'Candidatus Liberibacter solanacearum' Is Tightly Associated with Carrot Yellows Symptoms in Israel and Transmitted by the Prevalent Psyllid Vector Bactericera trigonica.

Carrot yellows disease has been associated for many years with the Gram-positive, insect-vectored bacteria, 'Candidatus Phytoplasma' and Spiroplasma citri. However, reports in the last decade also link carrot yellows symptoms with a different, Gram-negative, insect-vectored bacterium, 'Ca. Liberibacter solanacearum'. Our study shows that to date 'Ca. L. solanacearum' is tightly associated with carrot yellows symptoms across Israel. The genetic variant found in Israel is most similar to haplotype D, found around the Mediterranean Basin. We further show that the psyllid vector of 'Ca. L. solanacearum', Bactericera trigonica, is highly abundant in Israel and is an efficient vector for this pathogen. A survey conducted comparing conventional and organic carrot fields showed a marked reduction in psyllid numbers and disease incidence in the field practicing chemical control. Fluorescent in situ hybridization and scanning electron microscopy analyses further support the association of 'Ca. L. solanacearum' with disease symptoms and show that the pathogen is located in phloem sieve elements. Seed transmission experiments revealed that while approximately 30% of the tested carrot seed lots are positive for 'Ca. L. solanacearum', disease transmission was not observed. Possible scenarios that may have led to the change in association of the disease etiological agent with carrot yellows are discussed. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Characterization of isolates of Citrus tristeza virus by sequential analyses of enzyme immunoassays and capillary electrophoresis-single-strand conformation polymorphisms.

Citrus tristeza virus (CTV) is the causal agent of tristeza disease, which is one of the most devastating diseases of citrus crops worldwide. This paper describes a method for the rapid detection and genotyping of naturally spreading CTV isolates. This method uses ELISA or dot-blot immunological tests to detect trees infected with CTV. The reaction wells or membrane spots for which there is a positive reaction are sequentially treated by (i) washing and elution of viral RNA from the trapped samples, (ii) one-step synthesis of cDNA and PCR and (iii) automated fluorescence-based capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis of amplification products. Comparative CE-SSCP results are presented for CTV RNA extracted directly from infected leaves and ELISA plates or from membranes. In the analyses of all of these RNA samples, the p18, p27 and p23 CTV genes were targeted for amplification. Specific profiles of forward and reverse strands were obtained from a group of eight CTV isolates collected in Sicily, each with distinct biological characteristics, which were analyzed using the conventional two-step procedure (immunological detection followed by CE-SSCP molecular characterization after RNA isolation) or in a continuous process of ELISA/CE-SSCP or dot-blot/CE-SSCP starting from infected plant material. The combined method is simple, highly sensitive and reproducible, thus allowing the processing of numerous field samples for a variety of epidemiological needs. The sequential processing of an ELISA or dot-blot/ELISA followed by CE-SSCP is expected to allow the rapid detection of recent CTV infections along with the simultaneous characterization of the genetic diversity and structure of the population of newly invading CTV.

The complete nucleotide sequence of Passiflora latent virus and its phylogenetic relationship to other carlaviruses.

A virus identified as Passiflora latent virus (PLV) was isolated from passion fruit plants. Particle morphology, host range and serological properties suggested that this virus belongs to the genus Carlavirus. The complete genomic sequence of PLV was determined by sequencing overlapping cDNA fragments. The genome consisted of 8386 nt, excluding the poly (A) tail and contained six open reading frames, typical of carlaviruses. The overall similarities of the predicted amino acid sequence of PLV to those of other carlaviruses ranged from 25 to 73%. Phylogenetic analysis indicated that PLV was closely related to lily symptomless virus and blueberry scorch virus. This is the first report of the complete nucleotide sequence and genome structure of PLV.

The new plant virus family Flexiviridae and assessment of molecular criteria for species demarcation.

The new plant virus family Flexiviridae is described. The family is named because its members have flexuous virions and it includes the existing genera Allexivirus, Capillovirus, Carlavirus, Foveavirus, Potexvirus, Trichovirus and Vitivirus, plus the new genus Mandarivirus together with some related viruses not assigned to any genus. The family is justified from phylogenetic analyses of the polymerase and coat protein (CP) sequences. To help to define suitable molecular criteria for demarcation of species, a complete set of pairwise comparisons was made using the nucleotide (nt) and amino acid (aa) sequences of each fully-sequenced gene from every available accession in the family. Based on the distributions and on inspection of the data, it was concluded that, as a general rule, distinct species have less than ca. 72% identical nt or 80% identical aa between their entire CP or replication protein genes.

