Effect of cultivar and temperature on the synergistic interaction between panicum mosaic virus and satellite panicum mosaic virus in switchgrass

Panicum mosaic virus (PMV), the type member of the genus Panicovirus in the family Tombusviridae, naturally infects switchgrass (Panicum virgatum L.). PMV and its molecular partner, satellite panicum mosaic virus (SPMV), interact synergistically in coinfected millets to exacerbate the disease phenotype and increase the accumulation of PMV compared to plants infected with PMV alone. In this study, we examined the reaction of switchgrass cvs. Summer and Kanlow to PMV and PMV+SPMV infections at 24°C and 32°C. Switchgrass cv. Summer was susceptible to PMV at both temperatures. In contrast, cv. Kanlow was tolerant to PMV at 24°C, but not at 32°C, suggesting that Kanlow harbors temperature-sensitive resistance to PMV. At 24°C, PMV was readily detected in inoculated leaves, but not in upper uninoculated leaves of Kanlow, suggesting that resistance to PMV was likely mediated by abrogation of long-distance virus transport. Coinfection by PMV and SPMV at 24°C and 32°C in cv. Summer, but not in Kanlow, caused increased symptomatic systemic infection and mild disease synergism with slightly increased PMV accumulation compared to plants infected with PMV alone. These data suggest that the interaction between PMV and SPMV in switchgrass is cultivar-dependent, manifested in Summer but not in Kanlow. However, co-inoculation of cv. Kanlow with PMV+SPMV caused an enhanced asymptomatic infection, suggesting a role of SPMV in enhancement of symptomless infection in a tolerant cultivar. These data suggest that enhanced asymptomatic infections in a virus-tolerant switchgrass cultivar could serve as a source of virus spread and play an important role in panicum mosaic disease epidemiology under field conditions. Our data reveal that the cultivar, coinfection with SPMV, and temperature influence the severity of symptoms elicited by PMV in switchgrass.


Introduction
Switchgrass (Panicum virgatum L.) is a hardy perennial warm-season C 4 grass native to the region east of the Rocky Mountains in the United States. Switchgrass is being used for soil conservation, forage, ornamental grass, fiber, biosequestration of atmospheric carbon dioxide, and biofuels [1,2]. Switchgrass has gained prominence since the beginning of the 21 st century as research efforts have focused on lignocellulosic biomass crops for the generation of biofuels [1,3]. Switchgrass can be grown on marginal lands with minimal agronomic input, resulting in net bioenergy production output of more than five times the total resource input [3][4][5]. With the advancement of switchgrass cultivation practices and the development of processing technology, the lignocellulosic-based biofuel yield from switchgrass could be similar to the ethanol yield from maize [3,4]. However, biomass production from switchgrass is dependent on stable yields over several years and requires good local adaptation of cultivars [6].
In Nebraska, the tetraploid switchgrass upland cultivar (cv.) Summer and the lowland cv. Kanlow are the backbone of the USDA ARS breeding programs [7,8] and have been used to generate the dedicated bioenergy cv. 'Liberty' [2].

