Grant Information
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|---|---|---|
| 211 - Insects, Mites, and Other Arthropods Affecting Plants | 999 - Citrus, general/other | 1040 - Molecular biology | 50% |
| 212 - Pathogens and Nematodes Affecting Plants | 999 - Citrus, general/other | 1170 - Epidemiology | 50% |
According to the FAO, plant diseases cost the global economy $220 billion and invasive insects $70 billion, annually. Bacterial plant diseases spread by invasive vectors are especially difficult to study due to the intimate relationships between the vector, pathogen and host plant. Recent studies have suggested that vector genetics as well as the source of inoculum may play larger roles in determining pathogen transmission to new plants than thought. Huanglongbing (HLB), or citrus greening disease, is the most devastating disease of citrus worldwide. The HLB pathosystem involves three players: the bacterial pathogen called "Candidatus Liberibacter asiaticus" (CLas), the Asian citrus psyllid insect vector (Diaphorina citri), and the citrus trees (including all known varieties of citrus). Citrus in the US is a $3.40 billion dollar industry across four states and it has been hit hard by HLB, with thousands of jobs lost since 2005. Despite the global status of HLB, there is no cure, just costly and often ineffective disease prevention and mitigation. In California, intensive testing and "back-yard tree" removal programs are thought to be preventing the spread of HLB into commercial groves. However, growers and owners are often wary of removing productive trees because there are very few published studies showing trees are the main driver of HLB spread in the field. Additionally, evidence repeatedly shows that both vector and pathogen populations are genetically diverse and that this diversity has the potential to make control methods inefficient at best - including the use of insecticide sprays. The ultimate goals of this project are two-fold: 1) to clarify the relationship between the in-field diversity of D. citri populations to build control methods that are more targeted, and 2) to access which aspects of the HLB pathosystem are most important to target for control of HLB in the field. Using greenhouse studies based out of Cornell University, methods will utilize many replications of both single-leaf assays and whole plant studies. To track CLas in plants and insects over space and time a commonly used method of quantification - quantitative polymerase chain reaction (qPCR) - will be utilized. The highly detailed data will be analyzed using multivariate statistical tests, paired with a specially designed mathematical model, to evaluate significance and dependencies among the variables. Additionally, specially inbred vector populations will be crossed in traditional breeding experiments to determine dominance and heritability of the vector's ability to acquire and transmit CLas. This study straddles the line between basic and applied research making it relevant to both stakeholders and scientists. Results from this project have the potential for high impact in the relatively short-term of 5 years through updating policies that directly affect growers, through improving HLB management technologies and efficiency, and through demonstration of a method of studying a notoriously difficult insect-vector-plant pathosystem.
Bacterial plant diseases spread by insect vectors are especially difficult to study due to the intimate relationships between the vector, pathogen and host plant. Evidence repeatedly shows that D. citri and CLas populations are genetically diverse and that this diversity has the potential to make control methods inefficient at best. The ultimate goal of this project is to clarify the relationship between the genetic diversity of D. citri and the epidemiology of HLB disease by separating the effects of plant and pathogen on vector acquisition and transmission efficiency, potentially leading to better HLB control strategies.
