Genetic analysis of the emerging citrus yellow vein clearing virus reveals a divergent virus population in American isolates.

Citrus yellow vein clearing virus (CYVCV) is a previously reported citrus virus from Asia with widespread distribution in China. In 2022 the California Department of Food and Agriculture (CDFA) conducted a multi-pest citrus survey targeting multiple citrus pathogens including CYVCV. In March 2022, a lemon tree with symptoms of vein clearing, chlorosis and mottling in a private garden in the city of Tulare, California tested positive for CYVCV, which triggered an intensive survey in the surrounding areas. A total of 3,019 plant samples, including citrus and non-citrus species, were collected, and tested for CYVCV using conventional RT-PCR, RT-qPCR, and Sanger sequencing. Five hundred eighty-six citrus trees tested positive for CYVCV, including eight citrus species not previously recorded infected under field conditions. Comparative genomic studies were conducted using seventeen complete viral genomes. Sequence analysis revealed two major phylogenetic groups. Known Asian isolates and five California isolates from this study comprised the first group, whereas all other CYVCV isolates from California formed a second group, distinct from all worldwide isolates. Overall, CYVCV population shows rapid expansion and high differentiation indicating a population bottleneck typical of a recent introduction into a new geographic area. .

Unraveling the occasional occurrence of berry astringency in table grape cv. Scarlet Royal: a physiological and transcriptomic analysis.

Scarlet Royal, a mid-season ripening table grape, is one of the popular red grape varieties in California. However, its berries develop an undesirable astringent taste under certain conditions. Among the various factors contributing to the degradation of berry attributes, the levels and compositions of polyphenols play a fundamental role in defining berry quality and sensory characteristics. To comprehend the underlying mechanism of astringency development, Scarlet Royal berries with non-astringent attributes at the V7 vineyard were compared to astringent ones at the V9 vineyard. Biochemical analysis revealed that the divergence in berry astringency stemmed from alterations in its polyphenol composition, particularly tannins, during the late ripening stage at the V9 vineyard. Furthermore, transcriptomic profiling of berries positively associated nineteen flavonoid/proanthocyanidins (PAs) structural genes with the accumulation of PAs in V9 berries. The identification of these genes holds significance for table grape genetic improvement programs. At a practical level, the correlation between the taste panel and tannin content revealed a threshold level of tannins causing an astringent taste at approximately 400 mg/L. Additionally, berry astringency at the V9 vineyard was linked to a lower number of clusters and yield during the two study seasons, 2016 and 2017. Furthermore, petiole nutrient analysis at bloom showed differences in nutrient levels between the two vineyards, including higher levels of nitrogen and potassium in V9 vines compared to V7. It's worth noting that V9 berries at harvest displayed a lower level of total soluble solids and higher titratable acidity compared to V7 berries. In conclusion, our results indicate that the accumulation of tannins in berries during the ripening process results in a reduction in their red color intensity but significantly increases the astringency taste, thereby degrading the berry quality attributes. This study also highlights the association of high nitrogen nutrient levels and a lower crop load with berry astringency in table grapes, paving the way for further research in this area.

Automating Citrus Budwood Processing for Downstream Pathogen Detection Through Instrument Engineering.

Graft-transmissible, phloem-limited pathogens of citrus such as viruses, viroids, and bacteria are responsible for devastating epidemics and serious economic losses worldwide. For example, the citrus tristeza virus killed over 100 million citrus trees globally, while "Candidatus Liberibacter asiaticus" has cost Florida $9 billion. The use of pathogen-tested citrus budwood for tree propagation is key for the management of such pathogens. The Citrus Clonal Protection Program (CCPP) at the University of California, Riverside, uses polymerase chain reaction (PCR) assays to test thousands of samples from citrus budwood source trees every year to protect California's citrus and to provide clean propagation units to the National Clean Plant Network. A severe bottleneck in the high-throughput molecular detection of citrus viruses and viroids is the plant tissue processing step. Proper tissue preparation is critical for the extraction of quality nucleic acids and downstream use in PCR assays. Plant tissue chopping, weighing, freeze-drying, grinding, and centrifugation at low temperatures to avoid nucleic acid degradation is time-intensive and labor-intensive and requires expensive and specialized laboratory equipment. This paper presents the validation of a specialized instrument engineered to rapidly process phloem-rich bark tissues from citrus budwood, named the budwood tissue extractor (BTE). The BTE increases sample throughput by 100% compared to current methods. In addition, it decreases labor and the cost of equipment. In this work, the BTE samples had a DNA yield (80.25 ng/µL) that was comparable with the CCPP's hand-chopping protocol (77.84 ng/µL). This instrument and the rapid plant tissue processing protocol can benefit several citrus diagnostic laboratories and programs in California and become a model system for tissue processing for other woody perennial crops worldwide.

