TRANSFORM CITRUS REGULATORY DIAGNOSTICS AND PROVIDE INNOVATIVE TECHNOLOGIES FOR HUANGLONGBING MANAGEMENT

Sustainable perennial tree crops are an important component of agricultural economies. In California, 5 out of the 10 most valuable agricultural commodities are perennial crops: grapes, almonds, walnuts, pistachios and citrus. Citrus production in California is estimated at $3.4 billion with a total economic impact of $7.1 billion.Citrus is threatened by a number of diseases and pests including, but not limited to, Huanglongbing (HLB) and its vector the Asian citrus psyllid (ACP). In 2017, Florida's citrus production was reduced by 70% since the initial HLB detection in 2005. In California, ACP is widely spread and over 1,100 HLB positive trees have been identified so far in the state. Availability of pathogen-tested citrus propagative materials (i.e. clean citrus budwood free from all diseases) for over 4,500 California citrus growers and tens of thousands citrus enthusiasts as well as innovative and sustainable technologies for HLB and ACP management are critical for the survival of the iconic citrus trees of the state.Citrus orchards are destined to fail if they are not planted with clean disease-free trees. Disease-free citrus trees can be produced only with clean citrus budwood. Clean citrus budwood can be produced only under a strict regulatory frame within a germplasm program. The Agricultural Experiment Station (AES) at the University of California Riverside is the birth place of the first citrus germplasm program in the world, namely the Citrus Clonal Protection Program (CCPP). CCPP, a major hub of the National Clean Plant Network (NCPN) for Citrus, is the basis of the citrus nursery production that supplies growers and homeowners with healthy plants for fruit production or landscape use. The CCPP performs research that drives citrus regulatory changes based on the characterization of new citrus pathogens, development of high throughput diagnostic protocols and increased access to disease-free citrus propagative materials for decades. The current project will build on past CCPP research, to advance citrus regulatory diagnostics and innovate HLB management.A cohesive protocol of advanced biological, molecular and digital diagnostic assays for citrus diseases will reduce production time and cost for variety introduction into California and increase clean budwood availability. This is important for both commercial and non-commercial citrus production in California. In the last 5 years, there has been a 860% increase in the demand of clean citrus budwood from citrus hobbyists and enthusiasts while successful citrus products such as "ez peelers", now grown in tens of thousands of acres, can be traced back to a series of citrus varieties introduced into California by the CCPP. It has been estimated that every CCPP variety introduction that is commercially viable creates 1-2 jobs per acre including citrus nursery jobs, harvesting crews, field transportation, packing house staff, logistics personnel, sales & marketing staff, as well as cold storage and shipping positions.Innovative technologies utilizing naturally occurring citrus associated RNA molecules that dwarf citrus trees or express compounds that will maintain tree health, will allow for long-term sustainable HLB management. HLB management is currently based on a three part approach. Area wide insecticide sprays against the insect vector of the disease, removal of infected trees and replanting with disease-free trees. If successful, the two citrus RNA based technologies will reduce the number of insecticide sprays, allowing for organic production of citrus and treatment of existing orchards with therapeutic or prophylactic anti-HLB/ACP agents. This approach is very attractive since it does not require replanting the whole industry with an HLB resistant variety. Finally, citrus dwarfing technology could result to high density plantings, under protective structures or in the open field, that can be useful not only against the HLB disease, but could also help farmers to meet challenges such as water shortage, farm land reduction and last but not least labor cost and the potential for mechanized citrus harvesting.

