Effects of Sweet Potato Feathery Mottle Virus and Sweet Potato Chlorotic Stunt Virus on the Yield of SweetPotato in Uganda

Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) are the most common viruses infecting sweetpotato in Uganda. Field plots planted with graft inoculated plants of virus‐free cultivars Beauregard, Dimbuka, Ejumula, Kabode and NASPOT 1 were used to assess the effect of SPFMV and SPCSV on yield and quality of sweetpotatoes in two agro‐ecologies. SPFMV spreads rapidly to control plots at Makerere University Agricultural Research Institute Kabanyolo (MUARIK), and these plots had similar yields to those singly infected with SPFMV but at the National Semi Arid Resource Research Institute (NaSARRI) where SPFMV spreads slowly, plots infected with SPFMV yielded 40% less than the control. Recovery from SPFMV appeared to be more frequent at NaSARRI than at MUARIK. Infection by SPCSV alone resulted in yield losses of 14–52%, while mixed infections of SPFMV+SPCSV resulted in yield losses in both locations of 60–95% depending on the cultivar. SPCSV and mixed infections of SPFMV+SPCSV also reduced the number of roots formed as well as the diameter of the roots, resulting in a greater length to diameter ratio compared to the healthy control. This study, therefore, confirms that both SPFMV and SPCSV, both singly and when mixed, can reduce yield, the extent depending on the cultivar. To mitigate the effect of these viruses, farmers should use clean planting materials of resistant varieties.     

Identification and distribution of viruses infecting sweet potato in Kenya

Four hundred and forty-eight symptomatic and 638 asymptomatic samples were collected from sweet potato fields throughout Kenya and analysed serologically using antibodies to Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato mild mottle virus (SPMMV), Cucumber mosaic virus (CMV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato latent virus (SwPLV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato mild speckling virus (SPMSV) and C-6 virus in enzyme-linked immunosorbent assays (ELISA). Only SPFMV, SPMMV, SPCSV, and SPCFV were detected. Ninety-two percent and 25% of the symptomatic and asymptomatic plants respectively tested positive for at least one of these viruses. Virus-infected plants were collected from 89% of the fields. SPFMV was the most common and the most widespread, detected in 74% of the symptomatic plants and 86% of fields surveyed. SPCSV was also very common, being detected in 38% of the symptomatic plants and in 50% of the fields surveyed. SPMMV and SPCFV were detected in only 11% and 3% of the symptomatic plant samples respectively. Eight different combinations of these four viruses were found in individual plants. The combination SPFMV and SPCSV was the most common, observed in 22% of symptomatic plants. Virus combinations were rare in the asymptomatic plants tested. Incidence of virus infection was highest (18%) in Kisii district of Nyanza province and lowest (1%) in Kilifi and Malindi districts of Coast province.     

Development of transgenic sweet potato with multiple virus resistance in South Africa (SA)

Multiple infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KwaZulu-Natal, South Africa. In order to address the problem of multiple virus infections and synergism, this study aimed to develop transgenic sweet potato (cv. Blesbok) plants with broad virus resistance. Coat protein gene segments of SPFMV, SPCSV, SPVG and SPMMV were used to induce gene silencing in transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring the expression cassette. Polymerase chain reaction and Southern blot analyses showed integration of the transgenes occurred in six of the 24 putative transgenic plants and that all plants seemed to correspond to the same transformation event. The six transgenic plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV-infected Ipomoea setosa Ker. Although virus presence was detected using nitrocellulose enzyme-linked immunosorbent assay, all transgenic plants displayed delayed and milder symptoms of chlorosis and mottling of lower leaves when compared to the untransformed control plants. These results warrant further investigation on resistance to virus infection under field conditions.     

Resistance to sweet potato virus disease (SPVD) in wild East African Ipomoea

Sweet potato virus disease (SPVD), the most harmful disease of sweet potatoes in East Africa, is caused by mixed infection with sweet potato feathery mottle potyvirus (SPFMV) and sweet potato chlorotic stunt crinivirus (SPCSV). Wild Ipomoea spp. native to East Africa (J cairica, I. hildebrandtii, I. involucra and J wightii) were graft-inoculated with SPVD-affected sweet potato scions. Inoculated plants were monitored for symptom development and tested for SPFMV and SPCSV by grafting to the indicator plant J setosa, and by enzyme-linked immunosorbent assay (ELISA). Virus-free scions of sweet potato cv. Jersey were grafted onto these wild Ipomoea spp. in the field, and scions collected 3 wk later were rooted in the greenhouse and tested for viruses using serological tests and bioassays. In all virus tests, J cairica and J involucra were not infected with either SPFMV or SPCSV. J wightii was infected with SPFMV, but not SPCSV, in the field and following experimental inoculation; J hildebrandtii was infected with SPCSV, but not SPFMV, following experimental inoculation. These data provide the first evidence of East African wild Ipomoea germplasm resistant to the viruses causing SPVD.     

