Complete Nucleotide Sequence of a Novel Hibiscus-Infecting Cilevirus from Florida and Its Relationship with Closely Associated Cileviruses.

The complete nucleotide sequence of a recently discovered Florida (FL) isolate of hibiscus-infecting cilevirus (HiCV) was determined by Sanger sequencing. The movement and coat protein gene sequences of the HiCV-FL isolate are more divergent than other genes of the previously sequenced HiCV-HI (Hawaii) isolate.

Production of mono- and polyclonal antibodies to Citrus leprosis virus C2 and their application in triple antibody sandwich ELISA and immunocapture RT-PCR diagnostic assays.

The newly discovered Citrus leprosis virus cytoplasmic type 2 (CiLV-C2) is one of the causal virus of citrus leprosis disease complex; which leads to substantial loss of citrus production in the states of Meta and Casanare of Colombia. Specific and sensitive detection methods are needed to monitor the dissemination of CiLV-C2 in Colombia, and to prevent introduction of CiLV-C2 to other citrus growing countries. Toward this end, putative coat protein gene (CPG) of CiLV-C2 was amplified from CiLV-C2 infected citrus tissues. The CPG was cloned, expressed and purified a recombinant coat protein of ∼31kDa which used to generate monoclonal antibodies and polyclonal antisera. Four monoclonal antibodies and two polyclonal antisera were selected as being specific following Western blotting. The monoclonal antibody MAb E5 and polyclonal antiserum PAb UF715 were selected testing with an extract of CiLV-C2 infected leaves using triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). In addition, an immunocapture RT-PCR was standardized using MAb E5 for specific and sensitive detection of CiLV-C2. The standardized TAS-ELISA and IC-RT-PCR were able to detect CiLV-C2 in the extracts of symptomatic citrus leprosis tissues up to the dilutions of 1:160 and 1:2580, respectively. Result demonstrated that CiLV-C2 is present in citrus orchards in Meta and Casanare citrus growing areas of Colombia. TAS-ELISA could be used for routine detection of CiLV-C2, epidemiological studies, and for border inspections for quarantine purposes. IC-RT-PCR could be valuable for CiLV-C2 validation and viral genome analysis.

Detection of Citrus leprosis virus C using specific primers and TaqMan probe in one-step real-time reverse-transcription polymerase chain reaction assays.

Citrus leprosis virus C (CiLV-C), a causal agent of the leprosis disease in citrus, is mostly present in the South and Central America and spreading toward the North America. To enable better diagnosis and inhibit the further spread of this re-emerging virus a quantitative (q) real-time reverse transcription polymerase chain reaction (qRT-PCR) assay is needed for early detection of CiLV-C when the virus is present in low titer in citrus leprosis samples. Using the genomic sequence of CiLV-C, specific primers and probe were designed and synthesized to amplify a 73 nt amplicon from the movement protein (MP) gene. A standard curve of the 73 nt amplicon MP gene was developed using known 10(10)-10(1) copies of in vitro synthesized RNA transcript to estimate the copy number of RNA transcript in the citrus leprosis samples. The one-step qRT-PCR detection assays for CiLV-C were determined to be 1000 times more sensitive when compared to the one-step conventional reverse transcription polymerase chain reaction (RT-PCR) CiLV-C detection method. To evaluate the quality of the total RNA extracts, NADH dehydrogenase gene specific primers (nad5) and probe were included in reactions as an internal control. The one-step qRT-PCR specificity was successfully validated by testing for the presence of CiLV-C in the total RNA extracts of the citrus leprosis samples collected from Belize, Costa Rica, Mexico and Panama. Implementation of the one-step qRT-PCR assays for CiLV-C diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of CiLV-C in countries where it is present and to prevent further dissemination into citrus growing countries where there is no report of CiLV-C presence.

Production of monoclonal antibodies for detection of Citrus leprosis virus C in enzyme-linked immuno-assays and immunocapture reverse transcription-polymerase chain reaction.

