First Report of Cylindrocladium buxicola on Buxus sempervirens in Spain

Cylindrocladium buxicola Henricot, included in the EPPO alert list until November 2008, causes a dangerous foliar disease on Buxus spp. that has been recorded in several European countries and New Zealand (3,4). Buxus sempervirens L. (common boxwood) is one of the oldest ornamental garden plants in Europe. In September 2008, we received 10 2- to 3-year-old potted plants of B. sempervirens cv. Suffruticosa from a nursery in Galicia (northwest Spain) where ≈60% of the plants were affected and had finally defoliated. Diseased plants showed dark brown-to-black spots on the leaves and black streaks on the stems (3,4). To induce sporulation, diseased leaves and stem pieces were incubated in damp chambers at 22°C. A Cylindrocladium sp. was obtained. Four single conidial isolates were plated onto carnation leaf agar under near-UV light at 25°C for 7 days (2,3). Only conidiophores of the isolates growing on the surface of the carnation leaves were examined microscopically (1,3). Macroconidiophores were comprised of a stipe, a stipe extension, a terminal vesicle, and a penicillate arrangement of fertile branches (2). The stipe extension was septate, hyaline, and 90 to 165 × 2 to 4.5 μm (from the highest primary branch to the vesicle tip) (1) terminating in an ellipsoidal vesicle (6 to 11 μm in diameter) with a papillate apex. The widest part of the vesicle was above the middle. Primary branches were mainly aseptate or one septate (12 to 35 × 3 to 6 μm), secondary branches were aseptate (11 to 21 × 3 to 6 μm), and tertiary branches were rare. Each terminal branch produced two to five phialides (9 to 20 × 2.5 to 5 μm) that were reniform and aseptate. Conidia were cylindrical, straight, and one septate (56 to 75 × 4 to 6 μm). Chlamydospores were dark brown and aggregated to form microsclerotia. Cardinal temperatures of Cylindrocladium isolates on 2% malt extract agar ranged from 7 to 28°C (optimum 25°C). The 5' end of the β-tubulin gene was amplified using primers T1 and Bt2b (3), and PCR products were sequenced directly and deposited in GenBank (Accession No. FJ696535). Comparison of the sequence with others available in GenBank showed 100% homology with those previously identified as C. buxicola (Accession Nos. AY078123 and AY078118). Pathogenicity of one representative isolate was confirmed by inoculating stems and leaves of four 3- to 4-year-old plants of B. sempervirens cv. Suffruticosa. Leaves were inoculated by spraying a spore suspension of the fungus (1 × 10⁶ conidia per ml). For the stems, agar pieces of 1-week-old cultures grown on malt extract agar were placed and sealed with Parafilm. As a control, four plants were inoculated with agar malt plugs and sterile distilled water. Plants were incubated at 22°C and 95% humidity. Symptoms identical to ones previously described appeared 4 days after inoculation. C. buxicola was reisolated from inoculated plants but not from the controls. On the basis of morphological and physiological characteristics, pathogenicity, and the DNA sequencing of the β-tubulin gene, the isolates obtained from B. sempervirens were identified as C. buxicola (3). To our knowledge, this is the first report of C. buxicola on B. sempervirens in Spain. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) P. W. Crous and M. J. Wingfield. Mycotaxon 51:341, 1994. (3) B. Henricot and A. Culham. Mycologia 94:980, 2002. (4) B. Henricot et al. Plant Pathol. 49:805, 2000.