Attenuation of phytofungal pathogenicity by autophagy modulators

Abstract

The present invention is in the field of agricultural pathogen control. Methods and compositions are described for controlling plant diseases caused by pathogens, including fungi and oomycete phytopathogens. Chemicals were screened for activity as autophagy inhibitors. The screening method used a bioluminescence resonance energy transfer (BRET)-based high-throughput screening strategy to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 for autophagosome formation. Ebselen and ebselen analogs were identified as inhibitors of fungal pathogens ATG4-mediated ATG8 processing. Identified chemicals inhibit spore germination, hyphal development, and appressorium formation. The autophagy inhibitors significantly reduced the virulence of pathogens in their corresponding plant hosts. The invention also provides for compositions comprising the same, methods of making the same, and methods of controlling plant disease.

Description

ATTENUATION OF PHYTOFUNGAL PATHOGENICITY BY AUTOPHAGY MODULATORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Application 63 / 620655, filed January 12, 2024. The entire contents of which is incorporated by reference herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Nos. IOS-2126256, IOS- 1339185, MCB-EAGER- 1355459, MCB-EAGER- 1549580, EPS-1655726, awarded by the National Science Foundation (NSF); and Grant No. 2P20GM103432 awarded by the National Institute of General Medical Sciences (NIGMS) and IDeA Networks of Biomedical Research Excellence (INBRE). The U.S. government has certain rights in the invention.BACKGROUND

[0003] Fungal and fungus-like plant diseases lead to significant losses to agricultural and horticultural producers. Many existing methods and fungicidal compositions for controlling plant disease create risks to the environment or to human health. Accordingly, there is a need for safer methods and compositions for controlling plant disease.

[0004] The present specification generally relates to treatment of plant disease, and, more specifically, to the use of specific materials and methods to inhibit or treat disease caused by phytopathogens, including fungi and oomycetes.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The embodiments set forth in the drawings are illustrative and exemplary in nature and not intended to limit the subject matter defined by the claims. The following detailed description of the illustrative embodiments can be understood when read in conjunction with the following drawings, wherein like reference numerals designate corresponding parts throughout the views.

[0006] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0007] Fig. 1A schematically illustrates an example screening schematic of a BRET-based B. cinerea synthetic ATG8 (BcATG8)-sensor.

[0008] Fig. IB shows, left, BRET ratio of ebselen (EB) and control (DMSO); and, right, shows inhibition of the BcATG4-mediated BcATG8 processing by EB compared to the DMSO control.

[0009] Fig. 1C shows chemical structures of EB and its analogs, EO, PT, PID, and PIO.

[0010] Fig. ID shows assay results for selected chemicals.

[0011] Fig. IE shows IC50 values of EB, EO, and PT for the BcATG8 cleavage estimated by in vitro cleavage assay (left panels). The graph (right) shows mean and SE.

[0012] Fig. IF shows EB, EO, and PT inhibit MoATG4-mediated processing of M0ATG8.

[0013] Fig. 1G shows ITC data of EB with BcATG4 (left) and AfoATG4 (right).

[0014] Fig. 2A shows in vivo inhibition of autophagy by EB using transgenic Botrytis cinerea expressing GFP-BcATG8.

[0015] Fig. 2B shows images of incomplete autophagosomes under EB treatment.

[0016] Fig. 2C shows autophagy inhibition by level of lipidation on GFP-BcATG8.

[0017] Fig. 3A shows morphological differences of germinating conidia of B. cinerea in the presence of different concentrations of autophagy inhibitors EB, EO and PT.

[0018] Fig. 3B shows germination percentages of B. cinerea.

[0019] Fig. 3C shows suppression of conidial germination and appressorium formation of M. oryzae under EB treatment.

[0020] Fig. 3D shows delay in germination of Magnaporthe oryzae observed at low concentrations of EB, and germination rate significantly reduced above 7 M EB.

[0021] Fig. 3E shows suppression of appressorium formation of M. oryzae with EB treatment.

[0022] Fig. 4A shows EB inhibition of B. cinerea lesion development on grapes, strawberries, tomatoes, and rose petals, compared to control.

[0023] Fig. 4B shows inhibition of B. cinerea infection on tomato leaves by EB.

[0024] Fig. 4C shows inhibition of the growth of B. cinerea on N. benthamiana leaves by EB.

[0025] Fig. 4D shows inhibition of M. oryzae by EB on rice plants.

[0026] Fig. 5 shows EB suppresses the germination of nine isolates of B. cinerea.

[0027] Fig. 6A shows a comparison of Monilinia fructicola and B. cinerea response to EB.

[0028] Fig. 6B shows suppression of the infection of B. cinerea in N. benthamiana leaves.

[0029] Fig. 6C shows Sclerotinia sclerotiorum germination is inhibited by EB.

[0030] Fig. 6D shows hyphae growth of w.t. 5. sclerotiorum is inhibited by EB.

[0031] Fig. 7 shows loss of viability of M. oryzae conidia under EB treatment.

[0032] Fig. 8A shows suppression of conidial germination and appressorium formation of M. oryzae under EO treatment.

[0033] Fig. 8B shows dose-dependent delay in germination.

[0034] Fig. 8C shows inhibition of appressorium formation by EO.

[0035] Fig. 8D shows inhibition of conidial germination and appressorium formation of M. oryzae under PT treatment.

[0036] Fig. 8E shows complete suppression of germination with PT treatment.

[0037] Fig. 8F shows complete inhibition of appressorium formation with PT treatment.

[0038] Fig. 9A shows growth retardation of mycelia of B. cinerea, M. fructicola, and .S'. sclerotiorum with EB treatment.

[0039] Fig. 9B shows reduced sclerotial formation in .S', sclerotiorum and increased premature sclerotia in the presence of EB and EO.

[0040] Fig. 10A shows growth inhibition of M. fructicola in different concentrations of EB.

[0041] Fig. 10B shows lesion diameters from .S', sclerotiorum are reduced by EB treatment.

[0042] Fig. 10C shows curative activity of EB against B. cinerea.

[0043] Fig. 11A shows no significant effect of multiple applications of EB, as compared with control, on host plant autophagy.

[0044] Fig. 11B shows experimental results indicating host autophagy is not affected by exogenous application of EB .

[0045] Fig. 12A shows EB does not alter transcript levels of genes encoding known autophagy core components in B. cinerea.

[0046] Fig. 12B shows treatment with EB and its analogs inhibits growth of the transgenic B. cinerea on tomato leaves.DETAILED DESCRIPTION

[0047] Autophagy in eukaryotes functions to maintain homeostasis by degradation and recycling of long-lived and unwanted cellular materials. Autophagy plays important roles in pathogenicity of various fungal pathogens, suggesting that autophagy is a novel target for development of antifungal compounds. A bioluminescence resonance energy transfer (BRET)-based high-throughput screening (HTS) strategy was used to identify compounds that inhibit fungal ATG4 cysteine protease-mediated cleavage of ATG8 for autophagosome formation. Ebselen (EB), and its analogs ebselen oxide (EO),and 2-(4-methylphenyl)-l,2-benzisothiazol-3(2H)-one (PT) were identified as inhibitors of fungal pathogens Botrytis cinerea and Magnaporlhe oryzae ATG4-mediated ATG8 processing. The EB and its analogs inhibit spore germination, hyphal development, and appressorium formation in Ascomycota pathogens, such as, Botrytis cinerea, Magnaporthe oryzae, Sclerotinia sclerotiorum, and Monilinia fructicola. Treatment with EB and its analogs significantly reduced fungal pathogenicity. Described compositions may be used to target autophagy in important fungal pathogens and may be used as antifungal compounds.

[0048] Macroautophagy, hereafter referred to as autophagy, is a dynamic process that is conserved across eukaryotes and entails the engulfment of cellular components or cargoes in double membrane vesicles called autophagosomes. Autophagosomes are then targeted to the vacuole / lysosome for degradation or recycling their cargoes. It has been established that recycling of long-lived cellular proteins and organelles by autophagy is an important adaptive response to nutrient deprivation and fluctuation of environments. However, recent studies have revealed that autophagy participates in other diverse biological processes including cellular differentiation and development, cell and tissue homoeostasis, aging, senescence, innate and adaptive immunity, and programmed cell death (PCD).

