,04% p-toluenesulfonic acid acted antagonistically to the Cordyceps spores (Table 5).
Table 5, Synergistic and antagonistic action of various acids and C.fumosorosea spores for the control of white flies Abbott efficacy (expected Colby
Treatment efficacy) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,3
0,02% Blank type 1 formulation + 0,05% Oxalic acid 14 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Oxalic acid 62,3 (47,8) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,3
0,02% Blank type 1 formulation + 0,05% Dipicolinic acid 6,2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Dipicolinic acid 58,9 (43,1) l,0E+7 s/ml + 0,02% Blank type 1 formulation 35,8
0,02% Blank type 1 formulation + 0,05% Citric acid 9,5 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Citric acid 60,7 (42,0) l,0E+7 s/ml + 0,02% Blank type 1 formulation 31.6
0,02% Blank type 1 formulation + 0,1% Citric acid 14,8 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,1% Citric acid 64,7 (41.7) l,0E+7 s/ml + 0,02% Blank type 1 formulation 34,5
0,02% Blank type 1 formulation + 0,15% Citric acid 12 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,15% Citric acid 54,6 (42,4) l,0E+7 s/ml + 0,02% Blank type 1 formulation 34,5
0,02% Blank type 1 formulation + 0,2% Citric acid 34,4 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,2% Citric acid 61 (57) l,0E+7 s/ml + 0,02% Blank type 1 formulation 34,5
0,02% Blank type 1 formulation + 0,25% Citric acid 39,1 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,25% Citric acid 53 (60,1) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,4
0,02% Blank type 1 formulation + 0,05% Itaconic acid 35,6 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Itaconic acid 47 (61) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,4
0,02% Blank type 1 formulation + 0,05% Trimesic acid 6,2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Trimesic acid 50,2 (43,2) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,4
0,02% Blank type 1 formulation + 0,05% Aconitic acid 14 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Aconitic acid 62,2 (47,9) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,4
0,02% Blank type 1 formulation + 0,005% Phosphoric acid 9,3 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,005% Phosphoric acid 32,6 (45) l,0E+7 s/ml + 0,02% Blank type 1 formulation 33,5
0,02% Blank type 1 formulation + 0,05% Phosphoric acid 13,9 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Phosphoric acid 59,8 (42,7) l,0E+7 s/ml + 0,02% Blank type 1 formulation 33,5
0,02% Blank type 1 formulation + 0,01% Phosphoric acid 23,3 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,01% Phosphoric acid 43,2 (49) l,0E+7 s/ml + 0,02% Blank type 1 formulation 22,1
0,02% Blank type 1 formulation + 0,1% Benzoic acid 0,2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,1% Benzoic acid 55,3 (22,3) l,0E+7 s/ml + 0,02% Blank type 1 formulation 22,1
0,02% Blank type 1 formulation + 0,1% Sorbic acid 6,4 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,1% Sorbic acid 29,1 (27,1) l,0E+7 s/ml + 0,02% Blank type 1 formulation 22,1
0,02% Blank type 1 formulation + 0,1% Acetic acid 10,2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,1% Acetic acid 66,9 (30) l,0E+7 s/ml + 0,02% Blank type 1 formulation 22,1
0,02% Blank type 1 formulation + 0,1% Lactic acid 20,1 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,1% Lactic acid 48,8 (37,8) l,0E+7 s/ml + 0,02% Blank type 1 formulation 22,1
0,02% Blank type 1 formulation + 0,1% Ascorbic acid 20,1 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,1% Ascorbic acid 83,4 (37,8) l,0E+7 s/ml + 0,02% Blank type 1 formulation 37,3
0,02% Blank type 1 formulation + 0,08% Glutamic acid 9,6 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,08% Glutamic acid 68,4 (43,3) l,0E+7 s/ml + 0,02% Blank type 1 formulation 37,3
0,02% Blank type 1 formulation + 0,08% Lysine 6,7 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,08% Lysine 4,3 (41.5) l,0E+7 s/ml + 0,02% Blank type 1 formulation 37,3
0,02% Blank type 1 formulation + 0,04% Glycine 13,5 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,04% Glycine 54,4 (45,8) l,0E+7 s/ml + 0,02% Blank type 1 formulation 37,3
0,02% Blank type 1 formulation + 0,05% Serine 7,7 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Serine 53,2 (42,1) l,0E+7 s/ml + 0,02% Blank type 1 formulation 37,3
