University of Florida

University of Florida

University of Florida

University of Florida

1. Characterize the blockage materials with immunoassay techniques with TEM and test for induction signals and virulence factors.
2. Transgenic approaches to disrupt HLB-associated callose and phloem protein plugging by reducing their production.
3. Reduce sieve element blockage in field and greenhouse HLB-infected plants through treatment with chemicals that are expected to alter callose and p-protein production. The information learned from the proposed research may lead to alternative control methods and will contribute to the understanding of the virulence mechanisms of Las and will be distributed through an extension program.

1. Continue to examine the susceptibility of citrus genotypes to Las.
   1. Compare the response of commercial varieties and more citrus relatives to Las.
   2. Examine the sensitivity of elite lines and select hybrids for the Plant Improvement Group.
2. Examine the reduction in Las titer in Poncirus trifoliate.
3. Characterize the pathogen that induces symptoms in Citrus latipes.

1. Continue to screen peptides for activity against Las.

   1a. Screening against Las in citrus.

   1b. Screening against Liberibacter in tobacco.

2. Examine ability to export peptides from the rootstock.
3. Improve Citrus tristeza virus (CTV)-based vector.

   3a. Develop a vector to express 2–5 peptides.

   3b. Develop a vector to overcome cross protection.

   3c. Remove aphid transmissibility from the vector.

   3d. Develop a vector that is restricted to transgenic tree parts.

4. Examine survival of peptides in fruit and juice.
5. Develop transgenic citrus trees with effective peptides—mature transformation.

University of Florida

USDA

USDA

Cornell University

USDA

1. Develop a better method for monitoring both psyllids and HLB pathogens in the environment.
2. Identify attractants for the psyllids. Project will address the needs of regulatory agencies as well as citrus growers.

1. Development of HLB in young citrus subjected to intensive or moderate insecticide control programs or an oil program for psyllid control.
2. Development of HLB in young citrus subjected to an intensive insecticide program for ACP control compared to development of HLB in young citrus subjected to a moderate insecticide program in combination with growing Murraya paniculata as a potential trap plant or source of ACP natural enemies.
3. The effect of planting density on development of HLB in young citrus subjected to an intensive insecticide program for ACP control.
4. Protecting citrus resets from HLB.

1. To study pathogen–vector cellular interactions, transmission barriers, and other factors affecting vector specificity and vector competence.
2. To clarify various acquisition and transmission parameters between the psyllid vector and the HLB pathogen.

Induced citrus plants to mimic the molecular conditions associated with heat-induced Las resistance. We hypothesized that citrus plants can be genetically engineered to express the molecular mechanisms associated with heat-induced Las resistance.

Develop genetically modified citrus plants that will enhance defense response to HLB infection. We hypothesized that there will be a net gain in fitness and resistance to Las in genetically modified plants compared to non-genetically modified plants.

USDA

University of Florida

Objectives

Control of HLB using the putative Las LexA target

Control of HLB using the psyllid repressor as target

Control of HLB using Las peroxidase and Las lytic cycle activator(s) as targets

Field and greenhouse testing of lead compounds

USDA

University of Florida

University of Florida

To test the effect of AHL-producing citrus plants on the pathogencity of CLas; an AHL-producing citrus plant will be produced to study the effect of AHL on the pathogencity of CLas.

To prevent CLas from infecting citrus plants by jamming bacterial communication of CLas; quorum-sensing (QS) antagonists will be used to block the cell-to-cell signaling of CLas. Identification of AHLs in Asian citrus psyllids hemolymph using GC-MS. Because different types of symbionts are confined in a small environment “arena” within their insect host, cooperative interactions and communication among theses.

Test commonly used bioassay strains to find the best reporter strain for the AHLs present in ACP or AHL mimic compounds present in the phloem sap of citrus. In addition, we aim to develop a more sensitive reporter (double sensors).

Confirm HLB resistance or tolerance in citrus transgenic lines and putative mutants.

Stack the ELP3 and NPR1 genes in citrus.

Generate and test transgenic citrus plants overexpressing newly cloned disease resistance genes.

