Philip H. Berger

Volatiles from potato plants infected with potato leafroll virus attract and arrest the virus vector, Myzus persicae (Homoptera: Aphididae)

The influence of viral disease symptoms on the behaviour of virus vectors has implications for disease epidemiology. Here we show that previously reported preferential colonization of potatoes infected by potato leafroll virus (genus Polerovirus) (luteovirus) (PLRV) by alatae of Myzus persicae, the principal aphid vector of PLRV, is influenced by volatile emissions from PLRV–infected plants. First, in our bioassays both differential immigration and emigration were involved in preferential colonization by aphids of PLRV–infected plants. Second, M. persicae apterae aggregated preferentially, on screening above leaflets of PLRV–infected potatoes as compared with leaflets from uninfected plants, or from plants infected with potato virus X (PVX) or potato virus Y (PVY). Third, the aphids aggregated preferentially on screening over leaflet models treated with volatiles collected from PLRV–infected plants as compared with those collected from uninfected plants. The specific cues eliciting the aphid responses were not determined, but differences between headspace volatiles of infected and uninfected plants suggest possible ones.

Volatile Cues Influence the Response of Rhopalosiphum padi (Homoptera: Aphididae) to Barley Yellow Dwarf Virus–Infected Transgenic and Untransformed Wheat

Abstract The attractiveness of Barley yellow dwarf luteovirus (BYDV)–infected wheat plants to Rhopalosiphum padi L. was evaluated under laboratory conditions. Two untransformed wheat varieties, virus-susceptible Lambert and virus-tolerant Caldwell, and one transgenic wheat genotype (103.1J) derived from Lambert and expressing the BYDV coat protein gene, were tested in three bioassays. First, R. padi responses to BYDV-infected or noninfected Lambert and Caldwell were evaluated. Significantly more aphids settled onto virus-infected than noninfected plants when aphids were able to contact the leaves. Second, aphid responses to headspace from virus-infected or noninfected Lambert and Caldwell were tested. Significantly more aphids congregated on screens above headspace of BYDV-infected plants than above headspace of noninfected plants of both varieties. Third, aphid responses to headspace from virus-infected or noninfected and sham-inoculated (exposed to nonviruliferous aphids) Lambert and 103.1J plants were examined. Significantly more aphids congregated on screens above BYDV-infected than above noninfected or sham-inoculated Lambert. No significant differences in R. padi preferences for headspace above BYDV-infected compared with noninfected or sham-inoculated 103.1J plants were observed. The concentration of volatiles extractable from whole plant headspace was greater on BYDV-infected Lambert than on BYDV-infected 103.1J, noninfected, or sham-inoculated plants of either genotype. This is the first report of volatile cues associated with BYDV infection in wheat plants influencing the behavior of the vector R. padi. Additionally, these findings show for the first time that transgenic virus resistance in wheat can indirectly influence the production of volatiles making virus-infected plants less attractive or arrestant to aphids than are infected untransformed plants.

Phylogenetic analysis of the Potyviridae with emphasis on legume-infecting potyviruses

SummaryThe 3′-terminal nucleotide sequences of thirteen authenticated strains of bean common mosaic virus (BCMV) and one strain of bean common mosaic necrosis virus (BCMNV) were obtained. The regions sequenced included the coat protein coding sequence and 3′-end non-coding region. These data, combined with sequence information from other legume-infecting potyviruses and the Potyviridae were used for phylogenetic analysis. Evidence is provided for delineation of BCMNV as distinct from BCMV and the inclusion of azuki mosaic, dendrobium mosaic, blackeye cowpea mosaic, and peanut stripe viruses as strains of BCMV. This relationship defines the members of the BCMV and BCMNV subgroups. These data also provide a basis upon which to define virus strains, in combination with biological data. Other aspects and implications of legume-infecting potyvirus phylogenetics are discussed.

Using Real-Time PCR to Determine Transgene Copy Number in Wheat

Transgene copy number is usually determined by means of Southern blot analysis which can be time consuming and laborious. In this study, quantitative real-time PCR was developed to determine transgene copy number in transgenic wheat. A conserved wheat housekeeping gene,puroindoline-b, was used as an internal control to calculate transgene copy number. Estimated copy number in transgenic lines using real-time quantitative PCR was correlated with actual copy number based on Southern blot analysis. Real-time PCR can analyze hundreds of samples in a day, making it an efficient method for estimating copy number in transgenic wheat.

