First Report of Pseudocercospora angolensis Causing Fruit and Leaf Spot of Citrus in Ghana.

Citrus is one of the most important crops in Ghana, representing a large proportion of the fresh fruit consumed in the country. In 2004, symptoms consisting of necrotic leaf spots of about 1 cm in diameter with light brown centers and dark brown margins surrounded by a yellow halo were first observed in sweet orange (Citrus sinensis) and mandarin (C. reticulata) orchards in the Eastern Region of Ghana. Fruits with raised corky lesions of up to 3 to 4 cm in diameter with yellow halos were also observed. Affected fruit had longitudinal and transversal cracks in the rind with the internal locules exposed. Juice content in diseased fruit was strongly reduced, making them unsuitable for fresh consumption or processing. The disease expanded to the Central and Ashanti Regions, with incidences over 95% and estimated yield losses of about 50 to 90%. Symptomatic leaves and fruit collected in Kade in the Eastern Region were surface disinfested with 0.5% NaOCl for 5 min and small fragments from lesions were plated onto malt extract agar (MEA). Slow-growing fungal colonies were isolated from about 5% of the affected tissues plated after 5 days of incubation at 24 to 26°C, and were transferred to V-8 juice agar and MEA. Plates were incubated for 30 days at 24 to 26°C with 12 h of fluorescent light and 12 h of dark for morphological examination. Colonies were gray in the upper side and dark green on the underside. Conidia produced in culture were mostly solitary or in short chains of 2 to 6 spores, hyaline to pale brown, cylindrical, with rounded apex and base truncated. Conidia were 24 to 82 × 4 to 6 μm, with up to 3 to 5 transverse septa and no longisepta. The 5.8S, ITS2, and 28S ribosomal RNA (rRNA) regions were amplified using the primers ITS1 and ITS4 (3) and sequenced from DNA extracted from the isolate MC-39, obtained from sweet orange leaves in Kade (GenBank Accession No. KF111755). The sequence had 99% identity (total score 819, 85% coverage) with that of Pseudocercospora angolensis (T. Carvalho & O. Mendes) Crous & U. Braun epitype strain CBS 112933 (GU269836) (1). Pathogenicity tests were performed twice on 12-week-old detached fruit of sweet orange cv. Valencia Late of about 4 to 5 cm in diameter. Inoculations were performed using a conidial suspension (3.0 × 10 conidia/ml water) by spraying fruit to run off, brushing the rind, dipping for 6 min, or injecting 2 ml in the albedo. Twenty-two isolates were evaluated and 18 fruit were used for each inoculation technique, isolate and experiment. Fruit treated with sterile distilled water were used as controls. Inoculated fruit were maintained in humid chambers at 24 to 26°C. Disease incidence on inoculated fruit varied from 40.7% to 92.6% and severity from 2 to 3 to 3 to 11 lesions per fruit, depending on the isolate and inoculation technique. No symptoms were observed on control fruit. Fungal colonies morphologically identified as P. angolensis were reisolated from lesions on inoculated fruit, but not from asymptomatic control fruit. Based on these results, the disease was identified as Pseudocercospora fruit and leaf spot of citrus caused by P. angolensis. Until this present report, Ghana was considered one of the few countries in Central Africa that was still free of this citrus disease (2). References: (1) P. W. Crous et al. Stud. Mycol. 75:37, 2013. (2) A. A. Seif and R. J. Hillocks. Int. J. Pest Manag. 39:44, 1993. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.