Potato psyllid (Bactericera cockerelli) is an important pest of potato, tomato and pepper. Not on... more Potato psyllid (Bactericera cockerelli) is an important pest of potato, tomato and pepper. Not only could a toxin secreted by nymphs results in serious phytotoxemia in some host plants, but also over the past few years B. cockerelli was shown to transmit "Candidatus Liberibacter solanacearum", the putative bacterial pathogen of potato zebra chip (ZC) disease, to potato and tomato. ZC has caused devastating losses to potato production in the western U.S., Mexico, and elsewhere. New knowledge of the genetic diversity of the B. cockerelli is needed to develop improved strategies to manage pest populations. Mitochondrial genome (mitogenome) sequencing provides important knowledge about insect evolution and diversity in and among populations. This report provides the first complete B. cockerelli mitogenome sequence as determined by next generation sequencing technology (Illumina MiSeq). The circular B. cockerelli mitogenome had a size of 15,220 bp with 13 protein-coding gene (P...
Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD) and can be tra... more Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD) and can be transmitted from plant to plant by several species of phloem-feeding leafhoppers. CSD is an important disorder in certain warm and arid citrus-growing areas, and its agent has been recorded from several Mediterranean and Middle Eastern countries, including Syria. In September 2008, columella were collected from fruits of 130 symptomatic and symptomless trees, 102 were from 10 commercial fields in Lattakia and 28 were from 2 groves in Tartous. Silica gel-desiccated columella samples were brought to the University of Bari for S. citri detection. DNA extraction and polymerase chain reaction (PCR) were performed using the primer pairs P58-6f/4r and P58 3f/4r in conventional and real-time PCR, respectively. Twelve sweet orange trees (9.2%) from two different groves (11 in Lattakia and 1 in Tartous) were S. citri-positive in both assays. Nucleotide sequences of the P58-6f/4r amplicon of four sele...
... RK YOKOMI, R. LASTRA,1.2 MB STOETZEL,3 VD DAMSTEEGT,4 RF LEE,S 'so M. GARNSEY, TR GO... more ... RK YOKOMI, R. LASTRA,1.2 MB STOETZEL,3 VD DAMSTEEGT,4 RF LEE,S 'so M. GARNSEY, TR GOTTWALD, MA ROCHA-PENA,6 AND CL NIBLETT? ... In 1992, it was found in the Dominican Republic, Guadeloupe, Haiti, Martinique, Puerto Rico, St. Croix, St. ...
Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using pri... more Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using primers based on sequences of the P89 putative adhesin gene and the P58 putative adhesin multigene of Spiroplasma citri. Real-time PCR also was developed with detection limits estimated to be between 10–4 and 10–4 ng by serial dilution of a recombinant S. citri plasmid into DNA extracts from healthy Madam Vinous sweet orange. PCR for the detection of S. citri by these new primers was validated by comparing culturing of the pathogen, the traditional method of diagnosis, with PCR assays from samples taken from two citrus plots in Kern County, CA. Fruit columella was collected from 384 and 377 individual trees in each of two fields, respectively; one portion was used for culturing and the other for DNA extraction and PCR. PCR results matched those of culturing 85 to 100% of the time depending on the primers used. More importantly, PCR detected S. citri from culture-negative trees in 5 to 15% o...
The impact of citrus stubborn disease, caused by Spiroplasma citri, on citrus production is assoc... more The impact of citrus stubborn disease, caused by Spiroplasma citri, on citrus production is associated with the symptom severity of infected trees but its association with bacterial levels and virulence are unknown. Fifty-eight S. citri isolates were cultivated from severely and mildly symptomatic trees and randomly amplified polymorphic DNA and short-sequence repeat fingerprinting differentiated four major S. citri genotypes among these isolates. Each genotype was present in both mildly and severely symptomatic trees, suggesting that readily detectable genetic differences in the S. citri populations did not account for differences in disease severity. No variation in the size of amplicons of the pathogenicity-related fructose operon was observed in isolates from trees having varying degrees of symptom expression. Quantitative polymerase chain reaction demonstrated that spiroplasma titer is over 6,000 times higher in fruit from severely symptomatic than from mildly symptomatic trees...