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Switchgrass is susceptible to several fungal and viral pathogens, and these infections could cause significant losses to ethanol yields [9][10][11][12][13][14]. A multi-year survey of switchgrass breeding plots in Nebraska revealed a high prevalence of infections by panicum mosaic virus (PMV) and its satellite virus, satellite panicum mosaic virus (SPMV) [12]. In 2012, 72% of 139 symptomatic plants were infected by PMV, with 19% of plants coinfected with PMV and SPMV. Infections by PMV or PMV+SPMV were more prevalent on Summer or its progeny, with severer symptoms than on Kanlow [12], suggesting that cv. Kanlow harbors some level of resistance to PMV. However, the levels of cv. Kanlow resistance or susceptibility of Summer to PMV have not been comparatively demonstrated experimentally.
Although PMV and SPMV were first reported in 1953 and 1976, respectively, on switchgrass from Kansas in the USA [15,16], most research on interactions between PMV and SPMV has been conducted using other experimental hosts such as proso millet (Panicum miliaceum L.) and pearl millet (Cenchrus americanus (L.) Morrone). PMV is the type member of the genus Panicovirus in the family Tombusviridae. PMV has a single-stranded plus-sense RNA genome of ~4300 nucleotides (nt) encapsidated in a ~30-nm icosahedral virion [17]. PMV acts as a helper virus for its satellite virus, SPMV, and for two satellite RNAs, satS and satC [18,19]. SPMV is a member of the genus Papanivirus and has a single-stranded positive-sense RNA genome of ~820 nt encapsidated in a 16-nm icosahedral virus particle [18,20]. The SPMV genome contains a multifunctional ORF encoding a 17-kDa capsid protein (CP). In addition to virion assembly, the SPMV CP is also involved in synergistic interaction with its helper virus PMV [21,22]. PMV elicits mild mottling or symptomless infection in millets. In contrast, coinfection of millets with PMV and SPMV exacerbates symptoms, with drastically enhanced accumulation of PMV compared to infection with PMV alone [21][22][23].
Experimental evidence is lacking on whether coinfection of switchgrass by PMV and SPMV elicits a similar synergistic interaction like that observed previously in millets. A preliminary investigation of the effects of temperature, cultivar, and synergistic interaction between PMV and SPMV was conducted using anti-PMV sera [24]. These data suggested that Kanlow plants are more resistant than Summer plants, although accumulation of PMV and SPMV was not specifically quantitated. It has also been shown in other virus-plant interaction studies that an elevated temperature can inhibit the plant's defense response and facilitate systemic infection at higher temperatures [25][26][27]. Therefore, this study was conducted (i) to examine differences between Kanlow and Summer in their resistance to PMV, (ii) to determine whether the synergistic effect of coinfection with SPMV occurs in these two switchgrass cultivars, and (iii) to assess the effect of temperature on the reaction of the switchgrass cultivars to PMV and PMV+SPMV coinfection. We found that Summer was susceptible to PMV and PMV+SPMV, with a mild synergistic interaction. In contrast, Kanlow was tolerant to PMV at 24°C, but not at 32°C, suggesting temperature-dependent resistance. However, coinfection of Kanlow with PMV+SPMV at 24°C resulted in enhanced asymptomatic infection.

Propagation of switchgrass cultivars
The switchgrass cvs. Summer and Kanlow were grown in cone-tainers (Stuewe & Sons, Inc.; 3.8 cm diameter, 21 cm depth, and 164 ml volume) that were filled with a pasteurized greenhouse potting mix (1 part loam soil, 2 parts peat moss, 1 part sand, and 1 part vermiculite). Eight switchgrass seeds were initially planted per cone-tainer, and as seeds germinated, the seedlings were thinned to two per cone-tainer. Prior to inoculation, plants were grown in a greenhouse with an average temperature of 26°C for about three weeks, or until the second true-leaf stage. Cone-tainers were arranged on racks (30 cm W × 61 cm L × 17 cm H) and watered uniformly by placing trays under the racks of cone-tainers and filling the trays with water to keep the bottom of the conetainers submerged. Once a week, trays were filled with a low dose of fertilizer  at 250 ppm.

Experimental design
A 2 × 2 × 3 factorial experiment was conducted comparing infection of switchgrass cultivars (Kanlow and Summer) at two temperatures (24°C and 32°C) following three viral inoculation treatments (mock, PMV, and PMV+SPMV). The experiment was conducted twice, and the results from two independent experiments were combined for the analysis. In each experiment, 10 switchgrass seedlings per cultivar were inoculated with each of the two virus treatments, and five seedlings were used for mock inoculation with buffer. Inoculated switchgrass seedlings were transferred to growth chambers (Conviron A2000) at 24°C or 32°C with 16 h light (300 μmol m -2 s -1 ) and ~35% relative humidity. The inoculated plants were kept in growth chambers in a randomized array.

Virus inoculation
A Nebraska strain of PMV (PMV-NE) and a Kansas strain of SPMV (SPMV-KS) were used in this study [23]. In vitro-generated transcripts of PMV or PMV and SPMV (PMV+SPMV) were inoculated onto proso millet cv. Sunup seedlings at the two-leaf stage as described by Chowda-Reddy et al. [23]. PMV-or PMV+SPMV-infected proso millet leaves were harvested at 14 days postinoculation (dpi) and stored at -80°C until used. PMV or PMV+SPMV inoculum was prepared by grinding 1 g of virus-infected proso millet tissue in 19 ml of 20 mM sodium phosphate buffer, pH 7.0. Carborundum-dusted switchgrass leaves were rub-inoculated by dipping a pestle into the inoculum and rubbed on the adaxial side of the second leaf of each plant in an upward motion four times with medium pressure. Mock inoculations were performed with 20 mM sodium phosphate buffer, pH 7.0.

