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First Report of Postharvest Fruit Rot of Citrus reticulata ‘Kinnow’ Caused by Penicillium expansum in Pakistan

Kinnow mandarin (Citrus reticulata ‘Kinnow’) is cultivated predominantly in Pakistan and occupies 60% of total acreage planted to citrus (Khan et al. 2016). Three Penicillium species have been reported to cause postharvest infection of citrus fruit in Pakistan, which include P. italicum (blue mold) (Akhtar et al. 2013), P. digitatum (green mold) (Khokhar and Bajwa 2014), and P. ulaiense (whisker mold) (Khan et al. 2017). In January 2017, a postharvest survey for Penicillium-infected citrus fruit was conducted in mandarin fruit markets of Faisalabad, Punjab Province, Pakistan (31°25′15″ N, 73°5′21″ E). A total of 40 fruits with symptoms and signs of infections caused by Penicillium spp. were collected and processed for pathogen isolation. The symptoms on mandarin fruit were soft lesions covered with light bluish-green colored spore masses that later expanded to cause fruit rot. Samples of infected fruit peel were excised from the margin of the lesions, surfaced sterilized with 1% NaClO, and rinsed twice with sterile distilled water. The tissues from infected fruit were cultured on potato dextrose agar (PDA) for 3 days at 25 ± 2°C, and then 40 fungal isolates were purified through single-spore culture. On PDA and Czapek yeast extract agar (CYA), colonies of 16 of these 40 isolates first appeared as white mycelial growth that later turned into greyish-green spore masses with whitish edges. The colonies from the reverse side of the plate were brownish and yellowish green on CYA and PDA, respectively, at 25 ± 2°C. The colonies were circular, sulcate, and velutinous with dense velvety mycelium. After 7 days the average colony diameter was 43 mm on CYA and 45 mm on PDA. Conidia were ellipsoidal to subglobose, smooth and thin walled, borne on monoverticillate conidiophores measuring 3.05 to 3.78 × 2.3 to 3.12 µm (n = 100). Based on these morphological characters these 16 isolates were tentatively identified as Penicillium expansum L. (Palou et al. 2013). For three of these isolates (PEN-18, PEN-20, and PEN-27), the internal transcribed region (ITS) was amplified and sequenced with ITS1/ITS4 primers (White et al. 1990). The sequences of PEN-18, PEN-20 and PEN-27 were deposited to GenBank (accession no. MH578597, MH612893, and MH513319). The isolates showed 99% similarity with P. expansum GenBank accessions KX243329.1, KX243328.1, and DQ339562.1. The isolate PEN-27 was tested for pathogenicity on five mature healthy fruits of cultivar Kinnow. The mandarin fruits were surface disinfected with 1% NaClO, wounded, and inoculated, depositing 10 µl of conidial suspension (1 × 10⁵ conidia/ml) on 1- to 2-mm wounds made by a sterilized hypodermic needle on the surface of asymptomatic fruits. Disinfested, wounded, and inoculated with 10 µl of sterile water fruits were used as controls. After 7 days of incubation at 25 ± 2°C with 90% relative humidity, inoculated fruit had symptoms and signs consistent with the original infection from which the isolate was obtained. No symptoms were observed on control fruits. The reisolation was only from symptomatic (infected) fruits. Previously, P. expansum has been reported to infect citrus fruits in Spain (Vilanova et al. 2012) and Germany (Louw and Korsten 2015). To our knowledge, this is the first report of P. expansum causing a fruit rot on mandarin cultivar Kinnow in Pakistan. The prevalence of P. expansum was 40% of the Penicillium-infected fruits, reflecting the importance of P. expansum as a postharvest pathogen in mandarin fruits in Pakistan.