Justification for Introducing OFSP and the Need for Quality Seed and Virus-Resistant Varieties

For whatever reason, when sweetpotato was introduced to Africa from the Americas over 500 years ago (O’Brien 1972), the types of sweetpotato that came to dominate were white-fleshed, containing no beta-carotene, or yellow-fleshed , which can have limited amounts of beta-carotene. Today in sub-Saharan Africa (SSA), 48% of children under five years of age are estimated to be vitamin A deficient (Black et al. 2013). The main causes of vitamin A deficiency (VAD) are lack of intake of vitamin-A-rich foods and illness, which leads to poor absorption or the loss of vitamin A (Sommer and West 1996). Significant reduction of VAD in young children can lower the incidence of young child mortality by over 20% and improved access to vitamin-A-rich foods and reducing illness are part of a long-term sustainable solution (Stevens et al. 2015). Thus, given the high level of VAD in SSA and the fact that sweetpotato is widely grown throughout SSA as a food security crop, with 4.7 million hectares in production (FAO 2017), the rationale for introducing and promoting orange-fleshed sweetpotato (OFSP) on the continent is obvious. Moreover, sweetpotato is principally considered a woman’s crop and a crop of the poor in SSA (Low et al. 2009), so the intervention naturally targets the vulnerable groups most at risk of vitamin A deficiency.

One of the first studies to investigate the acceptability of OFSP varieties in SSA was undertaken among 20 women’s groups in Western Kenya in 1995–1996 (Low et al. 1997). The research demonstrated that the orange color of the root was highly acceptable to both children and women, but the challenge was the texture with young children preferring low dry-matter types (like those found in the USA), while adult women demanded high dry-matter types (>30%) with mealy textures similar to the dominant, local white-fleshed types (Hagenimana et al. 1999). However, nearly all OFSP varieties imported from outside of SSA that were introduced to East Africa failed within a few years due to Sweet Potato Virus Disease (SPVD) (Fig. 5.1) (Grüneberg et al. 2015). Initial progress in uptake and utilization of OFSP imported varieties was faster in Southern Africa, where virus pressure was lower and adult dry-matter preferences (26–28%) also lower than those found in East Africa (Low et al. 2017b). In East Africa, however, we were fortunate to locate two orange-fleshed landraces (Kakamega, Ejumula) that could be utilized as varieties and as parents in breeding programs.

Fig. 5.1
figure 1

Orange-fleshed sweetpotato variety ‘Kakamega’, showing severe symptoms of SPVD (right), next to a symptomless plant (left). (Photo: Segundo Fuentes)

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In part due to the swathe of bi-modal rainfall areas found in East and Central Africa, over half of sweetpotato production in SSA is concentrated in this zone. Sweetpotato is vegetatively propagated in this region, principally by taking cuttings from existing plants, or in cases where the dry season is longer, leaving a few roots unharvested, which sprout again when the next rains arrive. Traditionally, if one does not have sufficient planting material on one’s own field, one can easily receive planting material from a neighbor for free (McEwan et al. 2015). Clearly, this is the ideal environment for viruses to accumulate over time in a given variety. While most farmers do not know what a virus is, they note that the plant “is getting tired” or the plant is “sick”. Sales of planting material are mostly to those traveling from outside the immediate community.

In this context, during the past 15 years, the International Potato Center (CIP) and 13 national partners in SSA have developed a two-pronged approach for tackling the often devastating effects of SPVD: (1) Breeding for virus resistance, and (2) developing sustainable quality “seed” systems.

The CIP virus resistance breeding effort is concentrated in Uganda and undertaken in collaboration with the National Crops Resources Research Institute (NaCRRI). This effort can be described as searching for a needle in a haystack, given that sweetpotato is a hexaploid (2n = 6× = 90) and the virus resistance level found in breeding populations to date occurs at very low frequencies of ≤0.2%. The two viruses constituting SPVD are Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV), in which inheritance of resistance is supposedly recessive (Mwanga et al. 2017). Moreover, resistance working against one virus strain may not work against another strain of the same virus. In spite of this, several high-yielding, moderately resistant varieties have been released during the past 12 years (Mwanga et al. 2009, 2016), the most notable being Kabode (NASPOT 10 O), which has also been released by Kenya, Tanzania, and Rwanda.

