First Report of Cadophora luteo-olivacea Causing Side Rot on ‘Conference’ Pears in the Netherlands

Abstract

Pear (Pyrus communis) is an important fruit crop in the Netherlands. Symptoms of side rot disease of pear fruits were first observed in 2008 on cv. Conference in storage in the Netherlands. Typical round to oval, dark-brown, and slightly sunken spots (size 0.5 to 1.0 cm in diameter) appeared after six or more months of cold storage under controlled atmosphere. Lesions of rinsed pears were sprayed with 70% ethanol and tissue under the lesion was placed onto potato dextrose agar (PDA) at 20°C in the dark. Colonies obtained from single spores produced on PDA were flat, felty and cottony in the middle, with smooth margins, an even edge, and varying in color from white turning to gray/black-olivaceous. Under UV light, ellipsoid or elongate conidia were produced (2.2 to 2.3 × 4.9 to 6.5 µm). Both cultural and morphological characteristics of the pathogen were similar to those described for Cadophora sp. (Spadaro et al. 2011). Three representative isolates (PPO 11-1228, PPO 24-1234, and PPO 107-1267) were sequenced using primers ITS1/ITS4 and EF1-728F and EF1-986R (Carbone and Kohn 1999). MegaBLAST analysis revealed that the ITS sequences (GenBank accession nos. KT350591, KT350592, and KT350593) matched with 99.8 to 100% identity to Cadophora luteo-olivacea in GenBank (KU141394 and KU141395). The TEF1 sequences (KT350597, KT350598, and KT350599) were 100% identical with many other culture collection C. luteo-olivacea sequences in GenBank (HQ661071 and KF764576) and only 71 to 80% to other Cadophora species isolated from pear (KT350601 and KT350602). 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 C. luteo-olivacea prepared from a 14-day-old culture grown on PDA (isolates PPO 11-1228, PPO 24-1234, and PPO 107-1267); and ii) with 20 µl of a spore suspension (105 conidia ml–1) prepared from a 21-day-old PDA culture after wounding with a needle (isolates PPO 11-1228 and PPO 107-1267). Both experiments were performed at 5 and 15°C, on 10 ‘Conference’ pears per isolate-temperature combination. Inoculated fruits were sealed in plastic bags and were incubated in darkness. Typical symptoms appeared 7 to 14 days and 4 to 6 weeks later, for fruits incubated at 15 and 5°C, respectively. 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 the reisolations was confirmed as C. luteo-olivacea by sequencing, thus completing Koch’s postulates. Side rot of long-term stored pears has first been reported in Oregon, United States (Bertrand et al. 1977). The primary causal fungus was identified as C. malorum (syn. Phialophora malorum) (Sugar and Spotts 1992). Recently, a skin pitting disease of kiwifruit caused by C. luteo-olivacea has been reported from Italy (Spadaro et al. 2010). To our knowledge, this is the first report of side rot disease of pear fruits caused by C. luteo-olivacea.