Objective 1 will be addressed by careful tracking of Diaphorina citri individuals from 6 basic generations including P1 (L8 - good acquirer and transmitter of CLas) and P2 (L16 - poor acquirer and non-transmitter of CLas) parent isofemale lines, the F1 and F2 first and second progeny generations, and at least two back-crosses, BC1 and BC2. To produce F1 and an initial assessment of whether the L16 phenotype is sex-linked, I will reciprocally mate 50 CLas- virgin males and 50 CLas- virgin females from P1 (L8) and P2 (L16) populations. Males and females are morphologically distinct and easy to phenotype, and adults are sexually incapable for two days post molting allowing collection of virgins. These 100 healthy adults will produce F1 progeny on CLas- (healthy) citron (Citrus medica) plants for 7 days. There will be 10 replicates of these reciprocal crosses between P1 and P2. A total of 100, 4th-instar nymphal F1 progeny from each replicate will be placed in groups of 10 CLas+ (infected) citron detached leaf assays to acquire CLas until molting to adults. A total of 100 resulting F1 adult progeny will be scored by sex, color morphology, and then placed on new, healthy detached leaf assays in 5 groups of 10 females and 5 groups of 10 males to test transmission efficiency. After 7 days for inoculation, psyllids will be flash frozen and DNA extracted while leaves incubate for another 7 days. Leaves (excised midribs only) and F1 psyllids will be tested using qPCR for CLas titer, including the use of technical positives, negatives and a standard curve dilution series to allow for absolute quantification. To re-isolate the P2 (L16) acquisition and transmission phenotype, an additional 100 F1 adults from each reciprocal cross will be inbred and allowed to lay eggs for 7 days to generate a heterozygous population. A total of 50 resulting virgin F2 adults from each of the 10 replicates will be back-crossed (BC) with 50 virgin L16 adults, for at least two consecutive back cross events. For both BC1 and BC2, a subset of 100 nymphs will be allowed to acquire and subsequent adults will be tested for transmission and acquisition efficiency, as described above. A model will be developed to estimate narrow-sense trait heritability of the natural non-vector (L16) phenotype from the proportion of phenotypic variance that is due to genetic factors [14]. The model will be updated as each replicate and back-cross is processed. Specifically, I will test the generation means (F1, BC1,2) for goodness-of-fit to genetic models incorporating additive or dominant effects using the joint scaling test. The minimum number of segregating factors involved in genetic divergence in CLas acquisition will be estimated using models developed for non-homozygous populations to correct for sampling variances in the estimates of parental populations and to take into account possible linkage and inequality of allelic effects [15]. Possible genetic correlations between acquisition, titer, and transmission of CLas will be calculated as Pearson's product-moment correlations.Objective 2 will be addressed by testing whether the infection state of the plant or the insect plays a more significant role in regulating CLas acquisition and transmission rate. I will establish four treatments including positive and negative controls. A total of 10 CLas- and CLas+ citron (Citrus medica) plants will be used for each experiment. A total of 100 adult psyllids ("parents") collected randomly from colonies raised on CLas+ citron plants will be added to cages with either CLas- (treatment 1) or CLas+ citron (treatment 2) and allowed to randomly mate. Likewise, 100 random adult psyllids collected from CLas- citron plants will be added to cages containing either CLas+ (treatment 3) or CLas- trees (treatment 4) and allowed to randomly mate. For all treatments, adults (parents) will be removed after 15 days and 30 insects from each replicate will be saved for qPCR analysis to gauge CLas titer and proportion of the population that is infected. After 30 days post egg-laying, a random sample of 50 adult progeny will be assessed for their CLas titer (to measure acquisition) and transmission via detached leaf assay. Each transmission test will include 10 random adults in a 50 ml conical tube containing a single detached leaf. Adults will be removed after feeding for 7 days, and CLas titer will be accessed with qPCR. After incubation for another 7 days, qPCR analysis of CLas titer in leaf midribs will be tested. These experiments are routine for the Heck lab. All qPCR tests will include standard curve dilutions, positive and negative tests, and all samples will have three technical replications. Additionally, psyllid parameters such as sex, color morph, mortality and plant parameters such as tree age, leaf age, and mottling will be collected for each individual psyllid and leaf. Treatments 1-4. CLas-= uninfected, CLas+ = infected. B) Detached leaf assay set up. Qualitative data from psyllid and plant phenotypic observations will be tested using two-way multivariate analysis of variance (MANOVA) tests, followed by univariate ANOVA tests to determine the specific significant dependent variable. Standard error in CLas titer between the 10 biological replicates from each treatment will be calculated and if the values are skewed significantly, logarithmic transformation will be applied prior to comparing treatments. To analyze the qPCR results, a one-way analysis of variance (ANOVA) will test for differences in "parent" CLas titer across the four treatments, and again for differences in progeny CLas titer across treatments. The Tukey honestly significant difference (HSD) test will be applied to find the significant pair-wise interactions. Finally, a one-way MANOVA and a delta delta analysis of relative CLas titer will show whether "parent" infection significantly impacts transmission of CLas in each treatment, where the value of "parent" CLas titer in each treatment represents zero and the comparative value of the respective progeny CLas titer is represented as a bar above or below zero.