0,02% Blank type 1 formulation + 0,04% p-Toluenesulfonic acid -11.2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,04% p-Toluenesulfonic acid 29,2 (30,3) l,0E+7 s/ml + 0,02% Blank type 1 formulation 27
0,02% Blank type 1 formulation + 0,025% Phosphoric acid 14 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,025% Phosphoric acid 44,4 (37,2) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,5
0,02% Blank type 1 formulation + 0,09% Aconitic acid 11.1 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,09% Aconitic acid 47,6 (46,2) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,5
0,02% Blank type 1 formulation + 0,08% Tartaric acid -2,5 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,08% Tartaric acid 80,9 (38) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,5
0,02% Blank type 1 formulation + 0,06% Succinic acid 19,8 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,06% Succinic acid 81.4 (51.5) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,5
0,02% Blank type 1 formulation + 0,05% Malonic acid 10,2 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Malonic acid 68 (45,7) l,0E+7 s/ml + 0,02% Blank type 1 formulation 39,5
0,02% Blank type 1 formulation + 0,07% Malic acid 16,1 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,07% Malic acid 77,2 (49,2) l,0E+7 s/ml + 0,02% Blank type 1 formulation 32,9
0,02% Blank type 1 formulation + 0,05% Propionic acid 20,9 l,0E+7 s/ml + 0,02% Blank type 1 formulation + 0,05% Propionic acid 38,4 (46,9)
Correlation analysis revealed a highly significant negative relationship between insect mortality and pH after treatment with C. fumosorosea spores (Figure 7).
Figure 7 shows the correlation between pH of the spray liquid containing acids and C. fumosorosea spores and mortality of white flies. In total, 359 leaves which were all treated with l,0E+07 spores/ml with or without addition of various acids, were examined. All acids in their final concentrations and their pH values are listed in Table 4, The pH of the spray liquid containing l,0E+07 spores/ml without addition of acids was 7,8, The number of examined leaves per treatment were 64 for spores only and 0,05% citric acid, 23 for 0,1% citric acid, 10 for 0,05% malonic acid, 0,01% and 0,05% phosphoric acid, 3 for 0,09% aconitic acid and 5 for all remaining acids listed in Table 4, The regression line indicates negative correlation between pH and insect mortality. Pearson correlation coefficient (/?) and probability value (p) are depicted in the lower left comer, and the 95% confidence interval is indicated in light grey.
Example 8: Synergistic action of an enthomopathogenic fungus C. javanica strain GF 511 and citric acid, for insect control
Spores of C. javanica strain GF 511 were blended with distilled water containing Tween 80 (0,05% v/v). Pure spores in a concentration of l,0E+07 CFU/ml were either treated alone, or in composition with 0,05% citric acid. The 20 ml spray liquid containing l,0E+07 spores/ml and 0,05% citric acid was prepared as follows: 1 g of pure spores (l,0E+10) were suspended in 100 ml of Tween 80 (0,05% v/v) solution. After determining the number of spores per ml using a haemocytometer (e.g., a Thoma chamber), the amount of this suspension needed to reach l,0E+07 spores per ml in 20 ml was calculated. E.g., if the initial spore suspension contained l,0E+08 spores/ml, then 2 ml of this spore suspension was added to 17 ml of water followed by 1 ml of the 1% stock solution of citric acid. A solution of 1 ml of the 1% stock solution of citric acid in 19 ml of water and pure water served as control experiments.
For testing insecticidal activity against white flies, 9-day-old plants (Phaseolus vulgaris cv. Perola) with two primary leaves grown with Oxisol (2 L) with fertilizer NPK 5-30-15 were kept in a screenhouse covered with a fine screen fabric (50 mesh). Adult-infested plants with Bemisia tabaci were shaken and placed next to pest-free bean plants (9 days old with two primary leaves) for 6 to 8h to allow oviposition. This exposure provided more than 200 eggs per leaf. Afterwards the plants were transferred to a clean and insect-free room and incubated at 25°C until hatching and development of immobile N2 nymphal stages ( 10 to 12 days after infestation) . Per treatment, seven pots with four bean plants in the two-leave stage were used as biological replicates. The experiment was conducted in a randomized block design in five screenhouse benches, one for each repetition, with the treatments randomized within the block.
