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. Serological and molecular assays showed that the cytoplasmic types of Citrus leprosis virus (CiLV-C) often associated with leprosis symptomatic tissues were absent. However, using transmission electron microscopy, bullet-shaped rhabdovirus-like virions were observed in the nuclei and cytoplasm of the citrus leprosis-infected leaf tissues. An analysis of small RNA populations from symptomatic tissue was carried out to determine the genome sequence of the rhabdovirus-like particles observed in the citrus leprosis samples. The complete genome sequence showed that the nuclear type of CiLV (CiLV-N) present in the samples consisted of two negative-sense RNAs: 6,268-nucleotide (nt)-long RNA1 and 5,847-nt-long RNA2, excluding the poly(A) tails. CiLV-N had a genome organization identical to that of Orchid fleck virus (OFV), with the exception of shorter 5' untranslated regions in RNA1 (53 versus 205 nt) and RNA2 (34 versus 182 nt). Phylogenetic trees constructed with the amino acid sequences of the nucleocapsid (N) and glycoproteins (G) and the RNA polymerase (L protein) showed that CiLV-N clusters with OFV. Furthermore, phylogenetic analyses of N protein established CiLV-N as a member of the proposed genus Dichorhavirus. Reverse-transcription polymerase chain reaction primers for the detection of CiLV-N were designed based on the sequence of the N gene and the assay was optimized and tested to detect the presence of CiLV-N in both diseased and symptom-free plants.

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.

Molecular Analysis of a Plum pox virus W Isolate in Plum Germplasm Hand Carried into the USA from the Ukraine Shows a Close Relationship to a Latvian Isolate.

Four of 19 Prunus germplasm accessions hand carried from the Ukraine into the United States without authorization were found to be infected with Plum pox virus (PPV). Of the three isolates characterized, isolates UKR 44189 and UKR 44191 were confirmed to be isolates of PPV strain W, and UKR 44188 was confirmed to be an isolate of PPV strain D. UKR 44189 and UKR 44191 are very closely related to the PPV strain W isolate LV-145bt (HQ670748) from Latvia. Nucleotide and amino acid sequence identities between these three isolates were greater than 99%. This indicates that the isolates are very closely related and likely originated from a common source. The high genetic diversity among PPV-W strain isolates allowed the identification of potential recombination events between PPV isolates. It appears also that GF 305 peach and Prunus tomentosa are not hosts for the PPV isolate UKR 44189.

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. citrus industry. Many strains of X. fastidiosa are pathogens or endophytes in various plants growing in the U.S., and some strains cross infect several host plants. In this study, a TaqMan-based assay targeting the 16S rDNA signature region was developed for the identification of X. fastidiosa at the species level. Another TaqMan-based assay was developed for the specific identification of the CVC strains. Both new assays have been systematically validated in comparison with the primer/probe sets from four previously published assays on one platform and under similar PCR conditions, and shown to be superior. The species specific assay detected all X. fastidiosa strains and did not amplify any other citrus pathogen or endophyte tested. The CVC-specific assay detected all CVC strains but did not amplify any non-CVC X. fastidiosa nor any other citrus pathogen or endophyte evaluated. Both sets were multiplexed with a reliable internal control assay targeting host plant DNA, and their diagnostic specificity and sensitivity remained unchanged. This internal control provides quality assurance for DNA extraction, performance of PCR reagents, platforms and operators. The limit of detection for both assays was equivalent to 2 to 10 cells of X. fastidiosa per reaction for field citrus samples. Petioles and midribs of symptomatic leaves of sweet orange harbored the highest populations of X. fastidiosa, providing the best materials for detection of the pathogen. These new species specific assay will be invaluable for molecular identification of X. fastidiosa at the species level, and the CVC specific assay will be very powerful for the specific identification of X. fastidiosa strains that cause citrus variegated chlorosis.

Molecular, ultrastructural, and biological characterization of Pennsylvania isolates of Plum pox virus.

Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.

Multiplex Real-Time PCR Assays for the Identification of the Potato Cyst and Tobacco Cyst Nematodes.

TaqMan primer-probe sets were developed for the detection and identification of potato cyst nematodes (PCNs) Globodera pallida and G. rostochiensis using two-tube, multiplex real-time polymerase chain reaction (PCR). One tube contained a primer-probe set specific for G. pallida (pale potato cyst nematode) multiplexed with another primer-probe set specific for G. rostochiensis (golden potato cyst nematode). A second tube consisted of the G. pallida-specific primer-probe set multiplexed with a primer-probe set specific for G. tabacum (the morphologically similar tobacco cyst nematode). This internal transcribed spacer rDNA-based system was specific for the Globodera spp. of interest and successfully identified several populations of PCN. This rapid, sensitive, and specific quantitative PCR assay presents a useful tool for PCN regulatory response and management programs.