[0049] The core AuTophaGy (ATG) proteins first identified in yeast are conserved in higher eukaryotes including humans and plants. Among ATG proteins, the ubiquitin-like ATG8 plays an important role in autophagosome initiation and biogenesis. Furthermore, ATG8 serves as a docking site for adaptor proteins and also plays a role in autophagic cargo recruitment into autophagosomes. In the autophagy biogenesis, two ubiquitin-like conjugation systems facilitate delivery of phospholipids to autophagosomes. Ubiquitin-like ATG 12 is transferred to a target ATG5 in a conjugation process that is mediated by El-like ATG7 activity and E2-like ATG10. After the assembly of ATG5 with ATG12, the ATG5-12 complex interacts with ATG16. The ATG5-12-16 complex appears to promote lipidation on other ubiquitin-like proteins ATG8 or LC3. In the second ubiquitin-like conjugation process, the ATG4 cysteine proteases arc important for maturation and recycling of ATG8s. Ubiquitin-like ATG8s are cleaved at the catalytically important C-terminal Gly residue by ATG4 before being conjugated to PE. The conserved Gly at the carboxy terminus is also implicated in adduct formation with PE after ubiquitin-like conjugation reaction carried out by El- like ATG7, E2-like ATG3, and ATG5-12-16. ATG8-PE on the outside membrane can be recycled for delivery of new lipid molecules after additional catalysis by ATG4s. In contrast, the ATG8-PE in the inner membrane of the autophagosomes are degraded in the vacuolar / lysosomal lumen likeautophagic cargoes. Among ATG proteins in the vesicle formation, only ATG8 integrates into the phagophore and it marks the autophagosome until vacuolar fusion and cargo delivery. Because of the unique association of ATG8 / LC3 during autophagosome maturation, ATG8 / LC3 is presently the most widely used marker for visualizing autophagosomes.

[0050] ATG genes are also conserved in the sequenced plant pathogenic fungi and autophagy in fungi plays an important role in pathogenicity. During host-microbe interactions, conidia germination of pathogenic fungi is an initial step of the infection cycle and germ tubes further develop to find an infection site on hydrophobic surface of hosts for successful invasion. Once successful engagement between the pathogen and its host, conidia cell death is initiated and followed by an infection organelle formation such as appressorium. Cumulated evidence indicates that autophagy in a variety of fungi plays an important role in cellular differentiation, nutrient starvation, sporulation, and pathogenicity of fungal pathogens. It has been shown that the deletion of ATG1 in rice fungal pathogen Magnaphorthe oryzae causes to impair pathogenicity, conidia germination, higher turgor pressure of appressorium, and glycogen catabolism. Moatg8 mutant has also been shown similar phenotypes as Moatgl, indicating that autophagy in M. oryzae may play a role in pathogenicity. In addition, autophagy mutants of Aspergillus fumigatus and Fusarium graminearum fungi exhibit reduced conidiation, conidial germination, and virulence.

[0051] Leveraging the important functions of autophagy in phytofungal pathogens, manipulation of autophagy activity can be used by the described methods to control pathogenicity during hostmicrobe interaction. Botrytis cinerea is a phytopathogenic ascomycete known as grey mold. It is capable of infecting over two hundred plant species. Because the devastating fungal pathogen causes annual losses of up to $100 billion dollars worldwide, effective and safe fungicides are required to prevent Botrytis for food security. Like the other phytofungal pathogens, the deletion of Bcatgl showed defective appressorium resulting in both the reduction of pathogenicity and the defect in fungal life cycle. Therefore, experimental evidence indicates that the inhibition of autophagy in the fungal pathogen during host-microbe interaction can facilitate preventing the compatible interaction and improving food quality or grain yield.

[0052] The Bioluminescence Resonance Energy Transfer (BRET)-based synthetic ATG8 sensors were designed and used for dissecting molecular events in autophagy biogenesis and high-throughput screening (HTS) for characterization of new autophagy modulators. ATG4 is a cysteine protease that is essential in the biogenesis of autophagosomes by processing the ubiquitin-like ATG8 proteins at the evolutionally conserved glycine residue at the carboxyl terminus of ATG8s. ATG4-mediatedprocessing of ATG8 was selected as a target to apply intramolecular BRET for monitoring the kinetics of ATG4 because the enzymatic reaction induces separation of ATG8, resulting in reduction of intramolecular BRET ratio. Cross-kingdom assay reactivity has been shown using BRET-based synthetic substrates originated from different species. The BRET-based synthetic substrates of ATG8s in combination with in vitro cleavage assay was used to design and optimize target-based high-throughput screening for characterization of autophagy modulators in plant, fungal pathogen, and human. The identification of autophagy regulators can advance our mechanistic understanding of autophagy in eukaryotes and can facilitate modulation of autophagy for practical applications in agriculture and medical field.

[0053] Two thousand bioactive chemical compounds and seven-hundred-and-twenty drugs of NIH clinical trial collection were screened using ATG8 BRET-based sensor and HTS. From this screen, strong fungicide effects of EB and its analogs, PT and EO, were confirmed against fungal pathogens Botrytis cinerea, Sclerotinia sclerotiorum. and Monilinia fructicola as well as oomycete pathogen Phytophthora capsici. Moreover, efficacies of selected small molecules indicate that the compounds can be used against phytofungal pathogens as fungicides for increasing crop yield and food security.

[0054] Provided are treatment methods and compositions for prevention and treatment of plant disease.

[0055] Materials and methods to inhibit or treat disease caused by Ascomycota pathogens are described. Autophagy modulating agents are provided, including agents with anti-pathogen or antifungal effects. Antifungal agents can include fungicides and fungistats. Antifungal agents can include agents that inhibit fungal growth, fungal germination, fungal spread, fungal reproduction, fungal persistence in soil, fungal inoculation or infection of a host plant, and / or fungal viability. Methods of using and producing an autophagy modulating agent, including antifungal and anti- pathogcnic agents arc provided. Formulations and methods arc provided for preventing, inhibiting, controlling, and / or treating plant disease. Described compositions can include ebselen (2-pheny 1-1,2- benzoselenazol-3-one). Methods for modulating autophagy in Ascomycota pathogens, can include contacting the Ascomycota pathogen with a composition comprising an autophagy modulator.

[0056] In some embodiments, an autophagy modulator composition comprises ebselen (EB) or an analog thereof. Ebselen analogs may include, for example: ebselen oxide (EO), 2-(4- methylphenyl)-l,2-benzisothiazol-3(2H)-one (PT), 2-Phenyl-3H-indol-3-one (PIO), and 2-Phenyl-IH-isoindole- 1 ,3(2H)-dione (PID) .

[0057] Methods and treatments are provided for managing pathogens causing plant disease. The methods and treatments are suitable for treatment of phytopathogens, including fungi and oomycetes.

[0058] Methods are provided for controlling, preventing, and / or treating oomycete and / or fungal infections in plants. In an example, the method comprises applying an effective amount of a compound to a plant or part thereof, or to the soil, substrate or water surrounding the plant, wherein the compound is selected from the group consisting of: cbsclcn, or an cbsclcn analog, including a stereoisomer, tautomer, hydrate, salt, ester and / or solvate thereof. In an example, The method can include contacting the pathogen with an anti-pathogen composition, or contacting at least a portion of a susceptible plant with the anti-pathogen composition, wherein the anti-pathogen composition comprises an autophagy modulator, including ebselen and / or an analog thereof. The method can include applying a treatment agent comprising the composition.

[0059] Compositions are provided for preventing, treating, or ameliorating a plant disease caused by a pathogen, including pathogenic fungi and / or oomycetes. The composition includes a compound of ebselen and / or an ebselen analog. In some examples, the composition may be a foliar spray, a root drench, a growth media, a mulch, a crop dust, or a cleaning solution.

[0060] Methods are provided for the formulation and manufacture of an agrochemical composition comprising at least one autophagy modulating compound. In an example, the method includes providing at least one compound including ebselen and / or an ebselen analog, including a stereoisomer, tautomer, hydrate, salt, ester and / or solvate thereof. And the method includes combining the compound with at least one agrochemical auxiliary agent.