Citrus leprosis virus C (CiLV-C) causes damage in citrus production in the South and Central America. Since closely related types of citrus viruses have recently been described monoclonal antibodies (MAbs) are needed for accurate and sensitive diagnosis of CiLV-C. In this study, MAbs to the expressed coat protein of CiLV-C were produced for serological detection of CiLV-C in crude extracts of infected tissues in double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA), dot blot immunosorbent assays (DBIA) and immuonocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) procedures. Monoclonal antibodies were developed in mice to the purified expressed coat protein of CiLV-C. The published standard protocols of DAS-ELISA, DBIA and IC-RT-PCR were followed for the detection of coat protein p29 of CiLV-C in the crude extracts of CiLV-C infected tissues. Two monoclonal antibodies, designated G10 and C11, were identified from four potential candidates for the specific and sensitive detection of coat protein p29 of CiLV-C in the crude citrus extracts of CiLV-C infected tissues in DAS-ELISA, whereas G10 was also selected based on performance for use in the DBIA and IC-RT-PCR diagnostic assays. Sensitivity analysis comparing the three methods for detection of coat protein p29 of CiLV-C determined that IC-RT-PCR was more sensitive than DAS-ELISA and DBIA. The creation of MAbs to CiLV-C allows for the sensitive and accurate detection of the virus from CiLV-C infected citrus leaf tissues. Successful detection of the virus in three diagnostic assays formats provides flexibility to diagnosticians who can use either ELISA or DBIA for screening large numbers of samples, and IC-RT-PCR for rapid, sensitive confirmation testing.

First Report of Citrus leprosis virus Nuclear Type in Sweet Orange in Colombia.

Colombia is ranked 18th in the world in citrus production and contributed 0.9% of the total world share. Among four important citrus-producing regions of Colombia, the Orinoco region (3 to 6°N, 68 to 74°W) consists of two citrus-producing states, Meta and Casanare. Citrus leprosis is the most important viral disease of citrus in Colombia (1,3). Three types of Citrus leprosis virus (CiLV) infect citrus, producing leprosis-like lesion symptoms. Two of the three CiLV species, Citrus leprosis virus cytoplasmic type (CiLV-C) and cytoplasmic type 2 (CiLV-C2), produce particles only in the cytoplasm (3). The other species, Citrus leprosis virus nuclear type (CiLV-N), produces particles in both the cytoplasm and nucleus (4). CiLV-C is more prevalent and destructive while CiLV-N has been reported only in Brazil, Panama, and Mexico (4). Interestingly, both CiLV-C and -C2 were reported from the same regions of Meta and Casanare States in Colombia in 2004 and 2012 (1,3). CiLV-C lesions are usually rounded (initially 2 to 3 mm in diameter and extending up to 30 mm), have dark-brown or greenish central chlorotic spots, and are surrounded by yellow halos. CiLV-N lesions have been described as smaller in size and form three well-defined regions including a necrotic center with an intermediate orange color halo and an outer chlorotic halo (2). In 2013, 'Valencia' sweet orange (Citrus sinensis L.) leaves with suspected CiLV-N symptoms were collected from 8 plants in Casanare State and shipped under permit to the USDA-APHIS-PPQ-CPHST, Beltsville, MD. Total RNA from symptomatic and healthy sweet orange leaves were extracted using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA). RT-PCR primers specific to CiLV-C, CiLV-C2 (3), and CiLV-N nucleocapsid (N) (CiLV-N-NPF: 5'-ATGGCTAACCCAAGTGAGATCGATTA-3'; CiLV-N-NPR: 5'-AGTTGCCTTGAGATCATCACATTGGT-3') and putative matrix protein (M) genes (CiLV-N-MF: 5'-ATGTCTAAACAGATTAATATGTGCACTGTG-3'; CiLV-N-MR: 5'-CTAACCACTGGGTCCCGC-3') were utilized to identify the CiLV associated with the leprosis-affected leaf samples from Casanare. RT-PCR with CiLV-C primers failed to produce any amplicon, but CiLV-N primers successfully amplified the partial N gene (681 bp) and entire M gene (552 nt) amplicons from multiple leaves of all leprosis samples. In addition, a 795-bp amplicon specific to CiLV-C2 also was amplified from the CiLV-N suspected samples. Similar results were obtained when the vector, flat spider mite (Brevipalpus spp.) total RNA was used as template for RT-PCR. For further confirmation, each amplicon was cloned and sequenced. Sequencing of the N and M gene amplicons of CiLV-N (accession nos. KJ195893 and KJ195894) and coat protein gene of CiLV-C2 showed 97 to 99% nucleotide sequence identity with the CiLV-N M2345 isolate sequence (KF209275) from Mexico (4) and CiLV-C2 L147V1 isolate sequence (JX000024) from Colombia (3), respectively. Phylogenetic analyses of these N and M protein gene sequences confirmed a mixed infection of the same plant with two viruses, one from an unassigned new genus Dichorhavirus (CiLV-N) and another from genus Cilevirus (CiLV-C2). This is the first report of CiLV-N in Colombia, and also the first report of an occurrence of CiLV-N in mixed infection with CiLV-C2. All three known species of CiLV occur in the Orinoco region of Colombia. References: (1) M. G. León et al. Plant Dis. 90: 682, 2006. (2) J. P. R. Marques et al. Anais da Academia Brasileira de Ciências 82:501, 2010. (3) A. Roy et al. Phytopathology 103:488, 2013. (4) A. Roy et al. Genome Announc. 1(4): e00519-13, 2013.