In the future, we expect more rapid progress in virus resistance breeding through the use of breeding schemes exploiting heterosis that will allow breeders in high SPVD pressure zones to apply more inbreeding for SPVD resistance (two partially inbred genepools) without sacrificing heterozygosity (hybrid population) for yield and stability performance (Mwanga et al. 2017). In addition, breakthroughs in on-going molecular marker research could vastly accelerate progress (Gruneberg et al. 2015). Clearly, low-cost but accurate ways to screen for different viruses at scale would also contribute to more efficient and timely selection decisions.

Regardless of the variety used, timely access to quality seed (actually cuttings from a vine rather than true seed) is essential for high yields in any sweetpotato production system. Virus-free planting material is higher yielding than non-virus-free (Adikini et al. 2016). On women’s small landholdings in Rwanda, use of quality seed was associated with increased yield; there were then surplus roots to sell, generating on average $277 annually per household (Sindi et al. 2015). In China, the introduction of virus-free seed to 80% of a major growing area by public-sector extension led to an average yield increase of 30% (Fuglie et al. 1999) .

Our efforts have focused on collaborating with 11 national programs and two private-sector companies to improve the efficiency and sustainability of early-generation seed (EGS) production, which consists of tissue culture plantlet production, and subsequent multiplication in a screenhouse, following a business model (Rajendran et al. 2017). Clearly, ensuring that all pre-basic materials are virus-free is a core part of their mandate. Given the low multiplication rates of sweetpotato compared to grain crops and the perishability and bulkiness of the vines, coupled with limited willingness of growers to pay, companies specializing in grain seed sales have shown little interest in investing in sweetpotato seed (Low et al. 2017a). Hence, in collaboration with non-governmental organizations and government extension services, the focus has been on setting up a network of decentralized, trained vine multipliers (DVMS) to serve their surrounding communities. These DVMS, in turn, are served by a few larger, well-resourced basic multipliers in their districts (or equivalent administrative unit) (McEwan et al. 2015). Multipliers are encouraged to become commercial root producers as well, as demand for seed often fluctuates. Several SSA countries are now implementing more formal certification or quality-declared seed classifications at this stage in the multiplication process to ensure that farmers know what variety they are receiving and their level of quality (McEwan et al. 2012). To make such classification schemes work in the long-run, affordable and accurate diagnostic tools for virus detection are required, so that ensuring quality does not become a bottleneck, impeding farmer access to clean planting material of improved varieties.

Thus, to achieve progress in both breeding for virus resistance and establishing sustainable access to seed, a better knowledge of how viruses operate, and how the ones causing serious economic damage can be combatted, are urgent priorities.

Brief View of Progress Made in Detecting Viruses in Africa

Being a vegetatively propagated crop, sweetpotato is prone to the buildup of pathogens in planting material. Because planting material is largely produced through stem cuttings (even when roots are used as the primary multiplication material) these are largely limited to foliar diseases, and particularly viruses. Sweetpotato is known to be affected by over 30 viruses (Clark et al. 2012), most of which are recently described DNA viruses (Table 5.1). However, studies have consistently shown that the potyviruses SPFMV and SPCSV are the most widespread and damaging in tropical regions of the world. In Africa, besides SPFMV and SPCSV, the potyvirus Sweet potato virus C (SPVC , previously known as the C strain of SPFMV) is also widespread and often found in association with SPFMV. Other potyviruses such as Sweet potato virus G (SPVG) and Sweet potato virus 2 (SPV2) are also found, as are the ipomovirus Sweetpotato mild mottle virus (SPMMV) (Ateka et al. 2007; Rännäli et al. 2009; Tugume 2010), begomoviruses (Miano et al. 2006; Wasswa et al. 2011), the carlaviruses Sweet potato chlorotic fleck virus (SPCFV) (Aritua et al. 2007) and Sweet potato C6 virus (SPC6V) (De Souza et al. 2013), the cavemovirus Sweet potato collusive virus (SPCV , previously known as Sweet potato caulimo-like virus) (Cuellar et al. 2011b), and the cucumovirus Cucumber mosaic virus (CMV) . The vectors of these viruses, where known, are aphids (potyviruses and CMV) and whiteflies (SPCSV and begomoviruses). Except for CMV, all these viruses are unique to sweetpotato, and sweetpotato (or related Ipomoea spp.) is not affected by viruses infecting other crops, nor are sweetpotato viruses found to infect other host plants, suggesting that the plant provides some unique cellular environment in which only specialized viruses can propagate.

