Development of a TaqMan assay for the detection of 'Candidatus Phytoplasma brasiliense' and assessment by high resolution melt-curve analysis (HRMA).

'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.

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.

Acquisition and Transmission of the Lethal Bronzing Phytoplasma by Haplaxius crudus Using Infected Palm Spear Leaves and Artificial Feeding Media.

Lethal bronzing (LB) is a fatal palm disease caused by the phytoplasma 'Candidatus Phytoplasma aculeata'. This disease causes significant economic losses in palm industries and landscapes. The American palm cixiid, Haplaxius crudus, recently was identified as the vector of the phytoplasma. However, knowledge about LB phytoplasma transmission is limited due to the lack of a method to generate phytoplasma-infected insects in the laboratory. In this study, the acquisition and transmission of the LB phytoplasma by H. crudus were investigated. Successful acquisitions of the phytoplasma by H. crudus were observed at 2 days acquisition access period on LB-infected palm spear leaves. Analyses revealed increased phytoplasma infection rates of H. crudus with longer acquisition access periods and latent periods. A significantly higher phytoplasma infection rate was shown after various acquisition access periods and latent periods than the infection rate of the field-collected H. crudus population. Transmission of the phytoplasma from LB-infected spear leaves to sucrose media by H. crudus also was observed using digital PCR assays. These results further support the vector status of H. crudus and offer valuable information to understand LB phytoplasma transmission. Additionally, these results generate a critical baseline for future LB phytoplasma-vector research by providing a way to generate vectors with high phytoplasma infection rates in the laboratory setting.

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.

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.

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.

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.

The CpnClassiPhyR Is a Resource for cpn60 Universal Target-Based Classification of Phytoplasmas.

Phytoplasmas are plant-pathogenic bacteria that are associated with yield losses in many crop plants worldwide. Phytoplasma strain differentiation is accomplished using in silico restriction fragment length polymorphism (RFLP) analysis of 16S ribosomal RNA-encoding gene sequences, which has resulted in the definition of ribosomal groups and subgroups of phytoplasmas. Due to limitations associated with this approach, a complementary classification scheme was recently developed based on RFLP analysis of the single-copy, protein-encoding gene chaperonin-60 (cpn60). We present the CpnClassiPhyR, software that facilitates phytoplasma strain classification using both RFLP and automated phylogenetic analysis of cpn60 sequences. This software is available through a web interface at http://cpnclassiphyr.ca.

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.

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.

Digital PCR Technology for Detection of Palm-Infecting Phytoplasmas Belonging to Group 16SrIV that Occur in Florida.

Phytoplasmas are an economically important group of plant pathogens that negatively impact a wide variety of plants in agricultural and natural ecosystems. In Florida, palm trees are essential elements in the nursery and landscaping industries that suffer from diseases caused by phytoplasmas that are related to each other but are classified in two different subgroups, 16SrIV-A and 16SrIV-D. In this study, a TaqMan assay was developed for digital polymerase chain reaction (dPCR) to detect both palm-infecting phytoplasmas found in Florida. When compared with real-time PCR assays and nested PCR assays, dPCR was capable of detecting the phytoplasmas at much lower concentrations than was possible by using real-time PCR and nested PCR. Additionally, the assay was capable of detecting 16SrIV-B phytoplasma as well as isolates representing the 16SrI and 16SrIII phytoplasma groups. Due to sequence identity of primer annealing regions across diverse phytoplasmas, the assay is likely to be successful for detection of a wide variety of phytoplasmas. The increased sensitivity of this dPCR assay over real-time PCR will allow for earlier detection of phytoplasma infection in palm trees, as well as for screening of salivary glands of candidate insect vector species. These advantages should aid timely management decisions to reduce disease spread and rapid determination of phytoplasma transmission by vectors.

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).

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.

Phylogeny of Geminivirus Coat Protein Sequences and Digital PCR Aid in Identifying Spissistilus festinus as a Vector of Grapevine red blotch-associated virus.

Grapevine red blotch-associated virus (GRBaV) is a single-stranded DNA virus, proposed to be a member of the family Geminiviridae, and is associated with grapevines showing red blotch symptoms in North America. The existence of the virus was reported in 2012, and subsequently detected in grapevines in major grape production regions. We investigated if a vector exists that can transmit GRBaV in vineyards. Phylogenetic analysis of the predicted amino acid sequence of coat protein (CP) of GRBaV with the CP of 23 geminiviruses representing all seven genera of the family Geminiviridae revealed that GRBaV-CP was most similar to that of Tomato pseudo-curly top virus, a geminivirus known to be transmitted by a treehopper (Membracidae), a family that is closely related to leafhoppers (Cicadellidae). To identify vectors of GRBaV, hemipteran species within and nearby wine grape vineyards where virus spread was suspected were collected and transmission assays were conducted. Among the species tested, the three-cornered alfalfa hopper Spissistilus festinus (Hemiptera: Membracidae) was able to both acquire the virus from a grapevine infected with GRBaV and transmit the virus to healthy grapevines in the laboratory. In commercial vineyards, lateral shoots of grapevines girdled due to feeding injury by the adult three-cornered alfalfa hopper also tested positive for the virus using digital PCR. These findings represent an important step in understanding the biology of GRBaV and develop management guidelines.

An Evaluation of the Flora Adjacent to Wine Grape Vineyards for the Presence of Alternative Host Plants of Grapevine red blotch-associated virus.