[0061] In an example, the compound includes one or more of: ebselen (EB), ebselen oxide (EO), 2-(4-methylphenyl)-l,2-benzisothiazol-3(2H)-one (PT), 2-Phenyl-3H-indol-3-one (PIO), or 2- Phenyl-lH-isoindole-l,3(2H)-dione (PID), and the compound may be provided as a stereoisomer, tautomer, hydrate, salt, ester and / or solvate.

[0062] The method can include preventing, controlling, treating, or ameliorating an infection by a phytopathogen, including by fungi and oomycetes. In some embodiments, the pathogen is a funguslike organism such as, oomycete pathogen Phytophthora sp. In some embodiments, the pathogen is Phytophthora capsici.

[0063] In some embodiments, the pathogen is a fungus. In some embodiments, the pathogen is at least one species of the Ascomycota phylum.

[0064] In some embodiments, the pathogen is one of: Bolrylis sp., Magnaporthe sp., Sclerotiniasp., Monilinia sp., or Phytophthora sp. In some embodiments, the pathogen is at least one of: Botrytis cinerea, Magnaporthe oryzae, Magnaporthe grisea, Sclerotinia sclerotiorum, Monilinia fructicola, or Phytophthora capsici.

[0065] In some embodiments, the pathogen is a fungal pathogen including at least one of: Botrytis sp., Magnaporthe Sp., Sclerotinia sp., and Monilinia sp. In some embodiments, the pathogen is at least one of: Botrytis cinerea, Magnaporthe oryzae, Magnaporthe grisea, Sclerotinia sclerotiorum, or Monilinia fructicola .

[0066] In some embodiments, the fungus belongs to the genus selected from the group consisting of: Magnaporthe, Botrytis, Puccinia, Fusarium, Blumeria, Mycosphaerella, Colletotrichum, Ustilago, Melampsora, Phakopsora, Altemaria, Sclerotinia, Cladosporium and Rhizoctonia. In some embodiments, the pathogen is one or more of: Acremonium spp., Alternaria spp., Arkoola nigra, Blumeria sp., Botrytis sp., Botrytis cinerea, Cercospora kikuchii, Cercospora sojina, Choanephora infundibulifera, Choanephora trispora, Cladosporium sp., Colletotrichum sp., Colletotrichum dematium f. truncatum, Colletotrichum truncatum, Corynespora cassiicola, Cylindrocladium crotalarie, Dactuliochaeta glycines, Diaporthe phaseolorum, Diaporthe phaseolorum var. caulivora, Drechslera glycines, Fusarium sp., Fusarium solani f. sp. glycines, Glomerella glycines, Leptosphaerulina trifolii, Macrophomina phaseolina, Magnaporthe sp., Magnaporthe oryzae, Melampsora sp., Meocosmospora vasinfecta, Microsphaera diffusa, Monilinia sp., Monilinia fructicola, Mycoleptodiscus terrestris, Mycosphaerella sp., Peronospora manshurica, Phakopsora sp., Phakopsora pachyrhizi, Philophora gregata, Phoma spp., Phomopsis spp., Phyllosticta sojaecola, Phy matot richop sis omnivora, Phytophthora spp., Phytophthora sojae, Phytophthora capsici, Pyrenochaeta glycines, Pythium spp., Pythium aphanidermatum, Pythium debaryanum, Pythium irregulars, Puccinia sp., Pythium myriotylum, Pythium ultimum, Rhizoctonia spp., Rhizoctonia solani, Sclerotinia sp., Sclerotium rolfsii, Sclerotinia sclerotiorum, Septoria glycines, Spaceloma glycines, Stegophora ulmea, Stemphylium botryosum, Taphrina deformans, Thielaviopsis basicola, or Ustilago sp.

[0067] In some embodiments, a susceptible plant is a crop. In some embodiments, the crop is at least one of: rice, wheat, rye, barley, millet, corn, grapes, strawberries, raspberry, blackberry, citrus, lettuce, spinach, melon, squash, rhubarb, onion, leek, garlic, chickpeas, cabbage, broccoli, kale, mustard, canola, pea, bean, lentil, soybean, potato, tomato, capsicum pepper, cocoa, cannabis, tobacco, betel nut, oak, Douglas-fir, chestnut, apple, apricot, peach, plum, nectarine, or cherry.

[0068] In some embodiments, a susceptible plant is an ornamental. In some embodiments, theornamental is at least one of: geranium, begonia, rose, lily, dogwood, elm tree, rhododendron, dahlia, magnolia, camellia, peony, tulip, poinsettia, or chrysanthemum.

[0069] In some embodiments, the method is used to treat, prevent, mitigate, or respond to a pathogen or an outbreak of a plant disease in a plurality of susceptible plants. In some embodiments, the plant disease is at least one of: botrytis bunch rot, rice blast, rice blast fungus, rice rotten neck, rice seedling blight, blast of rice, oval leaf spot of graminea, pitting disease, ryegrass blast, Johnson spot, Imochi, neck blast, wheat blast, fungal blight, brown rot, grey mold, white mold, powdery mildew, fungal blister, apple scab, ergot fungi, blackspot, black knot, chocolate spot disease, cottony rot, canker rot, black root rot, wilt disease, Fusarium wilt disease, verticillium wilt, root rot, canker, or leaf blight.

[0070] In some embodiments, the method includes applying the composition at one or more times. In some embodiments, the method includes applying the composition to a susceptible plant during seeding, after germination, during a growing season, prior to harvest, or at harvest. In some embodiments, the method includes applying the composition to a diseased plant. In some embodiments, the method includes applying the composition to soil.

[0071] In some embodiments, the method includes applying the composition to agricultural or horticultural implements, including tools, equipment, and storage containers. In some examples, the composition is applied to an air filter or humidifier in a greenhouse.

[0072] In some embodiments, the method includes applying the composition to harvested plant products. In some embodiments, the harvested plant products can include fruit, seeds, nuts, grain, legumes, leaves, stems, tubers, bulbs, corms, rhizomes, roots, flowers, bark, or wood. In some examples, aqueous solutions are used on harvested plant products; these may dry onto a surface to provide protection from pathogens, but are easily removed before consumption by rinsing, including rinsing only with room-temperature or cold water. Experimental data show that the compounds arc safe and non-toxic at levels described. In another example, the composition can be incorporated into a coating, such as a food wax or shellac, and the coating applied to an exterior surface, such as the rind of a citrus fruit; because this type of product is typically pealed, residual compounds pose no health concern from ingestion.

[0073] In some examples, the composition is applied to, or incorporated into, a growth media. In some examples the growth media comprises at least one of: soil, rock wool, peat moss, hydroponic culture, agar, perlite, vermiculite, or other growth substrate. In some examples, the composition is applied to a plant part during propagation, in an example, the propagative plant part can include:seeds, fruits, stems, buds, branches, tubers, bulbs, corms, rhizomes, and / or roots. In some examples, the composition is applied to a scion and / or stock during a grafting process. In some examples, the composition is applied in a tree wound dressing or sealant.

[0074] In some examples, the compound or composition is contacted to a plant surface. In some examples, the composition is contacted to between 10% to 100% of an above-ground plant surface in a growing plant. In some examples, the composition is contacted to between 10% to 100% of a surface of a harvested plant part, such as a fruit.

[0075] Experimental data indicate that there is negligible absorption of the compounds into plant tissues and the ebselen and ebselen analogs pose no health or safety risk for consumption of treated plant tissues at the dosages provided. Additionally, aqueous solutions which have been dried onto plant surfaces can be removed by rinsing in water, including cold water and room-temperature water.

[0076] In some examples, the compound or composition is applied to the leaves of the plant, as a coat to the seeds of the plant, as a soil drench, or as a root drench. In some examples, the compound or composition is applied by spraying, immersion, atomizing, foaming, fogging, coating, or encrusting.

[0077] In an example, the composition further comprises a coating enhancer to modify the rheometric properties and viscosity of the composition. The coating enhancer may include starch, amylose, amylopectin, dextrin, maltodextrin, polydextrose, syrup, cellulose, gum Arabic, gum tragacanth, gum karaya, mesquite gum, galactomannan, pectin, carrageenan, alginate, dextran, xanthan, gellan, whey protein, silk protein, casein, gelatin, gluten, fatty acids, fatty alcohols, wax, beeswax, carnauba wax, candellia wax, glyceride, and phospholipid, PVP, paraffin, shellac, or a solgel. In an example method, the composition substantially coats a surface and forms a dried residue on the surface. In an example method, the composition may be applied to a surface with a coating enhancer to form a coating, film, or residue on the surface.