5'-coterminal subgenomic RNAs in citrus tristeza virus-infected cells.

Three unusual 5' coterminal positive-stranded subgenomic (sg) RNAs, two of about 0.8 kb and one of 10 kb (designated LMT1, LMT2, and LaMT, respectively), from Citrus spp. plants and Nicotiana benthamiana protoplasts infected with Citrus tristeza virus (CTV) were characterized. The 5' termini of the LMT RNAs were mapped by runoff reverse transcription and found to correspond with the 5' terminus of the genomic RNA. The LMT 5'-coterminal sgRNAs consisted of two modal lengths of 744--746 and 842--854 nts. The 3' of the LaMT RNAs terminated near the junction of ORF 1b and ORF 2 (p33). None of the 5' sgRNAs had detectable amounts of corresponding negative-sense RNAs, as occurs with the genomic and 3' coterminal subgenomic RNAs of CTV. The abundance of the short and long 5' sgRNAs differed considerably in infected cells. The LMT RNAs were considerably more abundant than the genomic RNAs, while the larger LaMT RNA accumulated to much lower levels. The kinetics of accumulation of LMT1 and LMT2 in synchronously infected protoplasts differed. The larger RNA, LMT1, accumulated earlier with a strong hybridization signal at 2 days postinfection, a time when only traces of genomic and 3' sgRNAs were detected. The lack of corresponding RNAs, that could be 3' cleavage products corresponding to the 5' coterminal sgRNAs and the lack of complementary negative strands, suggest that these sgRNAs were produced by termination during the synthesis of the genomic positive strands.

Amplification of Citrus tristeza virus from a cDNA clone and infection of citrus trees.

Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the approximately 20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts ( approximately 0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population.

The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies.

Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins. The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23. The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another. The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells. The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus. Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus. Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells. Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP. The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape. Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells.

Both point mutation and RNA recombination contribute to the sequence diversity of citrus viroid III.

Field-grown citrus trees often harbor complex mixtures of 4-5 different viroid species, and the presence of citrus viroid III (CVd-III) has been shown to reduce the rate of tree growth without inducing disease. To more fully define the structure of its quasi-species, we have examined nine citrus viroid complexes for the presence of previously undescribed sequence variants of CVd-III. Analysis of 86 full-length cDNAs generated from these nine viroid complexes by RT-PCR revealed the presence of 20 new CVd-III variants. Chain lengths ranged from 293-297 nucleotides, and sequence changes were confined largely to the lower portions of the central conserved region and variable domain. The previously described variants CVd-IIIa (297 nt) and CVd-IIIb (294 nt) were clearly predominant, but phylogenetic analysis indicated that certain isolates may contain representatives of two additional fitness peaks. At least one group of CVd-III variants appears to have arisen as a result of RNA recombination. Populations recovered from diseased/declining trees were the most diverse, but even dwarfing isolates originating from old line Shamouti trees showed considerable variability.

D-RNA molecules associated with subisolates of the VT strain of citrus tristeza virus which induce different seedling-yellows reactions.

Citrus tristeza virus (CTV) strains were previously catalogued as seedling-yellows (SY) and non-SY (nSY) types, according to their yellowing and stunting effects on indicator seedlings. Among subisolates of the VT strain, which were selected from chronically infected Alemow plants, there was a correlation between the presence of 2.4-, 2.7- and 4.5-kb D-RNAs, and SY and nSY reactions, respectively. Similarly, plants infected with Mor-T subisolates, which cause SY, contained D-RNAs of 2.6 to 2.8 kb, while nSY subisolates from recovered sour orange tissue contained a major D-RNA of 5.1 kb. Plants harboring the 2.7-kb D-RNA were protected against challenge inoculation with a subisolate harboring the 4.5-kb D-RNA. This study suggests that the nSY reaction results either from the absence of SY gene(s) in the genomes of certain CTV strains or through the suppression of the effects of SY gene(s) by D-RNAs with 5' parts larger than 4000nt.

An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication.

Citrus tristeza virus (CTV) populations in citrus trees are unusually complex mixtures of viral genotypes and defective RNAs developed during the long-term vegetative propagation of the virus and by additional mixing by aphid transmission. The viral replication process allows the maintenance of minor amounts of disparate genotypes and defective RNAs in these populations. CTV is a member of the Closteroviridae possessing a positive-stranded RNA genome of approximately 20 kilobases that expresses the replicase-associated genes as an approximately 400-kDa polyprotein and the remaining 10 3' genes through subgenomic mRNAs. A full-length cDNA clone of CTV was generated from which RNA transcripts capable of replication in protoplasts were derived. The large size of cDNA hampered its use as a genetic system. Deletion of 10 3' genes resulted in an efficient RNA replicon that was easy to manipulate. To investigate the origin and maintenance of the genotypes in CTV populations, we tested the CTV replicase for its acceptance of divergent sequences by creating chimeric replicons with heterologous termini and examining their ability to replicate. Exchange of the similar 3' termini resulted in efficient replication whereas substitution of the divergent (up to 58% difference in sequence) 5' termini resulted in reduced but significant replication, generally in proportion to the extent of sequence divergence.

