Soybean Dwarf Virus (SbDV), first identified as a pathogen of soybean plants in northern Japan in 1969, is capable of causing serious yield losses in soybean [1
]. The host range of SbDV is largely limited to members of the Fabaceae except for a few species in Chenopodiaceae
]. Currently, four distinct strains of SbDV are recognized in Japan based on sequencing, symptomatology in infected soybean plants and aphid vector specificity [3
]. The yellowing strains, SbDV-YS and SbDV-YP, cause severe interveinal chlorosis, rugosity and thickening of leaves in soybeans. The dwarfing strains, SbDV-DS and SbDV-DP, cause stunting with shortened internodes and brittle curled leaves. The SbDV-YS and -DS strains are transmitted specifically by the aphid Aulacorthum solani
Kaltenbach, while SbDV-YP and -DP strains are transmitted by both Acyrthosiphon pisum
Harris, and Nearctaphis bakeri
Cowen. In the United States, SbDV-like isolates were identified in asymptomatic white clover (Trifolium repens
), symptomatic red clover (Trifolium pratense
), yellow sweet clover (Melilotus officinalis
), and subterranean clover (Trifolium subterraneum
L.) from 11 mid-western and eastern states [4
]. In 2000, it was reported that soybean (Glycine max
L Merr.) crops were infected by a SbDV-Y strain in Virginia [5
]. During 2003, dwarfing strains of SbDV emerged in soybean plants accompanied by the soybean aphid, Aphis glycines
Matsumura, in Wisconsin [6
]. A survey of soybean diseases in northern Illinois also identified two SbDV-D strains [7
]. Recently, the presence of mixed infections of both D and Y strains were confirmed in the eastern United States [8
], and several U.S. isolates were found to be transmitted by A. glycines
] Currently, there are no SbDV resistant commercial cultivars available. More recently, it has been found that the Rsdv1
quantitative trait loci gene was responsible for resistance of an Indonesian cultivar ‘Wilis’ [10
], but the mechanism of such gene is still unclear [11
]. It raises the interesting question that if the rapid evolved plant virus could emerge in a new environment and cause disease outbreak in the soybean field.
SbDV is a luteovirus, with a positive sense single-stranded RNA genome. The genomic RNAs of SbDV range from 5.7 to 5.9 kb, comprising five open reading frames (ORFs) and three untranslated regions (UTRs). ORF 1 and ORF 2 encode the replication-related proteins. ORF 3 encodes the 22-kiloDalton (kDa) major coat protein (CP) that is the major component of the capsid, and ORF 4, which is nested within ORF 3, putatively encodes a movement protein. ORF 5 encodes a 65–88 kDa Read-Through Protein (RTP), the minor capsid protein, formed by the in-frame translational readthrough of the ORF3 stop codon. ORFs 3, 4 and 5 on the 3′-end of SbDV show similarities to genus Polerovirus
, while ORFs 1 and 2 show similarities to the genus Luteovirus
Plant viruses have several mechanisms to generate genetic diversity both within and between species. Plant RNA viruses have highly error prone replication mechanisms, which result in numerous mutations and diverse populations. Mutation, recombination, and re-assortment are three major forces driving the evolution of viruses, and these forces generate diversity in viral genomes, providing variants to adapt to different environments [14
]. Viral emergence and adaptation to new hosts and/or new host resistances is one of the highest impact effects of plant virus evolution [15
]. Even minor changes in viral genomes can result in significant phenotypic effects. One study indicated that the site-specific mutant on amino acid 27 of NIaPro of Papaya Ringspot Virus (PRSV) determined the ability of infecting the host papaya [16
]. Another study on Soybean Mosaic Virus (SbMV) showed that precise mutations in the HC-Pro
gene were essential for virulence on different resistant genotypes of soybeans [17
]. However, there may be limitations to the amount of host adaptation a plant virus can tolerate. Adaptation to a new host could result in fitness losses in original hosts because mutations beneficial in the new host might be detrimental to infection of the original hosts, a phenomenon called the trade-off effect [18
]. For example, the Tobacco Etch Virus (TEV) adapted to a new host, pepper, as indicated by an increase in virulence and virus accumulation, whereas the viral fitness in the natural tobacco host was decreased [19
]. In the case of SbDV, a trade-off effect that increases fitness in soybeans while decreasing fitness in clover could have significant epidemiological impacts, as clover is the overwintering reservoir for the virus.
