ROOTING FOR THE FUTURE: ROOT AND RHIZOSPHERE ECOLOGY FOR SUSTAINABLE PRODUCTIONS IN A CHANGING WORLD

The long-term goal of this project is to improve sustainable agricultural practices, with emphasis on citrus, while improving and developing research tools to monitor plant root health and structure. The obtained results will facilitate the development of healthy agroecosystems, profitable agricultural production systems, and food security. Overall there will be tangible benefits to the community and improve living standards locally, nationally and internationally.

1. Determine optimal nutrient concentrations for multiple citrus varieties and rootstocks under HLB-endemic conditions to improve root health and increase tree productivity.Established, fruit-bearing groves, in the age range 4-7 years will be used for this study. They will include Hamlin (or Valencia) orange and grapefruit varieties grown in the Central Florida ridge, southwest Florida flatwoods, and the Indian River District. Replicated field plots for this proposed study will be established in these groves and current nutrient application rates assessed. The nutrient application procedures will then be modified to include a range of nutrient application rates applied via fertigation with and without foliar fertilization. Tree roots will be sampled seasonally and analyzed for all major and minor nutrients. Root health will be assessed monthly using minirhizotron scans, and semiannually with root collections for nutrient and Candidatus Liberibacter asiaticus (CLas) assessment.Visual HLB disease symptoms will be rated every 6 months along with leaf samples for CLas titer and activity (qPCR of DNA and RNA) and nutrient content. Yield per tree and per acre, fruit size and juice quality will be determined annually at harvest. Nutrient concentrations in roots for trees receiving a range of nutrient application rates from low to very high (overloaded) will be evaluated to determine the maximum potential uptake of individual macro and micronutrients beyond which further fertilization would not increase nutrient levels. By deploying a wide range of nutrient concentrations to leaves and soil from low to very high, we can determine the capacity of leaves of HLB-affected trees to take up fertilizer and/or soil nutrients.2. Develop new tools and approaches to characterize citrus root-system structure, architecture, and function in greenhouse and field trialsGreenhouse trials will compare one HLB-susceptible and several HLB-tolerant rootstocks. Rootstock seedlings about six inches high will be micro-grafted with either healthy or infected scion buds, resulting in healthy or HLB-affected plants for each rootstock type. After inoculation, leaf and root samples will be collected from all experimental plants, and DNA will be collected from leaves and roots. The extracted DNA will be used to quantify CLas-specific sequences by qRT-PCR. Although these data will not necessarily reflect levels of living CLas, they will nonetheless provide an approximate value for bacterial titer in leaves and roots. Treatments will begin after testing all plants for presence or absence of CLas.Leaves and roots will be sampled for physiological, biochemical, and nutritional analyses as well as a determination of Differentially-Expressed Genes (DEGs). Leaf and root tissues will be frozen at -80 °C and stored for future use. Plant height, diameter, leaf number, and SPAD readings will be recorded for each plant monthly for six months. Root and shoot biomass will be quantified at the end of the experiment.Nutrient content in the leaves and roots of each rootstock will be analyzed, and results compared between tolerant and susceptible rootstocks, and between healthy and infected plants of the same genotype. In addition to the leaf nutrient analysis, pH and electrical conductivity of the soil will also be recorded to assess the uptake rate as well as nutritional status of the soil used in the experiment. This will also be carried out at the same time points as other measurements.This project will also take a phenomics approach to characterizing citrus root-growth dynamics. This approach will include the development of imaging technologies, computational infrastructure, and statistical methods that can capture and analyze morphologically-complex features over time and in a high-throughput fashion. Root-system architecture encompasses the spatial and temporal organization of roots in the growth medium, and thus greatly influences the resource-capturing capacity of a plant. To achieve this goal, a system to phenotypically characterize root systems in the field (mini-rhizotrones) and greenhouse (rhizotrones) will be adapted for use with citrus. This system will be accompanied by a 2-D and 3-D modeling approach. By comparing different genotypes tolerant or sensitive to HLBthe project aims to identify genes that can help generate more HLB- resistant and sustainable citrus.3. Define the extent to which progression of HLB may involve changes in the relationship between the plant and the soil microbial communityEstablished, fruit-bearing citrus groves from 4 to 7 years old will be used for this study. They will include Hamlin (or Valencia) orange and grapefruit varieties grown at three sites: in the Central Florida ridge, the southwestern Florida flatwoods, and the Indian River District. Replicated field plots will be established in these groves. Leaf and root samples will be collected in triplicate from each tree and brought to the laboratory for analyses. Soil samples will be also collected from the different regions a few meters distant from trees (bulk soil samples). Rhizosphere samples will consist of root system parts and soil. DNA will be extracted from roots and leaves, with presence of CLas determined by PCR.We will profile the assemblage of bacterial flora associated with CLas-infected and CLas-free rhizospheres using metagenomics based on 16S rRNA. PCR products will be cloned into Agrobacterium tumefaciens and introduced into chemically competent Escherichia coli TOP10 cells. Transformed cells will be incubated on selective media, transferred to culture blocks, and aliquots grown for sequencing. Sequences will be analyzed for orientation and detection of non-16S rDNA sequences and chimeras. They will then be aligned, evaluated by principal-coordinate analysis (PCA), and phylogenetic composition evaluated.The data will be used to compare rhizosphere microbiomes of healthy and HLB-affected trees. Additional studies will include experimental perturbation of the rhizosphere of HLB-affected trees. These will be inoculated with bacterial strains obtained from healthy citrus root-associated microbiomes and the effects on the plants will be observed.4. Identify and quantify the potential impact of emerging contaminants on rhizosphere ecology, root physiology and root anatomy.Responses to different types of ENPs (i.e., CeO2NPs, TiO2NPs, etc.) will be tested in this project. Electron microscopy will be used to determine the average size and the zeta potential. Citrus and other horticultural species (i.e., tomato, lettuce, soybeans) seedlings will be grown in the greenhouse and controlled chambers under combined stresses: heavy metals + ENPs, or salinity and drought + ENPs. After a pre-determined time (variable for each tested species), the plants will be carefully removed from the growth media, rinsed with deionized water, divided into roots and leaves, and tapped dry with a paper towel. The root and leaf tissues from each replicate will be dried in an oven at 70 ºC for 7 days to determine the dry biomass and concentrations of ions and heavy metals by ICP-MS. Root samples from each plant will also be collected and stored at 4 ºC in pure methanol. Physiological measurements to correlate root health with leaf physiology will also be carried out each week using a portable photosynthesis system. At the end of the experiment, chlorophyll content will also be quantified. Roots will be analyzed under a light microscope for development of apoplastic barriers. A series of free-hand radial sections from roots from each plant will be used to visualize suberin lamellae using the fluorescence protocol developed by LUX et al. (2005). All anatomical observations will be performed at the Indian River and Research Center, University of Florida, IFAS, Fort Pierce, FL.