Alternative Tissue Sampling for Improved Detection of 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 ' Liberibacter asiaticus' (Las) 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.

Molecular and biological characterization of a novel citrus tristeza virus isolate that causes severe symptoms in Citrus junos cv. Ziyangxiangcheng.

The complete genomic sequence of a novel citrus tristeza virus (CTV) isolate, CT91-A1, from Orah tangor grafted on Citrus junos cv. Ziyangxiangcheng rootstock in China was determined by transcriptome sequencing. Sequence alignments showed that isolate CT91-A1 shared 83.

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

Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ' Liberibacter asiaticus' (Las), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of Las is real-time quantitative polymerase chain reaction (qPCR) using either the Las 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe.

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.

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.

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.

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.

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.

Knock-down of δ-aminolevulinic acid dehydratase via virus-induced gene silencing alters the microRNA biogenesis and causes stress-related reactions in citrus plants.

The δ-aminolevulinic acid (δ-ALA) is an intermediate in the biosynthetic pathway of tetrapyrroles. Tetrapyrroles play vital roles in many biological processes such as photosynthesis, respiration, and light-sensing. ALA-dehydratase (ALAD) combines two molecules of δ-ALA to form porphobilinogen.

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.

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.

Effects of -aminolevulinic acid dehydratase silencing on the primary and secondary metabolisms of citrus.

-aminolevulinic acid dehydratase (ALAD) is an important enzyme in tetrapyrrole synthesis. ALAD combines two -aminolevulinic acid (-ALA) molecules to form the pyrrole molecule, porphobilinogen, an important precursor for plant pigments involved in photosynthesis, respiration, and nutrient uptake. In this study, we investigated the effects of silencing of gene on citrus leaf pigments and metabolites.

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.

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).

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.

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.

Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri.

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri.

Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing).

A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D.

Silencing abnormal wing disc gene of the Asian citrus psyllid, Diaphorina citri disrupts adult wing development and increases nymph mortality.

Huanglongbing (HLB) causes considerable economic losses to citrus industries worldwide. Its management depends on controlling of the Asian citrus Psyllid (ACP), the vector of the bacterium, Candidatus Liberibacter asiaticus (CLas), the causal agent of HLB. Silencing genes by RNA interference (RNAi) is a promising tool to explore gene functions as well as control pests.

The destructive citrus pathogen, 'Candidatus Liberibacter asiaticus' encodes a functional flagellin characteristic of a pathogen-associated molecular pattern.

Huanglongbing (HLB) is presently the most devastating citrus disease worldwide. As an intracellular plant pathogen and insect symbiont, the HLB bacterium, 'Candidatus Liberibacter asiaticus' (Las), retains the entire flagellum-encoding gene cluster in its significantly reduced genome. Las encodes a flagellin and hook-associated protein (Fla) of 452 amino acids that contains a conserved 22 amino acid domain (flg22) at positions 29 to 50 in the N-terminus.

In vivo generated Citrus exocortis viroid progeny variants display a range of phenotypes with altered levels of replication, systemic accumulation and pathogenicity.

Citrus exocortis viroid (CEVd) exists as populations of heterogeneous variants in infected hosts. In vivo generated CEVd progeny variants (CEVd-PVs) populations from citrus protoplasts, seedlings and mature plants, following inoculation with transcripts of a single CEVd cDNA-clone (wild-type, WT), were studied. The CEVd-PVs population in protoplasts was heterogeneous and became progressively more homogeneous in seedlings and mature plants.

Nucleotide sequence and genome organization of Dweet mottle virus and its relationship to members of the family Betaflexiviridae.

The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the families Alphaflexiviridae and Betaflexiviridae. The DMV genome has 8,747 nucleotides (nt) excluding the 3' poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5' and 541 nt at 3' termini.