Antigen-coated Plate Enzyme-linked Immunosorbent Assay Research Articles

Biological, Serological and Molecular Characterization of Cucumber mosaic virus (CMV) Infecting Cucurbita moschata (Duchene ex. Poir) (Summer Squash) in Itu and Uyo Local Governments Areas, Akwa Ibom State, Nigeria

Cucurbita moschata is an edible member of the family Cucurbitaceae. The plant is commonly observed showing typical virus-like symptoms during the growing season in Akwa Ibom State, Nigeria. A total of 7 leaf samples showing typical virus-like symptoms were collected from C. moschata in different locations in Itu and Uyo Local Government, Akwa Ibom State. All the virus isolates used in this study were sap transmissible with a wide host range. The virus isolates irrespective of the place of collection were transmitted in a fore-gut manner by A. spiraecola. Aphis citricida did not transmit the viruses. All the virus isolates reacted positively to cucumovirus polyclonal antibody in antigen coated plate-ELISA (ACP-ELISA). Gel electrophoresis of the cDNA of the virus isolates produced 500 base pairs typical of Cucumber mosaic virus. The gene sequence analysis revealed that all the isolates had sequence homology of 95% and above with Cucumber mosaic virus (CMV) and are therefore considered strains of CMV. The results also showed that CMV isolates investigated in this study shared common ancestry, though belonging to different clusters. The results also indicated that CMV could be widespread and therefore a threat to C. moschata production in Itu and Uyo Local Government Areas of Akwa Ibom State. This is the first report of CMV infecting C. moschata in Akwa Ibom State, Nigeria. Further work should be carried out to cover the entire state.

Causal agent for disease symptoms on passionflower in Adim, Cross River State, Nigeria

The stinking passionflower (Passiflora foetida L.) is generally regarded as a weed, although its tender fruits are consumed by a few; while the plant is mostly regarded for its medicinal properties. Cucumber mosaic virus is known not just to cause significant losses among crops, but also present in many weeds species. The objective of this study was to identify the causal agent of the disease symptoms observed from P. foetida in Adim, Cross River State, Nigeria. Leaves of P. foetida with virus symptoms were collected from various locations in Adim. Antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and reverse transcription polymerase chain reactions (RT-PCR) were used for virus detection and characterisation. The symptomatic leaves reacted positively with the CMV antisera in ACP-ELISA and sequences from RT-PCR products showed the highest identity (98.5%) with a CMV isolate from Xanthosoma sp. (MG021460) in Uganda. Phylogenetic reconstruction aggregated the isolates with other Nigerian CMV sequences in a separate uncharacterised subgroup, closest to MH798809 (Nigeria), MW655577 (Nigeria) and HQ874434 (Austria). The tree also revealed the isolates as members of subgroup IB. This study provides the first molecular evidence of CMV infection on P. foetida in Nigeria and adds to the list of possible natural host for the virus in the country.

Production of polyclonal antibodies against leek yellow stripe virus (LYSV) coat protein expressed in Escherichia coli and its application in serological diagnostics.

Leek yellow stripe virus (LYSV) is one of the most important potyviruses, associated with garlic throughout the world, including India. LYSV causes stunting and yellow streaks in garlic and leek leaves and with other coinfecting viruses leading to severe symptom expression and yield reduction. In this study, we have made the first reported attempt to produce specific polyclonal antibodies to LYSV using expressed recombinant coat protein (CP), which would be useful for screening and routine indexing of the garlic germplasm. The CP gene was cloned, sequenced, and further subcloned in pET-28a(+) expression vector, which yielded ∼35 kDa fusion protein. The fusion protein was obtained in insoluble fraction after purification and its identity was confirmed by SDS-PAGE and western blotting. The purified protein was used as immunogen for production of polyclonal antisera in New Zealand white rabbit. Antisera raised, was able to recognize the corresponding recombinant proteins in western blotting, immunosorbent electron microscopy and dot immunobinding assay (DIBA). Developed antisera to LYSV (titer 1:2000) was used for screening of 21 garlic accessions in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA) and 16 accessions were found positive for LYSV, indicating its widespread presence within the collection tested. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in-vitro expressed CP of LYSV and its successful application in diagnosis of LYSV in garlic accessions in India.

