Sweet Potato Chlorotic Stunt Virus Research Articles

A synoptic review of plant disease epidemics and outbreaks published in 2022

This scientometric study reviews the scientific literature and CABI distribution records published in 2022 to find evidence of major disease outbreaks and first reports of pathogens in new locations or on new hosts. This is the second time we have done this, and this study builds on our work documenting and analysing reports from 2021. Pathogens with three or more articles identified in 2022 literature were: Xylella fastidiosa, Bursaphelenchus xylophilus, Meloidogyne species complexes, Candidatus Liberibacter asiaticus, Raffaelea lauricola, Fusarium oxysporum formae specialis and Puccinia graminis f. sp. tritici. Our review of CABI distribution records found 29 pathogens with confirmed first reports in 2022. Pathogens with four or more first reports were: Meloidogyne species complexes, Pantoea ananatis, grapevine red globe virus and Thekopsora minima. Analysis of the proportion of new distribution records from 2022 indicated that grapevine red globe virus, sweet potato chlorotic stunt virus and Ca. Phytoplasma vitis may have been actively spreading. As we saw last year, there was little overlap between the pathogens identified by reviewing scientific literature versus distribution records. Strikingly, too, there was also no overlap between species assessed to be actively spreading in this year’s study and those identified last year. In general, introduction of new pathogens and outbreaks of extant pathogens threaten food security and ecosystem services. Continued monitoring of these threats is essential to support phytosanitary measures intended to prevent pathogen introductions and management of threats within a country.

First Report of Sweet Potato Virus E(SPVE) Infecting Sweet Potato in China.

Sweet potato (Ipomoea batatas [L.] Lam.) is a versatile crop, cultivated in the subtropical and tropical areas, as food, fodder, and industrial raw material crop. In China, sweet potato has been used as a health-care food in recent years, as it contains a wide range of nutrients and xenobiotic phytochemicals. However, viral diseases are major constraint for the sweet potato yield and quality, especially the seed production and quality. Over 30 species of viruses infect sweet potato worldwide (Clark et al. 2012). More recently, a few new viruses infected sweet potato were identified, such as sweet potato virus E (SPVE), which was reported in Korea(Jo et al. 2020). In May 2022, a sweet potato sample (JSXZ1) with virus-like symptom, such as mosaic and vein clearing were collected from sweet potato germplasm Xuzhou resource nursery, Jiangsu Province, China (N34˚16', E117˚18') (Fig. S1A). To investigate the virus disease, the sample JSXZ1 showing the typical symptoms of disease was prepared for Small-RNA (sRNA) deep-sequencing. The sRNA library was constructed using TruSeq™ Small RNA Sample Prep Kits (Illumina, San Diego, USA) and sequenced using the Illumine Hiseq 2500 platform by LC-Bop Technologies (Hangzhou) CO., LTD. The sample was sequenced to obtain 26, 358, 439 raw reads and 22, 969, 139 clean reads after quality control trimming and analysis. The Velvet 1.0.5 software was used to de novo assemble the clean reads (18 to 28 nt) into larger contigs, which were then compared with the nucleotide sequences in the National Center for Biotechnology Information (NCBI) database using the BLASTn algorithm. Viruses found in the sample were sweet potato latent virus (SPLV), sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato badnavirus A (SPBV-A) and sweet potato badnavirus B (SPBV-B). Surprisingly, besides the viruses listed above, 28 contigs matched sequences of SPVE isolate GS (MH388502). To verify the result, total RNA was extracted from the sample JSXZ1 and from other leave samples (JSXZ2-JSXZ5) that contained SPFMV, SPVC, SPLV, SPVG respectively stored in lab using FastPure Universal Plant Total RNA Isolation Kit (Vazyme Biotech Co., LTD, Nanjing, China). cDNA was synthesized using random primer (hexadeoxyribonucleotide mixture; pd(N)6). The cDNA serves as template in PCR using a newly designed primer pairs based on SPVE p1 gene (SPVE-F: 5'- TCACCAAAAAGAATGCTACAAC-3'/SPVE-R: 5'-GAAATCCTCCCACTCTCCATA-3'). An expected ~500-bp PCR fragment was obtained in JSXZ1, while none of the fragment was obtained from JSXZ2-JSXZ5 (Fig. S1B). The PCR fragment was cloned into pMD18-T vector (Takara Bio Inc., Beijing, China) and plasmid DNA from transformed Escherichia coli DH5α cell (n=3) were commercially sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequences of the three fragment clones we obtained were 100% identical when compared. A BLASTN analysis of the sequences revealed that they are specific to SPVE and shared 98.62% nucleotide identity to SPVE GS isolate (MH388502) and one sequence was submitted to GenBank (Accession number OQ948331). To determine the occurrence of SPVE in infected sweet potato plants, a total of 37 leaves samples with viral symptom collected from Shandong Province (n=6) and Jiangsu Province (n=31) were indexed by RT-PCR as described before. Only 9 (24.3%) out of 37 from Shandong (n=1) and Jiangsu (n=8) were positive to SPVE respectively. In addition, five additional viruses (SPFMV, SPVC, SPVG, SPLV, SPCSV) were detected among these 37 samples and always in a mixed infection of two or more viruses. To our knowledge, this is the first report of SPVE infecting sweet potato in China. Sweet potato is an important crop in China and other countries (Zhang et al. 2023). China is the largest sweet potato producer all over the world. In addition, as sweet potato is produced through the vegetative propagation mode, thus, more attention should be paid to detection and monitoring of occurrence of SPVE in China.

