Robert R. Martin

Impacts of molecular diagnostic technologies on plant disease management

Detection and diagnosis of plant viruses has included serological laboratory tests since the 1960s. Relatively little work was done on serological detection of plant pathogenic bacteria and fungi prior to the development of ELISA and monoclonal antibody technologies. Most applications for laboratory-based tests were directed at virus detection with relatively little emphasis on fungal and bacterial pathogens, though there was some good work done with other groups of plant pathogens. With the advent of molecular biology and the ability to compare regions of genomic DNA representing conserved sequences, the development of laboratory tests increased at an amazing rate for all groups of plant pathogens. Comparison of ITS regions of bacteria, fungi, and nematodes has proven useful for taxonomic purposes. Sequencing of conserved genes has been used to develop PCR-based detection with varying levels of specificity for viruses, fungi, and bacteria. Combinations of ELISA and PCR technologies are used to improve sensitivity of detection and to avoid problems with inhibitors or PCR often found in plants. The application of these technologies in plant pathology has greatly improved our ability to detect plant pathogens and is increasing our understanding of, their ecology and epidemiology.

De Novo Reconstruction of Consensus Master Genomes of Plant RNA and DNA Viruses from siRNAs

Virus-infected plants accumulate abundant, 21–24 nucleotide viral siRNAs which are generated by the evolutionary conserved RNA interference (RNAi) machinery that regulates gene expression and defends against invasive nucleic acids. Here we show that, similar to RNA viruses, the entire genome sequences of DNA viruses are densely covered with siRNAs in both sense and antisense orientations. This implies pervasive transcription of both coding and non-coding viral DNA in the nucleus, which generates double-stranded RNA precursors of viral siRNAs. Consistent with our finding and hypothesis, we demonstrate that the complete genomes of DNA viruses from Caulimoviridae and Geminiviridae families can be reconstructed by deep sequencing and de novo assembly of viral siRNAs using bioinformatics tools. Furthermore, we prove that this ‘siRNA omics’ approach can be used for reliable identification of the consensus master genome and its microvariants in viral quasispecies. Finally, we utilized this approach to reconstruct an emerging DNA virus and two viroids associated with economically-important red blotch disease of grapevine, and to rapidly generate a biologically-active clone representing the wild type master genome of Oilseed rape mosaic virus. Our findings show that deep siRNA sequencing allows for de novo reconstruction of any DNA or RNA virus genome and its microvariants, making it suitable for universal characterization of evolving viral quasispecies as well as for studying the mechanisms of siRNA biogenesis and RNAi-based antiviral defense.

A discovery 70 years in the making: characterization of the Rose rosette virus.

Rose rosette was first described in the early 1940s and it has emerged as one of the most devastating diseases of roses. Although it has been 70 years since the disease description, the rosette agent is yet to be characterized. In this communication, we identify and characterize the putative causal agent of the disease, a negative-sense RNA virus and new member of the genus Emaravirus. The virus was detected in 84/84 rose rosette-affected plants collected from the eastern half of the USA, but not in any of 30 symptomless plants tested. The strong correlation between virus and disease is a good indication that the virus, provisionally named Rose rosette virus, is the causal agent of the disease. Diversity studies using two virus proteins, p3 and p4, demonstrated that the virus has low diversity between isolates as they share nucleotide identities ranging from 97 to 99%.

Survey for Viruses of Grapevine in Oregon and Washington

Grapevines (Vitis spp.) in Washington and Oregon were surveyed for the prevalence of key grapevine viruses. Samples collected from 1,522 vines in Washington were tested for Rupestris stem pitting associated virus (RSPaV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Tomato ringspot virus (ToRSV), and Grapevine leafroll associated virus-3 (GLRaV-3). Tests were also conducted for GLRaV-1 and -2 on 420 samples from Washington. Two hundred forty samples collected from wine grape vineyards in Oregon were tested for GLRaV-1, -2, and -3, and an additional 2,880 samples were collected from 40 vineyards known to have high populations of Xiphinema americanum nematodes. The latter were tested for ArMV, ToRSV, and GFLV. GLRaV-1, -2, and -3 were detected in 2.6, 0.2, and 6.5% of the Washington samples and in 3.0, 0.4, and 4.4% of the Oregon samples. RSPaV was detected in 4.6% of the samples from Washington. No ToRSV, ArMV, or GFLV was detected in any of the samples from Oregon or Washington. Transmission of field isolates of GLRaV-3 from Washington by the grape mealybug also was demonstrated.

Viruses and Virus Diseases of Rubus

Blackberry and raspberry are members of the family Rosaceae. They are classified in the genus Rubus, which comprises hundreds of species and has a center of origin in the Far East. Rubus is divided into 15 subgenera with blackberries classified in the Rubus (formerly Eubatus) and raspberries in the Idaeobatus subgenera. Rubus species are propagated vegetatively and are subject to infection by viruses during development, propagation, and fruit production stages. Reports of initial detection and symptoms of more than 30 viruses, virus-like diseases, and phytoplasmas affecting Rubus spp. were reviewed more than 20 years ago. Since the last review on Rubus viruses, significant progress has been made in the molecular characterization of many of the viruses that infect Rubus spp. Currently, reverse transcription-polymerase chain reaction detection methods are available for most of the viruses known to infect Rubus. The goals of this article are to update the knowledge on previously characterized viruses of Rubus, highlight recently described viruses, review the virus-induced symptoms, describe the advances made in their detection, and discuss our knowledge about several virus complexes that cause serious diseases in Rubus. Virus complexes have been identified recently as the major cause of diseases in blackberries and raspberries.

