Improved Microassays Used to Test Natural Product-Based and Conventional Fungicides on Plant Pathogenic Fungi
Originally published in: Plant Disease (2008) 92 ( 1 ): 106-112
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Interpretive Summary: New effective fungicides are needed to control fungal pathogens that have developed resistance to currently used fungicides or to replace fungicides that have lost their registration for use on certain crops. Natural products are one source of these new fungicides. Prospective new fungicides are screened in vitro to identify products which inhibit germination of fungal conidia or prevent normal growth of fungal hyphae. In this paper we established standard conditions that improved the in vitro micro titer assays used in the initial screening of natural products as potential fungicides. The simple step of washing fungal spores prior to inoculation greatly increased spore germination times and the synchrony of the cultures. Using light microscopy to observe fungal hyphae after exposure to natural products documented the physical mode of action of one of the products which inhibited germ tube elongation. Other products inhibited conidial germination. This information is useful to plant pathologists and chemists developing new fungicides.
Technical Abstract: Identification and standardization of several parameters to ensure reproducible conditions for fungal germination valuable in screening fungicides in vitro were established in this study. Washing spores prior to inoculation significantly reduced germination times, increased cultural synchrony, and improved reproducibility of microtiter assays. The natural product-based fungicide, sampangine and a sampangine analog, 4-bromosampangine, plus seven commercial fungicides (captan, kresoxim-methyl, fenhexamid, iprodione, benomyl, fenbuconazole, and cyprodinil) were screened in vitro (microtiter assay) for their ability to inhibit germination and growth of seven important plant pathogenic fungi (Botrytis cinerea, Colletotrichum fragariae, C. acutatum, C. gloeosporioides, Fusarium oxysporum, Phomopsis obscurans, and P. viticola). Germination and morphology bioassays corroborated the results of microtiter assays where sampangine was effective at arresting fungal development. We hypothesize that sampangine has the same physical mode of action, germination inhibition, as the commercial fungicides captan, kresoxim-methyl, and iprodione. 4-Bromosampangine caused morphological anomalies including excessive branching of germ tubes of C. fragariae and splaying and branching of germ tubes of B. cinerea. These observations suggest that this compound may protect the host after fungi have already established themselves in or on the host. Our hypothesized physical mode of action of 4-bromosampangine, inhibition of germ tube elongation, but not conidial germination, is comparable to that of the commercial fungicides fenhexamid, benomyl, and cyprodinil.