Table 5.1 Viruses reported to infect sweetpotato worldwide
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Whereas SPFMV by itself usually causes only mild or no symptoms and limited or no yield loss in plants when it is the only virus infecting the plant, in combination with SPCSV it generates the severe sweetpotato virus disease complex (SPVD), which can lead to yield losses of close to 100% when grown from infected planting material (Clark et al. 2012; Gibson and Kreuze 2015). SPCSV can also cause synergistic diseases with other viruses, including SPMMV, SPCFV, CMV, begomoviruses, SPCV and the Solendovirus sweet potato vein clearing virus (SPVCV) (Untiveros et al. 2007; Cuellar et al. 2011a, 2015). This makes SPCSV the most significant virus contributing to sweetpotato yield losses worldwide. However, recent studies have provided evidence that SPFMV and begomoviruses by themselves may also cause significant yield losses in several different varieties (Ling et al. 2010; Mulabisana et al. 2019).

Despite this, except for Uganda and to a lesser extent Kenya, Tanzania and Rwanda, where surveys have been performed, until recently it was unclear how frequently all the different viruses occur and to what extent they contribute to yield losses throughout the African continent. The African sweetpotato virome project (Kreuze and Fei 2019) (http://bioinfo.bti.cornell.edu/virome/) applied high-throughput sequencing and assembly of small RNAs (sRSA) for virus detection to field-collected samples from 11 different African countries across the continent from 2012 to 2014. Results (Fig. 5.2) revealed that the most frequently occurring viruses were the recently discovered badnaviruses, Sweet potato pakakuy virus (SPPV ; 76% of samples), and the mastrevirus Sweet potato symptomless mastrevirus, which was also extremely common (45% of samples). Little is known about these viruses, but since their discovery through sRSA a decade ago they have been reported from sweetpotatoes around the world. They are not associated with any symptoms or disease, and our work (Kreuze et al. 2020) indicates that badnaviruses are not genome integrated, and occur in extremely low titers, and cause no appreciable symptoms or interaction with other viruses in sweetpotato or the indicator plant Ipomoea setosa. Thus, they may represent a new class of commensal or endosymbiotic viruses that are likely to exist and be discovered as new ultrasensitive virus detection approaches are applied more widely. Hence, when considering only viruses known to cause disease in sweetpotatoes, SPFMV was found to be the most common virus (55%) followed by SPCSV (25%), begomoviruses (18%) and SPVC (17%). In contrast, SPVG, SPV2 and a new potyvirus identified and named SPVZ appeared to be locally common in some countries but were not widespread over the continent. Several new viruses were also identified, but most represented only a fraction of the total and thus are not likely to be significant.

Fig. 5.2
figure 2

Viruses identified in African sweetpotato samples indicating the percentage of all viruses identified in the study. Viruses are organized according to taxonomic classification

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The begomoviruses were among the most common viruses found throughout the continent, something that was not known previously, yet little is known about their impact on yield. Studies performed previously in the USA showed they could have significant yield impacts, depending on the varieties, but are largely symptomless (Ling et al. 2010). Similar results were recently reported from different varieties in South Africa (Mulabisana et al. 2019). Recent results from yield trials in Kenya show a similar picture with apparently strong yield impacts in one variety, but none in another (Wanjala et al. 2019). Interestingly, the variety most affected by begomovirus was more resistant to SPVD, suggesting there might be a tradeoff for resistance to SPVD and begomoviruses, as previously suggested (Wasswa et al. 2011). Research should be prioritized to elucidate this point, because if it is confirmed, breeding for resistance to SPVD might inadvertently lead to selection of susceptibility to begomoviruses, replacing one problem with another. An additional result from the sweetpotato virome project was that visual symptoms on the plant, as determined by an expert, had little diagnostic value in relation to the viruses found in the plants under African field conditions.

Based on the studies described above, it can be concluded that the most important viruses to consider in Africa are SPFMV, SPVC, SPCSV and probably begomoviruses, although the extent of their yield impact seems highly variable. Nevertheless, other viruses can be locally common and should be considered in these locations when testing for viruses in production of clean planting material. To enable this, specific, sensitive, easy and affordable diagnostic tests need to be developed for these viruses, which are discussed in another section of this chapter.