Inheritance of resistance to three endemic viral diseases of cowpea in Nigeria

ABSTRACT Mosaic diseases, caused by bean common mosaic virus-blackeye cowpea mosaic strain (BCMV-BlCM), southern bean mosaic virus (SBMV), and cucumber mosaic virus (CMV), hamper the productivity of cowpea (Vigna unguiculata (L.) Walp.). Under single or mixed infections, these endemic viruses significantly reduce cowpea yield in sub-Saharan Africa. Planting resistant varieties is the most effective control method. Knowledge of the mode of inheritance of viral resistance is crucial in developing resistant varieties. Inheritance of resistance to BCMV-BlCM, SBMV, and CMV was investigated in two improved cowpea breeding lines. For BCMV-BICM, crosses were made between resistant IT97K-1042-3 (female) and susceptible IT99K-1060 (male); for SBMV, between resistant IT98K-1092-1 (male) and susceptible IT99K-1060 (female); and for CMV, between tolerant IT98K-1092-1 (female) and susceptible IT99K-573-1-1 (male). The F1 progenies were advanced to F2, and some F1 plants were backcrossed to the two parental lines. Reciprocal crosses were made and the 7-day-old seedlings of P1, P2, F1, F2, BCP1, and BCP2 were phenotyped by mechanical inoculation with BCMV-BlCM, SBMV, and CMV under screenhouse conditions. Data on disease incidence and severity were taken at weekly intervals for 5-week post-inoculation. Virus infections were confirmed via antigen-coated plate enzyme-linked immunosorbent assay or reverse transcription-polymerase chain reaction. Chi-square analysis of the genetic segregation indicated that a recessive gene pair in IT97K-1042-3 controlled the inheritance of resistance to BCMV-BlCM. Duplicate dominant genes conditioned the resistance to SBMV and tolerance to CMV in IT98K-1092-1. The backcrosses confirmed the monogenic and digenic inheritance patterns, whereas reciprocal crosses indicated absence of cytoplasmic effects.

Detection of Tomato Spotted Wilt Virus (TSWV) Infection in Plants Using DAS-ELISA and Dot-ELISA.

Plant viruses cause severe damages to crop productions each year worldwide. To prevent the losses caused by plant viruses, it is necessary to develop specific and efficient diagnostic tools to detect viruses. Among the current virus detection techniques, serological detection methods are considered to be rapid, simple, sensitive, and high throughput. Therefore, serological detection methods such as double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), triple antibody sandwich ELISA (TAS-ELISA), antigen coated plate-ELISA (ACP-ELISA), Dot-ELISA and tissue print-ELISA as well as colloidal gold immunochromatographic strip are now wildly used to detect viruses in plants. In this chapter, we describe the DAS-ELISA and Dot-ELISA methods, and their applications in the detection of Tomato spotted wilt virus (TSWV) infection in plants. These two methods can be easily adapted for diagnosis of other plant viruses.

Evidence of expanded diversity in weeds as reservoir host of viruses in pepper fields across southwestern Nigeria

This study aimed to investigate virus occurrence in weed species in the main pepper-growing areas in Southwest Nigeria. The weed samples collected from pepper farms were identified and subjected to Antigen-Coated Plate Enzyme-Linked Immunosorbent Assay using antibodies specific eight different viruses. Results showed that the Weed species collected contain 17 families, 33 genera and 36 plant species of which 83.33% of the plant species tested positive to one or more plant viruses. The results indicate that potato virus Y (PVY) and potato virus X (PVX) infected more weed species (24). Also, Ageratum conyzoides serve as host to 8 viruses while Alchornea cordata, Corchorus olitoris and Talinum triangulare serve as host to 7 viruses respectively. These results provide information on weeds as virus reservoirs and contribute to the knowledge of epidemiology of these diseases, enabling a proper weed management aiming at reducing the secondary spreading control of viruses.

Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus

In recent years, tomato mottle mosaic virus (ToMMV) has become one of the most important viral pathogens affecting solanaceous crop production in Yunnan, Hainan, and Shandong provinces of China, often causing huge yield reductions. To provide farmers and vegetable industry with reliable and easy-to-use ToMMV detection methods, we immunized BALB/c mice with purified ToMMV and obtained six hybridoma cell lines (i.e., 2D6, 9C12, 26A10, 3A4, 23A4 and 17B11) that secrete anti-ToMMV monoclonal antibodies (MAbs) through the hybridoma technology. Using these MAbs as the detection antibody, we developed three serological assays: antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue print enzyme-linked immunosorbent assay (tissue print-ELISA) for ToMMV detection. Our test results showed that these three newly developed serological methods can be used to specifically detect ToMMV infection in plant samples, but not tobacco mosaic virus, tomato mosaic virus, cucumber green mottle mosaic virus and cucumber mosaic virus. Sensitivity analyses further showed that ACP-ELISA and dot-ELISA can be used to detect ToMMV infection in plant crude extracts diluted at 1:81,920 and 1:40,960 (weight/volume, g/mL), respectively. Surprisingly, the detection limit of the developed dot-ELISA was 26 times higher than that of traditional RT-PCR. Using field-collected plant samples, we have demonstrated that these three new serological methods are accurate and easy-to-use for large-scale detection of ToMMV in fields.

Three sensitive and reliable serological assays for detection of potato virus A in potato plants

Vegetative propagation of seed potato often allows passaging of viruses to seed tubers, resulting in significant yield losses and reduction of potato tuber quality. Thus, virus detection approach is crucial for effective virus management programs and the production of virus-free seed potatoes. Among the reported potato-infecting viruses, potato virus A (PVA) is considered as one of the most important viruses in potato-growing regions worldwide. This study prepared four hybridoma lines secreting PVA-specific monoclonal antibodies (MAbs) (2D4, 8E11, 14A6 and 16H10) using purified PVA virions as an immunogen. Western blotting results indicated that all the four MAbs reacted strongly and specifically with the putative capsid protein of PVA. Using these four MAbs, this study developed antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), Dot-ELISA and Tissue print-ELISA for detection of PVA infection in potato plants. The results indicated that PVA can be detected in crude tissue extracts from infected potato plants diluted up to 1:327680 (w/v, g mL–1) by ACP-ELISA or up to 1:10240 by Dot-ELISA. The Tissue print-ELISA is the quickest and easiest approach among the three serological assays, and is more suitable for onsite large-scale potato screening programs. Further analyses of field-collected potato samples showed that the sensitivities and specificities of the three serological approaches were similar to those of RT-PCR in PVA detection and confirmed that PVA is currently widespread in Yunnan and Zhejiang provinces of China. Hence, the results strongly suggest that these highly sensitive serological approaches based on PVA-specific MAbs are useful and powerful for PVA-free seed potato production programs and PVA field surveys.

Mixed infection of a potyvirus and cucumber mosaic virus on Ocimum gratissimumin Okoyong and Boje, Cross River State, Nigeria