Transcriptome Analysis of Citrus Dwarfing Viroid Induced Dwarfing Phenotype of Sweet Orange on Trifoliate Orange Rootstock.

Dwarfed citrus trees for high-density plantings or mechanized production systems will be key for future sustainable citrus production. Citrus trees consist of two different species of scion and rootstock. Therefore, any observed phenotype results from gene expression in both species. Dwarfed sweet orange trees on trifoliate rootstock have been produced using citrus dwarfing viroid (CDVd). We performed RNA-seq transcriptome analysis of CDVd-infected stems and roots and compared them to non-infected controls. The identified differentially expressed genes validated with RT-qPCR corresponded to various physiological and developmental processes that could be associated with the dwarfing phenotype. For example, the transcription factors MYB13 and MADS-box, which regulate meristem functions and activate stress responses, were upregulated in the stems. Conversely, a calcium-dependent lipid-binding protein that regulates membrane transporters was downregulated in the roots. Most transcriptome reprogramming occurred in the scion rather than in the rootstock; this agrees with previous observations of CDVd affecting the growth of sweet orange stems while not affecting the trifoliate rootstock. Furthermore, the lack of alterations in the pathogen defense transcriptome supports the term "Transmissible small nuclear ribonucleic acid," which describes CDVd as a modifying agent of tree performance with desirable agronomic traits rather than a disease-causing pathogen.

Development of a one-step RT-qPCR detection assay for the newly described citrus viroid VII.

An apscaviroid, tentatively named citrus viroid VII (CVd-VII), was recently discovered in citrus in Australia. A diagnostic assay using real-time reverse transcription polymerase chain reaction was developed and validated to detect the viroid in citrus plants. The assay showed a high level of sensitivity, reliably detecting 2000 plasmid copies per reaction, while down to 20 plasmid copies per reaction were occasionally detected. The assay showed high specificity, producing no false positives or cross-reactivity with a range of other citrus graft-transmissible pathogens, including viroids, viruses and bacteria. The real-time assay was also found to be more sensitive than the available end-point reverse transcription polymerase chain reaction assay by a factor of 100,000 and could be a useful tool for the rapid detection of CVd-VII in diagnostic and research environments.

Study and Detection of Citrus Viroids in Woody Hosts.

Biological indexing is based upon the ability of certain plants, referred to as indicator plants or indicators, to produce specific symptoms when inoculated with a pathogen using mechanical means or grafting. In the case of citrus viroids, clonal indicators are grafted on to vigorous rootstocks such as rough lemon (Citrus × granulata Raf.). The 'Arizona-861-S-1' citron clonal indicator (C. medica L.) can detect and bioamplify all citrus viroids; however, for specific citrus variants of the hop stunt viroid (i.e., CVd-IIb and CVd-IIc), the clonal indicator 'Parson's special # 9' mandarin (C. reticulata Blanco) is preferred. Inoculation techniques and symptom expression are described in detail. Other supporting elements, such as greenhouse conditions and propagation techniques, are also presented.

Isolation and Transfection of Citrus Protoplasts with Citrus Exocortis Viroid.

Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non-coding, and naked. Unlike RNA viruses, which at least encode proteins for replication, encapsidation, and movement, lack of protein-coding capacity of viroids makes them completely reliant on host for replication and movement. The high genetic diversity in viroids is believed to be due to the absence of proof-reading activity of the host RNA polymerases, the large population size, and the rapid rate of replication. Protoplasts are viable plant cells that are prepared by enzymatic removal of cell walls. Plant protoplasts provide a synchronous single-cell system for studying early events of viroid infection such as replication and genetic diversity at the cellular level. A simple and efficient method to isolate and transfect citrus protoplasts with transcript RNA of citrus exocortis viroid is described in this chapter.

High-Throughput RNA Extraction from Citrus Tissues for the Detection of Viroids.

High-throughput nucleic acid extraction is critical for the implementation of modern viroid detection assays. Successful large-scale nursery, field surveys, and other regulatory, quarantine, or research diagnostic programs are increasingly dependent on high-throughput tissue pulverization and nucleic acid extraction protocols. Magnetic bead-based approaches using semi-automated robotic equipment allow high-throughput extraction and purification of high-quality uniform total nucleic acids for each individual sample. Here, we describe a streamlined and optimized protocol for citrus tissue processing and RNA extraction that can be used for downstream applications such as viroid detection by reverse transcription-quantitative polymerase chain reaction.

Detection of Viroids Using RT-qPCR.

Viroids are the smallest known infectious pathogens. They are nonprotein-encoding, single-stranded, circular, naked RNA molecules that can cause several diseases in economically important crops. With the advent of thermal cyclers incorporating fluorescent detection, reverse transcription coupled to the quantitative polymerase chain reaction (RT-qPCR) has transformed the way the viroids are detected. The method involves using sequence-specific primers that anneal to the viroid RNA of interest. The viroid RNA serves as a template during reverse transcription, in which the enzyme reverse transcriptase generates a cDNA copy of a portion of the target RNA molecule. After first-strand cDNA synthesis, RNA template from cDNA:RNA hybrid molecule is removed by digestion with RNase H to improve the sensitivity of PCR step. This cDNA is then be used as a template for amplification of viroid sequence in PCR.

Real-Time Detection of Viroids Using Singleplex and Multiplex Quantitative Polymerase Chain Reaction.

Multiplex quantitative polymerase chain reaction (multiplex qPCR) enables the amplification of more than one target in a single reaction using different reporter dyes with distinct fluorescent spectra. The number of reporter fluorophores is typically restricted to three or four, depending upon the capability of the real-time PCR platform and software used. Each target is amplified by a different set of primers and a uniquely labeled probe that distinguishes each PCR amplicon. Thus, the levels of several targets of interest can be quantified in real time. By combining several reactions in a single tube, multiplex qPCR reduces the quantity, and cost of reagents needed to screen a sample for multiple targets. Specificity and efficiency are not affected by the inclusion of the three assays in a multiplex reaction.

SYBR® Green RT-qPCR for the Universal Detection of Citrus Viroids.

Quantitative polymerase chain reaction (qPCR) and reverse transcription (RT)-qPCR have now become the gold standard for molecular diagnostics because of its sensitivity, specificity, and reproducibility. In addition, qPCR diagnostics are flexible because they can be scaled for high- or low-throughput applications. Here we describe an optimized assay and workflow for the universal detection of eight citrus viroid species and their variants by RT-qPCR. The assay allows for quick and efficient molecular detection of viroids without the need to run RT-qPCR for each individual viroid species.

QuantiGene Plex Assay: A Method for High-Throughput Multiplex Citrus Viroid Detection and Identification.

The QuantiGene Plex assay is a molecular non-polymerase chain reaction (PCR)-based multiplex method adapted for citrus viroid detection and identification. Here, we describe the procedures to utilize the QuantiGene Plex assay as a high-throughput screening tool for viroids in purified or crude RNA extracts from citrus tissues.

An In Silico Detection of a Citrus Viroid from Raw High-Throughput Sequencing Data.

E-probe Diagnostic for Nucleic Acid Analysis (EDNA) is a user-friendly bioinformatic tool that has been adapted for the detection and identification of citrus exocortis viroid (CEVd). Here, we describe the procedures for RNA extraction from citrus tissues, library and sequencing preparation, and the utilization of EDNA Mi-Finder online platform on raw high-throughput sequencing (HTS) data.

