WO2023026281A1

WO2023026281A1 - A method of plant treatment with 8-geranyl-7-hydroxycoumarin and pesticide compositions thereof

Info

Publication number: WO2023026281A1
Application number: PCT/IL2022/050916
Authority: WO
Inventors: David Panik; Ido KORMAN
Original assignee: Metabolic Insights Ltd.
Priority date: 2021-08-27
Filing date: 2022-08-22
Publication date: 2023-03-02
Also published as: IT202100022448A1

Abstract

A method for controlling, preventing, reducing or eradicating the instances of plant-pathogen infestation or plant diseases on a plant, plant organ, plant part, or plant propagation material is provided. The method comprises applying to a plant, plant part, plant organ or plant propagation material, or to soil surrounding said plant, a pesticidally effective amount of 8-geranyl-7- hydroxycoumarin or a pesticide composition comprising it, wherein said plant-pathogen is a member selected from a Basidiomycota of the class Agaricomycetes or the genus Rhizoctonia, an Ascomycota of the class Sordariomycetes or a genus selected from Fusarium, Heterokontophyta of the class Oomycota and a Heterokontophyta of the order Peronosporales.

Description

A METHOD OF PLANT TREATMENT WITH 8-GERANYL-7-HYDROXYCOUMARIN

AND PESTICIDE COMPOSITIONS THEREOF

TECHNICAL FIELD

[0001] The present invention relates in general to pesticides and their agricultural uses.

BACKGROUND

[0002] Plant pests and diseases represent major challenges to productivity in modern agriculture. Soil-borne plant pathogens cause crucial damage to agricultural crops. The phytopathogenic fungus Rhizoctonia spp. belongs to phylogenetic lineage of Basidiomycota. It causes a wide range of commercially significant plant diseases, such as brown patch, damping off in seedlings, root rot and belly rot in vegetable crops and sheath blight in rice. All Rhizoctonia diseases, and subsequent secondary infections in plants are difficult to control (Erlacher et al., 2014).

[0003] Pythium spp. is phytopathogenic fungus-like organism which belongs to phylogenetic lineage of eukaryotic microorganisms called Oomycetes which causes the widespread “damping off’ disease of tobacco, tomato, mustard, chilies and cress seedlings (Martin and Loper, 2010).

[0004] Phytophthora spp. is an obligatory plant fungal like pathogen which belongs to phylogenetic lineage of eukaryotic microorganisms called Oomycetes. Phytophthora infestans is a serious potato disease known as potato blight resulting in foliage blight and rot of tubers. The disease can cause complete loss of a potato harvest (Sedlakova et al., 2012). Phytophthora attacks the aerial parts of many plant species and it is the major cause of leaf blight, canker fruit rot diseases in tomato, pumpkins and other crops.

[0005] Fusarium spp. is a large genus of filamentous fungi belonging to phylogenetic lineage of Ascomycetes. Many species of Fusarium are pathogenic to plants and cause serious diseases like wilt or ‘rot’ of economically important plants, mostly vegetables. In addition, Fusarium species infects cereals causing head blight and ear rot in maize and cause to mycotoxins accumulation under certain conditions (J. E. E. Jenkins, Y. S. Clark and A. E. Buckle, 1998).

[0006] The number of available active ingredients for crop protection purposes against these diseases is diminishing from year to year due to increasing pest resistance, erratic climatic conditions and mounting regulatory pressure. New active ingredients are urgently needed for development of novel environmentally sustainable crop protection solutions. SUMMARY OF THE INVENTION

[0007] In one aspect of the present invention, a method for controlling, preventing, reducing or eradicating instances of plant-pathogen infestation or plant diseases, on a plant, plant organ, plant part, or plant propagation material, comprises applying to a plant, plant part, plant organ or plant propagation material, or to soil surrounding said plant, a pesticidally effective amount of the plant metabolite compound 8-geranyl-7-hydroxycoumarin.

[0008] In some embodiments, 8-geranyl-7-hydroxycoumarin is applied to a plant-pathogen which is a member selected from: a Basidiomycota of the class Agaricomycetes or the genus Rhizoctonia, an Ascomycota of the class Sordariomycetes or a genus selected from Fusarium, Heterokontophyta of the class Oomycota and a Heterokontophyta of the order Peronosporales.

[0009] Various embodiments may allow various benefits and may be used in conjunction with various applications. The details of one or more embodiments are set forth in the accompanying figures and the description below. Other features, objects and advantages of the described invention will be apparent from the description and drawings and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

[0010] Disclosed embodiments will be understood and appreciated more fully from the following detailed description taken in conjunction with the appended figures.

[0011] Fig. 1 shows Experiment 538 results on the effect of 8-geranyl-7-hydroxycoumarin on the Phytophthora infestans disease severity in tomato seedlings, seven days after infection, under greenhouse conditions, determined as % disease severity, using preventative approach and application via spraying. Conditions of the experiment: Wettable Powder (WP) Fungicide containing 50% dimethomorph (Acrobat®, BASF); p < 0.05; **, p < 0.01; ***, p < 0.001; and non-significant (n.s.) difference vs. untreated control; ppm - parts per million; Formulation 21 at small scale (n = 24) (see Example 5).

[0012] Fig. 2 shows Experiment 563 results on the effect of 8-geranyl-7-hydroxycoumarin on the Phytophthora infestans disease severity in tomato seedlings, seven days after infection, under greenhouse conditions, determined as % disease severity, using preventative approach and application via spraying. Conditions of the experiment: Wettable Powder (WP) Fungicide containing 50% dimethomorph (Acrobat®, BASF); p < 0.05; **, p < 0.01; ***, p < 0.001; and non-significant (n.s.) difference vs. untreated control; ppm - parts per million; Formulation 21 at small scale (n = 24) (see Example 5).

[0013] Fig. 3 shows Experiment 591 results on the effect of 8-geranyl-7-hydroxycoumarin on the Phytophthora infestans disease severity in tomato seedlings, seven days after infection, under greenhouse conditions, determined as % disease severity, using preventative approach and application via spraying. Conditions of the experiment: Wettable Powder (WP) Fungicide containing 50% dimethomorph (Acrobat®, BASF); p < 0.05; **, p < 0.01; ***, p < 0.001; and non-significant (n.s.) difference vs. untreated control; ppm - parts per million; Formulation 21 at small scale (n = 24) (see Example 5).

[0014] Fig. 4 shows Experiment 661 results on the effect of 8-geranyl-7-hydroxycoumarin on the Phytophthora infestans disease severity in tomato seedlings, seven days after infection, under greenhouse conditions, determined as % disease severity, using preventative approach and application via spraying. Conditions of the experiment: Wettable Powder (WP) Fungicide containing 50% dimethomorph (Acrobat®, BASF); p < 0.05; **, p < 0.01; ***, p < 0.001; and non-significant (n.s.) difference vs. untreated control; ppm - parts per million; Formulation 21 at large scale (n = 60) (see Example 5).

[0015] Fig. 5 shows Experiment 664 results on the effect of 8-geranyl-7-hydroxycoumarin on the Phytophthora infestans disease severity in tomato seedlings, seven days after infection, under greenhouse conditions, determined as % disease severity, using preventative approach and application via spraying. Conditions of the experiment: Wettable Powder (WP) Fungicide containing 50% dimethomorph (Acrobat®, BASF); p < 0.05; **, p < 0.01; ***, p < 0.001; and non-significant (n.s.) difference vs. untreated control; ppm - parts per million; Formulation 21 at large scale (n = 60) (see Example 5).

DETAILED DESCRIPTION

[0016] It has been found in accordance with the present invention that the chemical compound 8 -gerany 1-7 -hydroxy coumarin, or agriculturally acceptable salts thereof, are potent pesticides against several Basidiomycota, Ascomycota and Heterokontophyta fungi.

[0017] The CAS registry identifies the compound 8-geranyl-7-hydroxycoumarin under the following names:

8-[(2E')-3 ,7 -dimethylocta-2,6-dien- 1 -yl] -7 -hydroxy-2/7- 1 -benzopyran-2-one; or (E)-8-(3,7-dimethylocta-2,6-dien-l-yl)-7-hydroxy-277-chromen-2-one.