Involvement of a subgenomic mRNA in the generation of a variable population of defective citrus tristeza virus molecules.

The fusion sites between the termini of naturally occurring defective RNAs (D-RNAs) from three citrus tristeza virus (CTV) isolates were sequenced. Seven of eight clones showed a common 3' terminus of 940 nucleotides (nt) fused to 5' termini with different sizes. An extra cytosine nucleotide was found at the junction site of the majority of the common 3' D-RNAs. Molecular analysis of the plus and minus strands of the 0.9-kbp double-stranded RNA, corresponding to the CTV open reading frame 11 subgenomic RNA (sgRNA), showed that they were identical in length and sequence to the common 3' sequence of the D-RNAs. These results imply that viral sgRNA messengers also function as building components for genomic rearrangement and exchange of complete viral genes.

Booster immunization with a partially purified citrus tristeza virus (CTV) preparation after priming with recombinant CTV coat protein enhances the binding capacity of capture antibodies by ELISA.

Groups of rabbits and young lambs were immunized subcutaneously and intramuscularly with a recombinant citrus tristeza virus (CTV) coat protein (rCTV-CP) antigen. Three weeks after primary immunization the animals were divided into two groups that were boosted either with rCTV-CP or with a partially purified preparation of CTV particles (ppCTV). Twelve and 15 days after the last injection, the animals were bled and the binding capacity of the antisera for CTV detection was examined for capture antibodies by the indirect ELISA. Considerably higher ELISA titers were obtained from animals that were boosted with ppCTV than with rCP. Boosting with partially purified native antigens after priming with recombinant antigens is expected to extend the applicability of the antisera for detecting other structural and non-structural viral antigens by trapping ELISA.

A cDNA clone from a defective RNA of citrus tristeza virus is infective in the presence of the helper virus.

A naturally occurring defective RNA of 2379 nt (D2.3) from the VT strain of citrus tristeza closterovirus (CTV) was cloned and sequenced. The D2.3 RNA is a fusion of two regions of 1521 and 858 nt from the 5' and 3' ends of the CTV genome, respectively. A cDNA clone of D2.3 RNA was tagged by the insertion of a 0.47 kb chimeric DNA fragment and the recombinant cDNA was inserted downstream of the cauliflower mosaic virus 35S promoter. The resulting construct was bombarded into CTV-infected tissue, which was then grafted onto virus-free plants. The presence of recombinant RNA in systemically infected leaves was demonstrated by RT-PCR. Sequencing the RT-PCR products synthesized from double-stranded RNA confirmed the presence of the chimeric segment used for tagging. This is the first report of an infectious cDNA molecule derived from CTV D-RNA.

Unusual sequence relationships between two isolates of citrus tristeza virus.

The complete, 19,226 nt sequence of the RNA genome from VT, a seedling yellows strain of citrus tristeza virus (CTV), was determined and found to have a genome organization identical with that of the previously determined CTV-T36 isolate, except that ORF 1 of CTV-VT was 70 nt shorter due to two widely separated 18 nt deletions. Sequence comparison of CTV-VT and CTV-T36 revealed approximately 89% identity throughout the ten 3' ORFs, but only 60-70% identity throughout ORF 1. The 5' nontranslated regions were only 60% identical whereas the 3' nontranslated regions were 97% identical. The transition between regions of similarity and deviation was gradual, suggesting that the sequence similarities and differences compared to CTV-T36 were unlikely to have arisen from a recent recombination event between a close T36 relative and a distantly related CTV isolate. This is the first attempt to compare in detail the variation between the genomes of two strains of a member of the closterovirus group. The observed deviation between the large RNA genomes of the two CTV strains is greater than that among different viruses of most other groups, raising the question of how to define the taxonomy of these viruses.

Multiple species of defective RNAs in plants infected with citrus tristeza virus.

