Kanlow was tolerant to PMV infection at 24°C but not at 32°C
The reaction of switchgrass cvs. Summer and Kanlow to systemic infection by PMV at 24°C and 32°C was determined at 21 dpi on the basis of symptomatology. Switchgrass plants exhibiting chlorotic streaks and spots, mosaic, and mottling symptoms in upper noninoculated leaves were considered positive for infection. At 24°C, 60% of Summer plants inoculated with PMV alone exhibited symptoms, whereas only 15% of Kanlow plants developed systemic symptoms (Fig. 1). These data indicated that, at 24°C, the Kanlow population exhibited a higher frequency of genotypes with resistance to PMV compared to the Summer population. In contrast, the infection frequency at 32°C for Summer (75%) and Kanlow (70%) plants inoculated with PMV alone was higher than the respective plants held at 24°C, with the difference between Kanlow populations being significant (Fig. 1). None of the mock-inoculated plants of Summer or Kanlow exhibited symptoms. These data suggest that irrespective of temperature, the Summer population was susceptible to systemic infection by PMV. In contrast, the Kanlow population was tolerant to PMV at 24°C but not at 32°C indicating temperature-sensitive resistance against PMV.
PMV elicited efficient local infection on switchgrass cv. Kanlow
The temperature-dependent resistance of Kanlow to PMV was further examined to detect if this virus can cause a local infection that facilitates cell-to-cell movement on inoculated leaves before entering the vasculature required for systemic infection. Total RNA extracted from inoculated leaves of both cultivars at 7 dpi was used for RT-PCR. The RT-PCR assay revealed that PMV was detected in 95% of inoculated leaves of Kanlow at both temperature regimes (Fig. 2), suggesting that PMV initiated local infection and established cell-to-cell movement in inoculated leaves. The local infection of PMV in Kanlow was found to be identical to that of 95% of infections observed in inoculated leaves of Summer at 24°C and 32°C (Fig. 2). PMV was not detected in any mock-inoculated leaf (data not shown). These data suggested that PMV facilitated efficient cell-to-cell movement in inoculated leaves of Kanlow.
Interaction between PMV and SPMV on systemic infection of Summer and Kanlow
Interaction between PMV and SPMV on systemic infection was discerned by comparing the results of inoculations with PMV alone with those with PMV+SPMV. At 24°C and 32°C, co-inoculation of Summer plants with PMV and SPMV caused a statistically significant increase in the number of plants systemically infected, as indicated by the presence of symptoms, compared to those with PMV alone. At 24°C, inoculation of Summer seedlings with PMV+SPMV elicited symptoms in 90% of plants compared to 60% plants exhibiting symptoms by PMV alone (Fig. 1). At 32°C, 75% and 100% of Summer plants inoculated with PMV or PMV+SPMV, respectively, exhibited chlorotic streaks and mottling symptoms (Fig. 1). In contrast, there was no significant difference in infection of Kanlow plants by PMV with or without SPMV at either temperature as to the percentage of plants exhibiting systemic symptoms (Fig. 1). At 24°C, PMV+SPMV elicited symptoms in 20% of Kanlow plants compared to 15% of symptomatic infection by PMV. At 32°C, 70% and 75% of Kanlow plants produced symptomatic infection by PMV or PMV+SPMV, respectively (Fig. 1). These data suggest that the interaction between PMV and SPMV in switchgrass is cultivar dependent, manifested in Summer but not in Kanlow.
Co-infection of PMV and SPMV augmented asymptomatic systemic infection in Kanlow
To verify systemic infection determined by symptomology and to examine the possibility of asymptomatic infection by PMV in both switchgrass cultivars, total RNA isolated from the 4th upper fully expanded leaf was used as a template for RT-qPCR . The RT-qPCR analysis revealed that all treatments have a higher number of infected plants than indicated by symptomatology (Fig. 1), except for PMV+SPMV-infected Summer at 32°C, in which 100% systemic infection was indicated by both methods. The additional plants shown to be infected by RT-qPCR were considered to be asymptomatically infected. For Summer plants incubated at 24°C, RT-qPCR revealed that 70% and 100% of plants were found to be positive for PMV and PMV+SPMV infections, respectively, compared to 60% and 90% of visually assessed symptomatic infections (Fig. 1); thus, 10% asymptomatic infections for both virus treatments. In Summer at 32°C, 100% of PMV inoculated plants were infected based on RT-qPCR assay compared to 75% of plants being symptomatic, leading to 25% additional asymptomatic infections (Fig. 1). All of the Summer plants inoculated with PMV+SPMV were infected at 32°C based on both assessment methods (Fig. 1).