Target Audience
The target audience for this research includes the scientific community, specifically those working on vector biology, vectorpathogen interactions, epidemiology of plant pathogens, and population genomics. This research also targets citrus growers and other citrus stakeholders. Efforts include training and mentorship of junior scientists, especially women and minorities.
Changes / Problems
Nothing Reported
Training & Professional Development
This project provided many opportunities for the PD's professional development and training. For objective 1, she continued a collaboration with Dr. Edward Buckler and members of his lab to learn computational pipelines for genome-wide association mapping and estimation of trait heritability (including specific programs, troubleshooting, and discussion of underlying assumptions behind the computational methods employed). Through their Slack channel, she posted questions, updates and other progress and received valuable feedback. She also continues to share results with collaborators in the Buckler lab, as they are co-authors on the manuscript and are helping with interpretation. The PD also continued mentoring and collaborating with an ORISE fellow (post-bac) computational biologist. She taught him the computational pipeline and now he helps run large analyses, and any issues and problems are discussed together. Through weekly meetings, they discussed all aspects of research, graduate school, pros and cons of computational vs. wet/bench work, and prepared presentations for scientific conferences. The PD is peer mentor to multiple graduate students in her department at Cornell, is an active member on multiple committees dedicated to diversity, equity and inclusion, as well as being the founder and chair of a mentoring and advising committee in the plant pathology section at Cornell University. Most notably, the PD successfully defended her Ph.D. in April of 2024.
Dissemination Streams
Scientific findings were orally and visually presented and disseminated to the grower and scientific communities at various conferences, academic seminars and lab meetings.
Next Reporting Steps
Nothing Reported
Target Audience
Through poster presentation at conferences, the citrus greening community including stakeholders, scientists and policy makers was reached, as well as the broader plant health research community. Through drafting manuscripts and two peer-reviewed publicationin the past year, target audiences reached include the scientific community of plant pathologists, vector biologists, vector-pathogen interactions researchers, and agricultural entomologists.
Changes / Problems
Nothing Reported
Training & Professional Development
This project provided many opportunities for Marina's professional development and training. For objective 1, she continued a collaboration with Dr. Edward Buckler and members of his lab to learn computational pipelines for genome-wide association mapping and estimation of trait heritability (including specific programs, troubleshooting, and discussion of underlying assumptions behind the computational methods employed). Through their Slack channel, she posted questions, updates and other progress and received valuable feedback. She also continues to share results with collaborators in the Buckler lab, as they are co-authors on the manuscript and are helping with interpretation. Marina also began mentoring and collaborating with Douglas Stuehler who is an ORISE fellow (post-bac) computational biologist. She taught him the computational pipeline and now he helps run large analyses, and any issues and problems are discussed together. Through weekly meetings, they discuss graduate school, computational vs. wet/bench work, as well as the hypotheses, methods and results of the they do together. Douglas will be attending the Plant pathology conference in Lyon, France this summer to present objective 1 work. Marina's interactions with Douglas and the Buckler lab have been entirely virtual, an experience and skill for the future. Marina also attended three conferences in 2022 where she presented a poster of Objective 1 work to peers and colleagues. She also maintained membership to multiple professional societies relating to her research field(s), (notably American Phytopathological Society, and Entomological Society of America). Lastly, Marina is peer mentor to two new graduate students in her department at Cornell, is an active member on multiple committees dedicated to diversity, equity and inclusion, as well as being the founder and chair of a mentoring and advising committee in the plant pathology section at Cornell University.
Dissemination Streams
Scientific findings from Objective 1 were orally and visually presented at three conferences in poster format. Two conferences were general plant science audiences, while the third was specific to the plant pathology and citrus greening field (Huanglongbing and phloem-limited bacteria). All conferences included scientists, growers, and members from funding agencies and other stakeholders.
