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Phytopathogenic Mollicutes
Year : 2019, Volume : 9, Issue : 1
First page : ( 125) Last page : ( 126)
Print ISSN : 2249-4669. Online ISSN : 2249-4677.
Article DOI : 10.5958/2249-4677.2019.00063.X

Isolating intact phytoplasma particles from plants for sequencing applications and for testing host responses to phytoplasmas

Assoline Nofar1, Santos-Garcia Diego2, Naor Vered3, Bahar Ofir1,*

1Department of Plant Pathology and Weed Research, Agricultural Research Organization-Volcani Centre, Rishon LeZion, Israel

2Department of Entomology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel

3Shamir Research Institute, Katzrin, Israel

*Corresponding author e-mail: Ofir Bahar (ofirb@agri.gov.il)

Online published on 25 July, 2019.

Abstract

‘Candidatus Phytoplasma’ species are obligate plant parasites limited to the phloem tissue. The difficulties to grow phytoplasma in culture seriously impedes the study of these prokaryotes. In this study, a protocol to extract intact phytoplasmas from infected periwinkle plants using filtration, differential and density-gradient centrifugations was optimized. Confocal and transmission electron microscopy was used to visualize intact phytoplasmas in the preparation. Quantitative-PCR and DNA-seq analyses revealed that phytoplasma DNA was significantly enriched following the extraction procedure. Challenging periwinkle plants with phytoplasma preparation induced the expression of one defence-related gene. This study provides an optimized procedure for phytoplasma DNA enrichment and for extracting intact phytoplasmas that can be used to examine host responses to them.

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Keywords

Phytoplasma, DNA enrichment, sequencing, microscopy.

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