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Abstract Background Cassava mosaic disease (CMD) of cassava (Manihot esculenta Crantz) has expanded across many continents. Sri Lankan cassava mosaic virus (SLCMV; family Geminiviridae), which is the predominant cause of CMD in Thailand, has caused agricultural and economic damage in many Southeast Asia countries such as Vietnam, Loas, and Cambodia. The recent SLCMV epidemic in Thailand was commonly found in cassava plantations. Current understanding of plant–virus interactions for SLCMV and cassava is limited. Accordingly, this study explored the metabolic profiles of SLCMV-infected and healthy groups of tolerant (TME3 and KU50) and susceptible (R11) cultivars of cassava. Findings from the study may help to improve cassava breeding, particularly when combined with future transcriptomic and proteomic research. Results SLCMV-infected and healthy leaves were subjected to metabolite extraction followed by ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS/MS). The resulting data were analyzed using Compound Discoverer software, the mzCloud, mzVault, and ChemSpider databases, and published literature. Of the 85 differential compounds (SLCMV-infected vs healthy groups), 54 were differential compounds in all three cultivars. These compounds were analyzed using principal component analysis (PCA), hierarchical clustering dendrogram analysis, heatmap analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. Chlorogenic acid, DL-carnitine, neochlorogenic acid, (E)-aconitic acid, and ascorbyl glucoside were differentially expressed only in TME3 and KU50, with chlorogenic acid, (E)-aconitic acid, and neochlorogenic acid being downregulated in both SLCMV-infected TME3 and KU50, DL-carnitine being upregulated in both SLCMV-infected TME3 and KU50, and ascorbyl glucoside being downregulated in SLCMV-infected TME3 but upregulated in SLCMV-infected KU50. Furthermore, 7-hydroxycoumarine was differentially expressed only in TME3 and R11, while quercitrin, guanine, N-acetylornithine, uridine, vorinostat, sucrose, and lotaustralin were differentially expressed only in KU50 and R11. Conclusions Metabolic profiling of three cassava landrace cultivars (TME3, KU50, and R11) was performed after SLCMV infection and the profiles were compared with those of healthy samples. Certain differential compounds (SLCMV-infected vs healthy groups) in different cultivars of cassava may be involved in plant–virus interactions and could underlie the tolerance and susceptible responses in this important crop.

Sri Lankan cassava mosaic virus (SLCMV) poses a significant threat to global food security by affecting the production of cassava (Manihot esculenta) in South‐east Asia. It causes mosaic patterns on the leaves, disrupts photosynthesis and respiration, and alters carbohydrate accumulation in the tubers. This study aimed to identify proteins associated with cassava mosaic disease symptoms in three cassava cultivars: resistant (TMEB419), tolerant (Kasetsart 50, KU50) and susceptible (Rayong 11, R11) to SLCMV. These cultivars were inoculated with SLCMV using top‐cleft grafting and monitored at 21, 32 and 67 days post‐inoculation (dpi). The disease severity differed among the cultivars following SLCMV inoculation. Liquid chromatography–tandem mass spectrometry (LC–MS/MS)‐based proteomics analysis identified 541 differentially expressed proteins (DEPs), including photosynthesis‐ and respiration‐associated proteins such as RuBisCO large subunit‐binding protein α (LSBP), chlorophyll a‐b binding protein, photosystem I reaction centre subunit III (PSI‐F), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) and fructose‐bisphosphate aldolase (FBA). A protein–protein interaction network revealed interactions among PSI, PSII, cytochrome complex proteins and electron transporter proteins. Chlorophyll(ide) b reductase NYC1, upregulated in R11 (susceptible) at 21 and 32 dpi, interacted with chlorophyllase‐related proteins, suggesting that it has a role in altering chlorophyll metabolism. Reverse transcription‐quantitative PCR gene expression analysis confirmed upregulation of genes related to photosynthesis and respiration in KU50 and R11, predominantly at 21 or 32 dpi. These findings provide valuable insights into cassava responses to SLCMV infection, especially at the protein level and are key for developing resistant cultivars and effective disease management strategies in future research. [ABSTRACT FROM AUTHOR]

