Isolation and Partial Nucleic Acid Characterization of a New Isolate of Potato virus V with Distinct Biological and Serological Properties.

In vitro cultures of potato (Solanum tuberosum cv. Papa Amarillo) were imported into the United States and were subjected to routine pathogen screening for newly introduced varieties. These cultures indexed positive for several of the six viruses tested and thus were treated using standard methods for eliminating viruses in in vitro cultures. Although the therapy apparently freed the potato of the tested viruses, some of the cultures continued to exhibit mosaic symptoms when planted in the greenhouse. When examined by electron microscopy, these plants contained flexuous rods. Tested samples did not index positive with any potato virus-specific polyclonal antisera but reacted with the "universal" potyvirus monoclonal antibody. These results indicated that these samples were likely infected with an as yet undescribed potyvirus. Further investigation indicated that this virus is a new strain of Potato virus V (PVV) that is serologically distinct from the common strain. This is the first description of a distinct strain of PVV. This particular strain has diverged sufficiently from other isolates of PVV to no longer be detectable by some commonly used antisera, and therefore is a concern for the future restriction of PVV spread into regions of the world where it is not currently present.

A new gene, Ny (tbr), for hypersensitivity to Potato virus Y from Solanum tuberosumMaps to Chromosome IV.

A diploid backcross population derived from a cross between Solanum tuberosum and Solanum berthaultiisegregated for monogenic dominant hypersensitivity to Potato virus Y(PVY). We propose the symbol Ny (tbr) for this locus because plants carrying this gene develop necrosis after inoculation with PVY and the allele originated in S. tuberosum. The gene mapped to chromosome IV between TG316 and TG208 at LOD=2.72. This location does not correspond to any other mapped resistance genes in potato.

Tissue culture methods for the screening and analysis of putative virus-resistant transgenic potato plants.

ABSTRACT Following regeneration, putative virus-resistant transgenic plants are usually transferred from tissue culture to a greenhouse or growth chamber to screen for resistance to infection and disease development using mechanical, graft, or insect vector inoculation methods. To reduce initial screening costs and time, we developed mechanical and graft inoculation methods suitable for tissue culture use. The in vitro methods were validated by comparing them with similar greenhouse screens using putative potato virus Y strain o (PVY degrees ) replicase-mediated resistant regenerants of the potato cultivar Atlantic. Five transgenic lines were tested, with similar results obtained from in vitro and greenhouse experiments. Two of the transgenic lines, A1 and A3, showed the greatest resistance to PVY degrees infection, as indicated by low enzyme-linked immunosorbent assay values and infection rates. In vitro mechanical inoculation methods were also used to infect wild-type tomato and tobacco plants with cucumber mosaic virus and potato virus Y. Potato plants were also infected with the phloem-restricted potato leafroll virus, a low-titer virus, using in vitro graft inoculation methods. These results suggest the potential usefulness of these simple, effective, and economical techniques for screening large numbers of putative virus-resistant plants.

A New Geminivirus Associated with Tomato in the State of São Paulo, Brazil.

