The First Report of Calonectria Pteridis Causing a Leaf Spot Disease on Serenoa repens in China.

Serenoa repens [(Bartr) J. K. Small] is an important medicinal plant with their extracts is one of the three most effective drugs to cure benign prostatic hyperplasia (BPH). Also it can be used as an ornamental plant for garden. In November 2010, a new leaf spot disease was found on S. repens in Danzhou, Hainan Province, China. Disease occurred very seriously, with the incidence close or up to 100%, even leading to plant drying and death. Initially, the leaves had circular water-soaked dots, and had an obvious yellow halo on the edge, then expanded into oval, circular, or irregular shaped spots. Eventually the spot was beige and gray in the center and dark brown and slightly concave on the edge. The pathogen was isolated following the method reported by Fang (3) and prepared for further characterization. On potato dextrose agar (PDA) medium, the pathogen formed round and red-brown colonies with neat edges of a sandy beige color. A white powdery substance was formed on the surface of the colony, and it produced reddish-brown pigment on the back. On carnation leaf agar (CLA), only large macroconidium was observed. Macroconidiophores containing a stipe bearing penicillate suites of fertile branches, terminating in a clavate vesicle (5.9-) 6.4 (-6.9) × (33.8-) 39.6 (-46.7) μm. Conidiogenous apparatus had primary branches aseptate or rarely 1-septate and were (21.8-) 28.7 (-38.6) μm long, secondary branches were aseptate and (18.8-) 29.9 (-39.9) μm long, and tertiary branches were aseptate and (14.2-) 17.4 (-19.9) μm long. Macroconidium and microconidium were observed on water agar (WA) at 30 days. Macroconidium was colorless, cylindrical, rounded at both ends, 1 to 3 hyaline septate, but mainly one, and (4.5-) 5.2 (-6.2) × (71.3-) 84.1 (-98.0) μm; microconidium was colorless, cylindrical, both ends obtuse, curved or straight, 1-septate, and (24.8-) 33.2 (-45.2) × (2.5-) 3.5 (-5.0) μm. It could produce microsclerotia on PDA, CLA, and WA media. Morphological characteristics of the specimen examined were similar to Calonectria pteridis. In the genus of Calonectria, only C. pteridis could produce bending microconidium on WA medium (2). To confirm the morphological identification, primer pair ITS1/ITS4 were used for amplification of the ITS region of rDNA. Its sequence (GenBank Accession No. KF994926) showed 99% identity with C. pteridis Crous, M.J. Wingf. & Alfenas. (GQ280617.1). In addition, the translation elongation factor 1-alpha gene sequence was amplified (KF994927) and it showed 100% identify with C. pteridis (FJ918564.1) (1). Thus, the pathogen was identified as C. pteridis. To confirm pathogenicity, conidial suspensions (10 conidia ml) of the pathogen were inoculated with healthy leaves of 10 plants by pinprick inoculation method. Control plants were inoculated with water. Plants were maintained at 28°C in a greenhouse with constant humidity (RH 90%) and a 12-h photoperiod of fluorescent light. Symptoms similar to the original ones appeared after 7 days, while the control plants remained healthy. The tests were repeated three times and the pathogen was re-isolated from the leaves of inoculated plants and confirmed to be C. pteridis by both morphology and molecular characterization. To our knowledge, this is the first report of leaf spot caused by C. pteridis on S. repens in China. References: (1) I. Carbone and L. M. Kohn. Mycologia 91:553, 1999. (2) P. W. Crous and M. J. Wingfield. Mycotaxon 51:341, 1994. (3) Z. D. Fang. Plant Disease Research Methods, 3rd edition. China Agriculture Press, Beijing, 1998.