Impact of Timing and Method of Virus Inoculation on the Severity of Wheat Streak Mosaic Disease.

Wheat streak mosaic virus (WSMV), transmitted by the wheat curl mite Aceria tosichella, frequently causes significant yield loss in winter wheat throughout the Great Plains of the United States. A field study was conducted in the 2013-14 and 2014-15 growing seasons to compare the impact of timing of WSMV inoculation (early fall, late fall, or early spring) and method of inoculation (mite or mechanical) on susceptibility of winter wheat cultivars Mace (resistant) and Overland (susceptible). Relative chlorophyll content, WSMV incidence, and yield components were determined. The greatest WSMV infection occurred for Overland, with the early fall inoculations resulting in the highest WSMV infection rate (up to 97%) and the greatest yield reductions relative to the control (up to 94%). In contrast, inoculation of Mace resulted in low WSMV incidence (1 to 28.3%). The findings from this study indicate that both method of inoculation and wheat cultivar influenced severity of wheat streak mosaic; however, timing of inoculation also had a dramatic influence on disease. In addition, mite inoculation provided much more consistent infection rates and is considered a more realistic method of inoculation to measure disease impact on wheat cultivars.

Effect of Temperature on Wheat Streak Mosaic Disease Development in Winter Wheat.

Temperature is one of the key factors that influence viral disease development in plants. In this study, temperature effect on Wheat streak mosaic virus (WSMV) replication and in planta movement was determined using a green fluorescent protein (GFP)-tagged virus in two winter wheat cultivars. Virus-inoculated plants were first incubated at 10, 15, 20, and 25°C for 21 days, followed by 27°C for 14 days; and, in a second experiment, virus-inoculated plants were initially incubated at 27°C for 3 days, followed by 10, 15, 20, and 25°C for 21 days. In the first experiment, WSMV-GFP in susceptible 'Tomahawk' wheat at 10°C was restricted at the point of inoculation whereas, at 15°C, the virus moved systemically, accompanied with mild symptoms, and, at 20 and 25°C, WSMV elicited severe WSMV symptoms. In resistant 'Mace' wheat (PI 651043), WSMV-GFP was restricted at the point of inoculation at 10 and 15°C but, at 20 and 25°C, the virus infected systemically with no visual symptoms. Some plants that were not systemically infected at low temperatures expressed WSMV-GFP in regrowth shoots when later held at 27°C. In the second experiment, Tomahawk plants (100%) expressed systemic WSMV-GFP after 21 days at all four temperature levels; however, systemic WSMV expression in Mace was delayed at the lower temperatures. These results indicate that temperature played an important role in WSMV replication, movement, and symptom development in resistant and susceptible wheat cultivars. This study also demonstrates that suboptimal temperatures impair WSMV movement but the virus rapidly begins to replicate and spread in planta under optimal temperatures.

Introgression of chromosome segments from multiple alien species in wheat breeding lines with wheat streak mosaic virus resistance.

Pyramiding of alien-derived Wheat streak mosaic virus (WSMV) resistance and resistance enhancing genes in wheat is a cost-effective and environmentally safe strategy for disease control. PCR-based markers and cytogenetic analysis with genomic in situ hybridisation were applied to identify alien chromatin in four genetically diverse populations of wheat (Triticum aestivum) lines incorporating chromosome segments from Thinopyrum intermedium and Secale cereale (rye). Out of 20 experimental lines, 10 carried Th. intermedium chromatin as T4DL*4Ai#2S translocations, while, unexpectedly, 7 lines were positive for alien chromatin (Th. intermedium or rye) on chromosome 1B. The newly described rye 1RS chromatin, transmitted from early in the pedigree, was associated with enhanced WSMV resistance. Under field conditions, the 1RS chromatin alone showed some resistance, while together with the Th. intermedium 4Ai#2S offered superior resistance to that demonstrated by the known resistant cultivar Mace. Most alien wheat lines carry whole chromosome arms, and it is notable that these lines showed intra-arm recombination within the 1BS arm. The translocation breakpoints between 1BS and alien chromatin fell in three categories: (i) at or near to the centromere, (ii) intercalary between markers UL-Thin5 and Xgwm1130 and (iii) towards the telomere between Xgwm0911 and Xbarc194. Labelled genomic Th. intermedium DNA hybridised to the rye 1RS chromatin under high stringency conditions, indicating the presence of shared tandem repeats among the cereals. The novel small alien fragments may explain the difficulty in developing well-adapted lines carrying Wsm1 despite improved tolerance to the virus. The results will facilitate directed chromosome engineering producing agronomically desirable WSMV-resistant germplasm.