Before spraying, whitefly nymphs were checked for good coverage on the leaves and the infected plants were sprayed with the prepared solutions as detailed above. The different treatments were applied to the abaxial side of primary leaves containing nymphs with a microsprayer (0.30 mm needle, Paasche® airbrush type H-set) connected to a vacuum pump and calibrated to 250 pl per leaf in an even coverage. The amount is adjusted in function of the size of the primary leaves in order to ensure a good coverage. Afterwards, the plants are incubated under controlled conditions (30 °C and 40 % RH). Fourteen days after treatment, one plant/pot (two leaves per treatment) were destructively evaluated for insect mortality. Nymphal mortality was assessed under a dissecting stereomicroscope (Leica) at x40 magnification. For confirming the fungal infection, a small red dot was marked on the leaf next to the dead nymphs. The leaves were then incubated inside Gerbox box with a wet cotton added to the leaf petiole for four days at room temperature. The dead marked nymphs presenting sporulation (i.e., mycosed nymphs) were considered infected by the fungus.
The efficacy of treatments was calculated using Abbott’s formula as described in Example 1.
To calculate treatment efficacy, Tween 80 (0.05% v/v) was set as control treatment and Abbott’s formula (Abbott, 1925) was used to calculate the efficacy of the treatment with pure spores and the efficacy of the treatment with citric acid and the efficacy of the compositions of both treatments (Table 6).
Table 6: Synergistic action of C. javanica strain GF511 and citric acid for the control of white flies
Treatment Abbott efficacy (expected Colby efficacy) l,0E+07 s/ml + 0,05% Tween 67,8
0,05% Citric acid + 0,05% Tween 3,56 l,0E+7 s/ml + 0,05% Citric acid + 0,05% Tween 79,3 (68,9)
The expected efficacy of both treatments was calculated with Colby’s formula (Colby, 1967) and this expected control was found to be 10,4% less than the observed level of control indicating that the treatment of spores and the treatment of citric acid are synergistic for insect control (Table 6).

Claims

55
Claims
1. A preparation comprising an acid and spores of an entomopathogenic fungus.
2. The preparation of claim 1, wherein the spores of an entomopathogenic fungus are in a wettable powder form, wettable granule form, dispersed in an aqueous liquid or dispersed in a non-aqueous liquid, optionally oil.
3. The preparation of claim 1, wherein the spores of an entomopathogenic fungus are in a wettable powder form.
4. The preparation of any one of the preceding claims, wherein the acid is sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p-toluenesulfonic acid, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, isocinchomeronic acid, isocitric acid, isonicotinic acid, lutidinic acid, maleic acid, mandelic acid, nicotinic acid, phtalic acid, picolinic acid, propionic acid, salicylic acid, tetronic acid or methane sulfonic acid.
5. The preparation of any one of the preceding claims, wherein the acid is an organic acid, optionally wherein the organic acid is oxalic acid, dipicolinic acid, citric acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, phosphoric acid, aconitic acid, tartaric acid, succinic acid, malonic acid or malic acid.
6. The preparation of any one of the preceding claims, wherein the entomopathogenic fungus is: Beauveria bassicma, optionally strain ATCC 74040, strain GHA (Accession No. ATCC74250), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339, strain PPRI 7315, strain R444, strains IL197, IL12, IL236, IL10, IL131, IL116, strain Bv025, strain BaGPK, strain ICPE 279, strain CG 716, strain HF23, strain ANT-03, strain NPP111B00, strain 203, strain BblO, strain ESALQ PL63, strain IMI389521, strain IBCB 66, strain K4B1, strain K4B3, or strain CBMAI 1306; or
Metarhizium brunneum (formely known as Metarhizium anisopliae), optionally strain Cbl5, strain ESALQ 1037, strain E-9, strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013-1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448, IDAC Accession No. IMI 507278), strain ICIPE 78, strain ESF1, strain IBCB 425 or strain LRC112. 56
7. The preparation of any one of claims 1 to 6 wherein the entomopathogenic fungus is:
Cordyceps fumosorosea, optionally strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367; or
Cordyceps javanica, optionally strain Apopka 97, strain GF 511 (ID AC Accession No. 080621-01), wf GA 17, strain GZQ-1, strain IjH6102, strain pfl85, strain pf212, strain JZ-7, or strain I JNL-N8 / CCTCC M-2017709; or
Isaria farinosa, optionally strain ESALQ1205.
