First Report of Colletotrichum higginsianum Causing Anthracnose of Arugula (Eruca sativa) in Florida.

Arugula (Eruca sativa) is grown in Florida and is an important component in packaged salad products. During spring 2013, leaf lesions on arugula caused significant economic losses in Miami-Dade County, Florida. Symptoms initially appeared as small water-soaked lesions that later became circular, sunken, and white in the center with a dark brown to black halo, up to 4 mm in diameter. Acervuli were found under a dissecting microscope on infected leaf lesions with black spines or setae. Occasionally, small, circular, often longitudinal dark brown spots appeared on leaf branches. Leaf tissues (5 × 5 mm) from lesion margins were surface sterilized in 0.9% sodium hypochlorite for 10 min, rinsed with sterile distilled water, and plated on potato dextrose agar (PDA). PDA plates were incubated at 21°C under 24-h fluorescent lights for 4 to 6 days. The fungus initially produced gray mycelium followed by orange conidial mass. Hyphae of the fungus were septate and hyaline. After 5 to 7 days, the fungus produced acervuli with dark brown to black setae (75 to 130 μm long) (n = 20). Conidia were found in the colonies, which were single celled, oblong, hyaline, and 12 to 25 × 4 to 6 μm (n = 20). The cultural and morphological characteristics of the conidia were similar to those for Colletotrichum higginsianum Sacc (1). To further confirm the species of the isolates, the sequence of the ITS region of rDNA, chitin synthase 1 (CHS1), and actin (ACT) was amplified from isolates 05131 and 05132 using primer pairs ITS 1 and ITS 4 (4), CHS-79F and CHS-354R, and ACT-512F and ACT-783R (3), respectively. The sequenced data of each locus were deposited in GenBank with accessions KF550281.1, KF550282.1, KJ159904, KJ159905, KJ159906, and KJ159907. The resulting sequence of ITS showed 100% identity with sequences of C. higginsianum in JQ005760.1, and sequence of ACT gene showed 100% identity with C. higginsianum in JQ005823.1. The sequence of ACT gene and ITS region had ≤99% identity with other closely related Colletotrichum spp. CHS1 gene had 100% identity with JQ005781.1 belonging to C. higginsianum, and one accession JQ005783.1 belonging to C. fuscum. However, ACT gene and ITS region does not share 100% identity with C. fuscum and therefore, sequence data from three loci proves that isolated pathogen is C. higginsianum. All the above mentioned accessions that shared 100% identity with sequences of isolates used in our study have been previously used to represent the species in the C. destructivum clade in a systematics study (2). To confirm its pathogenicity, a suspension of isolate 05132 at 5 × 10 conidia/ml was sprayed on leaves of five arugula plants until runoff. The other five arugula plants sprayed with water served as non-inoculated controls. Both inoculated and non-inoculated plants were separately covered with a plastic bag to maintain high humidity for 24 h at 27 ± 5°C under natural day/night conditions in the greenhouse. Symptoms first appeared 3 to 4 days after inoculation as small water-soaked lesions, which became sunken with dark brown to black margins. Small circular and longitudinal dark brown spots were also seen on leaf branches as seen initially on naturally infected arugula. No symptoms developed on non-inoculated control plants. C. higginsianum was re-isolated from the lesions with the same morphological characteristics as described above, fulfilling Koch's postulates. To our knowledge, this is the first report of C. higginsianum causing anthracnose of arugula in Florida. This pathogen may potentially affect the salad industry in the United States. References: (1) A. J. Caesar et al. Plant Dis. 94:1166, 2010. (2) P. F. Cannon et al. Stud. Mycol. 73:181, 2012. (3) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.