First Report of Cercospora citrullina Causing Spot Disease on Watermelon in China.

Abstract

Watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai], Cucurbitaceae family, is an important vegetable crop. It is believed to be native to Africa and is cultivated in the temperate regions of Africa, central Asia, Americas and the Mediterranean (Chomicki & Renner 2015). China is the largest producer and consumer of watermelon, with an annual production of about 60.25 million tons in 2020 (https://www.fao.org/faostat/en/#home). In May 2022, a new fungal disease was observed on the leaves, vines and fruits of watermelon (cv. Heimeiren, 8424, Qilin) with an incidence of up to 75% in greenhouses, in Gudi Industrial Park, Hanting district, Weifang City, Shandong Province, China. The symptomatic leaves, vines and fruits showed small circular black spots. The disease caused the leaves and vines to desiccate rapidly, and severely affected the fruit quality. Symptomatic leaves, vines and fruits were randomly collected, and isolations were performed from infected tissues. The edges of necrotic lesions were cut into small pieces (about 5 mm), surface sterilized with 2% NaClO for 2 min, followed by 75% ethanol for 30 s, rinsed three times in sterile distilled water and placed in Petri dishes on potato dextrose agar (PDA). The same fungus was isolated from all tissue pieces and formed colonies white fluffy on the surface, and dark gray on the reverse side after 7 days incubation at 25oC. Colonies were subcultured on PDA and Corn Meal Agar (CMA), respectively, and grew slowly (the diameter was approximately 2 cm in 10 days) on PDA showing a white edge, but they grew more rapidly on CMA (approximately 3.5 cm in diameter after 10 days incubation) showing an orange halo. Hyphae were branched, brown and smooth. Conidiophores were fasciculate, brown, straight, unbranched and measured 20.03 to 304.08 × 3.41 to 6.41 μm. Conidia were needle-shaped to clavate, colorless, erect or curved and measured 22.53 to 243.97 × 3.16 to 7.02 μm. According to these morphological characteristics, the fungus was tentatively identified as Cercospora spp. (Chupp 1954). To determine the species of the fungus, three representative isolates, UNL090101, UNL090102 and UNL090103 obtained from symptomatic leaves, vines and fruits, respectively, were characterized. The genomic DNA was extracted to amplify the nuclear ribosomal internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF-1), histone H3 (HIS), and actin (ACT) genes, using the following primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone & Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2004), ACT-512F/ACT-783R (Carbone & Kohn 1999), respectively. The ITS, EF-1, HIS, and ACT gene sequences were blasted and deposited in GenBank (accession numbers ON849061/OQ102622/OQ102623, ON890306/OQ108278/OQ108281, ON890307/OQ108279/OQ108282 and ON890308/OQ108280/OQ108283, respectively). A phylogenetic tree based on concatenated sequences of ITS-CAT-TEF-H3 from the genus Cerospora was constructed using the maximum likelihood method. Isolates from watermelon and C. citrullina formed a monophyletic group with 100% bootstrap support, which was in accordance with BLAST results. Therefore, the fungus associated to watermelon spot disease was identified as C. citrullina. To fulfill the Koch's postulates, each of the three isolates was artificially inoculated onto watermelon (cv. Qilin) detached expanded leaves, vines and fruits. Three wounds were made with sterilized entomological needles on each leaf, vine and fruit, and each wound was inoculated with 6 mm CMA medium with the fungus, and without fungus as control. All the experiments were conducted for three times. All the inoculated and control leaves were placed in an incubator and incubated at 28oC and 85% relative humidity, with a 12 h photoperiod. Three days after inoculation, the inoculated leaves showed similar symptoms to those observed on naturally infected plants, while the control leaves remained asymptomatic. C. citrullina was re-isolated from symptomatic artificially inoculated leaves and identified by microscopy and re-sequencing, thus fulfilling Koch's postulates. C. citrullina has been reported on several Cucurbitaceae plants worldwide, eg. on watermelon in South Carolina (Rennberger et al. 2019) and on Burcucumber (Sicyos angulatus L.) in Korea (Hong et al. 2014), but, to our knowledge, this is the first report of C. citrullina causing spot disease on watermelon in China.