Vector species, pasture legume host range, and impact on grain legumes of an Australian soybean dwarf virus isolate

Soybean dwarf virus (SbDV; family Tombusviridae, genus Luteovirus, species Soybean dwarf virus) can cause damaging disease epidemics in cultivated plants of the family Fabaceae. The biological characteristics of SbDV isolate WA-8, including its vector species, host range, and impact on Australian grain legume cultivars, were investigated in a series of glasshouse experiments. Isolate WA-8 was classified as the YP strain, as it was transmitted by Acyrthosiphon pisum (pea aphid) and Myzus persicae (green peach aphid) and infected known strain indicator species. Of the 18 pasture legume species inoculated with SbDV, 12 were SbDV hosts, including eight that had not been identified previously as hosts. When inoculated with SbDV, field pea (Pisum sativum), faba bean (Vicia faba), lentil (Lens culinaris), and narrow-leafed lupin cv. Jurien were the most susceptible (70 to 100% plant infection rates), and albus lupin (Lupinus albus), chickpea (Cicer arietinum), and narrow-leafed lupin cv. Mandelup were less susceptible (20 to 70%). Over the course of three experiments, chickpea was the most sensitive to infection, with a > 97% reduction in dry above-ground biomass (AGB) and a 100% reduction in seed yield. Field pea cv. Gunyah, faba bean, and lentil were also sensitive, with a 36 to 61% reduction in AGB. Field pea cv. Kaspa was relatively tolerant, with no significant reduction in AGB or seed yield. The information generated under glasshouse conditions in this study provides important clues for understanding SbDV epidemiology and suggests that it has the potential to cause damage to Australian grain legume crops in the field, especially if climate change facilitates its spread.