Sample collection and symptom severity rating
The treatment combinations were evaluated for their effects on local and systemic infection. For local infection, the inoculated leaf was collected from each plant at 7 dpi into a separate Ziploc® bag for examination of local infection of PMV. For systemic infection, the fourth upper uninoculated true leaf of each plant was rated at 21 dpi for symptom severity and collected into a separate Ziploc® bag. All samples were stored at -80°C until further processing. Symptom ratings were based on a 0-3 scale: 0 = no symptoms, 1 = ≤ 25% of the leaf exhibiting chlorotic spots and mottling symptoms, 2 = 25-75% of the leaf exhibiting chlorotic spots, mottling, and mosaic symptoms, 3 = > 75% of the leaf exhibiting chlorotic or bright chlorotic spots, mottling, mosaic symptoms.

Virus detection and quantification
Leaf tissue was ground in liquid nitrogen and processed for RNA extraction using a Direct-Zol RNA Miniprep Kit (Zymo Research, Irvin, CA), which requires the use of TRIzol Reagent (Life Technologies, Carlsbad, CA). PMV detection in inoculated leaf tissues was performed using reverse transcription, followed by polymerase chain reaction (RT-PCR). First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA) with a PMV-specific reverse primer (PMV-R2, 5'-CAC TGA ACT ACT CTG GAT TAG TAC -3', complementary to nt 4233 to 4210 of PMV) at 50°C for 60 min, followed by 70°C for 15 min. PCR was performed using the forward primer PMV-F2 (5'-AAG CCC ATT TAC T CGG GAA GTGC-3', corresponding to nts 3548 to 3570 of PMV) and the reverse primer PMV-R2 with the following amplification conditions: 2 minutes at 94°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 59°C, and 1 minute at 72°C, with final amplification for 5 minutes at 72°C. The RT-PCR products were examined by 1% agarose gel electrophoresis in 1X TAE (Tris-acetate-EDTA) buffer with a GeneRuler 1.0-kbp ladder (Thermo Scientific) as a DNA size marker.
To determine relative amounts of PMV and SPMV associated with systemic infection, total RNA extracted from the fourth upper uninoculated leaf was used for reverse transcription quantitative PCR (RT-qPCR). The first-strand cDNA was synthesized by using iScript TM Reverse Transcriptase Supermix (Bio-Rad, Hercules, CA) by incubating for 5 minutes at 25°C, followed by 20 minutes at 46°C and 1 minute at 95°C. The RT-qPCR was performed with Luna Universal qPCR Master Mix (New England Biolabs, Ipswich, MA) using forward and reverse primers specific for PMV and SPMV as described by Chowda-Reddy et al. [23]. The RT-qPCR amplification started with a hot start of 3 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C and 30 seconds at 60.3°C. Samples without RNA template and reverse transcription were included as negative controls. The switchgrass casein kinase-1-like protein 2 and 26S proteasome regulatory subunit S2 1A genes were used as internal controls for determining the relative expression of PMV and SPMV genomic RNA copies using the ΔC t method [28].

Data analysis
Data from the two repetitions of the experiment were pooled for statistical analysis. Quantitative data, i.e., disease severity ratings and virus titer measurements, were subjected to analysis of variance for a randomized design, using treatment means that were computed using the Lsmeans package (version 2.27-62). The least significant difference (LSD) was used for mean separation by using the LSD.test package, and differences between treatment means at the 95% confidence level were considered to be significant. For proportional data, i.e., infection incidence, chi-squared analysis of infection rates was performed using JMP (version 12.2.0), followed by analysis of means for proportions (ANOMP) for significance grouping.

Switchgrass cultivar Kanlow is 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 uninoculated 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 1 3 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 to PMV.

PMV elicits efficient local infection in switchgrass cv. Kanlow
The temperature-dependent resistance of Kanlow to PMV was further examined to determine whether this virus can cause a local infection that facilitates cell-to-cell movement in inoculated leaves before entering the vasculature, as is 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 present 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 leaves (data not shown). These data suggest that PMV facilitates efficient cell-to-cell movement in inoculated leaves of Kanlow.

Interaction between PMV and SPMV in systemic infection of Summer and Kanlow
The interaction between PMV and SPMV in systemic infection was examined by comparing the results of inoculation with PMV alone with those obtained 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 that were systemically infected, as indicated by the presence of symptoms, compared to those inoculated with PMV alone. At 24°C, inoculation of Summer seedlings with PMV+SPMV elicited symptoms in 90% of the plants, compared to 60% of plants exhibiting symptoms when inoculated with PMV alone (Fig. 1). At 32°C, 75% and 100% of Summer plants inoculated with PMV and PMV+SPMV, respectively, exhibited chlorotic streaks and mottling symptoms (Fig. 1). In contrast, there was no significant difference in the percentage of Kanlow plants exhibiting systemic symptoms after infection with PMV alone or together with SPMV at either temperature ( Fig. 1). At 24°C, PMV+SPMV elicited symptoms in 20% of Kanlow plants, whereas PMV elicited symptoms in 15% of symptomatic. At 32°C, PMV and PMV+SPMV induced symptoms in 70% and 75% of Kanlow plants, 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.

