First Report of Alternaria arborescens Species Complex Causing Leaf Blotch and Associated Premature Leaf Drop of ‘Golden Delicious’ Apple Trees in the Netherlands

Apple (Malus domestica) is an important fruit crop in the Netherlands, with a total production area of 7,600 ha in 2016, and 1,600 ha of fruit tree nurseries. For decades, fruit growers and nurseries have reported a leaf blotch, characterized by irregular light brown spots, bordered by a dark brown to purple margin, and premature leaf drop, mainly on ‘Golden Delicious’. The disease causes severe, sometimes almost complete defoliation. Leaf blotch typically appears in early July and is most severe in the latter part of the growing season. In a survey in August 2014, affected ‘Golden Delicious’ leaves were collected from different orchards and taken to the laboratory. Next, 20 leaves per location were rinsed with sterile water, sprayed with 70% ethanol until droplet runoff and rinsed again with sterile water, and tissue sections were excised from lesions using a sterile scalpel and placed onto potato dextrose agar (PDA). The PDA plates were incubated at 20°C in the dark, and single spore isolates were transferred to fresh PDA plates. These isolates produced fast-growing colonies of irregular shape, tan brown to black and felty. Sporulation patterns showed long conidiophores with extensive terminal branching. Conidia were ovoid with a tapering apical beak and a size range of 10 to 30 × 5 to 10 μm, with 1 to 5 septa. The isolated fungi were morphologically identical to small spored Alternaria spp. (Simmons 2007). However, small spored Alternaria spp. cannot be identified based on morphological characteristics (Woudenberg et al. 2015). The identity of 12 representative isolates from two different orchards in their 4th growing season, located in the central part of the Netherlands, was determined by multilocus gene sequencing. To this end, genomic DNA was extracted using the UltraClean Microbial DNA isolation kit (MoBio Laboratories, Carlsbad, CA). Sequences of the ITS region, the endoPG gene, and the anonymous region OPA10-2 locus were amplified and sequenced as described in Woudenberg et al. (2013, 2015) and deposited under GenBank accession numbers MG744449–MG744460 (ITS), MG744473–MG744484 (endoPG), and MG744461–MG744472 (OPA10-2). MegaBLAST analysis revealed that our ITS, endoPG, and OPA10-2 sequences matched with >99 to 100% identity to Alternaria arborescens species complex (SC) isolates in GenBank (AF347033 and KP124400 [ITS], AY295028 and KP124104 [endoPG], and KP124712 and KP124714 [OPA10-2]). Subsequently, Koch’s postulates for three A. arborescens SC isolates were performed in the laboratory on ‘Golden Delicious’ leaves. Surface sterilized leaves were inoculated on the abaxial side with 10 μl of a suspension of 10⁵ conidiospores ml–¹ water, prepared from a 14-day-old PDA culture, after wounding with a needle, with four inoculations per leaf. Inoculated leaves were sealed in a plastic box and incubated in darkness at 20°C. The experiment was carried out in five replicates. Symptoms appeared within 7 days on 100% of the leaves, while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions cultured on PDA morphologically resembled the original isolate from the infected leaves. The identity of the reisolations was confirmed as A. arborescens SC by sequencing. To our knowledge, this is the first report of A. arborescens SC causing leaf blotch and subsequent premature leaf drop of apple cultivars in the Netherlands. Multiple Alternaria species groups are associated with leaf blotch diseases of apple in Australia and Italy (Harteveld et al. 2012; Rotondo et al. 2012). A. arborescens-like isolates are most prevalent in Australia and are mostly associated with leaf blotch symptoms (Harteveld et al. 2012).