First Report of Circular Leaf Spot of Persimmon Caused by Mycosphaerella nawae in Spain.

Abstract

Production of persimmon (Diospyros kaki L. f.) has increased significantly during the last decade in Spain as a profitable alternative for fruit growers. In August 2008, after a mild and rainy spring, symptoms of a new disease were observed in commercial persimmon fields located in Valencia Province (eastern-central Spain). Symptoms included circular necrotic spots on the leaves and defoliation. Early fruit maturation and premature abscission were associated with early symptom development in the trees. A fungus was consistently isolated from the margins of leaf lesions. All isolates obtained were hyphal-tipped twice and transferred to potato dextrose agar (PDA). The cultures grew slowly and reached a diameter of 21 to 29 (mean 26) mm within 4 weeks on PDA at 25°C in the dark. Mycelium was initially dark green and ultimately became dark gray to black. Several media and incubation conditions were tested to induce sporulation, but conidia formation was not observed. In April 2009, mature spherical pseudothecia were observed in lesions on fallen leaves that had remained in affected fields during the winter. Ascospores were uniseptate and mostly spindle shaped, 10 to 11.5 (mean 10.3) μm long, and 3 to 3.9 (mean 3.4) μm wide. Fungal colonies obtained from the ascospores were identical to those isolated from the leaf lesions. Morphological characters observed matched those described for the pathogen Mycosphaerella nawae Hiura & Ikata (1). In Korea, the circular leaf spot of persimmon caused by M. nawae was considered an economically important disease in the 1990s, especially in the southern regions (2). Sequences of the internal transcribed spacer (ITS) region of the rDNA were obtained for isolates MY2 and MY3 and deposited in GenBank (Accession Nos. GQ465767 and GQ465768). These sequences were identical to each other and to the sequence obtained from a Korean isolate of M. nawae. Symptoms of the disease were reproduced after inoculation of 2-year-old persimmon trees growing in individual pots. A ground mycelial suspension (5 × 105 CFU ml-1) of strain MY2 was sprayed onto 20 potted trees (200 ml per individual tree) in late May of 2009. Ten trees were sprayed with sterile distilled water as a control. Trees were incubated at 20°C in a growth chamber with a 12-h photoperiod and covered with a semitransparent plastic hood for the first 10 days after inoculation, after which the plastic was punctured for ventilation and trees were incubated at 22°C. The first symptoms (small circular spots on the leaves) appeared on inoculated trees 15 days after inoculation. One month after inoculation, all inoculated trees showed circular leaf spots and severe defoliation, whereas noninoculated trees remained healthy. M. nawae was successfully reisolated from the lesions. To our knowledge, this is the first report of M. nawae causing circular leaf spot of persimmon in Spain.