Live Population Dynamics of 'Candidatus Liberibacter asiaticus', the Bacterial Agent Associated with Citrus Huanglongbing, in Citrus and Non-Citrus Hosts.

Citrus huanglongbing (HLB) is a century-old destructive disease which presents an unprecedented challenge to citrus industries worldwide. In Florida, HLB is associated with the phloem-limited bacterium 'Candidatus Liberibacter asiaticus' and is mainly transmitted by Asian citrus psyllid (Diaphorina citri). Quantification of the pathogen population in a host aids in investigation of virulence mechanisms and disease management. Recently a procedure was developed to detect live bacterial populations using a novel DNA-binding dye, propidium monoazide, in conjunction with real-time polymerase chain reaction (PMA-qPCR). Chinese box orange (Severinia buxifolia) is a common ornamental present in Florida which could host D. citri and 'Ca. L. asiaticus'. For 20 months, the change of the live 'Ca. L. asiaticus' populations in graft- and psyllid-transmitted Valencia sweet orange (Citrus sinensis 'Valencia') and S. buxifolia plants was monitored by PMA-qPCR. Our results showed that the live 'Ca. L. asiaticus' population was significantly lower in the months of December, January, and February than the rest of the year in both hosts. No statistically significant pattern in the total bacterial population was observed in either host. This pattern may indicate a seasonal growth of 'Ca. L. asiaticus' along with the growth of both plants. These new findings should provide useful information on HLB management.

Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

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.

A novel virus of the genus Cilevirus causing symptoms similar to citrus leprosis.

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; however, 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, to healthy citrus seedlings. A library of small RNAs was 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 (8,717 nucleotides [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 (4,989 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 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 was designed and validated to detect its presence in citrus leprosis samples collected from the Casanare and Meta states in Colombia.

Quantification of Live 'Candidatus Liberibacter asiaticus' Populations Using Real-Time PCR and Propidium Monoazide.

Huanglongbing (HLB) is a devastating citrus disease. It is associated with a phloem-restricted bacterium, 'Candidatus Liberibacter asiaticus', and primarily transmitted by Asian citrus psyllid in Florida. Because Liberibacter cannot be cultured, early diagnosis of HLB relies on DNA-based polymerase chain reaction (PCR), including real-time quantitative (q)PCR. Although estimating genomes from live bacteria (GLB) is critical for HLB research, PCR does not distinguish between live and dead cells and, thus, does not estimate GLB in hosts. Propidium monoazide (PMA), a novel DNA-binding dye, has been successfully used on many bacterial pathogens to effectively remove DNA from dead cells but there is no report of its use on uncultured bacteria. In this study, PMA-qPCR protocols were first optimized to work with plant and psyllid samples, respectively. Both TissueLyser treatment and plant tissue were demonstrated to have an insignificant impact on the GLB detected by PMA-qPCR. Finally, a standard curve for GLB determination was successfully established between PMA-qPCR results and microscopic counts and then applied in two studies with different greenhouse plant samples. This rapid qPCR method provides a more accurate way to determine GLB in HLB hosts which, in turn, should benefit disease epidemiology studies and serve as a crucial component in HLB management.