[0018] 8-geranyl-7-hydroxycoumarin is a member of the class of hydroxycoumarins that is umbelliferone in which the ring hydrogen at position 8 has been replaced by a geranyl group. Therefore, another trivial name of 8-geranyl-7-hydroxycoumarin is 8-geranylumbelliferone, and the structural formula is:

[0019] The present invention provides in one aspect a method for controlling, preventing, reducing or eradicating plant-pathogen infestation or instances thereof, or plant diseases, on a plant, plant organ, plant part, or plant propagation material, the method comprising applying to a plant, plant organ or plant propagation material, or to soil surrounding said plant, a pesticidally effective amount of the compound 8-geranyl-7-hydroxycoumarin or agriculturally acceptable salts thereof, wherein said plant-pathogen is a member selected from a Basidiomycota of the class Agaricomycetes or the genus Rhizoctonia, an Ascomycota of the class Sordariomycetes or a genus selected from Fusarium, Heterokontophyta of the class Oomycota and a Heterokontophyta of the order Peronosporales.

[0020] In another aspect, the present invention provides a method for controlling, preventing, reducing or eradicating instances of plant-pathogen infestation on a plant, plant organ, plant part, or plant propagation material, the method comprising applying to a plant, plant organ or plant propagation material, or to soil surrounding said plant, a pesticidally effective amount of the compound 8-geranyl-7-hydroxycoumarin or agriculturally acceptable salts thereof, wherein said plant-pathogen is a member selected from a Basidiomycota of the class Agaricomycetes or the genus Rhizoctonia, and a Heterokontophyta of the order Peronosporales.

[0021] The plant treatment method of the present invention according to anyone of the embodiments disclosed herein is useful for example against the following diseases selected from potato blight, Phytophthora palmivora in cacao, canker fruit rot diseases in tomato and pumpkins, Phytophthora spp. crown and collar rot in pome and stone fruit, “damping off’ disease caused by Pythium spp. in tobacco, tomato, cucumbers, mustard, chilies and cress seedlings, Fusarium spp. causing wilt or ‘rot’ of vegetables, bananas, Fusarium spp. head and ear rot in maize, Fusarium graminearum head blight in small grains, Rhizoctonia spp. causing brown patch, damping off in seedlings, root rot and belly rot in vegetables and sheath blight in rice. [0022] In one embodiment, the plant-pathogen is a member of the class Agaricomycetes of an order selected from Amylocorticiales, Atheliales, Boletales, Jaapiales, Lepidostromatales, Phallomycetidae, Geastrales, Gomphales, Hysterangiales, Phallales, Auriculariales, Cantharellales, Corticiales, Gloeophyllales, Hymenochaetales, Polyporales, Russulales, Sebacinales, Stereopsidales, Thelephorales and Trechisporales. In a specific embodiment, the plant-pathogen is a member of the order Cantharellales.

[0023] In another embodiment, the Cantharellales plant-pathogen is a member of a family selected from Aphelariaceae, Botryobasidiaceae, Cantharellaceae, Ceratobasidiaceae, Clavulinaceae, Hydnaceae and Tulasnellaceae . In a particular embodiment, the Cantharellales plant-pathogen is a member of the family Ceratobasidiaceae.

[0024] In some embodiments, the plant-pathogen is a member of the genus Rhizoctonia (which is in the Ceratobasidiaceae family of the order Cantharellales), such as Rhizoctonia solani, Rhizoctonia bataticola also known as Macrophomina phaseolina, Rhizoctonia carotae also known as Fibulorhizoctonia carotae, Rhizoctonia cerealis, Rhizoctonia crocorum also known as Thanatophytum crocorum, Rhizoctonia fragariae, Rhizoctonia goodyerae-repentis also known as Ceratobasidium cornigerum, Rhizoctonia oryzae also known as Wailea circinate, and Rhizoctonia ramicola also known as Ceratorhiza ramicola. In a particular embodiment, the plant-pathogen is Rhizoctonia solani.

[0025] In a particular embodiment, the present invention provides a method for controlling, preventing, reducing or eradicating any one of the Rhizoctonia plant-pathogens described above, and in particular Rhizoctonia solani, the method comprising applying to a plant, plant organ or plant propagation material, or to soil surrounding said plant, a pesticidally effective amount of the compound 8 -gerany 1-7 -hydroxy coumarin or agriculturally acceptable salts thereof.

[0026] In some embodiments, the plant-pathogen is a member of the class Sordariomycetes of an order selected from Coronophorales, Glomerellales, Hypocreales, Melanosporales, Microascales, Boliniales, Calosphaeriales, Chaetosphaeriales, Coniochaetales, Diaporthales, Magnaporthales, Ophio stomatales, Sordariales, Xylariales, Koralionastetales, Lulworthiales, Meliolales, Phyllachorales, and Trichosphaeriales . In other embodiments, the plant-pathogen is a member of the order Hypocreales. In certain embodiments, the Hypocreales plant-pathogen is a member a family selected from Bionectriaceae, Cordycipitaceae, Clavicipitaceae, Hypocreaceae, Nectriaceae, Niessliaceae, Ophiocordycipitaceae, and Stachybotryaceae. In a specific embodiment, the Hypocreales plant-pathogen is a member of the family Nectriaceae.

[0027] In a further embodiment, the Nectriaceae plant-pathogen is a member of the genus Fusarium. In certain embodiments, the Fusarium plant-pathogen is selected from Fusarium acaciae, Fusarium acaciae-mearnsii, Fusarium acutatum, Fusarium aderholdii, Fusarium acremoniopsis, Fusarium affine, Fusarium arthrosporioides, Fusarium avenaceum, Fusarium bubigeum, Fusarium circinatum, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium incarnatum, Fusarium langsethiae, Fusarium mangiferae, Fusarium merismoides, Fusarium oxysporum, Fusarium pallidoroseum, Fusarium poae, Fusarium proliferatum, Fusarium pseudograminearum, Fusarium redolens, Fusarium sacchari, Fusarium solani, Fusarium sporotrichioides, Fusarium sterilihyphosum, Fusarium subglutinans, Fusarium sulphureum, Fusarium tricinctum, Fusarium venenatum, Fusarium verticillioides and Fusarium virguliforme. In some embodiments, the plant-pathogen is the species Fusarium oxysporum.

[0028] In another embodiment, the plant-pathogen is a member of the class Oomycota of an order selected from Lagenidiales, Leptomitales, Peronosporales, Rhipidiales and Saprolegniales. In a particular embodiments, the plant-pathogen is a member of the class Oomycota of the order Peronosporales.

[0029] In still another embodiment, the Peronosporales plant-pathogen is a member of a family selected from Lagenidiaceae, Olpidiosidaceae, Sirolpidiaceae, Leptomitaceae, Albuginaceae, Peronosporaceae, Pythiaceae, Rhipidaceae, Ectrogellaceae, Haliphthoraceae, Leptolegniellaceae and Saprolegniaceae. In a specific embodiment, the plant-pathogen is a member of the family Peronosporaceae or Pythiaceae.

[0030] In certain embodiments, the Peronosporaceae plant-pathogen is a member of a genus selected from Baobabopsis, Basidiophora, Benua, Bremia, Calycofera, Eraphthora, Graminivora, Hyaloperonospora, Nothophytophthora, Novotelnova, Paraperonospora, Perofascia, Peronosclerospora, Peronospora, Phytophthora, Plasmopara, Plasmovema, Protobremia, P seudoperonospora, Sclerophthora, Sclerospora and Viennotia.

