First Report of Leaf Spot Caused by Stemphylium lycopersici on Zinnia elegans in China

Abstract

Zinnia elegans originated from Mexico and has been widely cultivated as an ornamental plant in China. In August 2018, leaf spots were observed on Z. elegans in Tai’an, Shandong province, China. The symptoms appeared on the edge of the leaves as small brown spots. In serious cases, the leaves were withered, which affected the ornamental value of the plants. Diseased leaf samples were cut into small segments, put onto moist filter papers within Petri dishes, and incubated at 25°C. Spores resembling Stemphylium developed from the tissues after 1 day and were singly picked up and transferred to potato dextrose agar (PDA). Morphologically, all the colonies were identical to each other, and the strain YZU 181216 was selected for further study. A pure culture of YZU 181216 was deposited in the Fungi Herbarium of Yangtze University, Jingzhou, Hubei, China. To determine the colony morphology of the species, mycelial plugs (6 mm in diameter) were transferred to fresh PDA and incubated at 23°C in darkness. After 7 days, the colony was vinaceous buff, reverse cinnamon to buff, 52 to 53 mm in size. To examine its conidial morphology, the mycelium was transferred onto potato carrot agar (PCA) and incubated at 23°C under an 8-h photoperiod for 7 days. On PCA, conidiophores were straight or curved, 88 to 179 × 3.5 to 6 µm in size; conidia were solitary, oblong, three to five transverse septa, 24 to 59 × 11.5 to 20.5 µm in size, with a conical apex and bluntly rounded base. Based on morphology, the fungus was identified as Stemphylium lycopersici (21 to 60 × 12 to 24 µm) described by Yamamoto (1960). To corroborate morphological identification, genomic DNA of the strain was extracted from 3-day-old mycelia grown on PDA. Three gene fragments, including the internal transcribed spacer rDNA region (ITS), glyceraldehydes-3-phosphate dehydrogenase (gapdh), and calmodulin (cmdA), were amplified with primer pairs ITS5/ITS4, gpd1/gpd2, and CALDF1/CALDR2, respectively (Woudenberg et al. 2017). Sequences of the strain were deposited in the GenBank with accession numbers MK895975 (ITS), MK895977 (gapdh), and MK895976 (cmdA). Relevant gene sequences of Stemphylium spp. were derived from GenBank, and the three gene sequences were combined after alignment. Maximum likelihood analysis based on the combined three gene sequences was conducted with an evolutionary model of GTR+I+G under 1,000 bootstrap replicates. The phylogenetic tree showed that the strain YZU 181216 clustered with S. lycopersici (CBS 124980 and CBS 124981) and was supported by a bootstrap value of 100%. To confirm its pathogenicity, mycelial plugs (6 mm in diameter) were inoculated on living leaves of Z. elegans. Control groups were inoculated with fresh PDA plugs. The plants were covered with plastic bags and incubated in a greenhouse at 25°C. Brown spots were observed on the leaves after 2 days. Infected tissues withered after 5 days, similar to the original symptoms that occurred in the field. No symptoms were observed on control groups. The fungus reisolated from infected leaves was identical to S. lycopersici based on conidial morphology and gapdh gene sequence. To our knowledge, this is the first report of Z. elegans leaf spot caused by S. lycopersici in China.