First report of Colletotrichum chlorophyti infecting soybean seed in Arkansas, United States.

Colletotrichum chlorophyti was first reported in the United States in 2009 on soybean petioles (Glycine max [L.] Merr.) collected from Alabama, Illinois, and Mississippi (4). This species has not been reported to infect seed, unlike other Colletotrichum spp. (2). From the 2012 growing season, soybean seeds obtained from the National Agricultural Statistics Service representing 151 seed lots from growers' fields in 11 states were assayed by plating them on acidified potato dextrose agar (APDA). Before plating, seeds were surface disinfected by sequential immersion in 50% ethanol for 30 s, 20% commercial bleach for 1 min, two 1 min rinses in sterile distilled water, and kept at 25°C in the dark for 1 week. Infected seeds from one seed lot from Arkansas produced colonies similar to Colletotrichum spp. This seed lot was visually examined and divided into asymptomatic or discolored symptomatic seeds. Because of the limited number of seeds in the seed lot, 20 seeds that asymptomatic and 40 seeds that appeared symptomatic were assayed on APDA as previously described. Asymptomatic seeds did not produce any dark fungal colonies. Among the symptomatic seeds, five appeared to have flecked light gray seed coats with some larger grayish to black and irregular spots where cracks were sometimes formed, and they developed small black fungal masses or became entirely dark on the surface. Five fungal isolates were obtained from these infected seeds. On APDA, the isolates initially produced white to pink smooth-margined colonies, turned black with age, produced no aerial growth, and filled a 9 cm diameter petri dish within 10 days. DNA of one isolate was extracted for PCR and sequencing of the ITS region with ITS1 and ITS4 primers (3). From the BLAST analysis, the sequence was 100% identical to C. chlorophyti isolates, IMI 103806, and CBS 142.79 (Accession Nos. GU227894 and GU227895, respectively). To test for pathogenicity, the fungus was sub-cultured on APDA and eight APDA discs (4 mm diameter) were set into 50 ml potato dextrose broth inside a 250-ml flask and shook at a speed of 100 rpm at room temperature (24 ± 1°C) for 10 days. The mycelium was then weighed, fragmented with a blender, and resuspended in sterile distilled water to a final concentration of ~40 mg/ml. The mycelial suspension was sprayed on soybean seedlings of cv. Williams 82 (two plants/pot) at growth stage V1 to V2 until runoff. The inoculated plants were kept in a moist chamber (>90% relative humidity) for 48 h at 24 ± 1°C in the dark, and then transferred to normal plant growing conditions. At 5 days post-inoculation (dpi), the leaves showed typical symptoms caused by C. chlorophyti, including necrosis on the edge of young leaves and petioles, formation of irregular dark brown lesions, and leaves became scrolled (4). Setose acervuli, curved conidia with tapered ends (21.4 ± 1.1 × 3.8 ± 0.3 μm), and chlamydospores were found on the detached symptomatic leaves after 12 dpi. No perithecia formed. The morphology matched the description of C. chlorophyti (1,4). To our knowledge, this is the first report of C. chlorophyti in Arkansas and the first time that this species has been reported infecting seed of any plant. References: (1) S. Chandra and R. N. Tandon. Curr. Sci. 34:565, 1965. (2) G. L. Hartman et al. Page 13 in: Compendium of Soybean Diseases, APS Press, St. Paul, MN, 1999. (3) T. J. White et al. Page 315 in: PCR Protocols. A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (4) H.-C. Yang et al. Plant Dis. 96:1699, 2012.