In November of 2007, dieback symptoms (basal needle dieback, wilting, and dieback of terminal shoot) were observed on plant groups of Pinus radiata and P. pinaster in a tree nursery located in Anadia in the central region of Portugal (40°26′N, 08°23′W). Two containers with a total of 112 plants per pine species (with and without symptoms) were collected. Small pieces (5 mm long; two from the roots, stem at the soil level, and the aerial part, totaling six pieces) of 20 symptomatic plants were sterilized with 3% sodium hypochlorite, and isolations were performed on potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate. A species of Fusarium was isolated from all infected tissues and pure cultures were obtained by single hyphal tip transfers on PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The species was identified as Fusarium circinatum Nirenberg & O'Donnell (= F. subglutinans Wollenweb & Reinking) on the basis of morphological and cultural characteristics (2). They produced white, aerial mycelia, violet pigment, typically three-septate macroconidia with slightly curved walls, single-celled microconidia, and characteristic sterile, coiled hyphae. Microconidia were ovoid or allantoid and born in false heads on aerial polyphialides. The identification was confirmed by PCR with specific primers CIRC1A/CIRC4A, resulting in a 360-bp DNA fragment of the two nuclear ribosomal intergenic spacer regions (3). Pathogenicity tests were performed by inoculating 5- and 9-month-old P. pinaster and P. radiata seedlings, respectively. Plants belonging to P. pinea species (8-month-old), the second most important pine in the country, were also included in the tests. Small strips of bark (10 × 1 mm) were cut from the stems and similar-sized pieces of PDA colonized by two isolates of F. circinatum were placed in contact with the open wounds and covered with Parafilm. Ten seedlings for each pine species, isolate, and control (with sterile PDA) were provided in a total of 90 plants. First symptoms, basal needle and shoot dieback, were observed in P. radiata 8 days after inoculation. One month later, all P. radiata and 70% of the P. pinaster plants were dead. In all P. pinea plants, needles turned red along the main stem, from center to periphery, but only 2% of these plants presented wilting of the terminal shoot after 1 month. No symptoms were observed on control seedlings. F. circinatum was reisolated from symptomatic plants of the three species tested. To our knowledge, this is the first report of F. circinatum in Portugal. Pitch canker, caused by Gibberella circinata (anamorph F. circinatum), is one of the most aggressive pathogens on several pine species in the world (1). In 2005, the fungus was detected in the European continent affecting P. radiata and P. pinaster in northern Spain. References: (1) E. Landeras et al. Plant Dis. 89:1015, 2005. (2) H. I. Niremberg and K. O'Donnell. Mycologia 90:434, 1998. (3) W. Schweigkofler et al. Appl. Environ. Microbiol. 70:3512, 2004.
First report and characterization of Fusarium circinatum, the causal agent of pitch canker in Brazil