Next Reporting Steps
During the next (and final) reporting period, work on both objectives will be wrapped up by June 2024: Objective 1: One manuscript on GWAS of Diaphorina citri - CLas interactions will be published in late 2023, and possibly a second, follow-up manuscript based on further results from D. citri or endosymbiont genetic data collected from the same batch of 500 individual insects, will be submitted by May 2024. Additionally, any loci or genes of interest resulting from this analysis can be used to develop RNAi targets. Objective 2: One manuscript will be submitted in summer 2023, covering four populations of wild D. citri and their variable vector capacities as determined by quantitative PCR. Additional studies may include isofemale line crosses and testing for sex-linked genes or collection and testing of more field populations to verify previously published work and expand our understanding of how vectors adapt to field conditions. Marina plans to attend the final AFRI NIFA project director's meeting to present final results from this fellowship. She will also present objective 1 results in November at the Entomological Society of America, and will be defending her PhD thesis and writing the dissertation starting in January 2024. <br><br>
<br>What was accomplished under these goals? Impact statement: Bacterial vector-borne diseases take a huge toll each year on agricultural production and the economy, but due to the intimate relationships between the insect vector, plant host, bacterial pathogen and the environment, these disease systems are difficult to study. Huanglongbing, a bacterial disease of all citrus varieties worldwide, is currently devastating the US citrus industry. The hemipteran insect vector, Diaphorina citri, transmits the phloem-limited, non-culturable "Candidatus Liberibacter asiaticus" (CLas) in a circulative, propagative manner. The few management options rely on insecticides and tree symptom alleviation. With the vision of sustainably and economically managing HLB, our mission is to halt the spread of HLB to new healthy trees by researching ways to disrupt D. citri acquisition and transmission of CLas. Prior research shows D. citri populations have highly variable vector acquisition phenotypes, but each population maintains unique, stable, acquisition rates across multiple generations, suggesting a genetic component may regulate vector capacity. In a short timeline of 2-3 years this project will determine whether D. citri transmission and acquisition traits are linked to dominant or recessive genes, estimate how many alleles may be involved, provide insight into the genetic architecture and specific genes involved in CLas acquisition, and provide insight that can be used to update policies on HLB prevention, mitigation and control in citrus groves. The potential impacts of this work on the scientific community and the vector-biology field are many. This work is collaborative and multidisciplinary, applying plant breeding bioinformatic pipelines to an insect vector of a plant bacterial pathogen. The project lead, Marina Mann, is receiving training in bioinformatics and computational biology, population genetics, vector biology, plant pathology and more. D. citri is a non-model species with a high-quality genome assembly, a valuable resource not available to most agricultural insect pests and a necessary ingredient for Objective 1 in this proposal. This work is potentially transformative, as genome wide association studies are still rare in vector biology and have the potential to discover more genes regulating transmission across many other vector pathosystems. Activities for each objective under the main goal: Objective 1 determines whether D. citri genetics affect CLas acquisition and transmission and investigates D. citri genetic architecture. We hypothesize the ability to acquire CLas is a heritable, quantitative trait regulated by D. citri genetics. Marina, in collaboration with Douglas Stuehler and members of the Buckler lab, completed genome-wide association studies (GWAS) and identified multiple regions of the D. citri genome strongly associated with high CLas titer (our acquisition phenotype). D. citri adults (n=500) from four citrus fields in Florida, USA, were assessed for absolute CLas titer using quantitative PCR, then DNA sequenced to 7x coverage yielding terabytes of raw data. After aligning to the D. citri genome (Diaci v3.0), calling haplotypes, genotyping, imputing over missingness, and then filtering to reduce false positives, we applied an association model (BLUE(phenotype + batch) ~ SNPs + Kinship + e) which accounts for sample handling bias, field site effect, kinship and residual error. Multiple associated loci have been identified across the genome with varying effect size. Work is ongoing to find and characterize genes under the association peaks, as well as conduct a Gene Ontology analysis to find functional pathways for significant genes. Additional analyses being conducted in parallel include aspects of population genetics (kinship, heritability metrics, sex determination effect on associations, linkage disequilibrium are top of the list). The analyses are nearly finished, and writing and manuscript prep has begun. Objective 2 determines