[0031] In some embodiments, the Peronosporaceae plant-pathogen is a member of the genus Phytophthora. In a specific embodiment, the Phytophthora plant-pathogen is selected from Phytophthora acerina, Phytophthora agathidicida, Phytophthora alni, Phytophthora x alni, Phytophthora alticola, Phytophthora amaranthi, Phytophthora amnicola, Phytophthora amnicola x moyootj, Phytophthora andina, Phytophthora aquimorbida, Phytophthora arecae, Phytophthora arenaria, Phytophthora cf. arenaria, Phytophthora aff. arenaria, Phytophthora asiatica, Phytophthora asparagi, Phytophthora aff. asparagi, Phytophthora attenuata, Phytophthora austrocedrae, Phytophthora balyanboodja, Phytophthora batemanensis, Phytophthora bilorbang, Phytophthora bisheria, Phytophthora bishii, Phytophthora boehmeriae, Phytophthora boodjera, Phytophthora borealis, Phytophthora botryosa, Phytophthora cf. botryosa, Phytophthora aff. botryosa, Phytophthora brassicae, Phytophthora cactorum, Phytophthora cactorum var. applanata, Phytophthora cactorum x hedraiandra, Phytophthora cajani, Phytophthora cambivora, Phytophthora capensis, Phytophthora capsici, Phytophthora aff. capsici, Phytophthora captiosa, Phytophthora castaneae, Phytophthora castanetorum, Phytophthora chlamydospora, Phytophthora chrysanthemi, Phytophthora cichorii, Phytophthora aff. cichorii, Phytophthora cinnamomi, Phytophthora cinnamomi var. cinnamomi, Phytophthora cinnamomi var. parvispora, Phytophthora cinnamomi var. robiniae, Phytophthora citricola, Phytophthora aff. citricola, Phytophthora citrophthora, Phytophthora citrophthora var. Clementina, Phytophthora aff. citrophthora, Phytophthora clandestina, Phytophthora cocois, Phytophthora colocasiae, Phytophthora condilina, Phytophthora constricta, Phytophthora cooljarloo, Phytophthora crassamura, Phytophthora cryptogea, Phytophthora aff. cryptogea, Phytophthora cuyabensis, Phytophthora cyperi, Phytophthora dauci, Phytophthora aff. dauci, Phytophthora drechsleri, Phytophthora drechsleri var. cajani, Phytophthora elongata, Phytophthora cf. elongata, Phytophthora erythroseptica, Phytophthora erythroseptica var. pisi, Phytophthora aff. erythroseptica, Phytophthora estuarina, Phytophthora europaea, Phytophthora fallax, Phytophthora flexuosa, Phytophthora fluvialis, Phytophthora fluvialis x moyootj, Phytophthora foliorum, Phytophthora formosa, Phytophthora formosana, Phytophthora fragariae, Phytophthora fragariaefolia, Phytophthora frigida, Phytophthora gallica, Phytophthora gemini, Phytophthora gibbosa, Phytophthora glovera, Phytophthora gonapodyides, Phytophthora gondwanensis, Phytophthora gregata, Phytophthora cf. gregata, Phytophthora hedraiandra, Phytophthora aff. hedraiandra, Phytophthora x heterohybrida, Phytophthora heveae, Phytophthora hibernalis, Phytophthora himalayensis, Phytophthora himalsilva, Phytophthora aff. himalsilva, Phytophthora humicola, Phytophthora aff. humicola, Phytophthora hydrogena, Phytophthora hydropathica, Phytophthora idaei, Phytophthora ilicis, Phytophthora x incrassata, Phytophthora infestans, Phytophthora aff. infestans, Phytophthora inflata, Phytophthora insolita, Phytophthora cf. insolita, Phytophthora intercalaris, Phytophthora intricata, Phytophthora inundata, Phytophthora ipomoeae, Phytophthora iranica, Phytophthora irrigata, Phytophthora katsurae, Phytophthora kelmania, Phytophthora kernoviae, Phytophthora kwongonina, Phytophthora lactucae, Phytophthora lacustris, Phytophthora lacustris x riparia, Phytophthora lateralis, Phytophthora lilii, Phytophthora litchii, Phytophthora litoralis, Phytophthora litoralis x moyootj, Phytophthora macilentosa, Phytophthora macrochlamydospora, Phytophthora meadii, Phytophthora aff. meadii, Phytophthora medicaginis, Phytophthora medicaginis x cryptogea, Phytophthora megakarya, Phytophthora megasperma, Phytophthora melonis, Phytophthora mengei, Phytophthora mexicana, Phytophthora cf. mexicana, Phytophthora mirabilis, Phytophthora mississippiae, Phytophthora morindae, Phytophthora moyootj, Phytophthora moyootj x fluvialis, Phytophthora moyootj x litoralis, Phytophthora moyootj x thermophila, Phytophthora x multiformis, Phytophthora multivesiculata, Phytophthora multivora, Phytophthora nagaii, Phytophthora nemorosa, Phytophthora nicotianae, Phytophthora nicotianae var. parasitica, Phytophthora nicotianae x cactorum, Phytophthora niederhauserii, Phytophthora cf. niederhauserii, Phytophthora obscura, Phytophthora occultans, Phytophthora oleae, Phytophthora ornamentata, Phytophthora pachypleura, Phytophthora palmivora, Phytophthora palmivora var. palmivora, Phytophthora parasitica, Phytophthora parasitica var. nicotianae, Phytophthora parasitica var. piperina , Phytophthora parsiana, Phytophthora aff. parsiana, Phytophthora parvispora, Phytophthora x pelgrandis, Phytophthora phaseoli, Phytophthora pini, Phytophthora pinifolia, Phytophthora pisi, Phytophthora pistaciae, Phytophthora plurivora, Phytophthora pluvialis, Phytophthora polonica, Phytophthora porri, Phytophthora primulae, Phytophthora aff. primulae, Phytophthora pseudocryptogea, Phytophthora pseudolactucae, Phytophthora pseudorosacearum, Phytophthora pseudosyringae, Phytophthora pseudotsugae, Phytophthora aff. pseudotsugae, Phytophthora psychrophila, Phytophthora quercetorum, Phytophthora quercina, Phytophthora quininea, Phytophthora ramorum, Phytophthora rhizophorae, Phytophthora richardiae, Phytophthora riparia, Phytophthora rosacearum, Phytophthora aff. rosacearum, Phytophthora rubi, Phytophthora sansomea, Phytophthora sansomeana, Phytophthora aff. sansomeana, Phytophthora x serendipita, Phytophthora sinensis, Phytophthora siskiyouensis, Phytophthora sojae, Phytophthora stricta, Phytophthora sulawesiensis, Phytophthora syringae, Phytophthora tabaci, Phytophthora tentaculata, Phytophthora terminalis, Phytophthora thermophila, Phytophthora thermophila x amnicola, Phytophthora thermophila x moyootj, Phytophthora trifolii, Phytophthora tropicalis, Phytophthora cf. tropicalis, Phytophthora tubulina, Phytophthora tyrrhenica, Phytophthora uliginosa, Phytophthora undulata, Phytophthora uniformis, Phytophthora vignae, Phytophthora vignae f. sp. adzukicola, Phytophthora virginiana and Phytophthora vulcanica. In a further specific embodiment, the plant-pathogen is the species Phytophthora infestans.

[0032] In yet further embodiment, the Peronosporaceae plant-pathogen is a member of the family Pythiaceae. In a certain embodiment, the Pythiaceae plant-pathogen is a member of a genus selected from Cystosiphon, Diasporangium, Globisporangium, Lagenidium, Myzocytium, Phytophthora, Pythium and Trachysphaera.