Abstract Background Sri Lankan cassava mosaic virus (SLCMV) is a plant virus causing significant economic losses throughout Southeast Asia. While proteomics has the potential to identify molecular markers that could assist the breeding of virus resistant cultivars, the effects of SLCMV infection in cassava have not been previously explored in detail. Results Liquid Chromatography-Tandem Mass Spectrometry (LC/MS–MS) was used to identify differentially expressed proteins in SLCMV infected leaves, and qPCR was used to confirm changes at mRNA levels. LC/MS–MS identified 1,813 proteins, including 479 and 408 proteins that were upregulated in SLCMV-infected and healthy cassava plants respectively, while 109 proteins were detected in both samples. Most of the identified proteins were involved in biosynthetic processes (29.8%), cellular processes (20.9%), and metabolism (18.4%). Transport proteins, stress response molecules, and proteins involved in signal transduction, plant defense responses, photosynthesis, and cellular respiration, although present, only represented a relatively small subset of the detected differences. RT-qPCR confirmed the upregulation of WRKY 77 (A0A140H8T1), WRKY 83 (A0A140H8T7), NAC 6 (A0A0M4G3M4), NAC 35 (A0A0M5JAB4), NAC 22 (A0A0M5J8Q6), NAC 54 (A0A0M4FSG8), NAC 70 (A0A0M4FEU9), MYB (A0A2C9VER9 and A0A2C9VME6), bHLH (A0A2C9UNL9 and A0A2C9WBZ1) transcription factors. Additional upregulated transcripts included receptors, such as receptor-like serine/threonine-protein kinase (RSTK) (A0A2C9UPE4), Toll/interleukin-1 receptor (TIR) (A0A2C9V5Q3), leucine rich repeat N-terminal domain (LRRNT_2) (A0A2C9VHG8), and cupin (A0A199UBY6). These molecules participate in innate immunity, plant defense mechanisms, and responses to biotic stress and to phytohormones. Conclusions We detected 1,813 differentially expressed proteins infected cassava plants, of which 479 were selectively upregulated. These could be classified into three main biological functional groups, with roles in gene regulation, plant defense mechanisms, and stress responses. These results will help identify key proteins affected by SLCMV infection in cassava plants.

【Objective】Sri Lankan cassava mosaic virus (SLCMV) is a circular single-stranded DNA virus, containing two components of DNA-A and DNA-B. SLCMV is a member of the genus Begomovirus in the family Geminiviridae, which is a persistent threat to the world's cassava industry. In 2018, the SLCMV was found and reported for the first time in cassava from Fujian and Hainan provinces, China. However, the viral resistance of cassava varieties in domestic was unknown and the germplasm resources for disease resistance was lacking. To further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the mechanism of interaction between virus and host, SLCMV infectious clone was constructed and verified.【Method】In this study, SLCMV TVM3 strain was used as the object, and its 1.10 mer DNA-A and 1.08 mer DNA-B components were respectively constructed into pCAMBIA1301 vector, and the viral infectious clone vectors pCAMBIA1301-DNA-A (pDNA-A) and pCAMBIA1301-DNA-B (pDNA-B) were obtained. Agrobacterium tumefacien GV3101 mediated pDNA-A and pDNA-B co-infected tobacco and Arabidopsis plants.【Result】At 14 d post inoculation, the systematic leaves of tobacco plants produced severe mosaic and distortion symptoms, while the systematic leaves of Arabidopsis plants showed mild distortion at 18 d post inoculation. Total DNA was extracted from SLCMV infected tobacco and Arabidopsis plants leaves, and viral DNA-A and DNA-B components were detected by PCR method. In addition, the virus infection rate of tobacco plants was 100%, while the virus infection rate of Arabidopsis plants was only 40%, and the difference between the two group was significant (P < 0.05).【Conclusion】The SLCMV infectious clone have been successfully constructed, which could infect tobacco and Arabidopsis plants, and the viral infection rate reached 96.67% in tobacco plants. Furthmore, the DNA sequences of infectious clone in pDNA-A and pDNA-B are 1.10 mer and 1.08 mer length of the original virus genomic DNA-A and DNA-B components, respectively, whichdid not contain repetitive coding region sequences. This study provides an important basis for the further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the pathogenesis of virus.

