Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico.

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit-specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit-specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

Enhanced Serologically Based Detection of Liberibacters Associated with Citrus Huanglongbing.

'Candidatus Liberibacter spp.' are associated with the most devastating disease of citrus Huanglongbing (HLB). In previous work, we established an in situ tissue print method for the detection of 'Ca. L. asiaticus' (CLas) in sweet orange. We optimized the protocol by preincubation of the anti-Omp antibody with 5% (w/v) extract of healthy rough lemon. This simple process eliminated cross reactions between citrus and the antibody. The optimized protocol enhanced the application of the polyclonal antibody, and we demonstrate detection of CLas from all parts of the world, including isolates from Japan, Thailand, Vietnam, Pakistan, Saudi Arabia, Brazil, the United States, and a selection of strains from China representative of the diversity extant there. The assay also was used to detect four isolates of 'Ca. L. africanus' (CLaf) representative of the diversity present in South Africa. The corresponding outer membrane genes of representative isolates were cloned and sequenced. The coding sequences were highly conserved, and isolates of CLas and CLaf shared 53.8 to 55.9% identity between species at the amino acid level. The optimized protocol is efficient for recognition of both CLas and CLaf in phloem cells of different citrus tissues regardless of geographic origin of the HLB samples. The method is simple and scales well to match the urgent need for accurate, sensitive, and high-throughput screening of HLB bacteria, and may play an important role especially for plant inspection and quarantine programs.

Transcriptomic analyses reveal physiological changes in sweet orange roots affected by citrus blight.

BACKGROUND: Citrus blight is a very important progressive decline disease of commercial citrus. The etiology is unknown, although the disease can be transmitted by root grafts, suggesting a viral etiology. Diagnosis is made by demonstrating physical blockage of xylem cells that prevents the movement of water. This test was used to identify symptomatic trees from four commercial groves in Florida. Total RNA extracts of phloem-enriched scaffold root tissues were prepared from seven trees that failed to take up water and from one healthy tree. These RNA extracts were used for transcriptomic analyses using paired end RNA-Seq from an Illumina 2500 system. The expression of transcripts annotated as polyprotein of citrus endogenous pararetrovirus were estimated by both RT-qPCR and RNA-Seq. RESULTS: Transcripts from seven RNA-Seq libraries from trees affected by citrus blight were compared to a control tree. 129-148 million RNA fragments (two paired-end reads/fragment) were generated per library and were mapped to the sweet orange reference genome. In response to citrus blight stress, genes encoding aquaporins, proteins with water channel activity and several cellulose synthase genes were down-regulated, whereas genes involved in lignin and glucosinolate biosynthesis were up-regulated. Transcripts encoding proteins in pathways of carbohydrate metabolism, nucleotide synthesis, signaling, hormone metabolism, secondary metabolism, transport, and biotic stress pathways were overwhelmingly down regulated in all libraries. CONCLUSION: Reduced water intake and xylem plugging were observed in the trees tested and the changes in their transcriptome were analyzed. Plants adapted to reduced water flow by regulating primary and secondary metabolism, nuclear transport and hormone associated pathways. The patterns of energy generation, transcription, translation and protein degradation were consistent with irreversible decline. The down regulation of cellulose synthase transcripts and up regulation of transcripts related to lignin production likely lead to an imbalance in the pathways leading to wood formation, and may lead to the blockage of the xylem vessels seen as the cardinal symptom of citrus blight. Transcripts of a pararetrovirus were elevated in the transcriptome of roots used in this study.

Transcriptional analysis of sweet orange trees co-infected with 'Candidatus Liberibacter asiaticus' and mild or severe strains of Citrus tristeza virus.

BACKGROUND: Citrus worldwide is threatened by huanglongbing (HLB) and tristeza diseases caused by 'Candidatus Liberibacter asiaticus' (CaLas) and Citrus tristeza virus (CTV). Although the pathogens are members of the α-proteobacteria and Closteroviridae, respectively, both are restricted to phloem cells in infected citrus and are transmitted by insect vectors. The response of sweet orange to single infection by either of these two pathogens has been characterized previously by global gene expression analysis. But because of the ubiquity of these pathogens where the diseases occur, co-infection by both pathogens is very common and could lead to increased disease severity based on synergism. We therefore co-inoculated sweet orange trees with CaLas and either a mild or a severe strain of CTV, and measured changes of gene expression in host plants. RESULTS: In plants infected with CaLas-B232, the overall alteration in gene expression was much greater in plants co-inoculated with the severe strain of CTV, B6, than when co-infected with the mild strain of CTV, B2. Plants co-infected with CaLas-B232 and either strain of CTV died but trees co-infected with CTV-B2 survived much longer than those co-infected with CTV-B6. Many important pathways were perturbed by both CTV-B2/CaLas-B232 and/or CTV-B6/CaLas-B232, but always more severely by CTV-B6/CaLas-B232. Genes related to cell wall modification and metal transport responded differently to infection by the pathogens in combination than by the same pathogens singly. The expressions of genes encoding phloem proteins and sucrose loading proteins were also differentially altered in response to CTV-B2 or CTV-B6 in combination with CaLas-B232, leading to different phloem environments in plants co-infected by CaLas and mild or severe CTV. CONCLUSIONS: Many host genes were expressed differently in response to dual infection as compared to single infections with the same pathogens. Interactions of the pathogens within the host may lead to a better or worse result for the host plant. CTV-B6 may exert a synergistic effect with CaLas-B232 in weakening the plant; on the other hand, the responses activated by the mild strain CTV-B2 may provide some beneficial effects against CaLas-B232 by increasing the defense response of the host.