[0033] In another embodiment, the Pythiaceae plant-pathogen is a member of the genus Pythium. In a specific embodiment, the Pythium plant-pathogen is a species selected from Pythium aphanidermatum, Pythium acanthicum, Pythium acanthophoron, Pythium acrogynum, Pythium adhaerens, Pythium amasculinum, Pythium anandrum, Pythium angustatum, Pythium apleroticum, Pythium aquatile, Pythium aristosporum, Pythium arrhenoman.es, Pythium attrantheridium, Pythium bifurcatum, Pythium boreale, Pythium buismaniae, Pythium butleri, Pythium camurandrum, Pythium campanulatum, Pythium canariense, Pythium capillosum, Pythium carbonicum, Pythium carolinianum, Pythium catenulatum, Pythium chamaehyphon, Pythium chondricola, Pythium citrinum, Pythium coloratum, Pythium conidiophorum, Pythium contiguanum, Pythium cryptoirregulare, Pythium cucurbitacearum, Pythium cylindrosporum, Pythium cystogenes, Pythium debaryanum, Pythium delicense, Pythium destruens, Pythium diclinum, Pythium dimorphum, Pythium dissimile, Pythium dissotocum, Pythium echinulatum, Pythium emineosum, Pythium erinaceum, Pythium flevoense, Pythium folliculosum, Pythium glomeratum, Pythium graminicola, Pythium grandisporangium, Pythium guiyangense, Pythium helicandrum, Pythium helicoides, Pythium heterothallicum, Pythium hydnosporum, Pythium hypogynum, Pythium indigoferae, Pythium inflatum, Pythium insidiosum, Pythium intermedium, Pythium irregulare, Pythium iwayamae, Pythium jasmonium, Pythium kunmingense, Pythium litorale, Pythium longandrum, Pythium longisporangium, Pythium lutarium, Pythium macrosporum, Pythium mamillatum, Pythium marinum, Pythium marsupium, Pythium mastophorum, Pythium megacarpum, Pythium middletonii, Pythium minus, Pythium monospermum, Pythium montanum, Pythium multisporum, Pythium myriotylum, Pythium nagaii, Pythium nodosum, Pythium nunn, Pythium oedochilum, Pythium okanoganense, Pythium oligandrum, Pythium oopapillum, Pythium ornacarpum, Pythium orthogonon, Pythium ostracodes, Pythium pachycaule, Pythium pachycaule, Pythium paddicum, Pythium paroecandrum, Pythium parvum, Pythium pectinolyticum, Pythium periilum, Pythium periplocum, Pythium perniciosum, Pythium perplexum, Pythium phragmitis, Pythium pleroticum, Pythium plurisporium, Pythium polare, Pythium polymastum, Pythium porphyrae, Pythium prolatum, Pythium proliferatum, Pythium pulchrum, Pythium pyrilobum, Pythium quercum, Pythium radiosum, Pythium ramificatum, Pythium regulare, Pythium rhizo-oryzae, Pythium rhizosaccharum, Pythium rostratifingens, Pythium rostratum, Pythium salpingophorum, Pythium scleroteichum, Pythium segnitium, Pythium speculum, Pythium spinosum, Pythium splendens, Pythium sterilum, Pythium stipitatum, Pythium sulcatum, Pythium terrestris, Pythium torulosum, Pythium tracheiphilum, Pythium ultimum, Pythium ultimum var. ultimum, Pythium uncinulatum, Pythium undulatum, Pythium vanterpoolii, Pythium viniferum, Pythium violae, Pythium volutum, Pythium zingiberis and Pythium zingiberum. In a specific embodiment, the plant-pathogen is the species Pythium aphanidermatum. [0034] In a particular embodiment, the present invention provides a method for controlling, preventing, reducing or eradicating any one of the Oomycota plant pathogens described above, in particular Phytophthora infestans and Pythium aphanidermatum, the method comprising applying to a plant, plant organ or plant propagation material, or to soil surrounding said plant, a pesticidally effective amount of the compound 8-geranyl-7-hydroxycoumarin or agriculturally acceptable salts thereof.

[0035] In certain embodiments, the pesticide composition or formulation of any one of the above embodiments further comprises an agriculturally suitable or acceptable solvent or solubilising agent. In other certain embodiments, the agriculturally acceptable solvent or solubilising agent is a water-miscible solvent capable of dissolving or solubilising 8-geranyl-7- hydroxy coumarin .

[0036] In some embodiments, the water-miscible solvent capable of dissolving or solubilising 8-geranyl-7-hydroxycoumarin is a polar solvent, such as an alcohol, a ketone, a lactone, a keto-alcohol, a glycol, a glycoether, an amide, an alkanolamine, a sulfoxide and a pyrrolidone. In particular embodiments, the composition of any one of the above embodiments comprises a solvent selected from dimethyl- sulfoxide or ethanol. In specific embodiments, the composition further comprises a polysorbate-type non-ionic surfactant, such as polysorbate 20.

[0037] The pesticide composition of the present invention may be formulated into a formulation to facilitate application of the active pesticidal ingredient. The formulation may be a water-miscible formulation, such as a suspension concentrate (SC), a capsule suspension (CS), water-dispersible granules (WG), an emulsifiable concentrate (EC), a wettable powder (WP), a soluble (liquid) concentrate (SL), or a soluble powder (SP).

[0038] The composition or formulation of the present invention may further comprise at least one adjuvant, carrier, diluent, and/or surfactant. Non-limiting examples of adjuvants are activator adjuvants, such as cationic, anionic or non-ionic surfactants, oils and nitrogen-based fertilisers capable of improving activity of the pesticide product. Oils may be crop oils, such as paraffin or naphtha-based petroleum oil, crop oil concentrates based on emulsifiable petroleum-based oil, and vegetable oil concentrates derived from seed oil, usually cotton, linseed, soybean, or sunflower oil, used to control grassy weeds. Nitrogen-based fertilisers may be ammonium sulphate or urea-ammonium nitrate.

[0039] A non-limiting example of a polysaccharide adjuvant used also as a thixotropic agent in the compositions of the present embodiments, is Xanthan gum (commercially available under trademark KELZAN® by CP Kelco), which is produced from simple sugars using a fermentation process, and derives its name from the species of bacteria used, Xanthomonas campestris. Oils used as adjuvants may be crop oils, such as paraffin or naphtha-based petroleum oil, crop oil concentrates based on emulsifiable petroleum-based oil, and vegetable oil concentrates derived from seed oil, usually cotton, linseed, soybean, or sunflower oil, used to control grassy weeds. Nitrogen-based fertilisers may be ammonium sulphate or urea-ammonium nitrate.

[0040] Non-limiting examples of solubilising agents or solvents are petroleum-based solvents, the aforementioned oils, liquid mixtures of fatty acids, ethanol, glycerol and dimethyl sulfoxide. The agriculturally acceptable solvent or solubilising agent may be a water-miscible solvent capable of dissolving or solubilising 8-geranyl-7-hydroxycoumarin, such as a polar solvent, e.g., an alcohol, a ketone, a lactone, a keto-alcohol, a glycol, a glycoether, an amide, an alkanolamine, a sulfoxide and a pyrrolidone. Non-limiting examples of carriers are precipitated silica, colloidal silica, attapulgite, china clay, talc, kaolin and combinations thereof.

[0041] The pesticide composition or formulation of the present invention may further comprise a diluent, such as lactose, starch, urea, water soluble inorganic salts and combination thereof. The pesticide composition or formulation may further comprise one or more surfactants, such as polysorbate-type non-ionic surfactant, for example Polysorbate 20 or trisiloxane nonionic surfactant, styrene acrylic dispersant polymers, acid resin copolymer based dispersing agents, potassium polycarboxylate, sodium alkyl naphthalene sulphonate blend, sodium diisopropyl naphthalene sulphonate, sodium salt of naphthalene sulphonate condensate, lignin sulfonate salts and combinations thereof.

[0042] Trisiloxane non-ionic surfactants or polyether dimethyl siloxanes (PEMS), often referred to as super- spreaders or super- wetters, are added to pesticides to enhance their activity and the rain fastness of the active substance by promoting rapid spreading over the hydrophobic surfaces of leaves. Some spreaders of the modified trisiloxane type combine a very low molecular weight trisiloxane with a polyether group and capable of reducing surface tension and rapidly spreading on difficult to wet surfaces.

[0043] The active agent, composition, or formulation comprising it, is applied in the method of any one of the above embodiments to the plant or part, organ or plant propagation material thereof by spraying, immersing, dressing, coating, pelleting or soaking.

[0044] In certain embodiments, the concentration of the 8-geranyl-7-hydroxycoumarin of the present invention, in the composition or formulation comprising it may be in the range of 10- 2000, 10-1500, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10- 100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-2000, 20-1500, 20-1000, 20- 900, 20-800, 20-700, 20-600, 20-500, 20-400, 20-300, 20-200, 20-100, 20-90, 20-80, 20-70, 20- 60, 20-50, 20-40, 20-30, 20-20. 30-2000, 30-1500, 30-1000, 30-900, 30-800, 30-700, 30-600, 30-500, 30-400, 30-300, 30-200, 30-100, 30-0, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30- 40, 40-2000, 40-1500, 40-1000, 40-900, 40-800, 40-700, 40-600, 40-500, 40-400, 40-300, 40- 200, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-2000, 50-1500, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60, 60-2000, 60-1500, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70, 70-2000, 70-1500, 70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70- 400, 70-300, 70-200, 70-100, 70-90, 70-80, 80-2000, 80-1500, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400, 80-300, 80-200, 80-100, 80-90, 90-2000, 90-1500, 90-1000, 90-900, 90-800, 90-700, 90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-2000, 100-1500, 100- 1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-2000, 200-1500, 200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300- 2000, 300-1500, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-2000, 400-1500, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500, 500-2000, 500-1500, 500- 1000, 500-900, 500-800, 500-700, 500-600, 600-2000, 600-1500, 600-1000, 600-900, 600-800, 600-700, 700-2000, 700-1500, 700-1000, 700-900, 700-800, 800-2000, 800-1500, 800-1000, 800-900, 900-2000, 900-1500, 900-1000, 1000-2000, or 1000-1500 ppm.