Abstract Background Cassava mosaic disease (CMD) is one of the most devastating viral diseases for cassava production in Africa and Asia. Accurate yet affordable diagnostics are one of the fundamental tools supporting successful CMD management, especially in developing countries. This study aimed to develop an antibody-based immunoassay for the detection of Sri Lankan cassava mosaic virus (SLCMV), the only cassava mosaic begomovirus currently causing CMD outbreaks in Southeast Asia (SEA). Methods Monoclonal antibodies (MAbs) against the recombinant coat protein of SLCMV were generated using hybridoma technology. MAbs were characterized and used to develop a triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for SLCMV detection in cassava leaves and stems. Assay specificity, sensitivity and efficiency for SLCMV detection was investigated and compared to those of a commercial ELISA test kit and PCR, the gold standard. Results A TAS-ELISA for SLCMV detection was successfully developed using the newly established MAb 29B3 and an in-house polyclonal antibody (PAb) against begomoviruses, PAb PK. The assay was able to detect SLCMV in leaves, green bark from cassava stem tips, and young leaf sprouts from stem cuttings of SLCMV-infected cassava plants without cross-reactivity to those derived from healthy cassava controls. Sensitivity comparison using serial dilutions of SLCMV-infected cassava sap extracts revealed that the assay was 256-fold more sensitive than a commercial TAS-ELISA kit and 64-fold less sensitive than PCR using previously published SLCMV-specific primers. In terms of DNA content, our assay demonstrated a limit of detection of 2.21 to 4.08 × 106 virus copies as determined by quantitative real-time PCR (qPCR). When applied to field samples (n = 490), the TAS-ELISA showed high accuracy (99.6%), specificity (100%), and sensitivity (98.2%) relative to the results obtained by the reference PCR. SLCMV infecting chaya (Cnidoscolus aconitifolius) and coral plant (Jatropha multifida) was also reported for the first time in SEA. Conclusions Our findings suggest that the TAS-ELISA for SLCMV detection developed in this study can serve as an attractive tool for efficient, inexpensive and high-throughput detection of SLCMV and can be applied to CMD screening of cassava stem cuttings, large-scale surveillance, and screening for resistance.

Investigation of the Sri Lankan Cassava Mosaic Virus (SLCMV) resistant cassava (Manihot esculenta Crantz 1766) varieties requires high-efficiency single-strand viral DNA (ssDNA) extraction and molecular detection procedures. In our study, we tested four different DNA extraction methods to isolate total DNA containing high concentration of ssDNA. In addition, using Polymerase chain reaction (PCR) and Real-time PCR (RT-PCR), we can detect SLCMV from infected samples by amplifying a region of the Rep/AC1 viral gene anda cassava’s housekeeping gene, Beta-Actin. As our results, a modified DNA extraction method, from Mason, Caciagli, Accotto and Noris (2008) and Osena, Nyaboga and Amugune (2017), namely R-kit, gained high ssDNA content from young lamina tissue (from lobes) leaf tissue. Therefore, R-kit is recommended to be used for further ssDNA-using studies. The infection of SLCMV in cassava samples can be detected by PCR and RT-PCR procedures. Moreover, in RT-PCR optimization processes we figured out that PCR efficiency of SqPCR1 primer set (1.851X) and Beta-Actin primer set (1.896X) is compatiple for being processed and analyzed at once. The RT-PCR procedure showed ability to detect lower level of SLCMV in samples than the PCR procedure. Our results provide some essential ideas for studying on low virus titer circumstances, especially for model plants studies, resistant varieties development, or clean germplasm maintenance.

