Performing Department
Plant Pathology
Non Technical Summary
This Standard Research and Extension Project (SREP) addresses two SCRI focus areas: 1) Pest Management; and 2) Plant Breeding and Genetics. A trans-disciplinary team of plant pathologists, entomologists, vector ecologists, a modeler, horticulturists, ecophysiologists, an economist, and extension specialists will investigate solutions to a serious and recalcitrant problem, laurel wilt, of an important specialty crop, avocado. Laurel wilt has spread throughout Florida since 2011 and recently reached Texas. This disease has caused the abandonment of avocado production wherever expensive control measures have not been used. If adequate solutions for this problem are not found, commercial avocado production may become impossible in Florida, and as the disease spreads significant avocado production could eventually be impacted in other states (e.g. California and Hawaii), US protectorates (Puerto Rico), and other countries. Cost-effective measures to manage laurel wilt are needed, but will require new information in several key areas. The proposed work will fill critical data gaps, utilize new information to manage this problem, and inform stakeholders in Florida, Texas and California of the disease's status and advances in its management. Information will be presented in diverse formats in both English and Spanish due to a significant Hispanic clientele. The proposed work has significant stakeholder support and interest in all US commercial avocado production areas. Substantial stakeholder engagement ensures broad and timely dissemination of project outputs, as well as stakeholder input on project goals and objectives.
Animal Health Component
0%
Research Effort Categories
Basic
20%
Applied
70%
Developmental
10%
Goals / Objectives
The overarching goal of this project is to better understand the pathogen and vector dynamics of laurel wilt disease, devise strategies for mitigating losses and spread of the disease and to engage stakeholders to understand their realities in dealing with laurel wilt disease and provide the latest science-based knowledge for disease management. We are pursuing this goal through several specific objectives:Objective 1. Identify existing ambrosia beetles associated with commercial avocado production in Texas and California and model continent-scale disease movementObj. 1a. Identify existing ambrosia beetles associated with commercial avocado production outside of Florida.Obj. 1b. Identify fungal symbionts of ambrosia beetles associated with commercial avocado production outside of FloridaObj. 1c. Model continental-scale disease movement (Obj1c).Objective 2. Propagate and screen for laurel wilt resistant, cold-tolerant germplasmObjective 2a. Determine laurel wilt tolerance of avocado cultivars bred for cold-toleranceObjective 2b. Evaluate cold-tolerance of LW-tolerant avocado cultivars on UCR advanced PRR-resistant clonal rootstocks and progenies of open pollinated LW-tolerant seedling trees under cool orchard conditionsObjective 3. Further develop biological and cultural management of beetle vectorsObjective 4. Establish mechanisms of host resistance and susceptibilityObjective 4a. Compare dynamics of colonization, tylose development and gene expression in North American susceptible versus SE Asia resistant Lauraceae species.Objectives 4b. Relate within-tree movement of H. lauricola to the xylem sap flow rateObjective 5. Utilize a model host species to test endogenous and exogenous RNAi strategies for developing disease resistance based on interference of factors required for pathogenicityObjective 6. Further develop fungicidal, cultural and biological management strategies for laurel wiltObjective 7. Conduct economic assessments of management optionsObjective 8. Engage growers and scientists through extension activities
Project Methods
Controlled experiments will be conducted to test specific hypotheses under each of our objectives. Methods employed will vary over the scale and locations of the experiment from field trials to molecular manipulations in the laboratory. All experiments will be conducted and analyzed with the rigor, objectiveness, skill and latest technology present in the programs of the world-class scientists involved. Specific methods are outlined here. Periodic ambrosia beetle trapping and collection of infested logs will be undertaken in commercial avocado orchards and surrounding. The contents of each field trap will be sorted into para-taxonomic "species" under a stereo dissecting microscope using taxonomic keys. Genetic information will be generated and used to document and compare species composition, and assess intraspecific genetic variation, between orchards, counties, and states. At least 20 females from the most common ambrosia species trapped in each site will be processed for fungal isolation and identification through DNA analyses. For modeling disease spread, R packages we have previously developed and new custom codes will be used to combine our previous models with updated data obtained from beetle identifications and scenarios to determine the likely risk of avocado losses to laurel wilt in other states and regions. Utilizing avocado selections from the cold-tolerance breeding program, we will test lines for resistance to laurel wilt under controlled and repicated conditions. To better understand the role of cultural interventions of laurel wilt vector behavior, individual plots within an avocado orchard will be subject to one of the four main canopy treatments used in avocado production including tree topping, hedging, selective pruning and no pruning. Each treatment will be replicated in ten different plots. In addition, tree skirting will be undertaken in half of the experimental plots. Ambrosia beetle flight activity will be monitored on a biweekly basis using