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

Background: Persea americana and Theobroma cacao plants can be infected by fungal species of the genus Lasiodiplodia, which cause the disease known as dieback. However, T. cacao can also be infected by Ceratocystis sp., which causes cocoa wilt. These fungi cause necrosis of the vascular tissue, leading to wilting and death of the plant. However, these symptoms are not observed in the early stages of infection, making timely diagnosis of these pathogens difficult. Therefore, in this study, a nested PCR technique was developed for the early detection of Ceratocystis sp. and Lasiodiplodia sp. from infected and asymptomatic seedling stem samples. Results: A standardized pre-treatment with glycerol and 96% ethanol to soften cocoa and avocado seedling stem tissue was effective before DNA isolation. Subsequent amplification of the actin gene from pre-treated stem tissues confirmed that this step is necessary to obtain nucleic acids free of PCR inhibitors. Furthermore, 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 according to fragment size differences, a 279 bp amplicon to samples artificially infected with C. cacaofunesta and a 397 bp amplicon to samples with L. theobromae and L. subglobosa . 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 stem seedlings.