[0045] In particular, the concentration of 8-geranyl-7-hydroxycoumarin in the composition or formulation comprising it may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 1000, 1500 or 2000 ppm.

[0046] Any one of the above concentration ranges or concentrations can be used in accordance with any one of the above embodiments relating to the method of the present invention, against any one of the aforementioned pathogens and by means of any one of the above mentioned applications.

DEFINITIONS

[0047] The term "plant organ" as used herein refers to the leaf, stem, root, and reproductive structures. The term "plant part" as used herein refers to a vegetative plant material such as a cutting or a tuber; a leaf, flower, bark or a stem. The term "plant propagation material" as used herein refers to a seed, root, fruit, tuber, bulb, rhizome, or part of a plant. The term "pesticidal effective amount" as used herein refers to an amount of the pesticide that is able to bring about death to at least one pest, or to noticeably reduce pest growth, feeding, or normal physiological development. The terms "class", "order", "family", "genus", and "species" are used herein according to Art 3.1 of the International Code of Nomenclature for algae, fungi, and plants.

[0048] The term "comprising", used in the claims, is "open ended" and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. It should not be interpreted as being restricted to the means listed thereafter; it does not exclude other elements or steps. It needs to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression "a composition comprising x and z" should not be limited to compositions consisting only of components x and z. Also, the scope of the expression "a method comprising the steps x and z" should not be limited to methods consisting only of these steps.

[0049] Unless otherwise indicated, all numbers used in this specification are to be understood as being modified in all instances by the term "about". Unless specifically stated, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within two standard deviations of the mean. In one embodiment, the term "about" means within 10% of the reported numerical value of the number with which it is being used, preferably within 5% of the reported numerical value. For example, the term "about" can be immediately understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In other embodiments, the term "about" can mean a higher tolerance of variation depending on for instance the experimental technique used. Said variations of a specified value are understood by the skilled person and are within the context of the present invention. As an illustration, a numerical range of "about 1 to about 5" should be interpreted to include not only the explicitly recited values of about 1 to about 5, but also include individual values and sub-ranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 3, and 4 and sub-ranges, for example from 1-3, from 2-4, and from 3-5, as well as 1, 2, 3, 4, 5, or 6, individually. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum.

[0050] Unless otherwise clear from context, all numerical values provided herein are modified by the term "about". Other similar terms, such as "substantially", "generally", "up to" and the like are to be construed as modifying a term or value such that it is not an absolute. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skilled in the art. This includes, at very least, the degree of expected experimental error, technical error and instrumental error for a given experiment, technique or an instrument used to measure a value.

[0051] As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well- known functions or constructions may not be described in detail for brevity and/or clarity.

[0052] Another aspect of the present invention is a kit comprising a pesticidally effective amount of the compound 8-geranyl-7-hydroxycoumarin, or agriculturally acceptable salts thereof, and instructions for using said compound or said agriculturally acceptable salts thereof to control, prevent, reduce or eradicate plant-pathogen infestation or instances thereof, or plant diseases, on a plant, plant organ, plant part, or plant propagation material, or on soil surrounding said plant, wherein said plant-pathogen is a member selected from a Basidiomycota of the class Agaricomycetes or the genus Rhizoctonia, an Ascomycota of the class Sordariomycetes or a genus selected from Fusarium, Heterokontophyta of the class Oomycota and a Heterokontophyta of the order Peronosporales.

[0053] The invention will now be illustrated by the following non-limiting Examples. EXAMPLES

List of abbreviations:

RPM - Revolutions per minute

RCF - Relative centrifugal force

CFU - Colony forming unit

PDBC - Potato dextrose broth with 20 pg/ml chloramphenicol

PDAC - Potato dextrose agar with 20 pg/ml chloramphenicol

PDAT - Potato dextrose agar with 12 pg/ml tetracycline

DMSO - Dimethyl sulfoxide

LB - LB broth

LBA - LB agar

SCH - Schmittner medium

2:PDBC - PDBC diluted 2 fold by sterile distilled water

PDA - Potato dextrose agar

PDBT - Potato dextrose broth with 12 pg/ml tetracycline

Example 1. Microplate-based assay of 8-geranyl-7-hydroxycoumarin bioactivity against

Rhizoctonia solani

Summary: Diluted in DMSO 8-geranyl-7-hydroxycoumarin was added to microplate wells and mixed with 50 pl of hyphae suspension and growth of the fungus, starting from blended hyphae, was monitored by plate reader and visual inspection.

The following materials, methods and equipment were used:

Materials: PDAC, PDBC, DMSO.

Equipment: Plate reader, Centrifuge, Shaker, Incubator.

Method:

A. Inoculum preparation of Rhizoctonia solani hyphae

1) Grow Rhizoctonia on PDAC in 90 mm petri plates to get growing hyphae within 1-4 days.

2) Add 50 ml of PDBC medium into a sterile 250 ml Erlenmeyer flask.

3) Cut the solid medium by scalpel to several small pieces and insert them into the Erlenmeyer flask.

4) Grow the culture for 2-4 days using shaker at 27°C and 150RPM.

5) Discard the liquid and pour the hyphae on an empty Petri dish.

6) Cut many small pieces from the hyphae using a scalpel and insert them into a sterile 250 ml Erlenmeyer flask with 50 ml of PDBC medium.

7) Prepare 4 bottles with culture and grow for 3 days at 27°C shaking at 150 RPM. 8) Chill the culture in the fridge for Ih.

9) Pour the cold culture into a 250 ml beaker.

10) Add 20 ml of cold PDBC, so that the mixture will cover the blender knife.

11) Blend the culture with a blender for 2 min on ice at maximum speed, move the blender up and down several times.

12) Keep the mixture on ice.

13) Transfer about 5 ml of the blended mixture into a 15 ml tube on ice.

14) Homogenize the culture in the 15 ml tube for 2 min on ice, move the tube up and down as needed.

15) Homogenize several batches of 5 ml as above to prepare the amount that is needed (5ml of homogenized culture would make about 100 ml of inoculum).

16) Dilute a portion of the homogenate 10-fold to check the concentration of the homogenate. The concentration of the suspension should be 4xl0⁴ CFU/ml (diluted 10-fold concentration should be 4000 CFU/ml).

17) Dilute the inoculum stock 1:20 in PDBC - 1 ml in 20 ml, or calculate the dilution needed, to prepare final concentration of 2000 CFU/ml. The amount in each well should be about 100 CFU.

B. Microplate preparation for 8-geranyl-7-hydroxycoumarin bioactivity experiment

1) Take a stock solution of one of the purified 1% 8-geranyl-7-hydroxycoumarin in DMSO from the -20°C freezer and thaw it on the bench.

2) Take 1 pl of stock solution of 1% 8-geranyl-7-hydroxycoumarin and dilute up to 250 ppm with 39 pl of water.

3) Take 10 pl of the diluted (250 ppm) 8-geranyl-7-hydroxycoumarin solution into the wells of the microplate using a multi-pipette.

4) Add 40 pl of vigorously mixed spore suspension inoculum to the wells of the microplate using a multi-pipette.

5) Shake the plate for lOmin at 2000 RPM to mix the 8-geranyl-7-hydroxycoumarin with the hyphae suspension.

6) Centrifugate the plate at 1000 RCF for Is and stop to collect the liquid at the bottom of the plate.

7) Keep the microplate on the bench until it is read by the plate reader.

8) Read the plate using the plate reader, and then collect the plates on the bench.

