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Simultaneous detection of coconut lethal yellowing phytoplasmas (group 16SrIV) by real-time PCR assays using 16Sr- and GroEL-based TaqMan probes

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Abstract

Lethal yellowing (LY) represents a serious threat to coconut in Mexico, Central America and the Caribbean islands. It is caused by a phytoplasma of group 16SrIV, and particularly subgroups -A and -D have been the major phytoplasmas affecting coconut palms grown in the southeast of Mexico. Therefore, is important to reliably detect the 16SrIV group of phytoplasmas not only for diagnosis purposes but also to improve the understanding of pathogen-plant-vector pathosystems. The 16S ribosomal operon genes have been particularly used as the targets for probes and primers for universal phytoplasma detection. However, its conservative nature has imposed the search of non-ribosomal genes to support a finer characterization and differentiation of phytoplasmas, particularly those closely related and one alternative has been the GroEL gene for the specific detection of subgroups 16SrIV-A and 16SrIV-D. Sensitive, rapid and reliable singleplex and duplex assays were developed based on real-time PCR (TaqMan) targeting both the GroEL gene and 16S rRNA. The assay performed with higher sensitivity compared to conventional nested-PCR when analysing DNA extracts obtained from LY symptom bearing palms. The LY 16S TaqMan probe assays amplified DNA from both subgroups, while the LY GroEL TaqMan probe assay only amplified DNA from subgroup 16SrIV-A. The duplex TaqMan probe assay is a novel option for the simultaneous detection and quantification of LY phytoplasmas of 16Sr subgroups -A and -D, which are the phytoplasmas that predominantly affect palms in Mexico.

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Funding

This research was partially funded by CONACYT (Grant No. CB 129717) and the Common Fund for Commodities, Stadhouderskade 55,1072 AB Amsterdam (FIG00/22).

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Authors and Affiliations

  1. Centro de Investigación Científica de Yucatán, Biotechnology Unit, Calle 43 No. 130. Colonia Chuburna de Hidalgo, 97200, Mérida, Yucatán, Mexico

    Iván Córdova, Carlos Oropeza, Sandra Ku-Rodríguez, Carlos Puch-Hau, María Narváez & Luis Sáenz

  2. Plant Pathology Department, Fort Lauderdale Research and Education Center, University of Florida, 3205 College Avenue, Fort Lauderdale, FL, 33314, USA

    Nigel Harrison

Authors
  1. Iván Córdova
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  2. Carlos Oropeza
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  3. Nigel Harrison
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  4. Sandra Ku-Rodríguez
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  5. Carlos Puch-Hau
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  6. María Narváez
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  7. Luis Sáenz
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Correspondence to Luis Sáenz.

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Conflict of interest

I. Cordova declares that he has no conflict of interest. C. Oropeza declares that he has no conflict of interest. N. Harrison declares that he has no conflict of interest. S. Ku-Rodríguez declares that she has no conflict of interest. C. Puch- Hau declares that he has no conflict of interest. M. Narvaez declares that she has no conflict of interest. L. Sáenz declares that he has no conflict of interest.

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Córdova, I., Oropeza, C., Harrison, N. et al. Simultaneous detection of coconut lethal yellowing phytoplasmas (group 16SrIV) by real-time PCR assays using 16Sr- and GroEL-based TaqMan probes. J Plant Pathol 101, 609–619 (2019). https://doi.org/10.1007/s42161-019-00249-y

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