Many RNA viruses have genetically diverse populations in a single host. Important biological characteristics may be related to the levels of diversity, including adaptability, host specificity, and host range. Shifting the virus between hosts might result in a change in the levels of diversity associated with the new host. The level of genetic diversity for these viruses is related to host, vector and virus interactions, and understanding these interactions may facilitate the prediction and prevention of emerging viral diseases. It is known that luteoviruses have a very specific interaction with aphid vectors. Previous studies suggested that there may be a tradeoff effect between the viral adaptation and aphid transmission when Soybean dwarf virus (SbDV) was transmitted into new plant hosts by aphid vectors. In this study, virus titers in different aphid vectors and the levels of population diversity of SbDV in different plant hosts were examined during multiple sequential aphid transmission assays. The diversity of SbDV populations revealed biases for particular types of substitutions and for regions of the genome that may incur mutations among different hosts. Our results suggest that the selection on SbDV in soybean was probably leading to reduced efficiency of virus recognition in the aphid which would inhibit movement of SbDV through vector tissues known to regulate the specificity relationship between aphid and virus in many systems.

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.

Within two decades of its discovery, Zucchini yellow mosaic virus (ZYMV) achieved a global distribution. However, whether or not seed transmission occurs in this economically significant crop pathogen is controversial, and the relative impact of seed transmission on the epidemiology of ZYMV remains unclear. Using reverse transcription polymerase chain reaction, we observed a seed transmission rate of 1.6% in Cucurbita pepo subsp. texana and show that seed-infected C. pepo plants are capable of initiating horizontal ZYMV infections, both mechanically and via an aphid vector (Myzus persicae). We also provide evidence that ZYMV-infected seeds may act as effective viral reservoirs, partially accounting for the current geographic distribution of ZYMV. Finally, the observation that ZYMV infection of C. pepo seeds results in virtually symptomless infection, coupled with our finding that an antibody test failed to detect vertically transmitted ZYMV in infected seed, highlights the urgent need to standardize current detection methods for seed infection.

Soybean dwarf virus (SbDV) exists as several distinct strains based on symptomatology, vector specificity, and host range. Originally characterized Japanese isolates of SbDV were specifically transmitted by Aulacorthum solani. More recently, additional Japanese isolates and endemic U.S. isolates have been shown to be transmitted by several different aphid species. The soybean aphid, Aphis glycines, the only aphid that colonizes soybean, has been shown to be a very inefficient vector of some SbDV isolates from Japan and the United States. Transmission experiments have shown that the soybean aphid can transmit certain isolates of SbDV from soybean to soybean and clover species and from clover to clover and soybean with long acquisition and inoculation access periods. Although transmission of SbDV by the soybean aphid is very inefficient, the large soybean aphid populations that develop on soybean may have epidemiological potential to produce serious SbDV-induced yield losses.

Cucumber mosaic virus (CMV) has become a major limiting factor in snap bean production in the Great Lakes region of North America, and epidemics have occurred more frequently since the soybean aphid, Aphis glycines Matsumura, was introduced. Major aphid vectors of CMV epidemics were identified by statistically relating their temporal dispersal trends to the incidence of CMV. Alates were monitored weekly using water pan traps in 74 snap bean fields in New York and Pennsylvania from 2002 to 2006. Plants were tested for CMV by ELISA one time during late bloom in 2002 and 2003 and weekly over the season from 2004 to 2006. Principal vectors of CMV included Acyrthosiphon pisum (Harris), A. glycines, Aphis gossypii Glover, and Therioaphis trifolii (Monell). Among these, A. glycines and T. trifolii were likely responsible for severe CMV epidemics because they were among the most abundant species captured, they efficiently transmit CMV, and their dispersal activity was positively correlated with periods when CMV incidence was highest. Moreover, because high numbers of A. glycines and T. trifolii disperse during July and August, snap bean fields planted beyond late June are at risk for infection during early vegetative stages and are subsequently more at risk for yield loss. In contrast, plantings up to late June are less likely to become infected during early developmental stages and should escape yield loss because major vectors are dispersing infrequently. CMV-resistant or tolerant snap bean varieties should be planted after late June to reduce the risk of yield loss.

