Economic Studies Reinforce Efforts to Safeguard Specialty Crops in the United States.

Pathogen-tested foundation plant stocks are the cornerstone of sustainable specialty crop production. They provide the propagative units that are used to produce clean planting materials, which are essential as the first-line management option of diseases caused by graft-transmissible pathogens such as viruses, viroids, bacteria, and phytoplasmas. In the United States, efforts to produce, maintain, and distribute pathogen-tested propagative material of specialty crops are spearheaded by centers of the National Clean Plant Network (NCPN). Agricultural economists collaborated with plant pathologists, extension educators, specialty crop growers, and regulators to investigate the impacts of select diseases caused by graft-transmissible pathogens and to estimate the return on investments in NCPN centers. Economic studies have proven valuable to the NCPN in (i) incentivizing the use of clean planting material derived from pathogen-tested foundation plant stocks; (ii) documenting benefits of clean plant centers, which can outweigh operating costs by 10:1 to 150:1; (iii) aiding the development of disease management solutions that are not only ecologically driven but also profit maximizing; and (iv) disseminating integrated disease management recommendations that resonate with growers. Together, economic studies have reinforced efforts to safeguard specialty crops in the United States through the production and use of clean planting material.

Characterization and comparative analysis of promoters from three plant pararetroviruses associated with Dahlia (Dahlia variabilis).

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus (DCMV), and an endogenous plant pararetroviral sequence (DvEPRS, formerly known as DMV-D10) were reported from dahlia (Dahlia spp). Promoter elements from these dahlia-associated pararetroviruses were identified and characterized. The TATA box, the CAAT box, the transcription start site, the polyadenylation signal, and regulation factors, characteristic of caulimovirus promoters, were present in each of these promoter regions. Each of the promoter regions was separately cloned into a binary vector containing ß-glucuronidase (GUS) reporter gene and delivered into Agrobacterium tumefaciens by electroporation followed by agroinfiltration into Nicotiana benthamiana. The activity of the 35S promoter homologs was determined by transient expression of the GUS gene both in qualitative and quantitative assays. The length of the promoter regions in DMV, DCMV, and DvEPRS corresponded to 438, 439, and 259 bp, respectively. Quantitative GUS assays showed that the promoters from DMV and DCMV resulted in higher levels of gene expression compared to that of DvEPRS in N. benthamiana leaf tissue. Significant differences were observed among the three promoters (p < 0.001). Qualitative GUS assays were consistent with quantitative GUS results. This study provides important information on new promoters for prospect applications as novel promoters for their potential use in foreign gene expression in plants.

Comparative analysis of endogenous plant pararetroviruses in cultivated and wild Dahlia spp.

Two distinct caulimoviruses, Dahlia mosaic virus (DMV) and Dahlia common mosaic virus, and an endogenous plant pararetroviral sequence (DvEPRS) were reported in Dahlia spp. DvEPRS, previously referred to as DMV-D10, was originally identified in the US from the cultivated Dahlia variabilis, and has also been found in New Zealand, Lithuania and Egypt, as well as in wild dahlia species growing in their natural habitats in Mexico. Sequence analysis of three new EPRSs from cultivated dahlias from Lithuania [D10-LT; 7,159 nucleotide level (nt)], New Zealand (D10-NZ, 7,156 nt), and the wild species, Dahlia rupicola, from Mexico (D10-DR, 7,133 nt) is reported in this study. The three EPRSs have the structure and organization typical of a caulimovirus species and showed identities among various open reading frames (ORFs) ranging between 71 and 97 % at the nt when compared to those or the known DvEPRS from the US. Examination of a dataset of seven full-length EPRSs obtained to date from cultivated and wild Dahlia spp. provided clues into genetic diversity of these EPRSs from diverse sources of dahlia. Phylogenetic analyses, mutation frequencies, potential recombination events, selection, and fitness were evaluated as evolutionary evidences for genetic variation. Assessment of all ORFs using phylogenomic and population genetics approaches suggests a wide genetic diversity of EPRSs occurring in dahlias. Phylogenetic analyses show that the EPRSs from various sources form one clade indicating a lack of clustering by geographical origin. Grouping of various EPRSs into two host taxa (cultivated vs. wild) shows little divergence with respect to their origin. Population genetic parameters demonstrate negative selection for all ORFs, with the reverse transcriptase region more variable than other ORFs. Recombination events were found which provide evolutionary evidence for genetic diversity among dahlia-associated EPRSs. This study contributes to an increased understanding of molecular population genetics and evolutionary pathways of these reverse transcribing viral elements.

