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First Report of Banana Anthracnose Caused by Colletotrichum gloeosporioides in Ecuador

Riera, N., Ramirez-Villacis, D., Barriga-Medina, N., Alvarez-Santana, J., Herrera, K., Ruales, C., Leon-Reyes, A.
Plant disease 2019 v.103 no.4 pp. 763
Bayesian theory, Colletotrichum musae, DNA, Glomerella cingulata, Musa, actin, anthracnose, bananas, computer software, conidia, culture media, ethanol, fruits, fungi, genetic analysis, glyceraldehyde-3-phosphate dehydrogenase, internal transcribed spacers, pathogenicity, pathogens, photoperiod, phylogeny, plantations, postharvest treatment, sequence homology, Ecuador
In Ecuador, banana (Musa spp.) is the most important crop owing to its importance as a widely marketable fruit. In May 2014, in order to find pathogens observed during postharvest, banana fruit (Musa AAA Cavendish) collected from four organic plantations (under low input management) in the province of Los Ríos (Ecuador) were directly harvested (without postharvest treatment) and set in a humid chamber to evaluate the presence of disease symptoms. After 15 days banana fruit showed anthracnose-like symptoms. The symptoms observed began with small brown to reddish spots on the surface of the fruit and then became dark brown with sunken lesions. Fungal colonies with similar appearance were consistently isolated from lesions and grown on potato dextrose agar (PDA) medium at 27°C. All the isolates were subcultured applying the single-spore method. After 2 days, the isolates were grayish white at the beginning, and then they turned dark gray at the end of day 7. Under the microscope, we determined that conidia were hyaline and cylindrical to oblong with obtuse ends, with 15.8 ± 1.3 µm of length and 4.6 ± 0.5 µm of width. DNA from three independent isolates was used for further genetic analysis. The internal transcribed spacer (ITS), actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences were amplified (Weir et al. 2012). DNA sequences obtained from each marker were identical for the three isolates. Consensus sequences were built using ClustalX/MUSCLE under MEGA version 6 software (Tamura et al. 2013). BLAST search revealed 100% sequence homology with ITS (e-value, 0.0; accession KY342379), ACT (e-value, 2e-131; accession MH594288), and GAPDH (e-value, 3e-119; accession KT343752) sequences of Colletotrichum gloeosporioides. The consensus sequences were deposited in GenBank with the following accession numbers: ITS, MG564348; ACT, MH594288; and GAPDH, MH594287. A multilocus Bayesian inference phylogenetic tree (concatenated) was constructed using Beast version 1.8.4 to confirm the isolate identity (Drummond et al. 2012; Hyde et al. 2014; Weir et al. 2012). The isolate from banana clustered with isolates of C. gloeosporioides (stricto senso), which confirmed the identity of the isolate. Banana fruit were inoculated with C. gloeosporioides isolate. Fruit were surface sterilized with 70% ethanol followed by a wash of sterilized water. A wound of 1-mm diameter was made with a sterilized needle, and a drop of 5 µl of spore suspension (5.0 × 10⁵ spores/ml) was placed on each wound independently. Six bananas per treatment were tested, and 5 µl of potato dextrose broth and sterile water were used as a control. All banana fruit were incubated in a humid chamber at 27°C under 12/12-h dark-light cycle. Similar symptoms were observed in all inoculated fruit after 3 days. Control banana fruit remained symptomless. The pathogen C. gloeosporioides was reisolated from the lesions, fulfilling Koch’s postulates. The same pathogenicity assay was performed twice, showing similar results. Anthracnose in banana is known to be caused by C. musae, C. gloeosporioides, C. siamense, C. tropicale, C. chrysophilum, C. theobromicola, and C. scovillei (Kumar et al. 2017; Peres et al. 2001; Vieira et al. 2017; Zakaria et al. 2009; Zhou et al. 2017). To our knowledge, C. gloeosporioides has never been reported as pathogen causing anthracnose in banana fruit in Ecuador.