First Report of Diaporthe foeniculina Associated with Branch Canker of Avocado in Greece

Abstract

Avocado (Persea americana Mill) is a new and rapidly expanded crop in the Mediterranean basin. Due to environmental conditions, its cultivation in Greece, is restricted mainly to Chania prefecture (Crete island), and there is limited data on the fungal diseases which can affect the plantations. In the autumn of 2018, in an orchard in Alikianos district of Chania (35°27′05.03″N, 23°54′49.82″E), approximately 8% of fifty 5 to 6 years-old avocado trees (cv. Hass) showed symptoms consisting of branch and twig cankers with brownish bark discoloration, resulting in twig dieback with brown leaves remaining attached to the trees. Small symptomatic bark tissue pieces from diseased trees (10 tissue pieces originating from two branches of each of the four diseased trees) were surface disinfested in 0.5% sodium hypochlorite followed by 70% EtOH for 2 min each step, rinsed twice with sterile distilled water and dried prior to culturing on potato dextrose agar (PDA), amended with 1 ml of lactic acid 10% per 100 ml PDA (pH 4.5), and incubated in the dark at 25οC for 2 to 3 days. 27 isolates out of 40 isolations were recovered from eight branches (68% isolation frequency) and had identical colony morphology. Mycelial plugs from the actively grown colony margins were transferred onto fresh PDA plates in order to obtain pure cultures. The morphological identification of the fungus was determined based on the pure cultures. Colonies on PDA were white slow-growing with sparse aerial mycelium on which globose to subglobose, dark brown to black, and pycnidia (420-670 μm in diameter) with an elongated neck (up to 100 μm) were produced after 2 weeks. Conidiophores were hyaline, septate and branched (18.0 to 24.0 × 1.9 to 3.0 μm). Alpha-conidia were aseptate, hyaline and ellipsoidal or fusiform and measured 8.0 ± 0.8 × 2.5 ± 0.6 μm (n = 50). Beta-conidia were hyaline, aseptate, filiform and slightly curved, measuring 26.9 ± 1.8 × 1.3 ± 0.2 μm (n = 50) and presented in larger numbers than alpha-conidia. The morphological and cultural characteristics of the isolates were congruent with those of Diaporthe spp. (anamorph Phomopsis spp.). To confirm the morphological identification, DNA from mycelium of four isolates (Av-1, Av-2, Av-6, Av-8) was extracted using the NucleoSpin Plant II kit (Macherey-Nagel). The ITS1-5.8S-ITS2 region was amplified with primers ITS1/ITS4 (White et al., 1990). The PCR products were sequenced, and BLASTn analysis revealed 99.8% similarity with sequences of various D. foeniculina isolates (eg. chestnut [LN651172], citrus [KC843294], soybean [JF430495], apple [MK370623]). All sequences were identical and one representative (isolate Av-1) was deposited in GenBank (MT374094). Partial amplification of β-tubulin (tub2) and translation elongation factor 1-alpha (EF1-a) was performed with Bt2a/Bt2b and EF1-728F/EF1-986R primers, respectively, using the Av-1 isolate (Carbone & Kohn, 1999; Glass & Donaldson, 1995). The amplicons were sequenced, deposited in GenBank (EF1-a: MT374092; tub2: MT374093), and showed 100% homology with sequences of D. foeniculina isolates (tub2: MF418578; EF1-a: LN651178). Thus, the isolate was identified as D. foeniculina (Sacc.) Udayanga & Castl. (Udayanga et al. 2015). Pathogenicity tests were conducted using 15 1.5-year old avocado trees (cv. Hass) under greenhouse conditions. PDA plugs, 4 mm in diameter, with actively growing mycelium of a 7-day-old culture, were transferred onto wounds made by a cork borer on previously sterilized trunk surfaces, covered with sterile paper and sealed with parafilm. Five trees inoculated with noncolonized PDA disks were kept as controls. Five months after inoculation lesions with brownish bark discoloration had developed on all inoculated trees, while control plants remained symptomless. D. foeniculina was re-isolated on PDA from all artificially inoculated plants. Thirty isolations were performed and D. foeniculina was recovered from all symptomatic inoculated plants (100% isolation frequency) and their identity was morphologically reconfirmed. D. foeniculina was further molecularly identified by PCR amplification and sequencing of the ITS and EF1-a regions of two isolates derived from artificially inoculated plants. Koch’s postulates were thus fulfilled with the above described procedure. On avocado, canker symptoms on shoots, branches or trunks are caused by several fungal species belonging to Botryosphaeriaceae and Diaporthiaceae families, and to date, in Europe, there is one report of D. foeniculina in Italy causing avocado canker (Guarnaccia et al., 2016). To our knowledge, this is the first report of D. foeniculina causing branch canker disease on avocado in Crete, Greece. This record provides significant information as the disease poses a new threat to the widely cultivated avocado crops in the area and draws the attention for the application of preventive measures to control spread should be considered.