First Report of Leaf Blight Caused by Arthrinium arundinis on Tea Plants in China

Abstract

Tea (Camellia sinensis [L.] Kuntze) is one of the most important economic crops in China. During a survey in July 2018, symptoms of brown leaf patches (3 to 4 cm diameter) with irregular margins were observed on tea leaves of Longjing43 cultivar in Huoshan tea farm, Anhui province, China. The disease incidence reached 18 to 20% in the most severely affected tea gardens. Lesions first appeared as gray-brown dots, subsequently expanding to irregular dark brown, and then ended with large brown patches on the tea leaves. The infected tea leaf samples were collected from the tea plants, and the pathogenic fungus isolation was performed according to the method described by Thangaraj et al. (2018). Briefly, the tea leaf samples washed with tap water followed by distilled water. The margins of infected leaves were cut into small pieces (4 to 5 mm²) and surface sterilized with 0.1% HgCl₂ for 1 min followed by 70% ethanol for 30 s and rinsed three times in sterile distilled water. Then, these aseptic tissues were blotted dry on sterilized filter paper, transferred onto 2% water agar, and incubated in the dark at 28°C until fungal hyphae started to grow. Single hyphal tips were transferred onto potato dextrose agar (PDA) plates and incubated for 7 days at 28°C for morphological observations. A total of five isolates were obtained and identified as the same fungus on the basis of phenotypic characteristics. The colonial characters of the isolated fungus on PDA were dirty white and moderate aerial mycelium with dark colored patches. Hyphae were smooth, hyaline, branched, and septate. Conidiophores were reduced to conidiogenous cells, size ranged from 4.2 to 11.0 × 2.7 to 5.7 μm (length and width, respectively) (average 7.4 × 3.8 μm; n = 30), pale brown, smooth, and ampulliform. Conidia were 4.5 to 12.2 × 3.4 to 9.4 μm (average 6.9 × 5.0 μm; n = 50), single celled, brown, smooth, anomalous shape (globose to ellipsoid shape), mostly globose in surface view and lenticular in side view with an equatorial slit. Based on morphological characteristics, the isolated plant pathogenic fungus, named ZZZ3, was identified as Arthrinium spp. (Corda) Dyko & B. Sutton (Crous and Groenewald 2013). To identify the isolate to the species level, genomic DNA was extracted (Fungal DNA extraction kit, BIOMIGA, China) from mycelial mats. The internal transcribed spacer region (ITS1/ITS4) (White et al. 1990) and D1/D2 region of 28S rDNA (LR0R/TW13) genes (Hamayun et al. 2009) were amplified. The obtained ITS (539 bp) and 28S rDNA (585 bp) (GenBank accession nos.: ITS, MN187298; 28S rDNA, MN187305) sequence BLAST analysis showed 100 and 99% similarity with Arthrinium arundinis (ITS, KF144883; 28S rDNA, KF144929), respectively. Based on these morphological and genetic characteristics, the fungus was identified as A. arundinis. To confirm its pathogenicity, ten 2-year-old tea plants were selected, and the adaxial surface of matured leaves was injured (two needle punctures per leaf, using a sterile needle). The conidial suspension (10⁶/ml) was spread over the wounds, and sporeless sterile water was spread on wounded control leaves. After inoculation, each plant was covered by a plastic bag containing moist cotton wool for 10 days at 28°C to maintain high relative humidity (85 to 90%). At 14 days following inoculation, lesions appeared on the wounded leaves, and the symptoms were similar to those described above, whereas the control and unwounded leaves remained healthy. The experiment was performed thrice, and the same fungus was reisolated from the infected leaves, showing similar morphological characteristics and molecular traits. Wang et al. (2018) identified eight new Arthrinium spp. from different host plants in China, of which A. xenocordella and A. camelliae-sinensis were isolated from tea plants. To our knowledge, this is the first report of A. arundinis causing tea leaf blight in China. The establishment of the disease in tea plants may require the development of effective control strategies to minimize its impact on the production of tea.