Molecular features and genomic organization were determined for Citrus yellow vein clearing virus... more Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus-induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviru...
The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ... more The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ by five to six nucleotides located within the variable (V) domain referred to as the "cachexia expression motif". Sensitive hosts such as mandarin and its hybrids are seriously affected by cachexia disease. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson's Special mandarin and/or direct nucleotide sequence analysis of amplicons from RT-PCR of HSVd-infected plants. Two independent high throughput assays to segregate HSVd variants by real-time RT-PCR and High-Resolution Melting Temperature (HRM) analysis were developed: one based on EVAGreen dye; the other based on TaqMan probes. Primers for both assays targeted three differentiating nucleotides in the V domain which separated HSVd variants into three clusters by distinct melting temperatures with a confidence level higher than 98%. The accuracy of the HRM assays were validated by nucleotide sequencing of representative samples within each HRM cluster and by testing 45 HSVd-infected field trees from California, Italy, Spain, Syria and Turkey. To our knowledge, this is the first report of a rapid and sensitive approach to detect and differentiate HSVd variants associated with different biological behaviors. Although, HSVd is found in several crops including citrus, cachexia variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid diagnosis for cachexia and non-cachexia variants is, thus, important for the management of HSVd in citrus and reduces the need for bioindexing and sequencing analysis.
Potato psyllid (Bactericera cockerelli) is an important pest of potato, tomato and pepper. Not on... more Potato psyllid (Bactericera cockerelli) is an important pest of potato, tomato and pepper. Not only could a toxin secreted by nymphs results in serious phytotoxemia in some host plants, but also over the past few years B. cockerelli was shown to transmit "Candidatus Liberibacter solanacearum", the putative bacterial pathogen of potato zebra chip (ZC) disease, to potato and tomato. ZC has caused devastating losses to potato production in the western U.S., Mexico, and elsewhere. New knowledge of the genetic diversity of the B. cockerelli is needed to develop improved strategies to manage pest populations. Mitochondrial genome (mitogenome) sequencing provides important knowledge about insect evolution and diversity in and among populations. This report provides the first complete B. cockerelli mitogenome sequence as determined by next generation sequencing technology (Illumina MiSeq). The circular B. cockerelli mitogenome had a size of 15,220 bp with 13 protein-coding gene (P...
Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD) and can be tra... more Spiroplasma citri, a phloem-limited pathogen, causes citrus stubborn disease (CSD) and can be transmitted from plant to plant by several species of phloem-feeding leafhoppers. CSD is an important disorder in certain warm and arid citrus-growing areas, and its agent has been recorded from several Mediterranean and Middle Eastern countries, including Syria. In September 2008, columella were collected from fruits of 130 symptomatic and symptomless trees, 102 were from 10 commercial fields in Lattakia and 28 were from 2 groves in Tartous. Silica gel-desiccated columella samples were brought to the University of Bari for S. citri detection. DNA extraction and polymerase chain reaction (PCR) were performed using the primer pairs P58-6f/4r and P58 3f/4r in conventional and real-time PCR, respectively. Twelve sweet orange trees (9.2%) from two different groves (11 in Lattakia and 1 in Tartous) were S. citri-positive in both assays. Nucleotide sequences of the P58-6f/4r amplicon of four sele...
... RK YOKOMI, R. LASTRA,1.2 MB STOETZEL,3 VD DAMSTEEGT,4 RF LEE,S 'so M. GARNSEY, TR GO... more ... RK YOKOMI, R. LASTRA,1.2 MB STOETZEL,3 VD DAMSTEEGT,4 RF LEE,S 'so M. GARNSEY, TR GOTTWALD, MA ROCHA-PENA,6 AND CL NIBLETT? ... In 1992, it was found in the Dominican Republic, Guadeloupe, Haiti, Martinique, Puerto Rico, St. Croix, St. ...
Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using pri... more Polymerase chain reaction (PCR)-based detection of citrus stubborn disease was improved using primers based on sequences of the P89 putative adhesin gene and the P58 putative adhesin multigene of Spiroplasma citri. Real-time PCR also was developed with detection limits estimated to be between 10–4 and 10–4 ng by serial dilution of a recombinant S. citri plasmid into DNA extracts from healthy Madam Vinous sweet orange. PCR for the detection of S. citri by these new primers was validated by comparing culturing of the pathogen, the traditional method of diagnosis, with PCR assays from samples taken from two citrus plots in Kern County, CA. Fruit columella was collected from 384 and 377 individual trees in each of two fields, respectively; one portion was used for culturing and the other for DNA extraction and PCR. PCR results matched those of culturing 85 to 100% of the time depending on the primers used. More importantly, PCR detected S. citri from culture-negative trees in 5 to 15% o...
The impact of citrus stubborn disease, caused by Spiroplasma citri, on citrus production is assoc... more The impact of citrus stubborn disease, caused by Spiroplasma citri, on citrus production is associated with the symptom severity of infected trees but its association with bacterial levels and virulence are unknown. Fifty-eight S. citri isolates were cultivated from severely and mildly symptomatic trees and randomly amplified polymorphic DNA and short-sequence repeat fingerprinting differentiated four major S. citri genotypes among these isolates. Each genotype was present in both mildly and severely symptomatic trees, suggesting that readily detectable genetic differences in the S. citri populations did not account for differences in disease severity. No variation in the size of amplicons of the pathogenicity-related fructose operon was observed in isolates from trees having varying degrees of symptom expression. Quantitative polymerase chain reaction demonstrated that spiroplasma titer is over 6,000 times higher in fruit from severely symptomatic than from mildly symptomatic trees...
Molecular features and genomic organization were determined for Citrus yellow vein clearing virus... more Molecular features and genomic organization were determined for Citrus yellow vein clearing virus (CYVCV), the putative viral causal agent of yellow vein clearing disease of lemon trees, reported in Pakistan, India, and more recently in Turkey and China. CYVCV isolate Y1 from Adana, Turkey, was used for deep sequencing analysis of the virus-induced small RNA fractions and for mechanical and graft inoculation of herbaceous and citrus indicator plants. A polyclonal antiserum was developed from CYVCV-Y1 purified from Phaseolus vulgaris and used in western blot assays to characterize the coat protein of CYVCV-Y1 and determine its serological relationship with related viruses. Contigs assembled from the Illumina sequenced short reads were used to construct the whole genome of Citrus yellow vein clearing virus (CYVCV), consisting in a positive-sense RNA of 7,529 nucleotides and containing six predicted open reading frames. The CYVCV genome organization and size resembled that of flexiviru...
The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ... more The RNA genome of pathogenic and non-pathogenic variants of citrus Hop stunt viroid (HSVd) differ by five to six nucleotides located within the variable (V) domain referred to as the "cachexia expression motif". Sensitive hosts such as mandarin and its hybrids are seriously affected by cachexia disease. Current methods to differentiate HSVd variants rely on lengthy greenhouse biological indexing on Parson's Special mandarin and/or direct nucleotide sequence analysis of amplicons from RT-PCR of HSVd-infected plants. Two independent high throughput assays to segregate HSVd variants by real-time RT-PCR and High-Resolution Melting Temperature (HRM) analysis were developed: one based on EVAGreen dye; the other based on TaqMan probes. Primers for both assays targeted three differentiating nucleotides in the V domain which separated HSVd variants into three clusters by distinct melting temperatures with a confidence level higher than 98%. The accuracy of the HRM assays were validated by nucleotide sequencing of representative samples within each HRM cluster and by testing 45 HSVd-infected field trees from California, Italy, Spain, Syria and Turkey. To our knowledge, this is the first report of a rapid and sensitive approach to detect and differentiate HSVd variants associated with different biological behaviors. Although, HSVd is found in several crops including citrus, cachexia variants are restricted to some citrus-growing areas, particularly the Mediterranean Region. Rapid diagnosis for cachexia and non-cachexia variants is, thus, important for the management of HSVd in citrus and reduces the need for bioindexing and sequencing analysis.
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