Differential Transmission of Two Isolates of Wheat streak mosaic virus by Five Wheat Curl Mite Populations.

Wheat streak mosaic virus (WSMV), type member of the genus Tritimovirus in the family Potyviridae, is an economically important virus causing annual average yield losses of approximately 2 to 3% in winter wheat across the Great Plains. The wheat curl mite (WCM), Aceria tosichella, transmits WSMV along with two other viruses found throughout the Great Plains of the United States. Two common genotypes of WSMV (Sidney 81 and Type) in the United States share 97.6% nucleotide sequence identity but their transmission relationships with the WCM are unknown. The objective of this study was to determine transmission of these two isolates of WSMV by five WCM populations ('Nebraska', 'Montana', 'South Dakota', 'Type 1', and 'Type 2'). Nonviruliferous mites from each population were reared on wheat source plants mechanically inoculated with either Sidney 81 or Type WSMV isolates. For each source plant, individual mites were transferred to 10 separate test plants and virus transmission was determined by a double-antibody sandwich enzyme-linked immunosorbent assay. Source plants were replicated nine times for each treatment (90 individual mite transfers). Results indicate that three mite populations transmitted Sidney 81 at higher rates compared with Type. Two mite populations (Nebraska and Type 2) transmitted Sidney 81 and Type at higher rates compared with the other three populations. Results from this study demonstrate that interactions between virus isolates and mite populations influence the epidemiology of WSMV.

Impact of Wheat streak mosaic virus and Triticum mosaic virus Coinfection of Wheat on Transmission Rates by Wheat Curl Mites.

Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are transmitted by the wheat curl mite (WCM, Aceria tosichella), and coinfections of wheat by these viruses are common in the field. Previous work has shown that mite genotypes vary in their ability to transmit TriMV. However, the degree to which coinfection of wheat modifies WCM vector competence has not been studied. The objective was to determine whether mite genotypes differed in virus transmission ability when feeding on wheat coinfected by WSMV and TriMV. First, WCM genotype type 2 was used to determine virus transmission rates from mock-, WSMV-, TriMV-, and coinfected wheat plants. Transmission rates were determined by using single-mite transfers from replicated source plants. Coinfection reduced WSMV transmission by type 2 WCM from 50 to 35.6%; however, coinfection increased TriMV transmission from 43.3 to 56.8%. Mite survival on single-mite transfer test plants indicates that the reduction in WSMV transmission may result from poor mite survival when TriMV is present. In a second study, two separate colonies of WCM genotype type 1 were tested to assess the impact of coinfection on transmission. Type 1 mites did not transmit TriMV from coinfected plants but the two colonies varied in transmission rates for WSMV (20.9 to 36.5%). Even though these changes in mite transmission rates are moderate, they help explain the high relative incidence of TriMV-positive plants that are coinfected with WSMV in field observations. These findings begin to demonstrate the complicated interactions found in this mite-virus complex.

The Effect of Temperature, Relative Humidity, and Virus Infection Status on off-host Survival of the Wheat Curl Mite (Acari: Eriophyidae).

The wheat curl mite, Aceria tosichella Keifer, is an eriophyid pest of wheat, although its primary economic impact on wheat is due to the transmission of Wheat streak mosaic (WSMV), Wheat mosaic (also known as High Plains virus), and Triticum mosaic (TriMV) viruses. These viruses cause significant annual losses in winter wheat production throughout the western Great Plains. Temperature and humidity are factors that often influence arthropod survival, especially during dispersal from their hosts, yet the impact of these two factors on off-host survival has not been documented for wheat curl mite. Pathogen-infected host plants often influence the biology and behavior of vectors, yet it is not known if virus-infected wheat affects off-host survival of wheat curl mite. The objectives of this study were to 1) determine if temperature, relative humidity, and mite genotype impact off-host survival of wheat curl mite and 2) determine the effect of WSMV- and TriMV-infected host plants on off-host survival of wheat curl mite. Temperature and relative humidity significantly affected off-host survival of wheat curl mite. Length of survival decreased with increasing temperature (106.2 h at 10°C and 17.0 h at 30°C) and decreasing relative humidity (78.1 h at 95 and 21.3 h at 2%). Mites from TriMV-infected host plants had ∼20% reduction in survival at 20°C compared with those from WSMV-infected plants. The duration of off-host survival of wheat curl mite is influenced by environmental conditions. Management strategies that target a break in host presence will greatly reduce mite densities and virus spread and need to account for these limits.

