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.

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.

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.

A new species of planthopper in the genus Melanoliarus (Hemiptera: Auchenorrhyncha: Fulgoroidea: Cixiidae) from coconut palms (Cocos nucifera) in Trinidad.

During a survey in Trinidad and Grenada for putative vectors of palm lethal decline phytoplasmas, three species of planthopper in the genus Melanoliarus (Hemiptera, Cixiidae) were collected. Melanoliarus maidis was collected from coconut palms in Grenada, M. kindli was collected from grasses in a coconut plot in Trinidad and a new species of Melanoliarus was collected from coconut palms in Trinidad. Herein the novel taxon is described with supplementary molecular data for the cytochrome c oxidase subunit I (COI) gene, 18S rRNA gene, and histone 3 (H3) gene to support placement in the genus Melanoliarus in the strict sense.

A new species of planthopper in the genus Shellenius (Hemiptera: Fulgoroidea: Derbidae) from palms in Costa Rica.

The genus Shellenius is a small taxon of planthoppers in the family Derbidae (Otiocerinae: Otiocerini) found in the eastern United States and Mesoamerica. A new species of Shellenius associated with palms is herein described from Costa Rica. Molecular data for the barcoding region cytochrome c oxidase subunit I (COI) and 18S rRNA gene is provided to produce a preliminary phylogenetic tree for related taxa and support placement of the novel taxon in Shellenius. A review of Fowler type material suggests that Otiocerus interruptus Fowler is a Shellenius species and is here transferred to that genus as Shellenius interruptus new combination.

Identification of Green-Leaf Volatiles Released from Cabbage Palms (Sabal palmetto) Infected with the Lethal Bronzing Phytoplasma.

Lethal bronzing (LB) is a fatal infection that affects over 20 species of palms (Arecaceae) and is caused by the phytoplasma 'Candidatus Phytoplasma aculeata'. This pathogen causes significant economic losses to landscape and nursery companies in Florida, USA. Recently, the vector was determined to be the planthopper Haplaxius crudus, which was more abundant on LB-infected palms. Herein, the volatile chemicals emitted from LB-infected palms were characterized using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS). Infected Sabal palmetto were identified and confirmed as positive for LB via quantitative PCR. Healthy controls of each species were selected for comparison. All infected palms exhibited elevated levels of hexanal and E-2-hexenal. Threatened palms showed high releasing concentrations of 3-hexenal and Z-3-hexen-1-ol. The volatiles characterized herein are common green-leaf volatiles (GLVs) emitted by plants under stress. This study considers the first documented case of GLVs in palms attributed to phytoplasma infection. Due to the apparent attraction of LB-infected palms to the vector, one or several of the GLVs identified in this study could serve as a lure for the vector and supplement management programs.

A new species of planthopper from Costa Rica in the genus Herpis (Hemiptera: Derbidae) from palms in the cloud forest.

Recent survey efforts in Costa Rica have documented many new species of planthoppers, primarily in the families Derbidae and Cixiidae, on palms. Recently, a specimen was collected sweeping palms in the Los Angeles cloud forest in Costa Rica and was identified as belonging to the genus Herpis (Derbidae). It was subsequently determined to represent a previously undescribed species. Herein, the new species, Herpis circumsoros Bahder & Bartlett sp. n. is described with supplemental molecular data for the cytochrome c oxidase subunit I (COI) gene and 18S rRNA gene to support placement of the new species in the genus Herpis.

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 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.

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 Oecleus (Hemiptera: Auchenorrhyncha: Fulgoromorpha: Cixiidae) from the Osa Peninsula in Costa Rica.

Recent palm survey work in Costa Rica focusing on planthoppers has resulted in the discovery of several new taxa, primarily in Cixiidae and Derbidae. In addition to sampling palms directly, light trapping has been utilized to collect a broader range of planthoppers that may not be found on palms. During a light trapping event at the Cotinga Biological station on the Osa peninsula in Costa Rica, a cixiid was collected and subsequently determined to be an unidentified species in the genus Oecleus Stl. Herein, the novel taxon, Oecleus urru sp. n., is described. Supplemental molecular data for the barcoding region (5 half) of the cytochrome c oxidase subunit I (COI) gene, 18S rRNA gene, and histone 3 (H3) gene is provided to support the placement of the novel taxon in the genus Oecleus.

A new species of planthopper in the genus Cobacella (Hemiptera: Auchenorrhyncha: Derbidae) from oil palm (Elaeis guineensis) in Costa Rica.

