Genetic Variability of Palm Lethal Decline Phytoplasmas in the Caribbean Basin and Florida, U.S.A., Based on a Multilocus Analysis.

Palm lethal decline phytoplasmas are an important group of plant pathogens that cause death in a variety of palm species throughout the Caribbean basin and the southeastern United States. The 16SrIV-D phytoplasma was introduced to the state of Florida, United States; it has since caused severe economic losses to the green industries of Florida and is threating natural ecosystems because of its ability to infect the native palm Sabal palmetto. In this study, the genetic variability of the 16SrIV-D phytoplasma was assessed over a 10-year period to determine if multiple introductions had occurred or if natural mutations were occurring. Furthermore, the genetic variability of the palm lethal decline phytoplasma group (16SrIV) was assessed with a multiple locus analysis using the 16S ribosomal RNA gene, the 16S-23S intergenic spacer region, and secA and groEL genes. Overall, no variability of the 16SrIV-D phytoplasma was documented in Florida over a 10-year period. The multilocus analysis showed support for three distinct species of the phytoplasma in the Caribbean basin that infect palms and further support that the 16SrIV-C from Tanzania is not closely related. Furthermore, 16SrIV-B and 16SrIV-D were found to be the same phytoplasma based on 100% identity between the two based on intergenic spacer region, secA, and groEL analysis. This study represents the first robust, multilocus analysis of palm-infecting phytoplasmas from the Caribbean and sheds light on the phylogeny and evolution of the group.

Effects of Lethal Bronzing Disease, Palm Height, and Temperature on Abundance and Monitoring of Haplaxius crudus.

Insect vector feeding preference and behavior play important roles in pathogen transmission, especially for pathogens that solely rely on insect vector transmission. This study aims to examine the effects of the 16SrIV-D phytoplasma, the causal agent of lethal bronzing (LB) disease of palms, on associated auchenorrhynchan insects. The numbers of auchenorrhynchans collected during weekly surveys during a yearlong study using yellow sticky traps were analyzed. The cumulative number of H. crudus was 4.5 times greater on phytoplasma-infected relative to non-infected palms. Other auchenorrhynchans showed no difference between phytoplasma-infected and non-infected palms or were greater on non-infected rather than on infected palms. Furthermore, we examined the effects of LB, palm height, temperature, and the interactive effects of these factors on H. crudus abundance. When the palms were infected with LB, at low temperature, H. crudus was more abundant on shorter than taller palms; however, H. crudus was more abundant on taller than shorter palms at the median and higher temperatures. These results may indicate that H. crudus prefers LB-infected palms over non-infected palms. The interactive effects of LB, palm heights, and temperature further suggest that vector monitoring and disease management should be optimized according to seasonal variation in temperature.

Effect of Oxytetracycline-Hydrochloride on Phytoplasma Titer and Symptom Progression of the 16SrIV-D Phytoplasma in Cabbage Palms from Florida.

Lethal bronzing (LB) is a phytoplasma disease of palms in Florida, U.S.A. Historically, the use of the antibiotic oxytetracycline-hydrochloride (OTC-HCl) was optimized to treat for LY, and currently label rates for OTC-HCl developed for LY are being used to treat palms preventatively against LB. Because of the economic impact of LB, assessing OTC-HCl against LB is essential for developing and optimizing management options for this disease. In this study, Sabal palmetto palms declining from LB were injected with OTC-HCl to assess efficacy on LB. Four groups of palms were selected that represented healthy palms, early symptomatic palms, palms with moderate symptoms, and palms with late-stage symptoms. Within each group, treatment palms injected with OTC-HCl and a control group with no antibiotics were tested weekly by quantitative PCR for 1 year. For asymptomatic palms, treated palms never developed symptoms or tested positive, whereas one of the control palms did. The early symptomatic palms that were treated had similar levels of phytoplasma to the control group but had much slower symptom development. Palms with moderate symptoms had no difference in titer between the treatment and the control group, but treated palms had much slower symptom development. Palms with late-stage symptoms showed no difference in phytoplasma titer or symptom progression between treated and control palms. These results suggest that label rates of OTC-HCl appear useful as a preventative against LB, but once symptoms develop, label rates cannot cure palms. In the meantime, removal of symptomatic palms is recommended.