Grapevine red blotch-associated virus (GRBaV) is a recently discovered virus of concern to wine grape production in North America. While the vector of this virus is unknown, other elements of virus epidemiology are essential to develop guidelines for the management of the virus as well as to assist in the search for its vector. The objective of this study was to evaluate vegetation within and surrounding GRBaV-infected vineyards to identify potential virus reservoirs that may serve as sources of inoculum. In this study, 13 plant species were sampled throughout the year and were tested for the presence of GRBaV. Of the 13 species tested, two species, Rubus armeniacus and wild grapes (Vitis californica × V. vinifera), tested positive by quantitative PCR. Of these two species, only wild grapes were determined to be a true host. This study documents the first time GRBaV has been confirmed in an alternative host or in a species outside of a commercial vineyard and suggests that a mechanism exists by which GRBaV moves between plant species that is not human-mediated. The precise role that wild grapes play in the epidemiology of GRBaV remains unknown.

A new species of planthopper in the genus Paraphenice (Hemiptera: Derbidae: Otiocerinae) from palms in eastern Madagascar.

A survey of planthoppers associated with palms in Madagascar was initiated to assess putative vectors of a phytoplasma causing palm decline. Here a derbid collected from a Chinese fan palm (Livistona chinensis) is described as Paraphenice fluctus sp. n., with supplemental molecular data for the cytochrome c oxidase subunit I (COI) gene, 18S rRNA gene, and D9D10 expansion region of the 28S rRNA gene.

Multilamina teevani gen. et sp. nov., a microsporidian pathogen of the neotropical termite Uncitermes teevani.

A new genus and species of microsporidia is described from adults of the termite Uncitermes teevani (Emerson) (n. comb., formerly Armitermes teevani), collected in Ecuador. Masses of elongate, ovoid, uninucleate spores were localized to the coelomic cavity of adult workers and measured 6.29×3.33µm (fresh) and 5.83×3.00µm (fixed). These spores were individually contained within a multi-layered sporophorous vesicle and contained an isofilar polar filament with 24-28 coils. Blast-n analysis revealed that the small subunit ribosomal DNA (ssrDNA) sequence of this new species exhibited 85% identity with that of a Varimorpha species from the fire ant, Solenopsis richteri, and slightly less (78-85% identity) to a large clade of microsporidian parasites from mosquitoes and microcrustacea. The morphological and sequence data support the conclusion that Multilamina teevani gen. et sp. nov. is a novel microsporidium and distinct from any previously described genera or species.

A new species of planthopper in the genus Haplaxius (Hemiptera: Fulgoroidea: Cixiidae) from disturbed submontane rainforest in Jamaica.

Haplaxius is a large genus of cixiid planthopper found in the New World. The genus is of particular interest due to the ability of H. crudus to transmit the phytoplasmas for lethal decline in various palm species, primarily in the Caribbean and Florida, U.S.A. During recent vector survey work in Jamaica, a specimen was collected at Castleton Botanic Garden and determined to be a new species of Haplaxius. The novel taxon is herein described, Haplaxius fornicus sp. n., and corresponding DNA sequence data is provided for the barcoding region of the cytochrome c oxidase subunit I (COI) gene, 18S rRNA gene, and histone 3 (H3) gene. An updated phylogeny of the genus is provided with currently available taxa demonstrating additional support for the placement of H. fornicus sp. n. in Haplaxius.

A new species of planthopper in the genus Bothriocera (Hemiptera: Auchenorrhyncha: Cixiidae) from coconut palm (Cocos nucifera) in Jamaica.

Recent survey work in Jamaica on palm-associated planthoppers seeks to identify putative vectors of the lethal yellowing phytoplasma. Herein, a new species of planthopper, Bothriocera harthi sp. n., is described from coconut palm. Molecular data for the cytochrome c oxidase subunit I (COI), 18S rRNA gene, histone 3 (H3) gene, and 28S rRNA gene is provided to support placement of the novel taxon in Bothriocera. These findings are important because it provides novel data to help better understand the diversity and evolution of this unique group of planthoppers.

Nested PCR-Based Rapid Detection of Phytoplasma Leaf Wilt Disease of Coconut in Sri Lanka and Systemic Movement of the Pathogen.

Phytoplasmas are associated with many plant diseases. In palms, lethal bronzing disease, Texas Phoenix palm decline, and coconut lethal yellowing decline are some of them. In Sri Lanka, coconut leaf wilt decline has been reported in the Weligama area of the Southern province, and the disease is called Weligama coconut leaf wilt disease (WCLWD). Unlike other phytoplasma diseases of palms, WCLWD shows slow disease progress. Pathogen detection entirely relies on nested polymerase chain reaction (PCR). However, inconsistencies in pathogen detection have been experienced, i.e., symptomatic plants often produce negative results. The objectives of this study were to reconsider the choice of primers and to determine the best sampling tissue types for consistent detection of the pathogen. Among the six universal primer combinations tested, P1/Tint nested with fU5/rU3 produced consistent results. BLASTn searches of the sequences showed 99-100% similarity to sugarcane white leaf disease (SWL) or grassy shoot (SGS) disease-causing phytoplasma. The optimized nested PCR protocol was successful, with the minimum success rating of 88% and 100% specificity. Midribs of milky white bud leaf samples were the best tissue type for rapid detection. Systemic movement of the pathogen and a tentative latent period were also reported. The findings are helpful in the early detection of the disease.

A new species of planthopper in the genus Jamaha (Hemiptera: Fulgoroidea: Nogodinidae) from the Northeastern coast of Jamaica.

A new species of Jamaha Gnezdilov & O'Brien, 2008, is described from Jamaica and compared with other species in the genus. A key to the species of Jamaha is provided. Molecular data for the five-prime region of the cytochrome c oxidase subunit I (COI) gene and 18S rRNA gene is provided and compared to available Colpopterini, with other Nogodinidae as the outgroup, for a preliminary phylogenetic analysis. While the Colpopterini grouped with high support, these data remain too sparse to draw firm conclusions.