In Vitro and In Vivo Techniques For Screening New Natural Product-Based Fungicides For Control of Strawberry Anthracnose
Seven plant pathogenic fungi (Botrytis cinerea, Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Fusarium oxysporum, Phomopsis obscurans, and P. viticola ) valuable in screening fungicide efficacy were tested. Optimal and reproducible conditions for germination of these selected fungi were established by incorporating Roswell Park Memorial Institute 1640 (RPMI) as a medium of known composition and washing conidia to remove innate germination inhibitors. This step reduced average fungal germination times between 3.5 h and 21.2 h. The natural product-based fungicide, sampangine, seven sampangine analogs (4-bromo-sampangine, 4-methoxysampangine, benzo[4,5]sampangine, liriodenine Mel AMC-XIII-103, onychine, cryptolepine, and liriodenine CDH-II-37), plus seven conventional fungicides (benomyl, captan, cyprodinil, fenbuconazole, fenhexamid, iprodione, and kresoxim-methyl) were tested in vitro for their ability to inhibit germination and growth of the targeted fungal species. Microtiter assays demonstrated that sampangine was more efficacious than any of its analogs and than most of the commercial fungicides. In addition, I documented through microbioassays morphological anomalies in germ tube development caused by 4-bromosampangine. Germ tubes of B. cinerea splayed and branched and C. fragariae produced germ tubes that branched more frequently than the usual. These anomalies indicate a physical mode of action where appresorial formation and hyphal growth, but not conidial germination, are impaired, suggesting the possibility that 4-bromosampangine may protect hosts after fungi have germinated. The natural product-based fungicides, sampangine and CAY-1, and the commercial fungicide, azoxystrobin, were tested using a detached leaf assay on strawberry ( Fragaria x ananassa cv. Chandler). All three fungicides reduced anthracnose lesions on the leaves when applied to the host prior to inoculation with the fungal pathogen, but not after inoculation. In planta screening procedures exhibited a consistent 10-fold reduction in the number of conida landing on the plant surface. In vitro screening procedures demonstrated that conidia adhered to the inner surfaces of spray delivery equipment in high numbers. Practical implications of these findings include incorrectly classifying a fungicide as efficacious or a cultivar as resistant when in reality the amount of inoculum was insufficient to elicit disease.