Vernon D. Damsteegt

Identification, characterization, and relatedness of luteovirus isolates from forage legumes.

ABSTRACT Virus isolates from forage legumes collected from eight different states were identified as luteoviruses closely related to soybean dwarf luteovirus dwarfing (SbDV-D) and yellowing (SbDV-Y) described in Japan. All isolates produced reddened leaf margins in subterranean clover and were transmitted in a persistent manner by Acrythosiphon pisum, but not by Aulacorthum solani. Specific monoclonal antibodies raised against SbDV-Y were differentially reactive with endemic isolates. Immunoblots probed with a SbDV-D polyclonal antiserum showed single 26-kDa coat protein bands, confirming close serological relatedness to SbDV. Analyses of genomic and subgenomic double-stranded RNAs and northern blot analyses confirmed genomic relatedness to SbDV. Based on our results, we conclude that the U.S. luteovirus isolates studied comprise a strain or strains of the soybean dwarf virus that have clovers as common hosts and the pea aphid as a common vector.

Suppression of PLRV Titer in transgenic Russet Burbank and Ranger Russet

Potato varieties Russet Burbank and Ranger Russet were transformed with a cDNA version of the 23 kD coat protein cistron (CP) of the potato leafroll virus (PLRV) using anAgrobacterium-mediated procedure. Clones were assayed for presence of thenpt- II and CP genes by Southern analysis, for expression of CP mRNA by Northern analysis, and for presence of PLRV coat protein in uninoculated and aphid inoculated plants by ELISA and Western blot analysis in uninoculated plants. Two putative Russet Burbank transformants were escapes, lacking eithernptll or CP, while one putative Ranger Russet transformant possessed thenpt- II gene but not the CP gene. In Russet Burbank and Ranger Russet, some transformants had statistically lower virus titer. The lowered titer was consistent throughout assays at three times during primary infection and one assay of secondary infection. The ranking of virus titer across all tests was statistically consistent. Transformants with the lowest secondary titers had virus contents 15 and 31 % of the titers of untransformed controls for Russet Burbank and Ranger Russet, respectively. The virus titer of the two Russet Burbank escapes and the Ranger Russet with only thenpt- II gene did not differ significantly from their respective untransformed controls.

Molecular, Ultrastructural, and Biological Characterization of Pennsylvania Isolates of Plum pox virus

Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.

Molecular analysis of soybean dwarf virus isolates in the eastern United States confirms the presence of both D and Y strains and provides evidence of mixed infections and recombination.

Soybean dwarf virus (SbDV), first identified as an agricultural problem in Japan, has emerged as a growing problem in the Midwestern United States. The majority of research on SbDV had been limited to four lab maintained strains from Japan. SbDV had been found in clover in the eastern United States, but these isolates rarely emerged into soybeans. These isolates were analyzed by multiplex PCR and sequencing, revealing that some were infections of both Y and D components, including a recombinant subisolate. Phylogenetic analyses for the US isolates revealed a broad diversity of SbDV, with selection pressure greater on the movement protein than the coat protein. The field isolates from the Eastern United States showed differences in symptoms, aphid transmission and host range, demonstrating that a study of field isolates is an important complement to laboratory maintained strains in understanding the biology and evolution of plant viruses.

Nucleotide sequence andE. coli expression of the coat protein gene of the yellowing strain of soybean dwarf luteovirus

SummaryThe nucleotide sequence of the coat protein gene of the yellowing (Y) strain of soybean dwarf virus (SbDV) was determined from cloned cDNA. The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated Mr of 22 200. The identity of the open reading frame (ORF) encoding the coat protein was confirmed by expression of anEscherichia coli β-galactosidase fusion protein and detection by a dot blot immunoassay. Sequence comparisons of the deduced coat protein amino acids to several luteoviruses demonstrated that the SbDV-Y coat protein ORF shares greatest similarity with bean leaf roll virus (BLRV) (65%).

Sequence and Expression in Escherichia coli of the Coat Protein Gene of the Dwarfing Strain of Soybean Dwarf Luteovirus

The nucleotide sequence of the coat protein gene of the dwarfing (D) strain of soybean dwarf luteovirus (SbDV) was determined from cloned cDNA. The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated molecular mass of 22.2 kDa. A major portion of the coat protein open reading frame (ORF) was expressed in Escherichia coli as a pET fusion protein and the product was detected by western blot analysis using SbDV-D polyclonal antibodies. Comparison of the deduced coat protein amino acid sequence to that from the yellowing (Y) strain of SbDV demonstrated 88% identity.

Host adaptation of soybean dwarf virus following serial passages on pea (Pisum sativum) and soybean (Glycine max)

Soybean Dwarf Virus (SbDV) is an important plant pathogen, causing economic losses in soybean. In North America, indigenous strains of SbDV mainly infect clover, with occasional outbreaks in soybean. To evaluate the risk of a US clover strain of SbDV adapting to other plant hosts, the clover isolate SbDV-MD6 was serially transmitted to pea and soybean by aphid vectors. Sequence analysis of SbDV-MD6 from pea and soybean passages identified 11 non-synonymous mutations in soybean, and six mutations in pea. Increasing virus titers with each sequential transmission indicated that SbDV-MD6 was able to adapt to the plant host. However, aphid transmission efficiency on soybean decreased until the virus was no longer transmissible. Our results clearly demonstrated that the clover strain of SbDV-MD6 is able to adapt to soybean crops. However, mutations that improve replication and/or movement may have trade-off effects resulting in decreased vector transmission.