Complete Nucleotide Sequence, Genome Organization, and Comparative Genomic Analyses of Citrus Yellow-Vein Associated Virus (CYVaV).

Citrus yellow-vein disease (CYVD) was first reported in California in 1957. We now report that CYVD is associated with a virus-like agent, provisionally named citrus yellow-vein associated virus (CYVaV). The CYVaV RNA genome has 2,692 nucleotides and codes for two discernable open reading frames (ORFs). ORF1 encodes a protein of 190 amino acid (aa) whereas ORF2 is presumably generated by a -1 ribosomal frameshifting event just upstream of the ORF1 termination signal. The frameshift product (717 aa) encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analyses suggest that CYVaV is closely related to unclassified virus-like RNAs in the family Tombusviridae. Bio-indexing and RNA-seq experiments indicate that CYVaV can induce yellow vein symptoms independently of known citrus viruses or viroids.

Genome analysis of Spiroplasma citri strains from different host plants and its leafhopper vectors.

BACKGROUND: Spiroplasma citri comprises a bacterial complex that cause diseases in citrus, horseradish, carrot, sesame, and also infects a wide array of ornamental and weed species. S. citri is transmitted in a persistent propagative manner by the beet leafhopper, Neoaliturus tenellus in North America and Circulifer haematoceps in the Mediterranean region. Leafhopper transmission and the pathogen's wide host range serve as drivers of genetic diversity. This diversity was examined in silico by comparing the genome sequences of seven S. citri strains from the United States (BR12, CC-2, C5, C189, LB 319, BLH-13, and BLH-MB) collected from different hosts and times with other publicly available spiroplasmas. RESULTS: Phylogenetic analysis using 16S rRNA sequences from 39 spiroplasmas obtained from NCBI database showed that S. citri strains, along with S. kunkelii and S. phoeniceum, two other plant pathogenic spiroplasmas, formed a monophyletic group. To refine genetic relationships among S. citri strains, phylogenetic analyses with 863 core orthologous sequences were performed. Strains that clustered together were: CC-2 and C5; C189 and R8-A2; BR12, BLH-MB, BLH-13 and LB 319. Strain GII3-3X remained in a separate branch. Sequence rearrangements were observed among S. citri strains, predominantly in the center of the chromosome. One to nine plasmids were identified in the seven S. citri strains analyzed in this study. Plasmids were most abundant in strains isolated from the beet leafhopper, followed by strains from carrot, Chinese cabbage, horseradish, and citrus, respectively. All these S. citri strains contained one plasmid with high similarity to plasmid pSci6 from S. citri strain GII3-3X which is known to confer insect transmissibility. Additionally, 17 to 25 prophage-like elements were identified in these genomes, which may promote rearrangements and contribute to repetitive regions. CONCLUSIONS: The genome of seven S. citri strains were found to contain a single circularized chromosome, ranging from 1.58 Mbp to 1.74 Mbp and 1597-2232 protein-coding genes. These strains possessed a plasmid similar to pSci6 from the GII3-3X strain associated with leafhopper transmission. Prophage sequences found in the S. citri genomes may contribute to the extension of its host range. These findings increase our understanding of S. citri genetic diversity.

Next-Generation Sequencing Identification and Characterization of MicroRNAs in Dwarfed Citrus Trees Infected With Citrus Dwarfing Viroid in High-Density Plantings.

Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.

ICTV Virus Taxonomy Profile: Pospiviroidae.

Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/pospiviroidae.

Linking metabolic phenotypes to pathogenic traits among "Candidatus Liberibacter asiaticus" and its hosts.

