Genotype Sequencing and Phylogenetic Analysis Revealed the Origins of Citrus Yellow Vein Clearing Virus California Isolates.

The Citrus yellow vein clearing virus (CYVCV) causes a viral disease that has been reported in some citrus-growing regions in countries in Eurasia including Pakistan, India, Türkiye, Iran, China, and South Korea. Recently, CYVCV was detected in a localized urban area in a town in the middle of California's citrus-growing region and marks the first occurrence of the virus in North America. CYVCV has been reported to be spread by aphid and whitefly vectors and is graft and mechanically transmitted. Hence, it is an invasive pathogen that presents a significant threat to the California citrus industry, especially lemons, which are highly symptomatic to CYVCV. To elucidate the origin of the CYVCV California strain, we used long-read sequencing technology and obtained the complete genomes of three California CYVCV isolates, CA1, CA2, and CA3. The sequences of these isolates exhibited intergenomic similarities ranging from 95.4% to 97.4% to 54 publicly available CYVCV genome sequences, which indicated a relatively low level of heterogeneity. However, CYVCV CA isolates formed a distinct clade from the other isolates when aligned against other CYVCV genomes and coat protein gene sequences as shown by the neighbor network analysis. Based on the rooted Maximum Likelihood phylogenetic trees, CYVCV CA isolates shared the most recent common ancestor with isolates from India/South Asia. Bayesian evolutionary inferences resulted in a spatiotemporal reconstruction, suggesting that the CYVCV CA lineage diverged from the Indian lineage possibly around 1995. This analysis placed the origin of all CYVCV to around 1990, with South Asia and/or Middle East as the most plausible geographic source, which matches to the first discovery of CYVCV in Pakistan in 1988. Moreover, the spatiotemporal phylogenetic analysis indicated an additional virus diffusion pathway: one from South Asia to China and South Korea. Collectively, our phylogenetic inferences offer insights into the probable dynamics of global CYVCV dissemination, emphasizing the need for citrus industries and regulatory agencies to closely monitor citrus commodities crossing state and international borders.

Alternative Tissue Sampling for Improved Detection of Candidatus Liberibacter asiaticus.

Early detection and prompt response are key factors in the eradication of 'huanglongbing' (HLB) in California. Currently, qPCR testing of leaf tissue guides the removal of infected trees. However, because of the uneven distribution of 'Candidatus Liberibacter asiaticus' (CLas) in an infected tree and asymptomatic infection, selecting the best leaves to sample, from a mature tree with more than 200,000 estimated leaves, is a major hurdle for timely detection. The goal of this study was to address this issue by testing alternative tissues that might improve the CLas detection rate. Using two years of field data, old and young leaves, peduncle bark of fruit, and feeder roots were evaluated for the presence of CLas. Quadrant-peduncle (Q-P) tissue sampling consistently resulted in better CLas detection than any other tissue type. Q-P samples had a 30% higher qPCR positivity rate than quadrant-leaf (Q-L) samples. No significant seasonal patterns were observed. Roots and single peduncles had similar detection rates; both were higher than single leaves or Q-L samples. If symptoms were used to guide sampling, 30% of infected trees would have been missed. Taken together, these results suggest that Q-P tissue sampling is the optimal choice for improved CLas detection under California growing conditions.

Whole genome sequence of Citrus yellow vein clearing virus CA1 isolate.

OBJECTIVES: Citrus yellow vein clearing virus (CYVCV) is an emerging disease that poses a significant threat to the citrus industry in California. In this study, the viral genomic RNA was isolated from Eureka lemon plants in the greenhouse exhibiting CYVCV symptoms. Subsequently, the corresponding DNA genome amplicon was sequenced and annotated. These efforts expand the genotype database of CYVCV, which aims to enhance detection assays, promote understanding of the virus's genetics and evolution, and support the management of this disease. DATA DESCRIPTION: In this report, we present the complete genome sequence of the CYVCV California isolate (CA1). The genome was found to be 7,530 bp in length, with a G + C content of 51.7%. The 5' and 3' termini were determined using 5' and 3' termini rapid amplification of cDNA ends (RACE) systems. Furthermore, our analysis revealed the presence of six open reading frames (ORFs) potentially encoding proteins. All sequence data and annotation have been deposited in GenBank under the accession number OR037276.1.

