Antifungal compounds and compositions and antifungal use thereof

The present invention relates to the use of certain compounds, known to inhibit the response of plants to ethylene, for the control of phytopathogenic fungi. The compounds are substituted cyclopropenes.

EXAMPLE 1—Activity Against Botrytis cinerea Compounds were evaluated for the ability to inhibit germination of Botrytis cinerea spores using an assay which measures the inhibition of germination-associated adhesion in microtiter plates. Encapsulated compound 1 (1-methylcyclopropene) was dissolved in Sabouraud dextrose broth (“SDB”, obtained from Difco Laboratories) at a concentration of 500 ppm. Compound 2 (1-n-octyl-3,3-difluorocyclopropene) was dissolved in DMSO at 50 mg/ml. This solution was then diluted with SDB to give a solution concentration of compound 2 of 1000 ppm. Comparison compound, AVG, was dissolved in SDB at 5 mg/ml. Two-fold serial dilutions of the solutions of these compounds in SDB were made in 100 &mgr;l aliquots of SDB in 96-well microtiter plates. Spore suspension of B. cinerea (100 &mgr;l at 2×10 5 spores/ml) was added to the wells. The plates were then incubated at 25° C. for 5.5 hours at which time spores in control wells had germinated. The medium containing unbound spores was removed by inverting and flicking the microtiter plate to remove as much liquid as possible. Quantitation of the adhered spores was achieved by measuring their cellular protein content using sulforhodamine B (see Skehan et al., Journal of the National Cancer Institute, 82, 1107-1112 (1990)), measuring absorbance at 564 nm. Inhibition of adhesion was determined by comparing the absorbance value in a well containing fungicide with the absorbance in control wells without fungicide. EC 50 values for germination inhibition were determined from dose-response curves. The results of this test are in Table 1. 1 TABLE 1 Compound EC 50 (ppm) 1 37 2 3.3 Comparison (AVG) >2,500 A solution of &agr;-cyclodextrin alone had no activity at 50 mg/ml. 
 EXAMPLE 2—Activity Against Various Fungi Compound 1 was tested for in vitro fungitoxicity towards a variety of fungi. The compound was encapsulated in &agr;-cyclodextrin (0.14% by weight) which was dissolved in yeast-extract/glucose medium (“YEG” made from 20 g of glucose and 4 g of yeast extract per liter of water) to give a concentration of compound 1 of 430 ppm. Two-fold serial dilutions were prepared in 100 &mgr;l aliquots of YEG medium in 96-well microtiter plates. The wells were inoculated with the various fungi prepared in YEG as either spore suspensions ( Septoria nodorum, Colletotrichum lagenarium, Pyricularia oryzae, and Phytophthora capsici ) or as mycelial homogenates ( Pythium ultimum and Rhizoctonia solani ). Mycelial growth was assessed by measuring absorbance at 570 nm after growth at 25° C. for 2 days ( S. nodorum and P. ultimum ) or 5 days ( C. lagenarium, P. oryzae, P. capsici, and R. solani ). EC 50 values were determined from dose-response curves obtained. The results of this experiment are in Table 2. 2 TABLE 2 Organism EC 50 (ppm) Pythium ultimum 2.6 Pyricularia oryzae 6.7 Phytophthora capsici 6.9 Rhizoctonia solani 7.4 Septoria nodorurn 22.5 Colletotrichum lagenarium >70 A solution of &agr;-cyclodextrin alone had no activity at 30 mg/ml. 
 EXAMPLE 3—Technical Material v. Formulation The above examples demonstrate fungitoxicity of compound 1 when provided as encapsulated material in &agr;-cyclodextrin. Since compound 1 is a gas at room temperature its activity as a technical material in solution was evaluated in sealed vials with a small head space as follows. Compound 1, formulated by encapsulation in a-cyclodextrin, was also tested for comparison. Compound 1 (technical material) was added to 21×50 mm glass vials on dry ice as the appropriate amount of a 14% (w/v) solution in acetone to provide the desired concentrations. (The acetone solutions were kept on dry ice or in a freezer at −80° C. until used.) Controls received the same volumes of acetone alone. A spore suspension (8 ml) of Botrytis cinerea at 2.5×10 4 spores/ml in two-fold diluted SDB was added to each vial. The vials were immediately sealed with polyethylene plugs and placed in a 250° C. incubator for 16 hours. The extent of fungal growth in the vials was determined by measuring their cellular protein content using sulforhodamine B (Skehan, et al., Journal of the National Cancer Institute 82, 1107-1112 (1990)), and the percent inhibition of growth was calculated by comparing growth in the treatments with growth in the controls. The desired amounts of Compound 1, formulated by encapsulation in &agr;-cyclodextrin (4% by weight) were added to glass vials. A spore suspension (8 ml) of Botrytis cinerea at 2.5×10 4 spores/ml in two-fold diluted SDB was added to each vial, and vials processed as described above. Values for percent inhibition of growth are presented in the table below. &agr;-Cyclodextrin alone did not cause any inhibition of growth. 3 Conc. % Inhibition Treatment (ppm) of growth Cpd. 1, technical material 175 30.1 350 99.4 525 99.8 Cpd. 1, &agr;-cyclodextrin formulation 40 14.1 80 43.8 160 99.5 200 99.5 
