D. M. Mathews

Molecular characterization of an isolate of citrus tristeza virus that causes severe symptoms in sweet orange.

The complete sequence (19,249 nucleotides) of the genome of citrus tristeza virus (CTV) isolate SY568 was determined. The genome organization is identical to that of the previously determined CTV-T36 and CTV-VT isolates. Sequence comparisons revealed that CTV-SY568, a severe stem-pitting isolate from California, has more than 87% overall sequence identity with CTV-VT, a seedling yellows isolate from Israel. Although SY568 has an overall sequence identity of 81% with CTV-T36, a quick decline isolate from Florida, the sequence identity in the 3′ half of the genome is over 90% while the sequence identity in the 5′ half of the genome is as low as 56%. Based on the sequence alignments of these three isolates, sequences in the 3′ half of the genome are generally well conserved, while the sequences in the 5′ half are relatively divergent. Sequence data of independent overlapping clones from the CTV-SY568 genome revealed two regions with highly divergent sequences. In open reading frame 1b (RNA dependent RNA polymerase), there were 118 nucleotide differences that lead to 16 amino acid changes. In the open reading frame of the divergent coat protein gene, 5 amino acid changes result from 48 nucleotide differences. Most differences occurred in the third position of the codons, and resulted in silent amino acid substitutions. RNase protection assays demonstrated that most of the clones obtained are representative of the major RNA species of this isolate. Northern analysis indicated that CTV-SY568 accumulated more viral RNA including genomic and certain subgenomic RNAs than isolates VT or T36 in sweet orange.

Comparison of Detection Methods for Citrus Tristeza Virus in Field Trees During Months of Nonoptimal Titer

Enzyme-linked immunosorbent assay (ELISA) can reliably detect citrus tristeza virus (CTV) in samples collected during approximately 6 months of a typical year. Two reverse transcriptase polymerase chain reaction (RT-PCR) methods (total nucleic acid extract and immunocapture based) were evaluated and compared to ELISA in order to develop a more sensitive assay for CTV. From May 1994 to October 1995, 6 sweet orange trees infected with CTV from each of 2 geographic areas (Riverside and the San Joaquin Valley) were tested monthly by each method. In the months of August (San Joaquin Valley samples) and September (Riverside and San Joaquin Valley samples) several of the trees had a significant loss of virus titer such that CTV was not reliably detected by ELISA. By contrast, the 2 PCR methods gave definitive positive results for CTV in samples collected during these months. Different tissue types were analyzed by each of the above assays. Petioles and midribs, both phloem-rich tissues, were each satisfactory for ELISA, while distal leaf tips did not always produce a positive result. All tissue types were equally efficient in producing a positive result in both PCR-based assays. The results of this study provide a basis for CTV testing by PCR in months when virus titer drops to a level generally unacceptable for using ELISA.

Ensemble of Secondary Structures for Encapsidated Satellite Tobacco Mosaic Virus RNA Consistent with Chemical Probing and Crystallography Constraints

Viral genomic RNA adopts many conformations during its life cycle as the genome is replicated, translated, and encapsidated. The high-resolution crystallographic structure of the satellite tobacco mosaic virus (STMV) particle reveals 30 helices of well-ordered RNA. The crystallographic data provide global constraints on the possible secondary structures for the encapsidated RNA. Traditional free energy minimization methods of RNA secondary structure prediction do not generate structures consistent with the crystallographic data, and to date no complete STMV RNA basepaired secondary structure has been generated. RNA-protein interactions and tertiary interactions may contribute a significant degree of stability, and the kinetics of viral assembly may dominate the folding process. The computational tools, Helix Find & Combine, Crumple, and Sliding Windows and Assembly, evaluate and explore the possible secondary structures for encapsidated STMV RNA. All possible hairpins consistent with the experimental data and a cotranscriptional folding and assembly hypothesis were generated, and the combination of hairpins that was most consistent with experimental data is presented as the best representative structure of the ensemble. Multiple solutions to the genome packaging problem could be an evolutionary advantage for viruses. In such cases, an ensemble of structures that share favorable global features best represents the RNA fold.

