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Title: Digital PCR technology for detection of palm infecting phytoplasmas belonging to group 16SrIV that occur in Florida

item BAHDER, BRIAN - University Of Florida
item HELMICK, ERICKA - University Of Florida
item MOU, DE-FEN - University Of Florida
item HARRISON, NIGEL - University Of Florida
item Davis, Robert

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2017
Publication Date: 5/1/2018
Citation: Bahder, B.W., Helmick, E.E., Harrison, N.A., Davis, R.E. 2018. Digital PCR technology for detection of palm infecting phytoplasmas belonging to group 16SrIV that occur in Florida. Plant Disease. 102:1008-1014.

Interpretive Summary: Phytoplasmas are minute bacteria that cause damaging diseases of plants worldwide. In the U.S., plants growing in agricultural and natural ecosystems are potentially vulnerable to infection by phytoplasmas that occur outside of the country. One such plant is palm. Palm trees are found as key features of natural ecosystems, as economically important ornamental elements in landscaping, and as the source of agricultural products, but palm trees can be killed by phytoplasmas. Diverse phytoplasma species that are lethal to palms are not currently found in the U.S. To facilitate the diagnosis of palm diseases, and to prevent “exotic” palm infecting phytoplasmas from entering the U.S. in imported plant materials, we investigated polymerase chain reaction (PCR)-based technologies for improved detection of phytoplasmas. We found that a technology known as digital PCR (dPCR) offers a superior approach for sensitive detection of palm infecting phytoplasmas, compared to end-point PCR and to real time PCR, and we describe our use of the technology in this manuscript. Our work will facilitate the application of quarantine measures designed to prevent the spread of palm and other phytoplasmas across national borders. Such progress will aid the causes of food security, sustainable agricultural production, and stewardship of natural ecosystems. This report will be of interest to diagnostics laboratories, research scientists, farmers, and APHIS and quarantine agencies internationally.

Technical Abstract: Phytoplasmas are an economically important group of plant pathogens that negatively impact a wide variety of plants in agricultural and natural ecosystems. In Florida, palms are essential elements in the nursery and landscaping industries that suffer from diseases caused by phytoplasmas that are related to each other but are classified in two different subgroups, 16SrIV-A and 16SrIV-D. In this study, a TaqMan assay was developed for digital PCR (dPCR) to detect both palm-infecting phytoplasmas found in Florida. When compared to real-time PCR assays, dPCR was capable of detecting the phytoplasmas at much lower concentrations than was possible by using real-time PCR. Due to sequence identity of primer annealing regions across diverse phytoplasmas, the assay is likely to be successful for detection of a wide variety of phytoplasmas. The increased sensitivity of this dPCR assay over real-time PCR will allow for earlier detection of phytoplasmal infection in palms, as well as for screening of salivary glands of candidate insect vector species. These advantages should aid timely management decisions to reduce disease spread and rapid determinations of phytoplasma transmission by vectors.