First Report of Neofusicoccum australe (Botryosphaeria australis), as a Branch Dieback Pathogen of Avocado Trees in Chile.

Since 2007, Chilean avocado (Persea americana Mill.) orchards have been exposed to several abiotic stress conditions, namely frost damage and drought, due to three consecutive seasons of cold winters and shortage of irrigation water. At the same time, a severe disease resulting in tree dieback of cv. Hass, specifically, was observed in north-central Chile. Symptomatic trees exhibited abundant dead twigs in the tree canopy, and wilted leaves remained attached to the twigs in autumn. Closer inspection revealed reddish-brown necrotic lesions on the bark of the dead twigs, which girdled these symptomatic branches. When the bark was removed, the wood below appeared dark brown, in contrast to the yellowish-green coloring of healthy. The fungus was also consistently isolated from rotted fruit. A Neofusicoccum sp. with a yellow colony was consistently isolated from the necrotic lesions on PDA and incubated at room temperature for 3 days. Conidia produced in black pycnidia growing on 2% water agar with sterilized pine needles were smooth, unicellular, hyaline, and with granular contents. One or two septa developed at germination, but rarely before. The average length of the conidia was 27.0 ± 0.9 μm, with a length/width ratio of 3.9 ± 0.2 μm. Based on culture and conidial morphology, the isolates were putatively identified as Neofusicoccum luteum (1). DNA sequence analysis of the rDNA internal transcribed spacer (ITS) region was conducted for four representative isolates using primers ITS1 and ITS4 (4). The sequence analysis of ITS region of kiwifruit isolate H1M4 (Accession No. KC330230) reveled 100% nucleotide identity to N. australe (FJ157187 to FJ157192) (3). Pathogenicity tests were conducted with stem inoculations of 2-year-old cv. Hass plants grow in plastic containers in a sand/lime/peat mixture. For each inoculated plant (n = 8), a 7-mm-diameter agar plug from the margin of a 3-day culture was used as inoculum after wounding the stem to the depth to 7 mm with a cork borer. Negative control (n = 8) were wounded and then 'mock-inoculated' with sterile agar plugs. The inoculation sites were wrapped with Parafilm. All plants were kept in a greenhouse. After 5 months, all inoculated plants showed bark cankers and necrotic lesions beneath the bark, which were 5.2 cm long (n = 8). No symptoms developed on the control plants. N. australe was recovered from the margin of the necrosis lesion of every inoculated plant, thus fulfilling Koch's postulates and confirming its pathogenicity. Botryosphaeraceae spp. are the commonly reported to have ability to survive endophytically in their host, causing disease only when the host is exposed to a stress condition (2). To our knowledge, this is the first report of N. australe as a pathogen of avocado in Chile. The fungal isolates (PaHass No. 1 to 4) were deposited in the Laboratorio de Fitopatología Frutal y Molecular, Departamento de Sanidad Vegetal, Facultad de Ciencias Agronómicas de la Universidad de Chile. References: (1) A. J. L. Phillips. http://www.crem.fct.unl.pt/botryosphaeria_site/ Accessed November 20, 2011. (2) B. Slippers and M. J. Wingfield. Fungal Biol. Rev. 21:90, 2007. (3) B. Slippers et al. Mycologia 96:1030, 2004. (4) White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.