Viruses have been known to cause considerable yield losses in <i>Ocimum gratissimum</i> (L.), a leafy vegetable commonly known as African basil but locally referred to as “scent leaf’. The crop is cherished for its essential oil, medicinal and agricultural benefits. Virus-like symptoms on leaves causing mosaic, yellowing, leaf malformation and stunting on <i>O. gratissimum</i> (L.) were observed in several farms during the 2018/19 crop season in two locations of Boje and Okoyong in Cross river state. Serological and molecular methods were used to detect viruses infecting the crop at the two locations. Preliminary studies showed the virus could be transmitted mechanically, affirming it was a viral infection. Symptomatic leaf samples from both locations tested positive in antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) to antibodies of genus-specific <i>Cucumber mosaic virus</i> (CMV) and <i>Potyvirus. </i>Primers specific to the coat protein of CMV and the cylindrical inclusion (CI) protein of potyvirus were used for detection of the viruses in reverse transcriptase-polymerase chain reaction (RT-PCR). Obtained sequences were compared to corresponding sequences in the GenBank using the BLASTn program, and the ocimum isolates showed nucleotide sequence similarities of between 89.20 -97.98 <i>%</i> to ocimum potyvirus (MT396942) from Calabar in both locations and two of the collected samples from Okoyong showed a sequence similarity of 100 % with Ocimum CMV isolate from India MN481937. This is the first evidence of potyvirus infection in both locations and mixed infection of CMV and potyvirus on <i>O. gratissimum</i> in Nigeria.

Assessments of Sweet potato Virus Disease and Influence of Mulching and Organic Insecticide on Sweet Potato Varieties in Kwara State of Nigeria

Assessments of 30 sweet potato farms in three districts of Kwara State of Nigeria were carried out to evaluate the incidence of sweet potato virus disease (SPVD). A serological study by Antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) was set up to assay six viruses on sweet potato samples. A field experiment to appraise the influence of variety, mulch and organic insecticide on virus incidence was performed on two genetically improved sweet potato varieties. The surveys revealed the occurrence of SPVD in all locations with incidence from 2.6% in Oyun (Kwara Central district) to 39.1% in Agbamu (Kwara South district). The serology test confirmed the predominance of Sweet potato feathery mottle virus (SPFMV) and Sweet potato chlorotic stunt virus (SPCSV) correspondingly as 100% and 30% respectively in the study. The field experiment indicated lower virus disease incidence (15.3%) and increased numbers in storage roots (7.5/plant) and weight of storage roots (32.4 kg/plant) in Orange Fleshed Sweet Potato variety, mulched with organic insecticidal treatment. This is the first authentication of the occurrence and prevalence of sweet potato virus disease in Kwara State, Nigeria. The study recommends Orange Fleshed Sweet Potato variety, mulching with guinea corn stalk and organic insecticide application for sustainable management of SPVD, particularly for resource-poor farmers.

Mixed infection of a potyvirus and cucumber mosaic virus on Ocimum gratissimumin Okoyong and Boje, Cross River State, Nigeria

Viruses have been known to cause considerable yield losses in <i>Ocimum gratissimum</i> (L.), a leafy vegetable commonly known as African basil but locally referred to as “scent leaf’. The crop is cherished for its essential oil, medicinal and agricultural benefits. Virus-like symptoms on leaves causing mosaic, yellowing, leaf malformation and stunting on <i>O. gratissimum</i> (L.) were observed in several farms during the 2018/19 crop season in two locations of Boje and Okoyong in Cross river state. Serological and molecular methods were used to detect viruses infecting the crop at the two locations. Preliminary studies showed the virus could be transmitted mechanically, affirming it was a viral infection. Symptomatic leaf samples from both locations tested positive in antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA) to antibodies of genus-specific <i>Cucumber mosaic virus</i> (CMV) and <i>Potyvirus. </i>Primers specific to the coat protein of CMV and the cylindrical inclusion (CI) protein of potyvirus were used for detection of the viruses in reverse transcriptase-polymerase chain reaction (RT-PCR). Obtained sequences were compared to corresponding sequences in the GenBank using the BLASTn program, and the ocimum isolates showed nucleotide sequence similarities of between 89.20 -97.98 <i>%</i> to ocimum potyvirus (MT396942) from Calabar in both locations and two of the collected samples from Okoyong showed a sequence similarity of 100 % with Ocimum CMV isolate from India MN481937. This is the first evidence of potyvirus infection in both locations and mixed infection of CMV and potyvirus on <i>O. gratissimum</i> in Nigeria.