Introduction
Soybean dwarf virus (SbDV; family Tombusviridae, genus Luteovirus, species Soybean dwarf virus) is a singlestranded positive-sense RNA plant virus. SbDV primarily infects members of the family Fabaceae and is transmitted solely by aphids in a persistent, circulative, and non-propagative manner [47]. SbDV isolates were first classified as yellowing (Y) or dwarfing (D) strains based on the symptoms they caused in soybean (Glycine max) and their host range [48]. Subsequently, isolates were further classified as Acyrthosiphon pisum Harris (pea aphid)-transmitted (P) strains [2,33,51] or Aulacorthum solani Kaltenbach (foxglove aphid)-transmitted (S) strains [49]. Therefore, isolates are currently given one of four strain designations; YP, YS, DP, or DS. Subsequent research demonstrated that only Y strain isolates readily infect white clover (Trifolium repens L.), albus lupin (Lupinus albus L.), and common bean (Phaseolus vulgaris L.), and only D strain isolates readily infect red clover (Trifolium pratense L.) [9,22,40,48]. Moreover, studies have suggested that Myzus persicae Sulzer (green peach aphid) transmits only P strain isolates [11,16,46]. When analysed at the molecular level, the four strains form separate phylogenetic groups [49,50]. Many SbDV isolates collected from Australian graingrowing regions that underwent whole-genome sequencing and phylogenetic analysis grouped with known YP strain isolates, suggesting that YP is a common strain in Australia [6]. However, no studies have used these isolates to challenge indicator host species or examine aphid vector species to confirm that they fit within the strain classification system. In Australian grain-growing regions, A. pisum, M. persicae, Acyrthosiphon kondoi Shinji (blue green aphid), and Aphis craccivora Koch (cowpea aphid) are the most common virus vectors of Fabaceae crops and are therefore primary candidates as SbDV vectors.
Legumes form an integral component of the pasture feed base of Australia's $12.3 billion wool, dairy, and red meat production industries [38]. Moreover, in Australia's broadacre cropping industry, grain legumes are worth approximately $2 billion in exports alone without accounting for their domestic use and increasingly crucial role as break crops [1]. A vast range of annual and perennial legume pasture species are grown, including clovers (Trifolium sp.), annual medics (Medicago spp.), lucerne (Medicago sativa L.), serradellas (Ornithopus spp.), and biserrula (Biserrula pelicinus L.), with subterranean clover (T. subterraneum L.) being the most widely grown [42]. Studies on SbDV in mainland Australia have primarily focussed on the substantial damage it causes to subterranean clover pastures [19,20,33]. Since the first report of it causing a serious 'red leaf disease' in northern Victoria, SbDV (initially known as 'subterranean clover red leaf virus') has caused sporadic disease epidemics across the country, occasionally resulting in total pasture collapse [20,29,31,33,45]. Host range studies and field surveys have shown that SbDV can infect a wide range of other pasture legume species, including red clover, white clover, strawberry clover (T. fragiferum L.), annual medics, lucerne, and vetch (Vicia spp.) [2,23,27,34,37,47]. However, it is unknown whether several other species (e.g., serradellas and biserulla) that are grown in pasture swards or act as weeds in grain-focussed systems are SbDV hosts. To better understand its epidemiology, the host range of SbDV requires further examination, as its hosts could act as infection reservoirs for epidemics in more economically important legume pastures and grain legume crops.
The predominant grain legume crops sown in Australia are narrow-leafed lupin (L. angustifolius L.), field pea (Pisum sativum L.), chickpea (Cicer arietinum L.), lentil (Lens culinaris L.), and faba bean (Vicia faba L.) [1]. SbDV can infect all these species [2,14,23,24,27,35]. The few studies examining the impact of SbDV on grain legumes suggest that infection causes severe losses. For example, field trials conducted in Syria [36] demonstrated that SbDV could cause up to 56% yield loss in faba bean and > 90% in lentil [36]. Studies have also demonstrated a similarly dramatic impact on the yield of vegetable varieties of P. sativum and V. faba [24,25]. Two important characteristics of a host species that greatly influence its potential for virus-induced losses in the field are (i) its susceptibility/resistance, i.e., the degree to which the virus can infect and multiply in the host and (ii) its sensitivity/tolerance, i.e., the disease reaction of the host once infected with a virus [7]. No studies have assessed these characteristics following SbDV inoculation in Australian grain legume cultivars. This would provide important baseline data to assess the potential impact of SbDV in Australian grain legume crops.
This study was designed (i) to determine whether SbDV isolate WA-8 fits within the strain classification system, by examining some potential vector species and inoculating indicator plant species, (ii) to assess the host range of WA-8 among 18 annual and perennial pasture legume species, and (iii) to assess the reaction of two cultivars each of field pea, faba bean, lentil, chickpea, narrow-leafed lupin, and albus lupin to SbDV inoculation.

Glasshouses, aphid colonies, SbDV cultures, and inoculation
All plants were grown in commercial premium potting mix (Baileys, Australia) inside a naturally lit air-conditioned glasshouse kept at 15°C to 25°C located at South Perth, Australia (31°59′22″S, 115°53′09″E). SbDV isolate WA-8 was obtained from subterranean clover in 2018 at Torbay, Western Australia (35°01′36″S, 117°38′57″E), and cultured in subterranean clover cv. Dalkeith. Leaf samples from plants infected with SbDV were used as positive controls, and uninfected plants were used as negative controls in quantitative reverse transcription polymerase chain reaction (qPCR) and reverse transcription loop-mediated isothermal amplification (LAMP). Colonies of A. pisum clone Flor1 and A. kondoi were maintained on faba bean cv. Fiord plants, and M. persicae on canola cv. Bonito plants, inside aphid-rearing cages (Bugdorm, Australia) kept in an air-conditioned controlled environment room (maintained at 20°C with a 16 h photoperiod). For inoculations, aphids were given a 48-h acquisition access period (AAP) on a SbDV culture plant before being transferred to test plants using a fine-tipped paintbrush and given a 48-h inoculation access period (IAP). These access periods are deemed optimal for SbDV transmission [8]. Following the IAP, plants were treated with an imidacloprid soil drench (0.125 g/litre) to eliminate aphids.