8. The preparation of any one of the preceding claims, further comprising at least one further component, wherein the further component is one or more liquids for spore formulations, an oil, antioxidant, emulsifier, or rheology modifier, one or more other components being present in a fungal spore formulation, or one or more impurities which can be found in water.
9. The preparation of any one of the preceding claims, wherein the entomopathogenic fungus is Cordyceps javanica Apopka 97.
10. The preparation of claim 9 wherein the acid is citric acid.
11. The preparation of any one of claims 1 to 8 wherein the entomopathogenic fungus is Cordyceps javanica GF 511 (IDAC Accession number 080621-01).
12. The preparation of claim 11 wherein the acid is citric acid.
13. The preparation of any one of claims 1 to 8 wherein the entomopathogenic fungus is Metarhizium brunneum F52 (IDAC Accession No. IMI 507278).
14. The preparation of claim 13 wherein the acid is citric acid.
15. A composition comprising water, an acid, and spores of an entomopathogenic fungus, wherein the amount of acid in the composition is between 0,0001% (w/w) and 2% (w/w), and wherein the water solubility of the acid is at least 1 g/1, and wherein the pH of the composition is between 2,0 and 7,0, and wherein the pH of said composition is lower than the pH of a comparative composition without said acid.
16. The composition of claim 15, wherein the pH of the composition is at least 0,1 pH units lower than the pH the comparative composition without said acid. 57
17. The composition of claim 15 or 16 , wherein the concentration of the fungus is between l,0*104 spores per ml composition and l,0*109 spores per ml composition.
18. The composition of any one of claims 15 to 17, wherein the pH of the composition diluted in water is between pH 3,0 and pH 6,0 at 25 °C and 1013 hPa.
19. The composition of any one of claims 15 to 18, wherein the acid is sulfuric acid, nitric acid, phosphoric acid, hydrochloric acid, boric acid, oxalic acid, dipicolinic acid, citric acid, malonic acid, malic acid, fumaric acid, adipic acid, pimelic acid, tartaric acid, alpha-ketoglutaric acid, itaconic acid, trimesic acid, aconitic acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, asparagine, alanine, phenylalanine, valine, leucine, isoleucine, histidine, arginine, tyrosine, threonine, cysteine, methionine, tryptophan, proline, selenocysteine, p-toluenesulfonic acid, succinic acid, acrylic acid, aspartic acid, chinolinic acid, cinchomeronic acid, dinicotinic acid, formic acid, glutaric acid, glycolic acid, isocinchomeronic acid, isocitric acid, isonicotinic acid, lutidinic acid, maleic acid, mandelic acid, nicotinic acid, phtalic acid, picolinic acid, propionic acid, salicylic acid, tetronic acid or methane sulfonic acid.
20. The composition of any one of claims 15 to 19, wherein the acid is an organic acid, optionally wherein the organic acid is oxalic acid, dipicolinic acid, citric acid, trimesic acid, aconitic acid, phosphoric acid, benzoic acid, sorbic acid, acetic acid, lactic acid, ascorbic acid, glutamic acid, glycine, serine, phosphoric acid, aconitic acid, tartaric acid, succinic acid, malonic acid or malic acid.
21. The composition of any one of claims 15 to 20, wherein the entomopathogenic fungus is: Beauveria bassicma, optionally strain ATCC 74040, strain GHA (Accession No. ATCC74250), strain ATP02 (Accession No. DSM 24665), strain PPRI 5339, strain PPRI 7315, strain R444, strains IL197, IL12, IL236, IL10, IL131, IL116, strain Bv025, strain BaGPK, strain ICPE 279, strain CG 716, strain HF23, strain ANT-03, strain NPP111B00, strain 203, strain BblO, strain ESALQ PL63, strain IMI389521, strain IBCB 66, strain K4B1, strain K4B3, or strain CBMAI 1306; or
Metarhizium brunneum (formely known as Metarhizium anisopliae), optionally strain Cbl5, strain ESALQ 1037, strain E-9, strain M206077, strain C4-B (NRRL 30905), strain ESC1, strain 15013-1 (NRRL 67073), strain 3213-1 (NRRL 67074), strain C20091, strain C20092, strain F52 (DSM3884/ ATCC 90448, IDAC Accession No. IMI 507278), strain ICIPE 78, strain ESF1, strain IBCB 425 or strain LRC112.