Recent survey work in Costa Rica has revealed a high diversity of planthoppers in the family Derbidae on palms (Arecaceae). During an expedition to Costa Rica in 2021, specimens were collected from African oil palm (Elaeis guineensis) along the pacific coast and determined to represent a new species of derbid in the genus Cobacella. Herein, the novel taxon, Cobacella palmensis sp. n., is described and compared with the two other species in the genus. Supplemental molecular data for the cytochrome c oxidase subunit I (COI) barcoding region, 18S rRNA gene and D9-D10 expansion region of the 28S rRNA gene are provided to test the placement of the novel taxon relative to available otiocerine planthoppers. We also present a preliminary key to the species of Cobacella and review all available specimen records of the genus.

A new species of planthopper in the genus Anotia (Hemiptera: Auchenorrhyncha: Derbidae) from the Los Angeles cloud forest in Costa Rica.

Recent survey work in Costa Rica has resulted in the discovery of new species of derbid and cixiid planthoppers associated with palms. During this survey, one species belonging to the genus Anotia has been discovered and described, A. firebugia. A second species was collected while sweeping trailside vegetation in the Los Angeles cloud forest in Costa Rica and determined to represent a new species of Anotia. Herein, Anotia cerebro sp. n. is described and supplemental molecular data for the cytochrome c oxidase subunit I (COI), 18S rRNA gene and D9-D10 expansion region of the 28S rRNA gene are provided.

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.

A new species of Oecleus (Hemiptera: Auchenorrhyncha: Fulgoroidea: Cixiidae) from the Caribbean coast of Costa Rica and a reassessment of the generic status of Nymphomyndus.

A new species of cixiid planthopper in the genus Oecleus Stl is described from Tortuguero, Limn Province, Costa Rica. This is the first Oecleus species reported from Costa Rica. The new species, Oecleus dormido sp. n., was collected sweeping grassy edge habitat near the Caribbean coast. Sequence data for COI, 18S, and H3 was generated for phylogenetic comparison of the new species with other available Oecleus species and New World Oecleini to test genus-level placement and provide a preliminary examination of the phylogenetic relationships among New World genera of Oecleini. The results indicate that Oecleus, Haplaxius and Myxia are monophyletic, at least among the included taxa, the genera (Myxia+((Nymphocixia+Nymphomyndus)+Haplaxius) may form a clade apart from Oecleus. The monobasic genera Nymphocixia+Nymphomyndus are closely allied, with a difference of 0.98% for 18S, compared to an average of 2.2% among other included oecleine genera. In view of the limited morphological and molecular differences between these genera, we propose Nymphomyndus as a junior synonym of Nymphocixia.

A new species of planthopper in the genus Myconus (Hemiptera: Auchenorrhyncha: Fulgoroidea: Achilidae) from the Los Angeles Cloud Forest, Costa Rica.

Recent survey work in Costa Rica has resulted in the discovery of a wide variety of undescribed species of planthoppers in the families Derbidae and Cixiidae. During a light trapping event in the Los Angeles cloud forest, a large planthopper was collected and determined to belong to the genus Myconus in the family Achilidae. Herein, the novel taxon is described with accompanying molecular data for the cytochrome c oxidase subunit I (COI) gene, 18S rRNA gene, and histone 3 (H3) gene and an updated key for the New World Myconus is provided.

A new species of planthopper in the genus Tico (Hemiptera: Auchenorrhyncha: Derbidae) on palms from lowland tropical rainforest in Costa Rica.

Recently, the genus Tico Bahder Bartlett was described as part of ongoing research focused on planthopper diversity on palms in Costa Rica to accommodate two new species and the transfer of one species from Cenchrea Westwood. Herein, a new species of Tico is described from palms and Heliconia spp. at the La Selva Biological Station in Costa Rica. Placement of the novel taxon is supported by molecular analysis of the cytochrome c oxidase subunit I (COI) gene and 18S rRNA gene.

A new species of planthopper in the genus Anchimothon (Hemiptera: Auchenorrhyncha: Derbidae) on palms from Costa Rica.

Anchimothon is a small genus of derbid planthoppers known from Mesoamerica and northern South America. Recent survey efforts in the Caribbean basin have documented many new taxa of fulgoroids from palms. During this survey, a novel taxon identified as Anchimothon was collected from palm seedlings at La Selva Biological Station in Costa Rica. Here, the novel taxon is described as A. myriei sp. n. with molecular data for the cytochrome c oxidase subunit I (COI) and18S rRNA providing support for placement of the new species in Anchimothon. An updated key is provided for the current species of Anchimothon.

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.