[0078] In an example, the composition further comprises an antimicrobial compound. In an example, the composition further comprises an antibacterial compound. In an example, the composition further comprises a detergent or surfactant. In an example, the composition comprises an auxiliary antifungal composition, such as natamycin. In some examples, the composition exclusively consists of food-safe or GRAS ingredients.

[0079] In some embodiments, the autophagy modulating compound is provided in a treatment composition, and the composition comprises a liquid, an aqueous solution, an emulsion, a foam, a suspension, a powder, aerosolized droplets, or a spray.

[0080] In some embodiments, the composition comprises a concentration of EB, EO, PT, PIO, or PID provided at concentration between 0.1 pM and 1000 pM, or in a range of: 1.0 pM - 800 pM. 5.0 pM - 500 pM. 5.0 pM - 300 pM. 2.5 pM - 250 pM. 2.5 pM - 150 pM. or 10.0 pM - 200 pM. In some embodiments, the composition comprises a concentration of EB, EO, or PT in a solution at a level in a range from 1.0 pg / L to 0.1 g / L. In some embodiments the concentration of ebselen and / or its analogs in the composition is greater than 1.0 microMolar (pM), greater than 5.0 pM, greater than or equal to 10.0 pM, greater than or equal to 15.0 pM, greater than or equal to 20.0 pM, greater than or equal to 30.0 pM, greater than or equal to 40.0 pM, greater than or equal to 50 pM, greater than or equal to 100.0 pM, greater than or equal to 200.0 pM, or greater than or equal to 500.0 pM. In some embodiments the concentration of ebselen and / or its analogs in the composition is less than 0.1 M, less than 0.05 M, less than to 800.0 pM, less than or equal to 500.0 pM, less than or equal to 300.0 pM, less than or equal to 200.0 pM, less than or equal to 150.0 pM, less than or equal to 100.0 pM, less than or equal to 3.0 g / L, or less than or equal to 75.0 pM.

[0081] In some examples, the ebselen or ebselen analog is provided in an aqueous solution at a concentration between 0.1 pM and 1000 pM, or at a concentration in a range from 5.0 pM to 300 pM. In some examples, the compound is in the composition at concentration between 10 pM and 500 pM.

[0082] The compound or a composition comprising the compound is applied to a plant, directly or indirectly. The treatment method can include a single treatment application, or repeated application. The frequency of application can be adjusted based on environmental conditions, such as temperature, humidity, precipitation, or regional prevalence of pathogens. The frequency of application can be adjusted based on crop susceptibility, infection risk, and timing of planting, sprouting, maturation, or harvest. The treatment may be applied in response to plant stressors, such as insect attack, nematode infection, sun scald, waterlogging, or drought. The period of time within which protection is effected may extend from 1 to 10 days after the treatment.

[0083] The dose of active compound / application rate can be applied in the method of treatment to field crops or orchards and measured in grams per hectare (g / ha). In an example, a foliar spray, can have a treatment application area dosage in the range of: from 0.1 to 10,000 g / ha; from 10 to 1,000 g / ha; from 0.1 to 5,000 g / ha; from 1.0 to 5,000 g / ha; from 5 to 1,000 g / ha; from 10 to 1,000 g / ha; from 25 to 300 g / ha; from 15 to 150 g / ha; from 25 to 100 g / ha; from 10 to 1,000 g / ha, from 15 to 800 g / ha; from 200 to 900 g / ha; from 100 to 800 g / ha; or from 250 to 750 g / ha. In an example, a soil treatment area dosage can be: from 0.1 to 5,000 g / ha; from 1.0 to 5,000 g / ha; from 5 to 1,000 g / ha; from 10 to 1,000 g / ha; from 25 to 300 g / ha; from 15 to 150 g / ha; from 25 to 100 g / ha; from 10to 1,000 g / ha, from 15 to 800 g / ha; from 200 to 900 g / ha; from 100 to 800 g / ha; or from 250 to 750 g / ha. For germination, root soak, drench, or drip application, the dose of the autophagy modulating compound can generally be reduced, for example while using inert substrates like rockwool or perlite. In an example for seed treatment, a dosage can be: from 1.0 to 900 g per 100 kilogram of seed, from 1.0 to 500 g per 100 kilogram of seed, or from 2.0 to 250 g per 100 kilogram of seed.

[0084] In an example method, an agrochemical composition comprising the compound can be applied at a frequency of once every 2-20 days during a growing season. In some examples the treatment is performed at a frequency of: once per growing season, twice per season, 2-30 times per season, 2-25 times per season, or 5-15 times per season.

[0085] Suitable application methods include high or low-pressure spraying, immersion, atomizing, foaming, fogging, coating, and encrusting. In some methods, the agrochemical composition comprising the compound can be applied to the soil, or to the parts of the plant above ground, or to the foliage of the plant, by spraying, such as by the use of mechanical sprayers. Sprayers convert a formulation or composition which can be mixed with a liquid carrier, such as water or fertilizer, into droplets. The composition can be contacted to plants directly or indirectly, and sprayers can be used to apply formulations to pre-emergent or post-emergent crops. Application methods can include dispersing the composition using one or more of: air blast sprayers, boom sprayers, aerial sprayers, ultra-low volume sprayers, drip irrigation, sprinkler irrigation, and foggers. Where the formulations of the composition are in a solid, powder or granule form, they can be applied with granule or dust application equipment. Formulations can be applied to soil, plant media, plants, plant tissues or seeds.

[0086] Provided are compositions for preventing, treating, or ameliorating a plant disease caused by a pathogen, including pathogenic fungi and / or oomycetes. The compositions include an effective amount of a compound of ebselen and / or an ebselen analog.

[0087] In an example, an agrochemical composition includes at least one autophagy modulating compound. In an example, an agrochemical composition includes at least one compound of ebselen and / or an ebselen analog. The agrochemical composition one or more agrochemical auxiliary agents. In an example, the agrochemical auxiliary agent comprises at least one of: buffering agents, acidifiers, surfactants, wetting agents, spreading agents, tackifiers, stickers, carriers, fillers, thickeners, emulsifiers, dispersants, sequestering agents, anti-settling agents, coalescing agents, rheology modifiers, defoaming agents, photo-protectors, anti-freeze agents, biocides, penetrants, mineral or vegetable oils, pigments, or drift control agents. In an example, the agrochemical composition furthercomprises at least one of: a diluent, an additive, a plant nutrient, an emulsion stabilizer, a surfactant, a buffer, a crop oil, a drift inhibitor, a substratum, an insecticide, an herbicide, a nematicide, a molluscicide, a bactericide, an acaricide, a fungicide, a plant growth regulator, a fertilizer, or a coating enhancer.

[0088] In an example, a plant seed is coated with a coating composition comprising an effective amount of the compound. In some examples, the coating composition includes at least one of: a plant nutrient, a stabilizer, a sticker, a spreader, a coating enhancer, a buffer, a nematicide, a molluscicide, a bactericide, or a plant growth regulator.

[0089] In an example, a foliar spray or root drench comprises an effective amount of the compound with a solvent or diluent. In some examples, the foliar spray or drench, includes at least one of: a plant nutrient, an emulsion stabilizer, a surfactant, a sticker, a spreader, a coating enhancer, a buffer, a nematicide, a molluscicide, a bactericide, or a plant growth regulator.Experimental Results

[0090] The deletion of genes encoding core components of autophagy in B. cinerea showed loss of pathogenicity and defect in fungal life cycle. For example, the genetic defects of BcATG8 and BcATG4 blocked autophagy, resulting in significant impairment of vegetative development and pathogenicity of the fungal pathogen. Similar phenotypes of autophagy mutants have been observed in another devastating fungal pathogen, M. oryzae. Autophagy in Moatg4 and Moatg8 mutants was severely impaired and the virulence was attenuated, indicating that autophagy has a significant role in fungal pathogenicity. Once successful engagement between the fungus and its host, development of an infectious structure such as appressorium is initiated and followed by autophagic cell death. Intriguingly, the phenotypes of Moatg4 and Moatg8 mutants indicate that MoATG4 and A / 0ATG8 contribute not to formation of the structures but to proper function and / or maturation of appressoria. Malfunctional appressoria of the autophagy mutants of M. oryzae cause to attenuate pathogenicity of the rice blast fungus.