Monoclonal Antibody-Based Serological Detection of Rice Stripe Mosaic Virus Infection in Rice Plants or Leafhoppers.

Rice stripe mosaic virus (RSMV) is a rhabdovirus recently found in southern part of China and can cause severe reduction in rice production. To establish serological methods for RSMV epidemiological studies and to establish a control strategy for this virus, we first purified RSMV virions from infected rice plants and then used them as an immunogen to produce four RSMV-specific monoclonal antibodies (MAbs) (i.e.,1D4, 4A8, 8E4 and 11F11). With these MAbs, we have developed a highly specific and sensitive antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA), a Dot-ELISA and a Tissue print-ELISA for rapid detections of RSMV infection in rice plants or in leafhoppers. Our results showed that RSMV can be readily detected in RSMV-infected rice plant tissue crude extracts diluted at 1:20,971,520 (w/v, g/mL) through ACP-ELISA or diluted at 1:327,680 (w/v, g/mL) through Dot-ELISA. Both ACP-ELISA and Dot-ELISA can also be used to detect RSMV infection in individual RSMV viruliferous leafhopper (Recilia dorsalis) homogenate diluted at 1:307,200 and 1:163,840 (individual leafhopper/µL), respectively. Detection of RSMV infection in field-collected rice samples or in RSMV viruliferous leafhoppers indicated that the three serological methods can produce same results with that produced by RT-PCR (19 of the 33 rice samples and 5 of the 16 leafhoppers were RSMV-positive). We consider that the four MAbs produced in this study are very specific and sensitive, and the three new serological methods are very useful for detections of RSMV infection in rice plants or in leafhoppers and the establishment of the disease control strategies.

English

This study characterized Cucumber mosaic virus (CMV) infecting pepper in Southwestern Nigeria with the aim of providing up-to-date information on the taxonomic subgroup(s) of the parading strains in the study area. Fifty pepper leaf samples with one or more symptoms of mottling, mosaic, necrosis, leaf distortion and stunting were collected from eight commercial farms across Southwestern Nigeria and screened for CMV using antigen coated plate enzyme linked immunosorbent assay (ACP-ELISA). From the seropositive samples, four representative samples were selected, labelled PSWN1-4 before subjected to reverse transcription polymerase chain reaction (RT-PCR) and nucleic acid sequencing using a pair of primers specific for the amplification of RNA 3 genomic fragment of CMV. These were followed by multiple nucleotide sequence alignment and phylogenetic estimations of the isolates alongside selected CMV strains that were reported in previous studies using the molecular evolutionary genetics analysis (MEGA) software. The result of the ACP-ELISA test revealed that 14 (28%) of the samples collected were positive for CMV infection. The resulting nucleotide sequences from RT-PCR revealed 98% nucleotide homologies with several corresponding sequences of CMV strains from the Genbank. Further analysis of the PSWN nucleotide sequences through multiple nucleotide sequence alignment revealed the absence of the EcoR1 restriction site found only within the examined genomic portion of RNA 3 of subgroup II strains. These features defined the detected isolates as subgroup I strains. Phylogenetic analysis and estimations of genetic distances, conclusively distinguished the pepper-infecting CMV isolates from Southwestern Nigeria as members of subgroups IA and IB, which are the most virulent subgroups. Key words: Cucumber mosaic virus (CMV), pepper, Southwestern Nigeria.