Quantification and Distribution of the 16SrIV-D Phytoplasma in the Wild Date Palm, Phoenix sylvestris, at Different Stages of Decline Using Quantitative PCR (qPCR) Analysis.

Lethal bronzing disease (LBD) is a fatal infection in a variety of ornamental palms in Florida caused by the 16SrIV-D phytoplasma. The disease was first found in 2006 in Florida and has since spread to 31 different counties, reaching as far north as Jacksonville and as far south as Key Largo. Stakeholders (nursery personnel, landscaping personnel, and extension agents) from across the state take and send samples for analysis. To provide better sampling recommendations and reduce the time associated with the sampling process, the distribution of the 16SrIV-D phytoplasma was examined by quantitative PCR analysis in various Phoenix sylvestris that displayed different stages of decline, including early symptoms, moderate symptoms, and late symptoms. A declining Sabal palmetto was also available for analysis and examined as well. The findings of this study revealed that regardless of the stage of decline, the highest amount of phytoplasma detected was nearest to the base of the trunk and gradually decreased further up the trunk. Also, in P. sylvestris it was found that with symptoms present, the entire trunk has a systemic infection, resulting in positive reactions for every sample taken. These data are useful to stakeholders because they provide useful sampling recommendations in that only a single sample is needed when sampling a palm suspected to be infected with phytoplasma. Also, based on these results, sampling lower on the trunk is advised.

A Survey of Declining Palms (Arecaceae) With 16SrIV-D Phytoplasma to Evaluate the Distribution and Host Range in Florida.

The 16SrIV-D phytoplasma was first identified in Florida in 2006. Since its discovery, it has spread throughout most of the state. It is most prevalent in the central part of Florida, from Hillsborough County on the west coast to St. Lucie County on the east coast. The 16SrIV-D phytoplasma is the causal agent of lethal bronzing disease (LBD), which is also known as Texas Phoenix palm decline (TPPD). It affects a variety of common and economically important ornamental palm species as well as the native and ecologically important species, Sabal palmetto. It has spread into the southern portions of Florida, where the palm species diversity is higher. The aims of this survey were to document the spread of disease in terms of geographic and host range one decade after its introduction into Florida, and to assess the risk that LBD poses to the nursery and landscaping industries. The survey included samples received from stakeholders throughout the state, covering 18 counties, as well as a systematic sampling of palms at the Fort Lauderdale Research and Education Center (FLREC), where the disease is spreading actively. The findings of this survey resulted in the detection of LBD in eight new counties, including Collier, Hernando, Jefferson, Martin, Miami-Dade, Monroe, Seminole, and St. Johns, and the expansion of LBD into four new host species, Cocos nucifera, Livistona chinensis, Butia capitata, and Carpentaria acuminata. These findings are crucial for stakeholders because they highlight new hosts of 16SrIV-D phytoplasma and the geographic expansion of the disease, indicating that vigilance is needed when surveying declining palms.

Detection and Quantification of the 16SrIV-D Phytoplasma in Leaf Tissue of Common Ornamental Palm Species in Florida using qPCR and dPCR.

Lethal bronzing disease (LBD) is a lethal decline of various palm species caused by the 16SrIV-D phytoplasma. The disease was described in Texas in 2002 but found in Florida in 2006. Since its introduction, the phytoplasma has spread throughout much of the state. Typically, sampling of infected palms involves taking trunk tissue; however, in some instances this is not possible so alternative protocols are needed. In this study, phytoplasma titers were measured in different leaf tissues of infected palm in order to provide stakeholders with more sampling options. In addition, understanding the phytoplasma distribution in the palm canopy can shed light on the pathogen's biology and aid in vector studies. Three species of palm, Phoenix roebelinii, Sabal palmetto, and Syagrus romanzoffiana, were identified and confirmed positive for infection by qPCR analysis. Leaf tissue from these species that represented different stages of decay were sampled and tested by qPCR and dPCR. For each species, phytoplasma was only detectable in the spear leaf tissue that was connected directly to the apical meristem by both qPCR and dPCR. These data are useful by demonstrating that the 16SrIV-D phytoplasma appears to be restricted to the spear leaf so stakeholders who wish to sample palms but cannot sample trunk tissue due to palm size or lack of permission to drill into the trunk have an alternative tissue type to reliably sample. In addition, this information will help improve vector research by knowing where to collect insects that have a higher probably of possessing the phytoplasma.