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

Abstract

<jats:p> 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. </jats:p>

Effect of azoxystrobin on tobacco leaf microbial composition and diversity

“…This is mostly related to the decline in the abundance of the target spot pathogen Thanatephorus at that time, which comprised a large proportion of the fungal microbiome at 0 dpt. Other genera of possible tobacco pathogens that showed declines were Cercospora which could include C. nicotianae that causes frogeye spot of tobacco ( Zhao et al., 2020 ), Phoma which could include P. sorghina that causes leaf spot of tobacco ( Yuan et al., 2016 ), Cladosporium that could include C. cladosporioides that causes seed infections of tobacco ( Wang et al., 2014 ) and Fusarium that could include F. oxysporum that causes fusarium wilt of tobacco ( LaMondia, 2015 ). For the other three trophic modes, it was notable that there were large increases in abundance of all of them only on diseased leaves at 9 or 18 dpt, whereas they remained low on healthy leaves.…”

Spatio-Temporal Variation in the Phyllospheric Microbial Biodiversity of Alternaria Alternata-Infected Tobacco Foliage

“…Crops may pose a risk of opportunistic fungal infection development in patients who are immunocompromised as the potential pathogenicity of those fungal crop contaminants cannot be disregarded (Ashley et al, 2018). The data signifies the presence of a large number of fungi; having phyto-pathogenic potential, the fungi are associated with tobacco phyllosphere during leaf senescence, for instance, the facultative necrotrophs, including A. alternata (the causal agent of brown spot) (Wang et al, 2016), Cercospora nicotianae that causes frogeye both on flue-cured and cigar tobacco (Fajola and Alasoadura, 1973;Zhao et al, 2020), Phoma omnivirens (the causal agent of black spot stalk) (Jiang et al, 2018), Stagonosporopsis cucurbitacearum that causes spot blight on tobacco (Wang et al, 2018), and D. segeticola that causes tobacco leaf spot (Xiang et al, 2020), along with the obligate biotroph, G. cichoracearum (the causal agent of powdery mildew) (Chen et al, 2019). Probably, those pathogens had arrived on tobacco leaves from diseased tissues in the field, whereas some could be growing either pathogenically inside the leaves or saprophytically on leaf surfaces during plant growth (Laforest-Lapointe et al, 2016).…”

Endosphere Microbiome and Metabolic Differences Between the Spots and Green Parts of Tricyrtis macropoda Leaves

“…Cercospora is believed to cause the formation of leaf spots in plants and eventually lead to leaf spot disease (Albert and Charles, 1950;Heng et al, 2020). Cercospora can cause necrotic damage to the leaves, thus leading to suborbicular, oil-stained brown spots on the leaf surface (Chupp, 1954;Xie et al, 2017), and Cercospora can cause frog eye spots in cigar tobacco in Hainan (Zhao et al, 2020). Therefore, we speculate that the formation of leaf spots may be closely related to the colonization of leaves by fungi.…”