Loop‐mediated isothermal amplification assay: A novel disease diagnostics tool in sweetpotato seed quality assurance

AbstractSweetpotato is an important food crop in global production. However, sweetoptato viruses pose a threat to sustainable agriculture and cause significant economic loss. More than 30 viruses have been reported to date, with sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV) occurring frequently and in combination, causing sweetpotato virus disease. The detection of viruses directly from sweetpotato is still a challenge, and efforts are being made toward this front. Therefore, rapid detection of viruses is critical for effective control. Current diagnostic tests are not sufficiently sensitive to reliably detect viruses directly from sweetpotato and require expensive laboratory equipment and a high level of skilled experience. Loop‐mediated isothermal amplification (LAMP) is sensitive and specific for both DNA/RNA amplification; it is affordable and its characteristics make it potentially suitable for on‐site testing. LAMP assays have been developed for several sweetpotato viruses: SPCSV, SPFMV, and sweet potato leaf curl virus (and related sweepoviruses). Laboratory validation showed a 100% diagnostic sensitivity for all three viruses. The LAMP on‐site testing results were comparable to those of polymerase chain reaction and reverse transcriptase polymerase chain reaction confirmatory laboratory tests. Interlab validation and packaging into affordable kits will ensure high adoption in sub‐Saharan Africa. The LAMP assay can be used for field surveys and monitoring the phytosanitary status of pre‐basic seed production in quarantine or certification program. This guarantees pathogen‐free planting material enters the seed system. We discuss opportunities for LAMP as a diagnostic assay for the rapid detection of sweetpotato viruses and its adoption as a quality control system for planting materials.

Detection of SPV2 and SPCSV and their possible influence on the yield in multiple virus infections

AbstractThe prevalence of Sweet potato virus 2 (SPV2) and Sweet potato chlorotic stunt virus (SPCSV) in the Limpopo, Mpumalanga, and Gauteng provinces of South Africa was determined using serological and nucleic acid based methods. Cloning and sequencing part of the coat protein (CP) gene and the complete genome using next generation sequencing (NGS) were also conducted to characterize and determine the sequence variability in SPV2 isolates. Primers designed in the current study successfully detected and confirmed the prevalence of SPV2 in the three provinces surveyed. The pairwise comparison of the amino acid (aa) sequences encoding the CP gene region confirmed that isolates in this study are related to SPV2, sharing a 94% to 99% CP aa identity with those originally reported in South Africa. The East African (EA) strain of SPCSV was also detected in the three provinces surveyed. Phylogenetic analysis using part of the CP encoding nucleotide (nt) sequences showed that the South African isolates correspond to but are distinct from the other EA strains of SPCSV used as reference sequences. Assessment of varieties and breeding lines’ response to multiple infection of potyviruses identified variety NC1560, breeding lines 2002‐21‐1 and 2006‐5‐4 as promising for inclusion in future breeding programs as sources of parent material for virus tolerance.

First Report of Calystegia hederacea as a new host of sweet potato leaf curl Hubei virus.