9) Insert collected plates to a plastic box with cloth cover and put the box in the incubator at 27°C. C. Screening of plates

1) Screen plate at 4 more dates: 3d, 7d, 14d and 21d following the assay start.

2) Calculate the difference of absorbance between each screen and the read at zero time.

3) Calculate the percentage of growth inhibition of each well at each time point. Use the results of the DMSO treatment of the control plate as 100% growth.

See results in Example 5.

Example 2. Microplate-based screening of 8-geranyl-7-hydroxycoumarin with potential bioactivity against Pythium aphanidermatum

Summary: Diluted in DMSO 8-geranyl-7-hydroxycoumarin was added to microplate wells and mixed with 50 pl of zoospores in PDBC suspension and the growth of the fungus, starting from zoospores, was monitored by plate reader and visual inspection.

The following materials, methods and equipment were used:

Materials: SCH, PDBC, DMSO.

Equipment: Plate reader, Centrifuge, Shaker, Incubator.

Method:

A. Inoculum preparation of Pythium hyphae

1) Grow Pythium aphanidermatum on SCH in 90 mm petri plates to get sporulating hyphae. Each plate will produce 50 ml of zoospores suspension which will be enough for bioactivity screening for ten 96- well plates.

2) Add 60 ml of sterile water into a sterile 250 ml Erlenmeyer flask.

3) Cut the solid medium of 2 plates by scalpel to 12 pieces (each plate) and insert them into the Erlenmeyer flask (the solid pieces should be covered by the water).

4) Let the hyphae sporulate overnight at 17 °C.

5) Shake the Erlenmeyer flask by hand to suspend the zoospores.

6) Filter the suspension into 50 ml tube through 16-layer gauze.

7) Transfer the suspension into a sterile 500 ml bottle.

8) Discard the solids and disinfect the Erlenmeyer flask with hypochlorite.

9) Chill the zoospore suspension on ice, and then evaluate the zoospores concentration in the suspension (the concentration should be 1000-4000 spores/ml).

10) Dilute the suspension by sterile fridge cold distilled water in a sterile 500ml bottle.

11) Add the same volume (as the suspension) sterile fridge cold 2:PDBC to get 500-2000 spores/ml inoculum. This dilution will result in the amount of 25-100 zoospores in each well. Finally, keep the zoospore suspension inoculum on ice. B. Microplate preparation for 8-geranyl-7-hydroxycoumarin bioactivity experiment

1) Take a stock solution of purified 1% 8-geranyl-7-hydroxycoumarin in DMSO and thaw it on the bench for at least 20 min.

4) Add 40 pl of zoospore suspension inoculum to the wells of the microplate using a multipipette. Mix the spore suspension vigorously by hand and decant amount needed for one plate (5 ml) to keep the zoospores well suspended.

5) Seal the plate with transparent sealer.

6) Shake the plate for 10 min at 2000 RPM to mix the 8-geranyl-7-hydroxycoumarin with the hyphae suspension.

7) Centrifugate the plate at 1000 RCF for 1 second and stop to collect the liquid at the bottom of the plate.

8) Keep the microplate on the bench until it is read by the plate reader.

9) Read the plate using the plate reader.

10) Collect the plates on the bench.

11) Insert collected plates to a plastic box with cloth cover and put the box in the incubator at 27°C.

C. Screening of plates

1) Read out the plate at 4 more dates: 3d, 7d, 14d and 21d following the assay start.

2) Calculate the difference of absorbance between each readout and the readout at zero time.

See results in Example 5.

Example 3. Microplate-based screening of 8-geranyl-7-hydroxycoumarin with potential bioactivity against Fusarium oxysporum

Summary: 8-geranyl-7-hydroxycoumarin diluted in DMSO was added to microplate wells and mixed with freshly prepared spore suspension and growth of the fungus, starting from frozen spores, was monitored using the plate reader and by visual inspection. Background: Fusarium is a fungus of belonging to the Ascomycetes, and it is a soil borne pathogen. It is quite easy to produce large amounts of spores of Fusarium and they survive in liquid 60% glycerol at -20°C. Thus, we used frozen spores’ stock in the bioactivity screening experiments rather than prepare fresh spores for each experiment.

Aim: To determine the effect of 8-geranyl-7-hydroxycoumarin on the survival and growth of Fusarium.

The following materials, methods and equipment were used:

Materials: PDAC, PDBC, DMSO.

Equipment: Plate reader, Centrifuge, Shaker, Incubator.

Method:

A. Fusarium spore suspension preparation

1) Put agar block of growing fusarium on PDAC in the middle of a PDAC plate and grow for 9 days at 25°C.

2) Chill the plate in the fridge for at least Ih.

3) Add 30 ml of fridge cold, sterile, 60% glycerol solution to the 50-ml tube.

4) Cut the agar with the hyphae and spores to small pieces by scalpel and insert them into the 50 ml-tube with 30 ml 60% glycerol.

5) Shake for 1 min at 3000 RPM.

6) Keep spores on ice during the whole process.

7) Transfer the liquid to a new 50 ml sterile tube - about 25 ml should be recovered.

8) Filter the spore suspension through 16 layer of gauze cloth directly into a clean sterile 50 ml tube to discard the hyphae.

9) Calculate the spore concentration (at 40X10 magnification) and dilute by cold sterile 60% glycerol solution to get 2xl0⁵ spores/ml.

10) Aliquot 1 ml of spore suspension into 1.5-ml tubes - each aliquot should yield 20 plates for screening, and then store the spore suspension at -20°C.

B. Spore suspension preparation for screening

1) Take 1ml frozen spore suspension from the freezer and thaw it on ice.

2) Mix 200 pl spore suspension with 20 ml fridge cold PDBC in a 50-ml tube to make 2000 spores/ml suspension.

3) Use this amount to screening of 4 microplates with 100 spores per well.

C. Microplate preparation for 8-geranyl-7-hydroxycoumarin bioactivity experiment

2) Take 1 of stock solution of the 1% 8-geranyl-7-hydroxycoumarin and dilute up to 250 ppm with 39 pl of water.

4) Add 40 pl of spore suspension inoculum to the wells of the microplate using a multi-pipette.

5) Mix the spore suspension vigorously by hand and decant amount needed for one plate (5ml) to keep the spores well suspended.

6) Seal the plate with transparent sealer.

7) Shake the plate for 10 min at 2000 RPM to mix the materials with the hyphae suspension.

8) Centrifugate the plate at 1000 RCF for Is and stop to collect the liquid at the bottom of the plate.

9) Keep the microplate on the bench until it is read by the plate reader.

10) Read the plate using the plate reader.

11) Collect the plates on the bench, and then insert collected plates to a plastic box with cloth cover and put the box in the incubator at 25 °C.

D. Readout of the plates

Collect the readout of the plate at 4 more dates: 3d, 7d, 14d and 21d following the assay start

1) Calculate the difference of absorbance between each readout and the readout at zero time

2) Calculate the percentage of growth inhibition of each well at each time course. Use the results of the DMSO treatment, of the control plate as 100% growth.

See results in Example 5.

Example 4. Microplate-based screening of 8-geranyl-7-hydroxycoumarin with potential bioactivity against Phytophthora inf estans

Background: Phytophthora infestans is an obligatory pathogen from Oomycetes which is very difficult to grow on synthetic medium. Therefore, the bioactivity screening system based on leaf discs prepared from detached tomato leaves were used.

Summary: 8-geranyl-7-hydroxycoumarin in DMSO was added to tomato leaf discs infected with Phytophthora and the disease progress was monitored by visual inspection.

General description: Inoculation and maintenance on tomato leaves, preparation of spore suspension, their growth on leaf discs in microplates and inspection by magnifying glass of Phytophthora infestans severity of infection.

The following materials, methods and equipment were used: Method:

A. Preparation of tomato seedling for leaves production for inoculation

1) Use seedling pots of size 120x80x80.

2) Use standard garden earth with fertilizer.

3) Use 4 weeks old tomato seedlings.

4) Put 6 pots in a small tray.

5) Put one seedling in each pot.

6) Add water into the tray - about 100 ml for each pot. Earth should be wet, and no water should be left in the trey after 24 h.

7) Grow the tomato plants in growth room at 22°C and 16h light/darkness conditions.

