Sweet Potato Chlorotic Stunt Virus Research Articles

SWEETPOTATO VIRUS DISEASES RESEARCH IN EAST AFRICA

Sweetpotato is an important staple food crop in Sub-Saharan Africa, with production being concentrated in East Africa, particularly around Lake Victoria. Productivity of the crop is greatly constrained by viral diseases. Four main viruses have consistently been detected from various surveys done in the region viz. sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato mild mottle virus (SPMMV), and sweet potato chlorotic fleck virus (SPCFV). Sweet potato caulimo-like virus (SPCaLV), sweet potato latent virus (SPLV), and cucumber mosaic virus (CMV) have also been detected though only in isolated cases. The most severe symptoms have been caused by co-infection with SPCSV and SPFMV, resulting in the synergistic Sweet potato virus disease (SPVD). Yield reductions due to virus infections have been estimated to be >90% in very severe cases. Virus detection has mainly been limited to the use of serological methods. Some plants have been observed with symptoms resembling those caused by viruses, but do not react with available antisera, indicating that the plants could be infected with viruses that have not been described, or not tested in the region. Use of other detection techniques such as PCR may result in identification of more viruses in the region. This report gives a summary of our research efforts towards detection of other viruses present in the region, and identification of resistant germplasm.

AN UPDATE ON EVALUATION OF VIRUS STATUS IN THE HEIRLOOM SWEETPOTATO GERMPLASM MATERIALS WITH REAL-TIME PCR TECHNOLOGY

Sweet potato virus disease (SPVD) is the most devastating virus disease on sweetpotato [Ipomoea batatas (L.) Lam] world wide, especially in East Africa. However, weather it is present in the U.S. is unknown. SPVD is caused by co-infection of sweetpotato feathery mottle virus (SPFMV) and sweetpotato chlorotic stunt virus (SPCSV). Presence of two other potyviruses, sweetpotato virus G (SPVG) and Ipomoea vein mosaic virus (IVMV) has also been confirmed in the U.S. Sweet potato leaf curl virus (SPLCV), a whitefly (Bemisia tabaci) transmitted Begomovirus, also has the potential to spread to commercial sweetpotato fields and poses a great threat to the sweetpotato industry. The U.S. collection of sweetpotato germplasm contains about 700 genotypes or breeding lines introduced from over 20 different countries. Newly introduced sweetpotato germplasm from foreign sources are routinely screened for major viruses with serology and graft-transmission onto indicator plants (Ipomoea setosa). However, a large portion of this collection including heirloom cultivars or old breeding materials has not been systemically screened for these major sweetpotato viruses. In this study, a total of 69 so-called heirloom sweetpotato PI accessions were evaluated for their virus status. We used Real-time PCR to detect five sweetpotato viruses, including four RNA viruses (SPCSV, SPFMV, SPVG, and IVMV) and one DNA virus (SPLCV). A multiplex Real-time RT-PCR system was developed to detect three RNA viruses (SPFMV, SPVG, and IVMV). Preliminary data indicated that about 15% of these heirloom sweetpotato germplasm carried at least one of these viruses tested. Details on virus infection status will be presented.

VIRUS DISTRIBUTION IN FIELD GROWN SWEETPOTATO VINES IN AFRICA

Sweet potato virus disease (SPVD) is a major constraint to sweetpotato production in East Africa. The disease is a result of co-infection with sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV). Some local sweetpotato genotypes have been reported to recover from, or have localized distribution of SPVD, suggesting that the disease is not fully systemic. This has led to the suggestion that uninfected cuttings may be obtained from previously infected plants. Experiments were set to determine the possibility of obtaining cuttings long enough for propagation that are free from virus infection. This would form a basis for recommending to the local small-holder farmers of a way to reduce losses due to the disease. Field grown sweetpotato vines were cut into three pieces (15, 15 to 30, and >30 cm from the apex) and tested for SPCSV and SPFMV. Nine genotypes were selected from a group of 21 local clones and used for this study. The two viruses were equally present in all the three sections of infected vines, indicating that it is not easy to obtain a virus free cutting for field propagation from an infected vine.