The apical growth of about 20% of young tomato plants in observed fields near Campinas, State of São Paulo, Brazil, had yellow streaking of veins. Leaf symptoms developed into patches of yellow mosaic and the leaves became wavy. The whitefly Bemisia tabaci Genn. transmitted a pathogen from the infected tomato plants to healthy tomato and potato plants, reproducing the original symptoms in tomato. The apical leaves of infected potatoes showed yellow or green mottle that developed into leaf distortion with yellow blotches, symptoms indistinguishable from potato-deforming mosaic disease (2). DNA was extracted from these tomato and potato plants (1). Using DNA from the infected tomato plant, polymerase chain reaction (PCR) with the degenerate primer pair PAC1v1978/ PAV1c715 (1), which amplifies part of the rep gene (AC1 ORF), the common region (CR), and part of the cp gene (AV1 ORF), and with the primer pair PBC1v2039/PBV1c800, which amplifies part of BC1 ORF, CR, and part of BV1 ORF, gave virus-specific DNA fragments of the sizes expected from a whitefly-transmitted geminivirus. These were cloned and the complete nucleotide (sequences for DNA-A (pToYA, GenBank accession no. U79998) and DNA-B (pToYB, GenBank accession no. U80042) fragments obtained. Nucleotide identity between the CRs (184 nucleotides) was 90%, strongly indicating that those fragments correspond to a bipartite subgroup III geminivirus. PCR with the DNA from infected potato gave the expected size fragment for DNA-A. The partial sequence of the rep gene was 100% identical to the homologous sequence from the PCR fragment from the infected tomato. A search in the GenBank, EMBL, DDBJ, and PDB databases, using the BLAST program, found no identical geminivirus. The highest identity for the CR was 75% to tomato mottle geminivirus-Florida (ToMoV) and 74% to bean golden mosaic virus-Brazil. For the rep gene, the highest identity was 73% to tomato yellow leaf curl virus-Israel, an Old World geminivi-rus, followed by 71% to tomato golden mosaic virus-Brazil (TGMV) and ToMoV. For the cp gene, the highest identity was 86% to TGMV, followed by 83% to squash leaf curl geminivirus. Therefore, we propose the name tomato yellow vein streak geminivirus (ToYVSV) for this distinct virus (2). References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) J. A. C. Souza-Dias et al. Summa Phytopathol. 22:57, 1996.

Replicase-mediated resistance to potato virus Y in transgenic tobacco plants.

Nicotiana tabacum 'Turkish Samsun NN' plants were transformed with nuclear inclusion b (NIb) gene sequences of potato virus Y, O strain (PVYO). The full-length construct included an additional in-frame initiation codon contiguous to the putative N-terminal amino acid codon and a stop codon replacing the C-terminal amino acid codon. Of 13 independently transformed lines, four yielded 37 (out of 100) plants in the R1 generation that were resistant to PVYO infection. Progeny of 13 out of 15 of R1 plants tested expressed resistance in the R2 generation. Conversely, 30 independently transformed tobacco lines expressing essentially the same sequence but deleted for the Gly-Asp-Asp (GDD) motif were not resistant. Two other constructs encoding either the 5'-deleted or 3'-truncated NIb gene, but harboring the GDD motif, conferred resistance to PVYO in some tobacco plants. Despite the high level of nucleotide and amino acid identity shared by strains PVYO and PVYN for the NIb gene, PVYN replication was found in all PVYO-resistant plants. However, plants of one R2 line showed reduced PVYN replication.

Phosphorus Distribution in Red Pine Roots and the Ectomycorrhizal Fungus Hebeloma arenosa.

Red pines (Pinus resinosa Ait.) were grown in a pasteurized sandy loam either unamended with phosphate or fertilized with one of two levels of phosphate (34 or 136 mg/kg) as superphosphate, and with and without addition of Hebeloma arenosa inoculum. Shoot and total dry weights of mycorrhizal seedlings grown in soil unamended with P were greater than those for nonmycorrhizal seedlings grown in the same soil, but less than the dry weights of seedlings grown in soil amended with middle to high levels of P. Mycorrhizal infection was inhibited at the highest level of P amendment. (31)P nuclear magnetic resonance spectra of intact mycorrhizal roots showed the presence of two dominant peaks, orthophosphate (Pi) and polyphosphate (polyP). The polyP peak was absent in spectra of nonmycorrhizal roots. The ratio for areas under the two peaks, Pi/polyP, was 1.8 for mycorrhizal roots grown in both unamended soil and soil that had received middle levels of superphosphate. Apparently, the fungus strongly mediates the supply of phosphate to the tree through the production of polyP, even at growth-limiting levels of soil P, and regulates compartmentalization of P in the mycorrhizal roots.