Sweet Potato Feathery Mottle Virus Research Articles

Spreading of RNA silencing and DNA methylation in transgenic hybrid plants with the coat protein gene of Sweet potato feathery mottle virus

We previously reported spreading of RNA silencing that occurred from 5′ to 3′ direction along the transgene without any effect on transgene methylation by grafting transgenic Nicotiana benthamiana carrying either entire or 5′ region of coat protein gene (CP) of Sweet potato feathery mottle virus (SPFMV). Here, spreading of RNA silencing and transgene methylation was investigated in hybrids between these transgenic lines. When silenced line carrying 5′ region of CP was crossed with a non-silenced line harboring the entire CP, progeny showed very little accumulation of transgene mRNA and siRNAs were detected not only from 5′ region but also from middle and 3′ region of CP. This suggested that RNA silencing spread from 5′ to 3′ region over the entire CP transcript in hybrids. In addition, increased level of cytosine methylation in both symmetrical (CG and CNG) and asymmetrical (CHH) contexts (in the order of frequency, CG > CNG > CHH) were observed in entire transgene in hybrids. Together with the results, it is postulated that transitive silencing can induce spreading of transgene methylation depending on the system implemented reflecting the extent of interaction between silencing signal and transgene DNA in nuclei.

Biodiversity risk evaluation of transgenic sweet potato ‘EP200’ and ‘EP220’ with coat protein (CP) gene of <i>sweet potato feathery mottle virus</i> server strain (SPFMV-S) in a closed and semiclosed greenhouse

Biodiversity risk evaluation of transgenic sweet potato ‘EP200’ and ‘EP220’ with coat protein (CP) gene of <i>sweet potato feathery mottle virus</i> server strain (SPFMV-S) in a closed and semiclosed greenhouse

An Online Database of Sweetpotato Germplasm Collection in Uganda

Sweetpotato [Ipomoea batatas (L.) Lam], the world’s seventh most important crop, is widely grown and consumed as a staple food crop in Uganda. Uganda is the third largest global producer after China and Nigeria (Food and Agricultural Organization, 2007). It lies within the East African region, which is considered a secondary center of diversity for the crop where the farmers grow and maintain large numbers of different cultivars and landraces (Mwanga et al., 2001; Villordon et al., 2006). Genetic erosion threatens this diversity as a result of sweetpotato virus disease (SPVD) caused by dual infection of Sweetpotato feathery mottle virus (Potyvirus; Potyviridae) and Sweetpotato chlorotic stunt virus (Crinivirus; Closteroviridae) (Gibson et al., 1998), Alternaria bataticola blight, and African sweetpotato weevils, Cylas puncticollis Boheman and Cylas brunneus Fabricius. Farmers are also known to abandon poor-performing traditional cultivars in favor of newly developed ones released by the national breeding program (Bashaasha et al., 1995; Mwanga et al., 2001). The National Sweetpotato Program based at the National Crops Resources Research Institute (NaCRRI), Namulonge, Uganda, undertook a rigorous germplasm collection, characterization, evaluation, and conservation program to mitigate sweetpotato genetic erosion and also to find superior germplasm for cultivar development in hybridization schemes. A total of 1303 accessions of sweetpotato germplasm were collected from 21 major sweetpotato-producing districts of Uganda from January to July 2005. Information about farmers’ indigenous knowledge of the cultivars was also collected. These accessions were assembled at NaCRRI and morphologically characterized using 40 standard root, vine, leaf, and flower descriptors of sweetpotato [International Potato Center (CIP), Asian Vegetable Research and Development Center, and International Board for Plant Genetic Resources, 1991] scored 90 to 100 d after planting in the field. A total of 946 morphologically distinct accessions were identified after removal of duplicates. Field evaluations were also conducted for SPVD, Alternaria blight, and yield at three sites for two seasons. Subsequently, 192 superior genotypes were selected from the germplasm evaluation of which 190 were genetically distinct on molecular characterization using 10 fluorescent-labeled simple sequence repeat markers, IB-R16 (VIC), IB-R19 (PET), IBCIP-13 (NED), IB-R03 (PET), IBCIP-9 (6-FAM), IB-S09 (NED), IB-R08 (PET), IB-R12 (NED), IB-S07 (6-FAM), and IB-S07 (PET), obtained from CIP, Lima, Peru. The database for sweetpotato germplasm collection in Uganda referred to as the ‘‘Uganda Sweetpotato Germplasm Database’’ (USGDB) is described. To our knowledge, the USGDB is the most comprehensive description of collected sweetpotato germplasm in the region. USGDB has all 946 morphologically distinct accessions fully described, including passport (accession identifiers and information recorded by collectors) data, morphological characterization, and evaluation data. This database has been made available online for public use at: http:// www.viazivitamu.org/ugasp_db/index.php. The database contains the names, accession number as assigned by collectors, geographical location (longitude and latitude), form of maintenance, and a total of 40 morphological descriptors. It further contains the total root yield, SPVD, and Alternaria bataticola blight disease resistance values for each accession. This online database is an important resource for enhancing the global exchange of sweetpotato germplasm. An online web-accessible database developed for the sweetpotato germplasm collection in Kenya called the ‘‘Viazivitamu database’’ is also available at http:// www.viazivitamu.org (Villordon et al., 2007). The Ugandan and Kenyan databases will eventually be merged and expanded to produce a comprehensive regional database of sweetpotato germplasm resources that is representative of the entire East African region after checking for duplicates in the two collections. This USGDB collection is being maintained in the screenhouse and field at NaCRRI. Some 30 accessions are being maintained in vitro at the CIP gene bank at Lima, Peru. Request for this germplasm should be directed to the National Sweetpotato Program, NaCRRI, P.O. Box 7084, Kampala, Uganda.