RNA extraction, qPCR, and LAMP
All RNA extractions of test plants in experiments were done using an RNeasy Plant Mini Kit according to the manufacturer's instructions (QIAGEN, Australia). Based on previous research indicating that SbDV reaches the highest concentration in younger tissue [21], only the younger leaves of plants were sampled for testing in this study.
The sets of qPCR primers and probe and LAMP primers developed and used to test for the presence of SbDV infection in this study were derived from the ORF1 gene nt sequence of SbDV isolate WA-8 (Table 1, Supplementary File S1). For qPCR, primers and 5′ nuclease probes with a fluorescent reporter dye and a quencher dye were developed using the PrimerQuest™ Tool available at https:// sg. idtdna. com/ Prime rQuest/ Home/ Index (Integrated DNA Technologies, USA). For LAMP, primers were developed using Prim-erExplorer V5 software available at https:// prime rexpl orer. jp/e/.
All qPCR reactions were done using a Rotor-Gene Multiplex RT-PCR Kit on a Rotor-Gene Q instrument (QIA-GEN, Australia). In a total volume of 25 µL, the reaction mixture contained 1 µL of total RNA extraction template, 12.5 µL of 2X QIAGEN Rotor-Gene Multiplex PCR Master Mix, 1 µL of Rotor-Gene Reverse Transcriptase Mix, 0.5 µL of each primer, 1.25 µL of 10 µM probe stock, and 5 µL of RNAse-free water. The SbDV assay was run on the orange channel, and an internal control assay that amplified the NADH-ubiquinone oxidoreductase chain 5 (nad5) mitochondrial gene was run on the yellow channel [4,39]. The cycling conditions were initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing and elongation at 60°C for 15 s. The threshold was manually set at 0.5, and the result regarded as positive if the Ct value was ≤ 30, indeterminate if it was 31 to 35, and negative if it was > 35 or no Ct value was given. Indeterminate samples were retested using LAMP.
All LAMP reactions were done using a dual-block (eight reaction wells per block) Genie® II instrument (Optigene, United Kingdom). In a total volume of 25 µL, the reaction mixture contained 1 µL of total RNA extraction template, 15 µL of ISO-004RT master mix (Optigene, United Kingdom), 0.5 µL each of primers F3 and B3, 2 µL of forward inner primer (FIP) and backward inner primer (BIP), 1 µL of 10 µM LF2 and LB2 primer stock, and 2 µL of RNasefree water. The reaction mixture was incubated at 65°C for 40 minutes, followed by an annealing step for 10 minutes. Results were analysed in real time via amplification and annealing graphs. A sample was considered positive if the fluorescence exceeded 10,000 and peaked within the incubation time and the annealing temperatures were within 1°C of those of the positive controls.

Exploring the host range of SbDV isolate WA-8 in pasture legumes
In experiment 2a, seedlings of 29 cultivars representing 18 pasture legume species (Table 3) were grown in small pots (160 mm high by 130 mm diameter). At the two-leaf growth stage, plants were inoculated with SbDV using five aphids per plant. Just one cultivar per species was used, except for subterranean clover (nine cultivars), French serradella (O. sativus, three cultivars), and red clover (two cultivars). Due to seed availability and germination rate differences, the number of plants inoculated varied between cultivars. In experiment 2b, cultivars with ≤ 2 plants infected in experiment 2a were inoculated with 10 aphids per plant. In both experiments, each set of plants was split into two and inoculated at separate times as repeats. Two plants per cultivar were left uninoculated as negative controls. At 21 and 35 DAI, all plants were tested for SbDV infection by LAMP or qPCR.