Quantification of Yield Loss Caused by Triticum mosaic virus and Wheat streak mosaic virus in Winter Wheat Under Field Conditions.

Triticum mosaic virus (TriMV) and Wheat streak mosaic virus (WSMV) infect winter wheat (Triticum aestivum) in the Great Plains region of the United States. The two viruses are transmitted by wheat curl mites (Aceria tosichella), which also transmit High Plains virus. In a field study conducted in 2011 and 2012, winter wheat cultivars Millennium (WSMV-susceptible) and Mace (WSMV-resistant) were mechanically inoculated with TriMV, WSMV, TriMV+WSMV, or sterile water at the two-leaf growth stage. Chlorophyll meter (soil plant analysis development [SPAD]) readings, area under the SPAD progress curve (AUSPC), grain yield (=yield), yield components (spikes/m2, kernels/spike, 1,000-kernel weight), and aerial dry matter were determined. In Millennium, all measured variables were significantly reduced by single or double virus inoculation, with the greatest reductions occurring in the double-inoculated treatment. Among the yield components, the greatest reductions occurred in spikes/m2. In Mace, only AUSPC was significantly reduced by the TriMV+WSMV treatment in 2012. There was a significant (P ≤ 0.05), negative linear relationship between SPAD readings and day of year in all inoculation treatments in Millennium and in the TriMV+WSMV treatment in Mace. There were significant (P ≤ 0.05), positive linear relationships between yield and SPAD readings and between yield and aerial dry matter in Millennium but not in Mace. The results from this study indicate that under field conditions, (i) Mace, a WSMV-resistant cultivar, is also resistant to TriMV, and (ii) double inoculation of winter wheat by TriMV and WSMV exacerbates symptom expression and yield loss in a susceptible cultivar.

Effects of Planting Date and Barley Variety on Russian Wheat Aphid (Hemiptera: Aphididae) Populations in Colorado, Kansas, and Nebraska.

The Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Hemiptera: Aphididae), is an important pest in the western Great Plains of the United States, where it causes hundreds of millions of dollars of losses to barley and wheat production through reduced yields. Experiments to evaluate the effect of early planting and resistance in barley (Hordeum vulgare L.) on D. noxia were conducted at Fort Collins, CO; Tribune, KS; and Sidney, NE, in 2007, 2008, and 2009. Treatments included two planting dates and four cultivars, the D. noxia-resistant barley cultivars 'Stoneham' (Otis*4/STARS 9577B) and 'Sidney' (Otis*4/STARS 9301B), the susceptible cultivar 'Otis', and Otis treated with thiamethoxam. In tiller samples collected from May through early July, consistently lower D. noxia populations were found in plots planted ≍30 d earlier than normal at Fort Collins in all three years, and at Tribune in 2007. With one location-year exception, lower D. noxia populations occurred on plants of resistant varieties or the susceptible variety Otis treated with thiamethoxam than on untreated Otis plants. There were no significant differences in D. noxia populations produced on plants of either resistant variety and susceptible Otis plants treated with thiamethoxam. Interactions between resistant varieties and early planting resulted in reduced D. noxia populations at Fort Collins in 2007 and 2009, and at Tribune and Sidney in 2007. Planting D. noxia-resistant barley varieties, planting varieties earlier than normal, and the synergistic effect of resistant variety and early planting can significantly reduce D. noxia infestations on barley in the western High Plains.

Occurrence and Distribution of Triticum mosaic virus in the Central Great Plains.

