A Rapid Microbioassay for Discovery of Novel Fungicides for Phytophthora spp.

Abstract

A microbioassay was developed for the discovery of compounds that inhibit Phytophthora spp. This assay uses a 96-well format for high-throughput capability and a standardized method for quantitation of initial zoospore concentrations for maximum reproducibility. Zoospore suspensions were quantifiable between 0.7 and 1.5 x 10(5) zoospores per ml using percent transmittance (620 nm). Subsequent growth of mycelia was monitored by measuring optical density (620 nm) at 24-h intervals for 96 h. Full- and half-strength preparations of each of three media (V8 broth, Roswell Park Memorial Institute mycological broth [RPMI], and mineral salts medium) and four zoospore concentrations (10, 100, 1,000, and 10,000 zoospores per ml) were evaluated. Both full- and half-strength RPMI were identified as suitable synthetic media for growing P. nicotianae, and 1,000 zoospores per ml was established as the optimum initial concentration. The assay was used to determine effective concentration values for 50% growth reduction (EC(50)) for seven commercial antifungal compounds (azoxystrobin, fosetyl-aluminum, etridiazole, metalaxyl, pentachloronitrobenzene, pimaricin, and propamocarb). These EC(50) values were compared with those obtained by measuring linear growth of mycelia on fungicide-amended medium. The microbioassay proved to be a rapid, reproducible, and efficient method for testing the efficacy of compounds that inhibit spore germination in P. nicotianae and should be effective for other species of Phytophthora as well. The assay requires relatively small amounts of a test compound and is suitable for the evaluation of natural product samples.

Effects of systemic and contact fungicides on life stages and symptom expression of Phytophthora ramorum in vitro and in planta

“…Effects of 11 fungicides (Table 2) on zoospore germination were evaluated using methods modified from Kuhajek et al (2003). Sporangia production was initiated from 14 day old mycelia grown on 15% V8A plates and incubated at 20 C with 24 h continuous light.…”

Antagonistic interaction between Trichoderma asperellum and Phytophthora capsici in vitro

“…In addition, the extensive use of fungicides has resulted in the development of fungicide resistance in the P. capsici in pepper and cucurbits (Parra and Ristaino, 1998;Pennisi et al, 1998;Lamour and Hausbeck, 2000;Qi et al, 2012). Concern has been raised about the major environmental and toxicological hazards associated with the use of fungicides and thus alternative environmentally friendly methods are needed to control the disease (Kuhajek et al, 2003). Biocontrol alternatives have been found in the use of fungi antagonists, such as Trichoderma species.…”

The disease reactions of heirloom bell pepper “California Wonder” to <i>Phytophthora capsici</i>

“…A mixture of zoospores from these isolates was used to inoculate the test plants. The zoospores were produced aseptically by transferring 10 agar plugs from the advancing portion of 5-day-old cultures (25˚C, under dark condition) of P. capsici in 5% (v/v) clarified V8 juice agar [7] to each 100 × 15 mm Petri dish and 10 ml of clarified V8 juice were added thereafter. After 24 h of incubation at 25˚C under dark condition, the V8 juice in each plate was replaced with 10 ml sterile mineral salt solution (MSS) [7] and incubated at 20˚C, 30 cm under two fluorescent lights (cool white, 20 W, 35 µmol·m -2 ·s -1 ) for 24 h. The MSS from each plate was then replaced with the same volume of fresh MSS and allowed to incubate for three more days.…”