Calystegia hederacea (Convolvulaceae) is one of the most problematic perennial weeds widely distributed around or in crop fields. Our previous studies showed that C. hederacea is natural reservoir of sweet potato chlorotic stunt virus isolate CH (SPCSV-CH) and sweet potato latent virus (SPLV) (Liu et al. 2020; Zhao et al. 2022). To shed further light on the role of C. hederacea in the epidemiology of sweet potato viruses, in May 2021, a total of seven C. hederacea plants (five asymptomatic, one curling and one mild vein-clearing) were collected from two different sweet potato fields in Xinxiang city of Henan Province in China. Total RNA was prepared from a pool of the seven leaf samples using the EZNA Plant RNA Kit (Omega Bio-Tek, Norcross, GA). A library was constructed from the ribosomal-depleted RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, MA, USA) and sequenced using the Illumina HiSeq platform (Novogene, Tianjin, China). A total of 139,057,020 paired-end clean reads of 150 bp were obtained after removing adaptor sequences and low-quality reads and used for de novo assembly using the Trinity (v2.2.0) software. Blast searches of the assembled contigs longer than 200 bp against NCBI nucleotide and protein sequence databases revealed the presence of 37 contigs (237 to 4885 bp) and 19 contigs (261 to 758 bp) with high nucleotide (nt) identity with SPLV and SPCSV-CH, respectively. The occurrence of SPLV and SPCSV-CH on C. hederacea was previously reported, and thus the contig sequences related to SPLV and SPCSV-CH were not subjected to further verification in this study. In addition, one contig (2,827 bp) with the highest nt sequence identity of 94.94% with sweet potato leaf curl Hubei virus (SPLCHbV, genus Begomovirus, family Geminiviridae, accession no. MK931304) was assembled from 16,592 reads, with average coverage depth of 740.5X. These results suggested the presence of SPLCHbV in C. hederacea. To further confirm the RNA sequencing result, each of the seven samples was tested by PCR using partially overlapping (italicized nucleotides) forward and reverse primers (SweeIn-F1, 5`-GGAGGAAGCTAAGTACGAGAATCAGTTAGAG-3`; SweeIn-R1, 5`-GCTTCCTCCTTGTGATTGTAAGTAACATGG-3`) that were designed based on the SPLCHbV-related contig for amplification of circular DNA viral genome (approximately 2.7 kb). Two symptomatic and three symptomless C. hederacea samples were SPLCHbV positive, indicating that virus-like symptoms of the two C. hederacea samples were probably not induced by SPLCHbV. Two of the five amplified products were completely sequenced and deposited to GenBank (accession nos. OQ551733 and OQ551734). Sequences analysis showed that the complete genome sequences of two SPLCHbV C. headrace isolates (2,763 nt and 2,761 nt) had 96.53% nt identity with each other and 95.92 to 97.70% nt identity with that of SPLCHbV isolate Shandong7-2017 (MK931304). In August 2021, fourteen C. hederacea plants (three symptomatic, 11 asymptomatic) collected from natural fields from Zhumadian and Pingdingshan cities in Henan Province, were tested by PCR using SweeIn-F1/R1 primers for SPLCHbV, showing that eight samples were SPLCHbV positive. SPLCHbV belongs to the sweepoviruses, a group of phylogenetically distinct begomoviruses infecting sweet potato, and was reported to infect sweet potato from many provinces of China (Wang et al., 2021). To the best of our knowledge, this is the first report of SPLCHbV infection in C. hederacea, which expands the natural host range of SPLCHbV.

Degeneration of cleaned-up, virus-tested sweetpotato seed vines in Tanzania

Viruses pose a major challenge to sweetpotato production in Tanzania. Use of cleaned-up, virus-tested seed vines distributed through a formal seed system is among the proposed strategies to address this challenge. However, virus-tested seed vines can get infected once in the field and it is not known how they will perform following several seasons of on farm propagation. We assessed the performance of virus-tested seed vines and farmer-sourced seed vines of a susceptible variety, Ejumula, and a relatively tolerant variety, Kabode, over five seasons to understand the trend in root yields, vine yields and virus incidences. The experiments were done in high and low virus pressure areas. The most prevalent viruses were sweet potato chlorotic stunt virus (SPCSV) followed by sweet potato feathery mottle virus (SPFMV) and sweet potato leaf curl virus (SPLCV), respectively. Both farmer-sourced and cleaned-up, virus-tested seed of cv. Ejumula were rapidly infected with SPCSV. The incidence of this virus on Ejumula's farmer-sourced material at the high-virus-pressure area reached 100% by the second season. The incidences for all three viruses remained stable for cv. Kabode across the five seasons. Plants generated from cleaned-up, virus-tested seed had lower incidences for all viruses compared to those from farmer-sourced planting material. Virus-tested seed produced significantly higher root yields for cv. Ejumula in the high-virus-pressure site, with a gradual drop across the seasons. The findings show that regular replenishment of clean, virus-tested seed is more economical in high-virus-pressure areas and for more susceptible varieties like cv. Ejumula. They also indicate that farmers may be reluctant to invest in cleaned-up, virus-tested seed in cases where they have virus-tolerant varieties such as cv. Kabode due to lack of obvious virus effect on yields.