Wheat curl mite (WCM)-transmitted viruses-namely, Wheat streak mosaic virus (WSMV), Triticum mosaic virus (TriMV), and the High Plains virus (HPV)-are three of the wheat-infecting viruses in the central Great Plains of the United States. TriMV is newly discovered and its prevalence and incidence are largely unknown. Field surveys were carried out in Colorado, Kansas, Nebraska, and South Dakota in spring and fall 2010 and 2011 to determine TriMV prevalence and incidence and the frequency of TriMV co-infection with WSMV or HPV in winter wheat. WSMV was the most prevalent and was detected in 83% of 185 season-counties (= s-counties), 73% of 420 season-fields (= s-fields), and 35% of 12,973 samples. TriMV was detected in 32, 6, and 6% of s-counties, s-fields, and samples, respectively. HPV was detected in 34, 15, and 4% of s-counties, s-fields, and samples, respectively. TriMV was detected in all four states. In all, 91% of TriMV-positive samples were co-infected with WSMV, whereas WSMV and HPV were mainly detected as single infections. The results from this study indicate that TriMV occurs in winter wheat predominantly as a double infection with WSMV, which will complicate breeding for resistance to WCM-transmitted viruses.

Western bean cutworm survival and the development of economic injury levels and economic thresholds in field corn.

Western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is a native pest of dry beans (Phaseolus vulgaris L.) and corn (Zea mays L.). Historically, the western bean cutworm was distributed in the western United States, but since 1999 eastward expansion has been observed. In corn, economic impact is caused by larval ear feeding. Information on western bean cutworm biology, ecology, and economic impact is relatively limited, and the development of economic injury levels (EILs) and economic thresholds (ETs) is required for more effective management. Studies during 2008-2011, across three ecoregions of Nebraska, sought to characterize western bean cutworm survival and development of EILs and ETs. Calculations of EILs and ETs incorporated the dynamics of corn price, management cost, and pest survival. The results from the current study demonstrated low larval survival of this species (1.51-12.82%). The mean yield loss from one western bean cutworm larva per plant was 945.52 kg/ha (15.08 bu/acre), based on 74,100 plants per ha. Economic thresholds are expressed as a percentage of plants with at least one egg mass. This study is the first study that explicitly incorporates variable management costs and crop values into western bean cutworm EIL calculations, and larval survival into ET calculations.

Effects of Single and Double Infections of Winter Wheat by Triticum mosaic virus and Wheat streak mosaic virus on Yield Determinants.

Triticum mosaic virus (TriMV) is a recently discovered virus infecting wheat (Triticum aestivum) in the Great Plains region of the United States. It is transmitted by wheat curl mites (Aceria tosichella) which also transmit Wheat streak mosaic virus (WSMV) and Wheat mosaic virus. In a greenhouse study, winter wheat 'Millennium' (WSMV susceptible) and 'Mace' (WSMV resistant) were mechanically inoculated with TriMV, WSMV, TriMV+WSMV, or sterile water at the two-leaf growth stage. At 28 days after inoculation, final chlorophyll meter (soil plant analysis development [SPAD]) readings, area under the SPAD progress curve (AUSPC), the number of tillers per plant, shoot and root weight, and total nitrogen and carbon content were determined. In Millennium, all measured variables were significantly reduced by single or double virus infections, with the greatest reductions occurring in the double-infection treatment. In Mace, only final SPAD readings, AUSPC, and total nitrogen were significantly reduced by single or double virus infections. There was a significant (P ≤ 0.05), positive linear relationship between SPAD readings and shoot weight in Millennium but not in Mace. The relationship between total nitrogen and shoot weight was positive, linear, and significant in both cultivars. The results from this study indicate that Mace, a WSMV-resistant cultivar, is also resistant to TriMV, and double infection of winter wheat by TriMV and WSMV exacerbates symptom expression and loss of biomass in susceptible cultivars.

Cost-effective binomial sequential sampling of western bean cutworm, Striacosta albicosta (Lepidoptera: Noctuidae), egg masses in corn.

Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is a native pest of dry beans (Phaseolus vulgaris L.) and corn (Zea mays L.). As a result of larval feeding damage on corn ears, S. albicosta has a narrow treatment window; thus, early detection of the pest in the field is essential, and egg mass sampling has become a popular monitoring tool. Three action thresholds for field and sweet corn currently are used by crop consultants, including 4% of plants infested with egg masses on sweet corn in the silking-tasseling stage, 8% of plants infested with egg masses on field corn with approximately 95% tasseled, and 20% of plants infested with egg masses on field corn during mid-milk-stage corn. The current monitoring recommendation is to sample 20 plants at each of five locations per field (100 plants total). In an effort to develop a more cost-effective sampling plan for S. albicosta egg masses, several alternative binomial sampling plans were developed using Wald's sequential probability ratio test, and validated using Resampling for Validation of Sampling Plans (RVSP) software. The benefit-cost ratio also was calculated and used to determine the final selection of sampling plans. Based on final sampling plans selected for each action threshold, the average sample number required to reach a treat or no-treat decision ranged from 38 to 41 plants per field. This represents a significant savings in sampling cost over the current recommendation of 100 plants.