Multiplex real-time PCR for detection, identification and quantification of 'Candidatus Liberibacter solanacearum' in potato plants with zebra chip.

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.

Quantitative distribution of 'Candidatus Liberibacter asiaticus' in citrus plants with citrus huanglongbing.

Citrus huanglongbing (HLB), or greening disease, is strongly associated with any of three nonculturable gram-negative bacteria belonging to 'Candidatus Liberibacter spp.' 'Ca. Liberibacter spp.' are transmitted by citrus psyllids to all commercial cultivars of citrus. The diseases can be lethal to citrus and have recently become widespread in both São Paulo, Brazil, and Florida, United States, the locations of the largest citrus industries in the world. Asiatic HLB, the form of the disease found in Florida, is associated with 'Ca. Liberibacter asiaticus' and is the subject of this report. The nonculturable nature of the pathogen has hampered research and little is known about the distribution of 'Ca. L. asiaticus' in infected trees. In this study, we have used a quantitative polymerase chain reaction assay to systematically quantify the distribution of 'Ca. L. asiaticus' genomes in tissues of six species of citrus either identified in the field during survey efforts in Florida or propagated in a greenhouse in Beltsville, MD. The populations of 'Ca. L. asiaticus' inferred from the distribution of 16S rDNA sequences specific for 'Ca. L. asiaticus' in leaf midribs, leaf blades, and bark samples varied by a factor of 1,000 among samples prepared from the six citrus species tested and by a factor of 100 between two sweet orange trees tested. In naturally infected trees, above-ground portions of the tree averaged 10(10) 'Ca. L. asiaticus' genomes per gram of tissue. Similar levels of 'Ca. L. asiaticus' genomes were observed in some but not all root samples from the same plants. In samples taken from greenhouse-inoculated trees, levels of 'Ca. L. asiaticus' genomes varied systematically from 10(4) genomes/g at the graft inoculation site to 10(10) genomes/g in some leaf petioles. Root samples from these trees also contained 'Ca. L. asiaticus' at 10(7) genomes/g. In symptomatic fruit tissues, 'Ca. L. asiaticus' genomes were also readily detected and quantified. The highest levels of 'Ca. L. asiaticus' in fruit tissues were found in the locular membranes and septa (10(8) genomes/g), with 100-fold lower levels of 'Ca. L. asiaticus' in the meso and pericarp of such fruit. Our results demonstrate both the ubiquitous presence of 'Ca. L. asiaticus' in symptomatic citrus trees as well as great variation between individual trees and among samples of different tissues from the same trees. Our methods will be useful in both the management and scientific study of citrus HLB, also known as citrus greening disease.

Optimized Quantification of Unculturable Candidatus Liberibacter Spp. in Host Plants Using Real-Time PCR.

Citrus huanglongbing (HLB) is caused by the phloem-limited and psyllid-vectored Candidatus Liberibacter spp. and is a destructive disease of citrus that is rapidly increasing in importance. The disease was reported recently in the principle citrus-producing areas of São Paulo, Brazil in 2004 and in Florida in 2005. A variety of laboratory methods have been developed to confirm a symptom-based disease diagnosis or for the detection or identification of the pathogen; however, no quantitative information has been available on the pathogen titer in either host or vector interactions because the pathogen remains unculturable in artificial media. We previously developed a quantitative polymerase chain reaction (PCR)-based assay for detection of Ca. Liberibacter spp. and, in this study, we evaluated the effects of sample composition on quantification of the pathogen in citrus plants by TaqMan real-time PCR. Standard curves were established using cloned plasmids containing target DNA from the pathogen and with total DNA samples from field-grown HLB-infected citrus plants. Regression analysis showed that a standard curve established with DNA extracted from naturally infected field-grown plants was more accurate than the standard curve constructed from plasmids containing the amplification targets as cloned inserts. Nontarget DNA and putative PCR inhibitors from citrus plants decreased the sensitivity and the amplification efficiency of real-time PCR when plasmids provided the template target in "spiked" healthy citrus DNA extracts. This effect varied among plant tissue types, citrus species, and geographic locations. Based on these sample effects, a universal standard curve has been established for quantification of the pathogen in various citrus tissues of different citrus species planted in different geographic locations. Sample storage at 4°C for 2 months prior to PCR assay did not affect subsequent quantification of the pathogen. The validated quantitative real-time PCR method and the universal standard curve will be very useful for studies of host-pathogen interactions and epidemiology, and in the development of control strategies for the disease.