ABSTRACT Poleroviruses are restricted to vascular phloem tissues from which they are transmitted by their aphid vectors and are not transmissible mechanically. Phloem limitation has been attributed to the absence of virus proteins either facilitating movement or counteracting plant defense. The polerovirus capsid is composed of two forms of coat protein, the major P3 protein and the minor P3/P5 protein, a translational readthrough of P3. P3/P5 is required for insect transmission and acts in trans to facilitate long-distance virus movement in phloem tissue. Specific potato leafroll virus mutants lacking part or all of the P5 domain moved into and infected nonvascular mesophyll tissue when the source-sink relationship of the plant ( Solanum sarrachoides ) was altered by pruning, with the progeny virus now being transmissible mechanically. However, in a period of months, a phloem-specific distribution of the virus was reestablished in the absence of aphid transmission. Virus from the new phloem-limited infection showed compensatory mutations that would be expected to restore the production of full-length P3/P5 as well as the loss of mechanical transmissibility. The data support our hypothesis that phloem limitation in poleroviruses presumably does not result from a deficiency in the repertoire of virus genes but rather results from P3/P5 accumulation under selection in the infected plant, with the colateral effect of facilitating transmission by phloem-feeding aphid vectors.

North American populations of 13 aphid species were tested for their a b i i to transmit isolates of Hum p a potyvirus recovered from infected trees in Adams, Franklin, and York Counties, PA. Seven species, Aphis fabae, A. spiraecolu, B s ~ c i a u d u s pmsiwe, Mabpolophiulls dizhodum, My= persime, Rhopalosiphum pad and Toxoptera dricida transmitted PPV in unrestricted probing tests utilizing 'Colmo' pea as both the PPV source and transmission bioassay indicator. A. spiraecola and M. persicae were the most efficient vectors with 86Y0 and 83Y0 of 100 seedlings infected when infested with 25-50 aphids each. M abhodwm and R pa& only occasionally transmitted PPV to pea (2%). Although T. citridu was an effective vector (36% in pea), it is restricted to Florida and does not occur in major stone fruit growing areas of North America The six aphids that did not transmit PPV included Acyrtlrosiphum pisum, Aphip gbcines, Aulacorthum solani, Macros@hum euphorbiae, Rhopalosiphum maidis, and Sitobion avenae. When given an acquisition period on PPV-infeeted peach seedlings and then allowed an unrestricted inoculation period on healthy peach seedlings, M. pwsicae, A. spiraecola, A. fjirbse, and persicwe transmitted PPV to 63%,31%, 38%, and 32% of the seedlings, respectively. When acquisition fed on infected peach fruit add allowed an unrestricted probing period on peach seedlings, the same aphid species transmitted PPV to 50%, 35%, 0%, and 0% of seedlings, respectively, in replicated tests. In summary, Pennsylvania isolates of PPV were transmitted effectively by indigenous aphid populations, were acquired and transmitted from fruit collected from infected orchard trees over two growing seasons, and had different degrees of transmission efficiency when acquired from foliar or fruit tissues. INTRODUCTION In 1999, Plum pox virus (PPV) was first identilied in North America infecting peach and plums in commercial orchards located in Adams County, Pennsylvania, USA. The following year, a second focus of PPV was discavered in Ontario, Canada. The origin and mode of entry of PPV to North America has not been determined. National and State surveys for PPV conducted by the Pennsylvania Department of Agriculture and the USDA Animal and Plant Health Inspection %Nice indicated a relatively localized focus of infection in southeastern Pennsylvania Therefore, a quarantine and eradication program was initiated in an attempt to eliminate the sources of infection. Biological studies were initiated to characterize the Pennsylvania PPV isolates, and to iden* potential vectors and alternate reservoir plant species. All PPV isolates collected in Roc. XIX? B on Fruit Tree V5-u~ Diseases

Plum pox (Sharka) is a serious virus disease of stone fruits caused by the Plum pox virus (PPV). To determine which species could function as potential hosts and virus reservoirs, we used aphid transmission and bud or chip grafting to evaluate the susceptibility of commercial, ornamental, and wild Prunus species to isolates of PPV found in Pennsylvania, USA. Following inoculation, test trees were observed for symptoms, analyzed by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), back-assayed to healthy peach, and followed through at least four cold-induced dormancy (CID) cycles over 4 years. Thirty-one of 33 Prunus species and cultivars were systemically infected following aphid transmission. Systemic infection could not be detected in P. cerasus (sour cherry) and P. × ‘Snofozam’ (Snow Fountains) despite repeated aphid inoculation attempts. Following grafting of PPV-infected budwood, all 40 species and varieties became infected, although species differed in their susceptibility. Within most species, some individual plants remained PPV negative throughout the study despite repeated inoculations. Infection in some species could be detected only through quantitative reverse transcription (RT)-PCR. Most species displayed clear symptoms, were highly positive by ELISA and RT-PCR, and could be back-inoculated into peach seedlings following CID. Our results indicate that a wide range of native and ornamental Prunus species are susceptible to U.S. isolates of PPV-D.