Characterization and purification of proteins suitable for the production of antibodies against 'Ca. Liberibacter asiaticus'.

The citrus disease huanglongbing (HLB), which is caused by 'Candidatus Liberibacter asiaticus' (CaLas), is one of the most devastating pathogens of citrus, and with no effective method of control, poses a serious threat to citrus production throughout the world. In a previous study we described the production of single chain antibodies against several CaLas proteins that provide the basis for efficient and accurate detection of CaLas in citrus tissues. The isolation of a sufficient amount of purified antigen is a key step in the production of functional antibodies. The current report details purification procedures for six protein antigens used to select recombinant and produce polyclonal antibodies. These proteins include a flagellar biosynthesis protein (FlhA), a dinucleoside polyphosphate hydrolase (InvA), a portion of the major outer membrane protein (OmpA), a component of type IV pilus (CapB), the polysialic acid capsule expression protein (KpsA) and the outer membrane efflux protein (TolC). Results of purification under completely native or denatured conditions were not satisfactory. Therefore different hybrid purification conditions were optimized for each of the different proteins. The results of bioinformatic analysis also indicated that the six proteins contained a great diversity of potential antigenic epitopes, which varied in number, and that the antigenic clusters were not uniformly distributed throughout the proteins. The purified proteins are useful for the development of highly specific antibodies capable of differentiating specific strains of Liberibacter.

Transcriptome analysis of sweet orange trees infected with 'Candidatus Liberibacter asiaticus' and two strains of Citrus Tristeza Virus.

BACKGROUND: Huanglongbing (HLB) and tristeza, are diseases of citrus caused by a member of the α-proteobacteria, 'Candidatus Liberibacter asiaticus' (CaLas), and Citrus tristeza virus (CTV) respectively. HLB is a devastating disease, but CTV strains vary from very severe to very mild. Both CaLas and CTV are phloem-restricted. The CaLas-B232 strain and CTV-B6 cause a wide range of severe and similar symptoms. The mild strain CTV-B2 doesn't induce significant symptoms or damage to plants. RESULTS: Transcriptome profiles obtained through RNA-seq revealed 611, 404 and 285 differentially expressed transcripts (DETs) after infection with CaLas-B232, CTV-B6 and CTV-B2. These DETs were components of a wide range of pathways involved in circadian rhythm, cell wall modification and cell organization, as well as transcription factors, transport, hormone response and secondary metabolism, signaling and stress response. The number of transcripts that responded to both CTV-B6 and CaLas-B232 was much larger than the number of transcripts that responded to both strains of CTV or to both CTV-B2 and CaLas-B232. A total of 38 genes were assayed by RT-qPCR and the correlation coefficients between Gfold and RT-qPCR were 0.82, 0.69, 0.81 for sweet orange plants infected with CTV-B2, CTV-B6 and CaLas-B232, respectively. CONCLUSIONS: The number and composition of DETs reflected the complexity of symptoms caused by the pathogens in established infections, although the leaf tissues sampled were asymptomatic. There were greater similarities between the sweet orange in response to CTV-B6 and CaLas-B232 than between the two CTV strains, reflecting the similar physiological changes caused by both CTV-B6 and CaLas-B232. The circadian rhythm system of plants was perturbed by all three pathogens, especially by CTV-B6, and the ion balance was also disrupted by all three pathogens, especially by CaLas-B232. Defense responses related to cell wall modification, transcriptional regulation, hormones, secondary metabolites, kinases and stress were activated by all three pathogens but with different patterns. The transcriptome profiles of Citrus sinensis identified host genes whose expression is affected by the presence of a pathogen in the phloem without producing symptoms (CTV-B2), and host genes whose expression leads to induction of symptoms in the plant (CTV-B6, CaLas-B232).

History and Diversity of Citrus leprosis virus Recorded in Herbarium Specimens.