University of Florida

University of Florida

University of Florida

1. Establish nutrient sufficiency guidelines for leaf tissues of HLB-affected trees that have successfully responded to nutritional programs in order to help growers replicate the successes and achieve higher nutrient efficiencies.
2. Determine soil conditions that favor root hair and VAM proliferation of citrus roots, thus maximizing root uptake of calcium and other deficient nutrients in HLB-affected citrus trees.
3. Establish testing protocols and remedial soil amendments that can be deployed to maximize root uptake of calcium and other deficient nutrients in nonresponsive HLB-affected groves.
4. Deliver results of the research project to the Florida citrus industry through extension/outreach to all stakeholders (growers, contractors, supporting industries).

Determine the fight initiation thresholds of ACP depending on temperature and humidity. Determine the effect of wind speed on flight and the direction of psyllid flight with respect to wind.

Determine the effects of barometric pressure changes on psyllid dispersal.

Measure how psyllid dispersal is affected by abiotic factors in the field.

Establish a model to predict the risk of ACP dispersal/invasion based on prevailing abiotic conditions. Deliver this model as an online tool for growers.

Continue to integrate multi-omics approaches to improve the medium for L. crescens, which, by extension, will get us closer to a robust medium for L. asiaticus.

Overarching—embedded with all other objectives. Test each new media design for its ability to sustain growth by L. asiaticus.

Characterization of the physiology and genome of the L. asiaticus culture.

University of Florida

University of Florida

University of Florida

University of Florida

Control HLB by optimization of application of SA and its analogs. Application of SA and its analogs have potential to neutralize the SA hydroxylase. Based on our previous study, foliar spray of SA and its analogs slowed down the increase of Las population in citrus and HLB disease severity, whereas trunk injection of SA and its analogs significantly reduced Las population. The previous study suggest that we can improve the HLB management by optimizing the application methods of SA and its analogs.

Control HLB using a combination of SA, SA analogs, or SA hydroxylase inhibitors. By combining SA or SA analogs with SA hydroxylase inhibitors, we could improve the efficacy of plant defense inducing to control HLB. SA hydroxylase inhibitors can directly counteract Las SA hydroxylase. We have identified six SA hydroxylase inhibitors in our previous studies. We will continue to optimize those SA hydroxylase inhibitors.

Human resources as well as equipment in different research groups can be used for other activities while CCTF produces transgenic material for them.

Timely realization of ideas into life through production of transgenic plants that can be challenged by pathogens/vectors to test their susceptibility to diseases within a short period of time.

USDA

University of Florida

University of Connecticut

University of Florida

University of Florida

To characterize the phytobiomes and endophytic microbes from HLB survivor trees and HLB diseased trees

To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB

Using transient down regulation of AGO2 and NRPD1a expression to improve

Agrobacterium stable transformation efficiency of mature citrus tissues

Using H2A co-expression to improve Agrobacterium stable transformation efficiency of mature citrus tissues

Using a combination of genes to develop a super-transformation-vector that is genotype independent, highly effective for transformation and regeneration of mature citrus Using root specific auxin biosynthetic gene to engineer rootstock of citrus to improve success rates of micrografting

1. GFP labeling of Candidatus Liberibacter asiaticus in vivo
2. Elucidation of plant–Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las
3. Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las

1. Determine the optimum adjuvant-kaolin rate combinations to improve rainfastness of dyed kaolin particle film on citrus foliage.
2. Determine the optimum concentration of red-dyed kaolin residue to deter ACP.
3. Determine the effect of dyed kaolin particle films on ACP populations and CLas transmission under field conditions.
4. Assess horticulturally relevant physiological impacts of optical properties of red-dyed kaolin particle films as compared to undyed films.

USDA

University of Florida

University of Florida

University of Florida

University of Florida

Culturing Las bacterium in vitro running metabolic pathway analyses.

Role of the microbial community and genetic diversity of Las bacteria in HLB development.

Seed transmission of Las bacterium.

Monitor Las population dynamics in citrus and periwinkle plants after chemical and heat treatment.

1. Evaluate and refine the use of the tap sample to monitor adult psyllids and methods of assessing flush density, and infestation rates in blocks of growing and dormant citrus trees of different ages and varieties planted at experimental and commercial groves.
2. Test the influence of these parameters on precision of estimated means and the distribution of the population within blocks using the appropriate aggregation and regression models.
3. Evaluate and integrate methods for assessing these parameters into a user friendly system accessible to consultants and managers.

