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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 reclassified as X. 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 xanthomonads (diverse representation), 32 DNA extractions from corn leaves confirmed as positive or negative for the bacterium, 41 X. vasicola pv. vasculorum isolates from corn in the United States and Argentina, and 31 additional bacteria associated with corn, sugarcane, or sorghum. In all cases the assay was shown to be specific for the X. vasicola pv. vasculorum isolates that cause more severe disease on corn. We then tested the LAMP 166 assay against the 27 xanthomonads and 32 corn leaf DNA samples, and we found this assay was also specific for this subset of X. vasicola pv. vasculorum isolates. We also developed a live/dead cells distinction protocol using propidium monoazide prior to DNA extraction for analyzing seed washes using these assays. These two detection assays can be useful for both diagnosticians and researchers to specifically identify the X. vasicola pv. vasculorum isolates that cause more severe symptoms on corn.

‘Candidatus Liberibacter asiaticus’ is the unculturable causative agent of citrus huanglongbing disease. Here, we report the first citrus root metagenome sequence containing the draft genome of ‘Ca. L. asiaticus’ strain AHCA17, obtained from a pummelo tree in California. The assembled genome was 1.2 Mbp and resulted in 37 contigs (N50 = 158.7 kbp) containing 1,057 predicted open reading frames and 45 RNA-coding genes. This draft genome will provide a valuable resource in further study of ‘Ca. L. asiaticus’ genome diversity and pathogen epidemiology.

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 genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

Citrus fibrous root tissue was evaluated as an alternative source material for huanglongbing (HLB) diagnosis by real-time PCR using primer-probe set TXCChlb, developed in the present study based on 16S rDNA of “Candidatus Liberibacter asiaticus” (CLas). Real-time PCR data obtained with DNA samples prepared from leaf and fibrous root tissue collected from mature (> 10 years old) and young (4–5 years old) citrus trees growing in Texas and Florida confirmed that root HLB test is more sensitive than the leaf HLB test in terms of the detection rate of HLB positive trees. In addition, the current study confirmed that HLB can be diagnosed at the pre-symptomatic stage using the root HLB test to facilitate efficient removal of HLB-positive, asymptomatic trees that could serve as a source for HLB. The new HLB diagnostic method using root tissue described in the current study can assist in deploying a more efficient disease management strategy to deter HLB spread, especially at the pre-symptomatic stage.

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. vasculorum and X. vasicola pv. holcicola from each other and from other Xanthomonas spp.

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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 multiplex assay is sensitive and reliable to detect Dickeya spp. and D. dianthicola with no false positives or false negatives. It was able to detect mixed infection from naturally and artificially infected plant materials.Significance and ImpactThe developed assay will serve as a practical tool for screening of propagative material, monitoring the presence and distribution, and quantification of target pathogens in a breeding program. The assay also has applications in routine diagnostics, biosecurity and microbial forensics.

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 carrot agar and incubated with 12 h of light and dark at 24°C. Cultures that grew colonies within a week were discarded. Fourteen single-spore cultures were obtained from the isolates that grew slower than the Guignardia mangiferae reference cultures, although pycnidia formed more rapidly in the G. mangiferae cultures (1). No sexual structures were observed. Cultures on half-PDA were black and cordlike with irregular margins with numerous pycnidia, often bearing white cirrhi after 14 days. Conidia (7.1 to 7.8 × 10.3 to 11.8 μm) were hyaline, aseptate, multiguttulate, ovoid with a flattened base surrounded by a hyaline matrix (0.4 to 0.6 μm) and a hyaline appendage on the rounded apex, corresponding to published descriptions of G. citricarpa (anomorph Phyllosticta citricarpa) (1). A yellow pigment was seen in oatmeal agar surrounding G. citricarpa, but not G. mangiferae colonies as previously reported (1,2). DNA was extracted from lesions and cultures and amplified with species-specific primers (2). DNA was also extracted from G. mangiferae and healthy citrus fruit. The G. citricarpa-specific primers produced a 300-bp band from fruit lesions and pure cultures. G. mangiferae-specific primers produced 290-bp bands with DNA from G. mangiferae cultures. The internally transcribed spacer (ITS) of the rRNA gene, translation-elongation factor (TEF), and actin gene regions were sequenced from G. citricarpa isolates and deposited in GenBank. These sequences had 100% homology with G. citricarpa ITS sequences from South Africa and Brazil, 100% homology with TEF, and 99% homology with actin of a Brazilian isolate. Pathogenicity tests with G. citricarpa were not done because the organism infects immature fruit and has an incubation period of at least 6 months (3). In addition, quarantine restrictions limit work with the organism outside a contained facility. To our knowledge, this is the first report of black spot in North America. The initial infested area was ~57 km2. The disease is of great importance to the Florida citrus industry because it causes serious blemishes and significant yield reduction, especially on the most commonly grown ‘Valencia’ sweet orange. Also, the presence of the disease in Florida may affect market access because G. citricarpa is considered a quarantine pathogen by the United States and internationally. References: (1) R. P. Baayen et al. Phytopathology 92:464, 2002. (2) N. A. Peres et al. Plant Dis. 91:525, 2007 (3) R. F. Reis et al. Fitopath Bras. 31:29, 2006.

