First Report of Fusarium euwallaceae on Persea americana in South Africa

Avocado is an important fruit commodity in South Africa for both the local and export market. A total of 17, 500 ha of avocado are grown, and production is expanding annually by approximately 1000 ha. Avocados in Israel (Mendel at al. 2012) and California (Eskalen et al. 2013) are threatened by the invasive polyphagous shot hole borer (PSHB, Euwallacea nr. fornicatus) and its pathogenic symbiont, known now as Fusarium euwallaceae (Freeman et al., 2013). In 2017, the PSHB and the pathogen were identified on London plane trees (Platanus x acerifolia) in Pietermaritzburg, KwaZulu Natal, South Africa (Paap et al., 2018). During a tree health survey in Sandton, Gauteng, in January 2018 a diseasedvocado tree was observed, in the backyard of a home owner. Affected branches showed symptoms of a white powdery exudate (sugar volcano) in association with a single small beetle exit hole (0.8 ? 1 mm). Some exit holes were surrounded by a wet discolored lesion. Examination of the underlying tissue revealed brown, necrotic lesions and galleries penetrating the sap wood. Symptomatic tissue from the beetle galleries was plated onto Fusarium selective media and malt extract agar. After 5-7 days incubation at 25 ?C fungal colonies with aerial mycelia and reddish-brown margins were produced. Single spore isolations were used to establish pure cultures of the fungus on potato dextrose agar. DNA was extracted from fresh mycelia scraped from actively growing cultures using Prepman? Ultra Sample Preparation Reagent (Applied Biosystems) according to the manufacturer?s instructions. The translation elongation factor 1? (TEF1 - MH823818), ?-tubulin (MH823816) and RNA polymerase II second largest subunit (RBP2 - MH823817) gene regions were amplified, sequenced and assembled into contigs. TEF1 sequence data were analyzed together with data generated by Eskalen et al. (2013), O?Donnel et al. (2015), Paap et al. (2018), and Na et al. (2018), using MEGA 7.0.26 (Kumar, et al. 2016) (Suppl. Fig.1). A culture was deposited in the CMW collection ? Culture collection Mike Wingfield (CMW 51808 - FABI, University of Pretoria) and the sequences were submitted to Genbank . A PSHB specimen could not be retrieved from avocado, but a confirmed PSHB specimen (COX1 ? MH823819) was retrieved from a diseased Chinese Maple (Acer buergerianum) tree approximately 2 km away harboring F. euwallaceae that was genetically identical to the isolate from avocado. The fungus produced characteristic clavate macroconidia when grown in culture (Freeman et al. 2013). Stems and shoots of seven healthy 1-year-old avocado trees were wounded (1-2 mm deep) with a cork borer, and 5-mm mycelial plugs from a 7-day-old culture were placed mycelial side down into the wound and wrapped with Parafilm. Three control plants were inoculated with sterile agar plugs. After the trees were maintained in a greenhouse (25 ?C) for 6 weeks, watering twice a week, external and internal lesions were measured. Brown wet lesions were observed on all pathogen-inoculated stems and shoots. Internal reddish-brown lesions extended into the tissue. Mean lesion length was 5.56 cm and was significantly longer (One-way Anova with a multiple comparison test) than lesions on control plants (1.37 cm). Control plants had brown lesions around the point of inoculation, but the pathogen was not re-isolated. Fusarium euwallaceae was re-isolated from all symptomatic material to fulfil Koch?s postulates. Similar results were obtained in a repeat experiment. To our knowledge, this is the first report of F. euwallaceae isolated from and causing Fusarium dieback symptoms on avocado in South Africa. This discovery is of major concern to the avocado industry but the PSHB and its symbiont also threatens other fruit trees, exotic and indigenous trees in urban areas and natural forests.