The nucleotide sequence and genome organization of strawberry mild yellow edge-associated potexvirus

The nucleotide sequence (5966 nucleotides) of cDNA clones of strawberry mild yellow edge-associated potexvirus was determined. The genome contains six open reading frames (ORFs) encoding putative proteins with Mrs of 149,423, 25,344, 11,576, 8079, 25,714 and 11,216. In the first three putative proteins and the coat protein considerable similarity was found to comparable polypeptides of the potexviruses potato virus X, clover yellow mosaic virus, narcissus mosaic virus, papaya mosaic virus, white clover mosaic virus and lily virus X.

Identification and Detection of a Virus Associated with Strawberry Pallidosis Disease

The etiology of pallidosis, a disease of strawberry identified more than 45 years ago, remains unknown. We report a putative agent of the disease, a virus belonging to the Crinivirus genus of the Closterovirideae family. A sensitive reverse transcription-polymerase chain reaction (RTPCR) test has been developed. Polyclonal antibodies that can be used to detect the virus in petiole tissue blots were developed using a recombinant virus coat protein. The nucleotide sequences of regions of the viral genome that encode the heat shock protein 70 homolog and the major coat protein were obtained. Alignments of the major coat protein show that the virus isolated from strawberry plants positive for pallidosis is most closely related to Cucumber yellows virus (syn. Beet pseudo-yellows virus) and Cucurbit yellow stunt disorder virus, members of the Crinivirus genus.

Blueberry latent virus: An amalgam of the Partitiviridae and Totiviridae

A new, symptomless virus was identified in blueberry. The dsRNA genome of the virus, provisionally named Blueberry latent virus (BBLV), codes for two putative proteins, one without any similarities to virus proteins and an RNA-dependent RNA polymerase. More than 35 isolates of the virus from different cultivars and geographic regions were partially or completely sequenced. BBLV, found in more than 50% of the material tested, has high degree of homogeneity as isolates show more than 99% nucleotide identity between them. Phylogenetic analysis clearly shows a close relationship between BBLV and members of the Partitiviridae, although its genome organization is related more closely to members of the Totiviridae. Transmission studies from three separate crosses showed that the virus is transmitted very efficiently by seed. These properties suggest that BBLV belongs to a new family of plant viruses with unique genome organization for a plant virus but signature properties of cryptic viruses including symptomless infection and very efficient vertical transmission.

Complete nucleotide sequence of a strawberry isolate of Beet pseudoyellows virus.

In our effort to identify the causal agent(s) of strawberry pallidosis we found a single pallidosis positive plant that did not give any amplicons after RT-PCR using primer sets representing multiple regions of Strawberry pallidosis associated virus (SPaV) genome and failed to react with antibodies directed against the recombinant coat protein (CP) of SPaV. DsRNA extracted from this plant showed a similar pattern to that of SPaV indicating that another crinivirus may infect strawberry. Sequence analysis of multiple cDNA clones corresponding to the heat shock 70 homolog gene (HSP70h) of the unknown virus indicated that it was Beet pseudoyellows virus (BPYV). Analysis of the complete nucleo- tide sequence of BPYV-strawberry revealed that this isolate has several distinct features when compared to Cucumber yellows virus (CuYV), a cucumber strain of BPYV, including an entire ORF not found in CuYV.

Transformation of tobacco and potato with cDNA encoding the full-length genome of Potato leafroll virus: evidence for a novel virus distribution and host effects on virus multiplication

A full-length cDNA copy of the genome of Potato leafroll virus (PLRV) was introduced into the genome of tobacco and potato plants by Agrobacterium tumefaciens-mediated transformation. Transgenic lines were obtained in which the transgene was readily detected by PCR with DNA extracted from T(1) tobacco seedlings and clonally multiplied potato plants. PLRV-specific genomic and sub- genomic RNAs, coat protein antigen and virus particles were detected in transgenic plants. Aphids fed on the transgenic tobacco plants readily transmitted PLRV to test plants. Infected transgenic tobacco plants, like non-transgenic (WT) PLRV-infected plants, displayed no symptoms of the infection but transgenic plants of potato were severely stunted. In parallel tests, the mean PLRV titres in WT tobacco plants and transgenic tobacco plants were 600 and 630 ng virus/g leaf, respectively, although differences in PLRV titres among transgenic plants were much greater than those among infected WT plants. In similar tests with potato, the mean PLRV titre of WT plants was 50 ng virus/g leaf whereas higher concentrations (up to 3400 ng virus/g leaf) accumulated in transgenic potato plants. In tissue prints of stems, PLRV was detected in similar proportions of phloem cells in transgenic and infected WT plants. In transgenic tobacco and potato plants, but not in infected WT plants, a few stem epidermal cells also contained virus. From tissue prints of transgenic tobacco leaves, it was estimated that about one in 40000 mesophyll cells contained virus, but in transgenic potato, a greater proportion of mesophyll cells was infected.