RT-qPCR analysis indicated that, at 24°C, PMV or PMV+SPMV infected 35% and 70%, respectively, of Kanlow plants compared to 15% and 20%, respectively, being infected based on symptoms (Fig. 1). These data revealed that, at 24°C, PMV and PMV+SPMV caused asymptomatic systemic infection in 20% and 50% of Kanlow plants, respectively. At 32°C, RT-qPCR detected systemic infections in 95% and 100% of Kanlow plants inoculated with PMV or PMV+SPMV, respectively, compared to 70% and 75% symptomatic infections (Fig. 1). These data indicated 25% of asymptomatic infections resulted from both virus treatments in Kanlow at 32°C. Taken together, the data indicate a proportion of the Summer and Kanlow population to be susceptible to asymptomatic infection by PMV. That proportion was substantially increased in Kanlow at 24°C by inoculation with PMV+SPMV, suggesting that co-infection of PMV and SPMV can augment asymptomatic systemic infection.
Co-infection by PMV and SPMV elicited mild synergistic interaction in Summer but not in Kanlow
At 21 dpi, the upper noninoculated 4th leaves were rated for symptom severity rating on a 0-3 scale. No symptoms were observed in mock-inoculated plants. Symptoms were evaluated from symptomatic plants of two independent experiments of 10 plants each. To compare symptom severity, a mean rating was calculated for each treatment, with plants exhibiting no visible symptoms (rating of 0) being excluded from the mean calculation. In Summer plants, the temperature had no significant effect on symptom severity when the plants were inoculated with PMV alone, with mean symptom scores of 1.4 resulting from infection by PMV at 24°C and 32°C (Fig. 3). Infection of Summer plants with PMV+SPMV resulted in mean symptom severity scores of 2.0 and 2.2 at 24°C and 32°C, respectively, which were a modest increase over the corresponding scores from infection with PMV alone (Fig. 3). The difference between PMV and PMV+SPMV treatments was statistically significant at 32°C but not at 24°C.
In Kanlow, the severity of symptoms resulting from infection by PMV alone was temperature-dependent with a symptom score of 1.0 at 24°C compared to a statistically significant symptom score of 2.3 at 32°C (Fig. 3). However, co-infection of Kanlow plants by PMV+SPMV at 24°C and 32°C resulted in no increase in symptom severity over infection with PMV alone (Fig. 3). Taken together, these data indicated that switchgrass cultivars, co-infection with SPMV, and temperature influenced the severity of symptoms elicited by PMV in switchgrass.
Synergistic interaction between PMV and SPMV caused slightly enhanced accumulation of PMV genomic RNA
The effect of interaction between PMV and SPMV in two switchgrass cultivars at 24°C and 32°C on the accumulation of genomic RNA copies of PMV was examined by RT-qPCR. Total RNA isolated from the true 4th leaves of switchgrass cultivars symptomatically infected by PMV or PMV+SPMV was used for RT-qPCR. The log2 fold change in accumulation of genomic RNA copies of PMV and SPMV was presented in Figure 4.
At 24°C, the genomic RNA copies of PMV accumulated at a statistically insignificant level in PMV+SPMV-infected Summer plants at 1.29 log2 fold more copies than those in PMV-infected plants. However, at 32°C, the change was only 1.06 log2 fold in co-infected plants compared to those of PMV-infected Summer plants (Fig. 4). These data suggest that co-infection of Summer plants by PMV+SPMV caused only a marginal increase in PMV genomic RNA accumulation.
In contrast, in Kanlow plants, co-infection by PMV+SPMV caused a statistically insignificant increase in PMV genomic RNA copies (1.13 log2 fold) at 24°C compared to infection only by PMV (Fig. 4). At 32°C, the PMV genomic RNA copies accumulated at a slightly decreased level (0.89 log2 fold) in PMV+SPMV-infected plants compared to those infected by PMV (Fig. 4). These data suggest that the co-infection of Kanlow by PMV and SPMV did not cause a significant increase in accumulation of PMV genomic RNA copies at 24°C and 32°C. Notably, viral loads of PMV and SPMV were significantly lower in singly and doubly infected Kanlow plants compared to those in Summer plants, suggesting that virus propagation was potentially suppressed in Kanlow plants.