First report of Didymella macrostoma causing calyx-end rot of pear (Pyrus communis) in The Netherlands.

ABSTRACT

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 400,000 tons in 2020, and 'Conference' is the main pear cultivar that comprises 80% of total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage (-0.5°C) up to 11 months after harvest. Calyx-end rot incidences of 1% to 5% were observed on 'Conference' pears from different orchards in surveys from 2019-2021 in packing houses in the Netherlands. Infections showed 1 to 3 cm brown necrosis. Lesions were round, slightly sunken and next to or including part of the calyx. 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) (Oxoid, UK). The PDA plates were incubated at 20°C in the dark, and hyphal tip isolates were transferred to fresh PDA plates. Colonies on PDA were rosy-whitish to peach-colored. Colonies grown on oat meal agar (OA) under UV light were peach to red color, aerial mycelium sparse, and produced a pink to salmon colored conidial matrix. Conidia were irregular-ellipsoidal to allantoid, smooth, hyaline and usually with one or several gutulles. Conidia were sometimes one septate and measured 15.2±2.8 x 4.0±0.7 µm (n =14), but mostly aseptate and measured 7.9±1.7 x 3.2±0.6 µm (n =100). The fungus was morphologically identical to Didymella macrostoma (syn. Phoma macrostoma) (Boerema et al. 2004; Hou et al. 2020). The identity of four representative isolates, WURR-206, WURR-223, WURR-227 and WURR-308, from affected pears from four orchards in the Netherlands, was determined by 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, MA). Sequences of the internal transcribed spacer (ITS) region of ribosomal DNA, the large-subunit rRNA (LSU) region, partial sequences of beta-tubulin (TUB) and the translation elongation factor 1-alpha (TEF1) gene region were amplified with primers ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and EF1-983F/EF1-1567R (Rehner and Buckley 2005), respectively. Sequences were deposited under GenBank accession numbers ON077588-ON077591 (ITS), ON113487-ON113490 (LSU), ON098515-ON098518 (TUB) and ON098519-ON098522 (TEF1). MegaBLAST analysis revealed that the ITS, LSU, TUB sequences matched with 100% identity to culture collection sequences of Didymella macrostoma in GenBank MH854841 (ITS), MH866341 (LSU), MN983895 (TUB). The TEF1 sequences matched with 99.7% identity to TEF sequence of Didymella macrostoma MT454020. Subsequently, Koch's postulates were performed on 10 'Conference' pears per isolate (WURR-206, WURR-223, WURR-227 and WURR-308). Fruits wiped with 70% ethanol were inoculated in pathogenicity tests with an agar disk (5 mm diameter) of D. macrostoma prepared from the actively growing edge of 14-day-old cultures grown on PDA. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared 7-10 days after inoculation on all pears. PDA-only controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. The identity of the re-isolations was confirmed as D. macrostoma by sequencing, thus completing Koch's postulates. To the best of our knowledge, this is the first report of D. macrostoma causing calyx-end rot of pears. The identification of this causal agent is important knowledge necessary for developing control measures for postharvest diseases of pear.