First report of Diaporthe rudis in Chile causing Stem-End rot on ‘hass’ avocado fruit imported from California

In June 2013, avocado fruit (Persea americana Mill cv. Hass) imported from California, USA, entered Chile for sale on the national market. These avocado fruits arrived at the phytopathology laboratory with clear symptoms of stem-end rot, a dark brown color in the peel, necrosis in the vascular strands, and light brown discoloration in the flesh. Pieces of peel and flesh were extracted from the margins of the lesions, washed, dried, and placed on acidified PDA (0.5 ml of lactic acid at 96% per liter, APDA), and were incubated in the dark for 5 to 7 days at 25°C. From these tissues, a white mycelium, moderately fluffy, slow growing, which turned whitish to gray over time, was recovered. Pieces of mycelium from the hyphal tips were transferred to the APDA medium and incubated for at least 30 days at 23°C in order to obtain reproductive structures. The formation of grayish-black, solitary, semi-immersed pycnidia 500 to 600 µm in diameter was observed. Conidiophores 18 to 22 × 1 to 2 µm bore alpha conidia that were hyaline, smooth, and ellipsoidal, 5.8 to 8.5 × 1.8 to 2.6 µm with a subtruncate base (n = 30); beta conidia that were hyaline, smooth, fusiform, 17.7 to 27.2 × 0.7 to 1.6 µm (n = 30) with a truncate base, and gamma conidia that were hyaline, smooth, fusiform, 10.2 to 16.2 × 1.5 to 1.8 µm (n = 30) with a subtruncated base. These spores are consistent with those of Phomopsis anamorph of Diaporthe (Udayanga et al. 2014). The pathogenicity test was conducted using four avocados for each isolate of Phomopsis (isolates 759.1 and 759.4). Each avocado was inoculated with a piece of actively growing mycelium from APDA (8 mm diameter), depositing the mycelium onto the flesh of each fruit. In addition, four avocado fruits were left as controls. The fruit was left in a humid chamber for 15 days of incubation at 25°C. After 7 days, dark brown lesions, 4 cm in diameter, developed on the skin of the inoculated avocados. Internally, severe necrosis in the vascular strands was observed, as well as a lighter brown rot of the flesh. No symptoms were observed on the controls. The Phomopsis sp. was reisolated from 100% of the inoculated avocados, completing Koch’s postulates. The molecular identification of the two isolates was conducted using mycelia obtained from a single colony. For the DNA extraction, the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used. The amplification of the ITS1-5.8S-ITS2 region of the rDNA for PCR was conducted using the primers ITS1 and ITS4 (Udayanga et al. 2014) and was entered into GenBank with accession no. KM396310. For the two isolates, a BLAST analysis revealed a similarity of 99% with the reference sequence of D. rudis in GenBank JX515702.1. This similarity was confirmed by the amplification of the elongation factor gene (EF1-728F to EF1-986R) and deposited in GenBank with no. KM396311, which showed 100% similarity with the reference sequence JQ697853.1 (Udayanga et al. 2014) and with the β-tubulin gene deposited in GenBank no. KM396309, which showed a 99% similarity with the reference sequence in GenBank KC843177 (Udayanga et al. 2014). To date, there is only one citation by Twizeyimana et al. (2013) associated with Phomopsis sp. causing lesions on avocado fruit. To our knowledge, this is the first report of D. rudis causing stem end rot in avocados in Chile or elsewhere.