Development of Primers and Probes for Genus and Species Specific Detection of 'Candidatus Liberibacter Species' by Real-Time PCR.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease impacting citrus production. The disease is associated with three different 'Candidatus Liberibacter species', 'Ca. Liberibacter asiaticus', 'Ca. Liberibacter americanus', and 'Ca. Liberibacter africanus', which induce similar and overlapping symptoms. When HLB-symptomatic trees are tested, one of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. The 16S rRNA-based assays to detect the three species are species-specific and must be performed sequentially. We describe a single assay that detected all species of 'Ca. Liberibacter' at the genus level, providing increased convenience. Recent molecular analyses of 'Ca. Liberibacter species' and other bacteria suggest that the rpoB gene (encoding the ß-subunit of RNA polymerase) provides an alternative target for bacterial identification. We report here the design of a single pair of degenerate primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three citrus 'Ca. Liberibacter species', enabling detection of 'Ca. Liberibacter' at the genus level. In addition, species-specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus- and species-specific assays were validated in both SYBR Green I and TaqMan formats, and with both plant and insect extracts that contained the pathogen. These one-step qPCR diagnostic methods are useful for the detection of all species of Liberibacter infecting citrus. In addition, the degenerate genus-specific primers and probe successfully detected 'Ca. Liberibacter solanacearum', a psyllid-transmitted pathogen associated with disease in tomato, carrot, and potato.

The Prevalence of the Citrus tristeza virus Trifoliate Resistance Breaking Genotype Among Puerto Rican Isolates.

Citrus tristeza virus (CTV) isolates have been grouped into six genotypes: T3, T30, T36, VT, B165, and resistance breaking (RB) based on symptoms, host range, and genomic sequence data. The RB genotype has recently been identified with the novel property of replicating in trifoliate orange trees, a resistant host for the other five genotypes. Puerto Rican CTV isolate B301 caused mild vein clearing symptoms in Mexican lime but did not induce seedling yellows or stem pitting reactions in appropriate indicator Citrus spp., which are typical host reactions of the isolate T30. The isolate B301 was not detected by the genotype specific primer (GSP), which identifies the CTV-T3, -T30, -T36, -VT, and B165 genotypes. A primer pair for reverse transcription polymerase chain reaction (RT-PCR) amplification of the CTV-RB genotype was designed from the heat shock protein (p65) region based on the complete genomic sequences of trifoliate RB isolates from New Zealand available in the GenBank databases. The amplicon sequence from isolate B301 was 98% identical to that of the other trifoliate RB isolates. In addition, B301 was successfully inoculated into 'Carrizo citrange' (a trifoliate hybrid) but did not induce any symptoms. Furthermore, the complete genome sequence of B301 followed by the phylogenetic analysis revealed that the isolate is part of the RB clade with other CTV-RB isolates from New Zealand and Hawaii. Additional CTV isolates obtained from Puerto Rico were tested with the RB-GSP and confirmed the presence of trifoliate RB isolates in mixed infection with known CTV genotypes. Although this is the first report of a CTV trifoliate RB genotype from Puerto Rico, this genotype was present there prior to 1992.

Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants.

'Mexican' lime (Citrus aurantifolia Swingle) was transformed with constructs that contained chimeric promoter-gus gene fusions of phloem-specific rolC promoter of Agrobacterium rhizogenes, Arabidopsis thaliana sucrose-H(+) symporter (AtSUC2) gene promoter of Arabidopsis thaliana, rice tungro bacilliform virus (RTBV) promoter and sucrose synthase l (RSs1) gene promoter of Oryza sativa (rice). Histochemical ß-glucuronidase (GUS) analysis revealed vascular-specific expression of the GUS protein in citrus. The RTBV promoter was the most efficient promoter in this study while the RSs1 promoter could drive low levels of gus gene expression in citrus. These results were further validated by reverse transcription real-time polymerase chain reaction and northern blotting. Southern blot analysis confirmed stable transgene integration, which ranged from a single insertion to four copies per genome. The use of phloem-specific promoters in citrus will allow targeted transgene expression of antibacterial constructs designed to battle huanglongbing disease (HLB or citrus greening disease), associated with a phloem-limited Gram-negative bacterium.

Stem-Pitting Citrus tristeza virus Predominantly Transmitted by the Brown Citrus Aphid from Mixed Infections Containing Non-Stem-Pitting and Stem-Pitting Isolates.