Occurrence and completed genomes of sweet potato chlorotic stunt virus infecting sweet potato in Korea

Occurrence and completed genomes of sweet potato chlorotic stunt virus infecting sweet potato in Korea

Advances in the etiology of sweet potato (Ipomoea batatas (L.) Lam) yellow curling disease in Argentina

Sweet potato yellow curling (YC), the most severe disease of sweet potato detected in Argentina, causes symptoms and damage to sweet potato crops in all cultivated regions. Since 2010/11, the presence of four viruses has been detected in symptomatic cv. Arapey INIA: two potyviruses non-persistently transmitted by Myzus persicae (sweet potato feathery mottle virus, SPFMV and sweet potato virus G, SPVG); a closterovirus, sweet potato chlorotic stunt virus (SPCSV) and a geminivirus, sweet potato leaf curl virus (SPLCV), both transmitted by Bemisia tabaci in a semi-persistent and persistent manner, respectively. All the plants were collected from fields in Colonia Caroya, Córdoba province, Argentina. The objectives of the present work are to isolate and identify the virus or viruses involved in YC disease of sweet potato, and to elucidate the viral combination that reproduces YC symptoms. The most severe YC symptoms for this genotype in the field were only reproduced by a combination of the four viruses. The symptoms include chlorosis, stunting, mosaic, blistering, leaf curling, chlorotic spots, chlorotic patterns, leaf area reduction and distortion, and upward curling of leaf edges. The presence of each virus was detected by serological (DAS, NCM and TAS-ELISA) and molecular (PCR) tests. It is concluded that the interaction of SPFMV, SPVG, SPCSV and SPLCV is needed for the development of YC symptoms. Highlights Four viruses involved in yellow curling disease, SPFMV, SPVG, SPCSV and SPLCV were isolated and identified. The first antiserum produced in Argentina allows the fast, safe and efficient diagnosis of SPVG. The interaction of SPFMV, SPVG, SPCSV and SPLCV is needed for the manifestation of yellow curling in Argentina.

Early-Stage Phenotyping of Sweet Potato Virus Disease Caused by Sweet Potato Chlorotic Stunt Virus and Sweet Potato Virus C to Support Breeding.

Sweet potato virus disease (SPVD) is a global constraint to sweetpotato (Ipomoea batatas) production, especially under intensive cultivation in the humid tropics such as East Africa. The objectives of this study were to develop a precision SPVD phenotyping protocol, to find new SPVD-resistant genotypes, and to standardize the first stages of screening for SPVD resistance. The first part of the protocol was based on enzyme-linked immunosorbent assay results for sweet potato chlorotic stunt virus (SPCSV) and sweet potato virus C (SPVC) with adjustments to a negative control (uninfected clone Tanzania) and was performed on a prebreeding population (VZ08) comprising 455 clones and 27 check clones graft inoculated under screenhouse conditions. The second part included field studies with 52 selected clones for SPCSV resistance from VZ08 and 8 checks. In screenhouse conditions, the resistant and susceptible check clones performed as expected; 63 clones from VZ08 exhibited lower relative absorbance values for SPCSV and SPVC than inoculated check Tanzania. Field experiments confirmed SPVD resistance of several clones selected by relative absorbance values (nine resistant clones in two locations; that is, 17.3% of the screenhouse selection), supporting the reliability of our method for SPVD-resistance selection. Two clones were promising, exhibiting high storage root yields of 28.7 to 34.9 t ha-1 and SPVD resistance, based on the proposed selection procedure. This modified serological analysis for SPVD-resistance phenotyping might lead to more efficient development of resistant varieties by reducing costs and time at early stages, and provide solid data for marker-assisted selection with a quantitative tool for classifying resistance.[Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Elucidating the sweet potato virome in Greece with the aid of high‐throughput sequencing technology