Real-Time PCR Assays for Detection and Quantification of Sweetpotato Viruses.

Viral diseases, especially those caused by mixed infections, are among the economically most important diseases of sweetpotato. The difficulties inherent in detecting, quantifying, and isolating viruses directly from sweetpotato have impeded progress in sweetpotato virus research. Real-time polymerase chain reaction (PCR) assays were developed for the detection and relative quantification in singleplex reactions of the potyviruses Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Ipomoea vein mosaic virus (IVMV); the crinivirus Sweet potato chlorotic stunt virus (SPCSV); and the begomovirus Sweet potato leaf curl virus(SPLCV) directly from infected sweetpotato plants. There was no significant effect from potential inhibitors in total nucleic acid extracts from sweetpotato leaves on the performance of the real-time PCR assays. Virus titers of SPFMV, IVMV, and SPVG were quantified using real-time PCR and found to be lower in singly infected sweetpotato plants compared with singly infected Brazilian morning-glory (Ipomoea setosa Ker.) and I. nil cv. Scarlet O'Hara plants. Real-time PCR was a more efficient detection method for SPLCV than conventional PCR assay.

Interactions between a crinivirus, an ipomovirus and a potyvirus in coinfected sweetpotato plants

Novel and severe symptoms of chlorosis, rugosity, leaf strapping and dark green islands, designated as sweetpotato severe mosaic disease (SPSMD), were caused by dual infection of Sweet potato mild mottle virus (SPMMV; Ipomovirus) and Sweet potato chlorotic stunt virus (SPCSV; Crinivirus) in three East African sweetpotato cultivars (Tanzania, Dimbuka and New Kawogo). The storage root yield was reduced by ∼80%, as compared with healthy plants under screenhouse conditions in Uganda. Plants infected with SPMMV or SPCSV alone showed nonsignificant or 50% yield reduction, respectively. SPCSV reduced resistance to SPMMV in sweetpotato, similar to the situation with resistance to Sweet potato feathery mottle virus (SPFMV; Potyvirus) that breaks down following infection with SPCSV, followed by development of sweet potato virus disease (SPVD). In single virus infections with SPMMV and SPFMV or their coinfection, cvs Tanzania and Dimbuka were initially systemically infected, displayed symptoms and contained readily detectable virus titres, but new leaves were symptomless with very low virus titres, indicating recovery from disease. In contrast, cv. New Kawogo remained symptomless and contained low SPMMV and SPFMV titres following graft inoculation. These moderate and high levels of resistance to SPMMV and SPFMV, respectively, were lost and cultivars succumbed to a severe disease following coinfection with SPCSV. The synergistic interactions increased titres of SPMMV and SPFMV RNA by ∼1000‐fold as quantified by real‐time PCR, whereas SPCSV titres were reduced twofold, indicating an antagonistic interaction. Coinfection with SPMMV and SPFMV caused no detectable changes in virus titres or symptom severity.

Complete nucleotide sequence of the RNA2 of the crinivirus tomato chlorosis virus

The complete sequence of genomic RNA2 of Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae), isolate AT80/99 from Spain, was determined and compared with those from the other members of the genus sequenced to date. RNA2 is 8244 nucleotides (nt) long and putatively encodes nine ORFs that encompass the hallmark gene array of the family Closteroviridae, which includes a heat shock protein 70 family homologue, a 59 kDa protein, the coat protein, and a diverged coat protein. Phylogenetic analysis confirmed assignment of ToCV in the genus Crinivirus, being most similar to sweet potato chlorotic stunt virus and cucurbit yellow stunting disorder virus.

Viral class 1 RNase III involved in suppression of RNA silencing.

