INTRODUCTION

The sweet potato (Ipomoea batatas [L.] Lam), a member of the Convolvulaceae family, originates from Central and South America and ranks as the sixth most important food crop globally (Mu and Li, 2019;O’Brien, 1972;Ogero and Vlugt, 2023). Its versatility and hardiness make it a vital crop for human consumption (Di Feo et al., 2018). Global production reached 86 million metric tons in 2022, with 7 million hectares under cultivation. The leading producers include China, Malawi, Nigeria, and Tanzania (FAO, 2024). In Paraguay, sweet potato is grown in more than 5,000 hectares, producing over 50,000 tons (DCEAMAG, 2023).

Sweet potato production faces challenges that can significantly impact both yield and quality. Pests and diseases can reduce yields by up to 98%, with vegetative propagation of tubers promoting the spread and accumulation of viral infections, which represent the most critical threat, negatively affecting yields in cultivated areas (Barkessa, 2018;Di Feo et al., 2018;Loebenstein, 2015).

Sweet potato, being a vegetatively propagated crop, can lead to the accumulation of systemic pathogens, especially viruses, many of which infect crops worldwide. Over 30 viruses infecting sweet potato have been identified worldwide, assigned to the following nine families: Geminiviridae with 15 viruses, Potyviridae with 9, Caulimoviridae with 3, and Luteoviridae, Bromoviridae, Bunyaviridae, Closteroviridae, Comoviridae, and Flexiviridae with one virus each (Clark et al., 2012;Kreuze et al., 2020). Viruses including Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato latent virus (SwPLV), Sweet potato caulimovirus-like virus (SPCaLV), Cucumber mosaic virus (CMV), and Sweet potato leaf curl virus (SPLCV) are economically relevant due to their impact on sweet potato productivity (Barkessa, 2018). Many of these viruses can co-infect sweet potato and Ipomoea setosa (an indicator plant) without causing symptoms. Some also exhibit synergistic interactions with SPCSV, which mediates severe viral diseases in this crop (Clark et al., 2012). The most significant disease is known as the Sweet potato virus disease (SPVD) complex, which is transmitted by aphids and is caused by the synergistic co-infection of SPCSV and SPFMV, sometimes aggravated by additional viral infections (CIP, 2015;Kreuze et al., 2020;Kwak et al., 2014).

Symptoms include small, thin leaves, stunted plant growth, chlorotic mottling, or vein clearing in leaves (Varela and Trejos, 2020). Moreover, SPVD leads to substantial y ield losses. In China, the l argest p rod ucer worldwide, an average of 20% of the sweet potato crop is lost due to SPVD (Loebenstein, 2015;Zhang et al., 2020).

In Paraguay, studies on identifying viruses in sweet potatoes are still scarce (Esquivel et al., 2024). Cuellar et al. (2015) detected the SPFMV in mixed infection with the SPVG in sweet potato samples by PCR from the Yety (Abayybo) accession, exhibiting symptoms of chlorotic spots, leaf shrinkage, dwarfism, and vein clearing. In the same study, the accessions of Japonesa I, Batata Bobora, Yety Azucena, Yety Pyta, and Moroti showed chlorosis and leaf shrinkage symptoms, but viral infections were not identified.

The present study investigated a viral disease outbreak in sweet potato plantations in Choré major sweet potato- producing areas in Paraguay.

MATERIALS AND METHODS

Sample collection: The samples for the virus testing were collected from symptomatic and symptomless sweet potato leaves from sweet potato plantations in late August 2024 in Choré in the San Pedro Department, the main sweet potato- producing areas in Paraguay. Samples were collected from 12 varieties. The 12 varieties include those that have been cultivated in Paraguay for a long time or were introduced from abroad. The growth stage of the plants sampled were about three months after planting, and the field relies on natural rainfall to grow. During this period, the ambient air temperature in S an P ed ro w as 2 4 ºC, with a maximum of 32 ºC and a minimum of 18 ºC. Precipitation in Jul, August and September in the Choré region was 15 mm, 12 mm and 7mm, respectively (DMH, 2024).