【Objective】Sri Lankan cassava mosaic virus (SLCMV) is a circular single-stranded DNA virus, containing two components of DNA-A and DNA-B. SLCMV is a member of the genus Begomovirus in the family Geminiviridae, which is a persistent threat to the world’s cassava industry. In 2018, the SLCMV was found and reported for the first time in cassava from Fujian and Hainan provinces, China. However, the viral resistance of cassava varieties in domestic was unknown and the germplasm resources for disease resistance was lacking. To further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the mechanism of interaction between virus and host, SLCMV infectious clone was constructed and verified.【Method】 In this study, SLCMV TVM3 strain was used as the object, and its 1.10 mer DNA-A and 1.08 mer DNA-B components were respectively constructed into pCAMBIA1301 vector, and the viral infectious clone vectors pCAMBIA1301-DNA-A (pDNA-A) and pCAMBIA1301-DNA-B (pDNA-B) were obtained. Agrobacterium tumefacien GV3101 mediated pDNA-A and pDNA-B co-infected tobacco and Arabidopsis plants. 【Result】 At 14 d post inoculation, the systematic leaves of tobacco plants produced severe mosaic and distortion symptoms, while the systematic leaves of Arabidopsis plants showed mild distortion at 18 d post inoculation. Total DNA was extracted from SLCMV infected tobacco and Arabidopsis plants leaves, and viral DNA-A and DNA-B components were detected by PCR method. In addition, the virus infection rate of tobacco plants was 100%, while the virus infection rate of Arabidopsis plants was only 40%, and the difference between the two group was significant (P < 0.05).【Conclusion】The SLCMV infectious clone have been successfully constructed, which could infect tobacco and Arabidopsis plants, and the viral infection rate reached 96.67% in tobacco plants. Furthmore, the DNA sequences of infectious clone in pDNA-A and pDNA-B are 1.10 mer and 1.08 mer length of the original virus genomic DNA-A and DNA-B components, respectively, whichdid not contain repetitive coding region sequences. This study provides an important basis for the further explore the genetic background of domestic cassava germplasm resources resistance to mosaic disease and the pathogenesis of virus. [ABSTRACT FROM AUTHOR]

Abstract Cassava (Manihot esculenta Crantz) is a major staple food crop for more than a billion people in the world. Cassava mosaic virus (CMV), belonging to the Geminiviridae family, is a primary threat to cassava production. Sri Lankan cassava mosaic virus (SLCMV) is the only emergent CMV prevalent in South Asia and Southeast Asia since its identification in 2002. We reported the identification of two invasive strains of SLCMV, Col and HN7, in China in 2018. However, the occurrence and distribution of these known SLCMV strains and the presence of unknown geminivirus in China are still elusive. In this study, we firstly reported an improved CMV detection system based on molecular and serological methods, which was further used to determine the distribution of CMV in major cassava plantations in China. Two optimized PCR primer pairs based on the conserved regions of AV1 and AC1 genes were designed to detect different CMV species and distinguish SLCMV simultaneously. For a serological method, a polyclonal antibody against SLCMV AV1-encoded capsid protein was raised and used for enzyme-linked immunosorbent assay (ELISA). Consistent detection results were achieved by PCR- and ELISA-based methods. Among 62 examined samples collected in 2018, 10 were SLCMV positive, with 4 coinfection cases of two strains (HN7 and Col) in the same cassava plant. Two primer pairs could also be used to detect the presence of CMV in whitefly (Bemisia tabaci) sensitively. All positive samples were from Fujian and Hainan Provinces, indicating a limited distribution of SLCMV in cassava plants in China. Our detection methods could be used for future surveillance system to control and manage cassava mosaic disease in China and other countries.

