Delano James

Standardizing the Nomenclature for Clonal Lineages of the Sudden Oak Death Pathogen, Phytophthora ramorum

Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.

A simple and reliable protocol for the detection of apple stem grooving virus by RT-PCR and in a multiplex PCR assay

Primers were identified which amplify specifically a 499 bp fragment in the coat protein coding region of apple stem grooving virus (ASGV) genome. These primers were used in various RT-polymerase chain reaction (PCR) analyses for the detection of ASGV in Chenopodium quinoa, Nicotiana occidentalis, and in species of Malus and Pyrus. Isolates of ASGV in Malus and Pyrus from locations in Canada, China, Israel, Japan, Nepal, Pakistan, South Africa, and the U.S.A. were reliably detected in leaf and bark (budwood) tissue. Storage of the tissues at -80 degrees C for more than 4 months did not affect the reliability of detection by immunocapture (IC) RT-PCR. Triton-X was not necessary for the detection of ASGV by IC/RT-PCR, and it was also possible to combine the antibody incubation and virus sap incubation into a single step without any obvious loss in sensitivity. A Tube Capture (TC) RT-PCR procedure was developed that eliminated the need for antibody binding of the virus. Phosphate buffered saline with 2% PVP was identified as the most effective sample-grinding buffer for the detection of ASGV by IC/RT-PCR and TC/RT-PCR. TC/RT-PCR facilitated the simultaneous detection (multiplex PCR) of ASGV and cherry mottle leaf virus.

First Report of Plum Pox Potyvirus in Ontario, Canada

Plum pox potyvirus (PPV) causes plum pox (sharka) disease, which is considered the most serious disease of stone fruits including peach, plum, nectarine, and apricot (2). The disease may cause losses as high as 80 to 100% of some crops (2). A survey was initiated in the Niagara region of Ontario, Canada, after it was reported that PPV was detected in Pennsylvania (1). The initial survey focused on Prunus material imported into Canada from the Pennsylvania region. Where imported trees could be identified, every tree was sampled. In cases where the imported trees were growing in mixed blocks with plants from other sources, 25% of the trees were sampled and tested as composites of four trees. PPV was detected in three symptomless Fantasia nectarine (Prunus persica var. nectarina) trees by triple-antibody sandwich (TAS) ELISA using the REAL Durviz kit (Valencia, Spain), which contains the universal PPV monoclonal 5B. PPV infection was confirmed by western blot analyses (a PPV polyclonal antibody and PPV 5B monoclonal were used as primary antibodies), reverse transcription polymerase chain reaction (RT-PCR), and TC/RT-PCR. In western blot analyses, the coat protein subunit sizes of the Canadian PPV isolates were estimated at 32 kDa based on electrophoretic mobility in 12% SDS-PAGE. RFLP analysis of the 243-bp fragment amplified using PPV specific primers P1 and P2 (4) indicated the presence of RsaI and AluI enzyme restriction sites, which is characteristic of PPV D strains. In RT-PCR analysis using D and M specific primers (3), only the D specific primers amplified a fragment 198 bp in size. This data provided conclusive evidence that the PPV isolates detected in Canada were PPV D, similar to the strain detected in Pennsylvania. The survey is continuing and is being expanded to determine the extent of spread and the exact distribution of the virus. References: (1) L. Levy et al. Phytopathology (Abstr.) 90:46, 2000. (2) M. Nemeth. Virus, Mycoplasma, and Rickettsia Diseases of Fruit Trees. Akademiai Kiado, Budapest. (3) A. Olmos et al. J. Virol. Methods 68:127-137, 1997. (4) T. Wetzel et al. J. Virol. Methods 33:355-365, 1991.

trategies for simultaneous detection of multiple plant viruses

Plants are infected by a wide range of viruses. Many cause devastation of plants and crops resulting in significant economic losses and threats to the viability of certain horticultural and agricultural industries. Resources available for routine detection of plant viruses tend to be limited. This means that techniques adopted for routine diagnosis must be of low cost yet sensitive and reliable. Approaches that allow simultaneous detection of multiple plant viruses (multiplexing) reduce the number of tests required, reagent usage, time for analysis, and consequently, the cost. Multiplex polymerase chain reaction (PCR), polyvalent PCR, nonisotopic molecular hybridization techniques, real-time PCR, and array technologies allow simultaneous detection of multiple plant viruses. The increased sensitivity achieved with some techniques, such as real-time PCR, permits the use of simple, low-cost target isolation methods such as direct binding, tissue printing, or immunocapture. These result in reduced overall cost. Multiplexing techniques have the capacity for simultaneous broad-spectrum and specific identification by combining primers and (or) probes that target various taxonomic levels such as family, genus, and species. Polyvalent PCR and broad-spectrum probes have the potential to detect unknown or uncharacterized viruses, improving our ability to monitor and successfully control these pathogens. Techniques such as microarray analysis offer the potential for development of a single biochip that may facilitate detection of all viruses affecting a particular crop (e.g., a cucurbit or potato biochip). This may be expanded in time to the detection of every pathogen, including viruses, affecting a particular plant.

