First Report of Pythium cylindrosporum Causing Black Leg on Geraniums in China

Geranium has been widely used in garden landscaping for its rich colors, long florescence, and easy reproduction. During the fall season of 2014 and 2015, the geranium seedlings in a nursery garden in the northern suburbs of Kunming City, Yunnan Province, showed symptoms of black leg. Five cultivars of geranium (Pelargonium × hortorum) including ‘Horizon’ and ‘Divas’ suffered, showing a maximum morbidity rate of 50%. The basal stems of the plant turned black and were water-soaked, and its leaves turned yellow from the bottom up. As the disease progressed, the affected plants wilted and collapsed. The diseased basal stem tissue was rinsed under running water, blotted dry, and cut into 0.5-cm pieces. The pieces were plated on the semiselective medium for oomycetes (17 g/liter of corn meal agar amended with nystatin 15 mg/liter, ampicillin 250 mg/liter, rifampicin 10 mg/liter, and miconazole 1 mg/liter) (Morita and Tojo 2007) and incubated at 25°C for 3 days. Colonies resembling Pythium sp. were transferred to obtain pure cultures. Sexual propagule of oomycetes was induced by the grass-leaf method and observed under a microscope (Yin et al. 2016). The colony developed white, cottony, and aseptate hyphae that were 2.3 to 6.8 μm (mean 4.9 μm) wide. Oogonia were globose with a smooth surface and a diameter ranging from 11.5 to 22.3 μm (mean 17.4 μm). Antheridia were clavate, whereas oospores were globose, with diameter ranging from 12.8 to 19.1 μm (mean 16.7 μm). According to the morphological features, the microorganism was identified as a Pythium sp. (van der Plaats-Niterink 1981). DNA was extracted respectively from mycelia of isolates G1 and G2. The internal transcribed spacer (ITS) region of ribosomal DNA was amplified using universal primers ITS1/ITS4 and sequenced. The BLAST analysis of the sequences (GenBank accession nos. MF144550 and MF919631) showed 100 and 99.9% similarity to the type strain of Pythium cylindrosporum (CBS 218.94, GenBank no. AY598643). Pathogenicity was tested on Divas geranium cuttings (approximately 10 cm in length). Wheat kernel colonized by the isolate G1 of P. cylindrosporum served as the inoculum. Five wheat kernels were buried approximately 2 cm deep in steam-disinfested soil (121°C for 30 min) in 10-cm-diameter pots. The soil was incubated for 48 h under 25°C, and geranium cuttings were inserted into inoculated soil about 2 cm deep. Each pot was inoculated with three cuttings, and four replicates were used (one pot per replicate). The cuttings were planted in noninoculated soil as a control treatment. Plants were kept in a greenhouse at 23 to 25°C and were irrigated daily to maintain the potting soil at field capacity. On the seventh day of inoculation, the basal stem of geranium turned black and hygrophanous, consistent with the behavior of the stems in the nursery garden. No symptoms were observed in the control group. P. cylindrosporum was reisolated consistently from the symptomatic basal stem of geranium. There are several Pythium spp. reported on this host; only P. ultimum and P. aphanidermatum have been reported in China (Yin et al. 2016). To our knowledge, this is the first report of P. cylindrosporum causing black leg of geranium in China. This pathogen may present a threat to the seedling production of geraniums in this area. Thus, identifying the cause of this disease is necessary to develop effective management strategies.