Double-stranded RNA (dsRNA)-specific endonucleases belonging to RNase III classes 3 and 2 process dsRNA precursors to small interfering RNA (siRNA) or microRNA, respectively, thereby initiating and amplifying RNA silencing-based antiviral defense and gene regulation in eukaryotic cells. However, we now provide evidence that a class 1 RNase III is involved in suppression of RNA silencing. The single-stranded RNA genome of sweet potato chlorotic stunt virus (SPCSV) encodes an RNase III (RNase3) homologous to putative class 1 RNase IIIs of unknown function in rice and Arabidopsis. We show that RNase3 has dsRNA-specific endonuclease activity that enhances the RNA-silencing suppression activity of another protein (p22) encoded by SPCSV. RNase3 and p22 coexpression reduced siRNA accumulation more efficiently than p22 alone in Nicotiana benthamiana leaves expressing a strong silencing inducer (i.e., dsRNA). RNase3 did not cause intracellular silencing suppression or reduce accumulation of siRNA in the absence of p22 or enhance silencing suppression activity of a protein encoded by a heterologous virus. No other known RNA virus encodes an RNase III or uses two independent proteins cooperatively for RNA silencing suppression.

Unravelling the genetic diversity of the three main viruses involved in Sweet Potato Virus Disease (SPVD), and its practical implications

SUMMARY Sweetpotato (Ipomoea batatas) is a widely grown food crop, in which the most important diseases are caused by viruses. Genetic variability of three widely distributed sweetpotato viruses was analysed using data from 46 isolates of Sweet potato feathery mottle virus (SPFMV), 16 isolates of Sweet potato mild mottle virus (SPMMV) and 25 isolates of Sweet potato chlorotic stunt virus (SPCSV), of which 19, seven and six isolates, respectively, are newly characterized. Division of SPFMV into four genetic groups (strains) according to phylogenetic analysis of coat protein (CP) encoding sequences revealed that strain EA contained the East African isolates of SPFMV but none from elsewhere. In contrast, strain RC contained ten isolates from Australia, Africa, Asia and North America. Strain O contained six heterogeneous isolates from Africa, Asia and South America. The seven strain C isolates from Australia, Africa, Asia, and North and South America formed a group that was genetically distant from the other SPFMV strains. SPMMV isolates showed a high level of variability with no discrete strain groupings. SPCSV isolates from East Africa were phylogenetically distant to SPCSV isolates from elsewhere. Only from East Africa were adequate data available for different isolates of the three viruses to estimate the genetic variability of their local populations. The implications of the current sequence information and the need for more such information from most sweetpotato-growing regions of the world are discussed in relation to virus diagnostics and breeding for virus resistance.

Incidence of Viruses Infecting Sweetpotato in Tanzania.

A survey for incidence of sweetpotato viruses was carried out in the three sweetpotato-producing districts (Bukoba, Misungwi, and Tarime) in the Lake Victoria basin and in the Indian Ocean coastal zone (Bagamoya on the mainland and Unguja on Zanzibar Island) in Tanzania in March and April 2003. A total of 170 plants from 43 sweetpotato fields were collected, established in an insect-proof screenhouse, and tested for viruses by nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). Scions from symptomless plants were grafted onto Ipomoea setosa, a nearly universal indicator plant for sweetpotato viruses, and leaves were tested by NCM-ELISA. Results were confirmed in several seropositive plants by cloning and partial sequencing of the viruses. Sweet potato feathery mottle virus (SPMFV), Sweet potato mild mottle virus, Sweet potato chlorotic stunt virus (SPCSV), and Sweet potato chlorotic fleck virus were detected serologically in single or mixed infections. Frequencies of occurrence of these viruses as determined by serological detection showed SPMFV to be the most prevalent virus in all the surveyed districts. Our study revealed a higher incidence and diversity of viruses in the Lake Victoria basin compared with the Indian Ocean coastal area. These results represent the first survey for sweetpotato viruses in Tanzania using accurate detection methods.