Susceptibility of grain legumes and common bean to infection with SbDV isolate WA-8
In experiments 3a, seedlings of field pea cvs. Kaspa and Gunyah, faba bean cvs. Fiord and Samira, chickpea cvs. Genesis and Hattrick, lentils cvs. Hurricane and Jumbo2, narrow-leafed lupin cvs. Mandelup and Jurien, albus lupin cvs. Luxor and Amira, and common bean vegetable cvs. Borlotti and Simba were grown in small pots (160 mm high by 130 mm diameter). Ten plants of each cultivar were  Rev  CAA ATT CGT GCT TGG CGA TTAG  Probe  TEX615/ACA GTT CTC AAG TGC CGA ACA CGA /BHQ_2  LAMP-WA-8  F3  GAG TTT GAG TGG GAA CTG CA  B3  GGA GGG TTC TGG GTC CAT T  LF2  GCG CTC AAA TTC GTG CTT GG  LB2 GCA

Sensitivity of grain legume cultivars to infection with SbDV isolate WA-8
In experiments 4a to 4c, seedlings of grain legume cultivars were grown in large pots (230 mm high by 270 mm diameter) and organised into a factorial randomized block design. In experiment 4a, field pea cv. Kaspa, faba bean cv. Fiord, lentil cv. Hurricane, chickpea cv. Hattrick, and subterranean clover cv. Dalkeith were tested. In experiments 4b and 4c, field pea cv. Gunyah, faba bean cv. Samira, lentil cv. Jumbo2, and chickpea cv. Genesis were tested in addition to those tested in experiment 4a. Subterranean clover was not tested in experiment 4c. The appropriate rhizobium (ALOSCA Technologies, Australia) for each species was applied immediately after sowing to the soil surface. Up to 10 plants of each cultivar were inoculated with SbDV at the two-to three-leaf growth stage, and an equal number of plants were left uninoculated as controls. At 21 and 35 DAI, all plants were tested for SbDV infection by LAMP and qPCR. Plants that were inoculated but did not become infected were treated as uninfected plants in statistical analysis. Plants were observed for symptoms throughout their life. Once plants senesced, they were harvested to measure growth variables. In all experiments, pods were removed, and the remaining oven-dried above-ground biomass (AGB) was measured. Furthermore, depending on the cultivar and the experiment, the pods were counted, and for each plant, 10 pods were randomly selected to measure their length and the number of seeds in each. Pods were threshed to obtain seed, and the weight of 20 randomly selected seeds and total seed yield were measured for each plant. Pod and seed data were not available or incomplete for some cultivars in some experiments because of rodent damage to pods (field pea, lentil, and chickpea in experiment 4b), poor growth and podding (lentil in experiment 4c), or because AGB was the primary focus (subterranean clover).
A paired two-sample t-test was used to analyse differences in individual variables between infected and uninfected plants of each cultivar. Multiple linear regression was used to examine relationships between growth variables for each species. To enable comparison across cultivars and experiments, the difference in AGB between the infected and uninfected plant in each block for all three experiments was calculated. Statistical analysis on mean AGB reduction across all cultivars was performed using analysis of variance (ANOVA) and Tukey's honest significant difference (HSD). All statistical analyses were conducted in RStudio 1.4.1717 (RStudio PBC, USA).

Examining some potential vector species of SbDV isolate WA-8
Experiments 1a and 1b were conducted to determine which aphid vectors could transmit SbDV isolate WA-8. Across the two experiments, SbDV was detected in all plants inoculated using A. pisum and in 20% and 30% of plants, respectively, inoculated using M. persicae. SbDV was not detected in plants inoculated using A. kondoi ( Table 2). All three aphid species accepted subterranean clover as a host during the IAP. Based on these results, A. pisum was used for inoculations for subsequent experiments.