[0091] To identify chemical modulators of fungal autophagy, a BRET-based sensor of BcATG8 (BcATG8-sensor) was generated, and the target-based high-throughput screening (HTS) was optimized for the BcATG4-mediated processing of the BcATGS-scnsor. In this assay, cleavage of the BcATG8-sensor by BcATG4 leads to separation of Citrine-BcATG8 and ShR and hence results in a low BRET ratio. In contrast, if BcATG4 fails to cleave the BcATG8-sensor, it will result in a high BRET ratio. Consistent with this, the BRET ratio was high when the non-cleavable BcATGS (G116A)mutant-sensor was used in the assay compared to the wild type BcATG8-sensor. These results indicate that hit compounds to control the BcATG4-mediated processing of BcATG8 can be easily identified by monitoring BRET ratios from the in vitro cleavage assay with the BcATG8-sensor and the recombinant Be ATG4 in the presence of the chemical modulators.

[0092] Fig. 1A schematically illustrates an example screening schematic of a BRET-based B. cinerea synthetic ATG8 (BcATG8)-sensor, in which citrine fluorescence protein and modified Renilla luciferase, SupcrhRLUC (ShR), arc fused to the N- and the C-tcrminus of BcATG8, respectively. BcATG4 cysteine protease-mediated cleavage of Citrine-BcATG8-ShR leads to generation of Citrine-BcATG8 and ShR byproducts. Citrine-BcATG8-ShR exhibits a higher BRET ratio in the presence of ShR substrate coelenterazine (CLZ) and the cleavage of the sensor leads to lower BRET ratio. Compounds with high and low BRET ratios are considered as inhibitors and activators, respectively.

[0093] Fig. IB shows BRET ratio of the ebselen (EB) treatment (left). The cleavage of Citrine- BcATG8-ShR by BcATG4 (right) confirms the inhibition of the BcATG4-mediated BcATG8 processing by EB compared to the DMSO control. The graph presents mean and SE. p < 0.0001 (****), two-tailed z-test.

[0094] Fig. 1C shows a chemical structure for ebselen (EB) and a few examples of ebselen analogs, ebselen oxide (EO), 2-(4-methylphenyl)-l,2-benzisothiazol-3(2H)-one (PT), 2-Phenyl-3H- indol-3-one (PIO), and 2-Phenyl-lH-isoindole-l,3(2H)-dione (PID). Fig. ID shows assay results for selected chemicals. EO and PT inhibit the BcATG8 cleavage similar to EB. PID and PIO analogs of EB have weak effects on the cleavage. Inhibition of the cleavage is reversible under reducing conditions (+DTT). Fig. IE shows ICso values of EB, EO, and PT for the BcATG8 cleavage estimated by in vitro cleavage assay (left panels). The graph (right) shows mean and SE. Inhibitory roles of selected chemicals were validated by in vitro cleavage assay. The inhibition of the M. oryzae ATG4 (Afr>ATG4)-mediated processing of Afr>ATG8 by EB, EO, and PT is shown in Fig. IF. Strong inhibition of the MoATG4-mcdiatcd cleavage of AfoATG8 by EB and its analogs was observed.

[0095] The equilibrium dissociation constant of EB is estimated by Isothermal Titration Calorimetry (ITC). ITC experiments validated that EB could directly bind to BcATG4 and AfoATG4 with the degree of dissociation strength (Kd) of 3.4 and 1 nM, respectively. Fig 1G shows the ITC data of EB with Be ATG4 (left) and AfoATG4 (right) are fitted to the model of multiple binding sites. Three independent experiments were performed with similar results in Fig. 1B-1G. A representative image is shown in each panel. Arrows and arrowheads represent the full-length BRET-sensors andthe cleavage byproducts, respectively. Results indicate that EB and its analogs, EO and PT, inhibit BcATG4- and MoATG4-medaited cleavage of BcATG8 and M0ATG8, respectively.

[0096] Figs. 2A-2C illustrate results showing EB inhibition of autophagy in Botrytis cinerea.

[0097] Fig. 2A shows in vivo inhibition of autophagy by EB using transgenic B. cinerea expressing GFP-BcATG8. GFP localization pattern in B. cinerea expressing GFP-BcATG8 in the mock control or the EB treatment under nutrient starvation, an autophagy inducing environment. GFP puncta indicative of autophagosomes arc observed in mock treatment (top second panel), compared to diffusion of GFP fluorescence in the cytoplasm under EB treatment (bottom second panel). Right panels are magnifications of white boxed areas of each panel. Scale bars, 20 pm. Fig. 2B shows superresolution images of incomplete autophagosomes under EB treatment. In mock treatment, the complete autophagosomes (white arrow) are observed while the EB-treated fungal cells show incomplete membranous structures stained with GFP-BcATG8 (yellow arrows) frequently under 20 pM EB treatment. White arrowheads indicate autolysosomes in the vacuole (V). Scale bar, 5 pm. Fig. 2C shows autophagy inhibition by detecting level of lipidation on GFP-BcATG8 under EB treatment. The fully grown transgenic B. cinerea expressing GFP-BcATG8 is treated with either 50 pM or 100 pM EB for 8 hrs and proteins are separated in 6 M urea SDS-PAGE. The lipidation on GFP-BcATG8 is reduced whilst the unmodified GFP-BcATG8 accumulates under EB treatment, compared to the mock (M) control (top panel), indicating that EB can inhibit autophagy. #1 and #2 of 50 pM EB represent technical replicates. The blot probed with a-H3 is used as a loading control (bottom panel). The three biologically independent experiments were conducted with similar results in Figs. 2A-2C. A representative image is shown in each panel.

[0098] Once ATG8 is processed by ATG4, the exposed Gly residue in the C-terminus of ATG8 is involved in adduct formation with a membrane lipid, PE. The lipidation status on ATG8 is a hallmark for autophagy biogenesis. In addition, the lipidation of GFP-ATG8 has also been reported and widely used as an autophagy marker in various eukaryotes. To further validate that EB inhibits autophagy in the fungal cells, shown in Figs 2A-2B, we performed a pulse-chase experiment to monitor the lipidation of GFP-ZAATG8 under EB treatment. Since the lipidated GFP-ATG8 migrates faster than the unmodified GFP-ATG8 in 6 M urea SDS-PAGE, we analyzed the lipidation status of GFP-BcATG8 under EB treatment for 8 h. The post-translational modification on GFP-BcATG8 was significantly inhibited in the presence of 50 pM EB and the inhibition was more severe in 100 pM EB, compared to mock treatment (Fig. 2C, top panel, bottom band). In parallel, we observed increased accumulation of the unmodified GFP-BcATG8 under EB treatment (Fig. 2C, top band) indicating thesuppression of the GFP-BcATG8 maturation. Together, these results indicate that EB inhibits the BcATG4-mediated processing of GFP-BcATG8 in vivo.

[0099] Figs. 3A-3E show experimental results for EB, EO, and PT inhibition of the conidial germination of B. cinerea and M. oryzae in a dose-dependent manner.

[0100] A conidium of M. oryzae consists of three cells and typically produces a single germ tube from the apical cell during germination. At the tip of a germ tube, a dome-shaped melanin-pigmented apprcssorium forms and penetrates rice cells. Autophagy plays a significant role in the appressorial maturation of M. oryzae. The effects of the autophagy inhibitors EB, EO, and PT were tested on early development of M. oryzae. A significant delay and reduction in germination was observed under EB treatment in a dose-dependent manner as shown as the decreased germination rate of the mutant of Moatg4.