Amino acid, sugar, phenolic, and terpenoid profiles are capable of distinguishing Citrus tristeza virus infection status in citrus cultivars: Grapefruit, lemon, mandarin, and sweet orange.

Citrus tristeza virus (CTV) is the most severe viral disease for citrus production. Many strains of CTV have been characterized and their symptomology widely varies, ranging from asymptomatic or mild infections to severe symptomology that results in substantial yield loss or host death. The capacity of the different CTV strains to affect the biochemistry of different citrus species has remained largely unstudied, despite that associated metabolomic shifts would be relevant toward symptom development. Thus, amino acid, sugar, phenolic, and terpenoid levels were assessed in leaves of healthy and CTV-infected grapefruit, lemon, mandarin, and two different sweet orange cultivars. Both mild [VT-negative (VT-)] and severe [VT-positive (VT+)] CTV genotype strains were utilized. When looking at overall totals of these metabolite classes, only amino acid levels were significantly increased by infection of citrus with severe CTV strains, relative to mild CTV strains or healthy plants. No significant trends of CTV infection on summed amounts of all sugar, phenolic, or terpenoid compounds were observed. However, individual compound levels were affected by CTV infections. Subsequent canonical discriminant analysis (CDA) that utilized profiles of individual amino acids, terpenoids, or phenolics successfully distinguished leaf samples to specific citrus varieties and identified infection status with good accuracy. Collectively, this study reveals biochemical patterns associated with severity of CTV infections that can potentially be utilized to help identify in-field CTV infections of economic relevance.

An Improved Recombinase Polymerase Amplification Coupled with Lateral Flow Assay for Rapid Field Detection of 'Candidatus Liberibacter asiaticus'.

Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. 'Candidatus Liberibacter asiaticus' (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either the CLas 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to CLas, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.

Immunocapture-Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for Detection of Plant RNA Viruses.

Loop-mediated isothermal amplification (LAMP) is a sensitive method that can rapidly amplify a specific nucleic acid target with high specificity. The LAMP reaction process has no denaturation step, instead DNA amplification occurs by strand displacement activity of the Bacillus stearothermophilus (Bst) DNA polymerase under isothermal conditions. It utilizes three sets of forward and reverse oligonucleotide primers specific to six distinct sequences on the target gene. These primers are used to generate amplification products that contain single-stranded loops, thereby allowing primers to bind to these sequences without the need for repeated cycles of thermal denaturation. For diagnosis of pathogens with RNA genome, LAMP has been merged with reverse transcription (RT) step to create RT-LAMP. To further reduce the cost of diagnosis and increase the throughput, immunocapture (IC) step was added to develop IC-RT-LAMP assay. Hence, this chapter focuses on utilizing IC-RT-LAMP assay to specifically identify severe strain of a plant virus from field samples.

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.

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.

Multiplex detection of "Candidatus Liberibacter asiaticus" and Spiroplasma citri by qPCR and droplet digital PCR.

"Candidatus Liberibacter asiaticus" (CLas) and Spiroplasma citri are phloem-limited bacteria that infect citrus and are transmitted by insect vectors. S. citri causes citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California. CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid. CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Therefore, the objective of this study was to develop a multiplex qPCR and duplex droplet digital PCR (ddPCR) assay for simultaneous detection of CLas and S. citri to be used where both pathogens can co-exist. The multiplex qPCR assay was designed to detect multicopy genes of CLas-RNR (5 copies) and S. citri-SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection of CLas and S. citri. ddPCR showed higher tolerance to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol for CLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection of CLas and S. citri at very low concentrations without any standards for pathogen titer.