(A) Reduce time for variety introduction and increase pathogen-tested budwood availability via improved regulatory diagnostics.Ai. In the MPGU in planta objective, we will identify the best technologies and methods for seed germination, light (LED & plasma) conditions, irrigation, nutrients, temperature and air quality management for indoor citrus seedlings production and will lead to the development of a MPGU tailored to quarantine and research for the CA citrus industry's needs. The MPGU will be used directly by CCPP for citrus variety introductions, and will become a long term showcase for the industry. Compare side-by-side and measure the effect of the new equipment i.e. MPGU and the BTE and sampling methods (temperature vs. 6 tissue types vs. 12 pathogens' single & mix infections vs. 19 citrus types) on sensitivity, specificity, and reproducibility of diagnostics.Aii. In the qPCR in vitro objective, we will develop 10 multiplex qPCR assays (i.e. adjust existing and design new qPCR primers and probes with sequence alignments and primer/probe design software) and initiate validation for the assays with side-by-side comparisons with current bioindexing and PCR assays using the novel sample collection and processing protocols so they can become official regulatory tests. In support of this objective we will also continue our efforts for the de novo discovery of the pathogens associated with not well characterized citrus diseases.Aiii. In the in silico objective, we will validate the 3 developed EDNA probes (collaboration with OSU-NIMFFAB) i.e. side-by-side comparison with bioindexing and qPCR to demonstrate that can detect equally well or better regulated citrus pathogens. Create sources for 11 virus, viroid and bacterial citrus diseases. Construct libraries and sequence with Illumina infected and healthy samples. Compare results with bioindexing (Ai) and qPCR (Aii) experiments. Work with NIMFFAB to develop EDNA probes for a more user friendly platform than raw NGS data. Complete the genome analysis and EDNA probes development for S. citri, tatter leaf virus and vein enation virus. Continue mining databases and produce sequence data for additional citrus pathogens.(B) Investigate two RNA based innovative technologies for HLB management.Bi. Citrus yellow vein independent-mobile RNA (iRNA) expression vector. Define the parameters necessary for introducing iRNA into citrus phloem. In the iRNA objective, we will introduce iRNA (wild type & engineered by the UM) into citrus phloem using Agro-infiltration or pure RNA utilizing multiple citrus species and several inoculation techniques (e.g. slashing, bark window, heterologous grafting). We will also test samples (8-12 weeks post-inoculation) for the presence and stability of iRNA. This objective will contribute to the overall goal to develop an CYVaV iRNA vector that can be used as a general platform for expression of anti-CLas and anti-ACP peptides, proteins and small RNAs in the phloem of citrus.Bii. Transmissible small nuclear RNAs (TsnRNAs) dwarfing agents. Study TsnRNAs effects on tree growth, water usage and cultivation practices and perform hormonal and gene expression analysis for underlying mechanisms. Measure shoot growth, hormone profile (e.g. cytokinins & giberellins) and gene expression on TsnRNA-IIIb dwarfed and full size non-TsnRNA trees. In the TsnRNA objective, we will calculate fertilizer requirements by analyzing the nutritional content of fruits and leaves and the known amount of fertilizer supplied to the trees. We will also calculate water-use efficiency directly (photosynthesis vs transpiration) and indirectly (sap flow sensors). Pesticide application efficiency and time for horticultural operations for labor cost estimations will also take place. Finally, long term experiments of TsnRNA-Ia, -IIa & -IIIb for effects on scion/rootstock combinations, flowering, fruit quality, yield, and high density plantings will be planted and monitored.Performance measurementsAi We anticipate that this project will accomplish its goals by the increased rate of citrus seedlings production used by CCPP for its germplasm and research operations and by the transfer of the newly developed seedlings production technologies to citrus nurseries. We will use three performance measures: (i) total number of seedlings produced, (ii) time required for citrus variety introduction by the CCPP and (iii) number of nurseries educated on novel MPGU technologies.Data will be collected on citrus seedling growth rate and availability for the expedited (less than 24-36 months) release of ~100 citrus accessions from the 200 currently at the CCPP-Rubidoux Quarantine Facility (RQF) using records of the CCPP Laboratory Information Management System (LIMS). Data will also be collected from CCPP-LIMS for the number of citrus seedlings produced at the CCPP-MPGU for use in citrus introductions (https://ccppdms.ucr.edu/ccppdms/upcoming_varieties).Aii & iii. We anticipate that this project will accomplish its goals by streamlining citrus pathogen detection assays and introducing novel diagnostic technologies. We will use seven performance measures: (i) effect on time and cost using the novel or streamlined assays, (ii) number of true and false positives and negatives, (iii) sensitivity = true positives / (true positives + false negatives), (iv) specificity (Sp) = true negatives / (true negatives + false positives), (v) positive likelihood ratio (LR+) = sensitivity / (1-specificity), (vi) negative likelihood ratio (LR-) = (1-sensitivity) / specificity and (vii) Youden's index (J) = sensitivity + specificity - 1.Bi. We anticipate that this project will accomplish its goals by identifying the appropriate method and developing the protocol for introduction of iRNA into citrus phloem.We will use three performance measures: (i) total number of plants successfully inoculated, (ii) time required for iRNA to reach detectable levels in the plant and (iii) time required for systemic infection with iRNA and number of plants systemically infected.Bii. We anticipate that this project will accomplish its goals by identifying the effects of TsnRNA on citrus trees.We will use five performance measures: (i) tree growth as defined by tree height, width and trunk circumference, (ii) water usage for irrigation and spray runoff, (iii) time for cultivation practices such fruit harvesting, (iv) time to flowering and (v) fruit quality and yield.Means of analysis, assessment, or interpretation of data to be produced.Standard statistical analysis packages will be used for the quantitative data (e.g. sigma plot & sigma stat). Interpretation of the qualitative data will take place based on prior established biological data.Indication of pitfalls, limitations, and contingency plans if needed over the project duration.We do not anticipate any major pitfalls or limitations. The researchers have been working on these topics for some time now and many issues have been troubleshot already.The biggest unknowns are with objective Bi. If the classical techniques of RNA inoculations fail, we will adapt to molecular approaches using citrus protoplasts and tissue culture regeneration.