Examine psyllid fitness on different commercial citrus varieties grown in Florida and the effects of rootstock selection on suitability of scion material for psyllid development.

Evaluate psyllid fitness when reared on non-commercial citrus species and other rutaceous noncitrus host plants.

Examine the effects of host plant quality on feeding behaviors of adult psyllids.

1. Import, release, and evaluate new strains and species of parasitoid specific to D. citri.
2. Identify genetic markers that can be used to track T. radiata in the environment.
3. Develop efficient methods for mass rearing and release of T. radiata and possibly other species to increase biological control through augmentation of natural populations.
4. Transfer technology to industry clientele.

Determine how flush availability impacts ACP movement and whether HLB-infected ACP disperse more than noninfected counterparts.

Quantify ACP dispersal distance within managed groves and distance of dispersal between managed groves.

Confirm the impact of abandoned groves on psyllid populations in neighboring managed groves.

Determine height at which psyllids disperse.

Develop management recommendations based on understanding of ACP dispersal behavior patterns.

University of Florida

University of Florida

Auburn University

University of Florida

Southwestern Medical

1. Transcriptional and microscopic analyses of different citrus varieties that are either susceptible or tolerant to Ca. L. asiaticus infection at different infection stages in greenhouse and citrus grove.

   1.1. Identify citrus genes with altered regulation caused by inoculation with Ca. Liberibacter asiaticus in susceptible and tolerant citrus cultivars.

   1.2. Characterize the relationship between the population of Ca. Liberibacter asiaticus in the phloem, anatomical changes of phloem, nutrient transport, and symptom development in susceptible and tolerant citrus cultivars.

2. Transcriptional and microscopic analyses of model system tobacco to Las infection.

Collect inoculum and phloem fluid from HLB-affected and healthy plants.

Observe movement and aggregation of Las inside microfluidic chambers.

1. Identify the putative effectors based on the genome sequence of Las and cloning into CTV vector.
2. Expression of selected putative effectors of Las in citrus using CTV vector and transcriptional analysis of the host in response to expression of Las effectors.

To compile a database from all the data obtained in the course of the project and make it available at our group website and at the central depository www.citrusgreening.org.

USDA

University of Florida

USDA

University of Florida

1. Determine the most effective rates and application methods for soil-applied systemic insecticides to control psyllids on nonbearing trees of different sizes.
2. Evaluate the use of Kaolin clay particle films in conjuction with various adjuvants/stickers to increase the rainfastness of these applications.
3. Determine the ability of soil-applied systemic insecticide applications in combination with Kaolin clay particle film or dimethyl disulfide (DMDS) to protect nonbearing trees from HLB in a commercial grove setting.

Identify specific salivary proteins secreted in both the gelling and the watery saliva. (year 1 and 2)

Use information from 1 and 2 above to construct a model describing the ACP feeding process. (end of year 2)

Use artificial diet system to screen for salivary sheath/feeding inhibitory compounds. (initiate in year 1 and continue through year 2)

Conduct plant spray tests to determine if inhibitory compounds currently identified (i.e., EDTA and related chelators) or identified in the future, will work to prevent ACP from feeding on plants. (initiate in year 1 and continue to field trials in years 2 and 3)

Use information to identify a transgenic plant approach to create citrus that block the ACP feeding process and thus are not at risk of becoming HLB infected. (year 3)

1. To determine whether healthy ACP distinguish between healthy versus HLB-infected citrus plants
2. To determine whether behavior of Ca. Las-infected ACP to healthy versus infected citrus plants differs from the behavior of ACP that are not carrying the pathogen
3. To determine whether the settling preference of healthy and Ca. Las-infected ACP on healthy versus HLB-infected citrus plants differs from the behavior of ACP that are not carrying the pathogen
4. To identify the active synthetic plant compounds that mediate ACP response to infected plants by GC-MS
5. To determine whether behavior of ACP to healthy versus HLB-infected trees differs in the field
6. To determine whether synthetic chemicals identified from HLB-infected plants attract ACP

University of Florida

University of Florida

University of Florida

USDA

1. Determine if field release of methyl salicylate (MeSA) will enhance biological control organisms.
2. Determine if MeSA is repellent to ACP.

