First Report of Neofusicoccum australe Causing Branch Dieback of English Walnut cv. Chandler in Central Chile

The English walnut (Juglans regia L.) is the second most important fruit crop of importance in Chile, with 43,700 hectares mainly in the Central Valley (www.odepa.cl, 2022). For several seasons symptoms of a branch dieback have been observed in walnut orchards with 3 to 50% of trees incidence levels. During the 2020 winter season (July) a total of 150 symptomatic spurs of 15 trees were sampled from an 8-year-old walnut cv. Chandler orchard located in Buin (33°42' S, 70° 42' W). The collected spurs showed external and internal brown necroses, starting from the tip with well-defined margins. The symptomatic tissue was cut in to small pieces (5 x 4 x 2 mm), surface disinfected by dipping in a 10% solution made from a commercial bleach solution (4,9% NaOHCl), rinsed twice in sterile water and plated on APDA (PDA Difco laboratories acidified with lactic acid (2,5 ml of 25% (vol/vol) per liter of medium). After five days at 20 °C in darkness, fast-growing, white-grey turning to black colonies were obtained, tentatively classified as a member of the Botryosphaeraceae family and two single-spore isolates (SS1, SS2) were selected for identification. Colony mycelia were first white and turned to light grey, dark grey or black, with tufts of mouse gray aerial mycelia. The pycnidia and conidia production was induced by inoculating autoclaved pine needles placed on APDA an incubation for 25 to 30 days at 20 °C in darkness. Black pycnidia solitary and globose were obtained producing hyaline, aseptate, fusiform to obovoid conidia with truncated ends with dimensions of (22.6-) 19.1 ± 1.4 (-13.3) x (6.7-) 5.5 ± 0.5 (-3.7) µm and 3.5 length/width ratio (n=100). Both isolates were identified using dichotomous keys confirming the description of Crous et al, 2006 as Neofusicoccum australe. The identification was molecularly confirmed by amplifying the nuclear ribosomal gene 5,8S (ITS1-5.8S-ITS2) using the ITS1/ITS4 primers, a partial region of β-tubulin gene (Bt2a/Bt2b), and the translation elongation factor 1-α gene (TEF1) with TEF1-728F/TEF1-986R primers. The BLASTn search revealed 100% of identity for ITS and TEF according to sequences of N. australe reference strains MT587467.1 and MK759852.1, respectively; and over 99% for β-tubulin compared to N. australe strain KX464929.1. The DNA sequences were submitted to the GenBank (ITS, OP142414, OP142416; BT, OP209981, OP209978; and TEF OP209979, OP209980) for SS1 and SS2 isolates, respectively, and deposited in the fungal collection of CChRGM - INIA, Chillán, Chile (RGM 3409 and 3410). Pathogenicity of both isolates was tested in 8-year-old asymtomatic English walnut cv. Chandler in the field during 2020 spring season, by cutting transversally 15 twigs of different tress and inoculating with a 5 day-old PDA plug. An equal number of wounded twigs were inoculated with a sterile PDA plug and served as control. After six months, all inoculated twigs developed the same necrotic lesions observed in field of 2.0 to 10.1 cm (SS1) and 1.9 to 10.8 cm (SS2) in length while control twigs showed only a scar without any dieback tissues. The inoculated pathogens of N. australe were recovered from the diseased tissues, thus fulfilling Koch's postulates. A similar dieback of walnut was reported in Chile, which caused Diplodia mutila (Díaz et al, 2018), and N. parvum (Luna et al, 2022) while N. australe has been reported in other hosts (Auger et al, 2013, Besoain et al, 2013). To the best of our knowledge, this is the first report of N. australe associated with walnut branch dieback in Chile.