Differential Gene Expression of Resistant and Susceptible Sweetpotato Plants after Infection with the Causal Agents of Sweet Potato Virus Disease

Sweet potato virus disease (SPVD) is one of the most devastating diseases affecting sweetpotato (Ipomoea batatas), an important food crop in developing countries. SPVD develops when sweetpotato plants are dually infected with sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV). To better understand the synergistic interaction between these viruses, global gene expression was previously studied in the susceptible cultivar Beauregard. In the current study, global gene expression between SPVD-affected plants and virus-tested control plants (VT) were compared in ‘Beauregard’ (Bx) and resistant ‘NASPOT 1’ (Nas) sweetpotato cultivars at 5, 9, 13, and 17 days post inoculation (DPI). Titer levels of SPFMV and SPCSV were significantly lower in inoculated resistant plants (Nas_SPVD) than in susceptible plants (Bx_SPVD) at most of the time points. Chloroplast genes and cell expansion-related genes (including xyloglucan endotransglucosylase/hydrolases) were suppressed in Bx_SPVD, while stress-related genes were induced. This trend was not observed in resistant NAS_SPVD. Genes related to protein synthesis (e.g., ribosomal proteins and elongation factor genes) were induced in resistant NAS_SPVD at 5 DPI before returning to levels comparable with NAS_VT plants. At this time (5 DPI), individual viruses could not be detected in NAS_SPVD samples, and no symptoms were observed. Induction of protein synthesis-related genes is common in susceptible plants after virus infection and is generally in proportion to virus accumulation. Our results show that induction of protein synthesis genes also occurs early in the infection process in resistant plants, while virus titers were below the level of detection, suggesting that virus accumulation is not required for induction.

Molecular Characterization of Sweet potato feathery mottle virus (SPFMV) Isolates from Easter Island, French Polynesia, New Zealand, and Southern Africa.

Strains of Sweet potato feathery mottle virus (SPFMV; Potyvirus; Potyviridae) infecting sweet-potato (Ipomoea batatas) in Oceania, one of the worlds' earliest sweetpotato-growing areas, and in southern Africa were isolated and characterized phylogenetically by analysis of the coat protein (CP) encoding sequences. Sweetpotato plants from Easter Island were co-infected with SPFMV strains C and EA. The EA strain isolates from this isolated location were related phylogenetically to those from Peru and East Africa. Sweetpotato plants from French Polynesia (Tahiti, Tubuai, and Moorea) were co-infected with SPFMV strains C, O, and RC in different combinations, whereas strains C and RC were detected in New Zealand. Sweetpotato plants from Zimbabwe were infected with strains C and EA and those from Cape Town, South Africa, with strains C, O, and RC. Co-infections with SPFMV strains and Sweet potato virus G (Potyvirus) were common and, additionally, Sweet potato chlorotic fleck virus (Carlavirus) was detected in a sample from Tahiti. Taken together, occurrence of different SPFMV strains was established for the first time in Easter Island, French Polynesia, and New Zealand, and new strains were detected in Zimbabwe and the southernmost part of South Africa. These results from the Southern hemisphere reflect the anticipated global distribution of strains C, O, and RC but reveal a wider distribution of strain EA than was known previously.

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

AbstractSweet 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.

Elimination of antiviral defense by viral RNase III

Sweet potato (Ipomoea batatas) is an important subsistence and famine reserve crop grown in developing countries where Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae), a single-stranded RNA (ssRNA) crinivirus, synergizes unrelated viruses in co-infected sweet potato plants. The most severe disease and yield losses are caused by co-infection with SPCSV and a potyvirus, Sweet potato feathery mottle virus (SPFMV; Potyviridae). Potyviruses synergize unrelated viruses by suppression of RNA silencing with the P1/HC-Pro polyprotein; however, the SPCSV-SPFMV synergism is unusual in that the potyvirus is the beneficiary. Our data show that transformation of an SPFMV-resistant sweet potato variety with the double-stranded RNA (dsRNA)-specific class 1 RNA endoribonuclease III (RNase3) of SPCSV broke down resistance to SPFMV, leading to high accumulation of SPFMV antigen and severe disease symptoms similar to the synergism in plants co-infected with SPCSV and SPFMV. RNase3-transgenic sweet potatoes also accumulated higher concentrations of 2 other unrelated viruses and developed more severe symptoms than non-transgenic plants. In leaves, RNase3 suppressed ssRNA-induced gene silencing (RNAi) in an endonuclease activity-dependent manner. It cleaved synthetic double-stranded small interfering RNAs (siRNAs) of 21, 22, and 24 bp in vitro to products of approximately 14 bp that are inactive in RNAi. It also affected total siRNA isolated from SPFMV-infected sweet potato plants, suggesting a viral mechanism for suppression of RNAi by cleavage of siRNA. Results implicate RNase3 in suppression of antiviral defense in sweet potato plants and reveal RNase3 as a protein that mediates viral synergism with several unrelated viruses, a function previously described only for P1/HC-Pro.

Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation

AbstractCryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra‐low temperature, usually that of liquid nitrogen (−196°C), and is considered as an ideal means for long‐term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature‐limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo‐treatment and regeneration of healthy shoots from the surviving pathogen‐free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen‐free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubus idaeus), potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing ‘citrus greening’. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy.

Impact of common sweetpotato viruses on total carotenoids and root yields of an orange-fleshed sweetpotato in Tanzania

The growing and consumption of orange-fleshed sweetpotato (OFSP) is considered as a mean to alleviate vitamin A deficiency in sub-Saharan Africa (SSA) region. However, majority of the field-tested OFSP varieties are susceptible to major diseases especially sweetpotato virus disease (SPVD), which is caused by co-infection of sweetpotato chlorotic stunt virus (SPCSV) with sweetpotato feathery mottle virus (SPFMV). A high beta-carotene content but susceptible variety Resisto was used in this study to evaluate the effects of SPVD on total carotenoids content and root yield. Compared with apparently healthy plants, reduction of 43, 16, and 37% of the total carotenoids content in the OFSP variety Resisto were observed in plants infected with SPCSV, SPFMV, and co-infection of both viruses. Storage root fresh weight was significantly ( P < 0.001) reduced due to virus infection with high reduction recorded for SPFMV infection followed by co-infection of SPFMV with SPCSV. The same case was for sweetpotato vine length. However, no major reductions were observed in the vine weights. Co-infection of SPFMV with SPCSV caused more severe symptoms than single infections of the two viruses and each isolate caused distinct disease symptoms on the infected sweetpotato plants. In general, there was no direct correlation between sweetpotato virus disease symptom severity and reduction in total carotenoids. To our knowledge, this is the first report of the negative impact of SPCSV and SPFMV on the total carotenoid accumulation in OFSP varieties. Therefore follow up studies in the area of biochemical analysis should be initiated to gain knowledge on the impacts of SPVD on the biochemical pathways of carotenoid accumulation.

Detection of Sweet potato virus 2 in Sweet Potato in New Zealand.

In New Zealand, sweet potato (Ipomoea batatas) is a crop of cultural importance and an important food source; it is grown mainly in the districts of Kaipara, Auckland, and the Bay of Plenty in the North Island. In January of 2008, virus symptoms that included chlorotic spots, ring spots, and mottling were observed on the leaves of commercial sweet potato crops (cvs. Beauregard, Owairaka Red, and Toka Toka Gold) growing in the three main production areas. A survey was done to determine the extent of virus infection in these crops. Fifty to one hundred leaves were collected randomly from each of 26 different fields. Leaves from each field were bulked into groups of 10, giving a total of 173 composite samples. All samples tested negative for Cucumber mosaic virus, C-6 virus, Sweet potato caulimo-like virus, Sweet potato chlorotic fleck virus, Sweet potato chlorotic stunt virus (SPCSV), Sweet potato latent virus, and Sweet potato mild specking virus by nitrocellulose membrane enzyme-linked immunosorbent assays (International Potato Center-CIP, Lima, Peru). Total nucleic acid was extracted from all 173 composite samples and used in real-time PCR assays specific for Sweet potato leaf curl virus (SPLCV) and real-time reverse transcription (RT)-PCR specific for SPCSV, Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Sweet potato virus 2 (SPV2; synonym Sweet potato virus Y) (1). No samples were positive for SPLCV or SPCSV, but 107 and 138 samples tested positive for SPFMV and SPVG, respectively. SPFMV and SPVG have been reported previously in New Zealand (2,3). Sixty four samples from 16 different fields tested positive for SPV2. Of the 64 samples, 52 were also infected with SPVG and SPFMV, and 10 were co-infected with SPVG but not SPFMV; no samples were co-infected with SPV2 and SPFMV when SPVG was absent. From a representative SPV2 positive sample, the 70-bp amplicon obtained by the real-time RT-PCR primers was cloned and sequenced A BLAST search showed 100% nucleotide sequence identity with SPV2 (GenBank Accession Nos. AM050887 and AY178992). Subsequently, primers (V2-F1c: 5'-AGAACAGGACAAACTCAACC-3'; V2-R1: 5'-TAATCACCCTTCACACCTTC-3') were designed to amplify an approximately 434-bp fragment within the SPV2 coat protein gene. One-step RT-PCR was done on four of the SPV2 positive samples and amplicons of the expected size were sequenced directly (GenBank Accession No. FJ461774). Sequence comparison showed 99% nucleotide sequence identity with SPV2 (GenBank Accession Nos. AM050886, AM050887, AY178992, and EF577437). SPV2 is a member of the genus Potyvirus but the virus has not been fully characterized. It is known that single-potyvirus infections cause mild or no symptoms in sweet potato, and consequently, no significant yield reduction is observed generally. However, co-infection with other viruses such as SPCSV produces a synergistic effect and more severe disease symptoms (4). To our knowledge, this is the first report of SPV2 infecting sweet potato in New Zealand.

Control of Russet Crack Disease in Sweetpotato Plants Using a Protective Mild Strain of Sweet potato feathery mottle virus.

A mild isolate (designated as 10-O) of Sweet potato feathery mottle virus (SPFMV) was obtained from Ipomoea batatas. This isolate rarely caused skin discoloration and did not cause russet crack disease on the storage roots of I. batatas that are typical of infection with the severe strain of SPFMV (SPFMV-S). The yield of the 10-O-infected I. batatas ranged from 92 to 105% of the yield of healthy I. batatas. The coat protein gene of 10-O encodes 315 amino acids and has sequence identities of 91.1% at the nucleotide level and 95.6% at the amino acid level with the corresponding region of SPFMV-S. When I. batatas cuttings were inoculated with 10-O and later challenged with SPFMV-S, russet crack symptoms were much reduced and SPFMV-S was not detected in the challenged plants. These results indicate that 10-O can be an effective biological control agent against russet crack disease.

Viruses infecting sweet potato in Rwanda: occurrence and distribution

AbstractA 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.

‘Covington’ Sweetpotato

‘Covington’ is an orange-fleshed, smooth-skinned, rose-colored, table-stock sweetpotato [Ipomoea batatas (L.) Lam.] developed by North Carolina State University (NCSU). ‘Covington’, named after the late Henry M. Covington, an esteemed sweetpotato scientist at North Carolina State, was evaluated as NC98-608 in multiple state and regional yield trials during 2001 to 2006. ‘Covington’ produces yields equal to ‘Beauregard’, a dominant sweetpotato variety produced in the United States, but it is typically 5 to 10 days later in maturity. ‘Covington’ typically sizes its storage roots more evenly than ‘Beauregard’ resulting in fewer jumbo class roots and a higher percentage of number one roots. Total yields are similar for the two clones with the dry matter content of ‘Covington’ storage roots typically being 1 to 2 points higher than that of ‘Beauregard’. ‘Covington’ is resistant to fusarium wilt [Fusarium oxysporum Schlect. f.sp. batatas (Wollenw.) Snyd. &amp; Hans.], southern root-knot nematode [Meloidogyne incognita (Kofoid &amp; White 1919) Chitwood 1949 race 3], and moderately resistant to streptomyces soil rot [Streptomyces ipomoeae (Person &amp; W.J. Martin) Wakswan &amp; Henrici]. Symptoms of the russet crack strain of Sweet Potato Feathery Mottle Virus have not been observed in ‘Covington’. The flavor of the baked storage roots of ‘Covington’ has been rated as very good by standardized and informal taste panels and typically scores as well or better in this regard when compared with ‘Beauregard’.

Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy

Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae) and Sweet potato feathery mottle virus (SPFMV; Potyviridae) interact synergistically and cause severe diseases in co-infected sweetpotato plants ( Ipomoea batatas). Sweetpotato is propagated vegetatively and virus-free planting materials are pivotal for sustainable production. Using cryotherapy, SPCSV and SPCSV were eliminated from all treated single-virus-infected and co-infected shoot tips irrespective of size (0.5–1.5 mm including 2–4 leaf primordia). While shoot tip culture also eliminated SPCSV, elimination of SPFMV failed in 90–93% of the largest shoot tips (1.5 mm) using this technique. Virus distribution to different leaf primordia and tissues within leaf primordia in the shoot apex and petioles was not altered by co-infection of the viruses in the fully virus-susceptible sweetpotato genotype used. SPFMV was immunolocalized to all types of tissues and up to the fourth-youngest leaf primordium. In contrast, SPCSV was detected only in the phloem and up to the fifth leaf primordium. Because only cells in the apical dome of the meristem and the two first leaf primordia survived cryotherapy, all data taken together could explain the results of virus elimination. The simple and efficient cryotherapy protocol developed for virus elimination can also be used for preparation of sweetpotato materials for long-term preservation.

Molecular Genetic Characterization of Sweet potato virus G (SPVG) Isolates from Areas of the Pacific Ocean and Southern Africa.

Sweet potato virus G (SPVG, genus Potyvirus, family Potyviridae) was detected in sweetpotato (Ipomoea batatas) storage roots sold in the local markets and storage roots or cuttings sampled directly from farmers' fields. Using serological and molecular methods, the virus was detected for the first time in Java, New Zealand, Hawaii, Tahiti, Tubuai, Easter Island, Zimbabwe, and South Africa, and also in an imported storage root under post-entry quarantine conditions in Western Australia. In some specimens, SPVG was detected in mixed infection with Sweet potato feathery mottle virus (genus Potyvirus). The coat protein (CP) encoding sequences of SPVG were analyzed for 11 plants from each of the aforementioned locations and compared with the CP sequences of 12 previously characterized isolates from China, Egypt, Ethiopia, Spain, Peru, and the continental United States. The nucleotide sequence identities of all SPVG isolates ranged from 79 to 100%, and amino acid identities ranged from 89 to 100%. Isolates of the same strain of SPVG had nucleotide and amino acid sequence identities from 97 to 100% and 96 to 100%, respectively, and were found in sweetpotatoes from all countries sampled except Peru. Furthermore, a plant from Zimbabwe was co-infected with two clearly different SPVG isolates of this strain. In contrast, three previously characterized isolates from China and Peru were phylogenetically distinct and exhibited <90% nucleotide identity with any other isolate. So far, the highest genetic diversity of SPVG seems to occur among isolates in China. Distribution of SPVG within many sweetpotato growing areas of the world emphasizes the need to determine the economic importance of SPVG.

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

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) is one of the most important pathogens of sweet potato (Ipomoea batatas L.). It can reduce yields by 50% by itself and cause various synergistic disease complexes when co-infecting with other viruses, including sweet potato feathery mottle virus (SPFMV; genus Potyvirus, family Potyviridae). Because no sources of true resistance to SPCSV are available in sweet potato germplasm, a pathogen-derived transgenic resistance strategy was tested as an alternative solution in this study. A Peruvian sweet potato 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.

Natural Wild Hosts ofSweet potato feathery mottle virusShow Spatial Differences in Virus Incidence and Virus-Like Diseases in Uganda

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is globally the most common pathogen of sweetpotato. An East African strain of SPFMV incites the severe 'sweetpotato virus disease' in plants co-infected with Sweet potato chlorotic stunt virus and threatens subsistence sweetpotato production in East Africa; however, little is known about its natural hosts and ecology. In all, 2,864 wild plants growing in sweetpotato fields or in their close proximity in Uganda were observed for virus-like symptoms and tested for SPFMV in two surveys (2004 and 2007). SPFMV was detected at different incidence in 22 Ipomoea spp., Hewittia sublobata, and Lepistemon owariensis, of which 19 species are new hosts for SPFMV. Among the SPFMV-positive plants, approximately 60% displayed virus-like symptoms. Although SPFMV incidence was similar in annual and perennial species, virus-like diseases were more common in annuals than perennials. Virus-like diseases and SPFMV were more common in the eastern agroecological zone than the western, central, and northern zones, which contrasted with known incidence of SPFMV in sweetpotato crops. The data on a large number of new natural hosts of SPFMV detected in this study provide novel insights into the ecology of SPFMV in East Africa.

Incidence of sweetpotato viruses in Central Luzon, Philippines

Sweetpotao commercial growing areas in Central Luzon particularly Tarlac and Bataan were surveyed for the incidence of sweetpotato viruses. Healthy-looking sweetpotato leaves and those showing symptoms of virus infection were collected and subjected to nitrocellulose membrane-enzyme linked immunoabsorbent serological assay (NCM-ELISA) for the detection of known viruses using the kit and protocol from Centro Internacional de la Papa, Lima Peru. The NCM-ELISA detected sweetpotato feathery mottle virus (SPFMV), sweetpotato mild mottle virus (SPMMV), sweetpotato latent virus (SPLV), sweetpotao chlorotic fleck virus (SPCFV), C-6 virus, sweetpotato mild speckling virus (SPMSV), sweetpotato caulimo-like virus (SPCalV), and sweetpotato chlorotic stunt virus (SPCSV). Six to 8 viruses were found in Tarlac and 3-4 viruses in Bataan. Highest infections were recorded for C-6, SPMSV, SPCalV and SPCSV reaching 100% in almost all areas. Infection of SPFMV ranged from 45-70%. The combination of SPFMV with other viruses was common registering 40-70% complex infection. Mixed infection of 3-6 viruses (SPFMV, SPMMV, SPLV, C-6, SPMSV and SPCalV) was found in Tarlac and 2-4 viruses (SPFMV, C-6, SPMSV and SPCalV) in Bataan. Practically all sweetpotato growing areas of Tarlac are “hotspot” areas of sweetpotato viruses.

Occurrence of Sweet potato feathery mottle virus and Sweet potato leaf curl Georgia virus on Sweet Potato in India

Viruses are a major biotic constraint on sweet potato (Ipomoea batatas (L.) Lam) production worldwide. In 2005, 10 to 60% viral disease incidence was observed in sweet potato fields. Symptoms include ring and chlorotic spots, puckering, feathering, vein clearing, and leaf curl with chlorotic specks and pink spots. Cuttings from symptomatic plants were collected from Kerala (two clones), Orrisa (eight clones), and Adrapradesh (three clones) and maintained in an insect-proof glasshouse. Leaves from symptomatic plants were mechanically inoculated to I setosa, I. nil, Nicotiana tabacum, N. benthamiana, Datura stramonium, and Chenapodium quinoa (12 seedlings each). Vein clearing, netting, and leaf distortion were observed in I. setosa and N tabacum 7 days postinoculation, chlorotic spots observed in N. benthamiana, and violet spots and violet margins on leaves observed on I. Nil. No symptoms were observed on D. stramonium and C. quinoa. When scions from the symptomatic sweet potato plants were graft inoculated onto I. setosa, vein clearing, leaf curl, and puckering-like symptoms were observed within 5 days. Mosaic and leaf curling symptoms were also observed on mechanically inoculated N. tabacum. Total nucleic acids isolated from the 33 field-collected sweet potato samples, graft inoculated I. setosa plants, and mechanically inoculated N. tabacum and I. nil plants were used for PCR and reverse transcription (RT)-PCR with geminivirus group specific (2) and potyvirus group specific primers (1). The expected 530-bp and 1.3-kb fragment were generated from the geminivirus and potyvirus primer sets, respectively. Potyvirus alone was detected in 7 of the 33 field-collected plants; geminivirus alone was detected in 7 other plants, while 19 plants contained detectible levels of potyvirus and geminivirus. To further identify the viruses, nested primers specific for the coat protein gene of Sweet potato feathery mottle virus (SPFMV) (CP1S 5'AGT GGG AAG GCA CCA TAC ATA GC 3', CP1A5' GCA GAG GAT GTC CTA TTG CAC ACC 3') (CP2S 5'TCT AGT GAA CGT ACT GAA TTC AAA GA 3', CP2A 5'ATT GCA CAC CCC TGA TTC CTA AGA 3') and Sweet potato leaf curl virus (SPLCV) (CP1- 5'ATG ACA GGG CGA ATT CGC GTT TC 3', CP2- 5'TTA ATT TTT GTG CGA ATC ATA 3') were designed. I. setosa and N. tabacum were amplified with SPFMV and SPLCV primers and the amplicons of 960 and 764 bp, respectively, obtained were subsequently cloned into pGEM-T Easy vector and sequenced. Nucleotide BLAST analysis revealed that the 960-bp fragment (GenBank Accession No. EF015398.) was 98% identical to two Egyptian isolates of SPFMV (Nos. AJ 515379 and AJ 515378). The nucleotide sequence of the 764-bp products (Nos. EF 151926 and EF15483) from the samples collected from Kerala and Orisa was 95% identical to each other. The sequence identity of EF 15483 with Sweet potato leaf curl Georgia virus (SPLCGV) isolate AF326775. was 91% and identity with China isolate DQ 512731 was 90% The isolate EF 151926 also was 91% identical to the SPLCGV with a high query and alignment score whereas identity with the China isolate was 91% with a low query coverage and alignment score. Phylogenic analysis with MEGA software program also showed the highest sequence similarity with SPLCGV, hence it is concluded that the geminivirus isolate under study is SPLCGV. To our knowledge, this is the first report of identification of SPFMV and SPLCGV occurring on sweet potato in India. Further study is required to understand the consequences of occurrence of these two viruses in India.

Analysis of gene content in sweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNA silencing suppressor in only a few isolates: implications for viral evolution and synergism

Sweet potato chlorotic stunt virus (genus Crinivirus) belongs to the family Closteroviridae, members of which have a conserved overall genomic organization but are variable in gene content. In the bipartite criniviruses, heterogeneity is pronounced in the 3'-proximal region of RNA1, which in sweet potato chlorotic stuat virus (SPCSV) encodes two novel proteins, RNase3 (RNase III endonuclease) and p22 (RNA silencing suppressor). This study showed that two Ugandan SPCSV isolates contained the p22 gene, in contrast to three isolates of the East African strain from Tanzania and Peru and an isolate of the West African strain from Israel, which were missing a 767 nt fragment of RNA1 that included the p22 gene. Regardless of the presence of p22, all tested SPCSV isolates acted synergistically with potyvirus sweet potato feathery mottle virus (SPFMV; genus Potyvirus, family Potyviridae) in co-infected sweetpotato plants (Ipomoea batatas), which greatly enhanced SPFMV titres and caused severe sweetpotato virus disease (SPVD). Therefore, the results indicate that any efforts to engineer pathogen-derived RNA silencing-based resistance to SPCSV and SPVD in sweetpotato should not rely on p22 as the transgene. The data from this study demonstrate that isolates of this virus species can vary in the genes encoding RNA silencing suppressor proteins. This study also provides the first example of intraspecific variability in gene content of the family Closteroviridae and may be a new example of the recombination-mediated gene gain that is characteristic of virus evolution in this virus family.