scholarly journals Development of a triple antibody sandwich enzyme-linked immunosorbent assay for cassava mosaic disease detection using a monoclonal antibody to Sri Lankan cassava mosaic virus

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Saengsoon Charoenvilaisiri ◽  
Channarong Seepiban ◽  
Mallika Kumpoosiri ◽  
Sombat Rukpratanporn ◽  
Nuchnard Warin ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Mosaic Disease ◽  
Cassava Mosaic Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Sri Lankan ◽  
Stem Cuttings ◽  
Field Samples ◽  
Cassava Stem ◽  
Cassava Mosaic 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.

scholarly journals First report of Sri Lankan cassava mosaic virus and Cassava Mosaic Disease in Laos

Plant Disease ◽  
2021 ◽  
Author(s):  
Khonesavane Chittarath ◽  
Jenyfer Jimenez ◽  
Pinkham Vongphachanh ◽  
Ana Maria Leiva ◽  
Somkhit Sengsay ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Agricultural Research ◽  
Rolling Circle Amplification ◽  
Mosaic Disease ◽  
Cassava Mosaic Disease ◽  
Bipartite Begomovirus ◽  
Sri Lankan ◽  
Virus Species ◽  
Bipartite Genome ◽  
Cassava Mosaic Virus

Cassava (Manihot esculenta Crantz) has been traditionally grown as a subsistence crop in Laos, but in recent years cassava cultivation in this country has expanded and is becoming a ‘cash crop’ for farmers (Malik et al., 2020). This also means that cassava vegetative seed (stakes) is rapidly multiplied and distributed. One of the most important diseases affecting cassava in the world is the Cassava Mosaic Disease (CMD), caused by several species of begomoviruses and disseminated by infected stakes or vectored by the whitefly Bemisia tabaci (Legg et al., 2014). Sri Lankan cassava mosaic virus (SLCMV), a bipartite begomovirus, is the virus species causing CMD in Southeast Asia (SEA) and is widespread in Cambodia, Vietnam, Thailand and south China (Siriwan et al., 2020). During field surveys on July 12 to 14, 2020, the team in south Laos, surveyed 8 fields along the border with Cambodia, in the southern provinces of Attapeu and Champassack and identified CMD symptoms (Supplementary Figure 1A) in only one of the fields, located at Kong District of the Champassack province (GPS coordinates 13.94325, 105.99102). From these 8 fields, samples were collected from every third plant in an X pattern. Photographs from each sampled plant were taken and uploaded into CIAT’s PestDisPlace platform (https://pestdisplace.org), for CMD symptom confirmation (Supplementary Figure 1B). Leaf samples were sent to the laboratory for PCR using primers SLCMV-F 5’-ATGTCGAAGCGACCAGCAGATATAAT-3’ and SLCMV-R 5’-TTAATTGCTGACCGAATCGTAGAAG-3’ targeting the AV1 gene (Dutt et al., 2005), following the protocol described in Siriwan et al. (2020) and primers SLCMV-B-F1 5’-ACCGGATGGCCGCGCCCCCCTCT-3’ and SLCMV-B-606R 5’-CACCTACCCTGTTATCGCTAAG-3’ targeting part of the BV1 gene. Out of 60 samples collected for the field in Kong district, eleven (18.3%) resulted PCR positive to SLCMV (to DNA-A and DNA-B) but only four plants (6.7%) showed symptoms of CMD (see Supplementary Figure 1B and 1C). None of the samples in the other seven fields had CMD symptoms nor was SLCMV detected in any of these plants. Furthermore, the presence of CMD symptoms in the old leaves of the plants in the affected field suggests that the virus was introduced with contaminated stakes. The complete bipartite genome of one isolate (Champ1), was amplified by Rolling Circle Amplification and sequenced with the nanopore MinION technology as described by Leiva et al. (2020). The sequences were submitted to GenBank under accession nos MT946533 (DNA-A) and MT946534 (DNA-B). A phylogenetic tree for SLCMV and a link to the open SLCMV Nextstrain map (Hadfield et al., 2018) is included in Supplementary Figure 2. The sequences of the DNA-A and DNA-B components of the Champ1 isolate were nearly identical to those of anisolate of SLCMV from Ratanakiri, Cambodia (99.72% for DNA-A and 99.82 for DNA-B; Wang et al., 2016). Phylogenetic analysis (Supplementary Figure 2), grouped isolate Champ1 with those that form the cluster of SEA isolates that contain the shorter version of the rep gene (Siriwan et al., 2020). This short version of rep present a deletion of 7 amino acids at the C-terminus, which is involved in host responses to SLCMV (Wang et al., 2020). The confirmation of CMD and SLCMV in the border between Laos and Cambodia should be followed by disease containment and management strategies, particularly given that the majority cassava varieties grown in Laos are from neighbor countries, most of which have already reported the presence of CMD. Acknowledgements We thank all staff from the CIAT’s Cassava Program and the Plant Protection Center of Laos in Vientiane. We acknowledge financial support from the Australian Centre for International Agricultural Research (ACIAR) and the CGIAR Research Program on Roots, Tubers and Bananas (RTB) (https://www.cgiar.org/funders/).

