[show abstract][hide abstract] ABSTRACT: Citrus leprosis virus C (CiLV-C), a causal agent for Citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with L-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.
Journal of Virological Methods 07/2013; · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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, but 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 (Geijskes), to healthy citrus seedlings. A library of small RNAs was made constructed from symptomatic leaves and used as the template for Illumina high-throughput parallel sequencing. The complete genome sequence and structure of a new bipartite RNA virus was determined. RNA1 (8717 nt) contained two open reading frames (ORFs). ORF1 encoded the replication module, consisting of five domains; namely methyltransferase (MTR), cysteine protease- like, FtsJ-MTR, helicase (Hel), and RNA dependent RNA polymerase (RdRp) whereas ORF2 encoded the putative coat protein. RNA2 (4989 nt) contained five ORFs that encode the movement protein (MP) and four hypothetical proteins (p7, p15, p24 and p61). The structure of this virus genome resembled that of CiLV-C except that it contained a long 3' untranslated terminal region and an extra ORF (p7) in RNA2. Both the RNA1 and RNA2 of the new virus had only 58% and 50% nucleotide identities, respectively, with known CiLV-C sequences and thus it appears to be a novel virus infecting citrus. Phylogenetic analyses of the MTR, Hel, RdRp and the MP domains also indicated that the new virus was closely related to CiLV-C. We suggest that the virus be called Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) and it should be unambiguously classified as a definitive member of the genus Cilevirus. A pair of CiLV-C2 genome specific RT-PCR primers were designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of microbiological methods 10/2012; · 2.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of microbiological methods 06/2009; 78(1):59-65. · 2.43 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 1010 '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 104 genomes/g at the graft inoculation site to 1010 genomes/g in some leaf petioles. Root samples from these trees also contained 'Ca. L. asiaticus' at 107 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 (108 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.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Microbiological Methods 08/2006; 66(1):104-15. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Citrus huanglongbing (HLB) is one of the most devastating citrus diseases in the world. The disease is associated with three species of Candidatus Liberibacter, "Ca. L. asiaticus", "Ca. L. africanus", and "Ca. L. americanus", and transmitted mainly by the Asian citrus psyllid (Diaphorina citri) and the African citrus psyllid (Trioza erytreae). Upon the first report of the disease in São Paulo, Brazil in 2004 and Florida, USA in 2005, HLB started to devastate the two largest citrus industries in the world. The disease continues to rapidly spread to the remaining citrus producing areas still free of the disease, such as Central America and Middle East, besides its presence in almost all Asian citrus producing nations. Early detection of the associated bacterium in host plants and vector insects is essential to HLB control and management. Sampling, sample processing and testing are three major processes in disease detection. The main objective of this invited talk is to compile recent advances in method development of sampling, sample processing and molecular testing of Candidatus Liberibacter species associated with the disease, and to make recommendations to increase the efficiency and ability to detect the associated bacterium in survey samples. Based on uneven distribution of the bacterium in plant tissues associated with irregular vector transmission, low bacterial titer during early stages of infection in host plants and vector insects, and recent quantitative data of the bacterial populations in infected plants and vectors, different sampling methods and sample sizes are recommended for host plant and vector insect surveys. These recommendations are useful for surveys in areas with HLB and HLB-like symptomatic, asymptomatic orchards and nurseries. In addition, adequate storage methods are also suggested for plant and insect samples prior to testing for the associated bacterium. Sample processing methods, especially of the bacterial DNA isolation from suspect plant and insect samples were compared. A closed mechanical sample homogenization system with commercial DNA extraction kits using DNA binding filters was highly advised for critical samples to avoid sample cross contamination and to obtain consistently high yield and purity of the bacterial DNA. Positive internal control primer and/or probe sets are proscribed for real-time monitoring on DNA extraction quality and PCR testing efficiency of Liberibacter species from suspect plant and insect samples. In comparison with traditional diagnostic methods including electronic microscopy, ELISA, use of fluorescent substance and starch accumulation in host plants, disease symptomology, and biological indexing, isotheral DNA amplification including the Loop-mediated isothermal amplification (LAMP) and the cycleave isothermal and chimeric primer-initiated amplification with probe technology (Cycleave ICAN), single and nested conventional PCR, and single and multiplex real-time PCR assays were analyzed for specificity, sensitivity and assay performance. Multiplex quantitative real-time PCR methods using the species-specific primers and/or probes for associated bacterium and the positive internal control primers and/or probes targeting the host plant DNA or vector psyllid DNA were appraised for HLB detection. The potential of 125 selected genes from the complete genome of "Ca. L. asiaticus" was also discussed for future use in multiple-loci detection.