traps. The experiment will follow a factorial block design with canopy cover and skirting as the main factors. The number of laurel wilt vectors captured in traps will be analyzed using multiple regression analysis with canopy cover, tree skirting, and their interaction as fixed factors, while sampling date and light intensity nested within the year as random effects. To identify potential biocontrol agents for the beetle vector, ambrosia beetles and associated mites will be collected from six commercial avocado orchards distributed throughout the tropical fruit-growing region in south Florida. The capacity of the different mites to colonize and disrupt ambrosia beetle breeding sites will be evaluated by releasing mites on avocado logs previously infested with ambrosia beetles. Beetle mortality data will be analyzed using a generalized linear mixed-effects model with negative binomial distribution. The spatio-temporal dynamics of H. lauricola colonization of the laurel wilt resistant Lauraceae species Cinnamomum camphora will be compare with established colonization dynamics of susceptible avocado and swampbay trees through recovery-plating assays utilizing a green-fluorescent protein (GFP) -tagged strain of the pathogen. Using histology and epi-fluorescence microscopy, we will compare the morphology and cell-specific interactions of H. lauricola with these resistant and susceptible hosts and we will conduct comparative transcriptomic analyses of susceptible and resistant host gene expression preceding and following inoculation with the pathogen. The number of vessels with and without tyloses will be quantified from these scanned images using an artificial intelligence model we recently developed using the convolutional neural network YOLOv8 (Ultarlytics, Los Angeles, CA) to expedite detection and quantification of vessels with and without tyloses. The percent of vessels with tyloses will be statistically analyzed using a Kruskall-Wallis test for nonparametric distributions with the software R. Significant differences in the distributions will be followed by a post-hoc Wilcoxon rank-sum nonparametric test using the "bh" option to control for p-value adjustment for multiple comparisons with R. To determine defense pathways, metabolic shifts, cell wall dynamics and other processes that are differentially regulated in resistant versus susceptible hosts of H. lauricola, we will inoculate and evaluate resistant C. camphora trees and susceptible 'Waldin' avocado trees. Following RNA extraction and sequencing, the mapped reads will be counted with featureCounts and differential expression statistics will be derived using the package DESeq2 with the statistical software R (4.3.0) using the function DESeq2. From the available genome annotations, analysis of up-regulated or down-regulated GO terms will be produced using the R package clusterProfiler with the enricher function. Using the GFP-labeled strain of H. lauricola we will determine if susceptibility to laurel wilt is related to the rate of xylem sap flow and within-tree movement of the H. lauricola. Xylem sap flow and symptom progression will be related to the within tree movement of H. lauricola conidia and hyphae by linear and/or non-linear regression analyses. Differences in disease severity, xylem sap flow transpiration and fungal movement among treatments will be determined by repeated measures ANOVA. Statistical analyses will be done with SAS statistical software. Experiments will begin with proof of principal utilizing exogenous application of double-stranded RNA (dsRNA) to block H. lauricola growth. dsRNA treated and control plants will be inoculated by stem injection and monitored for symptom development daily. Disease incidence and disease severity ratings will be recorded using the same external rating scale we use for avocado. Statistical differences among dsRNA treated and untreated plants will be determined by non-parametric Wilcoxon analysis followed by a Bonferroni correction. All statistical analyses will be conducted using SAS statistical software. To determine the efficacy of fungicides, Trichoderma and Tolypocladium culture filtrates and antimicrobial peptides, laboratory, greenhouse, and field trials will follow standard procedures to assure statistical power. The screening of fungicide formulations will be done through consultation with the IR4 program and the chemical companies. Formulations that have shown good uptake and translocation (based on residue analysis in potted avocado plants) will be tested under field conditions using mature trees. Residue testing will be conducted in all experiments, from vascular tissue and from fruits. The screening of active ingredients. and formulations will be expanded to compounds with different mode of action and include natural substances such as chitosan-based products. Economic tools will be developed to assess the cost of disease management for Florida avocado growers. Assessment tools will be part of the online resource to facilitate stakeholders and policy makers decision making based on the costs of the proposed control measures. The laurel wilt extension program will provide regular industry updates through the Florida Avocado Administration, industry surveys, hybrid-workshops and seminars, guest speaking engagements (local, regional, state, national and international), field days, Extension Agent and Master Gardener in-service training, extension videos and e-publications, and email updates. The existing laurel wilt website will continue to curate extension materials and PowerPoint presentations given by members of the project. Workshops, seminars, and field days will be evaluated with pre- and/or post-program surveys. The impact of the LW website, videos, and electronic publications will be determined by page views or downloads (or other appropriate parameters).