Studies on the physiological effects of viruses on sweet potato yield in Kenya

SummaryExperiments to determine the effect of the three viruses most common on Kenyan sweet potato varieties were carried out at the University of Nairobi Kabete farm. The sweet potato varieties were Bungoma, Kemb 10 and Ex‐Shimba hills which were either virus‐free or infected with Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV) or a combination of SPFMV and SPCSV. Canopy photosynthetically active radiation (PAR) interception and the relative chlorophyll content of the plants were determined. At harvest, marketable tuber yield, harvest index (HI), leaf area index (LAI) and specific leaf weight (SLW) were measured.Infection with single viruses caused no or mild symptoms and had no significant effect on chlorophyll content of leaves of any variety. Dual infection with SPFMV and SPCSV resulted in severe symptoms characteristic of sweet potato virus disease complex (SPVD) and significantly reduced chlorophyll content. Only SPCSV and SPCSV + SPFMV reduced PAR interception, the latter combination especially. Generally, infection with single viruses increased the SLW but a decrease occurred in plants dually infected with SPFMV and SPCSV. Whereas SPVD significantly reduced the harvest index (78%) and tuber yield (98%) in the three varieties, infection with single viruses caused a reduction only in the varieties Bungoma and Kemb 10. SPVD reduced yield through reducing leaf chlorophyll content, PAR interception, LAI and HI. The single virus infections possibly reduced assimilate translocation from leaves to tubers as is inferred from high SLW. Effects of viruses on physiological processes and yield are discussed.

Identification and distribution of viruses infecting sweet potato in Kenya

SummaryFour 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.

First Report of Sweet potato chlorotic stunt virus and Sweet potato feathery mottle virus Infecting Sweet Potato in Spain.

Sweet potato chlorotic stunt virus (SPCSV), family Closteroviridae and Sweet potato feathery mottle virus (SPFMV), family Potyviridae are whitefly and aphid transmitted, respectively, which in double infections cause sweet potato virus disease (SPVD) that is a serious sweet potato (Ipomoea batatas Lam.) disease in Africa (2). During the past decade, sweet potato plants showing symptoms similar to SPVD have been observed in most areas of Spain. Nevertheless, not much information is available about the identity of the viruses infecting this crop in Spain. During the summer of 2002, sweet potato plants with foliar mosaic, stunting, leaf malformation, chlorosis, and ringspot symptoms were observed in several farms in Málaga (southern Spain) and Tenerife and Lanzarote (Canary Islands, Spain). Vine cuttings were collected from 21 symptomatic plants in Málaga and from eight plants on Lanzarote and six on Tenerife. Scions were grafted to the indicator hosts, Brazilian morning glory (I. setosa) and I. nil cv. Scarlett O'Hara. Three weeks after graft inoculations, all plants showed various degrees of mosaic, chlorosis, leaf malformation, and stunting. Four field collections (two from Málaga, one from Tenerife, and one from Lanzarote) with severe symptoms on I. setosa were selected for whitefly (Bemisia tabaci biotype Q) transmission experiments. Acquisition and transmission periods were 48 h. I. setosa was the acquisition host, and I. nil was the transmission host. For each isolate, groups of 10 whiteflies per I. nil plant were used. All I. nil plants used as transmission hosts with the four, field collections showed chlorosis and leaf malformation. Reverse-transcription polymerase chain reaction (RT-PCR) was performed on I. setosa (grafted with the four selected field collections) and I. nil plants (from the whitefly transmission experiments) with primers for the HSP70h gene of SPCSV. A 450-bp DNA fragment was obtained with all I. setosa and I. nil samples. Sequencing of the 450-bp DNA from two samples from Málaga yielded a nucleotide sequence with 98 to 99% similarity to the HSP70h gene of West African SPCSV isolates. Foliar samples from I. setosa, originally grafted with the 21 vine cuttings, were used for nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA) testing with antiserum specific to SPFMV-RC (provided by J. Moyer, North Carolina State University, Raleigh). Positive control was sap extract from I. setosa that was infected with the common strain of SPFMV. Procedures for NCM-ELISA were as described (4). NCM-ELISA testing suggested that SPFMV was present in all samples. RT-PCR was conducted with degenerate primers POT1/POT2 (1). The nucleotide sequence that was amplified by these two primers spans part of the NIb protein and part of the coat protein gene of potyviruses. All samples yielded the expected 1.3-kb DNA. Sequencing of the RT-PCR products of two isolates from Malaga and sequence comparisons yielded nucleotide sequences with 97% similarity to two East African isolates (Nam 1 and Nam 3) of SPFMV (3). These results confirm the presence of SPCSV and SPFMV in sweet potato in Spain.

