Spot Blight on Tobacco (Nicotiana tabacum) Caused by Stagonosporopsis cucurbitacearum in Guizhou Province of China.

Abstract

Tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves. China accounts for approximately 40% of the global tobacco production. In August 2017, small (2 to 8 mm in diameter) water-soaked lesions were observed on the leaves of tobacco cultivar Yunyan 85 in Zheng’an (Guizhou, China, 28.56 N, 107.43 E). Subsequently, the affected areas turned tan-colored and formed irregular leaf spots with conspicuous yellow borders between symptomatic and healthy tissues. The spots frequently coalesced, commonly resulting in wilting of entire leaves within 20 to 30 days. The symptoms were observed in 30% of the tobacco plants in a 3-ha field. Symptomatic leaf samples were surface sterilized with 70% ethanol for 2 min, followed by 1% NaOCl for 40 s, and then rinsed three times with sterile distilled water. These leaf pieces were placed on potato dextrose agar medium and incubated at 28°C in the dark for 5 days. Pure cultures were obtained using a modified method (Chilvers et al. 2014), and single spores were obtained after 5 days of incubation on 10% V8 medium. One isolate (H-2-3) was selected for identification. Perithecia were globose to oblate and 80 to 130 μm in diameter. Asci measured 45 to 70 × 14 to 25 µm, were straight or slightly curved, bitunicate, and clavate. Pseudoparaphyses were filiform. Ascospores were 22 to 43 × 8 to 14 µm, fusoid to ellipsoidal, uniseptate, and hyaline. Conidia were globose and 18 to 35 µm in diameter. Based on these characteristics, the fungus was identified as Stagonosporopsis cucurbitacearum (Fr.) Aveskamp, Gruyter & Verkley (syn. Phoma cucurbitacearum [Fr.] Sacc.) in Punithalingam et al. (1972). Species verification was completed with representative isolate H-2-3 by polymerase chain reaction amplification of internal transcribed spacer (ITS) rDNA and β-tubulin (TUB) with universal primers ITS1F/ITS4 and Bt2a/Bt2b (Stewart et al. 2015). The ITS region was sequenced and showed 100% identity to S. cucurbitacearum (GenBank MG195974). The TUB region was 100% homologous to those of S. cucurbitacearum (GenBank MG866169). Pathogenicity of the fungus was confirmed by performing Koch’s postulates as follows. The agar plugs of three isolates (H-1-2, H-2-1, and H-2-3) and healthy tobacco (cv. Yunyan 85) leaves were prepared. Plugs were directly placed onto the adaxial surface of a leaf, and agar plugs without fungus were handled as a control treatment. For each treatment of each isolate, three plugs were conducted. After inoculation, leaves were maintained at 25°C, 100 Lux, >70% relative humidity, and 16-h light/day, and monitored for 7 days for symptoms. Symptoms similar to the original ones occurred on the points of the leaves, whereas control leaves remained symptomless. By morphological characteristics study, S. cucurbitacearum was reisolated from all symptomatic leaves. Based on the fungal morphology and sequence analysis of the ITS and TUB, this fungus was identified as S. cucurbitacearum, which has been reported from luohanguo (Siraitia grosvenorii) (Jiang et al. 2015) and water spinach (Ipomoea aquatica) (Liu et al. 2017) in China. However, to the best of our knowledge, this is the first report of S. cucurbitacearum causing spot blight on tobacco in China, and more attention should be given to other potentially susceptible host plants in the area.