First Report of Fusarium acuminatum Causing Damping-Off Disease on Aleppo Pine in Algeria.

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In autumn and spring of 2008 to 2009, a survey of Aleppo pine seedling diseases was carried out in three forest nurseries from the Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. Aleppo pine seedlings were potted from the soil. In all three nurseries, 1- to 2-month old seedlings showed symptoms of damping-off disease in pre- and post-emergence (collar rot) with a disease incidence of 64, 77, and 72%, respectively. Disinfected collar segments, about 5 mm in length, were plated on PDA and petri dishes incubated at 25°C. A Fusarium sp. was consistently isolated from tissues and all isolates were morphologically identified as Fusarium acuminatum Ellis & Everh. (teleomorph: Gibberella acuminata Wollenw.) according to Fusarium keys (2). Colony growth was 43 mm after 3 days on PDA; the aerial mycelium was white, developing a brownish tinge in the center on PDA; macroconidia were formed in orange sporodochia, broadly falcate, strongly septate, 3 to 5 septa, the apical cell with an incurved elongation, distinct foot shape, 3 to 4 × 20 to 50 μm; microconidia were usually absent for isolates other than F12SS1, reniform, septate, 5 to 6 × 6 to 10 μm, in monophialides; chlamydospores were formed in chains, 6 to 13 μm. For the molecular identification, ITS regions of Fusarium isolates were amplified with the primers ITS1 and ITS4, and products were directly sequenced in both strands using the same primers ITS 1 and ITS4. Sequences were compared to known sequences deposited in the NCBI non redundant database to confirm morphological identification. An NCBI BLAST search identified isolates F12SS1, F14SS3, F30SS3, and F25SR as F. acuminatum based on 100% similarity with corresponding sequences. GenBank Accession Nos. were JX114788, JX114785, JX114782, and JX114790, respectively. Pathogenicity tests were performed to fulfill Koch's postulates. Inocula were produced by adding a 5-mm diameter plug from a 7-day-old CMA petri dish culture to a previously sterilized 500-ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml SDW), shaken over 9 days, and mixed with sterile soil at 1:3 (v:v). The inocula were transferred to a 500-ml pot, and 10 Aleppo pine seeds were planted with three replicates. After 1 month, all tested isolates caused typical symptoms on seedlings and the proportion of infected seedlings per each isolate was 50, 53.33, 56.66, 60, and 63.33%, respectively. There are many reports of F. acuminatum associated to conifer seedlings in nurseries (1,3) and most of them are conflicting because in some reports this species is considered non-pathogenic or only a seed contaminant and others consider it as a pathogen. To our knowledge, F. acuminatum is a first report on the Aleppo pine in northwestern Algeria, northern Africa. It is also the first report of this fungal species affecting the Aleppo pine throughout the world, and on conifers in Africa and the Mediterranean region. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA., Bestville, Maryland, USA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , June 18, 2012. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa, USA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , June 18, 2012.