Understanding Viruses to Improve Breeding for Virus Resistance

Virus-infected plants can gain resistance to further infections by closely related viruses, a phenomenon known as ‘cross-protection ’, or they can gain susceptibility to viruses that otherwise would not cause disease in a single infection, a phenomenon known as ‘synergistic interaction’ (Kassanis 1964). The biological study and characterization of mixed virus infections versus host defence responses has revealed important genetic and biochemical phenomena common to all living organisms. Early studies identified that infection by a virus affects the physiology, metabolic activity or even structure of the plant and any of these induced changes will affect the ease with which the host will respond to a second virus challenge (Matthews and Hull 2002; Smith 1931, 1960). As mentioned above, sweetpotato is vegetatively propagated and mixed virus infections do accumulate over time, representing a growing challenge for the plant. One well characterized host response to virus infection is based on ‘RNA silencing’, whereby host proteins identify the viral RNA and cut it into small molecules of 21–24 nt. These viral-derived RNA molecules then trigger a sequence-specific host response that degrades the RNA of the invader virus (Baulcombe 2004; Ding and Voinnet 2007). Therefore, it is not unexpected that viruses have evolved to encode proteins that block RNA silencing. Such proteins are known as RNA silencing suppression (RSS) proteins and several of them had been identified in the past as virulence factors or pathogenicity determinants (Díaz-Pendón and Ding 2008). RSS proteins, belonging to different families of viruses, inactivate the RNA silencing response at different points. Some are more effective than others in counteracting this host defence. One such protein, HCpro, is encoded by viruses in the genus Potyvirus and has been known for more than 20 years as a pathogenicity determinant and mediator of viral synergisms (Vance et al. 1995; Pruss et al. 2004). At the turn of the century there were around 20 different virus species reported to infect sweetpotato in single or mixed infections, including several potyviruses such as SPFMV (Loebenstein et al. 2003). However, potyviruses in sweetpotato were not mediators of viral synergisms in this host.

The Biggest Challenge: Sweet Potato Virus Disease (SPVD)

Several complex viral diseases of sweetpotato have been characterized and SPCSV has been identified as one of the components of the mixed infection: SPCSV in complex with SPFMV and Sweet potato mild speckling potyvirus (SPMSV) causes a chlorotic dwarf disease reported in Argentina (Di Feo et al. 2000). SPCSV and Sweet potato mild mottle ipomovirus (SPMMV) cause a severe mosaic disease reported in Uganda (Mukasa et al. 2006). In experimental inoculation tests, SPCSV could enhance the severity of disease symptoms caused by several RNA viruses including carlaviruses and cucumoviruses (Untiveros et al. 2007). What is most impressive is that the effect SPCSV has on mixed infections is not limited to co-infections with RNA viruses, but also occurs with DNA viruses such as the caulimoviruses (Cuellar et al. 2011b) and begomoviruses (Cuellar et al. 2015). Molecular analyses of mixed-virus infections involving SPCSV revealed that the disease symptom severity increases as the accumulation of the co-infecting virus(es) rises, while the titres of SPCSV remain little affected (Gibson et al. 1998; Karyeija et al. 2000; Mukasa et al. 2006; Untiveros et al. 2007; Kokkinos and Clark 2006a).

SPFMV and SPCSV are distributed worldwide including the Americas (Gutiérrez et al. 2003; Kashif et al. 2012; Di Feo et al. 2000) and Asia (Milgram et al. 1996; Qiao et al. 2011), yet cause the most damage in Africa (Gibson et al. 1998; Ateka et al. 2004; Njeru et al. 2008; Njeru et al. 2004; Mukasa et al. 2003; Mukasa et al. 2006; Tairo et al. 2005; Fenby et al. 2002). In Africa, epidemics of SPVD have been associated with the disappearance of elite cultivars (Gibson et al. 1997). A significant rise in the number of scientific reports on SPVD over the last 20 years (Fig. 5.3), linked to advances in virus detection protocols and the functional characterization of the disease, is a reflection of the relevance that SPVD still has in the developing world. Particular focus has been on two RSS proteins encoded by SPCSV, p22 and p26 (Kreuze et al. 2005).