Exploring the host range of SbDV isolate WA-8 in pasture legumes
Experiments 2a and 2b were conducted to explore the host range of SbDV isolate WA-8 among a selection of pasture legume species. Following inoculation using five aphids per plant in experiment 2a, SbDV was detected in 30 to 100% of plants of the eight subterranean clover cultivars, and in 71 to 100% plants of the three French serradella cultivars (Table 3). SbDV was also detected in 20 to 83% of plants of strand medic (M. littoralis), Moroccan clover (T. isthmocarpum), barrel medic (M. trunculata), balansa clover (T. michelianum), strawberry clover, sulla (H. coronarium), and Persian clover (T. resupinatum). Following inoculation using five aphids per plant in experiment 2a, SbDV was detected in 4 to 15% of plants of biserrula, arrowleaf clover (T. vesiculosum), and white clover, and 20 to 100% of plants of these species when inoculated with 10 aphids per plant in experiment 2b. In experiments 2a and 2b, SbDV was not

Sensitivity of grain legume cultivars to infection with SbDV isolate WA-8
Experiments 4a to 4c were conducted to test the sensitivity of a subterranean clover cultivar and six grain legume cultivars to infection with SbDV isolate WA-8.

Subterranean clover
Infected plants of subterranean clover were stunted with chlorotic foliage and developed margin reddening of lower leaves, which progressed inward until the leaves senesced ( Fig. 1a and b). In experiments 4a and 4b, SbDV-infected subterranean clover cv. Dalkeith plants had 66% and 89% lower AGB (P < 0.001), respectively, than uninfected plants (Table 5).

Field pea
Infected plants of cv. Gunyah, but not cv. Kaspa, were often moderately stunted and chlorotic with distorted leaves (Fig. 2a). However, infected plants of both cultivars commonly developed interveinal chlorosis of lower leaves (Fig. 2b).
In experiments 4a and 4b, AGB, and in experiment 4a, seed yield and seed weight, of SbDV-infected cv. Kaspa plants did not differ significantly from uninfected plants (P > 0.15, Tables 5 and 6). In experiment 4c, infected cv.
Kaspa plants had 27% lower AGB than uninfected plants (P = 0.005) but no significant reduction in the number of pods and seed yield (P > 0.20). In experiment 4a, infected cv. Kaspa plants had 11% shorter pods and 20% fewer seeds per pod than uninfected plants (P < 0.001).

Faba bean
Infected plants of both faba bean cultivars were often moderately stunted (Fig. 2e) and developed interveinal chlorosis of lower leaves, which, more commonly in cv. Samira, developed into interveinal reddening (Fig. 2f).
In both faba bean cultivars across all experiments, seed yield was predicted by AGB (R 2 = 0.44, P < 0.001, Fig. 3c). The predictive strength of AGB differed significantly between experiments (P = 0.047), but not between cultivars. Seed yield was predicted by the number of pods (R 2 = 0.64, P < 0.001), but not by seed weight (P = 0.53). Seed yield was predicted by pod length in experiments 4a and b (R 2 = 0.15, P = 0.01), but not in 4c (P = 0.69). Seed weight was predicted by pod length (R 2 = 0.4, P < 0.001). There was no relationship between seed weight or pod length and AGB (P = 0.29 and 0.87, respectively).

