First Report of Fusarium equiseti 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 Relizane, Sidi Bel Abbes, and Tlemcen provinces in northwestern Algeria. Aleppo pine seedlings showed symptoms of pre- and post-emergence damping-off disease, with an incidence of 64 to 77%. Four composite samples were taken from each location. Disinfested root and root collar segments, approximately 5 mm in length, were cultured on potato dextrose agar (PDA) and incubated at 25°C, and hyphal tips were transferred to PDA. Fusarium equiseti (Corda) Sacc. (teleomorph: Gibberella intricans Wollenw.) was identified from roots of two seedlings from the Sidi Bel Abbes nursery. Morphological identification was done according to Fusarium keys (2). PDA colonies with abundant, loosely floccose, whitish aerial mycelium and beige pigmentation were observed. Macroconidia with usually 5 to 6 septa, 31 to 45 μm long. A pronounced dorsiventral curvature, tapered and elongated apical cell, and prominent foot shape were observed. Microconidia were absent. Chlamydospores were produced in hyphae, most often intercalary, solitary, in pairs, frequently forming chains or clusters, globose (7 to 13 μm). To confirm the identity of this fungus, the internal transcribed spacer of F3RS1 and F19RS1 isolates of F. equiseti were amplified and sequenced using ITS1 and ITS4 primers (4), GenBank accession nos. JX114784 and JX114791, respectively. Those sequences bore 100% (HQ671182) similarity with sequences of F. equiseti in GenBank. Pathogenicity tests were performed to fulfill Koch's postulates. Inoculum was 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 sterile distilled water), shaken over 9 days at 25°C, and mixed with sterile sandy clay soil at 1:3 (v:v). Infested soil was then transferred to 500 ml pots, and 10 Aleppo pine seeds were planted per pot. A completely randomized design was used with three replicates per isolate and three control pots with a similar non-infested soil. After 1 month at 25°C the two tested isolates caused typical damping-off symptoms (collar rot) on seedlings and were re-isolated from recently infected tissues. The percentages of the inoculated plants that became infected were 59 to 65% among isolates (0% in control pots). To our knowledge, infection by F. equiseti is a first report on Aleppo pine in northwestern Algeria, Northern Africa, and globally, and on conifers in the Mediterranean region (1,3). In Algeria, F. equiseti is associated with black pepper (Piper nigrum L.) (3). These findings highlight the moderate impact of F. equiseti on the production of Aleppo seedling stock for reforestation activities in northwestern Algeria. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. 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.