AbstractIn 2018, typical symptoms of sweet potato virus disease (SPVD) such as purple ringspots, distortion and crinkling of the leaves appeared on a sweet potato crop located in southern Greece. High‐throughput sequencing (HTS) analysis revealed the presence of seven viruses, sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato virus C (SPVC), sweet potato virus G (SPVG), sweet potato virus 2 (SPV2), sweet potato leaf curl virus (SPLCV) and sweet potato pakakuy virus (SPPV). In silico analysis of the HTS data in combination with Sanger sequencing succeeded in obtaining the complete genome sequences of all seven viruses. Nucleotide sequence comparisons of complete viral genomes showed high similarity of all detected potyviruses and SPLCV with homologous sequences retrieved from GenΒank, whereas a new variant of SPCSV RNA1 was characterized and successfully transmitted onto Nicotiana benthamiana plants with the whitefly species Bemisia tabaci MED and MEAM1. Moreover, the Greek strain of SPPV showed higher similarity with SPPV strain B, even though it possesses a variable RT‐RNase H domain. Further phylogenetic analysis clustered Greek sweet potato virus isolates into various phylogenetic groups, whereas few new clades were formed in the cases of SPFMV, SPVG and SPCSV RNA1. Small‐scale surveys on three different sweet potato crops exhibiting typical, mild or no symptoms also indicated the close association of SPCSV with SPVD. Taken together, this study characterizes for the first time the virome of sweet potato crops in Greece and provides a glimpse into the evolutionary relationships of the identified viruses.

Calystegia hederacea: a new natural host of Sweet potato latent virus in China.

Sweet potato is a global root crop, with a worldwide production of 91.5 million tons in 2019 (FAOSTAT, 2019). However, virus diseases cause significant yield losses and quality decline in sweet potato. Up to now, over 30 different viruses have been identified in sweet potato (Clark et al. 2012). Expanding knowledge of the host range of sweet potato viruses will provide a benefit for the understanding of virus occurrence and designing appropriate virus control measures. In August 2019, ten Calystegia hederacea and two Convolvulus arvensis (Convolvulaceae) weed plants with or without symptoms of leaf yellowing symptoms were collected from various virus disease-affected sweet potato fields in four cities (Jiaozuo, Xinxiang, Zhengzhou and Kaifeng) of Henan Province for virus detection. The leaves of these plants were harvested and pooled for total RNA extraction using a Plant Total RNA Purification Kit (GMbiolab, Taichung, Taiwan). A library for high-throughput sequencing (HTS) was constructed and sequenced using the Illumina HiSeq 2000 platform by BGI Tech (Shenzhen, China). Clean reads (n = 100,570,346), each 150 bp in length, were de novo assembled using CLC Genomics Workbench 9.5 (Qiagen, USA). The assembled contigs were analyzed against the viral reference genome database in GenBank using the BLASTN and BLASTX searches. Three contigs related to sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus, family Closteroviridae) were identified (Liu et al. 2021). In addition, a total of 20 contigs, ranging from 1,019 to 9,859 bp in length with an average depth of coverage of 1439.26, showed 74.80-87.59% nucleotide (nt) sequence identities with corresponding sequences of sweet potato latent virus (SPLV, genus Potyvirus, family Potyviridae). The sequence of the 9,859-bp contig covering nearly complete genome sequence for SPLV, was deposited in GenBank (accession no.OL625609). These results demonstrated the presence of genetically diverse isolates of SPLV in the pooled samples. To further confirm the HTS result, each of the 12 samples were tested by RT-PCR using SPLV primers (SPLV-F1: 5'-AATGCCAAGGCTACAAGGAGT-3' and SPLV-R1: 5'-CAAGTAGTGTGTGTATGTTCC-3') that targets a partial conserved region of the coat protein gene in SPLV and SPCSV primers designed based on three contigs (ctg1-F1/R1, ctg2-F1/R1, and ctg3-F1/R1) (Liu et al. 2021), respectively. As a result, four symptomless C. hederacea samples tested positive for SPLV, yielding the expected approximately 500 bp PCR fragment, and one leaf yellowing C. hederacea sample tested positive for SPCSV (Liu et al. 2021). The sequences obtained from two of the four amplicons of SPLV (MZ089700 and OM056706) showed 90.2 and 89.8% nt (100 and 99.4% amino acid) identities with the corresponding sequences of the SPLV isolate Shaanxi1 from sweet potato (HQ844148). In 2021, a further 45 C. hederacea plants collected from Shangqiu (n = 6), Xinxiang (n =30) and Pingdingshan (n = 9) cities in Henan Province, were screened by RT-PCR with SPLV-F1/R1 primers, giving an incidence of 33.33%. SPLV is an important potyvirus infecting sweet potato. SPLV is asymptomatic in most sweet potato cultivars in single infection but is able to mediate synergistic viral disease in co-infection with SPCSV (Untiveros et al. 2007). To the best of our knowledge, this is the first report of SPLV in C. hederacea. The finding reported here indicated that C. hederacea may act as a reservoir of SPLV and possible infection source for the sweet potato crop.