Fig. 5.3
figure 3

Global distribution of SPVD field reports published between 1940 and 2019. (Source: Google Scholar). (a) Country shading indicates number of reports; at every tenth report, the shading of the country increases; Uganda shows the highest number of SPVD reports (>30). (b) Cumulative number of SPVD field reports published per decade, between 1940 and 2019. The y axis indicates number of publications during the decade starting from the year indicated on the x axis

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The Role of RSS Proteins in Virus Susceptibility

Historically p22 protein was the first candidate for an RSS protein involved in SPVD. Several factors contributed to this. First, p22 was present in the first SPCSV isolate from Uganda that was completely sequenced (Kreuze et al. 2002). Because at the time only partial sequencing (mainly regions corresponding to the Coat Protein or the Replicase) was the minimum required to identify other isolates of the virus, the absence of p22 in most non-Ugandan isolates was overlooked (Cuellar et al. 2008). Second, like other RSS proteins, p22 lacks any detectable sequence homology with other proteins and was located in a genomic region where other RSS proteins had been identified in the Closteroviridae (the family of viruses to which SPCSV belongs). Third, p22 readily showed RSS activity in standard agro-infiltration tests done in Nicotiana benthamiana plants (Kreuze et al. 2005). Efforts to clone and identify the diversity of p22 sequences from different isolates of SPCSV unveiled the fact that p22 was absent in most SPCSV isolates known so far (Cuellar et al. 2008). Interestingly, all those isolates lacking p22 were known to mediate SPVD, therefore any role of p22 in the development of the diseases was discarded. After conducting larger surveys for SPCSV isolates, based either on sequencing of the targeted 3′ region of RNA1 of SPCSV and generic deep-sequencing protocols (Kreuze et al. 2009), it was found that p22 was encoded in most of SPCSV isolates from Uganda but was absent elsewhere (Kashif et al. 2012; Tugume et al. 2013). At the same time, agro-infiltration assays of several SPCSV proteins identified a weak but still detectable RSS activity for the \p26 protein. P26 encodes an RNase III-type of protein (RNase3). Proteins in this family contain a single ribonuclease domain and a single dsRBD domain. Bacterial and viral RNAse III belong to this class (MacRae and Doudna 2007). Unlike other viral silencing suppression proteins, homologs of the viral RNase3 enzymes exist in unrelated RNA and DNA viruses (Weinheimer et al. 2015). All SPCSV isolates characterized so far encode an RNase3; this also occurs in SPCSV-related species as shown by its detection in wild Ipomoea plants. In contrast, p22 has been found in few isolates so far (Tugume et al. 2013), which suggests an active genomic region in SPCSV (the 3′ end of RNA1) moulded by recombination that could serve as an important target for engineering resistance to SPVD. Experiments by Cuellar et al. (2009) unequivocally demonstrated that expression of p26 by itself was sufficient to generate synergistic diseases with all other viruses tested, implicating it in the inhibition of a key antiviral defence mechanism in sweetpotato. Whereas the exact molecular mechanism by which RNase3 suppresses the plant RNA silencing response has yet to be determined, we know it requires dsRNA ribonuclease activity (Cuellar et al. 2009; Weinheimer et al. 2015) and that it interacts with the plant-encoded antiviral defence protein SGS3 (Weinheimer et al. 2016). This protein domain is a prime target for biotechnological engineering of resistance and for developing strategies to control the diseases and minimize economic losses in sweetpotato production.

The other major component of SPVD, SPFMV has also been studied for its ability to suppress RNA silencing. SPFMV, together with SPVC, SPVG and SPV2 belong to the genus Potyvirus and are related to each other in having a significantly larger genome than other potyviruses due to an exceptionally large P1 protein. Untiveros et al. (2007) recognized the existence of two domains in the P1 protein, named P1-N and P1-Pro, separated by a hypervariable domain. Then Clark et al. (2012) recognized that the P1-Pro domain had an overlapping open reading frame, which they named PISPO. This was subsequently identified as an RNA silencing suppressor unique to Ipomoea-infecting potyviruses (Untiveros et al. 2016; Mingot et al. 2016). The P1 itself and HC-Pro of sweetpotato-infecting potyviruses, however, also show silencing suppressor activities (Rodamilans et al. 2018). This implicates at least three RSS proteins with different modes of action in SPFMV. In addition, the polymerase slippage rate, leading to production of P1N-PISPO, is reduced in SPVD-affected plants, adding to the complexity of the interaction of this disease.

The Role of Wild Ipomoea Weeds in Virus Epidemiology

Long before plants were domesticated, viruses co-evolved with their wild host plants (Lovisolo et al. 2003). This co-evolution was drastically affected following plant domestication, agricultural intensification of monocultures and trade (Stukenbrock and McDonald 2008). Several studies suggest that species of wild host plants play the role of reservoirs in the ecology of plant viruses and, therefore, in their epidemiology as sources of inocula and diversity in agroecosystems (Fargette et al. 2006). Over 80 species of the genus Ipomoea and other genera within the family Convolvulaceae occur in East Africa. They are important reservoirs of a larger diversity of SPFMV isolates and their corresponding RSS proteins (Tugume et al. 2010a, b, 2013, 2016, 2008). Furthermore, at least one new viral species related to SPCSV and encoding an RNase3-like RNA-silencing suppressor protein has also been detected in Uganda (Tugume et al. 2013) and Tanzania (African Sweetpotato Virome), although this virus seems to be currently rare in cultivated sweetpotato.