First Report of Sweet potato virus G and Sweet potato virus 2 Infecting Sweetpotato in North Carolina.

Sweet potato virus G (SPVG) and Sweet potato virus 2 (SPV2) are two members of the genus Potyvirus, distinct from Sweet potato feathery mottle virus (SPFMV) (1,2,4). The significance of SPVG and SPV2 to sweetpotato (Ipomoea batatas Lam.) is that each virus can synergistically interact with Sweet potato chlorotic stunt virus (SPCSV) inducing sweet potato virus disease (SPVD) (1,2,4). During the summer of 2012, susceptible indicator plants (I. setosa) were evenly distributed in sweetpotato experimental plots at two research stations (Clinton and Kinston) in North Carolina (NC). Naturally infected indicator plants (n = 129) showing virus-like symptoms including vein clearing, chlorotic mosaic, and chlorotic spots were collected and tested for the presence of viruses. Sap extract from plants tested positive for SPVG and SPV2 by nitrocellulose immune-dot blot, using SPVG antiserum obtained from the International Potato Center (Lima, Peru) and SPV2 antiserum kindly provided by C. A. Clark, Louisiana State University. Total RNA was extracted from 200 mg of symptomatic leaf tissue by using the QIAGEN RNeasy Plant Mini Kit (Hilden, Germany) adding 2% PVP-40 and 1% 2-mercaptoethanol to the extraction buffer. Multiplex RT-PCR was carried out using the SuperScript III One-Step RT-PCR System (Invitrogen, Carlsbad, CA) with specific primers designed for simultaneous detection and differentiation of four closely related sweetpotato potyviruses (3). Amplicons were cloned using the pGEM-T Easy cloning kit (Promega, Madison, WI) and sequenced. Quantitative RT-PCR was used for SPCSV detection. Results confirmed the presence of SPVG and SPV2 in single infections on 7% and 0.8% of samples, respectively; and in mixed infections on 54% and 3% of samples, respectively. SPVG was found as the most prevalent in all viral combinations where 14% of samples were infected with SPVG and SPFMV; and 15% of samples were infected with SPVG, SPFMV, and Sweet potato virus C (SPVC). SPV2 was detected in less common combinations (0.8%) associated with SPVG and SPFMV. The mixed infection SPVG and SPCSV as well as the combination SPV2 and SPCSV was detected in 0.8% of samples. Sequence analyses of the samples at nucleotide level (GenBank Accession Nos. KC962218 and KC962219, respectively) showed 99% similarity to SPVG isolates from Louisiana (4) and SPV2 isolates from South Africa (1). Scions from infected indicator plants were wedge grafted onto healthy sweetpotatoes (cvs. Beauregard and Covington). Eight weeks after grafting, chlorotic mosaic was observed on plants with mixed potyvirus infections whereas plants with single potyvirus infection showed no obvious symptoms. RT-PCR testing and sequencing of amplicons corroborate the presence of both viruses initially detected in indicator plants. Additionally, naturally infected sweetpotato samples (n = 102) were collected in the same experimental plots. SPVG and SPV2 were detected and identified following the described methodology. In the United States, SPVG has been shown to be prevalent in Louisiana (4) and the results presented here indicate that SPVG is spreading in NC. Our results confirm the presence of SPVG and SPV2 in NC. To our knowledge, this is the first report of SPVG and SPV2 in sweetpotato fields in NC. References: (1) E. M. Ateka et al. Arch Virol 152:479, 2007. (2) F. Li et al. Virus Genes 45:118, 2012. (3) F. Li et al. J. Virol. Methods 186:161, 2012. (4) E. R. Souto et al. Plant Dis 87:1226, 2003.

Genomic characterization of pararetroviral sequences in wild Dahlia spp. in natural habitats.

The genome structure and organization of endogenous caulimovirus sequences from dahlia (Dahlia spp), dahlia mosaic virus (DMV)-D10 from three wild species, D. coccinea (D10-DC), D. sherffii (D10-DS) and D. tenuicaulis (D10-DT), were determined and compared to those from cultivated species of dahlia, D. variabilis (DvEPRS). The complete ca. 7-kb dsDNA genomes of D10-DC, D10-DS, and D10-DT had a structure and organization typical of a caulimovirus and shared 89.3 to 96.6% amino acid sequence identity in various open reading frames (ORF) when compared to DvEPRS. The absence of the aphid transmission factor and the truncated coat protein fused with the reverse transcriptase ORF were common among these DMV-D10 isolates from wild Dahlia species.