[0101] Fig. 3A shows morphological differences of germinating conidia of B. cinerea in the presence of different concentrations of autophagy inhibitors EB, EO and PT at 6 hours post- incubation (hpi). Dead conidia are often observed at 15 pM and no germination is observed at 50 pM of all compounds, n = 3 biologically independent experiments. A representative image is shown. Scale bars, 20 pm. Fig. 3B shows germination percentages of B. cinerea in the presence of indicated concentrations of autophagy inhibitors EB, EO and PT, compared to the mock control at different time points. All chemicals tested significantly reduce germination at concentrations above 15 pM. Fig. 3C shows suppression of conidial germination and appressorium formation of M. oryzae under EB treatment. Within 4 hpi, one or two cells of three cells consisting of a conidium were darker in 20 pM EB treatment (Fig. 3C, red arrows). Propidium iodide staining revealed that these cells lost their viability. Photographs were taken at 4 and 8 hpi. Yellow arrowheads and red arrows indicate abnormal hyphae and dead conidia, respectively. The experiments were repeated three times and similar results were observed. A representative image is shown. Scale bars, 50 pm. Fig. 3D shows the delay in germination of M. oryzae observed at low concentrations of EB and the germination rate significantly reduced above 7 pM EB . M. oryzae was more sensitive to EB than B. cinerea because 20 pM EB completely inhibited the germination of M. oryzae at 8 hpi. In the control, almost all conidia germinated and initiated appressorium formation by 24 hpi in our experimental condition. Fig. 3E shows significant suppression of appressorium formation of M. oryzae was observed above 10 pM EB. Under 1-5 pM EB, M. oryzae developed abnormal conidia with long and / or bi-directional germ tubes (Fig. 3C, yellow arrowheads). Appressorium formation was also inhibited in a dosedependent manner (Fig. 3e). EO at low concentration also induced the morphological changesobserved in EB treatment.

[0102] The conidia germinated after 6 h in water. However, conidial germination was delayed in 5 pM EB, EO, and PT and abolished at higher concentration of the inhibitors as shown in Figs. 3A- 3B. Therefore, EB and its analogs efficiently inhibit protease activity, resulting in suppression of germination of B. cinerea.

[0103] Figs. 4A-4D show EB inhibits infection of fungal pathogens in Ascomycota on various host plants.

[0104] Fig. 4A EB (50 pM) inhibition of B. cinerea lesion development on grapes, strawberries, tomatoes, and rose petals, compared to the mock (M) control. Three repeat experiments were performed with similar results. A representative image is shown. Fig. 4B Inhibition of B. cinerea infection on tomato leaves by EB, compared to the mock (M) control. Trypan blue staining of leaves shows the region of necrosis. The corresponding image of the live leaf is shown on the left. Mean lesion diameters in mock- and EB-treated leaves are plotted (right panel). Error bar, SE. p = 0.0002 (***), two-tailed / -test with Welch’s correction. n = 4 biologically independent experiments. Fig. 4C Spray application of EB inhibits the growth of B. cinerea on N. benthamiana leaves. Each leaf image was taken under white light (WL) and UV light after 3 dpi. Lesion areas are plotted (right panel). Error bar, SE. p < 0.0001 (****), Dunnett’s multiple comparisons test with oneway ANOVA. n = 3 biologically independent experiments. Fig. 4D Inhibition of M. oryzae by EB on rice plants. The conidial suspension of M. oryzae was mixed with either mock (M, 1% DMSO) or 100 p M of EB and directly applied onto rice plants. Lesion types were defined after a week. Error bar, SE. p < 0.0001 (****), two-tailed / -test. All scale bars in images, 2 cm.

[0105] EB and its analogs significantly limited the growth of B. cinerea expressing GFP- BcATG8, indicating that the autophagy inhibition by the lead compounds compromises pathogenicity of the transgenic B. cinerea, as shown in Fig. 4B.

[0106] The inhibitory activities of EB and its analogs on the fungal infections in several hosts were examined, as the fungal mutants of atg4 and atg8 showed significant reduction of virulence. EB treatment abolished B. cinerea lesion development on the hosts, compared to the mock-treated samples (Fig. 4A). Furthermore, EB treatment significantly inhibited B. cinerea lesion development on a vegetative tissue such as tomato leaves (Fig. 4B).

[0107] To mimic typical fungicide application setting for investigation of the protective activity of EB against B. cinerea, we sprayed EB onto N. benthamiana, allowed it to dry, and then inoculated the conidia suspension of B. cinerea, Fig. 4C. The inhibition of lesion development on N.benthamiana leaves was monitored after spray application of EB following recent studies testing active ingredients of commercial fungicides such as difenoconazole and hexanoic acid against B. cinerea. Fig. 4C shows dose-dependent inhibition of lesion development of B. cinerea was observed on N. benthamiana leaves infected by the conidia suspension.

[0108] Fig. 4D shows application of 100 pM EB was sufficient to abolish the rice blast infection, M. oryzae, by the conidia suspension while severe blast disease was developed in the mock-treated rice plants.

[0109] Fig. 5 shows EB suppresses the germination of nine isolates of B. cinerea. EB inhibits growth of various isolates of B. cinerea. All isolates cannot germinate under 50 pM EB. These results are consistent across two experimental replicates. Scale bar, 1 cm. Severe impairment of conidial germination was observed under 20 pM and exacerbated under 50 pM EB in nine isolates of B. cinerea, as seen in Fig. 5. Thus, targeting autophagy is an excellent strategy to attenuate the fungal growth regardless of the genetic variation of B. cinerea.

[0110] Figs. 6A-6D show the effects of autophagy inhibitors EB and EO against fungal pathogens.

[0111] EB, EO, and PT were found to suppress the growth of Ascomycota fungal pathogens. EB, EO, and PT were tested to determine whether the compositions had broad effects on development and pathogenicity of Ascomycota pathogens including B. cinerea, M. oryzae, Sclerotinia sclerotiorum, and Monilinia fructicola since autophagy plays important roles in growth, hyphal development, and pathogenicity of Ascomycota pathogens. Interestingly, M. fructicola failed to germinate in the presence of 20 pM EB and EO while the germination of B. cinerea and M. oryzae were abolished under 50 pM EB and EO as shown in Fig. 6A. It has been shown that potato PKI1 and PPI3B2 protease inhibitors inhibit conidia germination of B. cinerea but direct targets of these inhibitors are not fully understood. In addition, the genetic defect of BcATG4 causes reduced germination. These findings indicate that the function of proteases is important during germination of the fungal pathogen. Germination rates of B. cinerea were tested under the lead compounds. The conidia germinated after 6 h in water. However, conidial germination was delayed in 5 pM EB, EO, and PT and abolished at higher concentration of the inhibitors, as shown in Figs. 3A-3B. Therefore, EB and its analogs could efficiently inhibit protease activity, resulting in suppression of germination of B. cinerea. These experimental conditions were applied to investigate whether infection of B. cinerea could be inhibited on leaves of Nicotiana benthamiana. Based on the result from the germination test of B. cinerea under EB treatment, as shown in Fig. 3B, the conidia suspension of B. cinerea was directly mixed with lowconcentrations of EB (either 5 or 10 pM) and then inoculated on N. benthamiana. After 3 days postinoculation (dpi), the disease symptom in N. benthamiana was monitored. Infection of B. cinerea was suppressed under 10 pM EB treatment, indicating a low-dose of EB can be effective to inhibit infection of B. cinerea on live plant tissues, as illustrated by Fig. 6B.

[0112] Fig. 6 A M. fructicola (left bottom) is more sensitive to EB than B. cinerea (left top). The germination of M. fructicola conidia is strongly inhibited in 20 pM EB. The germination of M. oryzae conidia also shows severe inhibition under 50 pM EB and EO treatments (right panel). Scale bars, 1cm. M. fructicola failed to germinate in the presence of 20 pM EB and EO while the germination of B. cinerea and M. oryzae were abolished under 50 pM EB and EO.

[0113] As shown in Fig. 6B, with S. sclerotiorum. only 22% of the ascospores germinated under 50 pM EB at 12 h post-incubation (hpi). Fig. 6B Suppression of the infection of B. cinerea under a low-dose treatment of EB. 10 pM EB is sufficient to inhibit the infection of B. cinerea in N. benthamiana leaves. Sample sizes for analysis of the disease frequency are 86 and 43 infection spots of mock and EB treatments (either 5 or 10 pM), respectively. Graph shows mean with SE. ****, p < 0.0001, one-way ANOVA with Dunnett’s comparison. Fig. 6C The S'. sclerotiorum germination is significantly inhibited under 50 pM EB at 12 hpi, compared to the mock control (M). The quantification is on the right. Graph shows mean with SE. ***, p < 0.001, two-tailed t-test. Scale bar, 20 pm.

