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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center Molecular Plant Pathology Laboratory Research





- Roy, Avijit
- 2023
- 2022
- Publications
- Plant Diseases (303)

Research Project: Discovery, Characterization, and Diagnostics of Endemic and Exotic Citrus Pathogens Using High Throughput Sequencing (HTS) **Location:
                            Molecular Plant Pathology Laboratory** 2023 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**
                                

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. The current NP303 research project is 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). The CiLD complex is associated with two ssRNA viruses; Cilevirus and Dichorhavirus. CiLD in Florida in the early to mid-1900s was associated with an extinct, extended family member of orchid fleck dichorhavirus (OFV). OFV have two orchid strains (OFV-Orc1 and OFV-Orc2) and two citrus strains (OFV-Cit1 and OFV-Cit2). All orchid strains infect citrus, but there is no report of citrus strains of OFV infecting orchids. During 2021-22, our group reported OFV in ornamental groundcovers (Liriope, Ophiopogon, and Aspidistra) in Florida. To identify the strain/s of OFV in ornamentals, the complete genome sequence of three OFV ornamental isolates were determined using high throughput sequencing (HTS) followed by bioinformatic analysis. The contigs of RNA1 from all three ornamentals shared 90-91% nucleotide (nt) identities to OFV-Orc1 and RNA2 shared 97% nt identity with both orchid strains of OFV sequences available in GenBank. Phylogenetic analysis of RNA1 genome sequences created a separate clade in the dendrogram and showed the new strain (OFV-Orn) to be more closely related to OFV-Orc1 than other strains infecting orchid and citrus. The emerging diversity of OFV strains has complicated detection and diagnostic measures and prompted to develop a sensitive and specific multiplex reverse transcription polymerase chain reaction (RT-PCR) assay targeting plants and OFV genes. In the regulatory environment, the one-tube reverse transcription quantitative polymerase chain reaction (RT-qPCR) is mostly used for RNA-virus detection due to its rapidity, sensitivity, reproducibility, and to reduce risk of carry-over contamination. Recent findings of orchid strains of OFV in citrus in South Africa and Hawaii and a novel strain in Florida in ornamentals highlighted the need for a sensitive and specific RT-qPCR method for OFV detection. To identify the presence of OFV and its strains in citrus, orchid and ornamentals, a panel of duplex and multiplex TaqMan RT-qPCR assays were designed. All primer design criteria (specificity, stability, and compatibility) were used to design primers based on the conserved and variable genome sequences. Optimization of the conventional and quantitative simplex (S)- RT-PCR has begun using symptomatic tissue associated with cilevirus and dichorhavirus infection. Dichorhavirus RT-qPCR assays were optimized using total RNA of all four OFV strains. Comparison between the Ct value of duplex and multiplex RT-qPCR assays of each strain indicates that there is no significant interference on assay sensitivities caused by multiplexing. The limit of detection (LOD) range between ~10-3 to 10-4 dilution. Specificity tests on 90 citrus leprosis samples, 40 orchid and ornamental host target samples and 20 non-target samples revealed that both duplex and multiplex RT-qPCR panels are 100% specific, and there is no cross amplification observed. Results of RT-qPCR were also verified by analyzing HTS data. The newly developed assay will be applied for detection and differentiation of OFV and its strains associated with CiLD complex. **Accomplishments**
                                

1. A High-Throughput Sequencing (HTS) protocol for virus detection was optimized and applied for discovery of novel viruses infecting multiple plant hosts. Genetic diversity in the virus genome associated with the citrus leprosis disease complex has complicated current detection and diagnostic measures that prompted the application of HTS for improved virus detection and diagnosis. The adopted HTS protocol for virus detection was optimized utilizing 3 plant species (Citrus spp., Swinglea glutinosa, and Hibiscus rosa-sinensis) infected with a flat mite-transmitted cilevirus. Although the HTS protocol was evaluated to detect several known plant viruses in the three studied hosts, ARS scientist in Beltsville, Maryland, identified mutiple novel hosts in nature infected with citrus leprosis viruses. Furthermore, the current protocol was also successfully applied to identify four new species of carlavirus, one potexvirus, one potyvirus in hibiscus. Overall,application of the HTS method will enhance the surveillance of regulated viruses by accurate detection of known viruses and identification of novel viruses infecting known or new host species. **Review Publications**
                            *Padmanabhan, C., Nunziata, S., Guillermo, L.M., Rivera, Y., Mavrodieva, V.A., Nakhla, M.K., Roy, A. 2023. High-throughput sequencing application in the detection and discovery of viruses associated with the regulated citrus leprosis disease complex. Frontiers in Plant Science. 13. Article e1058847. https://doi.org/10.3389/fpls.2022.1058847.*
                                *Roy, A., Guillermo, L.M., Nunziata, S., Padmanabhan, C., Rivera, Y., Brlansky, R.H., Hartung, J.S. 2023. First report of Passion fruit green spot virus in yellow Passion fruit (Passiflora edulis f. flavicarpa) in Casanare in Colombia. Plant Disease. 107:2270. https://doi.org/10.1094/PDIS-09-22-2267-PDN.*


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