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.

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.

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.

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.