Coinfection with PMV and SPMV augments asymptomatic systemic infection in switchgrass cv. Kanlow
To verify systemic infection by symptomology and to examine possible asymptomatic infection by PMV in both switchgrass cultivars, total RNA isolated from the fourth fully expanded upper leaf was used as a template for RT-qPCR [23], which revealed that all treatments resulted in a larger 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 positive for PMV and PMV+SPMV, respectively, compared to 60% and 90% of visually assessed symptomatic infections (Fig. 1), i.e., 10% asymptomatic infections for both virus treatments. In Summer at 32°C, 100% of PMV-inoculated plants were infected based on the 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 and PMV+SPMV infected 35% and 70%, respectively, of Kanlow plants, whereas 15% and 20%, respectively, exhibited 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 and PMV+SPMV, respectively, and symptomatic infections in 70% and 75%, respectively (Fig. 1). These data indicate that 25% of the Kanlow plants were asymptomatic after both virus treatments 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 coinfection with PMV and SPMV can augment asymptomatic systemic infection.

Coinfection with PMV and SPMV elicits a mild synergistic interaction in Summer, but not in Kanlow
At 21 dpi, the upper noninoculated fourth leaves were rated for symptom severity on a 0-3 scale. No symptoms were observed in mock-inoculated plants. Symptoms were evaluated in symptomatic plants from two independent experiments with 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 with 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 at 24°C and 2.2 at 32°C, which represented 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 with 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, coinfection of Kanlow plants with 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 indicate that the switchgrass cultivar, coinfection 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 PMV genomic RNA was examined by RT-qPCR. Total RNA isolated from the fourth true leaves of switchgrass cultivars symptomatically infected with PMV or PMV+SPMV was used for RT-qPCR. The log 2 fold change in accumulation of genomic RNA copies of PMV and SPMV is shown in Fig. 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-log 2 -fold more copies than those in PMV-infected plants. However, at 32°C, the change was only 1.06-log 2 -fold in coinfected plants compared to those of PMV-infected Summer plants (Fig. 4). These data suggest  In contrast, in Kanlow plants, coinfection with PMV+SPMV caused a statistically insignificant increase in PMV genomic RNA copies (1.13-log 2 -fold) at 24°C compared to infection with PMV alone (Fig. 4), whereas at 32°C, the PMV genomic RNA copies accumulated at a slightly decreased level (0.89-log 2 -fold) in PMV+SPMV-infected plants compared to those infected by PMV (Fig. 4). These data suggest that the coinfection of Kanlow with PMV and SPMV did not cause a significant increase in accumulation of PMV genomic RNA at 24°C and 32°C. Notably, the 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.

