Comparing qPCR and Nested PCR Diagnostic Methods for Aster Yellows Phytoplasma in Aster Leafhoppers

Abstract

The aster yellows phytoplasma (AYp) is a wall-less bacterium that causes damage in multiple crops. They are spread primarily by the aster leafhopper, Macrosteles quadrilineatus (Hemiptera: Cicadellidae). A total of 3,156 aster leafhoppers were collected during the 2014 and 2015 growing seasons in Michigan celery and carrot fields using sweep nets. The objective of this study was to test previously developed 16S rDNA phytoplasma gene primers to find the most reliable and least time-consuming method for AYp detection in leafhoppers. Nested polymerase chain reaction (PCR) was performed with universal primers P1/P7 and R16F2n/R16R2, and then, restriction enzymes AluI, MseI, and HhaI identified the phytoplasma to subgroup. Over the two years, 2.2% of samples were phytoplasma positive with nested PCR, classified in subgroups 16SrI-A or 16SrI-B. All samples were also tested with a TaqMan quantitative qPCR assay with universal phytoplasma primers and probe and 4.6% tested positive. A subset of samples were also tested with AYp-specific SYBR green qPCR, showing a >93% similarity between SYBR green and TaqMan qPCR assay results. The qPCR assays were more than two times faster than nested PCR. However, qPCR assays likely have specificity issues that need to be addressed before they can be used as a reliable method of detection for AYp in leafhoppers.