22. The composition of any one of claims 15 to 21 wherein the entomopathogenic fungus is: Cordyceps fumosorosea, optionally strain IF-BDC01, strain FE 9901 / ARSEF 4490, strain ESALQ1296 or strain CCM 8367; or 58
Cordyceps javanica, optionally strain Apopka 97, strain GF 511 (ID AC Accession No. 080621-01), wf GAI 7, strain GZQ-1, strain IjH6102, strain pfl85, strain pf212, strain JZ-7, or strain I JNL-N8 / CCTCC M-2017709; or
Isaria farinosa, optionally strain ESALQ1205.
23. The composition of any one of claims 15 to 22, further comprising at least one further component, wherein the further component is one or more liquids for spore formulations, an oil, antioxidant, emulsifier, or rheology modifier, one or more other components being present in a fungal spore formulation, or one or more impurities which can be found in water.
24. The composition of any one of claims 15 to 23, wherein the entomopathogenic fungus is Cordyceps javanica Apopka 97.
25. The composition of claim 25 wherein the acid is citric acid.
26. The composition of any one of claims 15 to 23 wherein the entomopathogenic fungus is Cordyceps javanica GF 511 (IDAC Accession number 080621-01).
27. The composition of claim 26 wherein the acid is citric acid.
28. The composition of any one of claims 15 to 23 wherein the entomopathogenic fungus is Metarhizium brunneum F52 (IDAC Accession No. IMI 507278).
29. The composition of claim 28 wherein the acid is citric acid.
30. Use of an acid as defined in any one of claims 4, 5, 10, 12, 14, 16, 18-20, 25, 27 or 29for increasing the efficacy of entomopathogenic fungal spores as defined in any one of claims 2, 3, 6, 7, 9, 11, 13, 17, 21, 22, 24, 26 or 28 to control insects and/or nematodes.
31. A method for enhancing the efficacy of fungal spores comprising contacting spores of an entomopathogenic fungus as defined in any one of claims 2, 3, 6, 7, 9, 11, 13, 17, 21, 22, 24, 26 or 28with an acid as defined in any one of claims 4, 5, 10, 12, 14, 16, 18-20, 25, 27 or 29.
32. A method for controlling insects and/or nematodes, in or on a plant, for enhancing growth of a plant or for increasing plant yield or root health comprising applying an effective amount of a preparation according to any one of claims 1 to 14 or a composition according to any one of claims 15 to 29 to said plant or to a locus where plants are growing or intended to be grown.
33. The use of claim 30 or the method of claim 31 or 32 wherein the entomopathogenic fungus is Cordyceps javanica Apopka 97 and the acid is citric acid, wherein the composition increases white fly (Aleyrodidae) mortality.
34. The use of claim 30 or the method of claim 31 or 32 wherein the entomopathogenic fungus is Cordyceps javanica GF 511 (ID AC Accession number 080621-01) and the acid is citric acid, wherein the composition increases white fly (Aleyrodidae) mortality.
36. The use of claim 30 or the method of claim 31 or 32 wherein the entomopathogenic fungus is Metarhizium brunneum F52 (ID AC Accession No. IMI 507278) and the acid is citric acid, wherein the composition increases white fly (Aleyrodidae) mortality.
37. The method of any one of claims 32-36, wherein the amount of preparation or composition is at least 0,05 1/ha.
38. A kit comprising spores of an entomopathogenic fungus as defined in any one of claims 2, 3, 6, 7, 9, 11, 13, 17, 21, 22, 24, 26 or 28 and an acid as defined in any one of claims 4, 5, 10, 12, 14, 16, 18- 20, 25, 27 or 29, wherein the entomopathogenic fungi and the acid are spatially separated, optionally wherein said kit is suitable for preparation of a composition according to any one of claims 15 to 29.
39. The kit of claim 38 further comprising a description of how to prepare a composition according to any one of claims 15 to 29.
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