Discussion
Although PMV and SPMV were first reported as pathogens of switchgrass [15,16], the majority of interaction studies between these two viruses were performed in other experimental hosts, such as proso millet or pearl millet [21][22][23][29][30][31]. Previously, Stewart et al. [12] surveyed switchgrass nurseries in Nebraska and found that fewer Kanlow plants were infected with PMV but not coinfected with SPMV. In contrast, a large proportion of Summer plants were coinfected with PMV and SPMV. The physiological reasons for these findings were not explored. In this study, we found that Kanlow was tolerant to PMV at 24°C, with only 15% of inoculated plants developing visible symptoms of viral infection. However, the level of Kanlow resistance was drastically reduced at 32°C, with 70% of plants showing visible symptoms of chlorotic streaks, mosaic, and mottling. These data suggest that Kanlow possesses temperature-sensitive resistance to PMV. As expected, cv. Summer was susceptible to PMV at both temperatures, with 60 to 100% of plants infected. The nature of the resistance of Kanlow to PMV is not known. However, extreme resistance to PMV was not observed in Kanlow, since it was detected in inoculated leaves at the site of infection, suggesting efficient cell-to-cell movement of PMV in Kanlow. It is unlikely that the virus would be detected by RT-PCR in inoculated leaves without cell-to-cell movement. For other viruses, resistance has been observed for long-distance movement of viral infections, but not at the cell-to-cell level [26,[38][39][40][41]. Our data suggest that resistance to PMV in Kanlow is temperature-dependent, similar to that reported for wheat cultivars with the Wsm1 or Wsm2 gene [26,42,43].
We observed a mild hypersensitive reaction to PMV infection on a few inoculated leaves of Kanlow, suggesting that Kanlow could harbor a dominant resistance (R) gene encoding nucleotide-binding proteins with leucinerich repeats [32,33]. The R-gene-mediated resistance in a majority of pathosystems leads to a hypersensitive reaction with localized cell death or extreme resistance [34,35]. The switchgrass genome encodes over 1000 R genes with variations in their expression across different switchgrass cultivars. Additionally, the expression of these genes was developmentally regulated in field-grown Summer plants [36]. More recently, a global transcriptomic study over a developmental time course of the fourth leaf of greenhouse-grown Kanlow and Summer plants was reported [37]. Under greenhouse conditions, both cultivar-dependent and independent gene networks were detected. Cultivar-independent gene networks included those governing normal leaf developmental processes such as photosynthesis and senescence. The cultivar-dependent gene networks included several genes coding for nucleotide-binding leucine-rich repeat (NB-LRR; R) proteins and cell wall kinases orthologous to genes that typically respond to environmental cues and influence plant defense responses. Notably, genomic resequencing data indicated that many of these NB-LRRs were not expressed or potentially absent in the genomes of Summer plants [37], raising the intriguing possibility that these differences could underlie the differential viral responses observed in the current study.
In this study, we found that the cultivar, coinfection with SPMV, and temperature played important roles in disease development in switchgrass infected with PMV. Coinfection with PMV+SPMV caused significantly enhanced symptomatic systemic infection in Summer, but not in Kanlow, compared to single infection with PMV, suggesting that the interaction of SPMV with PMV that results in enhanced systemic infections is cultivar-specific. Incubation of switchgrass cultivars at 32°C produced a similarly enhanced symptom phenotype with PMV or PMV+SPMV in Kanlow, but not in Summer, suggesting that Kanlow but not Summer produced high-temperature-specific interactions with host factors as has been reported in wheat [26]. PMV+SPMV interact synergistically at 24°C and 32°C in Summer, but not in Kanlow, resulting in an enhanced symptom phenotype, and a slight increase in PMV accumulation compared to infections with PMV alone. These data suggest that PMV and SPMV interact modestly, independent of temperature, with an enhanced symptom phenotype in Summer, but not in Kanlow.
Synergistic interaction between PMV and SPMV in proso millet has been shown to cause enhanced accumulation of PMV compared to infection with PMV alone [22,23]. A twoamino-acid difference in the CP of SPMV isolates was shown to be responsible for differential synergistic interaction with PMV [23]. In this study, we found that coinfection of Summer at 24°C and 32°C caused mild disease synergism with only a slight increase in symptoms compared to the severe disease synergism observed in coinfected millet. However, some Summer plants infected by PMV alone also showed symptoms similar to those caused by PMV+SPMV, pointing to differences in the underlying genetics of the population of switchgrass cultivars. PMV causes symptomless or mild mottling symptoms on proso millet, but coinfection with SPMV caused disease synergism with severe necrosis and occasional death of plants [22,23]. In contrast, PMV elicits mosaic, chlorotic streaks, and mottling symptoms with little or no synergistic interaction with SPMV in coinfections in switchgrass cultivars. Why the same viruses interact differently in two different hosts is not known. Perhaps these viruses interact differently with host factors in these two hosts, thus causing a differential synergistic interaction in these hosts.
The temperature-sensitive resistance of Kanlow to PMV and SPMV has two implications for the potential for disease development in the field. First, spring temperatures during the emergence of tillers (shoots) from virus-infected crowns could be a critical factor affecting the proportion of tillers in a plant that subsequently display panicum mosaic disease symptoms. Should high temperature occur during this early period, cv. Kanlow would likely display the same degree of disease as cv. Summer. Second, higher summer temperatures during the vegetative growth phase would increase virus replication and symptom severity, further limiting yield in virus-infected plants, regardless of the cultivar.
Our data indicate that there was a much greater number of coinfected Kanlow plants with no apparent disease symptoms. We observed drastically enhanced asymptomatic infections in Kanlow plants coinfected with PMV+SPMV compared to those infected with PMV alone. These data suggest that the coinfection of Kanlow by PMV and SPMV caused an enhanced asymptomatic systemic infection, which could be due to a different kind of synergistic interaction between PMV and SPMV in a resistant cultivar. Also, asymptomatic infections by PMV+SPMV in switchgrass cultivars could play a significant role in disease epidemiology because these infections could act as a natural reservoir for PMV and SPMV for other susceptible switchgrass cultivars.