First Report of Lasiodiplodia theobromae Causing Leaf Spot on Tea Plant in Guizhou Province of China

Abstract

Tea plant (Camellia sinensis) has been widely grown in Guizhou Province in China in recent years. A survey that was conducted from May to October 2016, in the main tea production fields in Huishui County (26.22 °N, 107.08 °E), southern Guizhou Province, revealed spots on tea leaves that were initially chlorotic and then brown, necrotic, and surrounded by slightly chlorotic halos. Spots gradually enlarged, formed larger irregular shapes, and covered the entire width of the leaves. The color of the lesion’s center changed to dark brown or black. Tea leaves with extensive lesions gradually dried and dropped off the plant. Disease incidence of leaves was estimated at 30 to 45%, depending on the field. Disease severity on a plant basis was estimated to be 32 to 38%. To identify the etiology of this disease, samples taken from the lesion margins were surface sterilized with 75% ethanol followed by 0.5% sodium hypochlorite for 30 s, rinsed with sterile water three times, and plated on potato dextrose agar (PDA). The plates were incubated at 25°C in darkness for 3 to 5 days, and then hyphal tips from the margin of a growing colony were successively transferred to fresh PDA plates to study their morphological and molecular characteristics. The fungal colonies were initially white, with sparse aerial mycelia 2 days after the inoculation, and they later turned gray, with woolly aerial mycelia 7 days after the inoculation. Globose to subglobose pycnidia were formed on 2- and 3-week-old cultures. The immature conidia were initially ovoid, hyaline, and aseptate, 25.83 (21 to 29) × 14.59 (12 to 15) μm (n = 50) single spores. Mature conidia became dark brown with striated cell walls and had a median septum. These observed morphological features are in accordance with those previously described for Lasiodiplodia theobromae (Pat.) Griffon & Maubl (Alves et al. 2008). The internal transcribed spacer (ITS1-5.8S-ITS2) and part of the translation elongation factor 1-alpha genes of the isolates were further amplified following the methods described in Alves et al. (2008) and then sequenced and deposited in GenBank. BLAST algorithm-based analyses showed 99 and 100% identities between six isolates (MH151161 and MH151160, etc.) and L. theobromae (KX270362.1 and KU886956.1, respectively). To fulfill Koch’s postulates, PDA plugs with actively growing mycelia from three isolates were placed on wounded tea leaves using a sterile needle or a sterile scalpel, covered with wet cotton, and kept under 80% relative humidity until symptom development (n = 10 for each isolate). The light brown leaf spots formed 1 to 2 days after inoculation and then gradually enlarged with the color becoming black in the center of the lesion. The spots resulted in a rapid necrosis and withered the entire leaf, as well as the top tender bud and fresh leaf, whereas the control leaves inoculated with PDA plugs without mycelia showed no symptoms. L. theobromae was consistently reisolated from inoculated leaves. L. theobromae is a plurivorous pathogen found worldwide, and it is transmitted in many ways. It has been previously reported to cause root disease in tea bushes after pruning (Petch 1923; Punithalingam 1976), foliar diseases on tea oil Camellia (Zhu et al. 2014) and on Parthenium (Kumar and Singh 2000), but not to cause disease on tea leaves in China. To our knowledge, this is the first report of L. theobromae causing leaf spot on tea plants in China. This identification will help producers in Guizhou Province seek and adopt appropriate field management measures to prevent and control this disease.