Resistance

Whereas a significant amount of research has been done to understand susceptibility to SPVD during the last 20 years, almost no work has been done to understand the mechanisms of resistance . This is probably largely due to the lack of highly resistant genotypes, the complex nature of the highly heterozygous hexaploid sweetpotato genome and lack of major genes contributing to virus resistance in sweetpotato germplasm. With more resistant genotypes now being produced through breeding efforts and genomic tools such as a reference genome for sweetpotato (Wu et al. 2018) available, new opportunities have arisen to address this lack of understanding and doing so should be a priority. Whereas resistance has been described in some related Ipomoea spp. (Karyeija et al. 1998), these do not cross with sweetpotatoes and are thus of limited value for breeding purposes.

Understanding Viruses to Improve Diagnostic Tools and to Support Breeding and Development of a Quality Seed System

Generally speaking, sweetpotato is rather resistant to most viruses: only specialized sweetpotato viruses seem to be able to infect it, and even they usually cause only mild or no symptoms and occur in only low titres when infecting sweetpotato alone. Many varieties also seem to be able to recover from single infections, producing virus-free branches from which cuttings can be taken, and this is more pronounced in varieties considered as resistant to SPVD. This phenomenon, which becomes more pronounced at higher temperatures, may explain why farmers in Africa can maintain planting material over many generations without it completely degenerating through virus infections (Gibson and Kreuze 2015). Whereas the mechanism for this recovery is not yet elucidated, it is likely to be related to the efficiency of resistance in the plant mediated by RNA silencing; this has also been shown to be temperature dependent (Szittya et al. 2003). If RNA-silencing-mediated resistance is critical for recovery and/or for virus resistance in the field, as is suggested by several lines of evidence (role of RNase3 in SPVD, temperature dependence of recovery), understanding its mechanism and the genes governing it should enable more efficient breeding for resistance and other control mechanisms.

Small RNAs are key molecules in the RNA silencing pathway and studying them might further contribute to understanding virus resistance and susceptibility in sweetpotato. Indeed, when the sRSA technology was invented and first applied to sweetpotato, an immediate result was the discovery of previously unknown viruses (Kreuze et al. 2009). Whereas sRSA has evolved into a widely adopted method for the discovery and identification of viruses in plants, it also enables the study of RNA-silencing-based antiviral defence in these same plants (Pooggin 2018). Indeed, the original intent of the experiment by Kreuze et al. (2009), was to try to understand how SPFMV and SPCSV were targeted by the plant RNA silencing machinery in order to enable more informed design of transgenic constructs for resistance to SPVD. Previous attempts to generate resistance to SPVD by targeting both viruses with RNA-silencing-inducing constructs had been only partially successful: these led to reduced titres of SPCSV, but were not sufficient to prevent the provocation of SPVD in co-infection with SPFMV (Kreuze et al. 2008). Based on that analysis, new constructs were designed and unpublished results from field trials in Kenya show promising levels of resistance. Likewise, experiments are ongoing in our lab to compare small RNA profiles/amounts, viral RNA concentrations and host gene expression in varieties considered susceptible, moderately resistant and resistant to SPVD. This will certainly lead to new insights that may be applied to guide and support breeding efforts.

The fact that most sweetpotato viruses occur at only low concentrations in plants when infecting alone, presents a diagnostic problem. Whereas the International Potato Center has for many years, produced virus detection kits for the most common sweetpotato viruses based on Enzyme linked immunosorbent assays (ELISAs), the reliability of detection directly from sweetpotato by ELISA is limited in single virus-infected plants. The only way to reliably detect such viruses is through grafting plants to the universal indicator plant I. setosa and performing the ELISA from that plant; this is a laborious and time-consuming procedure, only worthwhile for the most basic nuclear stock of planting material. More sensitive laboratory-based methods such as (multiplex) PCR (Kwak et al. 2014; Li et al. 2012; Opiyo et al. 2010) and qPCR (Kokkinos and Clark 2006b) have been published and are available for testing most sweetpotato viruses, but have the disadvantage of being relatively expensive, requiring laboratory conditions for sample preparation and running the actual test. Thus, like indicator grafting, these are typically only applied to nuclear stock material. However, new sensitive diagnostic tools are under development that could significantly reduce the time and cost of virus testing and could be used at the point of care, for example on seed production fields.