Detection and distribution of seed-borne viruses on commercial cowpea (&lt;i&gt;Vigna unguiculata&lt;/i&gt; (L.) Walp) cultivars in Ado-odo Ota, Ogun Sate, Nigeria

Cowpea is a major source of dietary protein in the nutrition of people in sub-Saharan Africa. Many viruses have been reported to cause economic reduction in cowpea productivity in Nigeria. However, their current distribution is not well documented while most farmers source their seeds from local markets. This study investigated the detection and distribution of seed-transmitted viruses on commercial cowpea cultivars in Ado-odo Ota Local Government area (LGA) of Ogun State. Cowpea seed samples were collected from eleven markets in the LGA. Coordinates of the locations were recorded with Geographical Positioning System (GPS). Thirty cowpea seeds from each market were sowed in planting pots under screenhouse conditions and seedlings were observed weekly for visible virus symptoms. Four-week old seedlings were tested for viruses using Antigen Coated Plate-Enzyme-Linked Immunosorbent Assay (ACP-ELISA) and Reverse Transcription Polymerase Chain Reaction (RT-PCR). Samples were tested for seven seed-transmitted viruses namely Blackeye cowpea mosaic virus (BlCMV), Cowpea aphidborne mosaic virus (CABMV), Cucumber mosaic virus (CMV), Southern bean mosaic virus (SBMV), Cowpea mottle virus (CMeV), Cowpea yellow mosaic virus (CYMV) and Cowpea mild mottle virus (CPMMV). Seed germination rates ranged from 63.3±15.3% of seeds from Joju to 96.7 ±5.8% for Atan and Iju markets. All samples tested negative to ELISA and were confirmed negative by RT-PCR. These findings suggest absence of seedtransmitted cowpea viruses in the study area as at the time of sampling. Periodic detection survey is recommended to assure the virus-free status of the area. Keywords: ACP-ELISA, Cowpea viruses, Diagnostics, RT-PCR, Seed transmission

First Report of Telosma Mosaic Virus Infecting Emperor’s Candlesticks (Senna alata) in China

Emperor’s candlesticks (Senna alata L.) is native to Mexico and is widely distributed in the tropics. It is an important medicinal tree, as well as an ornamental flowering plant in the family Caesalpinioideae. In July 2017, virus-like symptoms, including mosaic, mottle, and crumpling on leaves, were observed in emperor’s candlesticks in Xishuangbanna, Yunnan. Small RNAs (sRNAs) from the infected emperor’s candlesticks leaves were extracted and sequenced using an Illumina HiSeq 2000 system (Majorbio, China). All clean reads with lengths from 18 to 30 nucleotide sRNAs were assembled into contigs using Velvet software with k-mer value of 17. After searching against the GenBank database, 18 of 24 contigs were found that shared 84 to 100% nucleotide identities (approximately 38% coverage) with the full-length genome sequences of Telosma mosaic virus (TeMV) (GenBank accession no. DQ851493). The other six contigs showed more than 84% nucleotide identities with TeMV or more than 76% nucleotide identities with other potyviruses genome sequences owing to different program selection choices. For the amino acid sequence alignment, five of these six contigs shared 92% and above amino acid identities with the genome sequences of TeMV using BLASTx. To verify the result, reverse transcription polymerase chain reaction (PCR) was performed using a specific forward primer (TeMV-F, 5′-AGGAACTTGCAGCTGAAGGGAAAG-3′) designed based on the sequence of assembled contigs of virus-derived sRNAs, and the reverse primer M4 described before (Chen et al. 2001). Total RNAs were extracted from infected leaves using the RNeasy Plant Mini Kit (Qiagen, German), and reverse transcription was performed using M-MLV reverse transcription (RNase H-) (TaKaRa, Japan) and primer M4T, as described (Zhang et al. 2016). The 3′-proximal sequence (containing part of the NIb gene, the complete CP gene, and 3′-UTR) was amplified by PCR using Ex Taq polymerase (TaKaRa). Fragments of the expected size (1,300 bp) were observed after electrophoresis in 1.0% (w/v) agarose gels in infected but not in healthy plants. This PCR fragment was purified, cloned, and three independent clones were sequenced using Sanger methodology (GenBank accession no. MH137276). BLASTn analysis of the amplicon sequence revealed nucleotide identity of 89% (Hanoi-DQ851493) and 92% (GX1-KJ789129) with previously characterized TeMV isolates. Nucleotide and amino acid sequence analysis of the putative CP gene revealed 86.0 to 91.4% nucleotide and 85.3 to 94.9% amino acid identity, respectively, to TeMV isolates Hanoi, GX1, Pangda15 (AM409188), and Pangda12 (AM409187). The filamentous virus particles (700 to 800 × 13 nm) and pinwheel inclusions typical of members of the genus Potyvirus were observed in infected emperor’s candlesticks leaves by transmission electron microscopy. Four infected samples tested positive in antigen-coated plate ELISA with potyvirus group-specific antibodies (Agdia, U.S.A.),whereas three healthy plants tested were negative. These results further confirmed the presence of TeMV in infected emperor’s candlesticks leaves from Yunnan; hence, the isolate was named TeMV-YN. TeMV was reported to infect telosma (Telosma cordata), passion fruit (Passiflora edulis), and patchouli (Pogostemon cablin) in Asia (Chiemsombat et al. 2014; Ha et al. 2008; Noveriza et al. 2016). To our knowledge, this is the first report of TeMV infecting emperor’s candlesticks.