the role of inoculum source in CLas acquisition and transmission efficiency. Growers and current state policies for citrus management rely on testing trees for CLas infection. However, CLas is not uniformly distributed within a tree, yielding many false negative results. However, if there are CLas-positive adult D. citri present, they will be transmitting CLas to healthy leaves. Efforts focused on controlling vector populations are necessary for long term HLB management, but without studies of in-grove D. citri acquisition and transmission rates, management strategies risk underestimating natural variation and infection pressure from wild vector populations. Using over 1000 adult psyllids collected from four citrus groves of Citrus sinensis (sweet orange) in central Florida, each with unique management regimens, Marina applied quantitative polymerase chain reaction (PCR) methods to measure bacterial titer of both CLas and an endosymbiont, Wolbachia. With the help of an expert statistician, the infection results were fit to a mixed-effect linear model that accounted for much of the variation in the dataset. This work is currently in prep for submission to a peer reviewed journal. While there are not enough replicate fields sampled to determine the components of field management that influenced CLas titer in D. citri, there is a strong field effect on CLas titer which far outweighs any impact from Wolbachia. The data suggest that future studies should prioritize understanding the effect of citrus and psyllid management practices on the dynamics of D. citri infection by CLas. <br><br><b>Publications</b><br>
Target Audience
The target audience for this research includes the scientific community, specifically those working on vector biology, vector-pathogen interactions, epidemiology of plant pathogens, and population genomics. This research also targets citrus growers and other citrus stakeholders. Efforts also target training/mentorship of up-coming/junior scientists, especially women and minorities.
Changes / Problems
Objective 1 changes: While the hypothesis being tested remains the same - that distinct D. citri genes regulate the ability to acquire and transmit CLas and that this may include a combination of major and minor effect genes - a more targeted approach has been pursued for this objective. The proposed project narrative research plan for objective 1 involved inbreeding isofemale lines, but after discussion with my major advisor (Michelle Heck) and primary collaborator for this objective (Edward Buckler), objective 1 has shifted to focus efforts initially on computational methods utilizing genomic data via genome-wide association mapping (GWAS). Results from a genome-wide association analysis will be later complemented by the cross-variable correlations achieved from isofemale line inbreeding experiments. Objective 2 changes: With Michelle, my collaborator (Neil McRoberts), and mentee (Maida Ruiz) input, I have further developed my hypotheses, experimental plans and developed a deeper understanding of the current epidemiological modeling efforts available to the Huanglongbing pathosystem. This objective now has two specific aims: 1) Modeling HLB disease incidence in a citrus grove over time and predicting the differential impact of introducing CLas(+) trees vs CLas(+) D. citri, and 2) running targeted greenhouse experiments to clarify some gaps in the literature, provide numbers to inform my model, and help determine the role of inoculum source in CLas acquisition and transmission. Further, this project's hypotheses and goals overlap significantly with another on-going project in the Heck lab involving Citrus tristeza virus (CTV) construct silencing of psyllid genes. I am adding CTV constructs to my current experimental plans and I am excited to apply this technology to my models of HLB transmission in the field, adapting my predicted outcomes to include the effects of citrus groves implementing plant-modulated delivery of psyllid gene suppressor proteins via CTV constructs for suppressed vector transmission.
Training & Professional Development
This project provided many opportunities for my professional development and training. For objective 1, I developed and began learning to navigate a collaboration with Edward Buckler and members of his lab to learn computational pipelines for genome-wide association mapping and estimation of trait heritability (including specific programs, troubleshooting, and discussion of underlying assumptions behind the computational methods employed). I also attend and present in his lab meetings to gain wider feedback and connections, and I post questions, updates and other progress on Slack - the communication hub utilized by the Buckler lab. For objective 2, I began and developed collaboration with Neil McRoberts and began mentorship of an undergraduate - Maida Ruiz - from the University of California, Riverside who works in Dr. McRoberts' lab. This undergraduate helped (with my direction) accumulate a literature search and further develop my working hypothesis and experimental plans. Additionally, this fellowship allowed me to attend the Colman Inclusive Leadership Program at Cornell University, and to virtually attend the Arthropod Genomics Symposium.