Although sRSA is also relatively expensive, requiring laboratory conditions, skill and bio-informatics capacity, it has a tremendous potential to replace or accelerate virus indexing procedures to produce -virus-free nuclear stock material and to enable quarantine procedures. The current gold standard for virus indexing sweetpotato consists of two rounds of grafting to I. setosa, combined with PCR for DNA viruses and NCM ELISA for 10 other viruses. The whole procedure takes at least 6 months, a considerable amount of greenhouse space and skill – at a cost of more than 120 USD per sample. This procedure ensures that all possible viruses are reliably detected, including those variants that are as yet undescribed since they also may produce symptoms in the indicator plant. sRSA can likewise detect all known as well as unknown viruses, but directly from the query plant itself, avoiding the need for the use of an indicator plant. The procedure to prepare samples takes about a week; high-throughput sequencing takes a few days, and analysis a few hours using specialized software, thus providing a significant reduction in time to result, which is currently the biggest bottleneck in moving planting material of improved varieties and breeding materials between countries. Another benefit of sRSA is that data once generated can be saved and if new viruses are discovered in the future, material does not need to be re-tested; one can simply re-query the sequence data for its presence. With the cost being below 100 USD per sample combined with manifold reductions in time to results, sRSA is an obvious candidate to replace the current indicator-host-based indexing procedure. At present, validation data are being generated and standard operating procedures developed for sRSA to replace standard indexing, which is under ISO17025 certification at CIP headquarters in Lima. Once this has been done, sRSA can be fully implemented, replacing indicator host indexing. Training provided by CIP to national programs during recent years aims to ensure they become familiar with sRSA as well, and improvement of user-friendliness of analysis software may eventually lead to this technique being widely adopted throughout the world.

sRSA has already been widely applied to identify new viruses and as a survey tool to determine crop ‘viromes’, as described for sweetpotato above. The data also provide a valuable resource to design more specific and dedicated diagnostic assays that are fit for use in other settings. Using the sweetpotato virome data, CIP has been working on two particular assays over the last decade: diagnostic tube arrays and Loop mediated isothermal amplification (LAMP) assays.

Tube Arrays

With more than 30 viruses now known to infect sweetpotatoes, it is necessary to perform and combine multiple different assays to be certain that a plant is free of them all. Although sRSA provides an easier alternative, it is still relatively expensive and takes at least 2 weeks from initiation to final result. Thus, there is a scope for a multiplex assay that can detect all known sweetpotato viruses rapidly and with high sensitivity in one assay. To achieve this, CIP has been developing an approach combining multiplex PCR with microarray technology to create a rapid and sensitive assay for known sweetpotato viruses. To provide a cost-effective solution that meets the need for user-friendly processing via conventional lab equipment and high-volume manufacturing capacities that comply with in vitro diagnostic (IVD) regulations, the ArrayTube (AT) platform (Alere Technologies GmbH, Jena, Germany) was selected. This platform consists of a customizable microarray integrated into a 1.5 ml micro tube, which simplifies handling and is used for routing testing in the medical field (Braun et al. 2012; Schneeberg et al. 2015). The AT is printed with custom-designed probes corresponding to regions of the target virus, which are amplified prior to hybridization in the AT by a multiplex PCR reaction with primers corresponding to the same target viruses and a number of controls. PCR fragments are labeled with biotin during amplification, enabling their detection by ELISA after hybridization to the AT. Positive reactions will show up as dots at the corresponding probe position and can be documented and analysed with a dedicated AT reader, or even using a cellphone and a specific App designed by CIP. Design of an effective AT for multiple viruses is complex, requiring design of many primer-probe combinations and several iterations to optimize specificity. In the case of the sweetpotato virus AT that we developed, the sequence data obtained from the African sweetpotato virome project were used to design primers and probes able to detect all common sweetpotato viruses, and the samples were used for their validation. Unfortunately, Alere Technologies recently discontinued the production of the ArrayTube, highlighting the risk of utilizing technologies from a single provider for method development.

