First report of Diplodia mutila causing gummy canker in Araucaria araucana in Chile

Araucaria araucana (Molina) K. Koch (monkey-puzzle tree) is a conifer endemic to central and southern Chile and Argentina. This species has long been a staple food for the Araucanos, an indigenous group of southern Chile. It has a high timber value and has been used for ornamental purposes (^†). In the Araucaria garden at the School of Agronomy of the Pontificia Universidad Católica de Valparaíso, one specimen of A. araucana showed canker with branch dieback symptoms with a white gummy exudation and orange necrotic lesions on leaves. Another Araucaria plant showing the same symptoms was observed in a garden in Quilpué, both in the Valparaíso region. Both observations were done at the end of June of 2016. Samples of diseased branches were taken from the margins of the necrotic lesions, surface disinfected (75% ethanol, 30 s), dried and placed on acidified PDA (0.5 ml of lactic acid at 96% per liter, APDA), and were incubated in the dark for 4 to 7 days at 25°C. Colonies of whitish to gray mycelium were hyphal-tip purified to APDA. After 14 days, formation of black solitary pycnidia with conidiogenous cells and hyaline and aseptate round edge conidia (23.5 × 10.9 µm on average) were obtained. Dark brown septate conidia developed in cultures after 35 days. These isolates were tentatively identified as Diplodia mutila (^†). This morphological identification was confirmed by molecular analyses. For the DNA extraction, the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used. The amplification of the ITS1-5.8S-ITS2 and B-tubulin subunit 2 gene was conducted using ITS1/ITS4 and Bt2a and Bt2b, primer sets, respectively. The sequences showed 100% similarity to D. mutila KM580529 for ITS region, from Chile, and 100% similarity to KF778884 for Bt gene, from the U.S.A., and were deposited in GenBank with accession nos. KX901798–803. Pathogenicity test was conducted using two isolates on 10-year-old symptomless A. araucana plants (n = 3), which were inoculated into a wound made aseptically with a sterile scalpel on the branches and then sealed with Parafilm. Noninoculated controls (n = 3) were inoculated as described above but only with sterile agar plugs. After 2 months under controlled conditions (25 ± 5°C and 12 h dark and light cycle), orange necrotic lesions on leaves and white gummy exudation were observed in the inoculated plants. Noninoculated remain asymptomatic. D. mutila was reisolated from 100% of the inoculated plants, completing Koch’s postulates. To our knowledge, this is the first report of D. mutila causing gummy canker of A. araucana in Chile or elsewhere. This pathogen was previously reported on both table and wine grapes in the Valparaíso region of Chile (^†;^†). The report of this new disease will contribute to study and preserve this important national tree present in protected areas of Chile and Argentina.