A Leaf Spot Caused by Phoma novae-verbascicola on Black Mullein (Verbascum nigrum L.) in Italy.

Verbascum nigrum L., common name black mullein, family Scrophulariaceae, is a rustic perennial plant belonging to the native flora in Italy. The plant, which produces bright yellow flowers densely grouped on the tall stem, is used in low-maintenance gardens. During fall 2012, plants grown in mixed planting borders in a garden located in Biella Province (northern Italy) showed extensive foliar disease. Approximately 100 plants were affected by the disease. Early symptoms were small, light brown, necrotic spots on leaves, later reaching 10 mm diameter, with an irregular shape, showing a chlorotic halo. Necrotic areas often coalesced surrounded by yellowing. In some cases, the internal part of the necrotic areas dried with the appearance of holes. The disease progressed from the base to the apex of plants. In some cases, most of leaves turned completely necrotic and plants were severely damaged. Symptomatic tissues were immersed in a solution containing 1% sodium hypochlorite for 2 to 3 s and rinsed with sterile distilled water. Small fragments were excised from the margin of lesions and plated on potato dextrose agar (PDA) medium. Petri dishes were incubated at temperatures ranging between 20 and 25°C under alternating daylight and darkness (12 h light, 12 h dark). A single fungus was consistently isolated and subcultured on malt extract agar (MEA). On MEA, colonies were felty, white cream, and produced dark globose or subglobose pycnidia measuring 68 to 185 × 62 to 177 (average 122 × 113) μm, containing hyaline (light grey in mass), ellipsoid, non-septate conidia measuring 3.1 to 5.7 × 1.5 to 2.7 (average 4.0 × 2.0) μm after 15 days. The internal transcribed spacer (ITS) and D1/D2 regions of rDNA were amplified using the primers ITS1/ITS4 and NL1/NL4, respectively, and then sequenced (GenBank Accession Nos. KC411473 and KF041823). BLAST analysis of both fragments showed 99% homology with the sequences GU237753 and JQ768403 of Phoma novae-verbascicola Aveskamp, Gruyter & Verkley (Basionym: Phyllosticta verbascicola Ellis & Kellerm.). Morphological characteristics of the fungus also were consistent with the descriptions of P. poolensis var. verbascicola (Ellis & Kellerm.) Aa & Boerema (2) (Syn.: P. novae-verbascicola). Pathogenicity tests were performed by spraying a conidial suspension (4 × 10⁴ CFU/ml) obtained from 15-day-old PDA cultures of the fungus onto leaves of three healthy 3-month-old V. nigrum. Three plants inoculated with sterile water served as controls. Plants were maintained in a growth chamber for 5 days at 25 ± 1°C under 70 to 90% relative humidity. The first foliar lesions developed on leaves 2 days after inoculation and after 5 days, 80% of leaves were severely infected. Control plants remained healthy. The organism reisolated on PDA from leaf lesions was identical in morphology to the isolate used for inoculation. The pathogenicity test was carried out twice. Phoma spp. has been reported on Verbascum spp. P. novae-verbascicola has been very recently described (1). To our knowledge, this is the first report of the presence of P. novae-verbascicola on V. nigrum in Italy. At present, the economic importance of this disease is limited, but may become a more significant problem if the cultivation of this species increases. References: (1) M. M. Aveskamp et al. Studies in Mycology, 65: 1, 2010. (2) J. de Gruyter et al. Persoonia 15 (3): 369, 1993.