In Europe, chestnut blight caused by Cryphonectria parasitica (Murrill) Barr was first seen in Italy in 1938 (1). In Spain, the disease was first detected in Basque country in 1947 and later in other areas of northern Spain: Galicia, León, Navarra, and Catalonia, and in Trás-os-Montes in Portugal (2). In November 2012, in an orchard (2 ha) in Almonaster la Real (Huelva, Spain), approximately 20 cankered Castanea sativa (sweet chestnut) trees cv. Vazqueño, 40 to 50 years old, were observed. The trees were grafted 2 years before. In May and June 2013, six new disease focuses were detected near the first one. Five focuses were located in the same village and the other in Jabugo (a neighboring village). Diseased trees exhibited sunken cankers, cracked bark with mycelial fan spreads under the bark, and in some cases, orange fungal sporulation was visible on the bark. Samples were collected from two affected trees and symptom-bearing bark pieces were then placed in moist chambers at 20°C for up to 8 days to induce fungal sporulation. Cultures were made from spore masses extruding from the cankered bark and from the edge of necrotic lesions visible in the phloem of cankered bark tissue onto potato dextrose agar (PDA). Monoconidial fungal isolates were obtained from both trees. The morphological structure of two isolated fungi was identical to that described as C. parasitica (3). Species identity was confirmed by analysis of nucleotide sequences of the internal transcribed spacer (ITS) rDNA, using ITS1-ITS4 (4) as primer pairs, respectively. BLAST searches showed a high similarity between collected isolates' DNA sequences and C. parasitica sequences found on GenBank (96% coverage, 99% identity). Our isolates have been included in GenBank as KF220298 and KF220299. The pathogenicity assay of these two isolates was conducted using two cultivars of sweet chestnut (seedlings from Huelva and Granada nurseries). Isolate pathogenicity was tested on 3-year-old chestnut seedlings in a growth chamber at 25°C (day) and 20°C (night) with a 14-h photoperiod. The isolates were cultured on PDA at 25°C for 7 days. Stems were wounded at 10 cm height with a drill. Each isolate was inoculated to 25 replicates per cultivar by placing a mycelia agar plug (4 to 5 mm diameter) in the hole and wrapping the stem with Parafilm. Plants treated identically with sterile agar plugs were used as controls. Plants were then maintained at 100% relative humidity for 2 h. Both isolates induced diseases symptoms and death of seedlings of both cultivars at a mean time of 37.5 days after inoculation. No significant differences between isolates or between cultivars were detected. Twenty control plants similarly treated with sterile PDA discs did not display symptoms. C. parasitica was re-isolated from lesions, confirming Koch's postulates. Andalusia has 14,000 ha of chestnut crops with high commercial value due to their precocity. Dispersion of chestnut blight in this zone can reduce crop productivity. To our knowledge, this is the first report of C. parasitica causing chestnut blight in Andalusia (southern Spain), one of the few areas left in southwestern Europe free of chestnut blight. References: (1) A. Biraghi. Italia Agricola 7:1, 1946. (2) G. González-Varela et al. Eur. J. Plant Pathol. 131:67, 2011. (3) A. Sivanesan and P. Holliday. Cryphonectria parasitica. CMI Descriptions of Pathogenic Fungi and Bacteria. No. 704, Set. 71. Commonwealth Mycological Institute, Kew, UK, 1981. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Amplifications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.
The role of production site isolation in the plant health situation of sweet chestnut (Castanea sativa)