First Report of Coniella javanica Causing Blight on Roselle Leaves and Calyx in Guerrero, Mexico

Roselle (Hibiscus sabdariffa L.) is an annual plant, grown mainly for the use of its calyx in fresh drink production and in medicine as an antihypertensive and diuretic (Herrera-Arellano et al. 2007). During the summer of 2016, symptoms of leaf and calyx blight were observed in commercial roselle plantations located in Guerrero, the main producing state in Mexico. In December 2016, incidence of leaf and calyx blight in roselle was 100% for all production areas in Ayutla, Guerrero, México. The initial symptoms in leaves and calyces consisted of irregular, light brown leaf spots, which later increased in size causing a necrosis with the presence of pycnidia. In advanced stages of the disease, a general wilting of the plant was observed. Calyces and leaves with symptoms of the disease were collected from five fields. The samples were disinfested with 1% sodium hypochlorite, washed in autoclaved distilled water, and cultured in Petri dishes with potato dextrose agar medium. The plates were incubated at 25°C for 9 days in darkness. Forty-seven colonies that were recovered belong to the same morphotype. Fungal colonies produced white mycelium in a concentric ring pattern. Pycnidia were solitary, globose, black, and 315 to 380 μm in diameter. The conidia were unicellular, ellipsoidal to fusiform, hyaline to brown, and 11.5 to 13.5 × 3.2 to 4.6 μm. The conidiophores were light brown, densely aggregated, and ramified. These morphological characteristics were consistent with those described for Coniella javanica (Alvarez et al. 2016). A representative isolate was deposited in the Culture Collection of Phytopathogenic Fungi at the Postgrado en Fitosanidad-Fitopatología, Colegio de Postgraduados, Montecillo, as GUE-29. For molecular identification, the internal transcribed spacer (ITS) region and part of the 28S rRNA and translation elongation factor 1-α (EF1-α) genes were amplified by polymerase chain reaction and sequenced using the primer sets ITS1/ITS4 (White et al. 1990), LR0R/LR7 (Rehner and Samuels 1994), and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences were deposited in GenBank (accession nos. ITS, MH503948; 28S, MH578600; and EF1-α, MH613977). A phylogenetic analysis using maximum likelihood and including published ITS, 28S, and EF1-α sequences for C. javanica and other Coniella species was carried out. The phylogenetic analysis showed that the GUE-29 isolate belongs to the C. javanica clade. The pathogenicity test was carried out by inoculating 10 leaves and 10 calyces of 10 plants of roselle variety Tecoanapa that were 4 months old with a suspension of 10⁵ spores/ml of the representative isolate. Ten leaves and 10 calyces of 10 plants were sprayed with sterile distilled water as a control. The plants were kept in a greenhouse at 25°C for 12 days; after that time, all the plants that were inoculated with the fungus developed a leaf and calyx blight and the presence of pycnidia, whereas on the control plants no symptoms were observed. The pathogen was reisolated from plants that developed symptoms, and it was morphologically identified as C. javanica. To the best of our knowledge, this is the first report of C. javanica causing leaf and calyx blight in roselle plants in Mexico. This pathogen has previously been reported causing leaf spot on roselle leaves in Indonesia (Alvarez et al. 2016).