The coat protein (CP) of potato leafroll virus (PLRV) is the primary component of the capsid, and is a multifunctional protein known to be involved in vector transmission and virus movement within plant hosts, in addition to particle assembly. Thirteen mutations were generated in various regions of the CP and tested for their ability to affect virus-host and virus-vector interactions. Nine of the mutations prevented the assembly of stable virions. These mutants were unable to infect systemically four different host species. Furthermore, although virus replication and translation of the CP were similar for the mutants and wild-type virus in individual plant cells, the translation of the CP readthrough product was affected in several of the mutants. Four of the mutants were able to assemble stable particles and infect host plants systemically, similarly to the wild-type virus; however, two of the mutants were transmitted less efficiently by aphid vectors. Based on a computer-generated model of the PLRV CP, the mutations that prevented virion assembly were associated with subunit interfaces, while the amino acid alterations in the assembly-competent mutants were associated with surface loops. This and previous work indicates that the CP structural model has value in predicting the structural architecture of the virion.

ABSTRACT Sexual forms of two genotypes of the aphid Schizaphis graminum, one a vector, the other a nonvector of two viruses that cause barley yellow dwarf disease (Barley yellow dwarf virus [BYDV]-SGV, luteovirus and Cereal yellow dwarf virus-RPV, polerovirus), were mated to generate F1 and F2 populations. Segregation of the transmission phenotype for both viruses in the F1 and F2 populations indicated that the transmission phenotype is under genetic control and that the parents are heterozygous for genes involved in transmission. The ability to transmit both viruses was correlated within the F1 and F2 populations, suggesting that a major gene or linked genes regulate the transmission. However, individual hybrid genotypes differed significantly in their ability to transmit each virus, indicating that in addition to a major gene, minor genes can affect the transmission of each virus independently. Gut and salivary gland associated transmission barriers were identified in the nonvector parent and some progeny, while other progeny possessed only a gut barrier or a salivary gland barrier. Hemolymph factors do not appear to be involved in determining the transmission phenotype. These results provide direct evidence that aphid transmission of luteoviruses is genetically regulated in the insect and that the tissue-specific barriers to virus transmission are not genetically linked.

A Wheat streak mosaic virus (WSMV) genome lacking HC-Pro was constructed and confirmed by reverse transcription-PCR to systemically infect wheat, oat, and corn. Coupled in vitro transcription/translation reactions indicated that WSMV P1 proteinase cleaved the polyprotein at the P1/P3 junction of the HC-Pro null mutant. The WSMV HC-Pro null mutant was competent for virion formation, but the virus titer was reduced 4.5-fold relative to that of the wild type. Collectively, these results indicate that WSMV HC-Pro is dispensable for replication and movement, two essential processes that are disrupted by point and small-insertion mutations introduced into potyvirus HC-Pro.

Plum pox, an invasive disease recently identified in Pennsylvania stone fruit orchards, is caused by the aphid-transmitted Plum pox virus (genus Potyvirus, family Potyviridae, PPV). To identify potential vectors, we described the aphid species communities and the seasonal dynamics of the dominant aphid species within Pennsylvania peach orchards. Aphids were trapped weekly in 2002 and 2003 from mid-April through mid-November within two central Pennsylvania orchards by using yellow and green water pan traps. In total, 42 aphid species were identified from both orchards over 2 yr. Within orchards, actual species richness ranged from 24 to 30 species. The Abundance Based Coverage Estimator predicted species richness to range from 30 to 36 species, indicating that trap catches were identifying most aphid species expected to occur in the orchard. Three species, Rhopalosiphum maidis (Fitch), Aphis spiraecola Patch, and Myzus persicae (Sulzer), were consistently dominant across locations and years. Orchard-trapped populations of these three species peaked in a similar chronological sequence each year. As expected, trap color influenced the total number and distribution of the predominate species collected. However, the same dominant species occurred in both yellow and green traps. Based on the seasonal population dynamics reported here and on published vector efficacy studies, the most probable significant PPV vector was identified as A. spiraecola. If the PPV pathogen escapes current quarantine or if subsequent reintroductions of PPV occur, these data will be useful for developing plum pox management strategies.

