Research Project: Discovery, Characterization, and Diagnostics of Endemic and Exotic Citrus Pathogens Using High Throughput Sequencing (HTS)

Location: Molecular Plant Pathology Laboratory

2022 Annual Report

Objectives
Objective 1. Develop new diagnostic and research reagents to protect domestic citrus production with support of U.S. quarantine programs. Sub-objective A1: Design Cilevirus and Dichorhavirus genus and species-specific primers based on available genome sequences to detect all known CiLVs using conventional and quantitative RT-PCR. Sub-objective A2: Optimize the conventional and quantitative simplex (S)- and multiplex (M)-RT-PCR using symptomatic tissue associated with cilevirus and dichorhavirus infection. Sub-objective A3: Application of conventional RT-PCR and RT-qPCR for detection of cileviruses and dichorhaviruses in multiple hosts and in the Brevipalpus spp. Sub-objective B1: Optimization of rolling circle amplification (RCA) methods for the detection of pararetrovirus sequences associated with CBD. Sub-objective B2: Detection of active CBaPRV using Direct tissue blot immunoassay or tissue print. Sub-objective B3: Development of a conventional RT-PCR and a RT-qPCR assays for detection of active CBaPRV in blight affected citrus tissues and comparison with the antibody-based diagnostics. Objective 2. Apply High Throughput Sequencing to discover new viruses, investigate their genetic diversity, and evaluate changes in host gene expression upon exposure to citrus pathogens. Sub-objectives 2.1: Application of HTS to identify environmental hosts of viruses associated with Brevipalpus transmitted virus symptoms to enhance understanding of genetic diversity and evolution of pathogens. Sub-objectives 2.2: Application of HTS to identify the pathogen genome sequences associated with the selected accessions of Citrus tristeza virus (CTV) and Haunglongbing (HLB) maintained at the Exotic Pathogens of Citrus Collection (EPCC). Sub-objectives 2.3: Discovery of novel pararetrovirus sequences integrated within the citrus genome and their association with Citrus blight disease etiology. Sub-objectives 2.4: Evaluate patterns of gene expression in mixed infections of CBaPRV with CLas and CTV or both in citrus.

Approach
Objective 1 Genus and species-specific primers and probes will be developed for the conventional and quantitative RT-PCR assays. To optimize the protocols, dilutions of the primers and probes will be done with each primer/probe combination. All the BTV isolates tissue will be imported under permit granted by APHIS-PPQ and stored in the -80°C for downstream application. Established RCA protocol for Badnaviruses will be optimized for pararetroviruses amplification. Well established tissue printing protocol will be used as the antibodies have already been produced. Optimization of the tissue print protocols will be done with each antibody/antigen combination. Maximum number of heathy and Blight infected tree will be sequenced to redesign the primer and probe sequences, and optimize the conventional & real time PCR protocols. Objective 2 An optimized High-Throughput Sequencing (HTS)-Ribo-Zero protocol developed in our laboratory will be followed for the identification of novel hosts of CiLVs and the viruses present inside the Brevipalpus spp. Same HTS protocol will be used for detection of CTV, and will be optimized for HLB detection. Sweet orange scions on various rootstocks will be identified as blight-affected or healthy plants. The HTS libraries will be prepared using total or circular viral nucleic acid followed by sequencing on an Illumina platform. Bioinformatic analyses will be followed to determine the viral ID available in the GenBank. The clade specific generic primers will be designed for RT-PCR assays to identify the possible active pararetrovirus sequence/s. Citrus pathogens like CTV, CLas, and to a lesser extent active infection of CBaPRV are abundant in Florida citrus groves. Therefore, we will identify plants free from pathogen and a set of trees which contain (i) trees that are infected by CLas only, (ii) trees that are infected by CTV only, (iii) if possible, trees infected by infective EPRV (CBaPRV) only, (iv) trees that have infections by both CLas and active CBaPRV, (v) trees that have infections by both CTV and active CBaPRV, (vi) trees that have infections by both CTV and CLas, and (vii) trees that have active infections by CTV, CLas and CBaPRV. Detection of HLB and CTV infection will be evaluated by foliar symptoms, fruit drop and decline. Symptoms of CB will be monitored by using the water injection method. Both roots and leaves samples will be collected from the same infected plants from the same location in two different seasons in a year to minimize the effect of local environment on gene expression. Statistical tests will be performed to group these samples together and citrus cDNA libraries will be mapped. Differentially expressed transcripts (DETs) will be identified in each expression library by comparison against the healthy controls. RNA-seq results will be validated via RT-qPCR to identify differentially expressed genes (DEGs). All identified DEGs will be selected based on their predicted function in disease symptom development and fold changes (log2FC) will be estimated by Gfold and validated by RT-qPCR.

Progress Report
This is a bridge project. Please see final annual report for 8042-22000-319-00D. The goal of this project ‘Invasive Citrus Pathogens’ is to prevent the introduction or spread of a number of graft-transmissible and invasive pathogens of citrus within the citrus industry. Due to quarantine considerations this work is carried out at Beltsville, Maryland, far away from all the citrus growing states. This NP303 research project will be focusing on emerging exotic pathogens, including multiple viruses associated with citrus leprosis disease (CiLD) complex, the highly invasive and destructive pathogen ‘Ca. Liberibacter asiaticus’ (CaLas) and its interaction with an unknown etiological agent associated with citrus blight disease (CBD). CiLD complex is associated with two ssRNA viruses; Cilevirus and Dichorhavirus. Identifying these viruses associated with the CiLD syndrome. To identify these viruses primers and probes were designed for simplex (S), multiplex (M) conventional, and real-time RT-PCR. All primer design criteria (specificity, stability, and compatibility) was used to design primers using the conserved and variable genome sequences. Optimization of the conventional and quantitative simplex (S)- RT-PCR has been started using symptomatic tissue associated with cilevirus and dichorhavirus infection.

Accomplishments