Dissemination Streams
Scientific findings from both objectives 1 and 2 were orally communicated to a group consisting of citrus growers and scientists at the Citrus Research Board meeting, and feedback was acquired.
Next Reporting Steps
During the next reporting period: Objective 1 will be brought to publication-readiness and experimental work will commence on objective 2. Specifically, the objective 1 pilot analysis conducted during the past reporting period will be expanded with an additional 500 samples sequenced to 7x coverage and the computational pipeline will be repeated on this full dataset. Results analysis will dig deeper within the identified loci of interest to determine specific gene candidates involved in acquisition and transmission success. Depending on results from the genome-wide association study, the experimental plan outlined in the original project narrative for objective 1 will be pursued as additional support for the hypothesis. I will also attend the American Phytopathological Society (APS) Plant Health 2022 conference and present results from objective 1 as a poster and as an accompanying zine. Objective 2 will commence past the planning stage and will include closer collaboration and more frequent contact with the primary collaborator and mentee. Experiments testing and clarifying multiple insect-pathogen-plant interactions (filling current gaps in the literature or further verifying past results) will be conducted, analyzed and results will be integrated into the epidemiological model. Multiple simulations will be run to predict the effect of infected trees, the effect of infected vectors, and the effect of planting trees containing CTV constructs, on the spread of HLB in citrus groves. Mentoring of Maida Ruiz will continue. Results from the experiments and preliminary models will be written up and prepared for submission by the end of the next reporting period. <br><br>
<br>What was accomplished under these goals? Impact Statement Bacterial vector-borne diseases take a huge toll each year on agricultural production and the economy, but due to the intimate relationships between the insect vector, plant host, bacterial pathogen and the environment, these disease systems are difficult to study. Recent studies have suggested that vector genetics as well as the source of inoculum may play larger roles in determining pathogen transmission to new plants than thought. Huanglongbing (HLB), or citrus greening disease, is the most devastating disease of citrus worldwide. The HLB pathosystem involves three players: the bacterial pathogen called "Candidatus Liberibacter asiaticus" (CLas), the Asian citrus psyllid insect vector (Diaphorina citri), and the citrus trees (including all known varieties of citrus). The ultimate goals of this project are two-fold: 1) to clarify the relationship between the in-field diversity of D. citri populations to build control methods that are more targeted, and 2) to access which aspects of the HLB pathosystem are most important to target for control of HLB in the field. M. Mann worked with her supervisor, Michelle Heck and collaborators Edward Buckler from the USDA-ARS in Ithaca NY (objective 1) and Neil McRoberts from the University of California, Riverside CA (objective 2) to further develop her hypotheses and experimental designs, and to learn, apply and troubleshoot various methods and pipelines. While objective 2 experiments will commence in the next reporting period, objective 1 results have provided a computational pipeline that suggests the D. citri acquisition and transmission phenotype is heritable, and that three D. citri chromosomes have genetic regions that are strongly associated with increased CLas titer. NIFA Objective 1: Work this year was predominantly focused on this objective as outlined in my project narrative evaluation plan's timeline, and will continue into the 2nd year of my NIFA fellowship. The overarching hypothesis of my NIFA fellowship is that disrupting D. citri transmission of CLas will lead to long term management solutions. While the proposed research plan for objective 1 involved inbreeding isofemale lines, after discussion with my major advisor (Michelle Heck) and primary collaborator for this objective (Edward Buckler), objective 1 has shifted to focus efforts initially on computational methods utilizing genomic data via genome-wide association mapping (GWAS). Specifically, I aim to 1) describe heritability of the variable vector trait, and 2) identify specific genes or loci underlying the trait of interest along the entire chromosome. Due to the available high quality genome of D. citri, a method like GWAS is available for this non-model organism. Activities thus far include attending and presenting at Buckler lab meetings, and as a member of the Buckler lab Slack