Determine which fully sequenced, culturable bacterium is most closely related metabolically to Ca. L. asiaticus and then apply those growth conditions to culture Ca. L. asiaticus.

Perform the experiments necessary on citrus with the cultured pathogen to satisfy Koch’s postulates. Predict the antibiotic sensitivity of Ca. L. asiaticus based on its sequenced genome and verify this sensitivity in the cultured organism as well.

Identify genes in Ca. L. asiaticus that can be targeted for inhibition based on comparative genomic analyses with all other sequenced bacteria.

Evaluate the viability of extracted “Ca. L. asiaticus” cells.

Pennsylvania State University

University of Florida

University of Florida

University of Florida

1. Documentation of citrus greening management by application of the imidacloprid over time after selected irrigation applications.
2. Determine sorption and transformation of imidacloprid in soils.
3. Assess the soil transport and plant uptake of the applied imidacloprid and its metabolites over time after selected irrigation applications.
4. Characterize root zone water movement using bromide tracer.
5. Monitor psyllid populations and incidence of HLB over time.

1. Augment biological control of D. citri through mass production and release of previously established and imported colonies of T. radiata and D. aligarhensis.
2. Assess parasitism rates at release and control sites using feral populations of D. citri and infested sentinel plants.
3. Develop a system of genetic identification of the parasitoids recovered from the field to prove the establishment and effectiveness of released biotypes.
4. Transfer technology to industry clientele.

1. Evaluate psyllid populations, HLB incidence and intensity, gene expression, tree growth, and eventually yield in newly planted citrus blocks.
2. Assess separate contributions of vector control and foliar nutritional applications to the above parameters.
3. Evaluate the effectiveness of reflective mulch to control ACP.
4. Provide economic analysis of costs and projected benefits.
5. Extend results to clientele.

USDA

University of Florida

USDA

University of Florida

University of Florida

The second phase will be to choose from the effective genes ones that can be effectively marketed or to modify selected ones such that they are suitable.

1. Generate transcriptome profiles of both susceptible and resistant citrus responding to HLB infection using RNA-Seq technology.
2. Identify key resistant genes from differentially expressed genes and gene clusters between the HLB-susceptible and HLB-resistant plants via intensive bioinformatics and other experimental verifications, such as RT-PCR.
3. Create transgenic citrus cultivars with new constructs containing the resistant gene(s).

Cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter.

Cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter.

Cloning and expression of all four Las and the one possible Lam antirepressors, and determining responsiveness of the reporter and clones from Milestone 2.

Development of a chemical assay for Las-responsive SOS.

1. Phenotype 92 replicated progeny and determine the genetic basis and mode of inheritance of the HLB resistance in Poncirus.
2. Construct a linkage map of the Poncirus genome and locate and map the Poncirus genes conferring resistance to HLB.
3. Increase the marker density and mapping resolution to refine the map location of identified resistance genes.

University of Florida

USDA

University of Florida

University of Florida

University of Florida

Characteristics of the female reply that most effectively induce attraction of calling males will be identified in a laboratory setting.

Optimal replies will be tested in a field or greenhouse environment.

The best reply will be incorporated into a sticky (or alternative) trap with a piezoelectric buzzer that plays back the reply whenever a male call is detected.

The trap will be tested and modified as needed in laboratory and field tests with male ACP to determine whether infestations can be detected and targeted more precisely than with current methods.

If the trap is demonstrated to have enhanced efficacy, additional devices will be constructed for general field use.

Determine the capacity of alternative hosts as food sources and oviposition sites.

Directly sample ACP in citrus tree canopies and surrounding habitats all season long.

Determine effects of cold and heat acclimation on susceptibility of ACP to insecticides.

University of Florida

University of Florida

University of Florida

Objective 1, an electrical penetration graph (EPG) monitor will be used to determine whether insecticides can disrupt the psyllid feeding behaviors responsible for pathogen transmission. For insecticides that disrupt psyllid feeding behavior, the longevity of feeding disruption provided will be determined.

Objective 2 will examine the duration of psyllid control provided by foliar-applied insecticides in a typical grove setting. This objective will address whether certain insecticides perform better at certain times (seasons) of the year and examine the use of adjuvants to increase the longevity of psyllid control provided.