Reports indicate that SbDV is widely prevalent in clover in North America [20
]. There are currently only limited reports of SbDV emergence in soybean fields [6
], despite the high profile and acreage of this important crop. It is quite probable that SbDV is undergoing adaptation in the transition from clover to soybean. The risks of SbDV outbreaks in soybean would seem to be higher following the recent introduction of the soybean aphid (A. glycines
), and the establishment of this potential SbDV-vector species on soybean crops. Therefore, a better understanding of the capacity for SbDV host adaptation is important to evaluate potential risk of SbDV epidemics in commercial soybean crops in the U.S. The objectives of this study are to evaluate the SbDV fitness in different plant hosts and identify critical mutations of SbDV selected by such new host adaptations.
In initial characterization studies, a number of SbDV isolates (VA-20, MD6, MD8, MD10, MD11 and MD16) from the eastern United States were transmitted to soybean [9
]. However, for all of these strains, attempts at serial transmissions were unsuccessful. Typically, the SbDV strain introduced into soybean could be transmitted once or twice, but transmission efficiency decreased dramatically with serial passage on soybean until transmission eventually failed. In order to investigate this observation, a single well characterized isolate was chosen for controlled passage studies on both soybean and pea. The isolate used for this study was SbDV-MD6, a Y strain isolate collected from Prince George’s county in Maryland [9
]. Multiple SbDV-MD6 infected white clover were collected and identified from the edge of a soybean field, but there were no SbDV symptoms observed in the adjacent soybeans. Both A. pisum
and N. bakeri
were present in the clover surrounding the soybean fields, but neither of these aphids was observed feeding on soybeans. Based on this information, the natural history of SbDV-MD6 was assumed to be limited to a clover infection, transferred from clover to clover by A. pisum
and/or N. bakeri
. There were no peas (Pisum sativum
L.) observed anywhere within a 1 km radius of the originally infected white clover.
In the serial transmission experiments, SbDV-MD6 was transmitted to peas with high efficiency (67%) from the beginning of the passaging when using 10 A. pisum
per pea plant, and maintained a high level of transmission efficiency thereafter. When three aphids were used for the inoculation, the most dramatic improvement of transmission efficiency was observed after the third transmission (Figure 1
a). It would appear that the efficiency of transmission to pea was so good that the only way to see the subtle improvement with continued passage was to limit the inoculum load presented at each passage. qRT-PCR suggested that viral titers in pea passage lines were increasing by the second passage and significantly increasing by P7 (Figure 4
a). Symptom severity on infected peas increased with serial transmissions, as the later passages of infected peas became significantly shorter and smaller than the initial passage (Figure 2
a). The increases in titer, disease symptom severity, and transmission efficiency all suggested that SbDV-MD6 was adapting to the peas.
In contrast to the pea serial transmission lines, the transmission efficiency on soybeans was initially low, and it decreased with serial transmission on soybean, despite the fact that a significantly higher number of aphids were used in soybean inoculations. The competency of the vector could be a factor in the poor transmission efficiency. However, N. bakeri
typically transmitted SbDV from infected clover to healthy clover with an average of 66% efficiency (data not shown) and transmitted from infected clover to soybean with 33–53% efficiency following the first or second passage to soybean (Figure 1
b). Fluctuations in transmission efficiency might result from inconsistent aphid feeding behavior on the non-preferred host (soybean). Although soybean was not the preferred host, N. bakeri
was able to feed on soybean to acquire and transmit the virus, and were observed to survive and feed on soybean for the duration of the 5-day inoculation access period. Furthermore, if the aphid vector competency was the sole factor limiting transmission efficiency, one would expect that the transmission efficiency would remain relatively constant. The qRT-PCR data indicated that SbDV-MD6 could be adapting to soybeans as a host during the serial transmission process, since viral titers in soybeans improved dramatically after the third passage. Overall, SbDV-MD6 populations in soybeans demonstrated an approximately 6-fold increase in viral concentration based on qRT-PCR (Figure 4
b). An alternative explanation for the reduction of transmission efficiency in soybean passage lines was the possibility that host adaptive mutations in viral populations passaged on soybean had deleterious effects for the aphid vector transmission. Aphid transmission of luteoviruses requires very specific interactions between aphids and viruses [23
]. Although it would seem likely that transmission efficiency was not necessarily related to the host adaptation, it was not out of the realm of possibility that host adaptive mutations could have a deleterious effect on a separate biological process, aphid transmission.
Sequence analysis of SbDV-MD6 genomes recovered from the pea and soybean host serial passages might suggest possible mechanisms for the observed increases in virus titers along with the reduction of virus transmission in soybean. The non-synonymous mutations that change amino acids would be expected to affect biological functions. In pea passages, three non-synonymous mutations were located in ORF1. This protein is directly involved in viral replication and is likely to interact with host proteins. An additional non-synonymous mutation was found in ORF4, which has been identified as the movement protein, another likely source of host directed selection. The CP ORF3 had no non-synonymous mutations identified in any of passaged lines for both pea and soybean, suggesting the conserved function of CP, where only synonymous mutations were identified. Two more non-synonymous mutations were found in the RTP ORF5, which had been suggested to also contribute to tissue specificity within the plant host [24
]. In addition, it is important to remember that SbDV-MD6 may also be undergoing selection for transmission by A. pisum
as the viral population was serially transmitted. There were four additional mutations in the 3’-UTR of pea-passaged SbDV-MD6. The 3’-UTR is related to replication initiation [25
], although this portion of the genome is rather variable amongst SbDV isolates.
