First report of leaf spot of Hylotelephium erythrostictum caused by Alternaria alternata in Heilongjiang province of China

Abstract

Leaf spot of garden stonecrop (Hylotelephium erythrostictum [Miq.] H. Ohba) was observed at two commercial flower nurseries in Harbin, Heilongjiang Province, China, in July of 2015, 2016, and 2017. Disease symptoms were observed on approximately 100% of the plants and 80% of leaves were infected. Initial symptoms were small, round, gray, sunken leaf spots (0.2 to 0.6 cm in diameter) with concentric rings, which occurred on the entire leaf as disease progressed. Symptoms were first observed on the lower leaves and progressed to the upper portion of the plant. Some infected leaves turned chlorotic and were covered with dark gray mycelium. Diseased leaves were collected and surface-sterilized with 0.1% HgCl₂ for 1 min, cultured on potato dextrose agar (PDA), and incubated at 26°C. After 5 days, a fungus with dark gray mycelium and black pigmentations in the medium was isolated from all the samples. Conidia were pale brown to dark brown, straight or flexuous, obclavate to obpyriform or ellipsoid, with short conical beak at the tip or beakless. The spores had smooth or verruculose surface and were 3.9 to 12.6 × 7.4 to 33.3 μm in size, with 0 to 4 transverse septa and 0 to 1 longitudinal septa. According to these morphological characteristics, the fungus was identified as Alternaria alternata (Simmons 2007; Woudenberg et al. 2013). Genomic DNA was extracted from single conidial cultures of a representative isolate ‘B.3027820’ and internal transcribed spacer regions (ITS), translation elongation factor 1-alpha gene (TEF-1a), and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were amplified using primers ITS1 and ITS4, TEF-1a, and GAPDH (White et al. 1990; Woudenberg et al. 2013). The PCR products were sequenced and deposited in GenBank (accession nos. KU203775, KY062988, and KY062989, respectively). BLAST analysis showed that they were 99% identical with A. alternata isolates As2 (JN618076), GLYK2 (KU356176), and XN2 clone A2 (KP851969), respectively. To determine pathogenicity, garden stonecrop plants (cv. Babaojingtian) were grown in 15-cm pots containing a commercial potting mix (one plant/pot). After 3 months of growing, three plants (two leaves/plant) were inoculated with 50 μl of a conidial suspension (4 × 10⁶ spores/ml) of the isolate. Plants treated with sterile distilled water served as a control. All plants were placed in a humidity chamber (>95% relative humidity) for 48 h before they were kept in a greenhouse at 22/28°C (night/day) after inoculation. All inoculated leaves showed symptoms similar to those observed in the nurseries 7 days after inoculation, while no symptoms were observed on the control plants. The fungus was reisolated from the diseased leaves and confirmed to be A. alternata based on morphological characteristics and sequence analysis. To our knowledge, this is the first report of A. alternata causing leaf spot on garden stonecrop in China. Garden stonecrop is a common ornamental crop in China, and occurrence of this disease needs to be considered for developing and implementing disease management programs in garden stonecrop production.