Cacao (Theobroma cacao) plantations are spread throughout Venezuela, but the cultivar “Sweet cacao” or “Criollo (= Creole)” is concentrated in the states of Mérida, Táchira, and Zulia (Palma, 1953). In April 2016, brown and black sunken lesions typical of anthracnose symptoms were observed on cacao fruits in plantations from the municipalities Alberto Adriani, Andrés Bello, Antonio Pinto Salinas, Obispo Ramos de Lora, and Sucre in Mérida State, Venezuela (Figure 1). For isolation of the pathogen, fruits were cut into c. 5 mm2 sections at the healthy and diseased margin. Tissues were then surface disinfected for 30 seconds in 70% ethanol, 60 seconds in 0.5% sodium hypochlorite, washed three times with sterilised water for 60 seconds, and dried with sterilised filter paper. Disinfected samples were plated onto 2% malt extract agar and incubated at 25°C for seven days or until mycelia were observed growing from the samples. The fungal mycelium was sub-cultured onto potato dextrose agar (PDA), and hyphal tipping methods were performed to obtain pure cultures that were used for morphological analysis. Nine strains were isolated and evaluated. Colonies on PDA grew to 60–80 mm (mean = 70), in diameter in seven days, and >90 mm diameter in 10 days at 25°C. After 15 days, colonies were flat with an entire edge, with aerial mycelium that were dense, cottony, pale olivaceous grey on the surface and buff to pale luteous on the underside of the culture (Figure 2). Conidiomata were acervular with conidiophores formed on a cushion of roundish and medium olive-brown cells; setae were not observed. Conidiogenous cells were hyaline, cylindrical to ampulliform, simple, short, straight, measuring 9–14 × 1.1–1.2 µm. Conidial mass orange in colour and visible through the mycelium in the reverse of the culture. Conidia were hyaline, smooth-walled, cylindrical, aseptate, with ends rounded and often tapering slightly from the middle towards the base or only sometimes narrowed at the centre (sub-cylindrical), 8–13 × 2.4–3.8 µm, mean ± SD = 10.1 ± 1.4 × 3 ± 0.3 µm, L/W ratio = 3.4 (Figure 3). Appressoria measuring 5–9 µm diameter, terminal, subglobose or with one or two borders lobed, brown, solitary were observed (Figure 4). No chlamydospores were observed. The morphological characteristics of these isolates placed this fungus within the Colletotrichum gloeosporioides species complex (Weir et al., 2012). The glutamine synthetase (GS) and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) were amplified using the primer pairs GSLF2/GSLR1 (Liu et al., 2016) and AMF1/AMR1 (Silva et al., 2012), respectively. The ApMat sequences were deposited in GenBank (Accession Nos. MF436054-MF436062) and GS sequences (MF436063-MF436071). Maximum likelihood (ML) was evaluated with a bootstrapping method and Bayesian inference (BI) analysis was performed with MrBayes v. 3.2.6 phylogenies, both calculated in Geneious 2021.0.3.0. The posterior probabilities from ML/BI analyses (87/0.99) in the combined dataset supported the inclusion of T. cacao isolates within the Colletotrichum siamense clade (Figure 5). Therefore, using phylogenetic analyses based on sequence data from the two loci (GS and ApMat) and morphological characteristics of the fungus, we identified the isolates as C. siamense. ApMAT and GS loci provided robust support to separate all Colletotrichum spp. inside the C. gloeosporioides complex species, confirming that these loci provide higher phylogenetic resolution compared to other standard markers (Silva et al., 2012; Liu et al., 2015). Colletotrichum gloeosporioides has been reported to cause anthracnose on leaves, flowers and unripe cacao fruits in Venezuela (Reyes & Capriles de Reyes, 2000), and was also present at high incidence on fruits in the municipality of Sucre, Merida state (Parra et al., 2009). Morphological similarities between C. gloeosporioides and C. siamense, such as the orange colour of conidial masses that developed in cultures, could have led to misidentification of the causal agent of the disease previously in Venezuela. To our knowledge, this is the first report of C. siamense associated with anthracnose on cacao fruits in Venezuela. We are grateful to the U. S. Department of State Fulbright Scholar Program for supporting a one-year exchange visit and providing financial support to Prof. Mohali Castillo, ID assigned: PS00236102, as researcher in the Department Agricultural Biology, Colorado State University. Further, we thank Kristen Otto for excellent laboratory assistance.
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Oct 1, 2024
Sergey G Ermilov 
Open Access