Citrus tristeza virus (CTV) is a phloem-limited Closterovirus that produces a variety of symptoms in various Citrus spp. One of these symptoms is stem pitting (SP). SP does not occur in all Citrus spp. but when it does it may cause low tree vigor, decline, and an economic reduction in fruit size and yield. Historically, the first appearance of CTV-SP in a citrus area often occurs after the introduction of the most efficient CTV vector, the brown citrus aphid (BCA), Toxoptera citricida. Hypotheses for this association range from the introduction of these strains in new planting materials to the increased ability of BCA to transmit SP strains from existing CTV sources. It is known that CTV often exists as a complex of isolates or subisolates. Single and multiple BCA transmissions have been used to separate different genotypes or strains of CTV from mixed CTV infected plants. This study was initiated to determine what the BCA transmits when an exotic severe SP CTV isolate B12 from Brazil or B408 from Dominican Republic are mixed with a non-SP (NSP) isolate, FS627 from Florida. Biological and molecular data was generated from grafted mixtures of these isolates and their aphid-transmitted subisolates. Single-strand conformation polymorphism patterns of the 5' terminal region of open reading frame (ORF) 1a, the overlapping region of ORF1b and ORF2, and the major coat protein gene region of NSP and SP CTV-grafted plants remained unchanged but the patterns of doubly inoculated plants varied. The haplotype diversity within SP isolates B12, B408, and mixtures of NSP and SP isolates (FS627/B12 and FS627/B408) and aphid-transmitted subisolates from doubly inoculated plants was determined by analysis of the haplotype nucleotide sequences. Aphid transmission experiments, symptoms, and molecular analyses showed that SP-CTV was more frequently transmitted with or without NSP-CTV from mixed infections.

Transmission parameters for Candidatus liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae).

The purpose of this investigation was to evaluate acquisition and inoculation (together, transmission) efficiency of Candidatus Liberibacter asiaticus (Las), the pathogen associated with citrus huanglongbing (HLB) by the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae). In laboratory studies, nymphs reared on Las infected plants were more likely to acquire the bacterium than adults. Acquisition by nymphs ranged from 60 to 100%, whereas acquisition by adults only reached 40% after 5 wk of feeding on Las-infected plants. Similar rates of pathogen acquisition by psyllids after nymphal and adult feeding were observed in the field. Transmission of Las from parent to offspring (transovarial) occurred at a rate of 2-6%. One year after psyllid inoculations, successful transmission by individual D. citri ranged from 4 to 10%, whereas groups of 100 or more D. citri transmitted the pathogen at a rate of approximately 88%. In addition, the proportion of Las-positive adult psyllids, determined using quantitative real-time polymerase chain reaction, decreased over time when held on healthy plants. Due to the low rate of pathogen acquisition and long time period required for successful inoculation by adult D. citri, experiments designed to determine the latent period required for replication and successful inoculation of Las by D. citri did not result in Las-infected plants after >1 yr of incubation after inoculation. Collectively, these results indicate that adult D. citri which acquire the HLB pathogen as adults are poor vectors of the pathogen compared with adults that acquired the pathogen as nymphs.

Development and application of a multiplex reverse-transcription polymerase chain reaction assay for screening a global collection of Citrus tristeza virus isolates.

The emerging diversity of Citrus tristeza virus (CTV) genotypes has complicated detection and diagnostic measures and prompted the search for new differentiation methods. To simplify the identification and differentiation of CTV genotypes, a multiplex reverse-transcription polymerase chain reaction (RT-PCR) technique for the screening of CTV isolates was developed. Variable regions within the open reading frame (ORF)-1a of diverse CTV genotypes were identified to develop first a simplex (S) and then a hexaplex (H) RT-PCR. CTV isolates have been grouped previously into five genotypes (namely, T3, T30, T36, VT, and B165) based on the nucleotide sequence comparisons and phylogenetic analyses. Nucleotide sequences from GenBank were used to design species and genotype-specific primers (GSPs). The GSPs were initially used for reliable detection of all CTV genotypes using S-RT-PCR. Furthermore, detection of all five recognized CTV genotypes was established using the H-RT-PCR. Six amplicons, one generic to all CTV isolates and one for each of the five recognized genotypes, were identified on the basis of their size and were confirmed by sequence analysis. In all, 175 CTV isolates from 29 citrus-growing countries were successfully analyzed by S- and H-RT-PCR. Of these, 97 isolates contained T36 genotypes, 95 contained T3 genotypes, 76 contained T30 genotypes, 71 contained VT genotypes, and 24 contained B165 genotype isolates. In total, 126 isolates contained mixed infections of 2 to 5 of the known CTV genotypes. Two of the CTV isolates could not be assigned to a known genotype. H-RT-PCR provides a sensitive, specific, reliable, and rapid way to screen for CTV genotypes compared with other methods for CTV genotype detection. Efficient identification of CTV genotypes will facilitate a better understanding of CTV isolates, including the possible interaction of different genotypes in causing or preventing diseases. The methods described can also be used in virus-free citrus propagation programs and in the development of CTV-resistant cultivars.