Loop-Mediated Isothermal Amplification (LAMP) for Sensitive Field-Based Diagnostics

PCR-based methods have the great benefits of speed and sensitivity, being able to detect minute amounts of a target nucleic acid. However, these are offset by the need for physically large and power-hungry thermal cycling equipment and the need for laboratory conditions for nucleic acid extraction to achieve a sufficiently pure preparation. This combination of requirements makes the technique unsuitable for routine use under field conditions. Over the past 10 years, a number of isothermal amplification methods have been developed using various approaches and their use in diagnostics is reviewed in Boonham et al. (2013). The benefit of isothermal amplification is its requirement for much simpler equipment to run the reactions, and several field-portable battery-powered options are available (e.g. realtime Genie, Bio-ranger). Some of them are also considerably more robust to contaminants, enabling the use of rapid and simple nucleic acid extraction protocols directly in the field. CIP, together with partners, has been developing assays based on one of these: LAMP for the major sweetpotato viruses, including SPFMV, SPCSV and begomoviruses. LAMP reagents can be pre-loaded and lyophilized into reaction tubes to make them room stable. Extractions are done by macerating leaf punches in an alkaline PEG solution (Chomczynski and Rymaszewski 2006) and using disposable inoculation loops to transfer extract to reaction tubes (in which reagents have been reconstituted in molecular grade water). Assays take anything from 20 min to 1 h to perform depending on the virus titres in the plants and results are displayed in real time. Field testing of these assays, under various climatic conditions in Kenya, has shown them to be robust and reliable in detecting target viruses as compared to PCR (done from the same samples taken back to the lab). Thus, if the price can be reduced sufficiently through volume production, there is potential for such assays to be used in field inspection of certified planting material. Currently inspections are based purely on symptoms and thus can only eliminate SPVD-affected plants, leaving a significant reservoir of viruses in the seed plots, which can combine to form SPVD in production fields.

Lessons Learned and the Way Forward

Under the Sweetpotato for Profit and Health Initiative launched in 2009, CIP and over 30 partners were able to reach 6.2 million households with improved varieties of sweetpotato by July 2019. Along the way, we have been able to test different seed delivery approaches and continue to work on understanding the bottlenecks in the seed system and conducting research to address them. Clearly, addressing the virus issue has been a major focus.

The research described above has elucidated the extent to which different viruses contribute to yield reduction, has shed light on the mechanisms through which they interact, and has described more sensitive and rapid diagnostic methods. It has also suggested novel approaches to virus control through transgenic means. The continental surveys have confirmed the universal importance of SPCSV and SPFMV, but have also revealed several new viruses and viral strains, although most of them are only minor and local in occurrence. Widespread occurrence of novel badnavirus and mastrevirus seem to have only limited relevance since these viruses are not associated with any type of disease symptoms and occur only in very low titres. In contrast, begomoviruses were the third most prevalent viruses in sweetpotato. Because the sweetpotato begomoviruses cause almost imperceptible symptoms they have largely gone unnoticed until recently. Nevertheless, they can cause significant yield losses on their own, even in cultivars considered resistant to SPVD. Thus, breeders need to start taking begomovirus resistance into account, as do seed producers who will need inexpensive, sensitive and rapid diagnostic methods to be successful. The LAMP assays developed for sweetpotato viruses described above may provide a solution, particularly since they are also semi-quantitative and could enable breeders to detect partial resistance in their material.

Because production of virus-free seed vines is expensive, deploying molecular diagnostics in a sustainable way in sweetpotato seed systems is likely to occur only when a significant proportion of sweetpotato growers become highly commercial in their orientation, or when governments commit to using them to ensure high quality early-generation seed. This is beginning to emerge in some locations, either to provide fresh roots to urban centres in Africa, or to serve the export market to Europe through public-private partnerships. Deploying LAMP-type molecular tests to support resistance breeding may be more straightforward, as the cost of the test would be offset by the increased speed and accuracy of the result. The current approach is visual assessment using a largely subjective scale from 1 to 5 for virus resistance.

At a time when international funding agencies are focusing on short-term impacts and outcomes it is noteworthy that basic and applied virology research has revealed previously unsuspected virus problems in sweetpotato and has helped to provide solutions to them. We propose that continued surveillance of sweetpotato viruses should be conducted to monitor the emergence of new viruses and variants that can be expected as a result of increasing global trade and climate change . Further investment into the largely unknown mechanisms of virus resistance to the different viruses, a truly underinvested area of research, is also needed to assist breeding efforts to develop resistant varieties more efficiently.