Detecting and Differentiating Phytoplasmas Belonging to Subgroups 16SrIV-A and 16SrIV-D Associated With Lethal Declines of Palms in Florida Using qPCR and High-Resolution Melt Analysis (HRMA)

Abstract

Lethal yellowing (LY) and Texas Phoenix palm decline (TPPD) are two important phytoplasma diseases of palms in Florida. Both have been responsible for major economic losses historically and remain a constant threat to the sustainability of palm production in the landscaping and nursery industries in Florida. These two diseases cause rapid, lethal decline in afflicted palms, so rapid detection and identification is crucial to implement appropriate management strategies to reduce further spread and losses. In this study, a qPCR assay was developed to detect and identify the causal agents of LY and TPPD. Based on sequence data of the 16S gene for the 16SrIV-A phytoplasma (LY) and the 16SrIV-D phytoplasma (TPPD), two regions were identified in the gene that possessed sufficient variation to yield amplicons with measurable differences in melting temperature based on high resolution melt analysis (HRMA). One region was in the 5′ region and the other was located in the 3′ region of the gene. Products from both regions yielded amplicons with significantly different melting temperatures between the two phytoplasma strains. This research allows for the detection and identification of phytoplasmas in palms rapidly by eliminating many lengthy and post-PCR steps commonly used in phytoplasma identification.