First Report of Cladosporium Leaf Spot of Spinach Caused by Cladosporium variabile in North Carolina.

Cladosporium leaf spot of spinach, caused by Cladosporium variabile, can result in significant economic losses in the United States (2). In March 2014, symptoms consistent with Cladosporium leaf spot (4) appeared on the spinach cultivar Tyee in a greenhouse located in Rowan County, NC. Of 1,080 spinach plants, 90 to 100% were infected. Symptoms consisted of small (1 to 3 mm in diameter), circular, tan lesions each outlined with a dark margin on the adaxial surface of the leaf. On severely infected foliage, lesions coalesced to produce relatively large necrotic regions. Profuse fungal sporulation was observed on the lesion surface with a dissecting microscope at 40× magnification. Using a dissecting microscope, conidia were collected with a sterile needle and transferred to petri plates containing potato dextrose agar. Plates were then incubated at 23 ± 2°C under continuous fluorescent light, and fungal growth was apparent after 24 h. Isolations from leaves of six infected plants produced slow-growing, dark green to brown fungal colonies that reached only 31 mm in diameter after 14 days, which is characteristic of C. variabile (4). Colonies contained dense masses of dematiaceous, septate, unbranched conidiophores with conidial chains, each containing up to five conidia. Conidia were ovate to elongate, with some being septate. The length of individual conidia ranged from 10 to 19 μm. Conidial septa were distinctly dark when observed at 100× magnification, which is a defining feature of C. variabile vs. the conidia of C. macrocarpum (4). The surface of the conidia appeared verrucose at 100× magnification, and conidia were each distinctly darkened toward the base. A single isolate obtained through single-spore transfer was used for DNA extraction, and the histone 3 (H3) gene sequence was amplified using the primers CYLH3F and CYLH3R (1). Sequence analysis of the amplified product using BLAST analysis indicated that the H3 sequences had 100% identity to that of a C. variabile isolate (GenBank Accession No. EF679710.1), and 99% identity to a C. macrocarpum isolate (EF679687.1). The H3 sequence from a representative isolate was deposited in GenBank (KJ769146). To our knowledge, this is the first report of Cladosporium leaf spot on spinach in North Carolina based on morphological evaluation and H3 sequencing results. C. variabile is a seedborne pathogen, so it is possible inoculum was introduced into the greenhouses in North Carolina on infected seed (3). Seeds can be treated with hot water or chlorine to reduce the risk of disease outbreaks caused by infected seed (2). Furthermore, Cladosporium leaf spot may be controlled with the use of fungicides (3). References: (1) P. Crous et al. Stud. Mycol. 50:415, 2004. (2) L. J. du Toit and P. Hernandez-Perez. Plant Dis. 89:1305, 2005. (3) L. J. du Toit et al. Fung. Nemat. Tests 59:V115, 2004. (4) Schubert et al. Stud. Mycol. 58:105, 2007.