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.

Spread of Citrus Tristeza Virus in Citrus Orchards in Central California.

In California, citrus tristeza virus (CTV) is regulated by a State Interior Quarantine. In CTV abatement districts in central California, trees with CTV that react to MCA13 (MCA13-positive [MCA13+]), a strain-discriminating monoclonal antibody, are rogued to prevent virus spread. The Tulare County Pest Control District, however, does not participate in this abatement program except for a 1.6-km2 zone around the Lindcove Research and Extension Center, Exeter, CA. To quantify CTV spread under these two disparate management programs, CTV surveys were conducted in abatement plots with mandatory aphid control and nonabatement plots. Abatement plot surveys used hierarchical sampling of 25% of trees with samples pooled from four adjacent trees. Detection of MCA13+ CTV in a sample prompted resampling and testing of individual trees. From 2008 to 2018, incidence of CTV increased by an average of 3.9%, with only two MCA13+ samples detected. In contrast, in nonabatement plots, incidence of CTV increased by an average of 4.6% between 2015 and 2018. Increase in MCA13-negative (MCA-) isolates was 11 times greater than that of MCA13+ isolates, with the number of MCA13+ trees increasing by 19 trees between 2015 and 2018. MCA13- isolates were more randomly distributed, suggesting primary spread, whereas MCA13+ CTV isolates were more aggregated, suggesting some secondary spread. These results suggest that spread of MCA13+ isolates was limited by a combination of tree removal and aphid vector suppression. MCA13+ samples were VT isolates with some mixtures with T30 isolates. Despite the presence of VT isolates, all CTV-infected trees were asymptomatic.

Genome Sequence Resource for Spiroplasma citri, Strain CC-2, Associated with Citrus Stubborn Disease in California.

Spiroplasma citri is a bacterium that causes stubborn disease of citrus and infects other crops, ornamentals, and weeds. It is transmitted by leafhoppers in a circulative manner. Due to limited sequence data on S. citri, the bacterium was isolated from naturally infected Chinese cabbage grown on a farm in Fresno County, CA. DNA from S. citri CC-2 was extracted from a pure culture in LD8 and subjected to PacBio sequencing. Four contigs were obtained with a single circular chromosome of 1,709,192 bp and three plasmids of 40,210, 39,313, and 2,921 bp in size. The genome developed herein extends the sequence database of S. citri and is the first whole-genome sequence record of S. citri from California.

A rapid detection tool for VT isolates of Citrus tristeza virus by immunocapture-reverse transcriptase loop-mediated isothermal amplification assay.

Severe strains of Citrus tristeza virus (CTV) cause quick decline and stem pitting resulting in significant economic losses in citrus production. A immunocapture reverse-transcriptase loop-mediated amplification (IC-RT-LAMP) assay was developed in this study to detect the severe VT strains that are typically associated with severe CTV symptoms. The sensitivity of RT-LAMP assay was determined by ten-fold serial dilutions of CA-VT-AT39 RNA, in comparison to one-step RT-droplet digital (dd) PCR. RT-LAMP detected up to 0.002 ng RNA with an amplification time of 10:35 (min:sec.), equivalent to 11.3 copies as determined by one step RT-ddPCR. The RT-LAMP assay specifically detected CA-VT-AT39 RNA and did not cross react with other CTV genotypes tested (T36, T30, RB, S1 and T68). To facilitate rapid on-site detection, the RT-LAMP assay was improved by first capturing the CTV virions from citrus crude leaf sap using CTV-IgG (IC-RT-LAMP), thereby eliminating nucleic acid extraction steps. IC-RT-LAMP assay was optimized with two-fold dilutions of CTV-IgG ranging from 1:500 to 1:16,000. The IC-RT-LAMP assay detected the CA-VT-AT39 virions in all dilutions tested. The minimum amplification time was 6:45 (min:sec) with 1:500 and 1:1000 of CTV-IgG dilutions. The limit of detection of IC-RT-LAMP assay with crude leaf sap of CA-VT-AT39 was 1:320 with a maximum amplification time of 9:08 (min:sec). The IC-RT-LAMP assay was validated for VT genotype by comparing to IC-RT-qPCR using the CTV from 40 field tree samples. A 100% agreement was observed between tests, regardless of single or mixed infections of CTV VT with other genotypes. Therefore, the IC-RT-LAMP assay can serve as a useful tool in the management of potentially severe strains of CTV.