The Effect of the Sequence of Infection of the Causal Agents of Sweet Potato Virus Disease on Symptom Severity and Individual Virus Titres in Sweet Potato cv. Beauregard

Sweet potato virus disease (SPVD) is caused by dual infection of plants with Sweet Potato Feathery Mottle Virus (SPFMV) and Sweet Potato Chlorotic Stunt Virus (SPCSV). Because SPFMV and SPCSV are transmitted by aphids and whiteflies, respectively, infection in nature occurs independently rather than simultaneously. To investigate the effect of consecutive infection on symptom development and individual virus titres, plants infected with a single virus were later inoculated with the second virus. Symptoms were significantly more severe in plants infected with SPCSV followed by SPFMV compared to plants infected with SPFMV followed by SPCSV. Virus titres were not significantly different for SPCSV, but SPFMV titres, in plants infected with SPCSV followed by SPFMV, were significantly higher than all other treatments. The results indicate that the sequence of infection of sweetpotato plants with the causal agents of SPVD influence the severity of symptoms and SPFMV titres in SPVD affected plants.     

Genetic Variability and Evolutionary Implications of RNA Silencing Suppressor Genes in RNA1 of Sweet Potato Chlorotic Stunt Virus Isolates Infecting Sweetpotato and Related Wild Species

Background

The bipartite single-stranded RNA genome of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus; Closteroviridae) encodes a Class 1 RNase III (RNase3), a putative hydrophobic protein (p7) and a 22-kDa protein (p22) from genes located in RNA1. RNase3 and p22 suppress RNA silencing, the basal antiviral defence mechanism in plants. RNase3 is sufficient to render sweetpotato (Ipomoea batatas) virus-susceptible and predisposes it to development of severe diseases following infection with unrelated virus. The incidence, strains and gene content of SPCSV infecting wild plant species have not been studied.

Methodology/Principal Findings

Thirty SPCSV isolates were characterized from 10 wild Ipomoea species, Hewittia sublobata or Lepistemon owariensis (family Convolvulaceae) in Uganda and compared with 34 local SPCSV isolates infecting sweetpotatoes. All isolates belonged to the East African (EA) strain of SPCSV and contained RNase3 and p7, but p22 was not detected in six isolates. The three genes showed only limited genetic variability and the proteins were under purifying selection. SPCSV isolates lacking p22 synergized with Sweet potato feathery mottle virus (SPFMV, genus potyvirus; Potyviridae) and caused severe symptoms in co-infected sweetpotato plants. One SPCSV isolate enhanced accumulation of SPFMV, but no severe symptoms developed. A new whitefly-transmitted virus (KML33b) encoding an RNase3 homolog (<56% identity to SPCSV RNase3) able to suppresses sense-mediated RNA silencing was detected in I. sinensis.

Conclusions/Significance

SPCSV isolates infecting wild species and sweetpotato in Uganda were genetically undifferentiated, suggesting inter-species transmission of SPCSV. Most isolates in Uganda contained p22, unlike SPCSV isolates characterized from other countries and continents. Enhanced accumulation of SPFMV and increased disease severity were found to be uncoupled phenotypic outcomes of RNase3-mediated viral synergism in sweetpotato. A second virus encoding an RNase3-like RNA silencing suppressor was detected. Overall, results provided many novel and important insights into evolutionary biology of SPCSV.     

Viruses infecting sweet potato in Rwanda: occurrence and distribution

A survey of sweet potato virus diseases was conducted in the major sweet potato production areas in low, medium and high altitude zones of Rwanda. A total of 205 symptomatic and 103 asymptomatic samples were collected from 51 sweet potato fields and assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato latent virus (SwPLV), Sweet potato caulimo‐like virus (SPCaLV) and Cucumber mosaic virus (CMV) using nitrocellulose membrane enzyme‐linked immunosorbent assay. The viruses detected in the samples were SPFMV, SPMMV, SPCSV, SPCFV and SwPLV. Viruses were detected in 83% and 31% of the symptomatic and asymptomatic samples, respectively. SPFMV was detected in 49% of the samples. SPCSV, the second most common virus, was detected in 28% of samples collected from 73% of the fields. About 19% of the samples were tested positive for SPMMV. Thirteen combinations of multiple virus infections were detected in the samples. Viruses were detected in samples from all the fields surveyed, and the frequency of detection was greatest in samples from low altitude zones.     

Synergistic interactions of begomoviruses with Sweet potato chlorotic stunt virus (genus Crinivirus) in sweet potato (Ipomoea batatas L.)

Three hundred and ninety‐four sweet potato accessions from Latin America and East Africa were screened by polymerase chain reaction (PCR) for the presence of begomoviruses, and 46 were found to be positive. All were symptomless in sweet potato and generated leaf curling and/or chlorosis in Ipomoea setosa. The five most divergent isolates, based on complete genome sequences, were used to study interactions with Sweet potato chlorotic stunt virus (SPCSV), known to cause synergistic diseases with other viruses. Co‐infections led to increased titres of begomoviruses and decreased titres of SPCSV in all cases, although the extent of the changes varied notably between begomovirus isolates. Symptoms of leaf curling only developed temporarily in combination with isolate StV1 and coincided with the presence of the highest begomovirus concentrations in the plant. Small interfering RNA (siRNA) sequence analysis revealed that co‐infection of SPCSV with isolate StV1 led to relatively increased siRNA targeting of the central part of the SPCSV genome and a reduction in targeting of the genomic ends, but no changes to the targeting of StV1 relative to single infection of either virus. These changes were not observed in the interaction between SPCSV and the RNA virus Sweet potato feathery mottle virus (genus Potyvirus), implying specific effects of begomoviruses on RNA silencing of SPCSV in dually infected plants. Infection in RNase3‐expressing transgenic plants showed that this protein was sufficient to mediate this synergistic interaction with DNA viruses, similar to RNA viruses, but exposed distinct effects on RNA silencing when RNase3 was expressed from its native virus, or constitutively from a transgene, despite a similar pathogenic outcome.     

RNA silencing-mediated resistance to a crinivirus (Closteroviridae) in cultivated sweetpotato (Ipomoea batatas L.) and development of sweetpotato virus disease following co-infection with a potyvirus

Sweetpotato chlorotic stunt virus (SPCSV; genus Crinivirus , family Closteroviridae) is one of the most important pathogens of sweetpotato ( Ipomoea batatas L.). It can reduce yields by 50% by itself and cause various synergistic disease complexes when co-infecting with other viruses, including sweetpotato feathery mottle virus (SPFMV; genus Potyvirus , family Potyviridae). Because no sources of true resistance to SPCSV are available in sweetpotato germplasm, a pathogen-derived transgenic resistance strategy was tested as an alternative solution in this study. A Peruvian sweetpotato landrace 'Huachano' was transformed with an intron-spliced hairpin construct targeting the replicase encoding sequences of SPCSV and SPFMV using an improved genetic transformation procedure with reproducible efficiency. Twenty-eight independent transgenic events were obtained in three transformation experiments using a highly virulent Agrobacterium tumefaciens strain and regeneration through embryogenesis. Molecular analysis indicated that all regenerants were transgenic, with 1–7 transgene loci. Accumulation of transgene-specific siRNA was detected in most of them. None of the transgenic events was immune to SPCSV, but ten of the 20 tested transgenic events exhibited mild or no symptoms following infection, and accumulation of SPCSV was significantly reduced. There are few previous reports of RNA silencing-mediated transgenic resistance to viruses of Closteroviridae in cultivated plants. However, the high levels of resistance to accumulation of SPCSV could not prevent development of synergistic sweet potato virus disease in those transgenic plants also infected with SPFMV.     

Small RNA deep sequencing‐based detection and further evidence of DNA viruses infecting sweetpotato plants in Tanzania

Small interfering RNA deep sequencing (SRDS) was used to detect viruses in 23 sweetpotato plants, collected from various locations in Tanzania. Alignment of small RNA reads using a MAQ program recovered genomes of viruses from five families, namely Geminiviridae (2), Closteroviridae (1), Betaflexiviridae (1), Caulimoviridae (1) and Potyviridae (1). This was in agreement with the variation of symptoms observed on sweetpotato plants in fields and screen house, which included leaf curl, vein yellowing, chlorosis, stunted growth and brown blotches. PCR was also used to confirm the occurrence of viruses associated with leaf curl and symptomless infections. A complete genome (2768 nucleotides) was obtained for a sweepovirus that was 89.9% identical to the strain of Sweet potato leaf curl Sao Paulo virus (SPLCSPV; Begomovirus) reported in South Africa. Sweepoviruses are known to undergo frequent recombinations and evidence for this was found in the SPLCSPV sequence studied. The SRDS‐based results indicated occurrence of the poorly studied Sweet potato badnavirus B (SPBV‐B) and Sweet potato badnavirus A (collectively known as Sweet potato pakakuy virus; SPPV; Caulimoviridae) in sweetpotato plants in Tanzania. A 5′‐end partial sequence (3065 nucleotides), encoding hypothetical, movement and coat proteins, was obtained and found to be 86.3% and 73.1% identical to SPBV‐B and SPBV‐A, respectively. A survey for the distribution of SPPV and Sweet potato symptomless mastrevirus 1 (SPSMV‐1) showed that these viruses were wide spread and co‐infecting sweetpotato plants in Tanzania. The importance of East Africa as a hot spot for the diversity and evolution of sweet potato viruses is discussed.     

Aspects of resistance to sweet potato virus disease in sweet potato

In field trials during the first and the second rainy season of 1996 in Uganda, whiteflies were similarly abundant and aphids were absent on three clones of sweet potato (NIS-93–63, cv. Tanzania and cv. New Kawogo) although the three clones differed considerably in their resistance to sweet potato virus disease (SPVD), a complex disease resulting from infection by both the aphid-borne sweet potato feathery mottle virus (SPFMV) and the whitefly-borne sweet potato chlorotic stunt virus (SPCSV). This suggests that vector resistance does not determine the relative SPVD resistance of these genotypes. SPFMV alone had only a low virus titre in sweet potato cvs Tanzania and New Kawogo, became increasingly difficult to detect in plants of these cultivars and was seldom acquired by aphids. However, this resistance to SPFMV was not apparent in plants which were also infected with SPCSV. Plants then had a high SPFMV titre, appeared unable to eliminate SPFMV and provided good sources for aphids to acquire it.     

Variation in Virus Symptom Development and Root Architecture Attributes at the Onset of Storage Root Initiation in ‘Beauregard’ Sweetpotato Plants Grown with or without Nitrogen

It has been shown that virus infections, often symptomless, significantly limit sweetpotato productivity, especially in regions characterized by low input agricultural systems. In sweetpotatoes, the successful emergence and development of lateral roots (LRs), the main determinant of root architecture, determines the competency of adventitious roots to undergo storage root initiation. This study aimed to investigate the effect of some plant viruses on root architecture attributes during the onset of storage root initiation in ‘Beauregard’ sweetpotatoes that were grown with or without the presence of nitrogen. In two replicate experiments, virus-tested plants consistently failed to show visible symptoms at 20 days regardless of nitrogen treatment. In both experiments, the severity of symptom development among infected plants ranged from 25 to 118% when compared to the controls (virus tested plants grown in the presence of nitrogen). The presence of a complex of viruses (Sweet potato feathery mottle virus, Sweet potato virus G, Sweet potato virus C, and Sweet potato virus 2) was associated with 51% reduction in adventitious root number among plants grown without nitrogen. The effect of virus treatments on first order LR development depended on the presence or absence of nitrogen. In the presence of nitrogen, only plants infected with Sweet potato chlorotic stunt virus showed reductions in first order LR length, number, and density, which were decreased by 33%, 12%, and 11%, respectively, when compared to the controls. In the absence of nitrogen, virus tested and infected plants manifested significant reductions for all first order LR attributes. These results provide evidence that virus infection directly influences sweetpotato yield potential by reducing both the number of adventitious roots and LR development. These findings provide a framework for understanding how virus infection reduces sweetpotato yield and could lead to the development of novel strategies to mitigate virus effects on sweetpotato productivity.     

Molecular genetic analysis of virus isolates from wild and cultivated plants demonstrates that East Africa is a hotspot for the evolution and diversification of Sweet potato feathery mottle virus

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is globally the most common pathogen of cultivated sweet potatoes (Ipomoea batatas; Convolvulaceae). Although more than 150 SPFMV isolates have been sequence‐characterized from cultivated sweet potatos across the world, little is known about SPFMV isolates from wild hosts and the evolutionary forces shaping SPFMV population structures. In this study, 46 SPFMV isolates from 14 wild species of genera Ipomoea, Hewittia and Lepistemon (barcoded for the matK gene in this study) and 13 isolates from cultivated sweet potatoes were partially sequenced. Wild plants were infected with the EA, C or O strain, or co‐infected with the EA and C strains of SPFMV. In East Africa, SPFMV populations in wild species and sweet potato were genetically undifferentiated, suggesting inter‐host transmission of SPFMV. Globally, spatial diversification of the 178 isolates analysed was observed, strain EA being largely geographically restricted to East Africa. Recombination was frequently detected in the 6K2‐VPg‐NIaPro region of the EA strain, demonstrating a recombination ‘hotspot’. Recombination between strains EA and C was rare, despite their frequent co‐infections in wild plants, suggesting purifying selection against strain EA/C recombinants. Positive selection was predicted on 17 amino acids distributed over the entire coat protein in the globally distributed strain C, as compared to only four amino acids in the coat protein N‐terminus of the EA strain. This selection implies a more recent introduction of the C strain and a higher adaptation of the EA strain to the local ecosystem. Thus, East Africa appears as a hotspot for evolution and diversification of SPFMV.     

The incidence of sweet potato virus disease and virus resistance of sweet potato grown in Uganda

Sweet potato virus disease (SPVD) was common (25–30% average incidences), and farmers recognised it as an important disease, in sweet potato crops in southern Mpigi, Masaka and Rakai Districts in Uganda, but SPVD was rare in Soroti and Tororo Districts. Whiteflies, which are the vector of sweet potato chlorotic stunt crinivirus (SPCSV) a component cause of SPVD, were correspondingly common on sweet potato crops in Mpigi and rare on crops in Tororo. However, aphids, which are the vectors of sweet potato feathery mottle potyvirus (SPFMV), the other component cause of SPVD, were not found colonising sweet potato crops, and itinerant alate aphids may be the means of transmission. Different sweet potato cultivars were predominant in the different districts surveyed and four local cultivars obtained from Kanoni in S. Mpigi, where whiteflies and SPVD were common, were more resistant to SPVD than four cultivars from Busia in Tororo District, where whiteflies and SPVD were rare. However, nationally released cultivars were even more resistant than the local cultivars from Kanoni. Yield results and interviews with farmers indicated that farmers in S. Mpigi were making compromises in their choice of cultivars to grow, some key factors being SPVD susceptibility, and the yield, taste, and marketability, duration of harvest and in-ground storability of the storage roots. These compromises need to be included in an assessment of yield losses attributable to SPVD.     

Targeting of SPCSV-RNase3 via CRISPR-Cas13 confers resistance against sweet potato virus disease

Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co-infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis-related factors (i.e., SPCSV-RNase3) by using the CRISPR-Cas13 technique. First, the RNA targeting activity of four CRISPR-Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV-RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3-targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV-GFP and cucumber mosaic virus CMV-RNase3 co-infection. Moreover, transgenic sweet potato plants carrying an RNase3-targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD-prevalent regions.     

The Response of Lettuce Cultivars against Two Lettuce mosaic virus isolates,One Able to Systemically Infect the Resistant Cultivar Salinas 88

The response of seven lettuce cultivars to two geographically different Lettuce mosaic virus (LMV) isolates (LMV‐A, LMV‐T) was statistically evaluated based on infection rate, virus accumulation and symptom severity in different time trials. LMV‐A is characterized by the ability to systemically infect cv. Salinas 88 (mo1²‐carrying resistant cultivar), and inducing mild mosaic symptoms. Among lettuce cultivars, Varamin (a native cultivar) similar to cv. Salinas showed the most susceptibility to both LMV isolates, whereas another native cultivar, Varesh, was tolerant to the virus with minimal viral accumulation and symptom scores, significantly different from other cultivars at P < 0.05. LMV‐A systemically infects all susceptible lettuce cultivars more rapidly and at a higher rate than LMV‐T. This isolate accumulated in lettuce cultivars at a significantly higher level, determined by semiquantitative ELISA and induced more severe symptoms than LMV‐T isolate at 21 dpi. This is the first evidence for a LMV isolate with ability to systemically infect mo1²‐carrying resistant cultivar of lettuce from Iran. In this study, accumulation level of LMV showed statistically meaningful positive correlation with symptom severity on lettuce plants. Based on the results, three evaluated parameters differed considerably by lettuce cultivar and virus isolate.     

Failure of British isolates of beet western yellows virus to infect potato

Potato cultivars were tested for susceptibility to two British isolates of beet western yellows virus originally obtained from sugar beet and oil seed rape. Neither isolate was transmitted by Myzus persicae to virus-free potato plants, either by itself or in association with potato leafroll virus.