Material And Methods

2.1. Pathogen Production

Isolates of Colletotrichum acutatum, C. fragariae and C. gloeosporioides, Phomopsis viticola and P. obscurans, Botrytis cinerea and Fusarium oxysporum are maintained on silica gel at 4-6 °C. The three Colletotrichum species and P. obscurans strain were isolated from strawberry (Fragaria x ananassa Duchesne). Phomopsis viticola and B. cinerea were isolated from commercial grape (Vitis vinifera L.) and F. oxysporum from orchid (Cynoches sp.). Fungal cultures were grown on potato-dextrose agar (PDA, Difco, Detroit, MI) in 9-cm petri dishes and incubated in a growth chamber at 24 ± 2 °C under cool-white fluorescent lights (55 ± 5 |imols/m2/sec) with a 12h pho-toperiod.

Conidia were harvested from 7-10 day-old cultures by flooding plates with 5 mL of sterile distilled water and softly brushing the colonies with an L-shaped glass rod. Aqueous conidial suspensions are filtered through sterile Miracloth (Calbiochem-Novabiochem Corp., La Jolla CA) to remove hyphae. Conidial concentrations were determined photometrically (Espinel-Ingrof and Kekering, 1991; Wedge and Kuhajek, 1988) from a standard curve based on the absorbance at 625 nm, and suspensions are then adjusted with sterile distilled water to a concentration of 1.0x106 conidia/mL.

Standard conidial concentrations are determined from a standard curve for each fungal species. Standard turbidity curves were periodically validated using both a Bechman/Coulter Z1 (Fullerton, CA) particle counter and hemocytometer counts. Conidial and mycelial growth are evaluated using a Packard Spectra Count (PerkinElmer Life and Analytical Sciences, Inc., Boston, MA). Conidial growth and germ tube development were evaluated using an Olympus IX 70 (Olympus Industrial America, Inc., Melville, New York) inverted microscope and recorded with a Olympus DP12 digital camera as appropriate for compounds that affect spore germination and early germ tube development.

2.2. Direct Bioautography

A number of bioautography techniques were used as primary screening systems to detect antifungal compounds. Matrix, one-dimensional, and two-dimensional bioau-tography protocols on silica gel thin layer chromatography (TLC) plates and Colletotrichum spp. as the test organisms were used to identify the antifungal activity according to published methods (Homan and Fuchs, 1970; Moore, 1996; Wedge and Nagle, 2000). Matrix bioautography is used to screen large numbers of crude extract at 20mg/mL. One-dimensional TLC (1D-TLC) and two-dimensional TLC (2D-TLC) are subsequently used to separate and identify the number of antifungal agents in extract. Modification of these procedures can be used to visually evaluate natural chemical defenses in disease resistant and susceptible plant cultivars (Vincent et al. 1999).

A 2D-TLC direct bioautography method was used to evaluate active crude or partially purified extracts. This protocol utilizes two sequential TLC runs in which the TLC plates are developed once with a polar solvent, turned 90o, and then developed a second time with a non-polar solvent system (Wedge and Nagle, 2000). The method takes advantage of the resolving power of 2D-TLC to separate chemically diverse mixtures found in crude extracts. Two-dimensional TLC bioautography is well suited for resolving extracts containing lipophilic natural products that are difficult to separate by single elution TLC.

Each plate was subsequently sprayed with a spore suspension (105 spores/mL) of the test fungus and incubated in a moisture chamber for 3 days at 26 °C with a 12h photoperiod. Clear zones of fungal growth inhibition on the TLC plate indicate the

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presence of antifungal constituents in each extract. Inhibition of fungal growth was evaluated 3-4 days after treatment. Antifungal metabolites can be readily located on the plates by visually observing clear zones where the active compounds inhibit fungal growth (Tellez, 2000,Vincent et al., 1999). The 2-D TLC method eliminates the need for the development of large numbers of plates in multiple solvent systems, reduces the amount of waste solvents for disposal, and substantially reduces the time required to identify active compounds.

2.3. 96-Well Microbiassay

The quick discovery and evaluation of natural product fungicides is heavily dependent upon miniaturized antifungal bioassay techniques. A reference method [M27-A from the National Committee for Clinical Laboratory Standards (NCCLS)] for broth-dilution antifungal susceptibility testing of yeast was adapted for evaluation of antifungal compounds against sporulating filamentous fungi (Wedge and Kuhajek, 1998).

This standardized 96-well microtiter plate assay was developed for the detection of natural product fungicidal agents, and can also be used to evaluate purified antifungal agents. In our study a 96-well microtiter assay was used to determine sensitivity of C. acutatum, C. fragariae, C. gloeosporioides, F. oxysporum, B. cinerea, P. obscurans, and P. viticola to the various antifungal agents in comparison with the commercial fungicides. Fungicides such as benomyl, azoxystrobin and captan with different modes of action were used as standards in these assays. Each fungal species was challenged in a dose-response format so that in the final test, compound concentrations of 0.3, 3.0, and 30.0 |M were achieved (in duplicate) in the different columns of the 96-well plates.

Fungal growth was evaluated by measuring absorbance of each well at 620 nm at 0, 24, 48, and 72 hr, with the exception of tests with P. obscurans and P. viticola, where the data are recorded for up to 120 hr. Treatments are repeated so that mean absorbance values and standard errors can be calculated and are used to evaluate fungal growth. Differences in spore germination and mycelial growth in each of the wells in the 96-well plate demonstrate sensitivity to particular concentrations of compound and can indicate fungistatic or fungicidal effects. The microtiter assay can also be used to compare the sensitivity of fungal plant pathogens to natural and synthetic compounds with industry standards (Wedge et al., 2001).