Candidatus Liberibacter asiaticus (CLas) has been associated with Huanglongbing, a lethal vector-borne disease affecting citrus crops worldwide. While comparative genomics has provided preliminary insights into the metabolic capabilities of this uncultured microorganism, a comprehensive functional characterization is currently lacking. Here, we reconstructed and manually curated genome-scale metabolic models for the six CLas strains A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy, in addition to a model of the closest related culturable microorganism, L. crescens BT-1. Predictions about nutrient requirements and changes in growth phenotypes of CLas were confirmed using in vitro hairy root-based assays, while the L. crescens BT-1 model was validated using cultivation assays. Host-dependent metabolic phenotypes were revealed using expression data obtained from CLas-infected citrus trees and from the CLas-harboring psyllid Diaphorina citri Kuwayama. These results identified conserved and unique metabolic traits, as well as strain-specific interactions between CLas and its hosts, laying the foundation for the development of model-driven Huanglongbing management strategies.

Whole genome sequence of five strains of Spiroplasma citri isolated from different host plants and its leafhopper vector.

OBJECTIVES: Spiroplasma citri is a bacterium with a wide host range and is the causal agent of citrus stubborn and brittle root diseases of citrus and horseradish, respectively. S. citri is transmitted in a circulative, persistent manner by the beet leafhopper, Neoaliturus (Circulifer) tenellus (Baker), in North America. Five strains of S. citri were cultured from citrus, horseradish, and N. tenellus from different habitats and times. DNA from cultures were sequenced and genome assembled to expand the database to improve detection assays and better understand its genetics and evolution. DATA DESCRIPTION: The whole genome sequence of five strains of S. citri are described herein. The S. citri chromosome was circularized for all five strains and ranged from 1,576,550 to 1,742,208 bp with a G + C content of 25.4-25.6%. Characterization of extrachromosomal DNAs resulted in identification of one or two plasmids, with a G + C content of 23.3 to 27.6%, from plant hosts; and eight or nine plasmids, with a G + C content of 21.65 to 29.19%, from N. tenellus. Total genome size ranged from 1,611,714 to 1,832,173 bp from plants and 1,968,976 to 2,155,613 bp from the leafhopper. All sequence data has been deposited in DDBJ/ENA/GenBank under the accession numbers CP046368-CP046373 and CP047426-CP047446.

An In Vitro Pipeline for Screening and Selection of Citrus-Associated Microbiota with Potential Anti-"Candidatus Liberibacter asiaticus" Properties.

Huanglongbing (HLB) is a destructive citrus disease that is lethal to all commercial citrus plants, making it the most serious citrus disease and one of the most serious plant diseases. Because of the severity of HLB and the paucity of effective control measures, we structured this study to encompass the entirety of the citrus microbiome and the chemistries associated with that microbial community. We describe the spatial niche diversity of bacteria and fungi associated with citrus roots, stems, and leaves using traditional microbial culturing integrated with culture-independent methods. Using the culturable sector of the citrus microbiome, we created a microbial repository using a high-throughput bulk culturing and microbial identification pipeline. We integrated an in vitro agar diffusion inhibition bioassay into our culturing pipeline that queried the repository for antimicrobial activity against Liberibacter crescens, a culturable surrogate for the nonculturable "Candidatus Liberibacter asiaticus" bacterium associated with HLB. We identified microbes with robust inhibitory activity against L. crescens that include the fungi Cladosporium cladosporioides and Epicoccum nigrum and bacterial species of Pantoea, Bacillus, and Curtobacterium Purified bioactive natural products with anti-"Ca. Liberibacter asiaticus" activity were identified from the fungus C. cladosporioides Bioassay-guided fractionation of an organic extract of C. cladosporioides yielded the natural products cladosporols A, C, and D as the active agents against L. crescens This work serves as a foundation for unraveling the complex chemistries associated with the citrus microbiome to begin to understand the functional roles of members of the microbiome, with the long-term goal of developing anti-"Ca Liberibacter asiaticus" bioinoculants that thrive in the citrus holosystem.IMPORTANCE Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens, a culturable surrogate for the unculturable HLB-associated bacterium "Candidatus Liberibacter asiaticus," we tested the hypothesis that members of the citrus microbiome produce potential anti-"Ca Liberibacter asiaticus" natural products with potential anti-"Ca Liberibacter asiaticus" activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens.