In 2009, beds of Buxus sempervirens and newly planted B. sempervirens‘Suffruticosa’ in a park in Opatija, Croatia, showed extensive leaf and twig blight. Leaves with dark roundish spots turned red-brown and grey, then dropped. Shoots and stems had dieback with narrow blackish streaks on the bark surface. Two widely occurring fungal pathogens of common box, Volutella buxi and Puccinia buxi, were sporulating abundantly on the diseased plants. However, the presence of dark spots on the leaves and blackish streaks on the bark suggested Cylindrocladium buxicola (Henricot & Culham, 2002). The symptoms we observed were similar to C. buxicola infections observed in other European countries (Brand, 2005; EPPO, 2005; AGES, 2009). Detached stems kept in a moist chamber at 20°C for 5 days developed fruiting structures consistent with C. buxicola: hyaline cylindrical conidia (50–58 × 4–6 μm), rounded at both ends, with a single septum, produced on stipes with conidiogenous cells arranged in penicils. Sterile hyphae widened towards their tips developed among the conidiophores. Cultures grown from transfer of conidia to carrot agar (Leslie, 2006) were used to sequence the rDNA ITS regions and deposited in GenBank (Accession No. HM749646). Our sequence was identical to published sequences of C. buxicola (e.g. AY078112 from type strain RHS-9934 (Henricot & Culham, 2002)).
Spore suspensions (ca. 3 × 107 spores mL−1) of Cylindrocladium buxicola were sprayed on 4-year old common box plants (B. sempervirens var. sempervirens). The plants developed leaf and dieback symptoms within 14 days and C. buxicola was confirmed sporulating on stems and leaves. Control plants sprayed with sterile water remained healthy. This is the first report of C. buxicola from Croatia. In 2005, B. sempervirens‘Suffruticosa’ plants from outside of Croatia were planted in Opatija and it is possible this is how C. buxicola was introduced.