Evidence of synergism between African cassava mosaic virus and a new double-recombinant geminivirus infecting cassava in Cameroon

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 287-297 ◽  
Author(s):  
V. N. Fondong ◽  
J. S. Pita ◽  
M. E. C. Rey ◽  
A. de Kochko ◽  
R. N. Beachy ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Mosaic Disease ◽  
Bipartite Begomoviruses ◽  
African Cassava Mosaic Virus ◽  
Cassava Mosaic Disease ◽  
Stem Cuttings ◽  
East African ◽  
Infectious Clones ◽  
Viral Dnas ◽  
Cassava Mosaic Virus

Stem cuttings were collected in Cameroon from cassava plants displaying cassava mosaic disease (CMD) symptoms. The nature of the viruses present was determined by using the PCR with primers specific for the coat protein (CP) genes of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV). All samples were infected by ACMV and eight of the 50 samples were infected by both ACMV and an EACMV-like virus. The complete nucleotide sequences of DNA-A and -B of representative ACMV and EACMV-like viruses were determined. The DNA-A component of the EACMV-like virus contained evidence of recombination in the AC2–AC3 region and DNA-B also contained evidence of recombination in BC1. However, both components retained gene arrangements typical of bipartite begomoviruses. When Nicotiana benthamiana plants were doubly inoculated with these Cameroon isolates of ACMV and EACMV (ACMV/CM, EACMV/CM) by using sap from cassava plants or infectious clones, the symptoms were more severe than for plants inoculated with either virus alone. Southern blot analysis of viral DNAs from infected plants showed that there were significantly higher levels of accumulation of both ACMV/CM components and, to a lesser extent, of EACMV/CM components in mixed-infected plants than in singly infected plants. These results strongly suggest the occurrence of a synergistic interaction between the two viruses.

scholarly journals Survey and Molecular Detection of Sri Lankan Cassava Mosaic Virus in Thailand

2021 ◽  
Author(s):  
Wanwisa Siriwan ◽  
Kingkan Saokham ◽  
Nuannapa Hemniam ◽  
Sukanya Roekwan ◽  
Sirikan Hunsawattanakul ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Disease Severity ◽  
Bemisia Tabaci ◽  
Rolling Circle Amplification ◽  
Mosaic Disease ◽  
Cassava Mosaic Disease ◽  
Pcr Analysis ◽  
Nucleotide Sequencing ◽  
Sri Lankan ◽  
Cassava Mosaic Virus

Cassava plantations in an area of 458 ha 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 severity was generally scored as 2–3. The highest average disease severity was in Sakaeo province (3.7), followed by Buriram (3.6), Prachinburi (2.88), Surin (2.5), and Sisaket (2.4) provinces. Asymptomatic plants were identified in Surin (12%), Prachinburi (5%), Sakaeo (0.2%), and Buriram (0.1%) by PCR analysis. Interestingly, cassava cultivars CMR-89 and Rayong 11 were susceptible to CMD. In approximately 95% of cases, the infection was transmitted by whitefly ( Bemisia tabaci ), which had a high population density in Prachinburi but was sparse in Surin, with the largest populations observed in May and June. Nucleotide sequencing of the mitochondrial cytochrome oxidase 1 ( mtCO1 ) gene of whitefly ( Bemisia tabaci ) in Thailand revealed a similarity to the Asia II 1 whitefly gene. Furthermore, the AV1 gene—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.

scholarly journals Survey and molecular detection of Sri Lankan cassava mosaic virus in Thailand

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0252846
Author(s):  
Kingkan Saokham ◽  
Nuannapa Hemniam ◽  
Sukanya Roekwan ◽  
Sirikan Hunsawattanakul ◽  
Jutathip Thawinampan ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Disease Severity ◽  
Disease Incidence ◽  
Rolling Circle Amplification ◽  
Mosaic Disease ◽  
Cassava Mosaic Disease ◽  
Pcr Analysis ◽  
Nucleotide Sequencing ◽  
Sri Lankan ◽  
Cassava Mosaic 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.

