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

The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In 2008 and 2009, a survey of Aleppo pine seedling diseases was performed in three forest nurseries from the Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. One- to two-month-old Aleppo pine seedlings showed symptoms of damping-off in pre- and post-emergence (typical seedling collar rot). The problem was widespread with a disease incidence of 64 to 77% and an annual impact of US$50,000. Disinfested root and root collar segments (from four composite samples per location), approximately 5 mm in length, were cultured on PDA and incubated at 25°C and day/night light. Two (from 21) isolates were identified morphologically (2) as the anamorph Fusarium chlamydosporum Wollenw. & Reinking and isolated from collar rots of Relizane forest nursery seedlings. Colony development on PDA media was fast; 32 mm diameter colonies developed after 3 days. Colonies were white. Mycelia were floccose, fairly dense, off-white, and turned a lilac color in older portions of the colony. Macroconidia were thick-walled and moderately curved with unequal dorsiventral curvature (the lower wall is almost straight), short, curved and pointed apical cell, usually notched, but occasionally foot shaped basal cell, 3- to 5-septate, and 2 × 8 to 21 μm. Microconidia were abundant, 0-septate, and 2 × 6 to 9 μm. Chlamydospores were abundant, formed rapidly in single chains or clusters, and 8 to 15 μm diameter. To confirm the identity of this fungus, the internal transcribed spacer of F12RR and F4SR isolates of F. chlamydosporum were amplified and sequenced using ITS1 and ITS4 primers (4). Sequences were deposited in GenBank under accessions JX114795 and JX114789, respectively. Those sequences bore 99% similarity with reference sequence AY213655 (2) and 100% with HQ671187, also found 99 to 100% similarity with F. equiseti (Corda) Sacc. but with different conidia. Pathogenicity tests were performed to fulfill Koch's postulates. Inoculum was produced by adding a 5 mm diam. 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). Infested soil was then transferred to 500 ml pots, and 10 seeds were planted. A completely randomized design was used with three replicates per isolate and three control pots. After 1 month, two tested isolates caused typical damping-off symptoms on seedlings. The percentage of the plants that became infected was 65 to 77%. To our knowledge (1,3), this is the first report of F. chlamydosporum on Aleppo pine in northwestern Algeria. 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 (1,3). References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Syst. Mycol. Microbiol. Lab. ARS, USDA, Beltsville, MD. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , February 20, 2013. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , February 20, 2013. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.