Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience of this research project are stakeholders in the nursery and landscaping industry. These professionals are in the field collecting samples from symptomatic palms and sending to my lab for analysis and verification of disease status. Additionally, county extension agents are another target audience that serve as a point of contact between myself and the various stakeholders who are collecting samples in the field. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Two virtual in-service trainings were offered this year. Half-day palm school covering palm nutrition, fungal issues, and phytoplasma issues was done on Dec. 2 2020 and a two hour long workshop on Dec. 16 2020 to teach stakeholders how to identify and sample for the vector of palm phytoplasmas. How have the results been disseminated to communities of interest?Extension publications and presentations in various workshops across the state. What do you plan to do during the next reporting period to accomplish the goals?1) Developing a novel assay to amplify all 16SrIV phytoplasma and distinguish each based on melt curve analysis. 2) Conduct transmission assays to determine optimal AAP and IAP times and locations on palms.
Impacts
What was accomplished under these goals?
1) Phytoplasma titer was quantified along the length of the entire trunk of various palm species at different stages of decline. This technique documented that there are higher levels of phytoplasma lower to the ground early in the disease cycle and as symptoms progress, the level increases. Once physiological function ceases in the palm, the titer drops significantly. Additionally, high levels were found just below the crown of the palm (near the apical meristem). 2) dPCR assay developed in previous years has successfully been adapted to screen salivary glands of candidate vector species collected in the field. Using this technology, we were able to detect phytoplasma in salivary glands of one species (Haplaxius crudus?), implicating it as a vector under controlled conditions. 3) Continual testing of field specimens shows about 0.6% ofH. crudus? to be carrying the phytoplasma and is the only species to carry it in a field setting. Combined with findings from objective 2, there is ample support thatH. crudus? is the true vector of lethal bronzing in Florida. 4) Two new invasive species were confirmed to be established during survey efforts (Patara guttata? andColpoptera?acutata?).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Mou, D.F., Humphries, A.R., Soto, N., Helmick, E.E., Ascunce, M.S., Goss, E.M. and Bahder, B.W., 2020. A survey of auchenorrhynchan insects for identification of potential vectors of the 16SrIV-D phytoplasma in Florida. Florida Entomologist, 103(3), pp.344-352.
Mou, D.F., Lee, C.C., Hahn, P.G., Soto, N., Humphries, A.R., Helmick, E.E. and Bahder, B.W., 2020. Effects of Lethal Bronzing Disease, Palm Height, and Temperature on Abundance and Monitoring of Haplaxius crudus. Insects, 11(11), p.748.
Bahder, B.W., Soto, N., Mou, D.F., Humphries, A.R. and Helmick, E.E., 2020. 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. Plant Disease, 104(5), pp.1328-1334.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience of this research project are stakeholders in the nursery and landscaping industry. These professionals are in the field collecting samples from symptomatic palms and sending to my lab for analysis and verification of disease status. Additionally, county extension agents are another target audience that serve as a point of contact between myself and the various stakeholders who are collecting samples in the field. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?post doctoral researcher has begun and is currently being trained in plant pathology How have the results been disseminated to communities of interest?A day long workshop was offered to stakeholders and University of Florida faculty to educate them about the disease, the current data and research efforts (Sept. 25th, 2019 at FLREC) What do you plan to do during the next reporting period to accomplish the goals?Continue assessing populations of?Haplaxius crudus?throughout the state, establish a breeding a colony, develop nymph sampling strategy
Impacts
What was accomplished under these goals?
Objective 1: Phytoplasma distribution in infected trunk tissue and leaf tissue was quantified using qPCR, demonstrating higher amounts of phytoplasma lower on the trunk and absence of phytoplasma in the leaf tissue, with the exception of the spear leaf. In the trunk, once symptoms are present, the phytoplasma is fully systemic and detectable at every location, eliminating the need to sample from multiple sites. These finding help optimize sampling efforts in the field for stakeholders. Objective 2: For further assessing the utility of dPCR, pooled samples of trunk tissue were tested and it was found that even at 1 positive sample mixed with 299 negative samples, the phytoplasma was stil detectable by dPCR whereas in qPCR, results were inconsistent. Objective 3. Screening of salivary glands of Haplaxius crudus by digital PCR demonstrated that the phytoplasma was present in a small percentage of individuals collected from areas with high disease incidence. Objective 4. A planthopper belonging to the genus Colpoptera was found to be established in Florida and isolated from the plant?Rivina humilis?. This represents the second invasive species as a result of these survey efforts.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Bahder, B.W., Bartlett, C.R., Helmick, E.E., Barrantes, E.A.B., Echavarria, M.A.Z., Goss, E.M., Ascunce, M.S. 2020. Revised status of Omolicna subgenus Agoo (Hemiptera: Auchenorrhyncha: Fulgoroidea: Derbidae) with a new species from Costa Rica and new country records. Zootaxa 4718(4):521-535.