Natural occurrence of cucumber mosaic virus in Ogeira (Eleutheranthera ruderalis) in Nigeria

Plant viruses are difficult to control and cause increasing losses of horticultural crops as many weeds species are implicated as reservoir hosts of a number of plant viruses. A viral disease causing yellow mosaic Ogeira (Eleutheranthera ruderalis) was prevalent at the experimental fields of the National Horticultural research Institute (NIHORT), Ibadan Nigeria. To detect the virus, leaf samples were collected across different locations in NIHORT. It was found that cucumber mosaic virus (CMV) was detected in 10 of 15 symptomatic samples by Antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis with CMV-specific primers was positive in all tested samples. Nucleotide sequences of the obtained amplicons showed 95–88% similarity to corresponding sequences of CMV isolates deposited in the GenBank. Phylogenetic analysis was performed using 10 CMV sequences obtained from the Genbank which include the CMV-Ogeira Isolate obtained from this study. The CMV-Ogeira, isolate from this study stood alone in a biphyletic clade.

Impact of single and double infection with Cucumber mosaic virus and Potato virus Y on growth and yield of pepper

ABSTRACTPepper (Capsicum spp. L.) production is constrained by Cucumber mosaic virus (CMV) and Potato virus Y (PVY). Local pepper varieties may confer some degree of resistance against these viruses which needs to be evaluated. A screen house study using potted plants was carried out to determine performance of pepper accessions in response to single, and double, inoculation with CMV and PVY. The accessions were inoculated at 4 weeks after planting and growth and yield components recorded. Virus detection and quantification were with antigen-coated plate enzyme-linked immunosorbent assay, which also provided resistance classification. The accessions were rated based on loss incurred in growth and yield. Pepper accessions receiving no viral treatment generally performed better than viral infected plants. Accessions inoculated with CMV were most severely affected, followed by accessions inoculated with CMV+PVY. Pepper accessions inoculated with PVY had higher values than CMV infested accessions for plant height, number of leaves and number of branches but lower values for yield. Accession NCR/AA/MAY/09/051 is classified as very tolerant while accessions NCR/AA/MAY/09/013, NCR/TB/OCT/09/006, NCR/MR/MAY/09/021 are classified as moderately tolerant. For the mixed infection, all accessions were classified as very susceptible. Some accessions exhibited some level of tolerance and may be useful in the search of genotypes that could incorporate resistance.