Evaluation of DNA Amplification Methods for Improved Detection of "Candidatus Liberibacter Species" Associated with Citrus Huanglongbing.

Citrus huanglongbing (HLB), also known as citrus greening or citrus yellow shoot, is considered the most serious disease of citrus worldwide. The disease has Asian, African, and American forms caused by "Candidatus Liberibacter asiaticus", "Ca. L. africanus", and "Ca. L. americanus", respectively, which can be spread efficiently by the psyllid vectors Diaphorina citri and Trioza erytreae and through contaminated plant materials. Infected citrus groves are usually destroyed or become unproductive in 5 to 8 years. The presumed low concentration and uneven distribution of the pathogens in citrus plants and vector insects make the phloem-limited bacterium difficult to detect consistently. In this study, we compared and validated four conventional polymerase chain reaction (PCR)-based protocols, one loop-mediated isothermal amplification (LAMP) protocol, and three TaqMan real-time PCR protocols. The detection sensitivity of the validated conventional PCR assays reported are improved compared with the original protocols. All of the validated conventional and the newly developed real-time methods were reliable for confirmatory tests for the presence of "Ca. Liberibacter spp." in symptomatic samples. There were no differences in assay specificity among the standard format PCR-based methods evaluated. The TaqMan real-time PCR was 10- to 100-fold more sensitive than conventional PCR and LAMP, showing the potential to become a valuable tool for early detection and identification of "Ca. Liberibacter spp." prior to the appearance of disease symptoms. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a "Ca. Liberibacter spp."-free nursery system.

Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing.

Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1:13,000 (w/w) and 1:1000 (c/c: target copy/target copy) in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter species has been successfully used in the confirmation of HLB caused by Las in Florida, and will be very useful for a broad range of research programs as well as the regulatory response and management of HLB disease.

Plum pox in north america: identification of aphid vectors and a potential role for fruit in virus spread.

ABSTRACT Thirteen aphid species were tested for their ability to transmit Pennsylvania isolates of Plum pox virus (PPV) collected in Columbia (PENN-3), Franklin (PENN-4), and York (PENN-7) Counties, PA. Four species, Aphis fabae, A. spiraecola, Brachycaudus persicae, and Myzus persicae, consistently transmitted PPV in preliminary transmission tests. Two species, Metopolophium dirhodum and Rhopalosiphum padi, were occasional inefficient vectors. Toxoptera citricida, from Florida, also was an effective vector but it does not occur in major stone-fruit-growing states. Species not transmitting PPV in parallel tests included Acyrthosiphon pisum, Aphis glycines, Aulacorthum solani, Macrosiphum euphorbiae, Rhopalosiphum maidis, and Sitobion avenae. When given a 3-day probing access period simultaneously on PPV-infected peach seedlings and healthy peach seedlings, Myzus persicae, Aphis spiraecola, A. fabae, and B. persicae transmitted PPV to 63, 31, 38, and 32% of the healthy peach seedlings, respectively. When given a similar probing period on PPV-infected peach fruit and healthy peach seedlings, the same aphid species transmitted PPV to 50, 35, 0, and 0% of seedlings, respectively. Results support the hypothesis of secondary PPV spread by indigenous aphids in Pennsylvania, and suggest that PPV-infected fruit has the potential to function as a virus source for long-distance dispersal.

Improved sampling methods for real-time polymerase chain reaction diagnosis of citrus canker from field samples.

ABSTRACT Citrus bacterial canker disease has been introduced at least three times into Florida in the last 15 years and, despite federal and state quarantine and eradication efforts, continues to spread in Florida. Accurate, fast, and reliable detection of the causal agent is of great importance. However, citrus bacterial canker is caused by at least two groups of phylogenetically distinct Xanthomonas citri strains, and there is host range variation within both groups. We developed a fast, sensitive and reliable real-time polymerase chain reaction (PCR) assay using a portable, field-hardened RAPID machine and primers designed to detect all canker-causing strains. Single-lesion sampling methods were developed that required minimal handling and allowed complete real-time PCR diagnosis in a total time of 4 h and with an apparent sensitivity of less than 10 CFU of target cells from diseased lesions. This sensitivity allowed molecular detection for the first time of X. citri in a herbarium sample from a 1912 canker outbreak. Sensitivity was improved significantly by the use of CaCO(3) and Silwet L-77, and by either minimizing the amount of citrus lesion tissue sampled or by soaking or swiping but not grinding the lesions. Primer design also was of significant importance in both specificity and sensitivity.