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First Report of Colletotrichum acutatum sensu lato Causing Leaf Curling and Petiole Anthracnose on Celery (Apium graveolens) in Michigan

In September 2010, celery plants with leaf cupping and petiole twisting were observed in commercial production fields located in Barry, Kent, Newago, and Van Buren Counties in Michigan. Long, elliptical lesions were observed on petioles but signs (mycelia, conidia, or acervuli) were not readily observed. Celery petioles were incubated in humid chambers (acrylic boxes with wet paper towels). After 24 h, conidia corresponding to the genus Colletotrichum were observed. Isolations were performed by excising pieces of celery tissue from the lesion margin and placing them on potato dextrose agar (PDA) amended with 30 ppm of rifampicin and 100 ppm of ampicillin. Plates were incubated at 21 ± 2°C under fluorescent light for 5 days. Fungal colony morphology was gray with salmon-colored masses of spores when viewed from above, and carmine when viewed from below. Isolates were single-spored and placed on 30% glycerol in –20°C, and cryoconservation media (20% glycerol, 0.04% yeast extract, 0.1% malt extract, 0.04% glucose, 0.02% K₂HPO₄) at –80°C. Conidia were 8.5 to 12.0 × 2.8 to 4.0 μm and straight fusiform in shape. Three isolates were confirmed as C. acutatum sensu lato based on sequences of the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA and the 1-kb intron of the glutamine synthase gene (3), both with 100% similarity with Glomerella acutata sequences. Sequences were submitted to GenBank (Accession Nos. JQ951599 and JQ951600 for ITS and GS, respectively). Additionally, C. acutatum specific primer CaIntg was used in combination with the primer ITS4 on 54 isolates from symptomatic celery plants, obtaining the expected 490-pb fragment (1). Koch's postulates were completed by inoculating 4-week-old celery seedlings of cultivars Sabroso, Green Bay, and Dutchess using three plants per cultivar. Prior to inoculation, seedlings were incubated for 16 h in high relative humidity (≥95%) by enclosing the plants in humid chambers. Seven-day-old C. acutatum s. l. colonies were used to prepare the inoculum. Seedlings were spray-inoculated with a C. acutatum s. l. conidial suspension of 1 × 10⁶ conidia/ml in double-distilled water plus Tween 0.01%. Two control seedlings per cultivar were sprayed with sterile, double-distilled water plus 0.01% Tween. Plants were enclosed in bags for 96 h post inoculation and incubated in a greenhouse at 27°C by day/20°C by night with a 16-h photoperiod. Leaf curling was observed on all inoculated plants of the three cultivars 4 days after inoculation (DAI). Petiole lesions were observed 14 to 21 DAI. Conidia were observed in lesions after incubation in high humidity at 21 ± 2°C for 24 to 72 h. Symptomatic tissue was excised and cultured onto PDA and resulted in C. acutatum colonies. Control plants remained symptomless. C. acutatum (4) and C. orbiculare (2) were reported to cause celery leaf curl in Australia in 1966 (2,4). To our knowledge, this is the first report of C. acutatum s. l. infecting celery in Michigan.

References: (1) A. E. Brown et al. Phytopathology 86:523, 1996. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., USDA-ARS. Retrieved from http://nt.ars-grin.gov/fungaldatabases/, 10 September 2010. (3) J. C. Guerber et al. Mycologia 95:872, 2003. (4) D. G. Wright and J. B. Heaton. Austral. Plant Pathol. 20:155, 1991.