First Report of Leaf Spot Caused by Alternaria longipes on Atractylodes macrocephala in China.

Abstract

Atractylodes macrocephala Koidz. is a perennial herb that has been used in Chinese herbal medicine for centuries. During the summer of 2011, leaf spots were observed on leaves of A. macrocephala in Panan County of Zhejiang Province, China. Approximately 40% of the plants surveyed showed severe symptoms of leaf spot. At the initial stage of the infection, small, light brown spots appeared on the leaves that gradually became semicircular, oval, or irregular-shaped with dark brown or black centers surrounded by brown or light brown margins. The lesions enlarged and coalesced to form large areas of necrosis on leaves until entire leaves died. Eight fungal isolates were obtained from diseased A. macrocephala leaves by a tissue isolation method (1). Isolates were cultured on potato dextrose agar (PDA) plates at 25°C in darkness. Colonies on PDA were initially white and became grayish brown over time. Conidiophores were light brown with one or a few regular septa, mostly unbranched. Conidia were obclavate, dark brown, with three to eight transverse and zero to two longitudinal or oblique septa, and on average measured 35.1 (20 to 53) × 9.8 (5.8 to 13.3) μm (n = 20). The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS1F/ITS4 and sequenced (GenBank Accession No. JQ004404). The ITS sequence had 99% identity over 545 bp with Alternaria longipes (Ellis & Everh.) E. W. Mason (GenBank Accession No. AY278835), a fungal pathogen reported to cause leaf spot on Smilax china in China (3). To further identify the fungus, we chose the Alternaria allergen gene (Alt a 1 gene), which aids in identifying species of Alternaria (2). Amplification of the Alt a 1 gene was conducted using primers Alt-for/Alt-rev and sequenced (GenBank Accession No. JQ004405). Sequence comparisons showed there was 97% sequence identity with Alternaria longipes (GenBank Accession No. AY563304). Pathogenicity tests were performed on detached healthy A. macrocephala leaves. Nine leaves were inoculated by placing a PDA plug (0.5 cm2) of mycelia on upper surfaces of the leaves. Another nine leaves treated with sterile PDA plugs served as a control. Leaves were incubated in three petri dishes with a 12-h photoperiod at 25°C and 95% relative humidity. After 7 days, the symptoms described above were observed on all inoculated leaves, whereas no symptoms developed on control leaves. Reisolation of the fungus from symptomatic leaf tissues on PDA confirmed that the causal agent was Alternaria longipes. This pathogenicity test was performed three times. The genus Alternaria is ubiquitous and includes both plant pathogenic and saprophytic species that may cause leaf spot and blight on numerous plants (3,4), but to our knowledge, our research demonstrated this is the first report of leaf spot on A. macrocephalae caused by Alternaria longipes in China.