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

Location: Molecular Plant Pathology Laboratory

2024 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 focused 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 progress on CiLD complex is described below. CiLD is caused by two taxonomically distinct groups of Brevipalpus transmitted viruses (BTV), belonging to the families Kitaviridae and Rhabdoviridae, respectively. Recent research indicates that at least five Brevipalpus species, serve as vectors of CiLD complex. All the vectors are present in the U.S. and thus posing a risk of reemergence and spread. CiLD produces a range of foliar and fruit symptoms in multiple Citrus sp., from chlorotic to necrotic spots, and advanced infection may lead to plant death. CiLD in Florida in the early to mid-1900s was associated with an extinct, extended family member of orchid fleck dichorhavirus (OFV. To protect the billion-dollar U.S. citrus industry and prevent the introduction of CiLD, we have developed and validated three quadruplex real time RT-PCR assays for the detection of cileviruses [citrus leprosis virus C, CiLV-C2, CiLV-C2H] dichorhaviruses [citrus leprosis virus N, citrus chlorotic spot virus and orchid fleck virus] with internal control Nad5 and OFV orchid strains (OFV-Orc1 and OFV-Orc2) and citrus strains (OFV-Cit1 and OFV-Cit2) specific OFV RT-qPCR assays. All three assays were optimized using total RNA of all above mentioned viruses and its strains. Comparison between the Ct value of duplex and multiplex RT-qPCR assays of each virus/strain indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 90 citrus leprosis samples, 40 orchid and ornamental host target samples and 20 non-target samples (Including 14 other citrus infecting viruses) revealed that both duplex and multiplex RT-qPCR panel are 100% specific, and there is no cross amplification observed. The assays’ selectivity against common plant matrices like healthy sweet orange, orchids, and hibiscus leaf was evaluated and no performance differences were detected between tissue type matrices. These assays will be effective screening tools for detection and to prevent the spread of associated CiLVs in citrus, orchid and hibiscus growing regions. Since the discovery of type species citrus leprosis virus C (CiLV-C) in several regions of Colombia, two additional CiL causing viruses (CILV-C2 and OFV) have been identified belonging to the genus Cilevirus and Dichorhavirus, respectively. During this study, along with sweet orange (Citrus sinensis) leaf samples, several other Breviaplpus transmitted virus suspected symptomatic samples were collected from several departments in Colombia and optimized RT-PCR protocols were utilized to identify viruses associated with citrus leprosis disease complex. Interestingly, none of the samples were positive for CiLV-C, but single or mixed infections of CiLV-C2 with its hibiscus strain (CiLV-C2H) were detected. Even though only OFV was detected in Orchids but CiLVC2 and its hibiscus strains were detected in multiple hosts plants, belonging to the families Araceae, Apocynaceae, Ariliaceae, Asparagaceae, Cannaceae, Clusiaceae, Costaceae, Commelinaceae, Cycadaceae, Heliconiaceae, Hydrangeaceae, Iridaceae, Marantaceae, Orchidaceae, Passifloraceae, Rosaceae, Smilacaceae, Solanaceae, Strelitziaceae and Zingiberaceae. Most botanical families diagnosed with CiLV infections were associated with more than one virus. The families Araceae, Orchideaceae, Rutaceae and Zingiberaceae were the ones with the highest incidence in this type of mixed infections. Optimized bioinformatic pipeline in our lab (Roy et al., 2024) was utilized to analyze archived HTS data from 2019 citrus leprosis-like symptomatic grapefruit samples and identified the few Caulimoviridae and Retroviridae contigs along with a known temperate fruit virus contigs. Mostly abundant virus contigs presence in the high-throughout sequencing (HTS) library shared maximum nucleotide identity with the nectarine marafivirus M (NeVM) (GenBank accession KT273413). Multiple primer pairs were also designed for overlapping RT-PCR to bridge the gap between contig sequences retrieved from the HTS data, and the entire genome sequence was determined. Comparison of complete genome sequence of the grapefruit and nectarine NeVM isolates revealed 89% nucleotide and 97% amino acid sequence identities between them. This data confirmed the association of a distinct strain of NeVM with chlorotic blotch symptoms in grapefruit. Similarly, during the BTV surveys in the citrus growing regions in Colombia, several hibiscus samples were collected for virus testing as hibiscus is a well-known host for viruses associated with citrus leprosis disease syndrome. Hibiscus leaf samples in Risaralda, Colombia, showing black spots on upper and lower sides was selected for virome analysis using High-throughput metagenomic sequencing. Bioinformatic and phylogenetic analysis revealed the presence of four novel Carlavirus, one Potexvirus species along with known Betacarmovirus, Nepovirus, Tobamovirus and three cileviruses in mixed infection. To confirm the presence of known nepovirus and the novel species of carla- and potex- virus in the same sample, RT-PCR specific primers were designed, and amplified products were sequenced. This is first time any carla-, nepo- and potexvirus infection has been identified in hibiscus.

