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<p><i>Persea americana</i> and <i>Theobroma cacao</i> plants can be infected by species of the genus <i>Lasiodiplodia,</i> which cause the dieback disease, and <i>T. cacao</i> can also be infected by <i>Ceratocystis</i> 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 <i>Ceratocystis</i> and <i>Lasiodiplodia</i>. 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 added to facilitate DNA isolation from the samples. Then, the amplification of the <i>actin</i> 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 <i>Ceratocystis</i> and <i>Lasiodiplodia</i>, respectively, with a sensitivity ranging from 2–4 ng/μL. By using the primers ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for <i>Ceratocystis</i>, and ITS1/ITS4, and Las.ITS77-F/Las.ITS452-R for <i>Lasiodiplodia</i> 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 <i>C. cacaofunesta</i> and a 397 bp amplicon observed in samples with <i>L. theobromae</i> and <i>L. subglobosa</i>. In conclusion, the use of the nested PCR protocol with the primer sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for <i>Ceratocystis</i> and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for <i>Lasiodiplodia</i>, allowed early diagnosis of both pathogens in asymptomatic cocoa and avocado seedling stems.</p>

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Nested PCR for early detection of Lasiodiplodia and Ceratocystis in avocado and cocoa seedlings

  • 期刊论文

  • 发表日期：

    2026年5月7日

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  • Laura Valentina Laverde-Arias,
  • Yeirme Yaneth Jaimes-Suarez,
  • Adriana González-Almario

摘要

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 and Lasiodiplodia. 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 added to 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 and Lasiodiplodia, 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, and ITS1/ITS4, and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia 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 with the primer sets ITS1/ITS4 and Cer.ITS119-F/Cer.ITS379-R for Ceratocystis and ITS1/ITS4 and Las.ITS77-F/Las.ITS452-R for Lasiodiplodia, allowed early diagnosis of both pathogens in asymptomatic cocoa and avocado seedling stems.