First Report of Leaf Spot Caused by Cylindrocladium pauciramosum on Dwarf Willow Myrtle in Italy.

Abstract

Dwarf willow myrtle (Agonis flexuosa (Willd.) Sweet) cv. Nana, an evergreen ornamental shrub belonging to the Myrtaceae, is grown in Italy as an ornamental potted plant. In November 2008, a widespread new leaf spot disease was noticed on ~80% of 5,000 6-month-old potted plants. Plants were obtained from cuttings and produced by a commercial nursery in Catania Province. Symptomatic leaves showed minute, reddish brown spots that enlarged (3 to 5 mm in diameter) and then darkened, presenting a necrotic center defined by a dark purple halo. Leaf spots were surface disinfested with 0.8% NaOCl and plated on potato dextrose agar. Twenty isolates of the fungus that was consistently isolated from the spots were selected and cultured for 8 days at 25°C on carnation leaf agar (CLA). Macroconidiophores consisted of a stipe, a penicillate arrangement of fertile branches, and stipe extension terminating in an obpyriform to ellipsoidal vesicle (6 to 10 μm in diameter). Cylindrical conidia were rounded at both ends, straight, one-septate, and ranged from 44 to 60 × 4 to 5 μm. The fungus was tentatively identified as Cylindrocladium pauciramosum based on these morphological characteristics (2). All single-conidium isolates were mated with tester strains of Calonectria pauciramosa C. L. Schoch & Crous, telomorph of C. pauciramosum, on CLA and produced fertile perithecia (4). Perithecia were solitary or in groups, orange to red-brown, subglobose to ovoid, and ranged from 280 to 400 μm long × 180 to 290 μm in diameter. Further confirmation of species was obtained by amplification and sequencing of the intergenic spacer (IGS) region of rDNA, using M13 Forward (-20) and M13 Reverse primers. On the basis of the complete IGS sequence, two primer sets (218F/218R and 106F/106R) were designed and successfully used in a nested-PCR protocol for the detection of C. pauciramosum from tissues of infected plants (3). On the basis of the combination of morphological characters, mating type, and molecular data, the isolates were identified as C. pauciramosum C.L. Schoch & Crous. One representative isolate (DISTEF-Af1) was deposited at Centraalbureau voor Schimmelcultures open fungi collection (Fungal Biodiversity Centre, Utrecht, the Netherlands; CBS 124659). Pathogenicity tests were performed by adding sterile water to CLA cultures of C. pauciramosum from a single-conidium isolate (DISTEF-Af1) and spraying the resulting spore suspension (105 conidia per ml) on the leaf surface of 20 6-month-old A. flexuosa cv. Nana potted plants. The same number of plants served as noninoculated controls. Following inoculation, plants were kept in plastic bags in a growth chamber at 25 ± 1°C. All inoculated plants developed circular, brown leaf spots identical to those observed in the nursery 5 to 7 days after inoculation. Control plants remained symptomless. C. pauciramosum was always reisolated from the infected plants and identified as previously described. Leaf spotting in seedlings of A. flexuosa was previously associated with infections by C. scoparium in Australia (1). To our knowledge, this is the first record in the world of leaf spots caused by C. pauciramosum on A. flexuosa.