Nested PCR methodology for early detection of Lasiodiplodia sp. and Ceratocystis sp. in avocado and cocoa seedling samples

Persea americana and Theobroma cacao plants can be infected by species of the genus Lasiodiplodia , which cause the dieback disease, and T. cacao can also be infected by Ceratocystis sp., which causes cocoa wilt. These fungi begin by necrotizing the internal vascular tissues and thus, symptoms are not observed in the early stages of infection, making the diagnosis difficult. Therefore, in this study we developed a nested PCR for the early detection of Ceratocystis sp. and Lasiodiplodia sp. from infected and asymptomatic seedling stem samples. A pre-treatment with glycerol and 96% ethanol to soften lignified samples from cocoa and avocado seedlings was standardized and facilitated facilitate DNA isolation from the samples. Then, the amplification of the actin gene from the pre-treated stems confirmed that nucleic acids were free of PCR inhibitors to apply the diagnosis technique and avoid false negative results. The Cer.ITS119-F/Cer.ITS379-R and Las.ITS77-F/Las.ITS452-R primer sets designed in this study demonstrated to be specific for the detection of Ceratocystis sp. and Lasiodiplodia sp., respectively, with a sensitivity ranging from 2–4 ng/µL. By using the primers ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis sp., and ITS1/ITS4, and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia sp. in a nested-PCR, both pathogens were successfully detected directly from infected stem samples without a fungal isolation step in culture media. The results were easily interpreted by agarose electrophoresis based on fragment size differences, since a 279 bp amplicon was obtained in samples artificially infected with C. cacaofunesta and a 397 bp amplicon observed in samples with L. theobromae and L. subglobosa . In conclusion, the use of the nested PCR protocol standardized in this study with the primers sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis sp., and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia sp., allowed early diagnosis of both pathogens in asymptomatic cocoa and avocado seedling stem samples.