Development of a dual RT-RPA detection for Sweet potato feathery mottle virus and Sweet potato chlorotic stuntvirus

The disease co-infected by Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) is devastating in sweet potato, as it would give rise to the serious losses in both production and quality. Consequently, it is conducive for preventing and controlling this disease to detect these two viruses accurately and timely. Here we developed and optimized a dual reverse transcription recombinase polymerase amplification (RT-RPA) for rapid and accurate detection of SPFMV and SPCSV. Four special primers were designed based on the conserved sequences of SPFMV and SPCSV, respectively. The sensitivity of dual RT-RPA for SPFMV and SPCSV was 10−4 ng/μL at the optimal conditions in which the primer ratio between SPFMV and SPCSV was 2:1, and the reaction incubated for 25 min at a temperature of 39 °C. Both 61 sweet potato samples and 5 morning glory samples collected from China were tested using the dual RT-RPA successfully. Therefore, the dual RT-RPA is a reliable, rapid, sensitive method to detect these two viruses in sweet potato. It is the RT-RPA that was used for detection of SPFMV and SPCSV simultaneously firstly. This dual RT-RPA, as a convenient and powerful tool, will be useful to diagnose SPFMV and SPCSV.

First report of sweet potato chlorotic stunt virus infecting sweet potatoes in Hungary.

Sweet potato chlorotic stunt virus (SPCSV), a crinivirus in the family Closteroviridae, is a quarantine pest in Europe and one of the most economically important viruses of sweet potato (Ipomoea batatas (L.) Lam) crops globally. It forms synergies with other viruses in sweet potato, leading to yield loss of 30-100% (Qin et al., 2014). In summer 2020, 62 symptomatic and 38 symptomless sweet potato vines were randomly collected in farmers' fields in the south (Ásotthalom, Szeged) and central (Galgahévíz) parts of Hungary and transplanted in an insect-proof greenhouse. Six of the plants expressed SPCSV-like symptoms, including stunting, vein clearing and leaf purpling (Suppl1). To check for common viruses of sweet potato (Suppl2), total RNA and DNA were extracted from leaves of each of the 100 plants using Trizolate reagent (UD-GenoMed, Debrecen, Hungary) and Zenogene kit (Zenon Bio, Szeged, Hungary), respectively. Primer pair Ch2N (Suppl2) was designed using Primer3 (v. 0.4.0) to amplify a 194 bp fragment of SPCSV RNA1. Presence of the RNA viruses was checked by qPCR using qPCRBIO SyGreen 1-step qPCR kit (PCR Biosystems, London, UK), while DNA viruses were checked by PCR using DreamTaq DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), followed by 1% agarose gel electrophoresis. Four samples (labelled A5.1, A6.1, A6V9-1, A6V9-2) out of the 100 tested positive for SPCSV. Two of them (A6V9-1 and A6V9-2) were co-infected with SPCSV, a badnavirus sweet potato pakakuy virus (SPPV) and a potyvirus sweet potato virus 2 (SPV2), while the other two (A5.1 and A6.1) lacked SPV2. Plants infected with SPCSV, SPV2 and SPPV displayed more severe symptoms. To confirm the results, cDNA synthesized from the four SPCSV positive samples using RevertAid first strand cDNA synthesis kit (Thermo Scientific, Vilnius, Lithuania) underwent PCR (94oC 4 min, 94oC 1 min, 53oC 30 s, 72oC 70 s and 72oC 10 min for a total of 30 cycles) using primers CL43U and CL43L for the viral heat shock protein 70 gene (Maliogka et al., 2020). An expected band size of 486 bp was obtained in all cases. The amplicon from sample A6.1 was sequenced and found to be identical to SPCSV Guatemalan isolate GT:B3:08 (acc. JF699628). RNA1 and RNA2 complete sequences from sample A6.1 were obtained via PCR amplifications of cDNA using primers (Suppl2) designed (from acc. KC888966 for RNA1 and acc. KC888963 for RNA2) to amplify overlapping fragments of West African strain of SPCSV. QIAquick gel extraction kit (QIAGEN, Hilden, Germany) was used to purify the PCR fragments, which were then cloned into pGEM-T Easy Vector (Promega, Madison, USA) and sequenced using Sanger sequencing technique (Biomi, Gödöllő, Hungary). BLASTn search revealed that RNA1 of our isolate Hun_01 (acc. MW892835) had 99.63% sequence identity to SPCSV isolate su-17-10 (acc. MK802073), while RNA2 of Hun_01 (acc. MW892836) was 99.68% similar to SPCSV isolate min-17-1 (acc. MK802078) and isolate 24-1 (acc. MK802080). Phylogenetic analysis using MegAlign (v. 7.1.0, 44.1) showed a close relationship between our isolate and those isolated in China, suggesting that they may have a common origin (Suppl1). Severe stunting and leaf yellowing symptoms developed in I. setosa indicator plants grafted with SPCSV infected sweet potato scions. qPCR test for the virus confirmed its presence in the I. setosa leaves. To the best of our knowledge, this is the first report on the occurrence of SPCSV in Hungary and the third in Europe (Valverde et al. 2004; EPPO 2021).

Detection and elimination of viruses infecting sweet potatoes in Hungary

AbstractSweet potato has been grown in Hungary for the last three decades, and its popularity is increasing among farmers and consumers. Its production is hampered by pests and diseases due to poor agricultural practices, such as the use of virus‐infected propagation materials. We tested the presence of 15 viruses by PCR and quantitative PCR in 110 sweet potato plants collected from seven regions in Hungary. Seven viruses in single or multiple infections associated with a wide range of foliar symptoms were detected: sweet potato chlorotic stunt virus (SPCSV), sweet potato virus G (SPVG), sweet potato virus C (SPVC), sweet potato feathery mottle virus (SPFMV), sweet potato virus 2 (SPV2), sweet potato leaf curl virus (SPLCV), and sweet potato pakakuy virus (SPPV). This is the first report on the occurrence of the begomovirus SPLCV in sweet potatoes in Hungary. The infectivity and identity of these viruses were confirmed through bioassays (grafting to Ipomoea setosa) and sequencing of the PCR‐amplified sections of their genomes, respectively. Due to the necessity for virus‐free sweet potato propagation material in Hungary, virus elimination was carried out successfully in five out of six genotypes important for Hungarian farmers using heat treatment and meristem tip culture. All five viruses detected in the plants before heat treatment were removed except SPPV, which persists after heat treatment. Production and strict regulation of virus‐free sweet potato propagation materials are recommended to avoid exacerbating the virus situation and protect Hungarian farmers from further losses.

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.

Loop-Mediated Isothermal Amplification assays for on-site detection of the main sweetpotato infecting viruses

Globally, Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) occur frequently and in combination cause sweetpotato virus disease (SPVD). Many viral diseases are economically important and negatively impact the production and movement of germplasm across regions. Rapid detection of viruses is critical for effective control. Detection and quantification of viruses directly from sweetpotato remains a challenge. Current diagnostic tests are not sensitive enough to reliably detect viruses directly from the plant or require expensive laboratory equipment and expertise to perform. We developed a simple and rapid loop‐mediated isothermal amplification (LAMP) assay for the detection of SPFMV, SPCSV and begomoviruses related to sweet potato leaf curl virus (SPLCV). Laboratory validation recorded 100 % diagnostic sensitivity for all the three viruses. The LAMP assays were customized for field testing using a lyophilized thermostable isothermal master mix in a ready-to-use form that required no cold chain. The average time to positivity (TTP) was: SPFMV 5−30 min, SPCSV 15–43 min s and begomoviruses 28−45 mins. LAMP on-site testing results were comparable to PCR and RT-PCR confirmatory laboratory tests. The LAMP assay is a powerful tool for rapid sweetpotato virus detection at a reasonable cost and thus could serve as quality control systems for planting materials.

Species and genetic variability of sweet potato viruses in China

China is the world’s largest producer of sweet potato (Ipomoea batatas (L.) Lam.). Considering that there are numerous sweet potato-producing regions in China and sweet potato is a vegetatively propagated crop, the genetic diversity of sweet potato viruses could be high in the country. However, studies on species and genetic variabilities of sweet potato viruses in China are limited, making it difficult to prevent and control viral diseases in this crop. During 2014–2019, sweet potato samples with viral disease-like symptoms were randomly collected from sweet potato fields in 25 provinces in China. Twenty-one virus species, including 12 DNA and 9 RNA viruses, were identified in the samples using next-generation sequencing, polymerase chain reaction and rolling-circle amplification methods. One novel sweepovirus species, Sweet potato leaf curl Hubei virus (SPLCHbV), was identified. Two species, Sweet potato collusive virus and Tobacco mosaic virus, were identified for the first time in sweet potato in China. Full-length or nearly full-length genomic sequences of 111 isolates belonging to 18 viral species were obtained. Genome sequence comparisons of potyvirus isolates obtained in this study indicate that the genome of sweet potato virus 2 is highly conserved, whereas the other four potyviruses, sweet potato feathery mottle virus, sweet potato virus G, sweet potato latent virus and sweet potato virus C, exhibited a high genetic variability. The similarities among the 40 sweepovirus genomic sequences obtained from eight sweepovirus species are 67.0–99.8%. The eight sweepoviruses include 14 strains, of which 4 novel strains were identified from SPLCHbV and 1 from sweet potato leaf curl Guangxi virus. Five sweet potato chlorotic stunt virus (SPCSV) isolates obtained belong to the WA strain, and the genome sequences of SPCSV are highly conserved. Together, this study for the first time comprehensively reports the variability of sweet potato viruses in China.

In Vitro Identification and In Vivo Confirmation of Inhibitors for Sweet Potato Chlorotic Stunt Virus RNA Silencing Suppressor, a Viral RNase III.

ABSTRACTSweet potato virus disease (SPVD), caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV), is responsible for substantial yield losses all over the world. However, there are currently no approved treatments for this severe disease. The crucial role played by RNase III of SPCSV (CSR3) as an RNA silencing suppressor during the viruses’ synergistic interaction in sweetpotato makes it an ideal drug target for developing antiviral treatment. In this study, high-throughput screening (HTS) of small molecular libraries targeting CSR3 was initiated by a virtual screen using Glide docking, allowing the selection of 6,400 compounds out of 136,353. We subsequently developed and carried out kinetic-based HTS using fluorescence resonance energy transfer technology, which isolated 112 compounds. These compounds were validated with dose-response assays including kinetic-based HTS and binding affinity assays using surface plasmon resonance and microscale thermophoresis. Finally, the interference of the selected compounds with viral accumulation was verified in planta. In summary, we identified five compounds belonging to two structural classes that inhibited CSR3 activity and reduced viral accumulation in plants. These results provide the foundation for developing antiviral agents targeting CSR3 to provide new strategies for controlling sweetpotato virus diseases.IMPORTANCE We report here a high-throughput inhibitor identification method that targets a severe sweetpotato virus disease caused by coinfection with two viruses (SPCSV and SPFMV). The disease is responsible for up to 90% yield losses. Specifically, we targeted the RNase III enzyme encoded by SPCSV, which plays an important role in suppressing the RNA silencing defense system of sweetpotato plants. Based on virtual screening, laboratory assays, and confirmation in planta, we identified five compounds that could be used to develop antiviral drugs to combat the most severe sweetpotato virus disease.

Articles of Significant Interest in This Issue

Sweet potato virus disease (SPVD) caused by synergistic infection of Sweet potato chlorotic stunt virus (SPCSV) and Sweet potato feathery mottle virus (SPFMV) leads to dramatic yield loss. Wang et al. (e00107-21) report in vitro identification and in vivo confirmation of inhibitors targeting the SPCSV RNase III (CSR3), an RNA silencing suppressor. As a result, five compounds belonging to two structural classes which inhibited CSR3 activity and reduced viral accumulation in plants were identified. These results provide the foundation for developing antiviral agents targeting viral RNase III to provide new strategies for controlling plant viruses.

Complete genome sequence of a divergent sweet potato chlorotic stunt virus isolate infecting Calystegia hederacea in China.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) is one of the most destructive viruses infecting sweet potatoes. In this study, we determined the complete genome sequence of an SPCSV-like isolate (CH) from Calystegia hederacea Wall. (Convolvulaceae), a weed species related to sweet potato, by combining next-generation sequencing and rapid amplification of cDNA ends. Comparisons of genome sequences and organization confirmed the classification of CH as SPCSV. However, the sequences and phylogenetic data revealed substantial genetic divergence between CH and all known SPCSV isolates. The amino acid sequence identity between the putative proteins in SPCSV-CH and the corresponding proteins in other known SPCSV isolates in each case was less than 85.0%. Phylogenetic analysis indicated that SPCSV-CH is separate from the groups of the known SPCSV isolates. Additionally, SPCSV-CH RNA1 lacks a p22 gene. A 10.1-kDa putative protein (p10) encoded by a sequence in the 5'-terminal region of RNA2 in SPCSV-CH is much larger than the corresponding protein in all known SPCSV isolates.