CTV Vectors and Interactions with the Virus and Host Plants.

Citrus is a graft-propagated perennial crop, and Citrus tristeza virus (CTV) is readily graft-transmissible. CTV is comprised of a complex of strains and isolates and, in nature, is spread semi-persistently by aphid vectors. Therefore, citrus trees become infected with multiple CTV strains over time. An important step in characterizing a CTV field isolate is to use aphid vectors to "clean" up the CTV population of a source tree to separate strains and eliminate other graft-transmissible agents. Use of Toxoptera citricida or Aphis gossypii will expedite efficient CTV transmission. CTV vector studies require critical coordination of abundant robust and virus-free vector-competent aphid colonies and an insect-proof, climate-controlled greenhouse or growth chamber. CTV donor and healthy receptor plants with young flush growth must be available for virus acquisition and inoculation. Vector optimums for virus acquisition and inoculation are 24 h for each. CTV infection is readily determined by serology using a polyclonal antiserum or a monoclonal antiserum cocktail; whereas, molecular genotyping is conducted with reverse transcription polymerase chain (RT-PCR) or real time quantitavtive RT-PCR (RT-qPCR) with strain-specific primers and probes. However, the phenotype of the aphid-transmitted isolate still requires virus indexing by graft inoculation to a citrus host range and evaluating symptoms such as stem pitting, vein clearing, stunting, and chlorosis.

Identification and Characterization of Resistance-Breaking (RB) Isolates of Citrus tristeza virus.

Resistance-breaking (RB) strains constitute a clade of biological and genetically distinct isolates of Citrus tristeza virus (CTV) that replicate and move systemically in Poncirus trifoliata (trifoliate orange), resistant to other known strains of CTV. Molecular markers have been developed by comparative genome analysis to allow quick identification of potential RB isolates. Here, methods are described to identify and characterize RB strains by reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR (RT-qPCR), full-length genome sequencing, and biological indexing.

Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis.

BACKGROUND: The non-translated regions at the genome ends of RNA viruses serve diverse functions and can exhibit various levels of nucleotide (nt) heterogeneity. However, the extent of nt heterogeneity at the extreme termini of Citrus tristeza virus (CTV) genomes has not been comprehensively documented. This study aimed to characterize two widely prevalent CTV genotypes, T36-CA and T30-CA, from California that have not been sequenced or analyzed substantially. The information obtained will be used in our ongoing effort to construct the infectious complementary (c) DNA clones of these viruses. METHODS: The terminal nts of the viral genomes were identified by sequencing cDNA clones of the plus- and/or minus-strand of the viral double-stranded (ds) RNAs generated using 5' and 3' rapid amplification of cDNA ends. Cloned cDNAs corresponding to the complete genome sequences of both viruses were generated using reverse transcription-polymerase chain reactions, sequenced, and subjected to phylogenetic analysis. RESULTS: Among the predominant terminal nts identified, some were identical to the consensus sequences in GenBank, while others were different or unique. Remarkably, one of the predominant 5' nt variants of T36-CA contained the consensus nts "AATTTCAAA" in which a highly conserved cytidylate, seen in all other full-length T36 sequences, was absent. As expected, but never systematically verified before, unique variants with additional nt (s) incorporated upstream of the 5' terminal consensus nts of T36-CA and T30-CA were also identified. In contrast to the extreme 5' terminal nts, those at the extreme 3' termini of T36-CA and T30-CA were more conserved compared to the reference sequences, although nt variants were also found. Notably, an additional thymidylate at the extreme 3' end was identified in many T36-CA sequences. Finally, based on pairwise comparisons and phylogenetic analysis with multiple reference sequences, the complete sequences of both viruses were found to be highly conserved with those of the respective genotypes. CONCLUSIONS: The extreme terminal nts in the T36-CA and T30-CA genomes were identified, revealing new insights on the heterogeneity of these CTV genomic regions. T36-CA and T30-CA were the first and the second genotypes, respectively, of CTV originating from California to be completely sequenced and analyzed.

