Isolation and molecular identification of Curvularia lunata/Cochliobolus lunatus causal agent of leaf spot disease of cocoa.

Abstract

Cocoa is an important crop that provides to the food industry the chocolate, cocoa, and cocoa butter that are used in many applications. Globally, cocoa production is affected by fungal diseases such as frosty pod rot (Moniliophthora roreri), witches' broom (Moniliopthora perniciosa), black pod (Phytophthora spp.), and leaf spot (Curvularia lunata). Curvularia is a hyphomycete fungus, which is a facultative pathogen of many plant species. Curvularia are found in tropical regions, though a few are found in temperate zones. The teleomorphic state of the type species C. lunata is Cochliobolus lunatus. In Mexico, there aren't enough information about molecular identification of fungi cocoa diseases (i.e. M. roreri). Therefore, the objective of this study was the isolation and molecular characterization of four fungi strains that come from diseased fruits of cocoa plants. The methodology involved the molecular characterization of the strains HT-ITV03, HT-ITV40, HT-ITV45 and HT-ITV46. The amplification of the ITS I/5.8s/ITS II rDNA region of each strain was performed with the primers ITS1 and ITS4 described by White et al. (1991). The DNA sequences were analyzed with the Chromas software version 4.15 and aligned with sequences deposited in the GenBank database. Four sequences were amplified with a size of 605 bp, 598 pb, 621pb and 535 bp for strains HT-ITV03, HT-ITV40, HT-ITV45 and HT-ITV46, respectively. The partial sequences obtained for strains characterized were subjected to a search online with the program BLASTN. The Multiple alignments were carried out with ClustalX, selecting the sequences related deposited in the NCBI GenBank. The sense sequences of strains HT-ITV03, HT-ITV40, HT-ITV45 and HT-ITV46 were aligned with the sequences of C. lunatus (GQ836800, DQ836799, GQ280375) and C. hawaiiensis (AF071324) with a homology of 94%, 97%, 95% and 95% respectively. Finally, with data from the multiple alignment, was constructed the phylogenetic tree for strains characterized and the sequences of the phylogenetically related fungi. In conclusion, the molecular identification of the cocoa strains C. lunatus, C. hawaiiensis and B. tetramera confirmed its presence in the state of Tabasco, Mexico. In addition, the information obtained can be important for developing strategies for prevention and control of this fungus.