8) When plants are grown (4 weeks after planting) transfer them to a 5L pot and fertilize every week with.

B. Preparation of tomato leaves for inoculation

1) Put two pieces of sterile paper in a square Petri dish.

2) Work in sterile conditions.

3) Use leaves of 5 weeks old tomato plants or older.

4) Cut the leaves by a sterile scalpel.

5) Add 20 ml sterile distilled water to wet the paper (the paper should be maximally wet, but without additional dripping water).

6) Put about 6 lobes of leaves in a square Petri dish, on the wet paper (with the lower side of the leaves up).

7) Cover the plate with its lid.

C. Preparation of inoculum and leaf discs infection

Preparation of sporangium suspension

1) Put 10 lobes of tomato leaf infected with Phytophthora (4-6 days after infection), in a sterile 50ml tube, fill in 40 ml of fridge cold, sterile, distilled water.

2) Mix the tube gently by hand, to transfer sporangium into the water, but avoid disintegration of the leaf.

3) Filter the spore suspension through 161ayers of gauze into a 50-ml tube.

4) Calculate the spore concentration - use microscope with 200X magnification. A concentration of 6000 sporangium/ml is expected.

5) Chill the tube on ice. D. Sporangium wash and concentration by filtration

1) Prepare filtration system with membrane (0.65 micron - 5 micron) and wash the membrane with sterile cold water.

2) Suspend and decant the spore suspension from the 50 ml tube slowly, into the filtration system. Use low vacuum, don’t let the membrane dry - leave 4 ml unfiltered suspension on the filter.

3) Wash the spores to discard bacteria and other fungi spores (use 40 ml water to wash) - spray cold sterile water to suspend and wash the spores.

4) Repeat spore wash 5 more times. Do not let the membrane dry. Leave 4 ml unfiltered suspension.

5) Collect the spore suspension using a 1000 pl pipettor into a clean 50-ml tube.

6) Insert the membrane into the 50-ml tube mix gently to suspend the sporangium, which stick to the membrane, and then discard the membrane to be autoclaved.

7) Discard liquid, and disinfect the filtration system using hypochlorite for 30 min.

8) Wash the filtration system with tap water and dry the filtration system on a paper in a plastic basket.

9) Calculate the sporangium concentration - use microscope with 200X magnification - 10,000-50,000 sporangium / ml concentration is expected.

10) Keep the sporangium suspension on ice.

E. Inoculation of spores on detached leaves for maintenance of Phytophthora

1) Spray 1000 pl of Phytophthora spore suspension on all the lobes of leaves in one square dish and cover the dish.

2) Infect the leaves with the fungus on and keep the leaves into the incubator at 17°C in the dark for 24h.

3) Transfer the plates for additional 3-5 days into the incubator at 22°C, with 12h light, for sporangium growth.

F. Tomato leaf discs microplate preparation for screening

1) Take the 48 wells plate.

2) Prepare sterile water agar 0.5%, use it preheated, but cold.

3) Add 100 pl sterile water agar 0.5% to the microplate wells.

4) Put tomato leaf discs prepared from 3^rd, 4th or 5^th leaf, into microplate wells. Press the discs gently to ensure maximal contact with the liquid agar solution. G. Inoculation of spores on leaf discs microplate for materials screening

1) Insert the spore suspension into the microplate for testing.

2) Seal the chemicals microplate with transparent sealer.

3) Shake the microplate for 10 min at 2000 RPM to mix the materials with the added spore suspension.

4) Centrifugate the microplate at 1000 RCF for Is and stop to collect the liquid at the bottom of the plate.

5) Add 5 pl spore suspension onto the middle of each disc of the microplate.

6) Seal the leaf discs plate with transparent sealer.

7) Insert the sealed leaf discs plates into the incubator at 17°C for 24 h in the dark and then at 22°C, with 12h light/darkness regime for 3-5 days.

8) Perform the bioactivity evaluation.

H. Bioactivity evaluation

1) Screen plate at one time point: 5 days after suspension preparation using a X5 magnification glass.

2) Report infected discs in Excel sheet:

Type 1 - if discs are fully infected;

Type 2 - inconclusive;

Type 3 - if discs are not infected at all.

3) Calculate the number of repeats of scores of 3 for each material.

4) Calculate the sum of scores of 3 for each material.

5) Best score was calculated as following: number of repeats = 4, sum of scores = 12.

6) “Hits” are those materials which have 4 or 3 repeats, and sum of 6-12.

See results in Example 5.

Example 5. Results of in vitro experiments based on protocols of Examples 1-4.

In-vitro screening matrix

8 -gerany 1-7 -hydroxy coumarin was screened against selected agricultural pests (as indicated in the tables below). The bioactivity values are in % and reflect the potential of eradicating the target pests.

Rules for bioactivity relative value calculation (expressed in % from maximal value) a. Phytophthora infestans - activity grade (1/2/3) X repeats# /12 (maximal value 3x4=12) xlOO b. Rhizoctonia solani, Fusarium oxysporum, Pythium aphanidermatum - activity grade (1/2/3) X repeats# X days of activity /252 (maximal value 3x4x21=252) xlOO

Table 1. Bioactivity values of Compound 1 on various target pests

Pathogen Relative Activity value ( % )

Phytophthora infestans 100

Rhizoctonia solani 24

Pythium aphanidermatum 50

Fusarium oxysporum 14

In summary, 8-geranyl-7-hydroxycoumarin are demonstrated to be effective pesticides against the following pests: Phytophthora infestans (positive results in greenhouse in-vivo validation experiments provided), Rhizoctonia solani, Pythium aphanidermatum and Fusarium oxysporum.

Statistical analysis used for validation experiments.

To evaluate the effect of a tested compounds in infected plants compared to control plants (infected but not treated) the data was analysed by Student’s t-test and the p-value is calculated. The minimum number of repeats in each experiment was 3. Results were considered significant if p < 0.05. The data presented as mean with standard error mean from biological replicates. * means that p-value <0.05, ** means that p-value is <0.01, *** means that p-value is <0.001, # means that p-value <0.1, n.s. - means non-significant effect vs. control.

Formulation recipes used for validation experiments.

Preparation of Formulation 21

Three types of stock solutions were used for final 8 -gerany 1-7 -hydroxy coumarin formulation preparation at 400 ppm:

(A) 8-geranyl-7-hydroxycoumarin 40% solution in Genagen.

A 8-geranyl-7-hydroxycoumarin was grinded using grinder and 60 mg. was dissolved in lOOpl of Genagen. The solution was mixed by vortex and sonicated to clarity. 30pl of Soprophor® Bsu (emulsifier) was added and mixed. Thereafter, 15 l of Emulgit 60 (another emulsifier) was added and mixed with sonication until clear solution was obtained.

(B) 0.4% Xanthan Gum in water (w/w).

(C) 0.6% Silwet® in water (w/w).

The final formulation which was applied to plants is composed of:

0.1% of stock solution (A), 10% of stock solutions (B), 10% of stock (C), and 79.9% of water.

The final formulated 8-geranyl-7-hydroxycoumarin was applied as 400 ppm or diluted to the required concentrations and applied to plants. Preparation of Formulation 22

(D) 8 -gerany 1-7 -hydroxy coumarin solution in DMSQ.

A 8-geranyl-7-hydroxycoumarin was grinded using grinder, and 50mg of the grinded compound was dissolved in 500pl of DMSO to get the final concentration of 10%.

(E) 0.4% Xanthan Gum in water (w/w).

(F) 0.6% Silwet® in water (w/w).

The final formulation which was applied to wheat plants is composed of:

0.4% of stock solution (A), 10% of stock solutions (B), 3.3% of stock (C), and 86.3% of water.

The final formulated 8-geranyl-7-hydroxycoumarin was applied as 400 ppm or diluted to the required concentrations and applied to plants.

Example 6. Validation in-vivo experiments in tomato infected with Phytophthora inf estans under greenhouse conditions.

General description: Severity of late blight disease caused by Phytophtora infestans was evaluated following treatment with 8-geranyl-7-hydroxycoumarin. Sporangium was used to infect 3-4 weeks-old tomato young plants following curative treatment with 8-geranyl-7- hydroxy coumarin .

