First Report of Nothofagus macrocarpa Dieback Caused by Phytophthora citrophthora and P. nicotianae in Chile.

The genus Nothofagus, family Nothofagaceae, comprises 36 species of trees that are native to the Southern Hemisphere. N. macrocarpa (DC.) F.M. Vásquez & R.A. Rodríguez (Roble de Santiago) is an important deciduous tree, endemic to central Chile (32 to 35°S), and found above 800 m altitude. There is an increasing interest in N. macrocarpa as an ornamental. However, a general dieback (40 to 50% prevalence) was observed at a commercial nursery in Santiago in 2009, limiting its multiplication. Symptoms are wilting, partial defoliation, reddish brown cankers on the crowns, and root necrosis. The purpose of this work was to study the etiology of the dieback in nurseries. Phytophthora was isolated from the roots and cankers of symptomatic plants (n = 3) and soil samples (using apples and avocados as baits) on amended corn meal agar (3) at 20°C for 5 days in the dark. Morphologically, P. citrophthora (Smith & Smith) Leonian, and P. nicotianae Breda de Haan were identified (2). On V8 juice agar (V8) (1), P. citrophthora formed petaloid colonies, grew between 5 and 30°C (optimum of 25°C), and produced deciduous, mono- or bipapillated sporangia of (28.1) 45.0 to 64.1 × (18.8) 32.0 to 39.2 μm. On V8, P. nicotianae produced cottony colonies, grew between 10 and 30°C (optimum of 25°C), and produced spherical, intercalary chlamydospores (mean diameter of 19.6 μm) and persistent, papillate, spherical to ovoid, ellipsoid, obpyriform sporangia of (33.2) 47.5 to 67.6 × (24.1) 30.0 to 48.9 μm. Isolates of P. citrophthora were sexually sterile, but P. nicotianae formed oogonia with amphigenous antheridia in dual cultures with P. cinnamomi (A2 compatibility type). BLAST analysis of the internal transcribed spacer (ITS) region of rDNA of isolates identified as P. citrophthora (IMI 399056 and IMI 399054, GenBank Accession Nos. JF699756 and JF699755) and P. nicotianae (IMI 399055, Accession No. JF699757), amplified by PCR using ITS universal primers (4), revealed 100% similarity with reference isolates of P. citrophthora (Accession Nos. GU259324.1 and GU259317.1) and P. nicotianae (Accession No. GU983635.1). P. citrophthora (n = 2) and P. nicotianae (n = 1) were pathogenic when wounded detached twigs (n = 5) of N. macrocarpa and N. obliqua were inoculated with 20 μl of a mycelial suspension (10⁶ CFU/ml) of either Phytophthora spp. Twigs were placed in a moist chamber at 20°C for 12 days prior to determine the length of the necrotic lesions that developed. An equal number of noninoculated twigs were left as control. Reisolation of P. citrophthora and P. nicotianae from inoculated material was 100%. The length of the necrotic lesions (13 to 80 mm) from inoculated N. macrocarpa and N. obliqua was significantly greater (P < 0.05) compared with the controls. Regardless of Phytophthora isolates, necrotic lesions (53.9 ± 15.8 mm) in infected N. macrocarpa were significantly longer than in N. obliqua (28.6 ± 13.1 mm) (P < 0.0001). To our knowledge, this is the first report of P. citrophthora and P. nicotianae associated with dieback on N. macrocarpa in Chile. Therefore, there is a potential risk of Phytophthora dieback in N. macrocarpa in nature. References: (1) J. Ampuero et al. Plant Dis. 92:1529, 2008. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) B. A. Latorre and R. Muñoz. Plant Dis. 77:715, 1993. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.