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 the disease incidence about 22.5%, have been observed on about 2,000 plants in a field 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 gradually expanded 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 the pathogen at 28°C. Pure cultures were obtained by successively transferring hyphal tips to new PDA plates. A total of fifteen 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 used for further study on morphological and molecular characteristics. The fungal colony of isolate RiL-1 was about 4 cm in diameter on PDA in 5 days, dark-green with granular surface and irregular white (later turned black) edge. Conidia were single-celled, oval, and narrow end with single apical appendage, 8.9 to 12.6 µm × 4.0 to 7.5 µm (n = 40). Morphological characteristics of isolate RiL-1 were consistent with the description on Phyllosticta capitalensis Henn. (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 nos. 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. The neighbor-joining phylogenetic tree showed that isolate RiL-1 (MN548089) clustered together with P. capitalensis (MF800912) from China. In combination of the morphological and molecular characteristics, isolate RiL-1 was determined as P. capitalensis. A pathogenicity test was performed in a greenhouse at 25 to 30°C with 80% relative humidity. 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 (wound group and unwound group). Spore cotton balls were used to cover the inoculated sites on 4 plants in the wound group and 4 in the unwound group, and the same numbers of plants were treated with water cotton balls. The cotton balls were removed after 48 h. After 15 days, diseased symptoms were observed on both wound and no wound leaves, while control plants (no matter wound or no wound) remained healthy. Morphological characteristics and the ITS sequences of fungal isolates re-isolated from the diseased leaves were identical to that of RiL-1. P. capitalensis caused leaf spot on various host plants around the world (Wikee et al. 2013), including tea plant in China (Cheng et al. 2019) and oil palm in Malaysia (Abbas et al. 2019), but it has not been previously reported on castor. Thus, this is the first report of P. capitalensis causing leaf spot on castor. This study provides an important reference for the control of the disease. The epidemiology of P. capitalensis on castor should be further studied.