 Example 4—Suppression of Botrytis rot on rose petals Freshly cut white roses were purchased from Zieger & Sons, Inc. rose growers. Non senescing petals were chosen to perform the experiment. Petals were placed in plastic petri dishes (100×20 mm) each containing a moist &num;3 Whatman filter paper with one petal per petri dish. Compound 1, formulated as in Example 1 (0.778 g) was dissolved in 20 ml hot water in a 100 ml jar sealed with a cap. When the powder was dissolved, the jar was placed in a 4.8 liter cabinet containing three petri dishes (without the lids) with petals. The cap of the jar was quickly removed and the cabinets sealed for ninety (90) minutes. The concentration of Compound 1 in the gas phase was 100 ppm. After exposure to Compound 1 at 100 ppm the petals were inoculated with two 20&mgr;l drops of spore suspension containing 1×10 6 spores per ml. Controls were inoculated without prior exposure to Compound 1. Lids were placed on the petri dishes and the dishes were transferred to a plant growth chamber set at a 12 hour photoperiod, a 20° C. day temperature and 18° C. night temperature, and 70% relative humidity to allow disease development. The number of petals with sporulating lesions was determined after 10 days. Two trials were conducted with 3 petals per treatment in each trial. The combined results from the two trials were as follows: 4 Petals with sporulating lesions Treatment (%) Compound 1 16.7 (100 ppm) control 66.7 These data demonstrate the fungitoxic activity of the compounds of this invention and their ability to control fungal disease. The compounds of this invention are useful as agricultural fungicides and, as such, can be applied to various loci such as plant seed, the soil where plants to be protected grow, or the foliage of plants to be protected. Certain compounds of this invention which are gases at temperatures used for the particular application can be applied to the plant in a sealed area by release of the compound as a gas. The compounds of this invention can also be applied as fungicidal sprays by methods commonly employed, such as conventional high-volume hydraulic sprays, low-volume sprays, air-blast spray, aerial sprays and dusts. The dilution and rate of application will depend upon the type of equipment employed, the method of application, plants to be treated and diseases to be controlled. Generally, the compounds of this invention will be applied in amount of from about 0.005 kilogram to about 50 kilograms per hectare and preferably from about 0.025 to about 25 kilograms per hectare of the active ingredient. As a seed protectant, the amount of toxicant coated on the seed is usually at a dosage rate of from about 0.05 to about 20, preferably from about 0.05 to about 4, and more preferably from about 0.1 to about 1 grams per hundred kilograms of seed. As a soil fungicide the chemical can be incorporated in the soil or applied to the surface usually at a rate of from about 0.02 to about 20, preferably from about 0.05 to about 10, and more preferably from about 0.1 to about 5 kilograms per hectare. As a foliar fungicide, the toxicant is usually applied to growing plants at a rate of from about 0.01 to about 10, preferably from about 0.02 to 5, and more preferably from about 0.25 to about 1 kilograms per hectare. Inasmuch as the compounds of this invention display fungicidal activity, these compounds can be combined with other known fungicides to provide broad spectrum activity. Suitable fungicides include, but are not limited to, those compounds listed in U.S. Pat. No. 5,252,594 (see in particular columns 14 and 15). Other known fungicides which can be combined with the compounds of this invention are dimethomorph, cymoxanil, thifluzamide, furalaxyl, ofurace, benalaxyl, oxadixyl, propamocarb, cyprofuram, fenpiclonil, fludioxonil, pyrimethanil, cyprodinil, triticonazole, fluquinconazole, metconazole, spiroxamine, carpropamid, azoxystrobin, kresoxim-methyl, metominostrobin and trifloxystrobin. The compounds of this invention can be advantageously employed as fungicides on cereals including wheat, barley and rye, on rice, peanuts, beans and grapes, on turf, on fruit, nuts and vegetables, and for golf course applications. The compounds can be employed in both pre and post harvest applications. Causal agents of diseases against which the compounds of the invention are useful include Botrytis spp., Penicillium spp., Cladosporium spp., Phialophora spp., Phytophthora spp., Pezicula spp., Colletotrichum spp., Alternaria spp., Stemphylium spp., Phomopsis spp., Glomerella spp., Mucor spp., Monilinia spp., Rhizopus spp., Mycosphaerella spp., Dothiorella spp., Phoma spp., Sclerotinia spp., Typhula spp., Fusarium spp., Lasiodiplodia spp., Thielaviopsis spp., Saccharomyces spp., Verticillium spp., Nigrospora spp., Aspergillus spp., Geotrichum spp., Pythium spp., Helminthosporium spp., Venturia spp., Septoria spp., Pyricularia spp., and fungi which cause powdery mildew and rust diseases.