account, I share updates and communicate with various people in the lab who are experts in the computational methods behind GWAS and heritability studies in maize. I have moved through the computational pipeline with the help of multiple graduate students and postdocs in the Buckler lab, from raw reads to a Manhattan plot that shows SNP distribution across the entire genome. Preliminary results from the pilot study suggest there are three chromosomes with loci that strongly associate with increased CLas titer, the metric used to measure the variable vector phenotype of interest. This objective deals with genomic data from hundreds of D. citri, and the analysis methods require the presence of a high-quality, chromosomal-length genome assembly which is available for this insect vector. I have been added as a co-author on the manuscript publicizing this genome, which is available on bioRxiv and undergoing revisions before re-submission for publication. Additionally, once loci of interest are identified, other 'omics analyses will be pertinent to add depth of understanding. To this end, I have also published a transcriptomic analysis of D. citri, broken up by multiple specific organs and comparing healthy D. citri to CLas-infected D. citri. This manuscript has been published in GigaScience. NIFA Objective 2: As predicted in my project narrative evaluation plan's timeline, work this year was focused mainly on objective 1 and I will be shifting focus to objective 2 during the 2nd and 3rd years of my NIFA fellowship. However, I have continued to develop and maintain a collaboration with Neil McRoberts (University of California, Riverside), I began virtually mentoring an undergraduate student in his lab (Maida Ruiz), and continued developing the experimental plans for this objective. Additionally, my primary mentor Michelle Heck - through her conversations about this project at the annual Citrus Research Board meeting in California - added Robert Shatters (USDA ARS, Fort Pierce FL) to this project's collaborators. With Michelle, my collaborators, and mentee's input, I have further developed my hypotheses, experimental plans and developed a deeper understanding of the current epidemiological modeling efforts available in the Huanglongbing pathosystem. Further, this project's hypotheses and goals overlap significantly with another on-going project in the Heck lab involving Citrus tristeza virus (CTV) construct silencing of psyllid genes. I am adding CTV constructs to my current experimental plans and I am excited to apply this technology to my models of HLB transmission in the field, adapting my predicted outcomes to include the effects of citrus groves implementing plant-modulated delivery of psyllid gene suppressor proteins via CTV constructs for suppressed vector transmission. The overarching hypothesis of my NIFA fellowship is that disrupting D. citri transmission of CLas will lead to long term management solutions. The working hypothesis of objective 2 is that a CLas(+) citrus tree has a smaller impact on increasing HLB incidence than CLas(+) D. citri. This hypothesis has two specific aims: 1) Modeling HLB disease incidence in a citrus grove over time and predicting the differential impact of introducing CLas(+) trees vs CLas(+) D. citri, and 2) running targeted greenhouse experiments to clarify some gaps in the literature, provide numbers to inform my model, and help determine the role of inoculum source in CLas acquisition and transmission. Aim 1 requires the aid of Neil McRoberts who is an expert in epidemiological modeling and has previously worked on models of insect-vector diseases systems. Aim 2 will be conducted in Heck lab spaces with help from Heck lab members. Thus far, for aim 1 I have worked closely with Maida Ruiz to generate a list of model assumptions and key parameters. For aim 2, I have tested and successfully implemented a cutting-based plant propagation system (a new SOP for the Heck lab), which reduces the time for citrus plant propagation from 6+ months, to 3-6 weeks. Additionally, I trialed this method using multiple varieties of healthy citrus and CLas-infected citrus and all were successful in greenhouse and growth chamber conditions. Establishing cutting-based propagation of CLas-infected material is crucial to the experimental plans for this objective due to the large number of biological replicates required. It allows me to improve the estimated time needed to set up and run experiments and benefits other lab members experimental designs as well. Other Heck lab members have also shown that this cutting propagation method works with the citrus varieties containing CTV constructs. This SOP is being included in a manuscript currently under prep, detailing Heck lab plant, insect and CLas propagation methods. <br><br><b>Publications</b><br>