Bahder, B.W., Bartlett, C.R., Barrantes, E.A.B., Echavarria, M.A.Z., Humphries, A.R., Helmick, E.E., Goss, E.M., and Asunce, M.S. 2019. A new genus and species of cixiid planthopper (Hemiptera: Auchenorrhyncha: Fulgoroidea) from the Reserva Privada el Silencio de Los Angeles Cloud Forest in Costa Rica. Zootaxa
4701(1):65-81.
Bahder, B.W., Soto, N., Helmick, E.E., Dey, K.K., Komondy, L., Humphries, A.R.,
Mou, D., Bailey, R., Ascunce, M.S., and Goss, E.M. 2019. A survey of declining
palms (Arecaceae) with 16SrIV-D phytoplasma to evaluate the distribution and
host range in Florida. Plant Disease 103(10):2512-2519.
Bahder, B.W., Soto, N., Komondy, L., Mou, D., Humphries, A.R., and Helmick, 2019. Detection and quantification of the 16SrIV-D phytoplasma in leaf tissue of common ornamental palm species in Florida using qPCR and dPCR. Plant
Disease 103(8):1918-1922.
Bahder, B.W., Bartlett, C.R., Barrantes Barrantes, E.A., Zumbado Echavarria M.A., Humphries, A.R., Helmick, E.E., Ascunce, M.S. and Goss, E.M. 2019. A new
species of Omolicna (Hemiptera: Auchenorrhyncha, Fulgoroidea: Derbidae) from
coconut palm in Costa Rica and new country records for Omolicna brunnea and
Omolicna triata. Zootaxa 4577(3):501-514.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:The target audience of this research project are stakeholders in the nursery and landscaping industry. These professionals are in the field collecting samples from symptomatic palms and sending to my lab for analysis and verification of disease status. Additionally, county extension agents are another target audience that serve as a point of contact between myself and the various stakeholders who are collecting samples in the field. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A visiting graduate student from Puerto Rico visited our lab for one month training on detection of phytoplasmas in palm tissue. How have the results been disseminated to communities of interest?A day long workshop was offered to stakeholders and University of Florida faculty to educate them about the disease, the current data and research efforts (Sept. 26th, 2018 at FLREC) What do you plan to do during the next reporting period to accomplish the goals?The main focus of the lab for the upcoming year is to conduct transmission assays to evaluate the ability of?Haplaxius crudus?to transmit the phytoplasma. In addition to this, population data of?H. crudus? will be taken throughout the year in different parts of the state to provide useful data to stakeholders about when management strategies will likely be most effective and where.
Impacts
What was accomplished under these goals?
