First Report of Penicillium ulaiense Causing Postharvest Whisker Mold of Oranges (Citrus sinensis) in Spain

Abstract

In a survey of postharvest losses in citrus packinghouses located in Valencia province (Spain) conducted during the 2017 to 2018 citrus season, mold symptoms distinctive from those of the most frequent and well-known citrus green and blue molds (caused by Penicillium digitatum [Pers.] Sacc. and Penicillium italicum Wehmer, respectively) were observed in about 1 to 2% of the sampled molded orange fruits (Citrus sinensis L.). Distinctive symptoms on sporulated soft lesions were the grayish blue color of the conidial masses and the presence of whisker-like coremia with tall whitish stalks. This whisker mold was often found in mixed infections with green and/or blue molds in the same fruit. The potential causal agent (isolate IVIA NAV-1) was transferred to potato dextrose agar (PDA) plates and purified by consecutive replating after 7 days of incubation at 25°C. At this time, colonies were mostly circular, compact, 30 to 40 mm in diameter, with dense velvety grayish mycelium and abundant bluish conidia. Conidiophores were sinoid, mostly terverticillate, and conidia were cylindrical to ellipsoidal, smooth, and thin walled, measuring 4.8 to 8.4 × 2.0 to 3.2 μm. Based on these morphological characteristics, the isolate IVIA NAV-1 was tentatively identified as Penicillium ulaiense H.M. Hsieh, H.J. Su & Tzean (Frisvad and Samson 2004). To confirm the identity, molecular identification was performed in a specialized laboratory. The rDNA internal transcribed spacer regions ITS 1 and ITS 2 and the β-tubulin gene were amplified, sequenced, and deposited in GenBank (accessions nos. MH477872 and MH477873, respectively). A BLAST search for ITS showed 100% identity and 100% query coverage with P. ulaiense strain CBS 210.92 (accession no. KC411695.1). A BLAST search for the β-tubulin gene showed 99% identity and 100% query coverage with P. ulaiense isolate 1399 (accession no. LT629297.1). Selected healthy oranges of cultivar Valencia were surface disinfected by dipping in 0.5% sodium hypochlorite for 2 min followed by thorough rinsing in deionized water. Pathogenicity was tested by pipetting 30 µl of a spore suspension (1 × 10⁶ conidia/ml), prepared from 7- to 10-day-old PDA cultures, onto fresh rind wounds, which were made on the equatorial area of disinfected fruit using a sterile, stainless steel rod with a probe tip of 1 mm in width × 2 mm in length (one wound per fruit, total of 20 oranges). Additionally, 10 disinfected, wounded, noninoculated oranges were used as controls. The same procedure was repeated 15 days later with another lot of Valencia oranges. In both tests, disease symptoms were observed on all inoculated fruit after 7 days of incubation at 25°C, and P. ulaiense was consistently reisolated, thereby fulfilling Koch’s postulates. No decay was observed on any of the control fruit. To our knowledge, this is the first report of P. ulaiense causing whisker mold of citrus fruit in Spain. The disease has been previously reported in important citrus-producing areas such as California (Holmes et al. 1993), Egypt (Youssef et al. 2010), Pakistan (Khan et al. 2017), and Korea (Park et al. 2018).