Nucleotide sequence of both genomic RNAs of a North American tobacco rattle virus isolate

SummaryThe complete sequence of a North American tobacco rattle virus (TRV) isolate, ‘Oregon yellow’ (ORY), was determined from cDNA and RT-PCR clones derived from the two genomic RNAs of this isolate. The RNA-1 is 6790 bases and RNA-2 is 3261 bases. The sequence of TRV-ORY RNA-1 was similar to RNA-1 of TRV isolate SYM, and differs in 48 nucleotides. TRV-ORY RNA-1 was one base shorter than -SYM, and had 47 base substitutions resulting in 12 amino acid substitutions of which 4 were conservative. The RNA-2 of TRV-ORY was distinct from RNA-2 of other characterized TRV isolates and contained three open reading frames (ORFs) that could potentially code for proteins of MW 22.4 kDa, 37.6 kDa and 17.9 kDa. Based on the homology of the predicted amino acid sequence with those of other tobraviruses, ORF1 of RNA-2 encodes the coat protein (CP). The protein sequence of ORF2 had regions of limited similarity with those of ORF2 of two other TRV isolates and pea early browning tobravirus. The ORF3 was unique to TRV-ORY. Phylogenetic analysis of tobravirus CPs indicated that TRV-ORY was most closely related to pepper ringspot tobravirus and TRV-TCM. The relationship of tobravirus CPs to other rod-shaped tubular plant viruses vis also discussed.

In vitro destabilization of plant viruses and cDNA synthesis

DNA copies of a wide range of RNA viruses can be made by the direct addition of appropriately treated, purified virus particles to a reverse transcription reaction. Therefore, many problems associated with RNA isolation can be circumvented. Virus particles can be sufficiently destabilized by adjustments of salt content, buffer, pH or by the use of physical force supplied by a freeze/thaw cycle so that RNA in sufficient quantity and physical condition is available for the synthesis of in some cases, full length cDNAs. cDNAs have been made of viruses in the bromo-, poty-, carla-, ilar-, potex-, tobra and tobamovirus groups. Reported here are experiments with cowpea chlorotic mottle virus and bean common mosaic virus.

Three epitopes located on the coat protein amino terminus of viruses in the bean common mosaic potyvirus subgroup

SummaryTwenty-seven of 29 strains of viruses in the bean common mosaic virus (BCMV) subgroup of legume-infecting potyviruses reacted strongly with one or more of the monoclonal antibodies (MAbs) which are known to be specific for epitopes located along the 50 amino acids which constitute the N-terminal end of the viral coat protein. Approximately one half of the virus strains reacted with the N-terminal epitope specific (NTES) MAb 4G12 which is specific for epitope E/B4, while the other half reacted with NTES MAbs 4 Aff1 or 4F9 which are specific for epitope E/B3. All but two strains contained at least one of these epitopes while no strain contained both. Competitive assays using five sequential, non-overlapping, synthetic, 10mer peptides indicated that the amino acids critical for epitope E/B3 reaction were located at positions 5, 7, and 10 from the N-terminal end of the coat protein. By deduction we postulate that the amino acids critical for epitope E/B4 are located at positions 10, 16, and 17. Because epitope E/B3 requires isoleucine at position 10 for expression whereas epitope E/B4 requires valine to be expressed, no one strain can express both epitopes. Two viruses in our tests (azuki mosaic and Dendrobium mosaic viruses) had deletions in this portion of their sequence explaining their failure to react MAbs specific for either epitope. The critical amino acids for a third epitope, E/B3A, were located at positions 16 and 17. We found no correlation between any of the three N-terminal epitopes defined in this study and the presence or absence of any biological property that we could accurately measure: i.e., symptomatology, host range, or pathotype. However, when coat protein sequences were aligned according to epitope type E/B3 or E/B4, we found that sequences within groups had high levels of identity while between group identities were low. We also found that sequences in the 3′-end non-coding region exhibited similar relationships within and between epitope groups. Two strains of BCMV (NL-4 and RU-1) were found to possess coat protein sequences typical of epitope E/B4 but 3′-NCR sequences typical of epitope E/B3. These data suggest that both strains may be the result of natural recombinants between the two epitope groups.

Biotechnology and Resistance to Potato Viruses

Potatoes are susceptible to many viruses, a situation that is exacerbated by viruses that can be transmitted through tuber seed pieces. Such vegetative propagation readily perpetuates and disperses infectious viruses and the diseases they cause. In general, most viruses are controlled by a combination of strategies that include seed certification and agronomic practices (see chapters 14, 15, 17), the most effective of which is host plant resistance. However, obtaining virus resistance is problematic. It is difficult and sometimes impossible to instill sources of resistance into cultivated potato from wild relatives, and obtain or retain agronomically useful cultivars. As a consequence, seed potato certification programs expend significant time and resources to minimize the amount of virus inoculum that enters commercial production. The economic and environmental cost of these practices, such as the application of pesticides for vector control, is of serious concern both to potato producers and consumers.