Assembly and characterization of hybrid virus-inorganic nanotubes

Recently, rod-shaped viruses have attracted attention as biological templates for assembly of nanostructures. Tobamoviruses such as the type strain of Tobacco mosaic virus (TMV-U1, or -common) have a cylindrical shape and dimensions suitable for nanoelectronic applications: 300nm long and 18nm in diameter with a 4nm axial channel. TMV particles can be coated with metals, silica, or semiconductor materials and may also form end-to-end assemblies to be used as interconnects or device channels. In this letter, we report the preparation of TMV-U1 templated organic-metal nanotubes, and their structural characterization using transmission electron microscopy and micro-Raman spectroscopy. Reproducible phonon signatures different from that of native TMV-U1 were observed from the metal-coated TMVs. Our results indicate that Raman spectroscopy can be used for monitoring of the bio-assisted nanostructure assembly and for analyzing the vibrational modes of the resulting bio-inorganic junctions.

Segregation of distinct variants from Citrus tristeza virus isolate SY568 using aphid transmission.

A well-studied severe isolate of Citrus tristeza virus (CTV) known as SY568 has previously been shown to contain multiple variants of the virus which differ in their genetic and biological characters. Aphid transmission was used in an attempt to segregate some of these variants for further characterization. Resulting infections gave symptoms which varied from asymptomatic to more severe than the inoculum source. RNase protection assays (RPAs) were used to compare nine regions of the CTV genome and determine whether unique strains could be identified. Five aphid-transmitted subcultures, with fingerprints that were different from those of the inoculum sources in at least one genomic area, were then cloned, sequenced, and compared with known isolates. An asymptomatic strain was shown to be different in every area of the CTV genome when examined by RPA and sequencing of selected regions. Mixed-infection studies using graft transmission of the asymptomatic subculture and two of the more severe aphid-transmitted subcultures showed that the mild strain was not able to compete well when in the presence of any of the severe variants tested, and its titer was significantly reduced from that seen in single infection. The mild strain and a selected severe strain were singly graft inoculated into five different citrus hosts (sweet orange, grapefruit, sour orange, lemon, and lime), where they maintained their distinct biological and genetic characteristics.

Pathogenicity and Management of Olpidium bornovanus, a Root Pathogen of Melons

Greenhouse studies document, for the first time, that Olpidium bornovanus, an obligate, holocarpic, root-inhabiting zoosporic fungus heretofore regarded as a nonpathogenic parasite, is a root pathogen. Significant browning of the roots and reductions in shoot and root growth were recorded within 28 days following inoculation of melons with the fungus. Amending the recirculating nutrient solution with either a nonionic surfactant (Agral 90) or a strobilurin fungicide (azoxystrobin) resulted in efficacious management of the disease caused by the fungus.

Natural Incidence of Mixed Infections and Experimental Cross Protection Between Two Genotypes of Tobacco mild green mosaic virus

ABSTRACT Isolates of Tobacco mild green mosaic virus (TMGMV), a member of the genus Tobamovirus, from Nicotiana glauca in southern California fall into two major genotypes, large (TMGMV-L) and small (TMGMV-S), distinguishable by the size of the coat protein (CP) subgenomic RNA. Mixed infections in the field were rare (1.6%), even at sites where both genotypes were common in single infections (62% for TMGMV-S; 37% for TMGMV-L). When plants experimentally protected by TMGMV-L were challenged by TMGMV-S, almost complete cross protection (90% of total plants challenged) was observed regardless of the protective time period (minimum 12 h and maximum 14 days). When plants protected by TMGMV-S were challenged with TMGMV-L, complete cross protection was observed when the protective time was 5 to 14 days. However, when the protective time was 3 days or less, protection by TMGMV-S was greatly reduced (11%), with mixed infections of TMGMV-S and -L predominating (69%), and single infections of the challenge virus TMGMVL were frequently observed (20%). When TMGMV-S and -L virions were co-inoculated, the virus progeny from individual plants most often contained only the TMGMV-L genome (61%) or, less frequently (39%), both genotypes. Therefore, TMGMV-L was more competitive than TMGMV-S and was able to displace TMGMV-S in experimental situations. The results obtained from cross-protection experiments in the greenhouse would explain the low frequency of natural mixed infections. It is possible that the experimental superior competitiveness of the novel L genotype has already or will play a role in its abundance in southern California.

Functional analysis of the N-terminal basic motif of a eukaryotic satellite RNA virus capsid protein in replication and packaging.