In soybean passage lines, the SbDV-MD6 dN
values were higher than the dN
values observed in pea passage lines. Both ORF1 and ORF2 had been undergone purifying selection pressure during the new host adaptation. Interestingly, the mean value 0.59 of RT ORF5 in soybean was significantly higher than the mean value 0.22 in pea passages. This suggests that the RT ORF5 genes making the host shift from clover to soybean encountered less purifying selection pressures than the SbDV-MD6 populations making the shift from clover to pea. As ORF5 is one of key factors for luteovirus vector transmission and systemic infection [26
], higher genetic diversity in the soybean host may cause trade-off effects during the host shifting [27
]. The more dramatic improvements in viral titer but lower transmission efficiency over the course of serial passage on soybean would support this hypothesis. The sites where amino acids changed in SbDV-MD6 soybean passages were mainly located on replication and RT coding regions. Just as with the pea serial passage, SbDV-MD6 passaged on soybean may also be undergoing selection for transmission by N. bakeri
. The mutations in the replication related proteins (ORF1 and ORF2) and the 5′ UTR (important replication control sequence) could easily be related to host selection pressures, but it is hard to imagine the exact function and effects of mutations in the C-terminal of the RTP.
Previous research suggests that RTP is related to vector transmission [12
], and it is possible that the RTP mutations are related to selection for N. bakeri
transmission. However, in this work, the evidence indicated that transmission efficiency by N. bakeri
was decreasing in soybean serial transmissions. Therefore, there might be additional functions for the RTP, such as host tissue specificity and intercellular transport [24
], and the mutations adapting to plants have a trade-off effect on vector transmission. It was interesting to note that SbDV-MD6 accumulated non-synonymous mutations in the RTP serially passaged on peas. When these pea-adapted virus populations were back inoculated into clover again, most of the mutations (including all of the non-synonymous mutations) reverted to the original clover sequence, further suggesting that these mutations were likely related to host adaptation. It was also interesting that one of the non-synonymous changes in soybean passage lines introduced a stop codon in ORF1, a critical protein for viral replication. However, all of the sequencing was performed directly on RT-PCR products, so it would be impossible to determine if the mutations were not present in a minority of the population without a more detailed cloning analysis. In addition, an internal methionine codon was available for the initiation of translation, just downstream from the introduced stop codon. The significant improvement in viral titers suggested that at least a functional level of ORF1 protein products must be available to the viral population.
On the surface, the reduction of aphid transmissibility in SbDV-MD6 soybean serial transmissions does not make sense. Certainly any viral population making such an adjustment in a field setting would be rapidly out competed by neighboring transmissible populations, regardless of replication efficiency within the host. However, it is important to remember that the format of the serial transmissions done here was heavily biased towards generating viral populations that were strongly adapted to the host plants. Although luteoviruses only infect host phloem cells, the viral populations were exposed to host related selection pressures in every infected cell, from the very beginning to the very end of the passage experiment. In contrast, the aphid vector itself constitutes a temporal selection pressure that is never actually associated with a replicating viral population. Under these conditions, if there is a mutation that is beneficial to the virus in the new host regime but deleterious to aphid transmission, this mutation would become dominant in the population even if the advantage in the host was minimal and the cost to transmissibility drastic.
These experiments might lead to speculation that soybeans were not really a viable alternative host for SbDV-MD6. A host that selects for a non-transmissible population would certainly seem to be an evolutionary dead end. However, that point of view would be flawed because of the host selection biased limitations of the experimental design. The conditions used here and in many other experimental systems do not necessarily reflect the reality for viruses in the field. In the field, particularly on perennial pasture hosts such as clover, aphids are a near constant presence. As such, the selection pressures for maintaining aphid transmissibility would almost certainly be stronger, and have a greater influence on the eventual makeup of the viral population. The role of near-continuous aphid transmission as a selection pressure on clover isolates of SbDV to adapt to new aphid vectors species or to new plant hosts has yet to be determined. However, this work does demonstrate that the compact nature of viral genomes may in certain cases constrain the amount of allowable variability, as mutations that would be advantageous to one phase of the viral life cycle (replication of movement) may be deleterious to another phase of the viral life cycle (transmission). This may be especially true for multifunctional viral proteins and overlapping reading frames.