A. Pathogen sporangium preparation (grown on plates/ solid media)

Preparation of sporangium suspension

1) Put 10 lobes of 4 days old Phytophthora infected tomato leaves, in a sterile 50 ml tube.

2) Fill the tube with 40ml of 4ml of the cold sterile distilled water

3) Mix the tube gently, by hand, to release the sporangium into the water, but avoid damaging the leaf tissue

4) Filter the spore suspension through 16 layers of miracloth into 50-ml tube.

5) Calculate the spore concentration using a microscope with 200X magnification, which is expected to be 3000 sporangium/ml.

6) Chill the tube on ice.

B. Sporangium washing and concentration by filtration

1) Prepare filtration system with filter membrane (in range of 0.45 pM to 5 pM pore size) and wash the membrane with sterile cold water. 2) Suspend and decant the spore suspension from the 50-ml tube slowly into the filtration system. Use low vacuum, do not let the membrane get dry - leave 4 ml unfiltered suspension on the filter.

3) Wash the spore to discard bacteria and other fungal spores with 40 ml of sterile cooled distilled water.

4) Repeat 5 times the washing process. Make sure the membrane will not get dry between the washing steps.

5) Collect the spore suspension into a clean 50-ml tube.

6) Insert the membrane using for filtration into the tube with the sporangium and gently suspend the sporangium left on the membrane.

7) Discard the membrane and wash and sterilize the filtration-vacuum system by hypochlorite solution (0.1%) - allow to stand for at least 1 hour in the hypochlorite solution.

8) Calculate sporangium concentration- using a microscope haemocytometer slide with X200 magnification. The final concentration needed for inoculation is 6000 spore/ml.

9) Store the sporangium in the fridge.

C. Plant/seedling germination conditions

1) Tomato Ikram/Brigade/Shani cultivars (sensitive to Phytophthord) were germinated in seedlings tray using standard greenhouse soil mixture. Seedlings were grown in clean growing chamber with 24°C temperature under 12h light/12h dark regime. Seedlings of 3-4 weeks old with 4 true leaves were used for experiments.

2) Plants needed for each treatment were transferred from the seedling’ trays to a dedicated experimental tray.

3) Two leaves on each seedling were labelled by small plastic tags. On each labelled leaf the last largest 3 leaflets were used for the experiment.

D. Inoculation application (preventative approach)

In the preventative approach, inoculation was applied following two repeating treatments with 8- geranyl-7 -hydroxycoumarin: a) 10 pl drop of Phytophthora infestans freshly prepared (6000 spore/ml) spores’ suspension was applied on each labelled leaflet (6 leaflets on each plant). b) Inoculated plants were put into a humid box for 24h. c) After 24h, the tray with the inoculated plants were removed from the humid box and placed in the greenhouse for further growth and disease development. E. Treatment application (preventative approach)

1) 4 weeks-old healthy tomato seedlings with 4 true leaves were used. The last three leaflets of two mature leaves on each plant were labelled with a small plastic tag.

2) 48h before inoculation (Day -2), plants were treated with formulated 8-geranyl-7- hydroxycoumarin and respective control treatments using hand sprayer until the full drainage of the tomato leaves ( 1 ml/plant) on the upper and bottom side of the leaf.

3) 24h before inoculation (Day -1), the plants were treated again with the same treatments.

F. Growth and analysis

1) Following treatment and inoculations, plants were grown under normal greenhouse conditions and watered according to need.

2) Five days following inoculation, disease was observed on the labelled leaves.

3) The labelled leaves were cut and collected, each treatment separately, and moved to the lab to measure the decay percentage expressed as disease severity in %.

4) Late blight symptoms should be observed as brownish-green spots which appear on the infected spot, then large areas of the leaves turn brown completely.

Formulation preparation

See formulation preparation in Formulation section in Example 5.

Results

Five (5) independent experiments were conducted in tomato plants infected with Phytophthora under greenhouse conditions, where the potential of 8-geranyl-7-hydroxycoumarin to prevent and control Phytophthora infestans (Figs. 1-5) was estimated. 8-geranyl-7-hydroxycoumarin controlled the Phytophthora infection with the efficacy up to 86.79%.

[0054] While certain features of the present application have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will be apparent to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the present application. REFERENCES

Erlacher A., Cardinale M., Grosch R., Grube M., Berg G., “The impact of the pathogen Rhizoctonia solani and its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce microbiome” , Front Microbiology 2014; 5: 175. Published online 2014 Apr 21. DOI: 10.3389/fmicb.2014.00175.

Frank N. Martin and Joyce E. Eoper, “Soil borne Plant Diseases Caused by Pythium spp.: Ecology, Epidemiology, and Prospects for Biological Control” . Critical Reviews in Plant Sciences, 1999; 18:111-181.

Jenkins J. E. E., Clark Y. S. and Buckle A. E., “Fusarium diseases of cereals”, Research Review 4, October 1988.

Sedlakova V., Dejmalova J., Hausvater E., Sedlak P., Dolezal P. and Mazakova J., “Effect of Phytophthora infestans on potato yield in dependence on variety characteristics and fungicide control”, Plant Soil Environment, 57, 2011 (10): 486-491.

Claims

1. A method for controlling, preventing, reducing or eradicating plant-pathogen infestation or instances thereof, or plant diseases, on a plant, plant organ, plant part, or plant propagation material, the method comprising applying to a plant, plant organ or plant propagation material, or to soil surrounding said plant, a pesticidally effective amount of the compound 8-geranyl-7-hydroxycoumarin:

or agriculturally acceptable salts thereof, wherein said plant-pathogen is a member selected from a Basidiomycota of the class Agaricomycetes or the genus Rhizoctonia, an Ascomycota of the class Sordariomycetes or a genus selected from Fusarium, Heterokontophyta of the class Oomycota and a Heterokontophyta of the order Peronosporales.

2. The method of claim 1, wherein said plant diseases are selected from potato blight, Phytophthora palmivora in cacao, canker fruit rot diseases in tomato and pumpkins, Phytophthora spp. crown and collar rot in pome and stone fruit, “damping off’ disease caused by Pythium spp. in tobacco, tomato, cucumbers, mustard, chilies and cress seedlings, Fusarium spp. causing wilt or ‘rot’ of vegetables, bananas, Fusarium spp. head and ear rot in maize, Fusarium graminearum head blight in small grains, Rhizoctonia spp. causing brown patch, damping off in seedlings, root rot and belly rot in vegetables and sheath blight in rice.

3. The method of claim 1, wherein said plant-pathogen is a member of the genus Rhizoctonia, which is in the Ceratobasidiaceae family of the order Cantharellales, such as Rhizoctonia solani.

4. The method of claim 1, wherein said plant-pathogen is a member of the genus Fusarium. The method of claim 4, wherein said plant-pathogen is the species Fusarium oxysporum. The method of claim 1, wherein said plant-pathogen is a member of the class Oomycota of the order Peronosporales. The method of claim 6, wherein said Peronosporales plant-pathogen is a member of a family selected from Lagenidiaceae, Olpidiosidaceae, Sirolpidiaceae, Leptomitaceae, Albuginaceae, Peronosporaceae, Pythiaceae, Rhipidaceae, Ectrogellaceae, Haliphthoraceae, Leptolegniellaceae, and Saprolegniaceae. In a specific embodiment, the plant-pathogen is a member of the family Peronosporaceae or Pythiaceae. The method of claim 1, wherein said plant-pathogen is the species Phytophthora inf estans. The method of claim 1, wherein said plant-pathogen is the species Pythium aphanidermatum.

A kit comprising a pesticidally effective amount of the compound 8-geranyl-7- hydroxycoumarin:

or agriculturally acceptable salts thereof, and instructions for using said compound or said agriculturally acceptable salts thereof to control, prevent, reduce or eradicate plantpathogen infestation or instances thereof, or plant diseases, on a plant, plant organ, plant part, or plant propagation material, or on soil surrounding said plant, wherein said plantpathogen is a member selected from a Basidiomycota of the class Agaricomycetes or the genus Rhizoctonia, an Ascomycota of the class Sordariomycetes or a genus selected from Fusarium, Heterokontophyta of the class Oomycota and a Heterokontophyta of the order Peronosporales.