First Report of Neofabraea kienholzii Causing Bull’s Eye Rot on Pear (Pyrus communis) in the Netherlands

Pear (Pyrus communis L.) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014, and ‘Conference’ is the main cultivar. In the Netherlands, pears are kept in controlled atmosphere cold storage up to 11 months after harvest. Symptoms of bull’s eye rot were observed in 2015 on ‘Conference’ pears in storage in the Netherlands. Bull’s eye lesions on apple and pear fruits are generally caused by four Neofabraea species: N. alba Jacks, N. malicorticis Guthrie, N. perennans Kienholz, and N. kienholzii Seifert, Spotts & Levesque (Gariepy et al. 2005). N. alba is the major pathogen causing bull’s eye rot on pear fruits in the Netherlands. Independent of the species, the symptoms appear as flat or slightly sunken lesions, which are brown, often lighter brown in the center (Spotts et al. 2009). To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto potato dextrose agar (PDA). PDA plates were incubated at 20°C in the dark, and single spores were transferred to fresh PDA plates. The isolates produced colonies with white-yellowish to brownish mycelium. Microconidia were produced on feathery fascicles of aerial mycelium, with a white, powdery, or sugary appearance on the surface of the agar colony. Microconidia were 2.5 to 6.5 × 1.5 to 2.5 µm, ellipsoidal, slightly asymmetrical to a curved form. The identity of a representative isolate (PPO 45010) was confirmed by means of multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin) in combination with the Kingfisher method (Waltham, MA). Segments of the internal transcribed spacer region (ITS), 28S ribosomal RNA (28S rRNA) and beta-tubulin (TUB2) loci were amplified, sequenced with primers ITS1/ITS4, LR0R/LR5, and Btub2Fd/Btub4Rd (Chen et al. 2016), and deposited in GenBank under accession nos. KX424942 (ITS), KX424941 (28S rRNA), and KX424940 (TUB2). MegaBLAST analysis revealed that the ITS, 28S rRNA, and TUB2 sequences matched with 99 to 100% identity to N. kienholzii isolates in GenBank (KR859082 and KR859083 [ITS], KR858873 and KR858874 [28S rRNA], KR859288 and KR859289 [TUB2]). Alcohol surface sterilized fruits were inoculated in pathogenicity tests in two ways: (i) with an agar disk (10 mm diameter) with actively growing mycelium of N. kienholzii prepared from a 14-day-old culture grown on PDA; and (ii) with 20 μl of a spore suspension (10 5 conidia ml -1 ) prepared from a 21-day-old PDA culture after wounding with a needle. Both experiments were performed on 10 ‘Conference’ pears. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared between 7 and 14 days. Mock-inoculated controls with water and PDA-only controls remained symptomless. Fungi isolated from the lesions had morphological characteristics that resembled the original isolates from infected pears. The identity of these isolates was confirmed as N. kienholzii by sequencing, thus completing Koch’s postulates. Bull’s eye rot of apple and pear is an important postharvest disease, occurring in major fruit growing areas of North America, Chile, Australia, and Europe (Henriquez et al. 2004; Spotts et al. 2009). N. kienholzii was reported twice on apple in Europe (Michalecka et al. 2016). To the best of our knowledge, this is the first report of N. kienholzii causing bull’s eye rot of pear in Europe.