Occurrence of Botrytis prunorum Causing Calyx-End Rot in European Pear Fruits During Cold Storage in Chile

Abstract

The European pear tree (Pyrus communis L.) is an important fruit species, with 8,217 ha concentrated in central Chile. Previously, only Botrytis cinerea has been described causing calyx-end rot in pears in Chile. During the 2016 to 2017 postharvest season, pears with symptoms of calyx-end rot were observed from a commercial packinghouse in Curico, Maule Region, after 60 days at 0°C. The rot prevalence registered during cold storage was 3, 2, and 7% for cultivars Beurre Bosc, Forelle, and Packham’s Triumph, respectively. The symptoms started in the pear calyx end as a light brown to dark brown lesion with soft watery decay toward the endocarp. To isolate the causal agent, symptomatic pear fruits (n = 72) were surface disinfected with 75% ethanol, and then small fragments (5-mm length) from rotten internal tissue were placed on Petri dishes containing PDA acidified with 0.5 ml/liter of 92% lactic acid (APDA) and incubated for 7 days at 20°C. Fifty-one isolates of Botrytis were obtained and classified according to conidia production as high sporulating (HS) and low sporulating (LS) on pea agar medium (PAM) containing per liter 160 g of pea (liquefied for 2 min in 1 liter of distilled water, adjusted to pH 6.0 using HCl), 5 g of sucrose, and 25 g of agar. The HS isolates (n = 38) were morphologically and molecularly identified as Botrytis cinerea Pers. The LS isolates (n = 13) produced white to yellowish colonies, floccose, tufted, and scarce sporulation, with a mean of 0.6 and 7.0 conidia/cm² after 7 days at 20°C on APDA and PAM media, respectively. Conidia were ovoid, hyaline, smooth, and (5.9) 11.2 ± 2.0 (14.7) × (5.3) 6.3 ± 0.6 (8.1) µm (n = 40). Conidiophores were septate, dark, and constricted at the base, hyaline and branched at the apex, and shorter than would correspond to B. cinerea. The molecular identification of six representative isolates was made by analysis of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2) genes (Staats et al. 2005). BLAST search showed 99 to 100% identity with isolate type Bpru8 of GenBank accessions KP339979 (G3PDH), KP339993 (HSP60), and KP339986 (RPB2) of Botrytis prunorum (Ferrada et al. 2016). Sequences of isolates BEP-10-1 to BEP-10-6 were deposited in GenBank (MN136239 to MN136244, MN136245 to MN136250, and MN136251 to MN136256 for G3PDH, HSP60, and RPB2, respectively). Pathogenicity testing was performed using three isolates (BEP-10-1 to BEP-10-3) in ripe pears (n = 75 fruits), cultivar Beurre Bosc. An isolate of B. cinerea (BAP-4) was included in this test. Fruits were surface disinfected (75% ethanol, 2 min), wounded with a sterile hypodermic syringe in the calyx-end area, and inoculated with 50 µl of a conidial suspension (10⁶ conidia/ml). The control fruits were inoculated with distilled water. After 10 days at 20°C in a humid chamber (>85% relative humidity), all inoculated fruits with B. prunorum developed brown, watery soft rot in the calyx-end area with lesions from 15.7 to 38.6 mm. Fruits inoculated with B. cinerea showed a lesion from 37.8 to 41.9 mm. B. prunorum isolates had a significantly lower virulence than B. cinerea (P < 0.05). Control fruit remained healthy. The pathogen was reisolated from diseased fruit and molecularly identified as B prunorum. To our knowledge, this is the first report of B. prunorum causing pear calyx-end rot in Chile and worldwide. Recently, B. prunorum was described causing kiwifruit rot in Chile (Elfar et al. 2017). These results confirm the occurrence of B. prunorum, with an isolation frequency of 25% of the total Botrytis isolates obtained during cold storage in Chile.