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First Report of Neofusicoccum nonquaesitum in Chile Causing Branch Dieback and Decline in Araucaria araucana

Pérez, S., Guerrero, J., Galdames, R.
Plant disease 2018 v.102 no.7 pp. 1460
Araucaria araucana, DNA, Juglans regia, Malus domestica, Neofusicoccum, Persea americana, Prunus dulcis, Sequoiadendron giganteum, Umbellularia californica, Vaccinium corymbosum, agar, branches, chloramphenicol, common names, conidia, conifers, cotton, culture media, dieback, discoloration, endangered species, forests, greenhouses, growth chambers, inoculum, internal transcribed spacers, leaves, mycelium, pathogenicity, pathogens, pigmentation, pycnidia, relative humidity, sodium hypochlorite, spring, tissues, tree diseases, tree mortality, trees, Chile, United States
Araucaria araucana (Molina) K. Koch, common name monkey-puzzle, is one of the oldest conifers in South America and a symbol of the Chilean forest. It has been found to have precedents from the Mesozoic in Gondwana and Laurasia (Stockey et al. 1992). The A. araucana tree is considered sacred by the Mapuche Pewenche native population. It is listed as an endangered species on the Red Data List of the International Union for Conservation of Nature and is legally protected in Chile. During spring 2016, 3-year-old nursery-grown A. araucana trees (n = 4) located in Chile’s Araucaria Region exhibited discoloration of leaves, necrotic leaf symptoms, branch dieback, discoloration of internal stem tissues, and whole tree death. Small pieces of symptomatic branches were surface sterilized with 0.5% NaOCl, washed with sterile distilled water, and cultured on potato dextrose agar media (PDA) plus chloramphenicol. These were incubated in growth chambers at 25°C for 5 days (12/12 h dark/light). Aerial mycelia turned from white to black, and after 45 days, pycnidia were observed. Conidia were hyaline, fusiform to ellipsoidal, and base truncate. When mature, conidia showed light brown pigmentation and 1 to 3 septa. They were 18.6 to 36.8 µm in length and 5.2 to 9.0 µm in width, with a l/w ratio of 3.7 ± 0.6 µm (n = 125). DNA was extracted from four fungal cultures using the DNeasy Plant Mini Kit (Qiagen). The internal transcribed spacer (ITS) region was amplified using ITS1/ITS4 primers (White et al. 1990), and part of the amplicon (550 bp) was sequenced and deposited in GenBank (accession nos. MF795593, MF795594, MF795595, and MF795596). These showed 100% identity value with sequences from a similar region from Neofusicoccum nonquaesitum (Inderbitzin et al. 2010; KF778850, KX357186, and JX217819). A pathogenicity test was conducted. Four isolates (IAF-UFRO A1 to A4) were used to inoculate 3-year-old A. araucana trees (n = 3 plants/isolate) and maintained in greenhouse conditions (14/10 h dark/light, 20°C; 70% relative humidity). One branch per tree was inoculated at a fresh wound made with a sterile scalpel at the point where leaf and branch meet. The inoculum consisted of a plug of acidified PDA (5 mm) with 4-day-old mycelia. The wound was covered with moistened cotton and sealed with Parafilm. Control trees (n = 3) were inoculated as described above but only with agar. After inoculation, branch dieback, decline of Araucaria trees, and mature pycnidia were observed. Fragments of vascular system tissues from control and inoculated Araucaria trees were removed and incubated on PDA. Koch’s postulates were achieved with consistent recovery and reidentification of N. nonquaesitum from infected trees, while controls remained disease free. N. nonquaesitum has been reported on Sequoiadendron giganteum, Juglans regia, Malus domestica, Prunus dulcis, and Umbelluria californica in the United States. In Chile, it has been detected in Persea americana and Vaccinium corymbosum. Other members of the Botryosphaeriaceae family have recently been found to cause gummy cankers in Araucaria trees (Besoain et al. 2017). To our knowledge, this is the first report of N. nonquaesitum causing branch dieback and decline in A. araucana trees. We inferred that this pathogen could be also associated with the decline of this native conifer in Chile.