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ARS Home » Pacific West Area » Riverside, California » National Clonal Germplasm Repository for Citrus » Research » Publications at this Location » Publication #414532
Research Project: Citrus and Date Genetic Resource Conservation and Utilization

Location: National Clonal Germplasm Repository for Citrus

Title: Development of a TaqMan assay for the detection of ‘Candidatus Phytoplasma brasiliense’ and assessment by high resolution melt-curve analysis (HRMA)

Author
item LANE, JEREMY - University Of Florida
item BLOCH, MELODY - University Of Florida
item HELMICK, ERICKA - University Of Florida
item Krueger, Robert
item BAHDER, BRIAN - University Of Florida

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: ‘Candidatus Phytoplasma brasiliense’ (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp. where it causes witches broom symptoms and papaya where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. This report documents development of 2 real time PCR assays, one utilizing high resoltion curve analysis and the other utilizing TaqMan. Both assays allowed detection of CPB and did not detect non-CPB Phytoplasma isolates. These assays will allow improved monitoring of CPB and assist in developing improved understanding of CPB epidemiology.

Technical Abstract: ‘Candidatus Phytoplasma brasiliense’ (CPB) is a phytoplasma originally discovered in South America and is known to infect a wide variety of economically important crops. It is most prevalent in Hibiscus spp. where it causes witches broom symptoms and papaya where it causes bunchy top. Recently, CPB was documented for the first time in North America in a new host, globe sedge. In this study two qPCR assays are developed, one utilizing high resolution melt curve analysis (HRMA) based on the secA gene and the other a TaqMan assay based on the dnaK gene. The secA/HRMA and dnaK/TaqMana ssay successfully amplified isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI, 16SrIII and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting Tm products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C difference, easily distinguishing them by melt curves. The dnaK/TaqMan assay failed to amplify all non-CPB phytoplasma isolates in the study. The development of these assays provides a valuable tool that will significantly improve monitoring programs in Florida and will aid in developing a better fundamental understanding of the epidemiology of this phytoplasma.