Objective 1.Develop rapid and accurate testes to detect and identify different phytoplasmas in palms: A novel extraction protocol has been developed that allows for faster isolation of DNA from plant tissue that is sent in by stakeholders. This protocol also requires less plant material than before, only requiring as little as 100 mg of trunk tissue from palms in order to detect phytoplasma. Previously, 1 g was needed for the extraction time. The new extraction protocol utilizes guanidine buffer (non-noxious) the a table-top macerator to disrupt tissue then allows for use of commerical kits to complete DNA extraction. This novel technique will soon be published in a new host record for the phytoplasma causing the disease. Additionally, a primer and probe set published by Cordova et al. (2014) was used to develop a plasmid standard dilution series for quanitification of of phytoplasma in palm samples that test positive. Objective 2. Develop digital PCR assays to detect low titers of pathogens in plant and insect tissues: The dPCR assay described in the previous report has been fully integrated into the diagnostic clinic so that stakeholders can now use this as an option for testing samples. Currently, we are screening pooled samples to determine how many palms a nursery can sample and test and still reliably detect a single infected palm. Thus far, we have found detection possible at 1 infected individual per 300 samples. This could potentially allow nurseries to verify palms are free of phytoplasma at a significantly reduced cost in terms of time and money. So far 34 specimens of?Haplaxius crudus? have had salivary glands tested by dCPR and 3 have tested positive. This is significant because it is a strong indicator that this species is a vector. Objective 3. Evaluate candidate insect vectors for transmission of phytoplasma infecting palms: The population survey of auchenorrhynchan insects in palm canopies was conducted at the Fort Lauderdale Research and Education Center, FL where the disease is actively spreading. Yellow sticky traps were used for insect collection for the population survey to determine the insect species that have feed on the palm trees in the disease area. Moreover, the insects collected by the traps were tested by nested PCR assay to detect the phytoplasma. There are four families of auchenorrhynchans (Cixiidae, Derbidae, Membracidae, and Cicadellidae) collected by the traps constantly during the one-year collecting time. Among them, a cixiid planthopper, Haplaxius crudus, is the most abundant insect that collected by the traps and approximately 1.11% of the H. crudus tested positive of the phytoplasma. In addition, one leafhopper (Cicadellidae) was tested positive of the phytoplasma. Based on the results, we suggest H. crudus is the potential vector of LBD and further research is needed to examine the phytoplasma transmission ability. Objective 4. Survey Florida for auchenorrhynchan and sternorrhynchan insects: A new invasive species was discovered and determined to be established in south Florida,Petrusa epilepsis?. A DNA barcode was developed for this species and the findings were published in Florida Entomologist. This species represents the first invasive discovered as a result of our ongoing planthopper survey.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Bahder, B.W., Helmick, E.E., Chakrabarti, S., Osorio, S., Soto, N., Chouvenc, T. and Harrison, N.A., 2018. Disease progression of a lethal decline caused by the 16SrIV?D phytoplasma in Florida palms. Plant Pathology, 67(8), pp.1821-1828.
Bahder, B.W., Helmick, E.E., Mou, D.F., Harrison, N.A. and Davis, R., 2018. Digital PCR Technology for Detection of Palm-Infecting Phytoplasmas Belonging to Group 16SrIV that Occur in Florida. Plant Disease, 102(5), pp.1008-1014.
Bahder, B.W., Halbert, S., Mou, D.F., Helmick, E.E., Soto, N., Otero, M. and Segarra, A.E., 2018. Establishment of the sea grape flatid, Petrusa epilepsis (Hemiptera: Fulgoroidea: Flatidae), in Florida. Florida Entomologist, 101(4), pp.634-642.
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Progress 10/29/16 to 09/30/17
Outputs
Target Audience:The target audience of this research project are stakeholders in the nursery and landscaping industry. These professionals are in the field collecting samples from symptomatic palms and sending to my lab for analysis and verification of disease status. Additionally, county extension agents are another target audience that serve as a point of contact between myself and the various stakeholders who are collecting samples in the field. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A Fulbright Scholar from the Philippines has been present for a four month training period to provide samples from her home country to learn phytoplasma detection techniques to improve their quarantine service. The PI has attened a weeklong workshop and has been certified in performing CRISPR/Cas9 techniques to eventually adapt to palm phytoplasmas and their associated vectors How have the results been disseminated to communities of interest?A day long workshop was offered to stakeholders and University of Florida faculty to educate them about the disease, the current data and research efforts (Sept. 27th, 2017 at FLREC) What do you plan to do during the next reporting period to accomplish the goals?Sequence the entire genome of the phytoplasma to obtain regions of the genome that are better suited for development of diagnostic assays. This will allow us to target regions that differe among all subgroups rather then just two which will better prepare us in the event that other phytoplasmas are introduced to the state. Improving detection techniques will allow us to identify infections and spread faster allowing for more rapid implementation of management strategies. Begin screening salivary glands of insects that have been collected from infected plants to evaluate their potential as vectors. With the development of a highly sensitive detection technique, we can now confidently screen the salivary glands for phytoplasma. Once we can detect phytoplasma in salivary glands, we can use this as evidence that a given species is the vector and proceed with more in dpeth studies on the species in question but also begin developing management strategies that use data on the insects biology.
Impacts
What was accomplished under these goals?
