First Report of Castor Dark Leaf Spot Caused by Phyllosticta capitalensis in Zhanjiang, China

Abstract

Castor (Ricinus communis L.) is an important oil crop. Leaf spots on castor with disease incidence of about 22.5% have been observed on about 2,000 plants in a field located in Zhanjiang (21.17N, 110.18E), China, since 2016. Initial leaf symptoms were round spots with gray centers, surrounded by yellow halos. Ultimately, the spots then gradually spread and merged. Six samples of symptomatic leaves were collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 15 isolates were obtained from the affected leaves. Two single-spore isolates (RiL-1 and RiL-2) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate RiL-1 was selected for further study on the morphological and molecular characteristics. The colony of isolate RiL-1 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white (later turning black) edge. Conidia were single-celled, oval, narrow at the end with a single apical appendage, and 8.9 to 12.6 × 4.0 to 7.5 µm (n = 40). Morphological characteristics of isolate RiL-1 were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), and actin (ACT) were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R (Druzhinina et al. 2005), and ACT-512F/ACT-783R (Wikee et al. 2013), respectively. Sequences were deposited in GenBank with accession numbers MN548089 for ITS, MN565576 for tef1-α, and MN565573 for ACT. BLASTn analysis demonstrated that these sequences were 99 to 100% similar to the corresponding ITS (MF170677), tef1-α (KU716086), and ACT (KY855659) sequences of P. capitalensis. Also, by the neighbor-joining method, a phylogenetic tree showed that isolate RiL-1 (MN548089) clustered together with P. capitalensis (MF800912) from China. From the combination of the morphological and molecular characteristics, isolate RiL-1 was determined to be P. capitalensis. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Sterilized cotton balls were immersed in spore suspension (1 × 10⁶ spores/ml) or sterile water for 1 min. Sixteen healthy plants were divided into two groups (wounded group and unwounded group). Spore-containing cotton balls covered the inoculated sites on four plants in the wounded group and four in the unwounded group, with the same procedure for the water-only cotton balls, and kept for 48 h before removing the cotton. After 15 days, disease symptoms were observed on both wounded and unwounded leaves, whereas control plants (both wounded and unwounded) remained healthy. Morphological characteristics and the ITS sequences of the fungal isolates reisolated from the diseased leaves were identical to those of RiL-1. P. capitalensis has caused leaf spot on various host plants around the world (Wikee et al. 2013), including on tea plant in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on castor. Thus, this is the first report of P. capitalensis causing leaf spot on castor. This study will provide an important reference for the control of the disease. The epidemiology of P. capitalensis on castor should be investigated in further research.