Cassava mosaic disease (CMD) is a major disease affecting cassava production in Southeast Asia. This study aimed to perform an integrated proteomics and metabolomics analysis of cassava cv. Kasetsart 50 infected with Sri Lankan cassava mosaic virus (SLCMV). Proteomics analyses revealed that 359 proteins were enriched in the plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways. A total of 79 compounds were identified by metabolomics analysis of the healthy and SLCMV-infected cassava plants. Integrated omics analysis revealed that 9 proteins and 5 metabolites were enriched in 11 KEGG pathways. The metabolic pathways, plant hormone signal transduction, and plant–pathogen interaction pathway terms were specifically investigated. The findings revealed that caffeic acid and chlorogenic acid were associated with the plant–pathogen interaction pathway, histidine (HK3) was involved in plant hormone signal transduction, while citric acid and D-serine were associated with the metabolic pathways. KEGG functional enrichment analysis revealed that plant–pathogen interaction, plant hormone signal transduction, and metabolic pathways were linked via the enriched protein (protein phosphatase 2C) and metabolites (cyclic nucleotide-binding (AT2G20050) and D-serine). The available information and resources for proteomics and metabolomics analyses of cassava can elucidate the mechanism of disease resistance and aid in cassava crop improvement programs.

Sri Lankan cassava mosaic virus (SLCMV) is the principal causal agent of cassava mosaic disease in the Indian subcontinent. To gain resistance against the virus, the coat protein (CP) gene, namely the AV1 of SLCMV-Adivaram isolate, was cloned in either sense or antisense orientation under the Cauliflower mosaic virus 35S promoter, and transgenic Nicotiana benthamiana plants were obtained through Agrobacterium-mediated transformation. A total of eight T1 transgenic lines, four harboring the CP-sense construct and four harboring the CP-antisense construct were challenged with agro-infectious clones of SLCMV DNA-A and DNA-B. Based on symptom exhibition at 20 days post inoculation, 3 out of the 4 CP-sense transgenic lines and all 4 CP-antisense transgenic lines showed a high level of resistance against SLCMV. In addition, a delay in symptom initiation was observed in all the transgenic lines inoculated with a high viral load at agro-dilution 1:625 from an absorbance (A600) of 1. However, the resistance was more prominent at a lower viral load of 1:1000 agro-dilution. The viral titer was lower in the SLCMV-challenged transgenic lines compared to the non-transgenic N. benthamiana plants as confirmed by quantitative PCR and dot blot analysis. Furthermore, small RNA Northern blot analysis revealed lowered amounts of virus-specific small interfering RNAs in the resistant transgenic lines as compared to the non-transgenic plants upon SLCMV infection, which correlates to lower virus titers due to resistance against the virus.

Cassava plantations in an area of 458 hectares spanning five provinces along the Thailand-Cambodia border were surveyed from October 2018 to July 2019 to determine the prevalence of cassava mosaic disease (CMD) caused by Sri Lankan cassava mosaic virus (SLCMV) in the region. CMD prevalence was 40% in the whole area and 80% in Prachinburi, 43% in Sakaeo, 37% in Burium, 25% in Surin, and 19% in Sisaket provinces. Disease incidence of CMD was highest 43.08% in Sakaeo, followed by 26.78% in Prachinburi, 7% in Burium, 2.58% in Surin, and 1.25% in Sisaket provinces. Disease severity of CMD symptoms was mild chlorosis to moderate mosaic (2-3). The greatest disease severity was recorded in Prachinburi and Sakaeo provinces. Asymptomatic plants were identified in Surin (12%), Prachinburi (5%), Sakaeo (0.2%), and Buriram (0.1%) by PCR analysis. Cassava cultivars CMR-89 and Huai Bong 80 were susceptible to CMD. In 95% of cases, the infection was transmitted by whiteflies (Bemisia tabaci), which were abundant in Sakaeo, Buriram, and Prachinburi but were sparse in Surin; their densities were highest in May and June 2019. Nucleotide sequencing of the mitochondrial cytochrome oxidase 1 (mtCO1) gene of whiteflies in Thailand revealed that it was similar to the mtCO1 gene of Asia II 1 whitefly. Furthermore, the AV1 gene of SLCMV-which encodes the capsid protein-showed 90% nucleotide identity with SLCMV. Phylogenetic analysis of completed nucleotide sequences of DNA-A and DNA-B components of the SLCMV genome determined by rolling circle amplification (RCA) indicated that they were similar to the nucleotide sequence of SLCMV isolates from Thailand, Vietnam, and Cambodia. These results provide important insights into the distribution, impact, and spread of CMD and SLCMV in Thailand.