A novel application of the microbioassay was also developed for the discovery of compounds that inhibit Phytophthora spp. This protocol used the 96-well format for high-throughput capability and a standardized method for quantification of initial zoospore concentrations for maximum reproducibility. Zoospore suspensions were quantifiable between 0.7 and 1.5 x 105 zoospores/mL at an absorbance value of 620 nm. Subsequent growth of mycelia was monitored by measuring absorbance (620 nm) at 24-hour intervals for 96 hr. Full- and half-strength preparations of each of three media (V8 broth, Roswell Park Memorial Institute mycological broth, and mineral salts medium), and four zoospore concentrations (10, 100, 1000, and 10,000

zoospores/mL) were evaluated. Both full- and half-strength Roswell Park Memorial Institute mycological broth were identified as suitable synthetic media for growing P nicotianae, and 1000 zoospores/mL was established as the optimum initial concentration (Kuhajek et al., 2003).

2.4. Detached Leaf Assay for Fungicide Evaluations In Planta

Anthracnose susceptible 'Chandler' strawberry plants were grown in 10 x 10 cm plastic pots in a 1:1 (v/v) mixture of Jiffy-Mix (JPA, West Chicago, IL), and pasteurized sand in the greenhouse for a minimum of six weeks before inoculation. The plants were grown under standard conditions of a 16-hr day length and 24 oC temperature. Growth parameters are varied as needed to accommodate the needs of particular studies.

Whole leaves (petiole and leaflets) were cut from plants no more than four hr before treatment or inoculation. Only the second or third youngest leaf on a plant was used for the fungicide assay, and only leaves with no visible signs of injury or symptoms of disease were collected for testing. Immediately after collection, the leaves were placed in a tray lined with moist paper towels and the tray is closed to retained near 100% RH and maintained at a cool temperature (~ 12 °C). To test for protective fungicide activity, treatment compounds were applied to the upper surface of each of the three leaflets on a leaf using a chromatography sprayer. After treatment, the base of each leaf stem was inserted into sterile distilled water in a 100 x 10 mm tissue culture tube. Each upper surface of each treated leaflet was inoculated with conidia from the test fungal isolate within 24 hr of treatment. Inoculated leaves were subsequently incubated in a dew chamber for 48 hour at 100% RH, 30 oC and then maintained at 25 oC in a moist chamber at 100% RH for 10 days. Sterile distilled water was added to each tube as needed to maintain the surface of the water above the base of the petiole. If a compound was to be tested for curative activity, the leaflets were inoculated 24 hr before the fungicidal compound is applied.

Experimental compounds were evaluated in a dose-response format. Azoxystrobin or other fungicides that have both protective and curative activity were used as a standard, and a solvent control was used in every study. The number of disease lesions per leaf was used to determine the ability of the test compounds to prevent infections. The size of the lesions was used to determine the curative activity of compounds. Each fungicide concentration is replicated four times and the experiment is repeated at least once. This bioassay does not differentiate between direct effects on the fungus and indirect effects through induction of plant defenses. However, if a compound is much more active in this in vivo assay than than the in vitro microtiter assay, 'induction' activity is indicated.

2.5. 2002-03 Experimental Field Studies at Hammond, Louisiana

Strawberry plots were established and maintained following standard horticulture practices used by commercial strawberry farmers in Lousisana. The following fungi-

cides were applied to strawberries: fenhexamid (Elevate®, Arvesta, San Francisco, CA) at 1.6 kg/H, fenhexamid + captan (CaptEvate®, Arvesta, San Francisco, CA) at a low rate (3.9 kg/H) and a high rate (5.82 kg/H), azoxystrobin (Quadris®, Syngenta Crop Protection, Greensboro, NC) at 0.56 kg/H, cyprodinil + fludioxonil (Switch®, Syngenta Crop Protection, Greensboro, NC) at 0.8 kg/H, captan (Micro Flo Company, Memphis, TN) at 3.4 kg/H, mylclobutanil (Nova®, Dow AgroSciences, Indianapolis, IN) at 0.28 kg/H, pyrimethanil (Scala®, Bayer CropScience, Research Triangle Park, NC) at 1.9 kg/H, fenhexamid at 1.68 kg/H + captan at 3.4 kg/H, pyraclostrobin (Cabrio®, BASF, Research Triangle Park, NC) at 0.20 kg/H, pyraclostrobin + boscalid (Pristine, BASF, Research Triangle Park, NC) at 0.62 kg/ H, boscalid (Emerald®, BASF, Research Triangle Park, NC) at 0..59 kg/H, and the experimental fungicide, CAY-1 at 0.37 and 0.74 kg/H. Fungicide treatments were applied every 7-10 days (or as soon after rainfall as possible) starting at or near full-bloom stage and continuing until 1 week before harvesting ceased.

Berries were harvested twice a week throughout the entire season beginning on January 30 and continuing until April 24 in 2003. Total fruit count and weights were obtained for both marketable and cull fruits, along with average weight per berry and percentages of marketable, physical cull, and diseased cull fruit per acre. Fruit were separated into marketable and cull classes, with the culls being further divided into physical (deformed or small fruit) culls and diseased culls. Diseases were identified and counted on three occasions including berries with gray mold (B. cinerea), an-thracnose (C. acutatum), leather rot (Phytophthora cactorum), stem end rot (Gnomonia comari), and other rots that occurred during the picking season. Plants were rated on April 24, 2003 for incidence of foliar disease, number of dead plants due to anthra-cnose, crown rot and plant vigor. Diseases were rated on a scale of 0-5; 0 plants showed no leaf symptoms and 5 plants were completely defoliated. Plant vigor was rated on a scale of 0-5; 0 were dead plants and 5 were extremely vigorous plants.

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