[0114] The suppression of autophagy by EB resulted in the growth inhibition of wild type .S'. sclerotiorum. Fig. 6D shows Ssatg8 was resistant to EB treatment at time points 40 hpi and 65 hpi whereas the significant growth inhibition by EB was observed in wild type. The hyphae growth of wild type of S. sclerotiorum is significantly inhibited, compared to the vegetative growth of Ssatg8 observed under 5 pM EB. Due to difference of hyphae growth between wild type and the Ssatg8 mutant, the growth rates of Ssatg8 are measured at two time points (40 and 65 hpi). Graph shows means with SE. ****, p < 0.0001 and **, p < 0.01 , two-way ANOVA with Dunnett’s multiple comparison. Scale bar, 1 cm. The three biologically independent experiments were conducted with similar results in Figs. 6A-6D. A representative image is shown in each panel.

[0115] Figs. 5, and 6A-6D show EB, EO, and PT suppress the growth of Ascomycota fungal pathogens.

[0116] Fig. 7 shows loss of viability of M. oryzae conidia under EB treatment. The M. oryzae conidia are dead following exposure to 20 pM EB for 4 hours, compared to the DMSO control. Dead conidia are confirmed by staining with propidium iodide. Red fluorescence indicates loss ofmembrane integrity of dead conidia under EB treatment. Scale bar, 40 pm. A representative image is shown. n=12 samples.

[0117] Figs. 8A-8F show EO and PT inhibit conidial germination and appressorium formation of M. oryzae.

[0118] Fig. 8A Suppression of conidial germination and appressorium formation of M. oryzae under EO treatment. Fig. 8B The delay in germination is observed over 5 pM EO. Germination is significantly suppressed at 50 pM EO. n=3 biologically independent experiments. Fig. 8C Appressorium formation is also inhibited at EO concentrations greater than 5 pM in a dose-dependent manner. Only 10% of conidia forms appressoria under 50 pM EO at 24 hpi. Two-way ANOVA test with Dunnett’s multiple comparison. A different number of asterisks indicates statistically significant differences. n=3. Fig. 8D Inhibition of conidial germination and appressorium formation of M. oryzae under PT treatment. Fig. 8E 50 pM PT completely suppresses germination. n=3 biologically independent experiments. Fig. 8F 50 pM PT completely inhibits appressorium formation. Two-way ANOVA test with Dunnett’s multiple comparison. A different number of asterisks indicates statistically significant differences. n=3. In Fig. 8A and Fig. 8D, arrowheads indicate abnormal germ tubes of germinating conidia. Scale bars, 50 pm. All graphs show means with SE. The three biologically independent experiments were conducted with similar results in Fig. 8A and Fig. 8D. A representative image is shown in each panel.

[0119] Figs. 9A-9B show inhibition of mycelial growth and sclerotial development of Ascomycota pathogens by EB.

[0120] Fig. 9A Growth retardation of mycelia of B. cinerea, M. fructicola, and S. sclerotiorum is observed under 50 pM EB, compared to the mock control (M). Scale bar, 2 cm. The three biologically independent experiments were conducted with similar results. A representative image is shown. Fig. 9B S', sclerotiorum shows less sclerotial formation in the presence of autophagy inhibitors, EB and EO while premature sclerotia are increased. The quantification of sclerotia is shown on the right. Red and green arrowheads represent full-grown and premature sclerotia, respectively. All graphs show means with SE. Dunnett’s multiple comparisons test with one-way ANOVA. n=4 for DMSO, EB, and EO and n=2 for PT. Scale bar, 2 cm.

[0121] The phenotypes induced by PT were as similar to those observed by EO in M. oryzae as shown in Figs. 8D-8F. In addition to the inhibition of both germination and appressorium formation of M. oryzae, mycelial growth of B. cinerea, M. fruticola, and .S'. sclerotiorum was significantly reduced under 50 pM EB as shown in Fig. 9A. Similar’ to impaired sclerotial development of atg8mutants of both B. cinerea and .S'. sclerotiorum. 20 pM EB and EO significantly inhibited sclerotial development of 5. scleroliorum while 20 pM PT affected the development of sclerotia less than the others, Fig. 9B. There appeal’s to be different stability and permeability of PT due to the incubation and different cell wall composition of sclerotia with that of mycelia. Together, these results show that treatment with autophagy inhibitors EB, EO, and PT suppresses germination, mycelial development, appressorium formation, and sclerotial development in the fungal pathogens examined.

[0122] Figs. 10A-10C shows suppression of the growth of B. cinerea, M. fructicola, and .S'. scleroliorum on hosts by EB.

[0123] Fig. 10A Growth of M. fructicola in the presence of two different concentrations of EB. 20 pM EB effectively inhibits the growth. Fig. 10B Lesion diameters are significantly reduced by treatment with 50 pM EB, indicating that EB inhibits pathogenicity of .S'. sclerotiorum. Graph shows means with SE. ****, p < 0.0001, two-tailed t-test. Fig. 10C The curative activity of EB against B. cinerea. Application of EB on the infected N. benthamiana leaves inhibits the disease progression. The disease progression is defined as the ratio of area of 4 dpi divided by area of 3 dpi at the same spot. Scale bars, 1 cm. Graph shows means with SE. **, p < 0.01, two-tailed t test. The three biologically independent experiments were conducted with similar results in Figs. 10A-10C. A representative image is shown in each panel.

[0124] As in Fig. 6 A, showing 20 pM EB completely suppressed M. fructicola spore germination, 20 pM EB also efficiently inhibited the growth of M. fructicola on apple fruits, as shown in Fig. 10A. With 50 pM EB treatment, significant reduction of .S'. sclerotiorum infection was observed on tomato leaves, as shown in Fig 10B.

[0125] Fig. 10C shows the curative activity of EB in the live tissues. Prior to spray-application of EB, the conidia suspension of B. cinerea was inoculated and the disease symptom was incurred on the host tissues at 36 hpi. After development of the disease symptom, 300 pM EB was applied directly onto infected leaves once a day. The disease lesions were measured at 3 and 4 dpi. Compared to mock treatment, spray-application of EB showed significant inhibition of the disease progression, indicating curative activity of EB against the necrotrophic fungal pathogen.

[0126] Figs. 11A-11B indicate plant ATG4-mediated processing of ATG8 is not significantly inhibited by EB treatment.

[0127] Fig. 11 A shows the effect of EB on host autophagy by repeated applications over multiple days. 300 pM EB is applied to investigate the inhibition of host autophagy in the transgenic N. benthamiana expressing the AzATG8a-sensor once per two days for three times. 10% acetone as mocktreatment and EB are applied to different leaves with a similar age in the same plants. Samples are collected for western blot at 7 dpi. Similar processing activities are observed in mock and EB treatment. An arrow indicates the unprocessed A / ATG8-sensor while an arrowhead indicates the processed byproduct. #1, 2, and 3 indicate independent transgenic lines. Fig. 1 IB Minimal effect of host autophagy under exogenous application of EB. Transgenic Arabidopsis expressing the GFP- AtATG8 cassette is subjected to examine host autophagy by spray application of EB. The amount of stable GFP represents autophagy flux in plants. Host autophagy is not affected by exogenous application of EB. M indicates 1% DMSO as mock treatment. Ponceau S represents a loading control. The three biologically independent experiments were conducted with similar results in Figs. 11A- 1 IB. A representative image is shown in each panel.

[0128] Figs. 11A-11B indicate that levels of EB producing growth inhibition of B. cinerea by EB-spraying on plant leaves, did not detectably result in a significant inhibition of plant autophagy activity , indicating that the effect on the host autophagy inhibition by conventional application of EB appears to be minimal. This was further verified by multiple applications of EB to transgenic N. benthamiana expressing the Arabidopsis / \ / ATG8a-scnsor. Although the transgenic plants were exposed to 300 pM EB for 1 week, the maturation of the A / ATG8a-scnsor was not affected, as seen in Fig. 11 A. The physical barriers of plants that prevent water loss and protect pathogen invasion appear to prevent absorption of EB into plant cells, resulting in a minimal effect on host autophagy and negligible absorption by plant tissues.

