First Report of Neonectria candida Causing Postharvest Decay on ‘Conference’ Pears in the Netherlands

Abstract

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 349,000 tons in 2014, and ‘Conference’ is the main pear cultivar that occupies 75% of the total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage up to 11 months after harvest. Occasionally, storage rots are observed when storage crates are contaminated with orchard soil. In a storage trial (2012 to 2013), boxes with ‘Conference’ pears were amended with soil particles from the same orchard from which the pears were harvested (four orchards), and stored for 11 months. Boxes without amended soil were included as controls. In contrast to the control boxes, up to 15% of the pears stored in boxes with soil particles showed typical rot symptoms (lesions) of an unknown causal agent. The lesions showed brown and watery circular necrosis, were slightly sunken, and displayed whitish to yellowish mycelia covering the lesions. 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). 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 with white-yellowish mycelium. Conidia were hyaline, cylindrical, 1 to 3 septate, and 15.8 to 26.4 × 5.3 to 7.9 µm. The fungus was morphologically identical to Neonectria candida (syn. N. ramulariae; anamorph Cylindrocarpon obtusiusculum) (Lombard et al. 2015). The identity of a representative isolate (VTN10Bs3) was confirmed by means of multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Sequences of the ITS region, translation elongation factor 1-alpha (TEF1), and actin (ACT2) loci were amplified, sequenced, and deposited in GenBank under accessions KU588183 (ITS), KU588186 (TEF1), and KU588184 (ACT2). MegaBLAST analysis revealed that our ITS, TEF1, and ACT2 sequences matched with >99 to 100% identity to N. candida isolates in GenBank (KM249079 and JF735314 [ITS], JF735791 and HM054091 [TEF1], and KM231146 [ACT2]). Subsequently, Koch’s postulates were performed on 15 ‘Conference’ pears. Surface sterilized fruits were inoculated with 20 µl of a suspension of 105 conidiospores ml–1 water, 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. Symptoms appeared after 7 days on 100% of the fruits while mock-inoculated controls with water remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. Moreover, symptoms observed on artificially inoculated ‘Conference’ pear fruit were identical to the decay observed on ‘Conference’ pears that were obtained from the cold storage experiment. The identity of the reisolations was confirmed as N. candida by sequencing. N. candida (syn. N. ramulariae) is known as a globally distributed soilborne fungus (Domsch et al. 2007), but only few studies have identified the fungus as plant pathogen (Hirooka 2012). This is the first report of N. candida causing storage rot of pears. Importantly, we note that the occurrence of storage rots may be enhanced by contamination of storage crates or fruit with orchard soil.