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First Report of Diplodia mutila Causing Branch Dieback of English Walnut cv. Chandler in the Maule Region, Chile

Díaz, G. A., Latorre, B. A., Ferrada, E., Gutiérrez, M., Bravo, F., Lolas, Mauricio
Plant disease 2018 v.102 no.7 pp. 1451
Araucaria araucana, Bacillus thuringiensis, Botryosphaeria stevensii, DNA fragmentation, Juglans regia, Vitis, agar, branches, conidia, container-grown plants, dieback, economic impact, ethanol, genes, hyphae, industry, internal transcribed spacers, mycelium, orchards, pathogenicity, pycnidia, ribosomal DNA, species identification, tissues, trees, tubulin, walnuts, winter, wood, California, Chile
English walnut (Juglans regia) has become a very important nut crop during the last decade in the Maule region of Chile, with 4,367 ha currently planted (, 2017). A branch dieback was observed at a 10% level in a 10-year-old ‘Chandler’ walnut orchard in Parral (36°09′S; 71°50′W) in the Maule region. Diseased trees showed multiple twig and branch dieback. Internally, brown, hard, wedge-shaped cankers were consistently observed in cross sections. Six branch samples were taken for laboratory examinations in June (winter) 2015. Samples were surface disinfected in 96% ethanol for 3 s, flamed, and small (3 to 5 mm long) pieces of wood, taken from the edge of cankered tissues, were placed on PDA plus 0.1% Igepal CO-630 (Díaz and Latorre 2014). Plates were incubated at 20°C for 5 days in darkness, and then pure cultures were obtained by transferring a hyphal tip to PDA. Six isolates (DMnog-1 to 6) that developed white to white-gray, fast-growing colonies with abundant aerial mycelium were selected after 7 days at 20°C on PDA. These isolates showed a black reverse side after 20 to 30 days at 20°C and abundant aggregated black pycnidia were obtained. Conidia were hyaline, unicellular, thick-walled, ellipsoidal to cylindrical, and (26.3–) 24.0 ±1.4 (–22.1) × (15.3–) 12.9 ± 1.0 (–11.5) µm with a length/width ratio of 1.9. These isolates were tentatively identified as Diplodia mutila (Fr.) Mont. (Alves et al. 2004). The species identification was confirmed molecularly, using ribosomal DNA fragments (ITS1-5.8S-ITS2) and β-tubulin gene, amplified with ITS5/ITS4, Bt2a/Bt2b primers, respectively, and sequenced. The sequences showed a 99% similarity when compared with sequences of D. mutila CBS 112553 ex-type, deposited in GenBank. The sequences of D. mutila isolates DMnog-1 to DMnog-4 were deposited in GenBank (MG386821 to MG386824 and MG418832 to MG418835 for ITS and Bt, respectively). Pathogenicity tests were conducted using isolates DMnog-1 and DMnog-4 on plants of English walnut cv. Chandler during June. First, 20 twigs of 10-year-old trees were pruned and immediately inoculated with 40 µl of mycelial fragments (10⁵ fragments/ml) or 40 µl of conidial suspension (10⁵ conidia/ml). An equal number of twigs inoculated with sterile water were left as control. Second, six 2-year-old potted plants were pruned aseptically at the tip and a 5-mm mycelial plug, taken from 5-day-old cultures in PDA, was placed upper side down on each pruning wound. An equal number of plants inoculated with sterile agar plugs served as controls. Four months after inoculation with mycelial fragments and conidial suspension, the twigs developed necrotic lesions of 19.9 to 35.3 mm and 12.5 to 26.3 mm in length, respectively. Similar necrotic lesions of 40.5 to 56.3 mm in length and dieback were observed after 16 months in the 2-year-old potted plants. D. mutila was 100% reisolated from inoculated plants, fulfilling Koch’s postulates. Control plants remained symptomless and reisolations were unsuccessful. To our knowledge, this is the first report of D. mutila associated with walnut dieback in the Maule region, Chile. Previously, D. mutila was described causing cankers and branch dieback in California (Chen et al. 2014) and it was identified causing canker on grapevine and Araucaria araucana in Chile (Besoain et al. 2017; Morales et al. 2012). The economic impact of branch dieback of English walnuts for the Chilean industry remains unknown.