Types of viruses tested and primer sets for PCR or RT-PCR: Nine viruses were tested, three DNA viruses of Sweet potato leaf curl virus (SPLCV), Sweet potato pakakuy virus (SPPV), and Sweet potato symptomless virus (SPSMV), and six RNA viruses of Sweet potato chlorotic fleck virus (SPCFV), Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), Sweet potato virus C (SPVC), Sweet potato virus 2 (SPV2), and Sweet potato latent virus (SwPLV). The primer sets used for PCR or RT-PCR reactions were the same as those reported by Kwak et al. (2014).

Plant total DNA Extraction: The extraction of viral DNA was carried out using the BCSTM Sweet Potato Viral DNA Preparation Kit VDPR50-SP (Bio Cube, Korea). The protocol was executed according to the manufacturer’s technical instructions.

Plant total RNA Extraction: The BCSTM Plant RNA Kit (Bio Cube, Korea) was used for RNA extraction. The protocol was executed according to the manufacturer’s technical instructions.

Polymerase Chain Reaction (PCR): PCR reactions were conducted using the GoTaq G2 DNA polymerase (Promega, USA). PCR reaction contains 20 μl mixture (4 μl of 5x buffer, 0 .4 μ l of d NTP (d ATP, d CTP, dTTP, a nd d GTP are at 2.5 mM each), 1μl of 5 pmol each primer, 0.1 μl of GoTaq DNA Polymerase, 13.5 μl of H₂O, a nd 1μl o f DNA template).

Thermal cycling conditions were initial denaturation (95°C for 5 minutes), denaturation (95 °C for 30 seconds), annealing (60°C for 30 seconds), extension (72°C for 1 minute), and a final extension (72 °C for 5 minutes).

Reverse Transcription Polymerase Chain Reaction (RT-PCR): RT-PCR reactions were performed according to the manufacturer’s recommended method using a one-step RT-PCR product (Access RT-PCR System, Promega, USA).

Sequencing of PCR or RT-PCR products: The specificity of each primer pair was confirmed by sequencing the PCR or RT-PCR products. The amplified DNA fragments were purified using a QIAquick gel extraction kit (QIAGEN, USA) and sequenced using the Sanger sequencing method (Biomi, Gödöllő, Hungary). BLAST search confirmed the identity of each of the viruses detected.

RESULTS AND DISCUSSION

Symptoms. Representative viral symptoms from 12 sweet potato varieties are shown in Figure 1. The irregular purple circle pattern (A) and purple along the leaf veins (B) were the most common symptoms, observed in many varieties, including Chaco I, Chaco II, Japonesa I, Jety Paraguay, Princesa, Pyta Guasu, Roxa, and Taiwanes. In ‘Jety Uruguayo,’ green chlorotic circles were formed in areas where the leaf color had turned purple (C), or there were chlorotic double circles (G). The small chlorotic circle was only observed in ‘Japonesa I’ (D). Vein clearing (E, F) was shown in both ‘Roxa’ and ‘Br. Yryvukua,’ leaf shrinkage with vein necrosis in ‘Pyta Guazu’ (H), and chlorosis in ‘Br. Yryvukua’ (I).

Symptoms of irregular purple circle pattern (A), purple along the leaf veins (B), and vein clearing (E) were also suggested as representative virus symptoms observed in Korea in the previous report (Kwak et al., 2014).

PCR or RT-PCR results. There was a significant variation between the kinds of viruses detected and the symptoms in the 12 varieties (Table 1). The number of plants that resulted positive out of the total plants tested is as follows: Andai (0/1), Br. Yryvukua (4/13), Chaco I (4/9), Chaco II (4/6), Japonesa I (4/5), Japonesa II (3/3), Jety Paraguay (10/15), Jety Uruguayo (8/12), Princesa (3/3), Pyta Guasu (4/11), Roxa (5/7), Taiwanes (4/7). Symptoms observed in the 12 varieties tested were chlorosis, chlorotic circle, chlorotic double circle, leaf shrinkage, purpling on parts of the leaves, necrosis, irregular purple circle, purple along the vein, and vein clearing (Table 1).

Two DNA viruses, SPPV and SPSMV, and three RNA viruses, SPFMV, SPVG, and SPVC, were d etected out o f the nine viruses tested. They were also cloned and sequenced, and the sequences deposited in GenBank under the accessions of PQ687549 (SPPV-01Py), PQ687551 (SPSMV- 01Py), PQ687548 (SPFMV-01Py), PQ687552 (SPVG-01Py) and PQ687550 (SPVC-01Py). SPCFV, SwPLV, SPLCV, and SPV2 were not detected in any of the varieties tested.

From a total of 92 samples, viruses were detected in 53, and these sample units were either infected with one or co-infected with up to three viruses. Of the 53 samples in which viruses were detected, SPVG was detected in 29, representing 57.6% of the virus-detected samples. SPFMV was also d etected from 23 with h igh frequency in a ll varieties tested: Therefore, in Paraguay, SPVG is the most prevalent virus in sweet potatoes, and SPFMV is the second most prevalent. However, as a comparison, in northern Uganda, SPFMV and SPMMV were the most f requent viruses detected (Wokorach et al., 2019).

From the results of virus detection based on foliar symptoms, it was found that there was no consistency in the presence of viruses by symptom. Despite some plants having the same symptoms, the virus was detected in some but not in others. This could be due to differences in virus concentration among plants, which may have resulted in undetectable virus in plants with lower concentrations. In particular, when viruses were tested from leaves showing necrotic symptoms, it is possible that the virus concentration was low due to the HR reaction and the virus was not detected (Moreal and Dangl, 1997). In a previous report (Kwak et al., 2014), viruses were detected in sweetpotato with the same symptoms as the “irregular purple circle” and the “purple along the leas veins symptoms” in Fig. 1 of this study. Viruses such as SPFMV, SPVG, SPVC, SPV2, SPLCV, and SPPV were detected in sweet potato with the s ame symptoms as C, E , F, G , H, a nd I in Fig. 1 of this study (Kiemo et al., 2022). Viruses were also detected in samples without symptoms. Even plants infected with the v irus m ay n ot s how symptoms or may have mild symptoms, depending on the environment or variety. Plant age and environmental conditions all influence symptom development. Light and temperature are essential for plant physiology and are associated with various signaling pathways (Qi et al., 2022). Therefore, it is assumed that the viral symptoms are hidden depending on the growth state of the plant as a result of environmental changes, as it has been reported that environmental requirements affect the expression of viral symptoms in plants (Ballaré, 2014;Kangasjarvi et al., 2012). Recent studies have indicated an interplay between light or temperature and viral infection, revealing that light or temperature can modulate the severity of viral symptoms (Godfrey et al., 2017;Harrison and Jones, 1971;Masinde et al., 2018). Sseremba et al. (2017) reported that temperature influenced the development of SPVD; low temperatures of 20 to 29°C produced more disease severities than high temperatures of 30 to 39°C.

This study cannot correlate cultivars with virus disease outbreaks because it cannot be certain that the virus-infected cultivars were infected in the field where they were sampled. The cultivars tested for viruses were either collected domestically or introduced from abroad, and since sweet potatoes are propagated by tubers or stems, it is impossible to rule out the possibility that those varieties are already infected with viruses when they were collected.

Despite the presence of symptoms, the failure to detect viruses in this study may be due to a low virus titer in leaves. Or, it is possible that these plants are infected with a virus that was not tested in this study. Therefore, it is important to note that the absence of a virus in a tested plant does not mean that the plant is free of that virus.

SPSMV and SPPV were detected in the Taiwanes. SPPV (genus Badnavirus, family Caulimoviridae) can be transmitted through seeds (Zhao et al., 2020). Sweet potatoes are propagated through the stems, and plant viruses spread using infected planting material. Therefore, it is imperative to ensure that the sweet potato mother plant used for c ross breeding is from a plant not infected with SPPV. The spread of plant viruses when trading sweet potato seedlings can be prevented by using certified seedlings that have been tested for viruses.

To prevent the spread and damage of viruses, be sure to use certified seedlings that have been tested for viruses when trading sweet potato seedlings.

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