Objective 1 - Develop rapid and accurate tests to detect and identify different phytoplasmas in palms Progress and Results: Using sequence data available in GenBank for the Texas Phoenix Palm Decline (TPPD) and Lethaly Yellowing (LY) phytoplasmas for the 16S gene, primers were designed in the 5' and 3' region of the gene. The 5' region of the 16S gene possessed an insertion of 13 base pairs (5'-GAAATCTTTTAGA-3') in LY but is absent in TPPD. The 3' region had four single nucleotide polymorphisms (SNPs) with the first being a change of T to G from TPPD to LY, respectively and the rest being T to C conversions from TPPD to LY. Both primer sets achieved successful amplification on initial screening. The 5' primer set in initial testing and after replicating in various isolates, consistently amplified target DNA and yielded a 0.6 degree C difference in amplicon melting temperate between TPPD and LY. This difference allows for detection and distinction between TPPD and LY in the same reaction without having to conduct post PCR analyses. Additionally, the primers developed for the 3' region successfully amplified both LY and TPPD but yielded a difference of 0.4 degree C. The results of this work were published in Plant Disease (https://doi.org/10.1094/PDIS-01-17-0023-RE). This assay distinguishes between LY and TPPD but will not allow the distinction of other phytoplasmas found throughout the Caribbean. Based on sequence similarities, other phytoplasmas would give a melting temperature that matched either LY or TPPD. As the genomes become available, new assays will be developed to be able to screen for other phytoplasmas and identify all subgroups based on melting temperatures in qPCR assays. Objective 2 - Develop digital PCR assays to detect low titers of pathogens in plant and insect tissues To develop a TaqMan assay to be tested by dPCR, the full 16S sequence for TPPD uploaded into the ThermoFisher custom assay design software. A region of 73 base pairs was identified as an appropriate region for primer and probe design. To test the effectiveness of this assay, one isolate of TPPD and one isolate of LY were diluted in a series of 9 factors at a 10X dilution factor. Both LY and TPPD successfully amplified using the TaqMan Assay. Both isolates were capable of being detected at two dilution factors lower that both nested PCR and qPCR, demonstrating an increased sensitivity over existing methods. This assay also amplified an isolate of the 16SrIV-B as well as isolates of phytoplasmas in other groups (16SrI and 16SrIII). Future studies will further optimize this assay to make it available as a diagnostic tool available to stakeholders, specifically nurseries to be able to screen large groups of palms at once. Additionally, it will be further optimized to use in testing insect salivary glands for the presence of phytoplasmas. The results of this study were recently accepted for publication in Plant Disease (https://doi.org/10.1094/PDIS-06-17-0787-RE). Objective 3 - Evaluate candidate insect vectors for transmission of phytoplasmas infecting palms. Two field sites where TPPD is currently active have been extensively sampled for Hemipteran insects. Sweep netting in vegetation surrounding infected palms and in palm canopies that were within reach was conducted bi-weekly. Additionally, yellow sticky traps were placed in canopy of an infected palm and monitored on a weekly basis. The most abundant species captured on sticky traps and by sweep netting was Haplaxius crudus with a total of 293 specimens sampled among all locations and methods. A subset of 50 individuals were screened for the presence of the TPPD with one individual testing positive. The second most common species encountered in palm canopies was the treehopper species Idioderma virescens. A total of 54 individuals of this species have been collected with all being tested and one individual testing positive for the presence of phytoplasma. The third most common species collected was Cedusa inflata, derbid planthopper, with a total of 24 individuals. All individuals of this species tested negative for the phytoplasma. Additionally, a variety of whiteflies were collected from sticky traps with two individuals from an identified species testing positive for phytoplasma. These result provide insight into which species are most likely to be vectors of the TPPD phytoplasma. Detecting phytoplasma in insects is not evidence that it is a vector but does provide some direction and allows to establish a list of candidate species that could potentially serve as vectors. Future studies will focus on the species identified in this study and evaluate their ability to transmit the phytoplasma. Objective 4 - Survey Florida for auchenorrhynchan and sternorrhynchan insects. Thus far, over 1,000 specimens have been collected within the auchenorrhyncha and sternorrhyncha and are being loaded into a database. Samples are predominantly from Broward County and St. Lucie County and in the years to come, more extensive sampling will be conducted in other nearby counties. A DNA barcoding assay is currently being optimized to aid in identification of all specimens collected.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Bahder BW, Helmick EE, Harrison NA. 2017. 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). Plant Disease 101(8):1449-1454.
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