The eriophyid mite transmitted Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) shares a common genome organization with aphid transmitted species of the genus Potyvirus. Although both tritimoviruses and potyviruses encode helper component-proteinase (HC-Pro) homologues (required for nonpersistent aphid transmission of potyviruses), sequence conservation is low (amino acid identity, approximately 16%), and a role for HC-Pro in semipersistent transmission of WSMV by the wheat curl mite (Aceria tosichella [Keifer]) has not been investigated. Wheat curl mite transmissibility was abolished by replacement of WSMV HC-Pro with homologues of an aphid transmitted potyvirus (Turnip mosaic virus), a rymovirus (Agropyron mosaic virus) vectored by a different eriophyid mite, or a closely related tritimovirus (Oat necrotic mottle virus; ONMV) with no known vector. In contrast, both WSMV-Sidney 81 and a chimeric WSMV genome bearing HC-Pro of a divergent strain (WSMV-El Batán 3; 86% amino acid sequence identity) were efficiently transmitted by A. tosichella. Replacing portions of WSMV-Sidney 81 HC-Pro with the corresponding regions from ONMV showed that determinants of wheat curl mite transmission map to the 5'-proximal half of HC-Pro. WSMV genomes bearing HC-Pro of heterologous species retained the ability to form virions, indicating that loss of vector transmissibility was not a result of failure to encapsidate. Although titer in systemically infected leaves was reduced for all chimeric genomes relative to WSMV-Sidney 81, titer was not correlated with loss of vector transmissibility. Collectively, these results demonstrate for the first time that HC-Pro is required for virus transmission by a vector other than aphids.

Natural spread of Plum pox potyvirus (PPV) occurs by aphid transmission, grafting, or movement of infected nursery stock. Two proven aphid vectors of Plum pox virus, Myzus persicae and/or Brachycaudus persicae were used to transmit North American isolates of PPV to a range of commercial and ornamental Prunus species to test the ability of these species to function as PPV reservoirs. Inoculum sources were 'Lovell' peach seedlings infected with the PPV-PENN-3 or PPV-PENN-4 isolates by previous aphid transmission. Aphids were starved for 30 min, placed onto either detached symptomatic PPV-infected peach leaves or intact peach seedlings, and then allowed a 3-day acquisition-inoculation feeding period from infected tissues to the healthy Prunus seedlings. Seedlings were sprayed with insecticide, placed onto greenhouse benches and observed for 6 to 8 weeks for development of symptoms. Test plants were analyzed by ELISA and PCR, and then back-assayed to 'Lovell' peach seedlings by healthy Myzuspersicae. Test plants then were vernalized at 4.1degrees C for 8-10 weeks. At four-weeks post-vernalization, plants were tested by ELISA and/or RT-PCR to verify systemic infection. Fourteen of the 15 Prunus species tested positive for PPV infection following the initial aphid transmission. Twelve of the 14 infected species were verified to function as potential PPV sources in subsequent aphid transmissions to peach. To date, at least 10 of the 14 Prunus species maintained systemic PPV infection following vernalization. Only A cerasus (sour cherry) tested negative to PPV infection. Other cherry species, including A avium, P. cistena, and A serrulata maintained systemic infection through vernalization. Results suggest that all species tested except A cerasus, could function as PPV hosts associated with unintentional movement of infected nursery stock and as reservoirs for aphid transmission.

North American populations of 13 aphid species were tested for their ability to transmit isolates of Plum pox potyvirus recovered from infected trees in Adams, Franklin, and York Counties, PA. Seven species, Aphis fabae, A. spiraecola, Brachycaudus persicae, Metopolophium dirhodum, Myzus persicae, Rhopalosiphum padi and Toxoptera citricida transmitted PPV in unrestricted probing tests utilizing 'Colmo' pea as both the PPV source and transmission bioassay indicator. A. spiraecola and M. persicae were the most efficient vectors with 86% and 83% of 100 seedlings infected when infested with 25-50 aphids each. M. dirhodum and R. padi only occasionally transmitted PPV to pea (2%). Although T. citricida was an effective vector (36% in pea), it is restricted to Florida and does not occur in major stone fruit growing areas of North America. The six aphids that did not transmit PPV included Acyrthosiphum pisum, Aphis glycines, Aulacorthum solani, Macrosiphum euphorbiae, Rhopalosiphum maidis, and Sitobion avellae. When given an acquisition period on PPV-infected peach seedlings and then allowed an unrestricted inoculation period on healthy peach seedlings, M. persicae, A. spiraecola, A. fabae, and B. persicae transmitted PPV to 63%, 31%, 38%, and 32% of the seedlings, respectively. When acquisition fed on infected peach fruit and allowed an unrestricted probing period on peach seedlings, the same aphid species transmitted PPV to 50%, 35%, 0%, and 0% of seedlings, respectively, in replicated tests. In summary, Pennsylvania isolates of PPV were transmitted effectively by indigenous aphid populations, were acquired and transmitted from fruit collected from infected orchard trees over two growing seasons, and had different degrees of transmission efficiency when acquired from foliar or fruit tissues.