Response of Pemphigus betae (Hemiptera: Aphididae) and Beneficial Epigeal Arthropod Communities to Sugarbeet Plant Density and Seed-Applied Insecticide in Western Nebraska.

This study investigated the impact of a neonicotinoid seed-applied insecticide (Poncho Beta) and two plant densities (86,487 and 61,776 plants per hectare) on the sugarbeet root aphid (Pemphigus betae Doane), beneficial epigeal arthropods, and selected crop yield parameters in sugarbeet (Beta vulgaris L. var. vulgaris). Ground beetles and centipedes were the most commonly collected taxa during 2012 and 2013, respectively. Centipede, spider, and rove beetle activity densities were not affected by the seed-applied insecticide, whereas plant density had a marginal effect on centipede activity density during 2012. Ground beetle species richness, diversity, and evenness were also not impacted by the seed treatments. However, during 2013, ground beetle activity density was significantly higher in plots planted with untreated sugarbeet seeds due to the abundance of Bembidion quadrimaculatum oppositum Say. Sugarbeet root aphid populations were significantly higher in the untreated plots during both years. In 2012, sugarbeet tonnage and sugar yield were higher under the low plant density treatment, while higher sugar content was recorded from the seed-applied insecticide plots (2013). Seed-applied neonicotinoids and plant density had little impact on beneficial epigeal arthropod activity density. Seed treatment did result in decreased root aphid populations; however, these reductions were not sufficient to be considered as an adequate control. This limited aphid control likely contributed to inconsistent effects on yield parameters.

Fully biologically active in vitro transcripts of the eriophyid mite-transmitted wheat streak mosaic tritimovirus.

ABSTRACT Infectious RNA of wheat streak mosaic virus (WSMV) has been produced using a full-length cDNA clone as a template for in vitro transcription with SP6 RNA polymerase. Infectivity was dependent on the use of template plasmid DNA that had not undergone spontaneous rearrangement during amplification in Escherichia coli. The presence of WSMV in systemically infected wheat plants inoculated with in vitro transcripts was confirmed by reverse-transcription polymerase chain reaction of the WSMV P3 gene and by accumulation of WSMV coat protein as detected by immunoblotting. Maintenance of the full-length WSMV cDNA in the high copy number plasmid pUC18 was problematic because of spontaneous rearrangement of WSMV sequences during growth in liquid media for more than 8 h or if the clone was subcultured. Stability of the WSMV cDNA clone was improved by the use of the low copy number plasmid pACYC177, and it could be grown in large scale volumes (up to 1 liter) of liquid culture for 14 h without noticeable rearrangements. Both the original WSMV culture and the progeny virus derived from infectious in vitro transcripts were efficiently transmitted by the natural eriophyid mite vector Aceria tosichella. This is the first report of infectious in vitro transcripts for any eriophyid mite-transmitted plant virus and represents the only monopartite member of the family Potyviridae infecting monocotyledonous hosts for which infectious in vitro transcripts are available.

Development of Serological Procedures for Rapid and Reliable Detection of Wheat Streak Mosaic Virus in a Single Wheat Curl Mite.

Wheat streak mosaic virus (WSMV) is transmitted by the wheat curl mite (WCM), Aceria tosichella. Immunofluorescent and dot-immunobinding assays were developed to detect the presence of WSMV in single WCM. Virus-specific immunofluorescent microscopy detected WSMV near the anterior end of viruliferous WCM. With dot-immunobinding assay, WSMV was detected in WCM fed on WSMV-infected wheat (Triticum aestivum) but not in WCM maintained on healthy plants. Both immunofluorescent and dot-immunobinding assays were sufficiently sensitive to detect WSMV in individual WCM, providing a means to determine the percentage of viruliferous WCM in field collections.

Mixed Infection of Hard Red Winter Wheat with High Plains Virus and Wheat Streak Mosaic Virus from Wheat Curl Mites in Nebraska.

A new disease of wheat and corn caused by the High Plains virus (HPV) has been observed in the High Plains region of western United States. HPV is transmitted by the wheat curl mite, Aceria tosichella, which is also the vector of wheat streak mosaic virus (WSMV). In the field it is extremely difficult to visually differentiate plants infected with WSMV from those with HPV. An indirect protein-A sandwich enzyme-linked immunosorbent assay (PAS-ELISA) and Western blot analysis were used to identify WSMV and HPV. Samples of wheat curl mites were collected from arbitrarily chosen sites from commercial wheat plantings in 1995 and 1996 and used to infest caged wheat plants. After 3 weeks, leaf samples were harvested and assayed. Both Western blot analysis and PAS-ELISA were effective at identifying samples positive for WSMV and HPV, both alone and in mixed infections. Western blot results showed that over the 2 years, 65% of the samples were positive for WSMV, 46% were positive for HPV, and mixed infections were found in 40% of the samples. HPV presence was verified with similar results from field collected plant samples. These levels of virus indicate an unexpectedly high incidence of HPV in wheat curl mite populations in Nebraska.

On-plant movement and feeding of western bean cutworm (Lepidoptera: Noctuidae) early instars on corn.

Western bean cutworm, Striacosta albicosta (Smith), has undergone a recent eastward expansion from the western U.S. Corn Belt to Pennsylvania and parts of Canada. Little is known about its ecology and behavior, particularly during the early instars, on corn (Zea mays L.). There is a narrow treatment window for larvae, and early detection of the pest in the field is essential. An understanding of western bean cutworm larval feeding and early-instar dispersal is essential to understand larval survival and establishment in corn. Studies were conducted in 2009 through 2011 in Nebraska to determine the feeding and dispersal of early-instar western bean cutworm on corn. The treatment design was a factorial with three corn stages (pretassel, tassel, and posttassel) and five corn plant zones (tassel, above ear, primary ear, secondary ear, and below ear) in a randomized complete block design. The effects of different corn tissues on larval survival and development were investigated in laboratory studies in a randomized complete block design during 2009 and 2011. Treatments were different corn tissues (leaf alone, leaf with developing tassel, pollen, pollen plus silk, and silk alone). Results demonstrated that neonate larvae move to the upper part of the plant, independent of corn stage. Larval growth was optimal when fed on tassel tissue. Overall results indicated a selective benefit for movement of the early instar to upper part of the plant.

Substitution of conserved cysteine residues in wheat streak mosaic virus HC-Pro abolishes virus transmission by the wheat curl mite.

Substitutions in the amino-proximal region of wheat streak mosaic virus (WSMV) HC-Pro were evaluated for effects on transmission by the wheat curl mite (Aceria tosichella Keifer). Alanine substitution at cysteine residues 16, 46 and 49 abolished vector transmission. Although alanine substitution at Cys(20) had no effect, substitution with arginine reduced vector transmission efficiency. Random substitutions at other positions (Lys(7) to Asn, Asn(19) to Ile, and Arg(45) to Lys) did not affect vector transmission. These results suggest that a zinc-finger-like motif (His(13)-X2-Cys(16)-X29-Cys(46)-X2-Cys(49)) in WSMV HC-Pro is essential for vector transmission.

Three distinct mechanisms facilitate genetic isolation of sympatric wheat streak mosaic virus lineages.

Cross-protection and vector transmission bottlenecks have been proposed as mechanisms facilitating genetic isolation of sympatric viral lineages. Molecular markers were used to monitor establishment and resolution of mixed infections with genetically defined strains of wheat streak mosaic virus (WSMV). Two closely related WSMV strains from the U.S. (Type and Sidney 81) exhibited reciprocal cross-protection in wheat, confirming this classic phenomenon as a mechanism of genetic isolation. In contrast, cross-protection between either U.S. strain and the divergent El Batán 3 strain from Mexico was unilateral, erratic, and only partially effective. Distribution of WSMV strains within individual leaves of plants supporting a mixed infection of Type and Sidney 81 was spatially nonuniform. Strain distribution among individual tillers of coinfected plants also was heterogeneous, with some containing either Type or Sidney 81 alone and some containing both. Transmission by wheat curl mites, acquiring virus from source plants simultaneously infected with both Type and Sidney 81, often resulted in test plants bearing only a single WSMV strain. Spatial subdivision of virus strains within coinfected plants likely contributed to vector transmission bottlenecks during acquisition. Collectively, these three distinct mechanisms enhance genetic isolation of individual viral lineages, and together with stochastic processes, may explain generation and maintenance of genetic diversity in field populations.