Discussion
In this study, we classified SbDV isolate WA-8 as belonging to the YP strain, as it was transmitted efficiently by A. pisum and infected the known indicator species white clover, albus lupin, and common bean, but not red clover. Using A. pisum to inoculate plants of 18 pasture legume species with this isolate, eight previously unknown SbDV host species were identified. These were balansa clover, biserrula, barrel medic, Persian clover, Moroccan clover, arrowleaf clover, sulla, and French serradella.
Furthermore, by inoculating grain legume species, susceptibility and sensitivity data for cultivars frequently sown in Australia is provided for the first time under glasshouse conditions. Field pea, faba bean, and lentil cultivars were highly susceptible to infection, and albus lupin and chickpea were less susceptible. Narrow-leafed lupin cv. Jurien was more susceptible than cv. Mandelup. The sensitivity of these cultivars to SbDV infection was also assessed by measuring growth variables, primarily AGB, in infected and uninfected plants across three experiments. Field pea cv. Kaspa was relatively tolerant in comparison to field pea cv. Gunyah, faba bean, cvs. Fiord and Samira, and lentil cvs. Hurricane and Jumbo2, which were sensitive, and chickpea cvs. Hattrick and Genesis, which were extremely sensitive. The spread of P-type SbDV strains such as WA-8 is likely to depend significantly on the local abundance and movement patterns of A. pisum. However, considering its abundance in several important Australian agricultural regions, M. persicae, confirmed to be a vector in this study, could be epidemiologically important. A. kondoi, a common aphid species in Australian grain and pasture legume crops, was not a vector of SbDV WA-8 and, in an earlier study, was not a vector of an S strain isolate [27]. Other aphid species that frequently occur in these systems such as A. craccivora, previously shown to transmit P strain isolates [46], and Megoura crassicauda (faba bean aphid) should also be tested. Aphid population studies in regions impacted by SbDV should be conducted to identify other locally important vector species and assess peak flight times and the epidemiological importance of each species. Although the results from this study support the established strain classification system, exceptions apparently can occur. Such anomalies could be due to the presence of other viruses or SbDV strains that facilitate the transmission of a strain to a non-host or by a non-vector [46,51]. Furthermore, such mixed infections could facilitate the generation of recombinant isolates with unique biological characteristics [46]. Biological characterisation of more isolates coupled with genomic studies on SbDV in Australia is required to determine the relevance of the classification system in Australia and to identify any important exceptions.
Of the pasture legume cultivars tested, 22 of 29 were hosts of SbDV isolate WA-8. They represented 12 of the 18 species tested, exemplifying the wide host range of SbDV among members of the family Fabaceae. Four of the eight new host species identified were from the genus Trifolium, a common host genus in the literature [2,9,13,47]. SbDV is often asymptomatic or produces mild symptoms on many important host species, including red clover, white clover, strawberry clover, annual medics, and lucerne [2,10,17,27], and a similar lack of obvious symptoms was observed in several of the host species tested in this study. Asymptomatic hosts would serve as concealed sources of infection to more economically important hosts such as subterranean clover and grain legumes. Field surveys utilising sensitive molecular assays should be conducted to examine the incidence of virus infection in such hosts at critical time-points of the growing season. French serradella is a particularly well-suited candidate as a reservoir host, as it is highly susceptible yet asymptomatic to SbDV infection and is relatively common as a summer-sown pasture or early-season germinating weed, thereby being established by the time autumn aphid flights begin [12]. Climate change could exacerbate the risk of SbDV epidemics if the frequency of summer rainfall events increases, thus increasing the abundance of virus reservoirs. On the other hand, an increase in summer temperatures may decrease aphid survival during the non-cropping period and thus decrease epidemic risk [32]. As non-Fabaceae plant families are also potential SbDV hosts [2,27], other regionally common weed and pasture species should be investigated as potential sources of SbDV infection. No plants of running postman, tiger-snake vine, lucerne, sainfoin, red clover, or bladder clover became infected with SbDV in this study, possibly because they are (i) non-hosts of SbDV, (ii) non-hosts of the strain used, i.e., red clover, or (iii) resistant and would have become infected with a higher inoculum load. Interestingly, lucerne has been reported both as a host and non-host of SbDV [27,30,33,47], which could indicate strain specificity. Although some subterranean clover cultivars had lower infection rates than others e.g., Seaton Park (30%) and Woogenellup (40%), strong resistance was not identified. This reflects results from more-extensive resistance screening studies done in the past, which did not uncover any promising resistance [26,28,33]. Tolerance to SbDV infection was reported in some cultivars, including Woogenellup, but this may have been resistance to virus accumulation [29], both of which would be worth assessing in future resistance screening efforts.