Objective 3 will examine the use of different application methods of neonicotinoid insecticides for young and intermediate-sized trees to maximize the level of psyllid control provided by soil-applied systemic products.

Objective 4 is a multiyear evaluation of 4 different season-long approaches to young tree care to determine which approach is most likely to prevent HLB in young trees in order to bring them into production.

Objective 5 will examine whether, once a young tree has become infected with the HLB pathogen, there is a benefit to continuing to control psyllids on those plants already infected.

1. Assess effects of abiotic factors (light quality, photoperiod, air flow, and temperature fluctuations) on psyllid movement.
2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density.
3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies.
4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field.
5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide-treated trap crops (such as Bergera koenigii) to attract and kill.

Subsequently, thorough field investigations will be conducted with the most promising chemicals under Florida conditions.

Objective 1. Determine the effect of non-neurotoxic compounds on mortality of ACP.

Objective 2. Investigate sublethal effects of non-neurotoxic compounds on development and fecundity of ACP.

Objective 3. Quantify the effect of non-neurotoxic compounds on ACP feeding behavior.

Objective 4. Determine the effectiveness of proven non-neurotoxic chemicals against ACP under field conditions in Florida citrus.

University of Florida

USDA

USDA

Objective 1. Evaluate factors that enhance visually induced flight behavior, including takeoff tendency, flight duration, and light choice of psyllids.

Objective 2. Optimize attraction of psyllids to visual targets to provide a pull system.

Objective 3. Develop and evaluate push-pull systems in small orchards and commercial groves.

Iowa State University

Texas AgriLife Research

Screen Bt strains for activity against the ACP and identify ACP-active Cry toxins. We will screen toxins from up to 200 Bt strains with diverse insect toxicities and toxin profiles for ACP toxicity.

Modify a Bt toxin for psyllid toxicity. We will isolate peptides that bind to ACP gut membrane by screening a phage display library, and confirm gut binding of selected peptides.

Therefore, full characterization is necessary before phages are released as biocontrol agents. All phages will be initially characterized as previously outlined for the ability to form clear plaques, not form lysogens and morphology. The genomes of candidate virulent phages will then be sequenced and annotated to ensure virulent status. Receptor diversity will be based on limited host range studies due to time limit.

Initiate phage persistence studies on plant tissue. Studies will be conducted as previously outlined in Objective IIIa using virulent phage Xfas303, a fully characterized KMV-like phage shown to have activity against Xac field strains (North 40, Block 22, Fort Besinger; see preliminary results) obtained from Florida. Phage will be sprayed with and without a protective carrier. The protective carriers developed by Balogh et al. (2003 and 2008) and along with others will be evaluated.

Determine therapeutic efficacy of Xac phages. Studies will be conducted as previously outlined in Objective IIIb in cooperation with Dr. Nian Wang.

Southwestern Medical

University of California, Riverside

University of Florida

Obj. 1. Using the computational pipeline developed for the analysis of CLas proteome, predict spatial structures and functions for all proteins in two available citrus genomes and drafts of the psyllid genome.

Obj. 2. Improve the prediction pipeline initially developed for prokaryotic genome and adapt it to the analysis of eukaryotic genomes.

Obj. 3. Present results of analysis as a comprehensive website with a dedicated webpage for each protein in these organisms, showing details of predictions.

Obj. 4. Compare analyzed genomes with each other. Find predicted physical and functional interactions and associations between proteins from different genomes.

Obj. 5. Foster collaborations with experimentalists, helping them with the analysis of proteins they have chosen as research targets.

1. Identify citrus proteins associating with three selected CLas effectors using yeast two hybrid screens.
2. Confirm the effector-host target interactions using a series of in vitro and in vivo assays.
3. Design control strategies aiming to enhance the resistance/tolerance of citrus to HLB based on effector activities and the functions of their targets.

Objective 1. Determine how biotic and abiotic parameters differ between resets, mature trees, and “party trees.”

Objective 2. Define the biotic factors that affect ACP host acceptance and fitness.

Objective 3. Investigate whether ACP population abundance differs among resets, mature trees, and party trees, and if party trees remain consistently attractive to ACP throughout seasons and years.

