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.

Monocot crop-aphid interactions: plant resilience and aphid adaptation.

Globally, aphids cause immense economic damage to several crop plants. In addition, aphids vector several plant viral diseases that accelerate crop yield losses. While feeding, aphids release saliva that contains effectors, which modulate plant defense responses. Although there are many studies that describe the mechanisms that contribute to dicot plant-aphid interactions, our understanding of monocot crop defense mechanisms against aphids is limited. In this review, we focus on the interactions between monocot crops and aphids and report the recently characterized aphid effectors and their functions in aphid adaptation to plant immunity. Recent studies on plant defense against aphids in monocot-resistant and -tolerant crop lines have exploited various 'omic' approaches to understand the roles of early signaling molecules, phytohormones, and secondary metabolites in plant response to aphid herbivory. Unraveling key regulatory mechanisms underlying monocot crop resistance to aphids will lead to deeper understanding of sap-feeding insect management strategies for increased food security and sustainable agriculture.

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.

Vector Transmission of Tomato Yellow Leaf Curl Thailand Virus by the Whitefly Bemisia tabaci: Circulative or Propagative?

Viruses that cause tomato yellow leaf curl disease are part of a group of viruses of the genus Begomovirus, family Geminiviridae. Tomato-infecting begomoviruses cause epidemics in tomato crops in tropical, subtropical, and Mediterranean climates, and they are exclusively transmitted by Bemisia tabaci in the field. The objective of the present study was to examine the transmission biology of the tomato yellow leaf curl Thailand virus (TYLCTHV) by B. tabaci, including virus-infected tissues, virus translocation, virus replication, and transovarial transmission. The results demonstrated that the virus translocates from the alimentary gut to the salivary glands via the hemolymph, without apparent replication when acquired by B. tabaci. Furthermore, the virus was detected in 10% of the first-generation progeny of viruliferous females, but the progeny was unable to cause the viral infection of host plants. There was no evidence of transovarial transmission of TYLCTHV in B. tabaci. When combined with the current literature, our results suggest that B. tabaci transmits TYLCTHV in a persistent-circulative mode. The present study enhances our understanding of virus-vector interaction and the transmission biology of TYLCTHV in B. tabaci.

Transmission mode of watermelon silver mottle virus by Thrips palmi.

Thrips and thrips-transmitted tospoviruses cause significant losses in crop yields worldwide. The melon thrips (Thrips palmi) is not only a pest of cucurbit crops, but also a vector that transmits tospoviruses, such as the watermelon silver mottle virus (WSMoV). Vector transmission of tospoviruses has been well studied in the tomato spotted wilt virus (TSWV)-Frankliniella occidentalis model system; however, until now the transmission mode of WSMoV by T. palmi has not been sufficiently examined. The results of the transmission assays suggest that T. palmi transmits WSMoV in a persistent manner, and that the virus is mainly transmitted by adults, having been ingested at the first-instar larval stage. Complementary RNAs corresponding to the NSm and NSs genes of WSMoV were detected in viruliferous thrips by reverse transcription-polymerase chain reaction; NSs protein was also detected in viruliferous thrips by western blotting, verifying the replication of WSMoV in T. palmi. Furthermore, we demonstrated that in thrips infected with WSMoV at the first-instar larval stage, the virus eventually infected various tissues of the adult thrips, including the primary salivary glands. Taken together, these results suggest that T. palmi transmits WSMoV in a persistent-propagative mode. The results of this study make a significant contribution to the understanding of the transmission biology of tospoviruses in general.

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.

A new species of planthopper in the genus Agoo Bahder amp; Bartlett (Hemiptera: Fulgoroidea: Derbidae) from coconut palm (Cocos nucifera L.) in Jamaica.

A new species of the genus Agoo Bahder Bartlett, Agoo beani sp. n. was found associated with coconut (Cocos nucifera L., Arecaceae) in Jamaica. This species was discovered as part of a survey of the Caribbean basin to document planthopper diversity on palms. Cytochrome c oxidase subunit I (COI) and 18S sequence data strongly support placement of the new species in Agoo. The morphological features of Omolicna cocoana Rodriguez-Leon Hidalgo-Gato from Cuba are reviewed and this species transferred into the genus Agoo.

A new species of planthopper in the genus Haplaxius (Hemiptera: Auchenorrhyncha: Fulgoroidea: Cixiidae) on palms in Costa Rica and a new country record for Haplaxius skarphion.

The genus Haplaxius is a large taxon of cixiid planthoppers that is of economic importance due to the ability of Haplaxius crudus to transmit lethal yellowing in coconut palms. Haplaxius dougwalshi sp. n. is established as a new taxon of Cixiidae in the tribe Oecleini collected from native palms in lowland tropical rainforest in Costa Rica. Placement in the genus Haplaxius is supported both by molecular evidence based on the COI and 18S genes as well as by morphological characters. This novel taxon was discovered during survey work in Costa Rica to look for phytoplasmas and document planthopper diversity on palms. Furthermore, Haplaxius skarphion was also collected from coconut palms during survey work and is reported for the first time in Costa Rica.

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.

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.