First Report of Cochliobolus sativus on Guzmania sp. in Belgium.

In 2001 and again in December 2005, an outbreak of leaf spots was observed on Guzmania sp. 'Gwendolyne' (Bromeliaceae) in a Belgian nursery. Typical disease symptoms were irregular spots with a grayish center and a narrow red-brown margin. Identification was based on morphological characteristics and molecular techniques. Isolations of diseased leaf tissues previously washed with sterile distilled water on potato dextrose agar (PDA) resulted in mycelial colonies after 7 to 8 days. Fungal mycelium grew at a linear rate of 30.4 mm per 24 h at 21°C in the dark. The pathogen produced aerial mycelium and sporulation was abundant. The color of the colonies on PDA was pale to dark brown and conidial characteristics similar to those of Cochliobolus sativus (anamorph Bipolaris sorokiniana) (1) were observed: brown ellipsoidal spores rounded at the top, 3 to 12 distoseptate, with average dimensions of 40 to 120 × 17 to 28 μm. The pathogen was also characterized with molecular tools. DNA was isolated from mycelium from a PDA plate. The ribosomal DNA region ITS1-5.8S-ITS2 was amplified and cloned. The ITS1 sequences (174 bp) of two independent clones were analyzed. The three highest similarity scores (E = 2e-⁷¹) obtained in BLAST were C. sativus (GenBank Accession Nos. AF158105 and AF071329) and B. sorokiniana strain BS11 (GenBank Accession No. AY372677). For these, pairwise alignments resulted in an identical score of 97.1% (169 identical bases, four indels, and one transversion). The new Genbank Accession No. of the ITS1 sequence is DQ 641269. To prove pathogenicity of the isolate, inoculations were done by spraying leaves of three young Guzmania sp. 'Gwendolyne' plants with a 20-ml spore suspension (10⁶ spores/ml). Three plants were sprayed with sterile distilled water as controls. The plants were kept for 48 h under a humid chamber and subsequently at room temperature (20 to 25°C) on the laboratory bench. Three to four days after inoculation, leaf spots were observed and C. sativus (anamorph B. sorokiniana) was reisolated, completing Koch's postulates successfully. On the basis of symptoms, morphological characteristics, and pathogenicity tests, the pathogen was identified as C. sativus (anamorph B. sorokiniana). To our knowledge, this is the first record of C. sativus (anamorph B. sorokiniana) on Guzmania sp. in Belgium. References: (1) A. Sivanesan and P. Holliday. Description of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, England, UK, 1981.