A serious vine decline of cucurbits known as cucurbit yellow vine disease (CYVD) is caused by rod... more A serious vine decline of cucurbits known as cucurbit yellow vine disease (CYVD) is caused by rod-shaped bacteria that colonize the phloem elements. Sequence analysis of a CYVD-specific polymerase chain reaction (PCR)-amplified 16S rDNA product showed the microbe to be a γ-proteobacterium related to the genus Serratia. To identify and characterize the bacteria, one strain each from watermelon and zucchini and several noncucurbit-derived reference strains were subjected to sequence analysis and biological function assays. Taxonomic and phylogenetic placement was investigated by analysis of the groE and 16S rDNA regions, which were amplified by PCR and directly sequenced. For comparison, eight other bacterial strains identified by others as Serratia spp. also were sequenced. These sequences clearly identified the CYVD strains as Serratia marcescens. However, evaluation of metabolic and biochemical features revealed that cucurbit-derived strains of S. marcescens differ substantially fr...
Huanglongbing (HLB) is a highly destructive citrus disease and is associated with a nonculturable... more Huanglongbing (HLB) is a highly destructive citrus disease and is associated with a nonculturable bacterium, ‘Candidatus Liberibacter asiaticus’. ‘Ca. L. asiaticus’ in the United States was first found in Florida in 2005 and is now endemic there. In California, ‘Ca. L. asiaticus’ was first detected in Hacienda Heights in Los Angeles County in 2012 and has now been detected in multiple urban locations in southern California. Knowledge of ‘Ca. L. asiaticus’ strain diversity in California is important for HLB management. In this study, genomic diversity among 10 ‘Ca. L. asiaticus’ strains from six California locations were analyzed using a next-generation sequencing (NGS) (Illumina MiSeq and HiSeq) approach. Draft genome sequences of ‘Ca. L. asiaticus’ strains were assembled. Sequences of the 16S ribosomal RNA gene and nrdB confirmed ‘Ca. L. asiaticus’ identity. Prophages were detected in all ‘Ca. L. asiaticus’ strains. The California ‘Ca. L. asiaticus’ strains formed four prophage typ...
In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Imm... more In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Immokalee, FL. Symptoms observed on fruit included hard spot, cracked spot, and early virulent spot. Hard spot lesions were up to 5 mm, depressed with a chocolate margin and a necrotic, tan center, often with black pycnidia (140 to 200 μm) present. Cracked spot lesions were large (15 mm), dark brown, with diffuse margins and raised cracks. In some cases, hard spots formed in the center of lesions. Early virulent spot lesions were small (up to 7 mm long), bright red, irregular, indented, and often with many pycnidia. In addition, small (2 to 3 mm), elliptical, reddish brown leaf lesions with depressed tan centers were observed on some trees with symptomatic fruit. Chlorotic halos appeared as they aged. Most leaves had single lesions, occasionally up to four per leaf. Tissue pieces from hard spots and early virulent spots were placed aseptically on potato dextrose agar (PDA), oatmeal agar, or...
We report here the draft genome sequence of "Candidatus Liberibacter asiaticus" strain ... more We report here the draft genome sequence of "Candidatus Liberibacter asiaticus" strain TX2351, collected from Asian citrus psyllids in south Texas, USA. The TX2351 genome has a size of 1,252,043 bp, a G+C content of 36.5%, 1,184 predicted open reading frames, and 52 RNA genes.
Huanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘C... more Huanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas...
Huanglongbing (HLB), also known as citrus greening, is a destructive citrus disease associated wi... more Huanglongbing (HLB), also known as citrus greening, is a destructive citrus disease associated with 3 α-proteobacteria species of Candidatus Liberibacter. The first report of HLB in the USA was from Florida in 2005 and Ca. L. asiaticus (Las) is the only species currently confirmed in the USA. In January 2012, a Valencia sweet orange tree in a commercial orchard in San Juan, Texas, tested positive for Las by real-time and conventional PCR assays and by the sequence of its partial 16S rRNA gene. The sample tested negative for Ca. L. americanus and Ca. L. africanus. All 4 Valencia sweet orange seedlings that were graft-inoculated using budwood from the first Texas HLB-infected tree showed typical HLB symptoms 3 months post-inoculation and tested positive for the pathogen. Such HLB typical symptoms as leaf blotchy mottle, twig die-back, veinal chlorosis, lopsided and greening fruits were observed on the Las-positive tree in the orchard, which immediately triggered an intensive survey of...
Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified ... more Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was re-classified as Xanthomonas campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonad...
AimsDickeya species are high consequence plant pathogenic bacteria listed among the quarantine pa... more AimsDickeya species are high consequence plant pathogenic bacteria listed among the quarantine pathogens of the European Union; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate, and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, D. dianthicola.Methods and ResultsA signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed detection limit of 10 fg DNA.ConclusionThe developed mult...
A pheromone-based system to locate and monitor Planococcus minor (Maskell), a pest of over 250 pl... more A pheromone-based system to locate and monitor Planococcus minor (Maskell), a pest of over 250 plants including citrus, grape, and cacao, was tested. The difficulty in distinguishing P. minor from the citrus mealybug, P. citri, makes finding and evaluating the impact of the pest challenging. Studies conducted in Puerto Rico determined that synthetic P. minor pheromone lures preaged 120 d in the field caught similar number of males as lures not aged (fresh). Molecular analysis of trapped mealybug males using mitochondrial cytochrome oxidase-1, the internal transcriber space two locus, and 28S-D2 gene showed the pheromone traps to be species specific. Traps baited with P. minor pheromone were used to monitor the pest in south Florida and to locate potential infestations. P. minor males were found at all locations studied in South Florida and were present in low numbers (1.03 +/- 0.69 mean +/- SE/trap/14 d). Over 14,000 terminals, fruit, and flowers were visually inspected over a 6 mo period of peak trap catches before the first adult P. minor female was found. The synthetic pheromone lures proved to be an effective tool to locate and monitor this pest new to the continental United States.
Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing s... more Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing states, and has been detected in another six states in the central United States. Xanthomonas vasicola was identified as the causal agent of this disease. A multilocus sequence alignment of six housekeeping genes and comparison of average nucleotide identity from draft genome sequence were used to confirm phylogenetic relationships and classification of this bacteria relative to other X. vasicola strains. X. vasicola isolates from Nebraska and South Africa were highly virulent on corn and sugarcane and less virulent on sorghum but caused water-soaking symptoms that are typical of X. vasicola infection on the leaves of all three hosts. Based on host range and phylogenetic comparison, we propose the taxonomic designation of this organism to X. vasicola pv. vasculorum ( Cobb 1894 ) comb. nov. Polymerase chain reaction-based diagnostic assays were developed that distinguish X. vasicola pv. va...
Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. T... more Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and ...
A serious vine decline of cucurbits known as cucurbit yellow vine disease (CYVD) is caused by rod... more A serious vine decline of cucurbits known as cucurbit yellow vine disease (CYVD) is caused by rod-shaped bacteria that colonize the phloem elements. Sequence analysis of a CYVD-specific polymerase chain reaction (PCR)-amplified 16S rDNA product showed the microbe to be a γ-proteobacterium related to the genus Serratia. To identify and characterize the bacteria, one strain each from watermelon and zucchini and several noncucurbit-derived reference strains were subjected to sequence analysis and biological function assays. Taxonomic and phylogenetic placement was investigated by analysis of the groE and 16S rDNA regions, which were amplified by PCR and directly sequenced. For comparison, eight other bacterial strains identified by others as Serratia spp. also were sequenced. These sequences clearly identified the CYVD strains as Serratia marcescens. However, evaluation of metabolic and biochemical features revealed that cucurbit-derived strains of S. marcescens differ substantially fr...
