Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing.

' Liberibacter spp.' are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of ' L.

Transcriptomic analyses reveal physiological changes in sweet orange roots affected by citrus blight.

Background: Citrus blight is a very important progressive decline disease of commercial citrus. The etiology is unknown, although the disease can be transmitted by root grafts, suggesting a viral etiology. Diagnosis is made by demonstrating physical blockage of xylem cells that prevents the movement of water.

Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico.

The genus s contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus , and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit-specific reverse transcription PCR (RT-PCR) assay.

Complete Nucleotide Sequence of a Novel Hibiscus-Infecting Cilevirus from Florida and Its Relationship with Closely Associated Cileviruses.

The complete nucleotide sequence of a recently discovered Florida (FL) isolate of hibiscus-infecting cilevirus (HiCV) was determined by Sanger sequencing. The movement and coat protein gene sequences of the HiCV-FL isolate are more divergent than other genes of the previously sequenced HiCV-HI (Hawaii) isolate.

Transcriptional analysis of sweet orange trees co-infected with 'Candidatus Liberibacter asiaticus' and mild or severe strains of Citrus tristeza virus.

Background: Citrus worldwide is threatened by huanglongbing (HLB) and tristeza diseases caused by 'Candidatus Liberibacter asiaticus' (CaLas) and Citrus tristeza virus (CTV). Although the pathogens are members of the α-proteobacteria and Closteroviridae, respectively, both are restricted to phloem cells in infected citrus and are transmitted by insect vectors. The response of sweet orange to single infection by either of these two pathogens has been characterized previously by global gene expression analysis.

Co-infection of Sweet Orange with Severe and Mild Strains of Is Overwhelmingly Dominated by the Severe Strain on Both the Transcriptional and Biological Levels.

Citrus tristeza is one of the most destructive citrus diseases and is caused by the phloem-restricted Closterovirus, . Mild strain CTV-B2 does not cause obvious symptoms on indicators whereas severe strain CTV-B6 causes symptoms, including stem pitting, cupping, yellowing, and stiffening of leaves, and vein corking. Our laboratory has previously characterized changes in transcription in sweet orange separately infected with CTV-B2 and CTV-B6.

Characterization and purification of proteins suitable for the production of antibodies against 'Ca. Liberibacter asiaticus'.

The citrus disease huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several CaLas proteins that provide the basis for efficient and accurate detection of CaLas in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies.

Immune Tissue Print and Immune Capture-PCR for Diagnosis and Detection of Candidatus Liberibacter Asiaticus.

'Candidatus Liberibacter asiaticus' (CaLas), associated with citrus Huanglongbing (HLB), is a non culturable member of the α-proteobacteria. In this study serologically based methods for the detection of CaLas were developed. An anti-outer membrane protein A (OmpA) polyclonal antibody previously produced (in our laboratory) was highly effective for the detection of CaLas from citrus tissues in a simple tissue printing format.

Serological detection of 'Candidatus Liberibacter asiaticus' in citrus, and identification by GeLC-MS/MS of a chaperone protein responding to cellular pathogens.

We describe experiments with antibodies against 'Candidatus Liberibacter asiaticus used to detect the pathogen in infected plants. We used scFv selected to bind epitopes exposed on the surface of the bacterium in tissue prints, with secondary monoclonal antibodies directed at a FLAG epitope included at the carboxyl end of the scFv. Unexpectedly, the anti-FLAG secondary antibody produced positive results with CaLas diseased samples when the primary scFv were not used.

Transcriptome analysis of sweet orange trees infected with 'Candidatus Liberibacter asiaticus' and two strains of Citrus Tristeza Virus.

Background: Huanglongbing (HLB) and tristeza, are diseases of citrus caused by a member of the α-proteobacteria, 'Candidatus Liberibacter asiaticus' (CaLas), and Citrus tristeza virus (CTV) respectively. HLB is a devastating disease, but CTV strains vary from very severe to very mild. Both CaLas and CTV are phloem-restricted.

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens.

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes.

Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves.

Identification and Molecular Characterization of Nuclear Citrus leprosis virus, a Member of the Proposed Dichorhavirus Genus Infecting Multiple Citrus Species in Mexico.

