First Report of <i>Alternaria arborescens</i> as the Causal Agent of Stem and Leaf Early Blight in Tomato ( <i>Solanum lycopersicum</i> cv. Pietro) in Cotopaxi-Ecuador

Tomato (Solanum lycopersicum) is a crop of economic importance in Ecuador, with the Pietro cultivar being widely grown in the Andean region, especially in the province of Cotopaxi (MAG, 2023). In February 2024, a high incidence (100%) of disease symptoms was observed in greenhouse-grown tomato plants (1,000 m²) in Salcedo, Cotopaxi Province (1°03'17.9"S, 78°34'43.2"W). The effects of the plants showed necrotic stem blight and dark brown foliar lesions with chlorotic halos. Six symptomatic plants were collected for pathogen isolation. Stem fragments (~5 mm²) were surface disinfected with 2.5% sodium hypochlorite and 70% ethanol, rinsed with sterile distilled water, and plated on potato dextrose agar supplemented with gentamicin (PDA+G, 160 mg/L). The plates were incubated at 28°C for 7 days. Fungal cultures were purified using the single-hyphal-tip method on PDA+ G. Two isolates were morphologically characterized and preliminarily assigned to the genus Alternaria spp., exhibiting grayish mycelia and ovoid to ellipsoidal, pale brown conidia, with longitudinal septa measuring 16.77 - 18.86 µm in length and 8.62 - 9.98 µm in width. These features are consistent with Alternaria spp. descriptions (Woudenberg et al. 2013). Pathogenicity tests were performed on healthy four-week-old S. lycopersicum cv. Pietro plants, with five replicates per treatment. Isolates A1 and A2, previously cultured on PDA+G for 15 days, were inoculated by spraying 10 ml of a conidial suspension (1 × 10⁷ conidia/ml) onto each plant. Control plants received distilled water only (Ramírez et al. 2023). Plants were kept in a humid chamber (18–29 °C, 12 h light/12 h dark), and symptom development was evaluated 30 days post-inoculation. Inoculated plants developed necrotic stem lesions and foliar spots with chlorotic halos, identical to those observed in the field, while control plants remained symptom-free. The experiment was repeated three times independently, and similar results were obtained. The pathogen was successfully re-isolated from symptomatic tissue, and its identity was confirmed morphologically by comparison with the original isolates. For molecular identification, genomic DNA from isolates A1 and A2 was used to amplify and further sequence three loci: the internal transcribed spacer (ITS) region using primers ITS1 and ITS4 (White et al. 1990), the translation elongation factor 1-alpha gene (tef-1α) using primers EF1-728F and EF1-986R (Carbone et al. 1999), and the second largest subunit of RNA polymerase II (rpb2) using primers RPB2-5F and RPB2-7cR (Liu et al. 1999). Genomic sequences were deposited in the GenBank under the accession numbers: ITS (PV704619, PV704770), TEF (PV726241, PV726242), RPB2 (PV726243, PV726244). Sequences were aligned using ClustalW, and a multilocus phylogenetic tree was generated by Bayesian inference using the GTR model in BEAST v10.5.0 based on the concatenated sequences (Woudenberg et al. 2015). Both isolates clustered within the Alternaria arborescens clade, confirming their identity. This study is the first report of Alternaria arborescens as the causal agent of necrotic lesions on tomato stems (Solanum lycopersicum cv. Pietro) in Ecuador. These results support future phytosanitary monitoring and sustainable disease management strategies for tomato production in the region.