Colonization of dodder, Cuscuta indecora, by 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus'.

Huanglongbing, or citrus greening, threatens the global citrus industry. The presumptive pathogens, 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus' can be transferred from citrus to more easily studied experimental hosts by using holoparasitic dodder plants. However, the interaction between 'Candidatus Liberibacter' spp. and the dodder has not been studied. We combined quantitative polymerase chain reaction with electron microscopy to show that only 65% of tendrils of Cuscuta indecora grown on 'Ca. Liberibacter' spp.-infected host plants had detectable levels of the pathogen. Among tendrils that were colonized by Liberibacter in at least one 2 cm segment, most were not colonized in all segments. Furthermore, the estimated population levels of the pathogen present in serial 2 cm segments of dodder tendrils varied widely and without any consistent pattern. Thus, there was generally not a concentration gradient of the pathogen from the source plant towards the recipient and populations of the pathogen were sometimes found in the distal segments of the dodder plant but not in the proximal or middle segments. Populations of the pathogens ranged from 2 x 10(2) to 3.0 x 10(8) cells per 2 cm segment. On a fresh weight basis, populations as high as 1.4 x 10(10) cells per g of tissue were observed demonstrating that 'Ca. Liberibacter' spp. multiplies well in Cuscuta indecora. However, 55% of individual stem segments did not contain detectable levels of the pathogen, consistent with a pattern of nonuniform colonization similar to that observed in the much more anatomically complex citrus tree. Colonization of dodder by the pathogen is also nonuniform at the ultrastructural level, with adjacent phloem vessel elements being completely full of the pathogen or free of the pathogen. We also observed bacteria in the phloem vessels that belonged to two distinct size classes based on the diameters of cross sections of cells. In other sections from the same tendrils we observed single bacterial cells that were apparently in the process of differentiating between the large and round forms to the long and thin forms (or vice versa). The process controlling this morphological differentiation of the pathogen is not known. The highly reduced and simplified anatomy of the dodder plant as well as its rapid growth rate compared with citrus, and the ability of the plant to support multiplication of the pathogen to high levels, makes it an interesting host plant for further studies of host-pathogen interactions.

Genome analysis of an orange stem pitting citrus tristeza virus isolate reveals a novel recombinant genotype.

An orange stem pitting citrus tristeza virus (CTV) isolate CTV-B165 was found to be symptomatically similar to other known CTV-VT isolates however molecular methods failed to classify it as an identifiable CTV genotype. The sequence variation of the Indian CTV-B165 isolate was compared to the three well known CTV genotypes, T36, T30, and VT. The genome of the predominant component of CTV-B165 was 19,247 nt in length with 12 open reading frames (ORFs) and was structurally identical to the other CTV isolates. All the completely sequenced CTV isolates except the VT isolate were 2-55 nt longer than the CTV-B165. In comparison to the other fully sequenced T36, T30 and VT genotypic isolates, CTV-B165 had nucleotide identity of 72-86% in ORF1 and 92-99% in ORFs 2-11. Sequence data of independent overlapping clones from the CTV-B165 genome showed highly divergent sequences of the overlapping region of 5'-UTR and ORF1a, the inter-domain region of ORF1a and the partial regions of ORF2. Phylogenetic analysis of five domains of ORF1a, ORF1b, and ORF2 revealed that CTV-B165 isolate distinctly segregates from the existing three genotypes in the dendrograms and was supported by high bootstrap values and robust tree topology. The PHYLPRO graphical analysis showed multiple recombination signals with significant correlation values. The precise detection of recombination sites for different genomic regions in CTV sequences was supported by several recombination-detecting methods. Collectively, the phylogenetic and recombination analyses suggest that the observed CTV-B165 genotype variation is an outcome of inter-genotype recombination. To determine the presence of the CTV-B165 genotype a pair of genome specific primers was designed and standardized for reliable detection of the novel CTV genotype by reverse-transcription polymerase chain reaction.