Citrus leprosis is one of the most destructive diseases of Citrus spp. and is associated with two unrelated virus groups that produce particles primarily in either the cytoplasm or nucleus of infected plant cells. Symptoms of leprosis, including chlorotic spots surrounded by yellow haloes on leaves and necrotic spots on twigs and fruit, were observed on leprosis-affected mandarin and navel sweet orange trees in the state of Querétaro, Mexico.

The Prevalence of the Citrus tristeza virus Trifoliate Resistance Breaking Genotype Among Puerto Rican Isolates.

Citrus tristeza virus (CTV) isolates have been grouped into six genotypes: T3, T30, T36, VT, B165, and resistance breaking (RB) based on symptoms, host range, and genomic sequence data. The RB genotype has recently been identified with the novel property of replicating in trifoliate orange trees, a resistant host for the other five genotypes. Puerto Rican CTV isolate B301 caused mild vein clearing symptoms in Mexican lime but did not induce seedling yellows or stem pitting reactions in appropriate indicator Citrus spp.

Genome assembly of citrus leprosis virus nuclear type reveals a close association with orchid fleck virus.

The complete genome of citrus leprosis virus nuclear type (CiLV-N) was identified by small RNA sequencing utilizing leprosis-affected citrus samples collected from the state of Querétaro, Mexico. The nucleotide identity and phylogenetic analysis indicate that CiLV-N is very closely related to orchid fleck virus, which typically infects Cymbidium species.

A novel virus of the genus Cilevirus causing symptoms similar to citrus leprosis.

Citrus leprosis in Colombia was previously shown to be caused by cytoplasmic Citrus leprosis virus (CiLV-C). In 2011, enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction (RT-PCR)-based diagnostic methods failed to identify CiLV-C from citrus samples with symptoms similar to citrus leprosis; however, virions similar to CiLV-C were observed in the cytoplasm of the symptomatic leaves by transmission electron microscopy. Furthermore, the causal organism was transmitted by the false spider mite, Brevipalpus phoenicis, to healthy citrus seedlings.

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis.

The xylem-limited, Gram-negative, fastidious plant bacterium Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease affecting approximately half of the citrus plantations in the State of São Paulo, Brazil. The disease was recently found in Central America and is threatening the multi-billion U.S.

'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interaction.

Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential 'accessory' genes for nitrogen fixation (nif), nodulation and host specificity (nod).

Conservation of gene order and content in the circular chromosomes of 'Candidatus Liberibacter asiaticus' and other Rhizobiales.

'Ca. Liberibacter asiaticus,' an insect-vectored, obligate intracellular bacterium associated with citrus-greening disease, also called "HLB," is a member of the Rhizobiales along with nitrogen-fixing microsymbionts Sinorhizobium meliloti and Bradyrhizobium japonicum, plant pathogen Agrobacterium tumefaciens and facultative intracellular mammalian pathogen Bartonella henselae. Comparative analyses of their circular chromosomes identified 514 orthologous genes shared among all five species.

Comparison of the 'Ca. Liberibacter asiaticus' genome adapted for an intracellular lifestyle with other members of the Rhizobiales.

An intracellular plant pathogen 'Candidatus Liberibacter asiaticus,' a member of the Rhizobiales, is related to Sinorhizobium meliloti, Bradyrhizobium japonicum, nitrogen fixing endosymbionts, Agrobacterium tumefaciens, a plant pathogen, and Bartonella henselae, an intracellular mammalian pathogen. Whole chromosome comparisons identified at least 50 clusters of conserved orthologous genes found on the chromosomes of all five metabolically diverse species. The intracellular pathogens 'Ca.

Lack of Evidence for Transmission of 'Candidatus Liberibacter asiaticus' Through Citrus Seed Taken from Affected Fruit.

Citrus huanglongbing, putatively caused by the associated bacterium 'Candidatus Liberibacter asiaticus', is the greatest threat to the world citrus industry today. The bacterium is spread locally and regionally by the citrus psyllid Diaphorina citri, and also can be disseminated by propagation of contaminated scion budwood that is grafted to the appropriate rootstock. The planting of 'Ca.

Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR.