IMPLEMENTING NOVEL CULTURAL PRACTICES FOR CROP PRODUCTIVITY AND FOOD SECURITY

California leads the United States in the production grapes and fresh market citrus. Invasive and endemic diseases, grapevine trunk diseases (TD) and Pierce's Disease (PD), and citrus Huanglongbing (HLB) are major limiting factors to the economic vitality of those crops. Public awareness of environmental risks has also expanded consumer demand for organic or sustainably grown food products. Hence, this research proposal is aimed at developing and implementing sustainable farming practices and reduced environmental contamination with synthetic agrochemicals. In this proposal, we will explore different strategies to manage fungal and bacterial vascular diseases of grapevine and citrus. First, we will evaluate the agricultural relevance and economic feasibility of an insect exclusion structure as a mean to control HLB. This project will strengthen the strategic position of the Lindcove Research and Extension Center and maintain UC Riverside at the forefront of citrus research innovation. Second, we will evaluate a microbe-based approach to citrus HLB and grape PD and TD management. This approach provides opportunities for the patenting of novel technologies, and for the development and commercialization of new science-based bioproducts. Lastly, we will screen V. vinifera cultivars from different genetic pools and evaluate their level of tolerance/susceptibility to the vascular pathogens causing TD and PD. This work may aid in the development of recommendations to industry stakeholders with the planting of tolerant grape cultivars when confronted with high diseases pressure. All the information collected will be extended to the industry so growers can make educated business decisions with regard to adoption of management strategies for TD, PD and HLB.

Objective 1 - Between 2015-2017, citrus samples were collected from 5 Florida groves and taken to a USDA lab in Florida for processing. A total of twenty trees were sampled per grove (10 survivor and 10 diseased trees) for a total of 100 samples. Roots, budwood and leaves were frozen and lyophilized prior to shipping as stipulated by our APHIS permit (#P526P-16-00352) to UC Riverside and stored at -80°C and total DNA was extracted from all samples. We used a combination of culture-based and amplicon metagenomics with Illumina sequencing to profile the bacterial (16S -V4) and fungal (ITS) communities of citrus tissues. After quality filtering of the sequences using the DADA2 pipeline, bacterial and fungal profiles were analyzed computationally utilizing the phyloseq package.Plant tissue samples were also plated on multiple media types routinely used for propagation of plant-associated microbes (LB, 523, R2A, TSA and PDA). All tissue types were ground in 2 ml of 1X PBS in plastic bags and the resulting slurries were serially diluted and plated on the specific media types listed above and incubated at 28°C for 2 days (bacteria) to 4 days (fungi). Culturable microbes were all harvested at once in sterile water and archived at -80°C for downstream experiments. DNA was also extracted from those microbial collections and taxa identified by Illumina sequencing as previously described. The beneficial bacteria or fungi (that we define as the taxa frequently associated with trees displaying a healthy phenotype) will be recovered from our archived culture collection and imported to California with granted APHIS permit. These microbes will be tested in in planta bioassays as biofertilizers for their ability to stimulate plant growth or biological control agents as their ability to mitigate disease development. In order to test microbes as biofertilizers we will co-inoculate them with citrus seeds/plantlets in sterile soils and place them in controlled conditions in a growth chamber. We will measure the impact of those organisms on plant growth and development (plant biomass, root and shoot length) and their ability to colonize plant bio-compartments by qPCR detection and quantification methods in above and below ground plant parts. To evaluate microbes as potential biological control agents (or BCAs) we will inoculate them on CLas-infected plants. Citrus plants will be infected with CLas by grafting infected plant material and the potential BCAs will be soil or foliar inoculated to the plants. BCAs efficacy will be evaluated as a measure of HLB symptom development and pathogen/BCA abundance with qPCR in plant biocompartments above and below ground. The BCA experiments will be carried out in the BSL3 facility at UC Riverside.Objective 2- The CUPS structure covers approximately a total of 5 acres and will be used to evaluate tree performance. This experiment is designed as a comparative study between indoor and outdoor production systems featuring two scion x rootstock combinations representative of the mainstream industry markets. In 2019, two one-acre blocks of Tango x C35 and Cara Cara x Rich 16-6 will be planted undercover and replicated outdoors at the Lindcove Research and Extension Center (4 acres total). Blocks will be planted at a density of 8' x 14' (390 trees per acre). Weather stations and soil sensors will be placed inside and outside so we can record environmental parameters including temperature, relative humidity, wind, soil moisture and irrigation water. In addition using a portable LICOR system we will record data on tree physiology (water consumption, water potential, stomatal conductivity, evapotranspiration, chlorophyll content, and photosynthesis) at key physiological tree stages. We will also record horticultural data (tree growth, flushing periods, timings of flowering and fruit set) and tree productivity data (fruit yield, and fruit quality) using the Lindcove packline. Finally we will collect data on pests and pathogens pressure at pre- and post-harvest. Data on those parameters will be recorded at the onset of the experiment (Summer 2019) and for the next 10 years.Starting in 2020, the remaining acreage in CUPS will be used to evaluate a range of rootstock x scion combinations and determine which of those combinations perform better under protective structures. For example we will specifically evaluate dwarfing rootstocks (Flying Dragon and TsnRNA viroid-inoculated trees) because those will be valuable traits to incorporate into the high density tree planting design required for CUPS. In this objective we will focus on tree horticultural (flushing periods, timing of flowering and fruit set, tree growth) and productivity (fruit yield, and fruit quality) parameters to evaluate individual rootstock x scion combination.The costs of CUPS relative to traditional citrus production are the up-front investment costs of the structure, ongoing structural maintenance costs, and any increased costs from operating within the structure. Particular attention will be paid to making sure that any cost reductions, such as reduced chemical costs and impacts on yields will be measured on an ongoing basis. Profitability and break-even analysis with respect to the level of cost savings relative to the yield will be conducted each year of the project using annually updated data.Objective 3- The goals are to (1) screen grapevine genotypes for tolerance to fungal and bacterial vascular diseases; and (2) characterize microbial profiles associated with healthy and diseased grapevines in order to identify beneficial microbes. Grapevine cuttings of specific genotypes will be screened for tolerance to Pierce's and trunk diseases using published protocols. Grapevines will be either needle-inoculated with a bacterial cell suspension of X. fastidiosa (1 x 108 CFU/ml) or drill-inoculated with a spore suspension (1 x 105 spores/ml) of Phaeomoniella chlamydospora, Macrophomina phaeosolina, and Neofusicoccum parvum. Grapevines will be incubated for 10 weeks for PD and 3 months for TD. Grapevine genotype resistance/tolerance will be rated based on disease symptoms appearance and pathogen abundance as measured by qPCR. Cane, sap and root samples of grapevines expressing a range of TD/PD symptoms will be collected from vineyards. To characterize microbial community profiles samples will be processed as described in objective 1 for citrus. The beneficial bacteria or fungi (defined as taxa commonly associated with healthy grapevine) will be isolated on cultural media by plating tissue samples. The culturable microbe will be tested in in planta bioassays as biofertilizers for their ability to stimulate plant growth or biological control agents as their ability to mitigate disease development. In order to test microbes as the biofertizers we will co-inoculate them with grape seeds/plantlets in sterile soils and place them in controlled conditions in a growth chamber. We will measure the impact of those organisms on plant growth and development (plant biomass, root and shoot length) and their ability to colonize plant biocompartments by measuring through qPCR their abundance in above and below ground plant parts. To evaluate microbes as BCAs we will co-innoculate those organisms with the pathogen of interest (e.g. X. fastidiosa) and measure disease symptom development and pathogen and BCA abundance by qPCR in plant shoots.