First Report of Seedling Damping-Off of Fennel Caused by <i>Alternaria petroselini</i> in the Netherlands

In April 2005, serious seedling damping-off was noted on fennel (Foeniculum vulgare Mill. cv. Rondo) in a transplant greenhouse facility in Maasdijk, the Netherlands. Symptoms appeared 3 to 4 weeks after sowing and included black, sunken lesions aboveground on the stem and belowground on the hypocotyls. Mortality of seedlings was 6 to 10% (10 to 15 seedlings per 150-plant tray). Following removal of diseased seedlings, further transplant mortality in the field was not evident. Samples of diseased tissue were collected, surface disinfested, and placed in petri dishes containing water agar. After 7 to 10 days of incubation at 25°C under fluorescent lights, an Alternaria sp. was growing from each sample. Single conidium cultures were obtained from representative colonies and cultured on potato dextrose agar (PDA) and potato carrot agar (PCA) for morphological examination. On PDA, colonies were dark olive brown, cottony, subsurface microsclerotia production abundant, and no production of pigments in the medium. On PCA, conidia were darkly pigmented, broadly ellipsoidal to subsphaerical, and produced singly. Mature conidia were 28 to 45 × 20 to 25 μm with two to four transepta and one to three longisepta. Characteristics were consistent with those of Alternaria petroselini (2,3). In subsequent freezer-blotter assays (ISTA blotter method; www.seedtest.org ) of seed lot used in the original planting, the same fungus was recovered at an infestation level of 30%, confirming that it was seedborne. To confirm pathogenicity on fennel, pathogenicity tests were conducted on a common fennel cultivar (Floro F1) in the greenhouse and on fennel stalks in the laboratory. Fennel seeds were soaked in a conidia suspension (10⁶/ml in sterile H₂O) for 10 min. Control seeds were soaked in sterile H₂O. Seeds were dried on paper, sown in soil plugs, and grown in the greenhouse at 16 to 20°C. After 4 weeks, black lesions were observed on the fennel stems and symptoms were similar to those observed on the original infected material. Control plants remained healthy. A fungus was reisolated from the lesions of symptomatic plants and was identical to the fungus isolated from the original infected material. For the fennel stalk assay, two surface-sterilized fennel stalks were sliced longitudinally and three 4-mm plugs from a 10-day-old culture of each isolate were placed along the fennel stalks. Sterile agar plugs were used as negative controls. After 7 to 10 days of incubation at 25°C in plastic boxes, test isolates grew extensively from agar plugs and resulted in extensive black necrosis of the fennel stalks. No necrosis resulted from control plugs. DNA was extracted from field isolates, and the nuclear internal transcribed spacer region was sequenced using protocol previously described (1). A representative sequence was deposited in GenBank (Accession No. EF636901). A BLAST search of the NCBI database revealed A. petroselini Accession No. AY154685 as the closest match (total score = 1,014, 100% coverage, 99% sequence identity). The next closest match was A. radicina Accession No. DQ394074 (total score 987, 100% coverage, 98% sequence identity). To our knowledge, this is the first report of A. petroselini causing disease of fennel and the fungus being seedborne on fennel seed. An isolate has been deposited at the Centraalbureau voor Schimmelcultures (Accession No. 118228). References: (1) B. M. Pryor and D. M. Bigelow. Mycologia 95:1141, 2003. (2) B. M. Pryor and R. L. Gilbertson. Mycologia 94:49, 2002. (3) E. G. Simmons. Mycotaxon 55:55, 1995.