First Report of Leaf Spot on Cherry Tomatoes Caused by Exserohilum rostratum in Hainan Province, China.

Abstract

Cherry tomatoes (Lycopersicon esculentum var. cerasiforme) is the main tomato variety planted in Hainan Province, China and is prized for its nutritional value and sweet taste (Zheng et al. 2020). During October 2020 to February 2021, a leaf spot disease was observed on cherry tomatoes (cultivar Qianxi) in Chengmai, Hainan Province. The disease incidence was approximately 40% in each of three fields in Yongfa (19°76'-21°08'N, 110°21'-110°51'E). Leaves were initially chlorotic before developing black, irregular-shaped lesions on the leaf margins or tips. After several days, lesions expanded along the mid-vein to encompass the entire leaf. Then, the affected leaves turned gray-brown, leading to defoliation. Severely affected leaves became dry and necrotic. Leaf tissues of 10 diseased plants samples collected from the fields were surface sterilized in 70% ethanol for 30 s, 0.1% HgCl2 for 30 s, rinsed thrice with sterile distilled water for 30 s, placed on a modified potato dextrose agar (PDA) with 30 mg/liter of kanamycin sulfate, and incubated at 28°C in the dark for 3 to 5 days. Three fungal isolates were obtained from the diseased leaves by single-sporing. The mycelia on PDA were white and later became gray or dark gray after 3 to 4 days. Conidia were rostrate, straight to slightly curved, ellipsoidal to narrowly obclavate, dark brown, protuberant with a darker and thicker wall at the basal end. Conidia were 4 to 12 distoseptate and measured 63.92 ± 5.77 × 13.47 ± 1.22 µm (n= 50) Conidiophores were single, cylindrical, dark brown, geniculate, with swollen conidiogenous cells containing a acircular conidial scar. Morphological characteristics of the isolates were similar to those of Exserohilum rostratum (Cardona et al. 2008). A representative isolate (FQY-7) was used for pathogenicity and genomic studies. Genomic DNA was extracted from the mycelium of a representative isolate (FQY-7). The internal transcribed spacer (ITS) region, actin (act), translation elongation factor 1-alpha (tef1-α), glyceraldehydes 3-phos-phate dehydrogenase (gapdh) and β-tubulin (tub2) genes were amplified with primers ITS1/ITS4 (White et al. 1990), Act1/Act4 (Voigt and Wöstemeyer 2000), EF1-728F/EF1-986R (Carbone and Kohn 1999), Gpd-1/Gpd-2 (Berbee et al. 1999) and T1 (O'Donnell and Cigelnik 1997) + Bt2b (Glass and Donaldson 1995). The consensus sequences (GenBank Accession No. MW036279 for ITS, MW133266 for act, MW133268 for tef1-α, MW133267 for gapdh, and MW133269 for tub2) were aligned using BLAST in GenBank obtaining 100%, 100%, 99%, 100%, and 99% identity to E. rostratum strain CBS706 (LT837842, LT837674, LT896663, LT882546, LT899350). Maximum likelihood analysis based on the combined five gene sequences was conducted under 1,000 bootstrap replicates. The Phylogenetic tree showed that FQY-7 and E. rostratum were located in one clade supported with 99% bootstrap values. Pathogenicity test was performed by depositing 10-µl droplets of a conidial suspension (1 × 106 per ml) into 5 noninoculated leaves (using a sterile needle) of 10 healthy 5-month-old cherry tomato (cv. Qianxi) plants. An equal number of artificially control leaves were received only sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 28°C with 80% humidity and observed for symptoms every day. Two weeks after inoculation, all the inoculated plants showed symptoms of black spots similar to those observed in the field. No symptoms were observed on the controls. FQY-7 was successfully re-isolated from the inoculated leaves and confirmed by morphological characterization and molecular assays as described herein. To the best of our knowledge, this is the first report of leaf spot of cherry tomatoes caused by E. rostratum in China. Confirming the existence of this pathogen in this area will be useful to adopt effective field management measures to control this disease on cherry tomatoes.