Murraya paniculata and Related Species as Potential Hosts and Inoculum Reservoirs of 'Candidatus Liberibacter asiaticus', Causal Agent of Huanglongbing.

Huanglongbing (HLB), considered to be the most serious insect-vectored bacterial disease of citrus, is transmitted in nature by the Asian citrus psyllid Diaphorina citri and the African citrus psyllid Trioza erytreae. D. citri was discovered in southern Florida in 1998 and the HLB disease in 2005. Both have become established throughout citrus-producing areas of Florida. Murraya species are widely grown in southern Florida as ornamental hedges and are readily colonized by D. citri vectors. Colonies of D. citri, isolates of 'Candidatus Liberibacter asiaticus' from Taiwan and Florida, and the Murraya species were established in the BSL-3 biosecurity facility at Fort Detrick. In controlled inoculation experiments, D. citri transmitted 'Ca. L. asiaticus' into M. paniculata (34/36 plants) and M. exotica (22/23 plants), but not into Bergera (Murraya) koenigii. Disease symptoms rarely developed in Murraya plants; however, positive infections were determined by conventional and real-time polymerase chain reaction (PCR). Back-inoculations of 'Ca. L. asiaticus' from M. paniculata to Madam Vinous sweet orange resulted in disease development in 25% of the inoculated plants. Considerable variability was observed in infection rates, titer, and persistence of 'Ca. L. asiaticus' in infected Murraya.

Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by reverse transcription-quantitative real-time PCR.

A multiplex real-time PCR assay was developed to detect and quantify the Citrus tristeza virus (CTV) genotypic mixture present in infected plants. CTV isolate FS627, a complex Florida isolate containing T36, T30 and VT genotypes and its aphid transmitted subisolates was used. The relative quantitative assay was carried out using specific primers and probes developed from the genotypes of three CTV virus isolates and included the coat protein region of isolate T36 and the 5' end, ORF 1a and ORF 2 region of isolates T36, T30 and VT. Among the three genotypes present in the aphid transmitted subisolates, the T30 genotype showed higher overall relative quantitation in all specific regions compared to other isolates. The profiles of the some aphid transmitted subisolates were different from the parent source from which they transmitted. The 2(-DeltaDeltaCt) method (the amount of target, normalized to an endogenous control and relative to a calibrator) was used to analyze the relative titers of the three reference genotypes in the aphid transmitted plants infected with FS627. This protocol enabled assessments of CTV genetic diversity in the aphid transmitted subisolates. This simple quantitative assay was sensitive, efficient, and took less time than other existing methods. This relative quantitative assay will be a reliable tool for diagnosis, detection and genetic diversity studies on CTV.

Population dynamics of a Florida Citrus tristeza virus isolate and aphid-transmitted subisolates: identification of three genotypic groups and recombinants after aphid transmission.

Tristeza is an important citrus disease affecting the viability and productivity of citrus worldwide. The causal agent, Citrus tristeza virus (CTV), usually occurs as a mixture of genotypes in nature, with one of the genotypes often dominating the population. CTV has a monopartite, positive-sense RNA genome of approximately 19.3 kb and exhibits over 30% diversity in the 5' half and less than 10% in the 3' half among different genotypes. A Florida CTV isolate, FS627, was selected for this study. Isolate FS627 was analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using primers to three regions: 788-bp region in the 5' (697 to 1,484 nucleotides), open reading frame (ORF)1a, 696 or 718 bp from the overlapping region of the RdRp (ORF1b) and p33 (ORF2) gene, and a 672-bp major coat protein gene (ORF7) in the 3' end of the CTV genome. The presence of T36, T30, and VT genotypes in isolate FS627 was confirmed utilizing the genotype specific overlapping region of RdRp primer pairs for RT-PCR amplification followed by cloning and sequence analysis. Analysis of single-strand conformational polymorphisms and sequences of RT-PCR-amplified products of the above regions were used to determine the presence of genotypes in both the parent and aphid-transmitted (AT) subisolates. Although the parent isolate had T36 as the major genotype, T30 was the major genotype in most of the AT subisolates. Some intermediate genotypes were detected that differed from the parental or AT subisolates. These intermediate genotypes were considered to be recombinants of the T30 and T36 genotypes and also were observed in the second level of AT subisolates generated from the of first-level AT subisolates of CTV-FS627. This work provides advance information on the population dynamics in CTV mixtures and the generation of virus recombinants after aphid transmission.