First Report of Diaporthe oxe on ‘Peroba Rosa’ in Brazil

“Peroba rosa” (Aspidosperma polyneuron Muell. Arg.) is a native tree species to the forests of Brazil, Venezuela, Argentina, Colombia, Peru, and Paraguay. The species has a high commercial value and, due to logging and destruction of its original ecosystems, is at extinction risk. Currently, the plant is listed for ex-situ and in-situ conservation in Brazil and Venezuela. In 2014, samples of peroba rosa seeds were collected from native trees in Parana State, southern Brazil, and analyzed to evaluate their sanitary quality (Mazarotto et al. 2019). Following germination under greenhouse conditions with average temperature of 24°C, Diaporthe sp. (TR-57) was transmitted from seeds to seedlings, causing necrotic spots on cotyledons, malformation of plants, and eventually their death. In PDA medium, this isolate showed sparse mycelium, white to olive, ochre reverse, presence of semi-immersed dark pycnidia, and alpha (7 × 3 µm) and beta conidia (28 × 2 µm), resembling Diaporthe oxe Gomes, Glienke & Crous (Gomes et al. 2013). The presence of perithecia was not observed. For species identification, the ITS and TEF-1α regions were partially sequenced using the primer pairs ITS4/ITS5 and EF1-728F/EF1-986R, respectively. The obtained sequences were compared phylogenetically with 90 congeneric species. The ITS (GenBank accession no. KX385041) and TEF-1α (KX385079) sequences had respectively 98 and 100% similarity with D. oxe (KC343166, KC343893) (Gomes et al. 2013). This isolate was deposited in the Collection of Forest Fungi and Oomycetes, Embrapa Florestas, Colombo, Parana State, Brazil. For the pathogenicity test in seedlings, disks (5 mm diameter) of active mycelium in PDA were removed and deposited on the adaxial side of the leaves with previous circular needle injuries (4 mm²). This procedure was repeated for 10 young leaves in three seedlings (1 year old), with one disk per leaf. The control consisted of a PDA disk deposited on the injured leaf. The seedlings were incubated in a humid chamber for 72 h and then kept in a greenhouse for 30 days. A darkening of the leaf blade was detected reaching an area of approximately 8 mm² and a white mycelial growth of the pathogen over the lesions in 20% of the leaves. The control group had no symptoms. The symptomatic leaves were plated in PDA for 7 days at 24°C. D. oxe was reisolated from 100% of the inoculated leaves, and identification was confirmed by molecular sequencing, fulfilling Koch’s postulates. In Brazil, D. oxe was isolated as an endophyte from Maytenus ilicifolia (Gomes et al. 2013) and, in Uruguay, was pathogenic to Pyrus communis (Sessa et al. 2017). This study represents the first report of D. oxe associated with peroba rosa seeds as pathogens, not only in Brazil but also worldwide.