First report of <i>Colletotrichum siamense</i> associated with anthracnose on <i>Theobroma cacao</i> fruits in Venezuela

Cacao (Theobroma cacao) plantations are spread throughout Venezuela, but the cultivar “Sweet cacao” or “Criollo (= Creole)” is concentrated in states of Mérida, Táchira, and Zulia (Palma, 1953). In April 2016, brown black sunken lesions typical anthracnose symptoms were observed on cacao fruits from municipalities Alberto Adriani, Andrés Bello, Antonio Pinto Salinas, Obispo Ramos de Lora, Sucre Mérida State, Venezuela (Figure 1). For isolation pathogen, cut into c. 5 mm2 sections at healthy diseased margin. Tissues then surface disinfected for 30 seconds 70% ethanol, 60 0.5% sodium hypochlorite, washed three times with sterilised water seconds, dried filter paper. Disinfected samples plated onto 2% malt extract agar incubated 25°C seven days until mycelia growing samples. The fungal mycelium was sub-cultured potato dextrose (PDA), hyphal tipping methods performed to obtain pure cultures that used morphological analysis. Nine strains isolated evaluated. Colonies PDA grew 60–80 mm (mean = 70), diameter days, >90 10 25°C. After 15 colonies flat an entire edge, aerial dense, cottony, pale olivaceous grey buff luteous underside culture 2). Conidiomata acervular conidiophores formed a cushion roundish medium olive-brown cells; setae not observed. Conidiogenous cells hyaline, cylindrical ampulliform, simple, short, straight, measuring 9–14 × 1.1–1.2 µm. Conidial mass orange colour visible through reverse culture. Conidia smooth-walled, cylindrical, aseptate, ends rounded often tapering slightly middle towards base only sometimes narrowed centre (sub-cylindrical), 8–13 2.4–3.8 µm, mean ± SD 10.1 1.4 3 0.3 L/W ratio 3.4 3). Appressoria 5–9 µm diameter, terminal, subglobose one two borders lobed, brown, solitary 4). No chlamydospores characteristics these isolates placed this fungus within Colletotrichum gloeosporioides species complex (Weir et al., 2012). glutamine synthetase (GS) Apn2-Mat1-2 intergenic spacer partial mating type (Mat1-2) gene (ApMat) amplified using primer pairs GSLF2/GSLR1 (Liu 2016) AMF1/AMR1 (Silva 2012), respectively. ApMat sequences deposited GenBank (Accession Nos. MF436054-MF436062) GS (MF436063-MF436071). Maximum likelihood (ML) evaluated bootstrapping method Bayesian inference (BI) analysis MrBayes v. 3.2.6 phylogenies, both calculated Geneious 2021.0.3.0. posterior probabilities ML/BI analyses (87/0.99) combined dataset supported inclusion T. siamense clade 5). Therefore, phylogenetic based sequence data loci (GS ApMat) fungus, we identified as C. siamense. ApMAT provided robust support separate all spp. inside species, confirming provide higher resolution compared other standard markers 2012; Liu 2015). has been reported cause leaves, flowers unripe (Reyes & Capriles Reyes, 2000), also present high incidence municipality Sucre, Merida state (Parra 2009). Morphological similarities between siamense, such conidial masses developed cultures, could have led misidentification causal agent disease previously Venezuela. To our knowledge, first report associated We grateful U. S. Department State Fulbright Scholar Program supporting one-year exchange visit providing financial Prof. Mohali Castillo, ID assigned: PS00236102, researcher Agricultural Biology, Colorado University. Further, thank Kristen Otto excellent laboratory assistance.