Detection of a new and unusual isolate of Plum pox virus in plum (Prunus domestica).

Plum pox virus (PPV) isolate 3174-01 was detected by triple-antibody sandwich enzyme-linked immunosorbent assay using the universal PPV monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) using primers that amplify a 243-bp fragment in the C-terminus of the coat protein coding region. The restriction sites RsaI and AluI were absent from this fragment, which is a feature unique to PPV-C isolates. The restriction sites in 3174-01 were replaced by GTAA/GTGA and GGCA, respectively. There was 95 to 99, 94, 91, and 92 to 94% identity of the 243-bp fragment of 3174-01 with the corresponding region of the strains C, D, EA, and M, respectively. Attempts to detect the virus by RT-PCR using strain C-specific primers in three different approaches were unsuccessful. All molecular techniques assessed in attempting to strain type isolate 3174-01 gave negative results, or results inconsistent for D or M in the case of P3-6K1 restriction fragment length polymorphism analysis. Isolate 3174-01 reacted in Western blot assay with MAb 5B, with an estimated molecular mass of 32 kDa. No reaction was observed with D-, M-, EA-, or C-specific monoclonal antibodies in Western blot or enzyme-linked immunosorbent assay. The molecular and serological data seem to indicate that PPV isolate 3174-01 does not belong to any of the recognized strains of PPV.

Use of Luminex xMAP-derived Bio-Plex bead-based suspension array for specific detection of PPV W and characterization of epitopes on the coat protein of the virus

A panel of monoclonal antibodies (MAbs) directed against the N-terminus region of the coat protein (CP) of strain PPV W (isolate 3174) was generated by immunizing mice with recombinant peptides. The best performing MAbs were identified as 2C3 and 10G7. MAb 2C3 was selected for comparison of a standard TAS-ELISA protocol with a Luminex xMAP technology-derived bead-based suspension array system described as a triple antibody sandwich-microsphere immunoassay (TAS-MIA). TAS-MIA was as sensitive as TAS-ELISA for the specific detection of PPV W in herbaceous and woody hosts. It was completed in 4h, and used less reagents. Epitope recognition analysis was carried out using a set of overlapping synthetic pin-bound peptides (Mimotopes). Peptides (2)DEEDD(6) and (46)MFNPV(50) were the epitopes recognized most commonly by the best performing MAbs. Linear epitope prediction of B-cell recognition sites confirmed that both peptides fall within highly antigenic and accessible regions. The second glutamic acid residue of the epitope is crucial for MAb recognition, and the context of the epitope is as important as the sequence of the epitope. The results obtained in ELISA, Western blot, and TAS-MIA correlated with B-cell recognition prediction. This is an effective approach to identify suitable antigenic epitopes that generate antibodies for use in reliable diagnostic procedures. This is the first report of the detection of a plant virus using the Luminex xMAP bead-based suspension array system.

Evaluation of several commercial biocontrol products on European and North American populations of Phytophthora ramorum

Abstract Five commercially available biological control products were tested in vitro with seven isolates of Phytophthora ramorum from North American (NA1, NA2), and European (EU1) populations. The in vitro tests included dual culture methods and detached leaf assays on wounded Rhododendron and Camellia leaves. Variability in response to biocontrol agents among isolates of P. ramorum from North American and European populations was examined. In dual culture tests, both Bacillus subtilis products (Companion® and Serenade®) resulted in better inhibition of the NA1 group than NA2 and EU1. Actinovate® (Streptomyces lydicus) was the least effective of the three bacterial biocontrol agents and there was no difference in percent inhibition among P. ramorum lineages. Two products containing Trichoderma spp. were tested: Plant Helper® (T. atroviride) caused 100% inhibition of all lineages of P. ramorum, while SoilGard™ (T. virens) was only about 30% effective. There was great variability among P. ramorum isolates in their response to biocontrol agents. All treatments reduced P. ramorum lesion size on both Rhododendron and Camellia. Combined treatments of Actinovate® with one other BCA did not perform as well as either treatment used individually. Best results were obtained with Serenade® on Rhododendron and Camellia foliage, especially against the NA1 group. Lack of a linear relationship between percent inhibition of P. ramorum by BCAs in vitro and foliar treatments on detached Rhododendron and Camellia leaves indicates that in vitro testing is a poor predictor of BCA performance on plant material.

