Efficacy of Bioindexing for Graft-Transmissible Citrus Pathogens in Mixed Infections.

Biological indexing for graft-transmissible pathogens of citrus in the presence of additional pathogens was investigated. The probability for symptom expression, the efficacy of the bio-indexing tests, and the number of citrus indicators required for pathogen detection were statistically evaluated. Multiple infections did not preclude symptom expression or reduce the diagnostic efficacy of the primary indexing hosts for Citrus tristeza virus (CTV), Citrus psorosis virus (CPsV), and Citrus tatter leaf virus (Apple stem grooving virus). Symptoms of Citrus vein enation virus (CVEV) and the diagnostic efficacy of Mexican lime were suppressed by the T30 group CTV isolates, but not by other CTV isolates tested. CPsV suppressed symptom expression and diagnostic efficiency of Dweet tangor and sweet orange for concave gum. The application of alternate bioassay hosts for indexing was also investigated. Dweet tangor, sweet orange, and Citrus excelsa are not typically used for bioindexing of CVEV, however, Dweet tangor and C. excelsa detected CVEV in single infections, whereas in sweet orange, CVEV was detected only when CPsV, concave gum, or citrus viroids were present. CTV was readily detected using the alternative indicator C. excelsa, whereas only shock reacting CPsV isolates were effectively indexed by Mexican lime.

Finger Imprint of Poncirus trifoliata: A Specific Interaction of a Viroid, a Host, and Irrigation.

The unusual symptom, "finger imprint", described exclusively on Poncirus trifoliata, has been reported in only a single field trial investigating the effects of citrus viroids on crop performance. With this, the question has persisted whether the observed growth abnormality was a disease symptom induced by Citrus viroid IIIb (CVd-IIIb) or a consequence of mechanical damage caused by the handling of young trees during propagation or cultural practices in the field. The recurrence of finger imprint symptoms on trees after 5 years in the field in which no abnormal growth features were previously noted now supports the proposition of a viroid-induced disease. The symptom expression results from an unusual etiology of a complex relationship of the specific viroid CVd-IIIb on the specific rootstock P. trifoliata only when supplemental water is applied by sprinkler irrigation.

Complete sequence of the citrus tristeza virus RNA genome.

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of citrus tristeza virus subgenomic RNAs in infected tissue.

Citrus tristeza virus (CTV) specific RNAs extracted from infected citrus tissue were analyzed by Northern blot hybridization. RNAs were characterized by size and identified using cDNA probes specific to nine open reading frames (ORFs) identified by the analysis of sequence obtained from cDNA clones of the T36 isolate of CTV. Sequence specific cDNA probes identified the genomic RNA as well as subgenomic RNAs representing the p33, p65, p61, p27, p25, p18, p13, p20, and p23 ORFs in extracts of total or double-stranded RNA (dsRNA) isolated from infected tissue. A probe derived from the 3' terminal ORF (p23) hybridized to each of these subgenomic RNAs, indicating that the RNAs are 3' coterminal. The relative amounts of the different subgenomic RNAs varied widely. The RNAs for the p20 and p23 ORFs were the most abundant and surpassed the amount of the p25 or capsid protein specific subgenomic RNA. The number and sizes of the CTV subgenomic RNAs were the same in total RNA and dsRNA preparations. Propagation of T36 in seven different citrus hosts did not alter the pattern of subgenomic RNAs.

Screening of the closterovirus genome by degenerate primer-mediated polymerase chain reaction.

The genome of beet yellows virus (BYV), the type representative of the closterovirus group, encodes a homologue of the cellular heat-shock protein (HSP) 70 family. A pair of degenerate primers targeted to motifs A and E, which are highly conserved in HSP70s, was synthesized. Genomes of several definite and possible members of the closterovirus group were screened for the presence of the HSP70 gene with PCR using these degenerate primers. BYV, citrus tristeza virus (CTV), beet yellow stunt virus (BYSV) and carnation necrotic fleck virus templates produced 1 kb amplification products, which were shown by sequencing to represent fragments of the respective HSP70 genes. Further screening was performed with an additional degenerate primer targeted to the motif IV of the putative viral polymerase. This degenerate primer and specific primers complementary to the 5' region of the HSP70 genes of the respective viruses were used to estimate the distance between polymerase motif IV and the start point of the HSP70 gene for BYV (approximately 1.1 kb), CTV and BYSV (around 2.0 kb) by PCR. The amplified genome regions of CTV (3026 nucleotides) and BYSV (2837 nucleotides) were cloned and sequenced. CTV and BYSV were found to encode the gene for an additional 30K (BYSV) or 33K (CTV) protein between the polymerase and the small hydrophobic protein genes, which was absent in BYV. These two 30K proteins displayed very weak similarity to each other, unlike the highly conserved polymerases, hydrophobic proteins and HSP70s of BYV, CTV and BYSV. Degenerate primer-mediated PCR proved to be an efficient tool for rapid screening and subsequent cloning of the viral genomes.

Production, characterization, and applications of monoclonal antibodies reactive with soybean nodule xanthine dehydrogenase.

Seven monoclonal antibodies were produced against soybean nodule xanthine dehydrogenase, an enzyme involved in ureide synthesis. Specificity of the seven monoclonal antibodies for xanthine dehydrogenase was demonstrated by immunopurifying the enzyme to homogeneity from a crude nodule extract using antibodies immobilized to Sepharose 4B beads. Each monoclonal antibody was covalently bound to Sepharose 4B beads for the preparation of immunoaffinity columns for each antibody. All seven antibodies were found to be of the IgG1,K subclass. A competitive, indirect enzyme-linked immunosorbent assay demonstrated that two of the seven antibodies shared a common epitope while the remaining five antibodies defined unique determinants on the protein. Rapid, large scale purification of active xanthine dehydrogenase to homogeneity was performed by immunoaffinity chromatography. The presence of xanthine dehydrogenase activity and protein in every organ of the soybean plant was determined. Crude extracts of nodules, roots, stems, and leaves cross-reacted with all seven monoclonal antibodies in an indirect enzyme-linked immunosorbent assay. A positive correlation was observed between the degree of cross-reactivity of a given organ and the level of enzyme activity in that organ. These data demonstrate that xanthine dehydrogenase is not nodule specific. Antigenic variability of xanthine dehydrogenase present in crude extracts from nodules of soybean, wild soybean, cowpea, lima bean, pea, and lupin were detected in the indirect enzyme-linked immunosorbent assay which corresponded to six binding patterns for xanthine dehydrogenase from these plant species. These results correspond well with the epitope determination data which showed that the seven antibodies bind to six different binding determinants on the enzyme.