First Report of Fusarium avenaceum Causing Wet Core Rot of ‘Elstar’ Apples in the Netherlands

Apple (Malus domestica) is an important fruit crop in the Netherlands. ‘Elstar’, the main cultivar, occupies 40% of the apple production area. Symptoms of apple wet core rot were observed on Elstar in January to March 2013 after 4 to 6 months’ storage in different packing houses at controlled atmosphere. The disease was present in a large number of lots harvested at orchards from different locations across the Netherlands, and incidences up to 25% were recorded. Apples exhibited light-brown wet rot, initially developing in the core and subsequently spreading into the surrounding cortex, often with a white to rose-reddish mycelium. Apples from five lots with infections (four apples per lot; 20 in total) were rinsed with sterile water, sprayed with 70% ethanol until droplet runoff and halved aseptically with a scalpel. The tissue next to the core rot was isolated and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single-spore isolates were propagated on fresh PDA plates. All cultures formed abundant white aerial mycelium with yellow to rose pigment and a dark pink to red reverse. Macroconidia were slightly falcate, thin-walled, usually 5 septate, with a tapering apical cell, and 40 to 80×3.5 to 5 μm. Pathogen characteristics were similar to those described for F. avenaceum, causal agent of wet core rot of apple (Sanzani et al. 2013;Sorenson et al. 2009). The identity of two representative isolates (Fu1-44145 and Fu2-44145) from different apple lots was confirmed by means of multilocus gene sequencing. Genomic DNA was extracted and sequences of ITS region, TEF1-α, and histone H3 loci were amplified and sequenced. The sequences have been deposited in GenBank (Accession Nos. KT350586 and KT350587 [ITS], KT350603 and KT350604 [TEF1], KT935567 and KT935568 [HIS3]). MegaBLAST analysis revealed 99.8 to 100% identity to Fusarium spp. belonging to the F. avenaceum-F. tricinctumspecies complex. The TEF1 and HIS3 sequences of both isolates were 100% identical with F.avenaceum sequences JQ429374, GQ915502, JQ435857, and GQ915469, confirming their identity as F. avenaceum. Koch’s postulates were satisfied in two experiments, with 15 Elstar apples per isolate. In the first experiment, surface-sterilized fruits were inoculated with 20 μl of a suspension of 10 5 conidiospores/ml prepared from a 15-day-old PDA culture after wounding with a needle. Inoculated fruits were sealed in a plastic bag and incubated in darkness at 20°C. In the second experiment, apples were surface sterilized, cut in half longitudinally, and one half was inoculated with 20 μl of the conidiospore suspension into the core of the apple. Control apple halves were inoculated with sterilized water. After inoculation, the halves were covered with plastic foil and incubated in darkness at 20°C. In both experiments, symptoms appeared after 4 to 6 days on 100% of the fruits; controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA plates morphologically resembled the original isolates. Fusarium avenaceum is a wound pathogen that has been isolated from apple fruit in Croatia and in the United States (Kou et al. 2014; Sever et al. 2012). Only few reports describe wet core symptoms associated with F. avenaceum on apple (Sanzani et al. 2013; Sorenson et al. 2009). This is the first report of wet core rot caused by F. avenaceum on apple fruit in the Netherlands. As wet core of apple is undetectable until the fruit is cut or consumed, it affects consumer confidence. Due to potential mycotoxin production during infection, F. avenaceum infections potentially pose a safety issue (Sorenson et al. 2009).