scholarly journals Viruses Infecting Cassava in Kenya

Plant Disease ◽  
2004 ◽  
Vol 88 (1) ◽  
pp. 17-22 ◽  
Author(s):  
H. K. Were ◽  
S. Winter ◽  
E. Maiss
Keyword(s):  
Mosaic Virus ◽  
Viral Disease ◽  
African Cassava Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Stem Cuttings ◽  
East African ◽  
Western Kenya ◽  
Disease Symptoms ◽  
Future Work ◽  
Cassava Mosaic Virus

A survey of cassava viruses was conducted in major cassava-growing regions of Kenya. A total of 185 leaf samples and 62 stem cuttings from plants with viral disease symptoms were collected and analyzed by biological, electron microscopy, enzyme-linked immunosorbent assay, and polymerase chain reaction. All samples from western Kenya had cassava begomoviruses (African cassava mosaic virus [ACMV], East African cassava mosaic virus [EACMV], and Uganda variant [EACMV-UG]) in either single or in mixed infection. However, all samples from the Coast region were infected with only EACMV, a begomovirus. In addition, 15 samples had mixed infections of EACMV and three other hitherto unidentified filamentous viruses. The viruses observed were 200, 500, 650, and 750 nm long, respectively. In addition to rod-shaped and some flexuous viruses, as seen in a crude sap preparation, pinwheels also were observed, indicating a possible association of some of the viruses with the Potyviridae family. The symptoms induced by these viruses in Nicotiana benthamiana were very severe and often caused about 50% death of the test plants. Back inoculation onto cassava resulted in 100% infections. This finding provides evidence that, other than begomoviruses that cause serious diseases of cassava in Africa, filamentous viruses also are present and, despite their limited distribution, they could reach local significance and, most probably, be as serious as begomoviruses. The implications of these findings are discussed and recommendations for future work suggested.

An enzyme-linked immunosorbent assay for the detection of apple mosaic virus in yellow birch

1980 ◽  
Vol 10 (3) ◽  
pp. 278-283 ◽  
Author(s):  
T. Hardcastle ◽  
A. R. Gotlieb
Keyword(s):  
Mosaic Virus ◽  
Immunosorbent Assay ◽  
Leaf Tissue ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Yellow Birch ◽  
Field Samples

An enzyme-linked immunosorbent assay (ELISA) method was developed to detect apple mosaic virus (ApMV) in Betulaalleghaniensis Britton. Tests for virus using bark and bud tissues of dormant trees were successful. ApMV was also detectable in old leaf tissue in August and September, as well as in newly emerging leaf tissue forced in a greenhouse in March. Whole crude antiserum used to coat ELISA plates in tests with bud tissues was a reliable substitute for purified immunoglobulin without loss of sensitivity or specificity. An attempt was made to use ELISA for quantifying virus concentrations in field samples of ApMV-infected birch leaves.

scholarly journals Variants of East African cassava mosaic virus and Its Distribution in Double Infections with African cassava mosaic virus in Nigeria

Plant Disease ◽  
2003 ◽  
Vol 87 (3) ◽  
pp. 229-232 ◽  
Author(s):  
F. O. Ogbe ◽  
G. Thottappilly ◽  
A. G. O. Dixon ◽  
G. I. Atiri ◽  
H. D. Mignouna
Keyword(s):  
Mosaic Virus ◽  
Mosaic Disease ◽  
African Cassava Mosaic Virus ◽  
Cassava Mosaic Disease ◽  
Single Infection ◽  
East African ◽  
Humid Forest ◽  
Forest Region ◽  
Agroecological Zones ◽  
Cassava Mosaic Virus

In a survey for cassava mosaic begomoviruses conducted in 1997 and 1998 in Nigeria, East African cassava mosaic virus (EACMV) was detected by the polymerase chain reaction together with African cassava mosaic virus (ACMV) in 27 out of 290 cassava leaf samples of infected plants from 254 farmers' fields in five agroecological zones. One plant was infected with EACMV only. Five variant isolates of EACMV were observed based on their reactions to primers that could detect Cameroonian and East African strains of EACMV. Isolates of variants 1 and 3 occurred mostly in the derived or coastal and southern Guinea savannahs, while variants 4 and 5 predominated in the humid forest region. Isolates of variant 2 were widely distributed across the three agroecologies. EACMV was not detected in the northern Guinea savannah and arid and semiarid zones. Most doubly infected plants showed more severe symptoms than plants with single infection. Occurrence of EACMV variants together with ACMV detection and information about their distribution in Nigeria could be used for the selection of cassava clones in cassava mosaic disease resistance programs.

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