Seed transmissibility of Cucumber mosaic virus in Capsicum species

ABSTRACT Cucumber mosaic virus (CMV) occurs worldwide and is a harmful pathogen for pepper (Capsicum spp.). Transmission of the virus is normally by insect. The possibility of transmission by seed is less clear. Accessions collected from eight states in Nigeria were evaluated to determine seed transmissibility of CMV in Capsicum species. Ten percent of the accessions were seed infected with CMV. Eight plants from each of 22 accessions were artificially inoculated with sap prepared from CMV-infected pepper plants and seed from resulting fruit from the inoculated plants were extracted, planted, and the resulting seedlings tested after 8 weeks. The CMV was detected in all four accessions selected randomly from plants that fruited in which accessions NHCrB/09/059, NCr/AA/MAY/09/015, NCr/SA/01/09/050, and NCr/AA/MAY/09/051 had CMV transmission percentage of 73.33%, 66.67%, 66.67%, or 16.67%, respectively. In another experiment, 100 seed of the pepper cvs. Tatase, Rodo, and Sombo extracted from fresh fruit obtained from local markets were randomly selected and sown in a plastic tray. Germinating seed, and developing plants, were maintained in screenhouse and assayed for symptom development. Three young leaf samples were randomly selected from each stand at 7 weeks after planting and subjected to antigen-coated plate enzyme-linked immunosorbent assay (ACP-ELISA). To evaluate the detection capacity of ELISA, and to prove its reliability, a reverse transcription polymerase chain reaction assay was performed for comparison. Natural seed transmission incidence of 57%, 86%, and 71% were determined for cvs. Tatase, Rodo, and Sombo, respectively. This implies that the ability to be transmitted by seed, and the rate of seed transmission of CMV, is cultivar dependent.

Monoclonal Antibody-Based Serological Detection Methods for Wheat Dwarf Virus.

Wheat dwarf disease caused by wheat dwarf virus (WDV) is currently present in wheat growing regions in China and causes serious losses in wheat yield. To develop reliable and effective serological detection methods for WDV, the coat protein (CP) gene of WDV was cloned and expressed in Escherichia coli. The purified recombinant CP protein was immunized to BALB/c mice, and four hybridoma cell lines (i.e. 18G10, 9G4, 23F4 and 22A10) secreting anti-WDV monoclonal antibodies (MAbs) were obtained through the hybridoma technique. Using the prepared MAbs, an antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA) and a dot-ELISA were established for detecting WDV in wheat samples. The most sensitive ACP-ELISA based on MAb 23F4 or 22A10 was able to detect WDV in 1:163,840 (w/v, g/mL) diluted WDV-infected wheat plant crude extracts. The dot-ELISA based on MAb 23F4 was the most sensitive and able to detect the virus in 1:5,120 (w/v, g/mL) diluted wheat plant crude extracts. A total of 128 wheat samples were collected from wheat growing regions in the Shaanxi and Qinghai provinces, China, and were screened for the presence of WDV using two developed serological assays. Results from the survey showed that approximately 62% of the samples were infected with WDV. PCR followed by DNA sequencing and sequence alignment validated the results from the two serological assays. Therefore, we consider that these two serological detection methods can be significantly useful for the control of WDV in China.

Serological detection of Grapevine leafroll-associated virus 4 in grapevine growing areas of India using polyclonal antiserum raised against the recombinant coat protein

Grapevine leafroll disease (GLD), caused by a complex of serologically and genetically distinct or related viral species termed as Grapevine leafroll associated viruses (GLRaVs), is a serious threat to the grape production worldwide. Recently, Grapevine leafroll-associated virus 4 (GLRaV-4) has been reported in the Indian vineyards, mostly as a mixed infection with GLRaV-3 and GLRaV-1. In order to screen the planting material, a polyclonal antiserum against the in vitro expressed coat protein (CP) of GLRaV-4 was produced. The antiserum, purified IgGs and conjugated IgGs detected GLRaV-4 in crude saps of infected grapevines and purified coat protein when used in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Western blotting and dot immunobinding assay (DIBA). GLRaV-4 could be detected in 54% (39 out of 72) of symptomatic grapevine samples from different grape growing regions of India using DAS-ELISA. The validation of DAS-ELISA results was done by RT-PCR using GLRaV-4 CP-specific primers. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in vitro expressed CP of GLRaV-4 and its successful application in diagnosis of the virus in grape vineyards of India.