With the economic importance of grain legumes likely to increase with rising nitrogen input costs, reductions in biomass, seed weight, and yield due to virus infection have significant implications. Firstly, impacts on seed yield and quality directly diminish the value of the grain, and secondly, reductions in AGB would likely result in reduced nitrogen deposition and weed competitiveness [5], greatly compromising their value as break crops. The sensitivity data provided show that SbDV has the potential to be as damaging in many grain legumes as it is in subterranean clover. SbDV had a significant impact on most growth variables measured in this study, including a consistently severe impact on AGB and seed yield, and in most cases when measured, the number and size of pods and seed weight. SbDV also has severe impact on some of these growth variables in other hosts [3,15,24,25]. Regression analysis indicated that the reduction in AGB causes reductions in seed yield. In this study, SbDV infection symptoms and associated losses in faba bean, field pea, lentil, and subterranean clover resembled those described in previous studies. Makkouk and Kumari [36] demonstrated up to 56% seed yield losses in faba bean, and Johnstone [24] and Johnstone and Rapley [25] demonstrated seed yield losses of > 80% in broad bean when infected early. In this study, AGB and seed yield reductions in SbDV-infected faba bean plants ranged from 27-67%, depending on the experiment. Johnstone [24] also found vast differences in SbDV infection tolerance of green pea varieties, which was also observed between the two field pea cultivars tested in this study. In the one experiment in which it was measured, SbDV reduced seed yield in lentil cv. Hurricane by 91%, similar to what has been described by Makkouk and Kumari [36], who also showed that using resistant 20 Page 12 of 14 lentil cultivars reduced yield losses to ~ 20%. This is the first study to demonstrate the extreme sensitivity of chickpea to SbDV infection. The sensitivity of the narrow-leafed and albus lupin cultivars was not tested -but should be in future research -as there are reports that SbDV causes disease in both [2]. The 60 to 80% reduction in AGB of SbDV-infected subterranean clover plants in this study is consistent with field reports and previous glasshouse studies in which fresh AGB was reduced by 50 to 90% [19,26]. Similar data in other potentially vulnerable and economically important cultivated legume species grown in Australia such as common bean, peanut (Arachis hypogaea), and vetches (Vicia spp.) should be collected.
There are many factors that would influence the likelihood of SbDV damage under field conditions that were not considered in the glasshouse experiments. Firstly, environmental factors such as temperature and light intensity greatly impact symptoms, virus accumulation, and infection severity in subterranean clover [18,19]. Whilst these factors could have contributed to differences in host susceptibility and sensitivity between experiments in this study, they vary to a far greater degree under field conditions and are therefore likely to play a significant role in SbDV epidemiology and impact. Evidently, multiple SbDV strains are circulating in Australian grain-growing regions, and they are likely to differ in their susceptibility and sensitivity [9]. Furthermore, plants were inoculated only at a single early growth stage, likely when they were most vulnerable, whilst in field crops, virus epidemics progress over multiple growth stages. The inoculation method used was artificial in that it circumvented any alighting preferences that aphids may have in the free-choice environment of the field. The differences in aphids remaining on plants following inoculation, e.g., 2% on albus lupin cv. Luxor and 22% on chickpea cv. Genesis compared to 100% on field pea cv. Kaspa in experiment 2a, likely reflect differences in host suitability, i.e., physical and biochemical traits that have antixenotic or antibiotic effects on the aphid [43,44]. Due to the nature of luteovirus transmission, crop colonisation by the aphid vector is vital in optimising virus spread [41], so unpalatable host crops may be less susceptible to widespread infection than palatable hosts. Host plant palatability may have influenced SbDV infection rates in this study and should be considered as a component of susceptibility and resistance [7]. Therefore, field-based research is now required to understand the epidemiology and impact of SbDV in Australian grain legume crops. Such research should include surveys of grain legume crops, especially during epidemics in pastures such as subterranean clover, and field experiments repeated under a range of conditions to examine the impact on biomass and seed yield and quality. If these field studies determine the risk of future SbDV epidemics to be high, breeders should focus on incorporating previously identified sources of resistance and tolerance [e.g., 36] into commercial cultivars. If such sources are unavailable or unsuitable, screening programs should aim to identify novel sources of SbDV resistance and tolerance among commercially available cultivars and germplasm collections.