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus".

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.

Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California.

Strain differentiating marker profiles of citrus tristeza virus (CTV) isolates from California have shown the presence of multiple genotypes. To better define the genetic diversity involved, full-length genome sequences from four California CTV isolates were determined by small-interfering RNA sequencing. Phylogenetic analysis and nucleotide sequence comparisons differentiated these isolates into the genotypes VT (CA-VT-AT39), T30 (CA-T30-AT4), and a new strain called S1 (CA-S1-L and CA-S1-L65). S1 isolates had three common recombination events within portions of genes from VT, T36 and RB strains and were transmissible by Aphis gossypii. Virus indexing showed that CA-VT-AT39 could be classified as a severe strain, whereas CA-T30-AT4, CA-S1-L and CA-S1-L65 were mild. CA-VT-AT39, CA-S1-L, and CA-S1-L65 reacted with monoclonal antibody MCA13, whereas CA-T30-AT4 did not. RT-PCR and RT-qPCR detection assays for the S1 strain were developed and used to screen MCA13-reactive isolates in a CTV collection from central California collected from 1968 to 2011. Forty-two isolates were found to contain the S1 strain, alone or in combinations with other genotypes. BLAST and phylogenetic analysis of the S1 p25 gene region with other extant CTV sequences from the NCBI database suggested that putative S1-like isolates might occur elsewhere (e.g., China, South Korea, Turkey, Bosnia and Croatia). This information is important for CTV evolution, detection of specific strains, and cross-protection.

Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

Droplet digital polymerase chain reaction (ddPCR) is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD) in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative) PCR (qPCR) for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

Identification and Characterization of Citrus tristeza virus Isolates Breaking Resistance in Trifoliate Orange in California.

Most Citrus tristeza virus (CTV) isolates in California are biologically mild and symptomless in commercial cultivars on CTV tolerant rootstocks. However, to better define California CTV isolates showing divergent serological and genetic profiles, selected isolates were subjected to deep sequencing of small RNAs. Full-length sequences were assembled, annotated and trifoliate orange resistance-breaking (RB) isolates of CTV were identified. Phylogenetic relationships based on their full genomes placed three isolates in the RB clade: CA-RB-115, CA-RB-AT25, and CA-RB-AT35. The latter two isolates were obtained by aphid transmission from Murcott and Dekopon trees, respectively, containing CTV mixtures. The California RB isolates were further distinguished into two subclades. Group I included CA-RB-115 and CA-RB-AT25 with 99% nucleotide sequence identity with RB type strain NZRB-G90; and group II included CA-RB-AT35 with 99 and 96% sequence identity with Taiwan Pumelo/SP/T1 and HA18-9, respectively. The RB phenotype was confirmed by detecting CTV replication in graft-inoculated Poncirus trifoliata and transmission from P. trifoliata to sweet orange. The California RB isolates induced mild symptoms compared with severe isolates in greenhouse indexing tests. Further examination of 570 CTV accessions, acquired from approximately 1960 and maintained in planta at the Central California Tristeza Eradication Agency, revealed 16 RB positive isolates based on partial p65 sequences. Six isolates collected from 1992 to 2011 from Tulare and Kern counties were CA-RB-115-like; and 10 isolates collected from 1968 to 2010 from Riverside, Fresno, and Kern counties were CA-RB-AT35-like. The presence of the RB genotype is relevant because P. trifoliata and its hybrids are the most popular rootstocks in California.