First Report of Cercospora nicotianae Causing Frog Eye Spot in Cigar Tobacco in Hainan, China

Cigar tobacco (Nicotiana tabacum L.), sun air-cured tobacco, originally from South America, with a main use to rolling cigar wrapper that is different from flue-cured tobacco. In April, 2018, diseased leaves were observed in cigar tobacco in some fields in Danzhou city (109.58°E, 19.53°N) and Wuzhishan city (109.52°E, 18.78°N), Hainan. 20 to 40% of plants were infected (total 8 ha), thereby affecting local leaf production. The symptoms appeared as small, circular or irregular, sunken, brown patches developing into white centers and obvious dark brown margins with necrotic spots of 0.2-0.8 cm in most middle and lower leaves at mature stage. To determine the causal agent, ten leaves from five cigar tobacco plants (cv. Nuowei 2) collected from Danzhou were used for pathogen isolation (Fig. 1). Sections of infected leaf tissues were surface-sterilized by 5% NaClO for 3 min, 70% ethanol for 40 sec, rinsed in sterilized distilled water (SDW) and then placed onto potato dextrose agar (PDA) plates, under aseptic conditions and incubated at 28°C. After 7 days, predominant and consistent colonies that were nearly circular, smooth edges, generally hard, leathery and wrinkled surface, dense aerial hyphae, producing red pigment, were obtained and purified by picking hyphal tips to PDA at 28℃ (Fig. 2). One culture, HN4-1-7 from Hainan were deposited in Chinese General Microbial Cultural Center (CGMCC, NO. 3.19604). Frogeye lesions can be coved by tiny black dots on two sides and the fruiting bodies was amphigenous. Conidiophores were bluish yellow brown and gradually lighten at tips, 0-4 knee points, apical or subapical section, 0-14 septa, measured 45.1-506.4×2.3-11.7 μm in size. Conidia were needle-shaped to clavate, colorless, erect or curved, measured 37.2-169.6×1.9-5.5μm (Fig. 2). Further comparisons were completed with CGMCC 3.19604 by PCR and BLAST sequence analyses of the patial ITS rDNA region (GenBank accession nos. MK752900), TEF gene (GenBank accession nos. MK881748), ACT gene (GenBank accession nos. MK881749), CAL gene (GenBank accession nos. MT127561), and HIS gene (GenBank accession nos. MT185579) as described by Groenewald et al (2012). The results showed high identity of all the five sequences to the Cercospora nicotianae isolates, DQ835073, DQ835099, DQ835119, DQ835146, DQ835173. Based on the microscopic observation and molecular characteristics, isolate CGMCC 3.19604 were identified as C. nicotianae. The pathogenicity of CGMCC 3.19604 was evaluated in greenhouse experiments. Twenty sixty-day old cigar tobacco leaves (cv. H211) and flue cured tobacco leaves (Honghuadajinyuan) were sprayed with hyphae suspensions of CGMCC, NO. 3.19604 until runoff, respectively, and the experiment was repeated once. For controls, leaves of two cultivars were similarly wounded and inoculated with SDW. All plants were incubated under 90% humidity and 28°C with a 12 h photoperiod/day. After 9 days, the same diseased symptom (Fig.3) was observed on inoculated leaves of cigar tobacco were identical to the natural infected leaves respectively, but not on control leaves. C. nicotianae was re-isolated from lesions by cultural and morphological characteristics, fufilling Koch's postulates. All tests were repeated once. To the best of our knowledge, this is the first report of C. nicotianae causing frog eye spot in cigar tobacco in Hainan, China. As we all known, appearance integrity and uniformity are playing an important role in high quality of cigar-wrapper, but the incidence of frog eye spot can seriously affect appearance quality of cigar-wrapper and led to increase direct losses to local cigar tobacco production. In addition, symptom of frog eye spot is similar to brown spot disease caused by Alternaria alternata, often cause their symptoms confused and then delay prevention at the right time. Since the cigar tobacco is a major industry in Hainan, better understanding of its diseases is relevant in order to establish disease control strategies.

Keywords: Cercospora nicotianae; Cigar tobacco; Frog Eye Spot.