First Report of Pseudocercospora oenotherae Causing Leaf spot in Hymenocallis littoralis in China.

Abstract

Hymenocallis littoralis (Jacq.) Salisb. is a common ornamental plant in China. In November 2021, leaf spots were observed on H. littoralis in a public garden in Zhanjiang, Guangdong Province, China (21°17'25″N, 110°18'12″E). Disease incidence was 82% (n = 100 investigated plants from about 10 ha). Initial small white dots densely distributed on the leaves and gradually expanded into round lesions with purple centers typically surrounded by yellow halos. The coalescence of the individual spot eventually led to leaf wilt. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The tissue surface was disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on potato dextrose agar (PDA), and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty-eight isolates were obtained (isolation frequency = 28/4 × 10 = 70%). Three representative single-spore isolates (HPO-1, HPO-2, and HPO-3) by a single-spore isolation method (Fang. 1998) were used for further study. The colonies of isolates on PDA were olive green in 7 days at 28 °C. Conidiogenous cells were unbranched, straight to geniculate-sinuous, tapered toward the apex, and 12-19 × 3 μm (n = 20). Conidia were solitary, smooth, straight or curved, pale brown, 3-8-septate, apex acute, base truncate, and 55.3-86.5 × 2.0-3.5 μm (n = 50). The morphological characteristics were consistent with the description of Pseudocercospora oenotherae (Guo and Liu. 1992; Kirschner. 2015). For molecular identification, the colony PCR method with Taq DNA polymerase and MightyAmp DNA Polymerase (Lu et al. 2012) was used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1), and actin (ACT) loci of the isolates using primer pairs ITS1/ITS4, EF1/EF2, and ACT-512F/ACT-783R, respectively (O'Donnell et al. 1998). Their sequences were deposited in GenBank under nos. OM654573-OM654575 (ITS), OM831379-OM831381 (TEF1), and OM831349-OM831351 (ACT). A phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, and ACT that clustered the isolates with P. oenotherae (the type strain CBS 131920). Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 28 °C to 30 °C. Healthy plants of H. littoralis were grown in pots, with one plant in each pot. They were inoculated with a spore suspension (1 × 105 per mL) of the isolates and sterile distilled water (control). Sterile cotton balls were immersed in the spore suspension and sterile distilled water for about 15 s before they were adhered to the leaves for 3 days. Each isolate was inoculated with three plants (1 month old), and each plant was inoculated with two leaves. The test was performed three times. Symptom were found on the inoculated plants after 2 weeks with the disease incidence 88.89%, whereas the control plants remained healthy. The fungus was re-isolated from the infected leaves and confirmed as the same isolates by morphological and ITS analyses. No fungus was isolated from the control plants. P. oenotherae caused leaf spot on OenotherabiennisL. (Guo and Liu. 1992). H. littoralis is the second host of the fungus investigated in this study firstly (Crous, et al. 2013). Thus, this work provides an important reference for the control of this disease in the future.