Huanglongbing (HLB) is a highly destructive citrus disease and is associated with a nonculturable... more Huanglongbing (HLB) is a highly destructive citrus disease and is associated with a nonculturable bacterium, ‘Candidatus Liberibacter asiaticus’. ‘Ca. L. asiaticus’ in the United States was first found in Florida in 2005 and is now endemic there. In California, ‘Ca. L. asiaticus’ was first detected in Hacienda Heights in Los Angeles County in 2012 and has now been detected in multiple urban locations in southern California. Knowledge of ‘Ca. L. asiaticus’ strain diversity in California is important for HLB management. In this study, genomic diversity among 10 ‘Ca. L. asiaticus’ strains from six California locations were analyzed using a next-generation sequencing (NGS) (Illumina MiSeq and HiSeq) approach. Draft genome sequences of ‘Ca. L. asiaticus’ strains were assembled. Sequences of the 16S ribosomal RNA gene and nrdB confirmed ‘Ca. L. asiaticus’ identity. Prophages were detected in all ‘Ca. L. asiaticus’ strains. The California ‘Ca. L. asiaticus’ strains formed four prophage typ...
In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Imm... more In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Immokalee, FL. Symptoms observed on fruit included hard spot, cracked spot, and early virulent spot. Hard spot lesions were up to 5 mm, depressed with a chocolate margin and a necrotic, tan center, often with black pycnidia (140 to 200 μm) present. Cracked spot lesions were large (15 mm), dark brown, with diffuse margins and raised cracks. In some cases, hard spots formed in the center of lesions. Early virulent spot lesions were small (up to 7 mm long), bright red, irregular, indented, and often with many pycnidia. In addition, small (2 to 3 mm), elliptical, reddish brown leaf lesions with depressed tan centers were observed on some trees with symptomatic fruit. Chlorotic halos appeared as they aged. Most leaves had single lesions, occasionally up to four per leaf. Tissue pieces from hard spots and early virulent spots were placed aseptically on potato dextrose agar (PDA), oatmeal agar, or...
We report here the draft genome sequence of "Candidatus Liberibacter asiaticus" strain ... more We report here the draft genome sequence of "Candidatus Liberibacter asiaticus" strain TX2351, collected from Asian citrus psyllids in south Texas, USA. The TX2351 genome has a size of 1,252,043 bp, a G+C content of 36.5%, 1,184 predicted open reading frames, and 52 RNA genes.
Huanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘C... more Huanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas...
Huanglongbing (HLB), also known as citrus greening, is a destructive citrus disease associated wi... more Huanglongbing (HLB), also known as citrus greening, is a destructive citrus disease associated with 3 α-proteobacteria species of Candidatus Liberibacter. The first report of HLB in the USA was from Florida in 2005 and Ca. L. asiaticus (Las) is the only species currently confirmed in the USA. In January 2012, a Valencia sweet orange tree in a commercial orchard in San Juan, Texas, tested positive for Las by real-time and conventional PCR assays and by the sequence of its partial 16S rRNA gene. The sample tested negative for Ca. L. americanus and Ca. L. africanus. All 4 Valencia sweet orange seedlings that were graft-inoculated using budwood from the first Texas HLB-infected tree showed typical HLB symptoms 3 months post-inoculation and tested positive for the pathogen. Such HLB typical symptoms as leaf blotchy mottle, twig die-back, veinal chlorosis, lopsided and greening fruits were observed on the Las-positive tree in the orchard, which immediately triggered an intensive survey of...
Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified ... more Xanthomonas vasicola pv. vasculorum (syn. X. campestris pv. vasculorum) was initially identified as the causal agent of bacterial leaf streak of corn in South Africa. The pathovar vasculorum causes disease on sugarcane and corn, but a subset of these strains was noted for its increased disease severity in corn. This subset was re-classified as Xanthomonas campestris pv. zeae in the early 1990s and was found to have slightly different biochemical and genetic properties than isolates from sugarcane. There has been an emergence of X. campestris pv. zeae-like strains of X. vasicola pv. vasculorum in both the United States and Argentina since 2010. We performed whole genome sequencing on U.S. isolates to confirm their identity. Informed by comparative genomics, we then developed specific TaqMan qPCR and loop-mediated isothermal amplification (LAMP) assays for the detection of this specific subset of X. vasicola pv. vasculorum strains. The qPCR 4909 assay was tested against 27 xanthomonad...
