DNA degradation in Haplaxius crudus (Hemiptera: Cixiidae) and Diaphorina citri (Hemiptera: Liviidae) on yellow sticky traps in Florida.

Lethal bronzing (LB) and huanglongbing (HLB) are harmful plant diseases causing significant economic losses in Florida agriculture. Both diseases are caused by bacteria that are transmitted by Hemipteran insect vectors. Accurate detection of pathogens within insect vectors can help provide a better understanding of disease epidemiology. Monitoring of the vector of LB is done primarily using sticky traps within palm canopies. However, it is unknown how long pathogen and vector DNA remain intact under field conditions. If significant DNA degradation takes place over the course of days or weeks, there is a possibility of false negatives occurring when detecting pathogens from these surveys. This study determined how long Haplaxius crudus Van Duzee (Hemiptera: Cixiidae) and LB DNA could remain detectable on sticky traps under field conditions in Florida in winter and summer, using PCR and qPCR. Additionally, this study compared the DNA degradation of Diaphorina citri Kuwayama (Hemiptera: Liviidae) and Candidatus Liberibacter asiaticus (CLas), the causal agent of HLB. The results showed that DNA concentration and amplification rate declined as time on sticky traps increased. Degradation varied between different target genes. The amplification rate of insect genes from sticky trap samples suggests that sticky traps should be changed weekly in summer, and every 2 wk in winter for accurate H. crudus detection. Traps should be changed every 4 days for phytoplasma detection. Traps can be changed monthly for accurate D. citri and CLas detection. Based on these results, standard monitoring protocols can be implemented to more accurately detect vectors and pathogens.

Differentiation of Palm-Infecting Phytoplasmas in the Caribbean Basin Using High Resolution Melt Curve Analysis of the secA Gene.

Palm lethal decline is a disease that is always fatal to infected palm hosts and is caused by three species of phytoplasma in the Caribbean basin: 'Candidatus Phytoplasma palmae', 'Ca. P. aculeata', and 'Ca. P. hispanola'. Movement of these pathogens throughout the Caribbean has been documented since their discovery in Jamaica. Over time, means of confirming infections in palms have improved. Current protocols utilize quantitative PCR (qPCR) for rapid amplification and distinction of these phytoplasmas using TaqMan probes and high-resolution melt-curve analysis (HRMA) of the 16S rRNA gene. These assays either do not detect all three phytoplasmas (HRMA) or do not distinguish between the three (TaqMan). In this study, a new qPCR-HRMA assay is developed that amplifies and distinguishes all three phytoplasmas currently known to kill palms in the Caribbean. Efficiency for the primer set secA614_F/secA759_R was shown to be consistent for all species at each concentration and yielded distinct melting temperature ranges for amplicons of 'Ca. P. palmae' (73.3 to 73.4°C), 'Ca. P. aculeata' (72.9 to 73.0°C), and 'Ca. P. hispanola' (73.5 to 73.6°C). This assay is a useful new tool not only for diagnostics that will contribute to monitoring and management programs, but it will also aid in basic research by allowing rapid screening of large samples in the context of vector surveys or identification of reservoir hosts.