Alemow (Citrus macrophylla) and sweet orange (C. sinensis) plants infected, respectively, with several Israeli and Florida isolates of the citrus tristeza virus (CTV) were found to contain multiple species of RNA molecules with features similar to defective-interfering RNAs. Northern blot hybridizations of dsRNAs extracted from serial passages of the Israeli VT isolate (CTV-VT) and from different plants infected with a single source of inoculum showed considerable variation both in the presence and in the relative abundance of the defective RNA (D-RNA) bands. The D-RNA molecules were found to be encapsidated in the CTV particles. Sequence analysis of two VT D-RNA molecules of 2.7 and 4.5 kb revealed that they were composed of two regions corresponding to 1818 and 4036 nucleotides from the 5' and 938 and 442 nucleotides from the 3' termini of the CTV-VT genomic RNA, respectively. A short (ca. 0.8 kb) nonencapsidated single-stranded positive-sense RNA species was also found in infected plants. This ssRNA, which copurified with dsRNAs, was shown by hybridization to encompass the 5'-terminal part of the CTV genome and might have an extensive secondary structure.

Defective RNA molecules associated with citrus tristeza virus.

Preparations of single-stranded (ss) RNA extracted from particles of the Israeli VT strain of citrus tristeza virus (CTV-VT), and ss- and double-stranded (ds) RNA preparations extracted from infected Alemow (Citrus macrophylla) plants, contained a population of molecules with features that suggest that they are defective RNAs. The prototype of 2424 nt was cloned and sequenced and was found to be composed of two genomic regions corresponding to the 5' (1151 nt) and the 3' (1259 nt) termini of the genomic CTV-RNA, with two perfect direct repeats of eight nucleotides of unknown origin at the junction site. Northern hybridization analysis demonstrated that this 2.4-kb defective RNA is an abundant species among the other CTV-specific ss- and ds-RNAs in infected plants. The 2.4-kb RNA was found encapsidated by the CTV coat protein indicating that the CTV origin of assembly is located close to the 5' or 3' terminus. This is the first defective RNA to be reported for a member of the closterovirus group.

Populations of citrus tristeza virus contain smaller-than-full-length particles which encapsidate sub-genomic RNA molecules.

Plants infected by citrus tristeza virus (CTV) contain, in addition to the 2000 nm full-length thread-like virions, a heterogeneous population of smaller particles. The CTV particles and RNA extracts from purified CTV preparations were fractionated by sucrose gradient centrifugation and the RNA molecules from different fractions translated in a reticulocyte translation system. Fractions containing predominantly an RNA band of approximately 3.2 kb directed the synthesis of CTV coat protein (CP), which in SDS-PAGE had an estimated molecular mass of 28 kDa. Three additional polypeptides, with estimated sizes of 21 kDa, 23 kDa and 27 kDa, were translated from a range of RNA molecules smaller than 3.2 kb. Hybridization with cDNA to the CP gene (CTV-CPG) and with a 350 base clone complementary to the 3' and 5' termini of the genes for CTV p20 and p23.5, respectively, indicated that preparations of CTV particles contain, in addition to the genomic (20 kb) RNA, two sub-genomic RNA molecules of 3.2 kb and 2.4 kb and probably also two smaller molecules of 1.6 kb and 0.9 kb. Only the 3.2 kb RNA, its corresponding dsRNA molecule and populations of larger RNAs, including the 20 kb genomic RNA, hybridized with a CTV-CPG probe, thus conflicting with our previous assignment of the CTV-CPG to the 0.8 kbp dsRNA. Based on these results we propose that distinct populations of CTV particles encapsidate smaller RNAs which were formed as a nested set of subgenomic RNAs. Sequence analysis of 2540 nucleotides downstream to CTV-CPG of strain VT revealed four open reading frames (ORFs) potentially encoding in the 5' to 3' direction, 18 kDa (p18), 13 kDa (p13), 20 kDa (p20) and 23.5 kDa (p23.5) proteins. The CTV-VT ORFs showed variable but usually close levels of homology with the corresponding ORFs of CTV-T36 from Florida.

The persistence of engineered Agrobacterium tumefaciens in agroinfected plants.

Several plant species, including tomato (Lycopersicon esculentum), Gynura aurantiaca, avocado (Persea americana), and grapefruit (Citrus paradisi) grafted on Troyer citrange (Poncirus trifoliata x C. sinensis) were "agro-infected" with Agrobacterium tumefaciens strain LBA-4404, carrying a mini-Ti plasmid with a dimeric cDNA of citrus exocortis viroid (CEVd). Extracts prepared from tissues of the agroinfected plants 38-90 days after inoculation were plated on selective media and found to contain large amounts of the engineered bacteria. These observations suggest the need for more stringent quarantine measures when handling A. tumefaciens cells harboring constructs for "agroinoculation" with plant viruses or viroids.