Leprosis refers to two diseases of citrus that present similar necrotic local lesions, often surrounded by chlorotic haloes on citrus. Two distinct viruses are associated with this disease, one that produces particles primarily in the nucleus of infected plant cells (Citrus leprosis virus nuclear type [CiLV-N]; Dichorhavirus) and another type that produces particles in the cytoplasm of infected plant cells (Citrus leprosis virus cytoplasmic type [CiLV-C]; Cilevirus). Both forms are transmitted by Brevipalpid mites and have bipartite, single-stranded, RNA genomes. CiLV-C and CiLV-N are present in South and Central America and as far north as parts of Mexico. Although leprosis disease was originally described from Florida, it disappeared from there in the 1960s. The United States Department of Agriculture-Agricultural Research Service maintains preserved citrus specimens identified at inspection stations 50 or more years ago with symptoms of citrus leprosis. We isolated RNA from these samples and performed degradome sequencing. We obtained nearly full-length genome sequences of both a typical CiLV-C isolate intercepted from Argentina in 1967 and a distinct CiLV-N isolate obtained in Florida in 1948. The latter is a novel form of CiLV-N, not known to exist anywhere in the world today. We have also documented the previously unreported presence of CiLV-N in Mexico in the mid-20th century.

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.

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease.

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

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.

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.

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.

Genetic diversity of citrus bacterial canker pathogens preserved in herbarium specimens.

Citrus bacterial canker (CBC) caused by Xanthomonas axonopodis pv. citri (Xac) was first documented in India and Java in the mid 19th century. Since that time, the known distribution of the disease has steadily increased. Concurrent with the dispersion of the pathogen, the diversity of described strains continues to increase, with novel strains appearing in Saudi Arabia, Iran, and Florida in the last decade. Herbarium specimens of infected plants provide an historical record documenting both the geographic distribution and genetic diversity of the pathogen in the past. However, no method was available to assess the genetic diversity within these herbarium samples. We have developed a method, insertion event scanning (IES), and applied the method to characterize the diversity present within CBC populations documented as herbarium specimens over the past century. IES is based on the specific amplification of junction fragments that define insertion events. The potential for IES in current forensic applications is demonstrated by finding an exact match of pathogen genotypes preserved in herbarium specimens from Japan and Florida, demonstrating the source of the original outbreak of citrus canker in Florida in 1911. IES is a very sensitive technique for differentiating bacterial strains and can be applied to any of the several hundred bacteria for which full genomic sequence data are available.

Lack of Evidence for Transmission of 'Candidatus Liberibacter asiaticus' Through Citrus Seed Taken from Affected Fruit.

Citrus huanglongbing, putatively caused by the associated bacterium 'Candidatus Liberibacter asiaticus', is the greatest threat to the world citrus industry today. The bacterium is spread locally and regionally by the citrus psyllid Diaphorina citri, and also can be disseminated by propagation of contaminated scion budwood that is grafted to the appropriate rootstock. The planting of 'Ca. Liberibacter asiaticus'-free trees is a component of a comprehensive strategy to manage huanglongbing. In contrast to the scion budwood, the rootstocks used to produce these trees are grown from seed. This research was undertaken to provide evidence as to whether or not 'Ca. L. asiaticus' can be transmitted through seed. Two groups of 360 or more seedlings each of various citrus species were grown from seed removed from fruit on trees that were symptomatic for huanglongbing and confirmed to be infected with 'Ca. L. asiaticus' by polymerase chain reaction (PCR) tests. These seedlings were tested multiple times over periods of up to 3 years. No symptoms typical of huanglongbing, such as blotchy leaf mottle, chlorotic shoots, or dieback of branches, were observed in these seedlings, and none of these 723 seedlings tested positive for the presence of 'Ca. L. asiaticus' even after repeated testing by sensitive quantitative PCR assays. Some sour orange seedlings did have quite pronounced and atypical growth, including stunting and mild to severe leaf malformation. These atypical growth habits were limited to seedlings that arose from zygotic embryos as determined by expressed-sequence tag simple-sequence repeat analyses. Thus, no evidence of transmission of 'Ca. L. asiaticus' via seed was obtained, and an earlier report of transmission of the pathogen through seed was not confirmed.

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.

Construction of infectious clones of double-stranded DNA viruses of plants using citrus yellow mosaic virus as an example.

Double-stranded DNA (dsDNA) viruses of plants are believed to be plant pararetroviruses. Their genome is replicated by reverse transcription of a larger than unit-length terminally redundant RNA transcript of the viral genomic DNA using the virus-encoded replicase. In order to produce a cloned, infectious viral genome, the clone must be constructed in a binary vector and be longer than the full, unit-length viral genome. The clone can then be transferred by Agrobacterium-assisted inoculation into a suitable host plant to induce virus infection.

Isolation and molecular characterization of Xylella fastidiosa from coffee plants in Costa Rica.

Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica's Central Valley. The symptoms are referred to by coffee producers in Costa Rica as "crespera" disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 microm in width and 1.5 to 3.0 microm in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-l-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.

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.