Efficient replication and assembly of virus particles are integral to the establishment of infection. In addition to the primary role of the capsid protein (CP) in encapsidating the RNA progeny, experimental evidence on positive sense single-stranded RNA viruses suggests that the CP also regulates RNA synthesis. Here, we demonstrate that replication of Satellite tobacco mosaic virus (STMV) is controlled by the cooperative interaction between STMV CP and the helper virus (HV) Tobacco mosaic virus (TMV) replicase. We identified that the STMV CP-HV replicase interaction requires a positively charged residue at the third position (3R) in the N-terminal 13 amino acid (aa) motif. Far-Northwestern blotting showed that STMV CP promotes binding between HV-replicase and STMV RNA. An STMV CP variant having an arginine to alanine substitution at position 3 in the N-terminal 13aa motif abolished replicase-CP binding. The N-terminal 13aa motif of the CP bearing alanine substitutions for positively charged residues located at positions 5, 7, 10 and 11 are defective in packaging full-length STMV, but can package a truncated STMV RNA lacking the 3′ terminal 150 nt region. These findings provide insights into the mechanism underlying the regulation of STMV replication and packaging.

First Report of Angelonia flower break virus in Nemesia spp. and Other Ornamental Plants in California

Between June 2006 and July 2007, ornamental plant samples were collected from four counties in California (Riverside, Sacramento, San Diego, and Santa Barbara) and tested for the presence of Angelonia flower break virus (AnFBV) using ELISA (Agdia, Inc., Elkhart, IN). Tissue samples were from propagation facilities or wholesale outlets except those from Riverside County, which were from retail stores. Thirteen positive samples were found in three varieties each of Angelonia and Nemesia spp. and seven varieties of Verbena spp., with at least one positive from each county. Foliar symptoms ranged from asymptomatic to a mild mosaic with distinct flower breaking in the Angelonia spp. Results were confirmed by reverse transcription (RT)-PCR of the coat protein gene (1) and the 1,172-bp amplicons were sequenced. Viral isolates from the three varieties of the Angelonia spp. had 98 to 99% nucleotide similarity and 99 to 100% amino acid identity to the Maryland strain of AnFBV (1; GenBank Accession No. DQ221212), with 91 to 92% nucleotide similarity and 96 to 97% amino acid identity to the Israel and Florida strains (GenBank Accession Nos. DQ223771 and DQ219415). All viral isolates from the Nemesia and Verbena spp. plants had nucleotide similarities of 96 to 98% and 98% amino acid identity to the Israel and Florida strains, with 91 to 92% nucleotide similarity to the Maryland strain. AnFBV has been previously reported in Angelonia and Verbena spp. among other hosts (1,2), but not in Nemesia spp. and not in California. This recently described carmovirus appears to be well established in the state in a variety of ornamental plant species. References: (1) S. Adkins et al. Phytopathology 96:460, 2006. (2) F. Assis-Filho et al. Plant Dis. 90:1115, 2006.

First report of Nemesia ring necrosis virus in North America in ornamental plants from California.

During the last several years, two California propagators have detected what was believed to be the tymovirus Scrophularia mottle virus (ScrMV) in several ornamental plant species on the basis of enzyme-linked immunosorbent assay (ELISA) using a ScrMV antibody system. Symptoms were generally mild, ranging from nonsymptomatic to a mild mosaic. Our laboratory confirmed the presence of a tymovirus in one Verbena sp. and two Diascia spp. cultivars on the basis of dsRNA analysis that showed bands of approximately 6,400 and 300 nucleotides representing the genomic and coat protein subgenomic RNAs, respectively. While these plants and those that were experimentally infected (Nicotiana benthamiana and N. clevelandii) also tested positive for ScrMV by ELISA, the host range did not match that published for ScrMV, notably the lack of symptoms in Chenopodium quinoa and the lack of systemic infection in Datura stramonium. A similar host range result was reported in Europe for another tymovirus that cross reacts with ScrMV antiserum, Nemesia ring necrosis virus (NeRNV) (2). Using NeRNV specific primers (1), we used reverse transcription-polymerase chain reaction (RT-PCR) to test plants that had previously tested positive for ScrMV by ELISA and had dsRNAs typical of a tymovirus. An amplicon of the appropriate size (960 bp) for NeRNV was obtained from each of five samples. Using ScrMV specific primers, the same samples failed to amplify the expected product. We have found NeRNV in three Diascia spp., one Verbena sp., and one Nemesia sp. plants in two counties in California (Riverside and San Diego). When the RT-PCR products were sequenced, they all had 99% sequence identities to NeRNV with 4 to 7 single nucleotide changes (GenBank Accession Nos. DQ648150 to DQ648154). Notably, each of the five amplicons had changes at nucleotides 5134 (G to C) and 5549 (G to T) when compared with the European isolates of NeRNV, which did not result in any amino acid changes. To our knowledge, this is the first report of NeRNV in North America and more specifically, in California. References: (1) R. Koenig et al. J. Gen. Virol. 86:1827, 2005. (2) A. L. Skelton et al. Plant Pathol. 53:798, 2004.