Cassava (Manihot esculenta Crantz) is a rainfed, smallholder-produced crop in mainland Southeast Asia, and is currently facing a serious challenge posed by the introduction of cassava mosaic disease (CMD). This study assessed the susceptibility of popular Asian varieties to CMD, yield penalties associated with the disease, and the efficacy of selecting clean or asymptomatic plants as seed for the following season. Field experiments evaluated agronomic management practices (i.e., fertilizer application, use of symptomatic and asymptomatic seed stakes) in Cambodia with six to nine popular varieties over three seasons under natural disease pressure. Popular cassava varieties KU50 and Huaybong60 showed superior CMD tolerance, with consistently fewer symptomatic plants, lower disease progress measures, and higher yields. Plants demonstrating symptoms at early stages of development, i.e., 60 days after planting, yielded significantly less than those developing symptoms later (i.e., 270 DAP) or not at all. Plants grown from clean stems yielded on average 20% to 2.7-fold higher than those grown from symptomatic planting material. A yield decline of ~50% was recorded with symptomatic planting materials of susceptible varieties (e.g., SC8, ~25 t ha−1) over successive years. The findings emphasize that farmers could use positive selection by choosing asymptomatic plants to significantly reduce yield losses.

Cassava mosaic disease (CMD) is a major disease affecting cassava production in Southeast Asia. This study aimed to perform an integrated proteomics and metabolomics analysis of cassava cv. Kasetsart 50 infected with Sri Lankan cassava mosaic virus (SLCMV). Proteomics analyses revealed that 359 proteins were enriched in the plant–pathogen interaction, plant hormone signal transduction, and MAPK signaling pathways. A total of 79 compounds were identified by metabolomics analysis of the healthy and SLCMV-infected cassava plants. Integrated omics analysis revealed that 9 proteins and 5 metabolites were enriched in 11 KEGG pathways. The metabolic pathways, plant hormone signal transduction, and plant–pathogen interaction pathway terms were specifically investigated. The findings revealed that caffeic acid and chlorogenic acid were associated with the plant–pathogen interaction pathway, histidine (HK3) was involved in plant hormone signal transduction, while citric acid and D-serine were associated with the metabolic pathways. KEGG functional enrichment analysis revealed that plant–pathogen interaction, plant hormone signal transduction, and metabolic pathways were linked via the enriched protein (protein phosphatase 2C) and metabolites (cyclic nucleotide-binding (AT2G20050) and D-serine). The available information and resources for proteomics and metabolomics analyses of cassava can elucidate the mechanism of disease resistance and aid in cassava crop improvement programs. [ABSTRACT FROM AUTHOR]