AimsDickeya species are high consequence plant pathogenic bacteria listed among the quarantine pa... more AimsDickeya species are high consequence plant pathogenic bacteria listed among the quarantine pathogens of the European Union; associated with potato disease outbreaks and subsequent economic losses worldwide. Early, accurate, and reliable detection of Dickeya spp. is needed to prevent establishment and further dissemination of this pathogen. Therefore, a multiplex TaqMan qPCR was developed for sensitive detection of Dickeya spp. and specifically, D. dianthicola.Methods and ResultsA signature genomic region for the genus Dickeya (mglA/mglC) and unique genomic region for D. dianthicola (alcohol dehydrogenase) were identified using a whole genome based comparative genomics approach. The developed multiplex TaqMan qPCR was validated using extensive inclusivity and exclusivity panels, and naturally/artificially infected samples to confirm broad range detection capability and specificity. Both sensitivity and spiked assays showed detection limit of 10 fg DNA.ConclusionThe developed mult...
A pheromone-based system to locate and monitor Planococcus minor (Maskell), a pest of over 250 pl... more A pheromone-based system to locate and monitor Planococcus minor (Maskell), a pest of over 250 plants including citrus, grape, and cacao, was tested. The difficulty in distinguishing P. minor from the citrus mealybug, P. citri, makes finding and evaluating the impact of the pest challenging. Studies conducted in Puerto Rico determined that synthetic P. minor pheromone lures preaged 120 d in the field caught similar number of males as lures not aged (fresh). Molecular analysis of trapped mealybug males using mitochondrial cytochrome oxidase-1, the internal transcriber space two locus, and 28S-D2 gene showed the pheromone traps to be species specific. Traps baited with P. minor pheromone were used to monitor the pest in south Florida and to locate potential infestations. P. minor males were found at all locations studied in South Florida and were present in low numbers (1.03 +/- 0.69 mean +/- SE/trap/14 d). Over 14,000 terminals, fruit, and flowers were visually inspected over a 6 mo period of peak trap catches before the first adult P. minor female was found. The synthetic pheromone lures proved to be an effective tool to locate and monitor this pest new to the continental United States.
Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing s... more Bacterial leaf streak of corn (Zea mays) recently reached epidemic levels in three corn-growing states, and has been detected in another six states in the central United States. Xanthomonas vasicola was identified as the causal agent of this disease. A multilocus sequence alignment of six housekeeping genes and comparison of average nucleotide identity from draft genome sequence were used to confirm phylogenetic relationships and classification of this bacteria relative to other X. vasicola strains. X. vasicola isolates from Nebraska and South Africa were highly virulent on corn and sugarcane and less virulent on sorghum but caused water-soaking symptoms that are typical of X. vasicola infection on the leaves of all three hosts. Based on host range and phylogenetic comparison, we propose the taxonomic designation of this organism to X. vasicola pv. vasculorum ( Cobb 1894 ) comb. nov. Polymerase chain reaction-based diagnostic assays were developed that distinguish X. vasicola pv. va...
Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. T... more Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperate climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is commonly used in federal and state diagnostic laboratories over conventional PCR due to its speed and sensitivity. We developed the Rs16S primers and probe set and compared it with a widely used set (RS) for detecting R. solanacearum species complex strains. We also developed the RsSA3 primers and probe set and compared it with the previously published B2 and RsSA2 sets for specific detection of r3b2 strains. Both comparisons were done under standardized qPCR master mix and cycling conditions. The Rs16S and RS assays detected all 90 R. solanacearum species complex strains and none of the five outgroups, but the former was more sensitive than the latter. For r3b2 strain detection, the RsSA2 and ...
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