First Report of Cladosporium ramotenellum Causing Sooty Spot on Postharvest Clementines in the United States.

Abstract

In 2022, post-harvest symptoms of black spots were observed at an incidence of 2-5% on easy peeling clementines (citrus × clementina) in a fresh fruit market in Phoenix metropolitan area, Arizona. Black lesions on the fruit rind were superficial, circular, dry, and firm with gray sporulation. Gray or black aerial mycelium was also noticed on top of the black spots. Black spots were distributed over the entire fruit surface without any regular patterns. Maceration of rind and flesh was also noticed under refrigerated storage conditions. Two isolates were obtained (S13 and S14) and cultured on potato dextrose agar (PDA) at 25oC for 14 days. The colony color and texture of both isolates were identical on PDA: olivaceous black with abundant sporulation, margin entire edge to slightly undulate. Conidia were 0-1 septate and globose or ellipsoid with sizes ranging from 4-10 × 2-4 μm (n = 15). Ramoconida were also 0-1 septate and ellipsoid to cylindrical with sizes ranging from 15-30 × 2-5 μm (n = 15). Conidiophore had a width of 3-6 μm (n = 15). Isolates were identified as Cladosporium ramotenellum based on these morphological features (Bensch et al. 2012). The IDs of these two isolates were further confirmed by genomic DNA isolation, PCR amplification and sequencing of the ITS1-5S-ITS2-28S region of rDNA (V9G/LR5 de Hoog and Gerrits van den 1998, primers ITS4/ITS5 White et al.), actin gene (ACT-512F/ACT-783R, Carbone and Kohn 1999), and elongation factor 1α gene (EF1-1018F/EF1-1620R, Stielow 2015). Sequences of the two isolates were identical and thus only one sequence of each gene was deposited in the GenBank. A BLASTn search of actin sequence (233-bp, OQ185511) revealed 99.1% match with ex-type sequence EF 679538 (strain CBS:121628) of C. ramotenellum holotype (query coverage: 97%). BLASTn analysis of a portion of EF-1α gene (579-bp, OQ185512) revealed more than 99.7% similarity with sequences KU933429 (ATCC strain 16022) and MT881827 (strain 18EPLE003) of C. ramotenellum (query coverage: 100%). The ITS sequence (1519-bp, OQ236707) was identical to the ITS sequences of C. ramotenellum strains in easy peeler mandarins from Peru (Murciano et al. 2021). Pathogenicity tests were carried out twice on fresh easy-peeling clementine fruit. The inocula (1 x 105 spores/ml) were prepared in sterile distilled water containing 0.1% Tween 20 (TW) by mixing the conidial suspensions of two isolates from 7-day-old PDA cultures. Ten fruit were washed, surface sanitized with 70% ethanol, and wound-inoculated by immersing five fruit in spore suspension for 1 min. Five control fruit were wound-inoculated with TW. Inoculated and control fruit were stored in separate zip-lock bags for one week. Black spots resembling those observed on naturally infected fruit were present on inoculated fruit, while control fruit remained symptomless. C. ramotenellum was reisolated and was morphologically identical to the original isolates, thus completing Koch's postulates. C. ramotenellum has been reported as a fungal pathogen causing sooty spots on easy peeler mandarins from Peru (Murciano et al. 2021). To our knowledge, this is the first report of C. ramotenellum causing postharvest sooty spot and decay on clementines in the United States. Results show that infected fruit is potentially a pathogen source for long-distance dispersal. This occurrence was communicated to the state regulatory agencies for regulatory actions on imports of citrus fruit from countries with the occurrence of C. ramotenellum.