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 RsSA3 sets specifically detected the 34 r3b2 strains and none of the 56 R. solanacearum non-r3b2 strains or out-group strains. The B2 set, however, detected five non-r3b2 R. solanacearum strains and was less sensitive than the other two sets under the same testing conditions. We conclude that the Rs16S, RsSA2, and RsSA3 sets are best suited under the standardized conditions for the detection of R. solanacearum species complex and r3b2 strains by TaqMan-based qPCR assays.

The draft genome sequence of “Candidatus Liberibacter asiaticus” strain TX1712, obtained from a Texas citrus tree, is reported here. Strain TX1712 has a draft genome size of 1,203,333 bp, a G+C content of 36.4%, 1,230 predicted open reading frames, and 41 RNAs and comprises 97.4% of the psy62 reference genome.

Here, we report two annotated draft genome sequences of Dickeya dianthicola isolates from potatoes collected in Delaware and West Virginia. The genomes of strains DE440 and WV516 show 99% similarity to each other and 96% and 95% similarity to the European strains IPO 980 and RNS04.9, respectively.

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 tra...

The article “A new diagnostic real-time PCR method for huanglongbing detection in citrus root tissue”, written by Jong Won Park, Eliezer S. Louzada, W. Evan Braswell, Philip A. Stansly, John V. da Graca, Greg McCollum, John E. Rascoe, and Madhurababu Kunta, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 13 June 2018 without open access.

The new Liberibacter species, 'Candidatus Liberibacter solanacearum' (Lso) recently associated with potato/tomato psyllid-transmitted diseases in tomato and capsicum in New Zealand, was found to be consistently associated with a newly emerging potato zebra chip (ZC) disease in Texas and other southwestern states in the USA. A species-specific primer LsoF was developed for both quantitative real-time PCR (qPCR) and conventional PCR (cPCR) to detect and quantify Lso in infected samples. In multiplex qPCR, a plant cytochrome oxidase (COX)-based probe-primer set was used as a positive internal control for host plants, which could be used to reliably access the DNA extraction quality and to normalize qPCR data for accurate quantification of the bacterial populations in environment samples. Neither the qPCR nor the cPCR using the primer and/or probe sets with LsoF reacted with other Liberibacter species infecting citrus or other potato pathogens. The low detection limit of the multiplex qPCR was about 20 copies of the target 16S rDNA templates per reaction for field samples. Lso was readily detected and quantified in various tissues of ZC-affected potato plants collected from fields in Texas. A thorough but uneven colonization of Lso was revealed in various tissues of potato plants. The highest Lso populations were about 3x10(8) genomes/g tissue in the root, which were 3-order higher than those in the above-ground tissues of potato plants. The Lso bacterial populations were normally distributed across the ZC-affected potato plants collected from fields in Texas, with 60% of ZC-affected potato plants harboring an average Lso population from 10(5) to 10(6) genomes/g tissue, 4% of plants hosting above 10(7) Lso genomes/g tissue, and 8% of plants holding below 10(3) Lso genomes/g tissue. The rapid, sensitive, specific and reliable multiplex qPCR showed its potential to become a powerful tool for early detection and quantification of the new Liberibacter species associated with potato ZC, and will be very useful for the potato quarantine programs and seed potato certification programs to ensure the availability of clean seed potato stocks and also for epidemiological studies on the disease.

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 the disease in the area. Typical HLB symptoms were found on 54 Valencia sweet orange trees in the same orchard and 18 Rio Red grapefruit trees in an adjacent orchard. All these symptomatic trees tested positive for Las by PCR and sequencing.

Elsinoe fawcettii causes citrus scab and E. australis causes sweet orange scab and there are different pathotypes of each species. Citrus scab is widely distributed, whereas sweet orange scab is limited mostly to southern South America. In 2010, E. australis was detected by PCR in Texas for the first time in the USA and subsequently found in commercial areas of Texas. In this study, cultures were obtained in Texas mostly from diseased fruit with symptoms similar to "late-season windscar" and identified as the Natsudaidai pathotype of E. australis by sequencing of the PCR amplification products and sequencing of the translation elongation factor and ITS regions. Inoculations of detached leaves and fruit of grapefruit, sweet orange, and tangerine produced scab-like symptoms on all organs and species. The fungus was re-isolated from the inoculated organs and PCR tests on re-isolated fungal colonies and lesions confirmed the presence of E. australis. The disease represents sweet orange scab of citrus with a new scab disease symptomatology, but the host range, ecology and epidemiology of this pathotype are still not completely understood. Thus far, Natsudaidai pathotype has been reported only from South Korea.

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.