Objective 4. Quantify how time since tree inoculation with the HLB pathogen and abundance of HLB-infected trees affects ACP host acceptance behavior within groves.

Objective 5. Determine if and where ACP disperse from resets and party trees and how this might contribute to HLB transmission within groves.

University of Florida

Stanford University

USDA

Determine the nutrient content of citrus and periwinkle phloem, which are well colonized by CLas, and murraya and tomato phloem, which are not good habitats for CLas. Team members involved: Nabil Killiny-Mansour (primary), Mark Hilf, and Eric Triplett.

Determine the nutrient content of the psyllid hemolymph. Team members involved: Kirsten Pelz-Stelinski (primary), Nabil Killiny-Mansour, David Hall, and Eric Triplett.

Prepare and test media based on the findings from Objectives 1 and 2 to attempt to culture CLas. Team members involved: Mike Davis (primary), Mark Hilf, and Eric Triplett.

Digitize the media recipes attempted to date by Prof. Davis into a searchable relational database. Team members involved: Eric Triplett (primary) and Mike Davis.

Once cultured, CLas will be characterized physiologically and made available to the world through two bacterial strain repositories. Team members involved: Eric Triplett (primary), Mike Davis, and Mark Hilf.

Determine the role of CLas prophage in the culture deterioration over time. If prophage activation is correlated with CLas culture deterioration, efforts will be made to inhibit prophage activation as well as search for CLas strains that lack the SC1 prophage in the genome. Team members involved: Eric Triplett (primary) and Dean Gabriel.

Use activator/promoter pairs to control expression of fluorescence in S. meliloti. Use these engineered strains to carry out high-throughput screen of compounds that inhibit the CLas regulatory protein.

1. To investigate the effect of heat stress on Las, associated prophages, and those genes involved in the phage lytic cycle.
2. To monitor healthy and HLB-affected citrus genome-wide response to heat stress.
3. To optimize field thermotherapy to produce a standard operating procedure.

University of Florida

USDA

University of Central Florida

University of Florida

1. Collect weather, flowering, and leaf flush data for recent 10 years and use to improve the Citrus Flowering Monitor System. Particular goals are to improve near-bloom temperature responses of the model and add leaf flushing time to the flowering time in the monitor system. Additionally, it would be desirable to add the 5% open flower point as the point of likely bee activity.
2. Establish a 4- to 5-week peak flowering period as part of the monitor and test it for use of chemicals with ACP effectiveness but minimum bee toxicity, after which all effective ACP control chemicals would again be used. Work with growers and beekeepers to see if this approach is feasible for both concern of the grower for adequate ACP control and beekeepers for adequate access to citrus flowers for their bees.

Test preference (antixenosis) and development (antibiosis) of ACP adults on susceptible and resistant host plants.

Identify attractive or repellent volatiles, metabolites, or their blends, and study the behavior of ACPs to these odorants or taste blends using Y-tube olfactometer assay, caged vial assay, flight tunnel assay, and SPLAT probing assay.

1. OSCF formulation development and optimization (Santra) In this objective we will perform the following tasks. (1) Prepare OSCF materials from readily available chemicals (such as silica precursors, acid/base catalyst, ionic cross-linker, and PAM), (2) Systematically characterize OSCF materials, (3) Optimize the synthesis protocol to produce OSCF formulations that will exhibit strong rainfastness, (4) Develop a scalable synthesis protocol and prepare formulations for field trials, and (5) Study OSCF liquid formulation stability and evaluate its shelf-life.
2. EPG testing to evaluate change of ACP feeding behavior (Rogers).
3. Field trial for evaluation of ACP population control (Graham and Rogers).
4. Evaluation of HLB infection using PCR method (Graham and Irey).

University of Florida

University of Florida

Glades Crop Care, Inc

Better Crops, LLC

University of Florida

University of Florida

Control HLB using antibacterial-producing bacteria.

Test how modification of the EBE of CsLOB1 gene affects citrus resistance against citrus canker and other important traits.

Test how modification of the EBE of CsLOB1 gene affects citrus resistance against citrus canker.

2.2 Experimental evolution analysis of the Xcc strain on the EBECsLOB1-modified grapefruit to investigate how stable is the resistance.

2.3 Test how modification of the EBE of CsLOB1 gene affects other important citrus traits.