Accomplishments
1. Developed and validated two quadraplex real-time PCR assays for detection cileviruses and dichorhaviruses. To prevent the introduction of viruses associated with citrus leprosis disease complex, ARS scientists in Beltsville, Maryland, have developed and validated two quadruplex real time RT-PCR assays for the detection of cileviruses [citrus leprosis virus C, CiLV-C2, CiLV-C2H] and dichorhaviruses [citrus leprosis virus N, citrus chlorotic spot virus and orchid fleck virus] with internal control Nad5. During surveys in different citrus growing regions in Colombia several plant species other than citrus were collected. Both protocols were applied to determine the diversity of CiLVs in the infected plant tissues and inside the mite vectors (Brevipalpus spp.). Newly developed RT-qPCR assays successfully detected CiLVs in multiple plants species for the first time. Those host species belong to the 20 families. Among them, the host species in families Araceae, Orchideaceae, Rutaceae and Zingiberaceae were the ones with the highest incidence in this type of mixed infections. To know the risk for citrus production in Colombia and the export agricultural products to other countries, HTS protocol and bioinformatic pipeline will be utilized to reveal the virome associated with each host symptoms.

2. Developed molecular diagnostic protocols for routine diagnostics of nectarine virus M in citrus and prunus species. A High-Throughput Sequencing (HTS) protocol optimized in our laboratory in Beltsville, Maryland, was applied for detection of nectarine virus M in the grapefruit trees in La Feria county, Texas, and a conventional RT-PCR protocol was developed for routine diagnostics of NeVM infection in citrus and prunus species. The same protocol was used for discovery of novel viruses including hibiscus carlavirus (HiCaV)-A, HiCaV-B, HiCaV-C, a distinct variant of HiCaV-C and the new hibiscus potexvirus X, infecting H. rosa-sinensis in Colombia. During this study, ARS scientists in Beltsville, Maryland, also reported the first incidence of hibiscus soymovirus, physalis vein necrosis nepovirus and passion fruit green spot cilevirus in hibiscus. Furthermore, the meta-transcriptomic approach revealed the possibility of a third hibiscus strain of citrus leprosis virus C2 (CiLV-C2H3) present in nature.

Review Publications
Roy, A., Grinstead, S.C., Leon, G.M., Juan Carlos, C.P., Nunziata, S., Padmanabhan, C., Hammond, J. 2024. Meta-transcriptomic analysis uncovers the presence of four novel viruses and multiple known virus genera in a single Hibiscus rosa-sinensis plant in Colombia. Viruses. 16:267. https://doi.org/10.3390/v16020267.
Roy, A., Grinstead, S.C., Juan Carlos, C.P., Hammond, J., Leon, G.M. 2024. First report of Hibiscus soymovirus in Hibiscus rosa-sinensis in Colombia in mixed infection. Plant Disease. 108:826. https://doi.org/10.1094/PDIS-10-23-2153-PDN.
Roy, A., Ouro-Djobo, A., Grinstead, S.C., Hammond, J., Setamou, M., Alabi, O.J. 2024. First report of nectarine virus M in grapefruit (Citrus x paradisi Macfad.) in association with citrus chlorotic blotch disease in Texas, USA. Plant Disease. https://doi.org/10.1094/PDIS-05-24-1024-PDN.