[0129] Figs 12A-12B show EB effects on transcription of known autophagy genes and pathogenicity of the transgenic B. cinerea.

[0130] Fig. 12A shows 20 pM EB does not alter transcript levels of genes encoding known autophagy core components in B. cinerea. Transcript levels of the autophagy genes are estimated by qRT-PCR. The transcription of BcATGl, BcATG3, BcATG4, BcATG6, BcATG7, and BcATG8 is not changed under EB treatment of which condition is the same as explained in the super-resolution microscopy experiment (Fig. 2b). n=3. Fig. 12B Treatment with EB and its analogs inhibits growth of the transgenic B. cinerea on tomato leaves. Treatment with 50 pM EB, EO, and PT suppresses the infection by B. cinerea harboring the GFP-BcATG8 cassette compared to the mock control (M) on tomato leaves (left panel). Quantification of disease lesion areas is shown in the right panel. WL, white light; UV, UV light. Scale bar, 1 cm. Error bar, SE. ****, p < 0.0001, Dunnett’s multiple comparisons test with one-way ANOVA. The three biologically independent experiments were conducted with similar results. A representative image is shown.

[0131] Throughout this disclosure, various publications, patents, or published patent specifications may be referenced. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe materials and methods which may be used in conjunction with aspects of the described invention.

[0132] Certain embodiments of the devices, apparatuses, and methods disclosed herein are defined in the above examples. It should be understood that these examples, while indicating particular embodiments, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the compositions and methods described herein to various usages and conditions. Various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the disclosure, and to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof.

Claims

CLAIMSWhat is claimed is:

1. A method for preventing, treating, or ameliorating a plant disease caused by a pathogen, comprising: contacting the pathogen with an autophagy modulating compound, or contacting at least a portion of a susceptible plant with the autophagy modulating compound, wherein the autophagy modulating compound comprises: ebselen, or an analog thereof, including a stereoisomer, tautomer, hydrate, salt, ester and / or solvate thereof.

2. The method of claim 1, wherein the pathogen is an oomycete or a fungus.

3. The method of claim 1, wherein the analog is 2-(4-methylphenyl)-l,2-benzisothiazol-3(2H)-one(PT).

4. The method of claim 1, wherein the analog is ebselen oxide.

5. The method of claim 1, wherein the pathogen is in the phylum, Ascomycota.

6. The method of claim 1, wherein the pathogen includes at least one of: Botrytis cine re a.Magnaporthe oryzae, Sclerotinia sclerotiorum, Monilinia fructicola, or Phytophthora capsici.

7. A composition for preventing, treating, or ameliorating a plant disease caused by a pathogen, wherein the pathogen is an oomycete or a fungus, the composition comprising: an autophagy modulating compound, wherein the autophagy modulating compound comprises: ebselen or an analog thereof, including a stereoisomer, tautomer, hydrate, salt, ester and / or solvate thereof.

8. The composition of claim 7, wherein the autophagy modulating compound comprises at least one of: ebselen (EB), ebselen oxide (EO), 2-(4-methylphenyl)-l,2-benzisothiazol-3(2H)-one (PT), 2-Phenyl-3H-indol-3-one (PIO), or 2-Phenyl-lH-isoindole-l,3(2H)-dione (PID).

9. The composition of claim 7, wherein the ebselen or ebselen analog is provided at concentration between 0.1 pM and 1000 pM.

10. The composition of claim 7, wherein the ebselen or ebselen analog is provided at concentration between 5.0 pM and 300 pM.

11. A method for controlling, preventing, and / or treating oomycctc and / or fungal infections in plants, the method comprising: applying an effective amount of a compound to a plant or part thereof, or to the soil, substrate or water surrounding the plant, wherein the compound is selected from the group consisting of: ebselen and / or an ebselen analog, including a stereoisomer, tautomer, hydrate, salt, ester and / or solvate thereof.

12. The method of claim 11, wherein the ebselen or the ebselen analog is selected from the group consisting of: ebselen (EB), ebselen oxide (EO), 2-(4-methylphenyl)-l,2-benzisothiazol-3(2H)-one (PT), 2-Phenyl-3H-indol-3-one (PIO), or 2-Phenyl-lH-isoindole-l,3(2H)-dione (PID).

13. The method of claim 12, wherein the compound includes the ebselen analog comprising at least one of: EO, PT, PIO, or PID.

14. The method of claim 12, wherein the compound includes 2-(4-methylphenyl)-l,2- benzisothiazol-3(2H)-one.

15. The method of claim 12, wherein the compound includes ebselen oxide.

16. The method according to any one of claims 1 1-15, wherein the fungus belongs to the genus selected from the group consisting of: Magnaporthe, Botrytis, Puccinia, Fusarium, Blumeria, Mycosphaerella, Colletotrichum, Ustilago, Melampsora, Phakopsora, Altemaria, Sclerotinia, Cladosporium, and Rhizoctonia.

17. The method according to any one of claims 11-15, wherein the oomycete is a phytopathogen belonging to the genera Pythium or Phytophtora.

18. The method according to any one of claims 11-15, wherein the oomycete and / or fungus includes at least one of: Bolrytis cinerea, Magnaporlhe oryzae, Sclerolinia scleroliorum, Momlinia fructicola, or Phytophthora capsici.

19. The method according to any one of claims 11-18, wherein the compound is formulated as part of a composition which further comprises: a diluent, an additive, a plant nutrient, an emulsion stabilizer, a surfactant, a buffer, a crop oil, a drift inhibitor, and / or a substratum.

20. The method of claim 19, wherein the compound is used in the composition at concentration between 0.1 pM and 1000 pM.

21. The method of claim 19, wherein the compound is used in the composition at concentration between 5.0 pM and 300 pM.

22. The method according to any one of claims 19-21, wherein the composition further comprises at least one of: an insecticide, an herbicide, a nematicide, a molluscicide, a bactericide, an acaricide, a fungicide, a fertilizer, and / or a plant growth regulator.

23. The method according to any one of claims 1-22, wherein the compound or composition is applied to the leaves of the plant, as a coat to the seeds of the plant, as a soil drench, or as a root drench.

24. The method of claim 23, wherein the compound or composition is applied by spraying, immersion, atomizing, foaming, fogging, coating, or encrusting.

25. A method for the formulation and manufacture of an agrochemical composition comprising: providing at least one compound comprising: ebselen and / or an ebselen analog; and combining the compound with at least one agrochemical auxiliary agent.

26. The method of claim 25, wherein the compound is used in the agrochemical composition at concentration between 0.1 pM and 1000 pM.

27. The method of claim 25, wherein the compound is used in the composition at concentration between 5.0 pM and 300 pM.

28. The method according to any one of claims 25-27, wherein the agrochemical auxiliary agent comprises at least one of: a buffering agent, an acidifier, a surfactant, a wetting agent, a spreading agent, a tackifier, a sticker, a carrier, a filler, a thickener, an emulsifier, a dispersant, a sequestering agent, an anti- settling agent, a coalescing agent, a rheology modifier, a defoaming agent, a photoprotector, an anti-freeze agent, a biocide, a penetrant, a mineral or vegetable oil, a pigment, or a drift control agent.

29. The method according to any one of claims 25-28, wherein the agrochemical composition comprises at least one of: a diluent, an additive, a plant nutrient, an emulsion stabilizer, a surfactant, a buffer, a crop oil, a drift inhibitor, a substratum, an insecticide, an herbicide, a nematicide, a molluscicide, a bactericide, an acaricide, a fungicide, a plant growth regulator, a fertilizer, or a coating enhancer.

30. A plant seed coated with a compound selected from the group consisting of: ebselen and / or an ebselen analog, including a stereoisomer, tautomer, hydrate, salt, ester and / or solvate thereof.

31. A foliar spray comprising: an autophagy modulating compound, wherein the autophagy modulating compound comprises ebselen and / or an ebselen analog, including a stereoisomer, tautomer, hydrate, salt, ester and / or solvate thereof.

32. The foliar spray of claim 31, further comprising at least one of: a plant nutrient, an emulsion stabilizer, a surfactant, a sticker, a spreader, a coating enhancer, a buffer, a nematicide, a molluscicide, a bactericide, or a plant growth regulator.

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