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First Report of Colletotrichum acutatum Causing Flower Anthracnose on Pomegranate (Punica granatum) in Southern Brazil

Bellé, C., Moccellin, R., Souza-Júnior, I. T., Maich, S. L. P., Neves, C. G., Nascimento, M. B., Barros, D. R.
Plant disease 2018 v.102 no.11 pp. 2373
Colletotrichum acutatum, DNA, Punica granatum, actin, anthracnose, chitin synthase, conidia, culture dishes, culture media, ethanol, flowers, fungi, genes, glass, glyceraldehyde-3-phosphate dehydrogenase, hyphae, internal transcribed spacers, orchards, pathogenicity, photoperiod, pomegranates, relative humidity, sequence analysis, sodium hypochlorite, tubulin, Brazil
Pomegranate (Punica granatum L.), a crop native to central Asia, has been cultivated in Brazil since the 1900s but has only recently become a commercial fruit crop (Watanabe and Oliveira 2014). In October 2017, typical symptoms of anthracnose were observed on pomegranate flowers (cv. Wonderful) in commercial orchards in Pelotas, Rio Grande do Sul state, Brazil. Initially, symptoms appeared as small, light brown, slightly sunken lesions, circular to irregular in shape. As infection continued, the lesions expanded and became dark brown to black, eventually forming orange conidial masses under high humidity, ultimately causing the flowers to abort. Flower lesion samples were surface sterilized (70% ethanol for 45 s, 1% NaClO for 2 min, rinsed three times in sterile water, and dried on sterilized filter paper) and placed on potato dextrose agar (PDA) at 25°C. The isolate produced was pale gray, with dense aerial hyphae, and was brown with concentric rings after 5 days. Conidia were hyaline, aseptate, straight, cylindrical to fusiform with both ends acute, and 11.0 to 17.2 (length) × 3.3 to 4.8 (width) μm, mean ± SD = 12.4 ± 1.9 × 3.8 ± 0.3 μm, length/width ratio = 3.2 (n = 100). Further diagnostic information was obtained by sequencing the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), histone3 (HIS3), actin (ACT), and β-tubulin (TUB2) genes of a representative isolate, using the method and primers described by Damm et al. (2012). Sequences of the studied DNA regions were submitted to GenBank (ITS, MG768913; GAPDH, MG775033; CHS-1, MG775034; HIS3, MG775035; ACT, MG775036; and TUB2, MG775037). BLAST searches showed 99 to 100% identity with sequences of Colletotrichum acutatum J.H. Simmonds (1968) (ex-type CBS 112996) (ITS, JQ005776; GAPDH, JQ948677; CHS-1, JQ005797; HIS3, JQ005818; ACT, JQ005839; and TUB2, JQ005860). Pathogenicity assays were performed on of detached flowers of pomegranate (cv. Wonderful). Flowers were surface disinfected with 0.1% NaClO for 3 min and rinsed with sterile water. A conidial suspension was adjusted to 1 × 10⁶ conidia/ml in sterile water for inoculation in 20 flowers, with atomizer. Control flowers (n = 20) were inoculated with sterile water suspension. After inoculation, the flowers were placed in glass culture dishes and maintained at 25°C in an incubator with constant relative humidity of 80% and a 12-h photoperiod. Five days later, 100% of inoculated flowers exhibited brown necrotic lesions, whereas the control flowers remained asymptomatic. The experiment was repeated. Cultures reisolated from inoculated flowers had the same morphological and molecular traits as the initial isolate. Based on the morphological characteristics and sequence analysis, the fungus was identified as C. acutatum (Simmonds 1968; Damm et al. 2012). To our knowledge, this is the first report of anthracnose of pomegranate flowers caused by C. acutatum. This disease can seriously affect pomegranate quality and yield, so effective measures should be implemented to control it.