Whitefly transmission of sweet potato viruses

Three genera of plant viruses, Begomovirus (Geminiviridae), Crinivirus (Closteroviridae) and Ipomovirus (Potyviridae), contain members that infect sweet potato ( Ipomoea batatas) and are transmitted by whiteflies. The begomoviruses, Sweet potato leaf curl virus (SPLCV) and Ipomoea leaf curl virus (ILCV), and the ipomovirus Sweet potato mild mottle virus are transmitted by Bemisia tabaci, the sweet potato whitefly. The crinivirus, Sweet potato chlorotic stunt virus (SPCSV), is transmitted by B. tabaci and Trialeurodes abutilonea, the bandedwinged whitefly. Transmission experiments were done with three of these viruses using laboratory-reared whiteflies and 2-day acquisition and transmission feeding periods. SPLCV and ILCV were transmitted from single and double infections by B. tabaci at rates of 5–10%. Transmission rates for SPLCV by B. tabaci were 15–20%. T. abutilonea transmitted SPCSV at a rate of ca. 3% but did not transmit ILCV or SPLCV.

Control strategies for sweet potato virus disease in Africa

Sweet potato virus disease (SPVD), caused by dual infection with the whitefly-borne Sweet potato chlorotic stunt virus (SPCSV) and the aphid-borne Sweet potato feathery mottle virus (SPFMV), is the most serious disease of sweet potato in Africa. SPVD has been known there since at least the 1940s, although it took several decades to elucidate its aetiology. It occurs throughout Africa and is particularly prevalent in the Great Lakes region. Production of sweet potato is largely by resource-poor farmers, growing mostly local landraces and for home consumption and so control strategies need to be appropriate to these circumstances. Most high yielding and/or early maturing landraces in Uganda are susceptible to SPVD and most resistant landraces are low-yielding, forcing farmers to compromise between the conflicting requirements of large and/or early yields, and food security. Accordingly, two strategies were tested to avoid the disadvantages associated with such compromises. These were deploying high-yielding SPVD-resistant cultivars and phytosanitation practices to enable susceptible landraces to be grown successfully. In on-farm trials in Masaka and Rakai Districts of Uganda, some SPVD-resistant cultivars bred at Namulonge Agricultural and Animal Research Station (NAARI), in Wakiso District, out-yielded local landraces. Other trials at NAARI and at nearby farms showed that roguing diseased cuttings within 1 month of planting and isolation from diseased crops, even by as little as 15 m, can considerably decrease spread of SPVD to susceptible cultivars. This indicates that phytosanitation can protect desirable susceptible cultivars, even if adopted only locally. A dual approach of deploying both resistant varieties and phytosanitation provides farmers with a valuable increase in their choice of control strategies for SPVD.

Within-crop spread of sweet potato virus disease and the population dynamics of its whitefly and aphid vectors

Sweet potato virus disease (SPVD) is the most damaging disease of sweet potato ( Ipomoea batatas (L) Lam) in Uganda and results from dual infections of Sweet potato feathery mottle virus (SPFMV), vectored by aphids, and Sweet potato chlorotic stunt virus (SPCSV), vectored by whiteflies. For three cropping cycles, whiteflies ( Bemisia tabaci), aphids ( Myzus persicae and Aphis gossypii) and the spread of SPVD were monitored within and outside the sweet potato crop containing a central plot of SPVD-infected plants. There were similar catches of whiteflies on sticky traps placed on poles within the crop at different distances and directions around the infected plot. Outside the crop, there were also similar catches of whiteflies near (15 m) and far away (100 m) from the sweet potato crop. However, more whiteflies were trapped within than outside the crop. Most whiteflies were trapped close to the canopy. Aphids were rarely found on sweet potato plants, although they were commonly found on the traps. The number of aphids trapped inside the crop did not differ significantly from those trapped outside the crop. Consistently, a higher proportion of plants developed SPVD symptoms in the inner square than plants located further away from the infected plot. Number of infected plants correlated with trap catches of whiteflies only for the first season.

Some molecular characteristics of three viruses from SPVD-affected sweet potato plants in Egypt.

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus, family Potyviridae), Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus, family Closteroviridae) and sweet potato virus G (SPVG) were detected in naturally infected sweet potato plants grown in the Delta region in Egypt. Before this study, SPVG was reported only from China. Two isolates of SPFMV and one isolate of SPVG were characterized for the 3'-proximal genomic sequences. Phylogenetic analyses indicated that the SPFMV isolates belong to the "russet crack" strain group (RC). Serological tests using monoclonal antibodies, and phylogenetic analysis of a partial sequence of the Hsp70 gene, indicated that the Egyptian SPCSV belongs to the so-called non-East African strain group of SPCSV.

Incidence of Viruses and Virus like Diseases of Sweetpotato in Uganda.

Sweetpotato plants were surveyed for viruslike diseases and viruses in the four major agroecological zones of Uganda. Testing of 1,260 sweetpotato plants, of which 634 had virus-like symptoms, showed that virus disease incidence ranged from 2.7% (Soroti district, short grassland-savannah zone) to 20% (Mukono district, tall grass-forest mosaic zone). Sweet potato chlorotic stunt virus (SPCSV), Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), and sweet potato chlorotic fleck virus (SPCFV) were serologically detected and positive results confirmed by immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) and subsequent sequence analyses of the amplified fragments, except SPCFV, which lacked sequence information. SPCSV and SPFMV were detected in all the 14 districts surveyed, whereas SPMMV and SPCFV were detected in 13 and 8 districts, respectively. Logistic regression analysis revealed that SPCSV and SPFMV, SPFMV and SPMMV, and SPFMV and SPCFV more frequently occurred together than any other virus combinations or as single virus infections. Co-infections of SPCSV with SPFMV and/or SPMMV were associated with more severe and persistent symptoms than infections with each of the viruses alone. Several plants (11%) displaying viruslike symptoms did not react with the virus antisera used, suggesting that more viruses or viruslike agents are infecting sweetpotatoes in Uganda.

Sweetpotato Virus Disease (SPVD): Distribution, Incidence, and Effect on Sweetpotato Yield in Peru.

Sweetpotato virus disease (SPVD), the most important disease affecting sweetpotato (Ipomoea batatas (L.) Lam), is caused by the synergistic interaction of the aphid-transmitted Sweetpotato feathery mottle virus (SPFMV) and whitefly-transmitted Sweetpotato chlorotic stunt virus (SPCSV). In this study, SPVD was the main disease in the Cañete Valley, the major sweetpotato-producing area in Peru. Studies on virus incidence showed that SPCSV and SPFMV were the most frequently identified viruses in Cañete Valley. Symptoms of different severity were associated with isolates of both viruses involved in the SPVD. Over 80% of plants infected with both SPFMV and SPCSV showed the symptoms (leaf reduction and deformation, vein clearing or mosaic, and stunting) typically attributed to SPVD elsewhere. SPFMV did not significantly affect the yield of the sweetpotato cultivars Jonathan and Costanero, but infection of these cultivars by SPCSV was associated with significant yield reduction. Double infection by the two viruses resulted in SPVD and greater yield reduction than for either alone. These results demonstrate that SPFMV and SPCSV interact synergistically and that the severity of SPVD symptoms also depends on the particular isolate of each virus.

First Report of Sweet potato leaf curl virus in Peru.

Leaf curling symptoms have been reported in sweet potato (Ipomoea batatas) plants infected with a geminivirus (1). Leaf curl disease appeared in Peru after the 1997 to 1998 El Niño, when the population and activity of whiteflies (Bemisia tabaci, B. argentifolii, and B. afer) increased. Approximately 6% of plants in farmers' commercial fields in San Ramón, Junín (September 2000) and Cañete, Lima (February 2001) showed typical leaf curling symptoms. Seventeen plants in total were collected from both places, and stem scions from those plants were graft-inoculated to I. setosa, which developed symptoms of leaf curling, interveinal chlorosis, and stunting. Total nucleic acid was obtained from infected sweet potato and I. setosa plants using cetyltrimethylammoniumbromide (CTAB) extraction, and primers PW285-3 (5'-CGT CGT TAG CAG TCT GCA GGC CTC CTC TAG-3') and PW285-4 (5' -AAC TGT AAA TAC GGA ACT GCA GTT CGA ATT-3') for Sweet potato leaf curl virus (SPLCV), developed and provided by R. Valverde and C. Clark of Louisiana State University (2), were used to amplify SPLCV by polymerase chain reaction (PCR). Expected DNA fragments of ca. 900 bp (in all samples) and 2.4 kp (in some samples), characteristic of the subgenomic and genomic DNAs of SPLCV respectively, were obtained from symptomatic but not from symptomless (uninfected) plants. This 2.4-kb fragment was amplified in relatively small amounts compared to the 900-bp fragment. Presence of SPLCV was also confirmed by nucleic acid spot hybridization using a full-length clone of SPLCV-US. Fourteen of 17 plants infected with SPLCV were also infected with Sweet potato chlorotic stunt virus (determined by nitrocellulose membrane enzyme-linked immunosorbent assay serological test), which is also transmitted by whiteflies. These viruses now seem to be common in farmers' fields in San Ramón and Cañete. To our knowledge, this is the first report of SPLCV in Peru.

Complete genome sequence and analyses of the subgenomic RNAs of sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus.

The complete nucleotide sequences of genomic RNA1 (9,407 nucleotides [nt]) and RNA2 (8,223 nt) of Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) were determined, revealing that SPCSV possesses the second largest identified positive-strand single-stranded RNA genome among plant viruses after Citrus tristeza virus. RNA1 contains two overlapping open reading frames (ORFs) that encode the replication module, consisting of the putative papain-like cysteine proteinase, methyltransferase, helicase, and polymerase domains. RNA2 contains the Closteroviridae hallmark gene array represented by a heat shock protein homologue (Hsp70h), a protein of 50 to 60 kDa depending on the virus, the major coat protein, and a divergent copy of the coat protein. This grouping resembles the genome organization of Lettuce infectious yellows virus (LIYV), the only other crinivirus for which the whole genomic sequence is available. However, in striking contrast to LIYV, the two genomic RNAs of SPCSV contained nearly identical 208-nt-long 3' terminal sequences, and the ORF for a putative small hydrophobic protein present in LIYV RNA2 was found at a novel position in SPCSV RNA1. Furthermore, unlike any other plant or animal virus, SPCSV carried an ORF for a putative RNase III-like protein (ORF2 on RNA1). Several subgenomic RNAs (sgRNAs) were detected in SPCSV-infected plants, indicating that the sgRNAs formed from RNA1 accumulated earlier in infection than those of RNA2. The 5' ends of seven sgRNAs were cloned and sequenced by an approach that provided compelling evidence that the sgRNAs are capped in infected plants, a novel finding for members of the Closteroviridae.