Detection and partial molecular characterization of two Plum pox virus isolates from plum and wild apricot in southeast Kazakhstan

Plum pox virus (PPV) was detected in wild apricot and cultivated plum maintained in a germ plasm collection in Kazakhstan. Both isolates were typed as D strain, with no evidence of recombination. The virus was detected by triple-antibody sandwich enzyme-linked immunosorbent assay (ELISA) utilizing the universal PPV-specific monoclonal antibody (MAb) 5B as the secondary antibody, and by reverse-transcription polymerase chain reaction (RT-PCR) assay using primers that amplified a 243-bp fragment in the C-terminus of the coat protein (CP) coding region. Immunocapture (IC) RT-PCR was used to detect PPV in nine wild apricot accessions, including eight ELISA-negative and one ELISA-positive. The plum and apricot isolates reacted positively in Western blot assay with the universal MAb 5B, and negatively with the strain-M-specific MAb-AL. Restriction fragment length polymorphism analysis applied to the amplified 243-bp fragment showed that restriction sites for AluI and RsaI were present in the were present in the plum and apricot samples. An amplified 836-bp cDNA fragment derived from the P3-6K1 coding region of both isolates had restriction profiles typical for strain D. Nucleotide identities of 99 to 100% were observed for the 243-bp fragments of the Kazakhstan isolates when compared with the corresponding regions of strain D, and 94 to 95% identity with strain M. Nucleotide sequence analysis of the entire CP coding region of the plum and apricotisolates resulted in the identification of a unique deletion of six nucleotides (two deduced proline amino acid residues) in the N-terminal region in the plum isolate. This is the first deletion of this nature observed among PPV isolates. The DAG motif was present in both isolates. Several nucleotide substitutions in the CP coding region were common to the plum and apricot isolates and appear to be unique to the Kazakstan isolates. This suggests a close relationship between the isolates.

Specific Detection of Cherry Mottle Leaf Virus Using Digoxigenin-Labeled cDNA Probes and RT-PCR

Cherry mottle leaf virus (CMLV)-associated double-stranded RNA (dsRNA) was isolated from the propagation host Chenopodium quinoa. The dsRNA band, with a molecular weight estimated at 7.0 × 106 Da, was used to produce cDNA. Two recombinant plasmids from the cloned cDNA library were identified that specifically bound with CMLV-associated RNA in dot blot hybridization studies. The cDNA inserts were sequenced, and oligonucleotide primers were designed that specifically amplify an 848-bp fragment of the CMLV genome by reverse-transcription polymerase chain reaction. Also, a poly(T) primer was reliably used for reverse transcription, with specific amplification using the CMLV primers, suggesting polyadenylation of the virus genome. Search of the database revealed some sequence homology of the partially characterized genome of CMLV with that of apple chlorotic leafspot virus. Additional sequence data are required, however, to establish the taxonomic position of the filamentous CMLV.

Genetic diversity of Plum pox virus: strains, disease and related challenges for control

Abstract Plum pox virus (PPV), a member of the genus Potyvirus, family Potyviridae, is a positive-sense single-stranded RNA virus. RNA viruses tend to be genetically diverse because the viral RNA-dependent RNA polymerase responsible for virus replication lacks proofreading capacity. Consequently, PPV is genetically diverse, and nine strains of the virus (D, M, EA, C, Rec, W, T, CR, and An) have been identified to date. At least two of these strains (Rec, T) are the direct products of recombination events, a contributor to genetic diversity and a common occurrence in the genus Potyvirus. The strains are genetically distinct and can be identified using phylogenetic analysis of the whole genome, the PIPO gene, the polymerase region, and the coat protein (CP) region. The unique genetic characteristics of each strain have facilitated the development of nucleic-acid-based and protein-based tools that allow strain identification. The strains are usually defined also by distinct biological properties that may include differences in host range, aphid transmissibility, disease symptomatology, and/or geographic distribution. There is genetic diversity within strains that affects these biological properties, and these differences may overlap with, and sometimes blur, the biological distinction between strains.