Florida, USA is a hotspot of biological invasions with over 500 non‐native species reported. Reptiles encompass most of the non‐native wildlife with over 50 species established, many of which are sympatric and are identified as invasive due to their impacts to the environment, economy, and human health and safety. Reports of new non‐native reptiles occur, and many established non‐native reptiles continue to expand their ranges in Florida, increasing the need for multitaxa detection and monitoring capabilities. Invasive constrictor snakes are a primary focus of management efforts due to life history traits that favor successful establishment and dispersal in Florida as well as their impacts to native wildlife and Everglades restoration efforts. While traditional survey methods that rely on visual detections fail to reliably detect invasive constrictors, environmental DNA (eDNA) has proven to be a promising method for detection of cryptic and rare species across the landscape. To address emerging needs for multispecies detection and monitoring in Florida, we developed the first tetraplex dPCR assay designed for detection of four species of invasive constrictor snakes, including Burmese pythons ( Python bivittatus ), northern African pythons ( P. sebae ), boa constrictors ( Boa constrictor ), and rainbow boas ( Epicrates cenchria ). In this tetraplex assay, no cross‐amplification across species was documented. This assay serves as a valuable tool for faster and more accurate monitoring efforts of these invasive species in South Florida. Additionally, eDNA samples comprised of soil and water both tested positive for Burmese python DNA under controlled and semicontrolled conditions with DNA being detectable up to 2‐weeks post removal in soil samples. Water samples yielded positive detection as quickly as 5 min after exposure to the organism. These data highlight the utility and sensitivity of this protocol for eDNA monitoring.

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.

Palm lethal yellowing phytoplasmas (PLYPs) are a group of phytoplasmas that cause death in infected hosts across the tropics. Historically, detection and identification has relied on standard PCR, nested PCR, and restriction fragment length polymorphism. While these approaches are generally good, they are prone to error and contamination that is significantly lower or absent in modern approaches using quantitative PCR (qPCR) and digital PCR (dPCR). Additionally, these modern approaches are more time-efficient and consume fewer resources, making them more cost-effective in the long term. Recent studies have adapted dPCR and qPCR coupled with high-resolution melt curve analysis (HRMA) for PLYPs in the Caribbean/New World; however, these tools have not been developed for phytoplasmas in Africa and Madagascar. In this study, a duplex dPCR assay was developed with two specific assays, one for ‘Candidatus Phytoplasma palmicola’ and one for ‘Ca. P. cocostanzaniae’ and isolates from Madagascar. Additionally, primers targeting the secA gene were optimized and allowed for the separation of ‘Ca. P. cocostanzaniae’ and Malagasy isolates by approximately one degree Celsius. New primers were developed based on secA for ‘Ca. P. palmicola’ that allowed for the separation of the two subgroups (A and B) by HRMA by a difference of approximately one degree Celsius. These assays provide a valuable resource to explore aspects such as vector discovery and host range.

‘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 quantitative PCR assays are developed: one using 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/TaqMan assay successfully amplified two of the three isolates of CPB. Both assays were screened against available isolates of 16SrI, 16SrII, and 16SrIV phytoplasmas. The secA/HRMA assay failed to amplify 16SrI and 16SrIV phytoplasmas but successfully amplified 16SrII phytoplasmas. The resulting melting point (Tm) products of CPB and 16SrII phytoplasmas displayed a difference of 0.5°C, 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.

Planthoppers are a diverse and interesting group of insects. In the tropics, there are many undiscovered species and recent efforts have uncovered many new taxa associated with palms. One species of Cixiidae, Haplaxius crudus (Van Duzee), is economically important due to its ability to transmit palm lethal decline phytoplasmas, which has caused significant economic losses to palm producers across the Caribbean and the United States. A survey was conducted in Trinidad and Tobago to assess the potential threat that lethal yellowing and lethal bronzing pose to Trinidad and Tobago by evaluating the status of Haplaxius crudus (and other putative vectors) on the islands and genetically characterize populations in the islands to determine if H. crudus was a distinct haplotype. Populations of H. crudus were sampled and analyzed to assess genetic variability. Specimens of H. crudus were homogenous for the COI gene but represent a novel haplotype of the species. All specimens of H. crudus were negative for phytoplasma. Additionally, 8 species of Derbidae were documented, including one new species, Oropuna tobagoensissp. n., 5 species of Cixiidae and one species of Delphacidae on coconut palms. These data provide a valuable baseline to aid in the development of a long-term, robust monitoring program that will allow for rapid and early detection of palm phytoplasmas should they be introduced to the islands and ultimately help prevent the establishment of the disease and economic losses to the coconut industry.

Invasive species are a significant threat to a variety of ecosystems in Florida, with freshwater habitats being of particular concern. Two nonnative species of fish with established populations that are priority organisms for monitoring and management programs are the bullseye snakehead (BS; Channa species complex) and Asian swamp eel (ASE; Monopterus species complex). Recent technological advances have seen the emergence of environmental DNA (eDNA) analysis as a useful tool for the detection and monitoring of target organisms and to assess removal efforts. In this study, a duplex digital polymerase chain reaction (dPCR) was developed, optimized, and validated on control samples and field samples from locations with documented occurrences of target organisms. The assay developed, which allows simultaneous detection of both fish species when present in a sample, demonstrated highly efficient amplification for the corresponding target species with individual assays failing to cross‐amplify. Under controlled conditions, high levels of eDNA were detected as early as five minutes post‐introduction of BS to water. Additionally, field eDNA samples yielded varying levels of positive amplification for both species based on comparison of fluorescence levels to positive controls (both tissue extract and plasmids with appropriate inserts). These data indicate that through careful assay design and stringent parameter optimization, eDNA results obtained for monitoring of these two fish species can be a viable and cost‐effective strategy to detect the presence of these species simultaneously as well as to evaluate the success of removal efforts.