Cassava mosaic disease, one of the ten most economically important crop viral diseases in the world, was first reported in Southeast Asia from a single plantation in Cambodia in 2015. To determine the presence and incidence of Sri Lankan cassava mosaic virus (SLCMV) one year after first detection, a total of 6,480 samples from 419 fields were systematically collected from cassava production areas across Cambodia (3,840 samples; 240 fields) and Vietnam (2,640samples; 179 fields) in the 2016 cropping season. Using PCR-based diagnostics, we identified 49 SLCMV-infected plants from nine fields, representing 2% of the total number of fields sampled. Infected fields were geographically restricted to two provinces of Eastern Cambodia, while no infection was detected from any of the other sampled sites in either country. Symptom expression patterns in infected plants suggested that SLCMV may have been transmitted both through infected planting materials, and by Bemisia tabaci, the known whitefly vector of SLCMV. In addition, 14% of virus infected plants did not express typical symptoms of cassava mosaic disease on their leaves, highlighting that molecular-based validation is needed to confirm the presence of SLCMV in the field. None of the owners of the SLCMV-infected fields indicated acquired planting materials from the plantation in Ratanakiri where SLCMV was first reported. The surveillance baseline data generated for both countries is discussed in light of future options to control and manage cassava mosaic disease.

Cassava mosaic disease (CMD), caused by Sri Lankan cassava mosaic virus (SLCMV), was first detected in Tay Ninh province in the southeast region of Vietnam in 2017 and is an emerging threat for cassava production in Vietnam. This study was conducted to characterize the causal agent of CMD in Nghe An province in the north-central region of Vietnam, which is more than 1,200 km away from Tay Ninh province. Genetic relationships between SLCMV isolates indicated that both DNA-A and DNA-B sequences from Nghe An province were identical with the isolates from Tay Ninh province and were highly homologous (>99.7%) with isolates from Cambodia, Thailand and China. This study confirms the gradual spread of SLCMV in Vietnam as an emerging threat to cassava production. In addition, the outcome of this study could help in the timely enforcement of effective management strategies for the cassava mosaic disease in Vietnam. [ABSTRACT FROM AUTHOR]

Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV) and Sri Lankan cassava mosaic virus (SLCMV). The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases.

Cassava plantations in an area of 458 hectares spanning five provinces along the Thailand-Cambodia border were surveyed from October 2018 to July 2019 to determine the prevalence of cassava mosaic disease (CMD) caused by Sri Lankan cassava mosaic virus (SLCMV) in the region. CMD prevalence was 40% in the whole area and 80% in Prachinburi, 43% in Sakaeo, 37% in Burium, 25% in Surin, and 19% in Sisaket provinces. Disease incidence of CMD was highest 43.08% in Sakaeo, followed by 26.78% in Prachinburi, 7% in Burium, 2.58% in Surin, and 1.25% in Sisaket provinces. Disease severity of CMD symptoms was mild chlorosis to moderate mosaic (2-3). The greatest disease severity was recorded in Prachinburi and Sakaeo provinces. Asymptomatic plants were identified in Surin (12%), Prachinburi (5%), Sakaeo (0.2%), and Buriram (0.1%) by PCR analysis. Cassava cultivars CMR-89 and Huai Bong 80 were susceptible to CMD. In 95% of cases, the infection was transmitted by whiteflies (Bemisia tabaci), which were abundant in Sakaeo, Buriram, and Prachinburi but were sparse in Surin; their densities were highest in May and June 2019. Nucleotide sequencing of the mitochondrial cytochrome oxidase 1 (mtCO1) gene of whiteflies in Thailand revealed that it was similar to the mtCO1 gene of Asia II 1 whitefly. Furthermore, the AV1 gene of SLCMV-which encodes the capsid protein-showed 90% nucleotide identity with SLCMV. Phylogenetic analysis of completed nucleotide sequences of DNA-A and DNA-B components of the SLCMV genome determined by rolling circle amplification (RCA) indicated that they were similar to the nucleotide sequence of SLCMV isolates from Thailand, Vietnam, and Cambodia. These results provide important insights into the distribution, impact, and spread of CMD and SLCMV in Thailand., Competing Interests: The authors have declared that no competing interests exist.