Direct Antigen-coated Enzyme-linked Immunosorbent Assay Research Articles

Germplasm Screening for Identification of BCMV Resistance Sources from Diverse Cowpea [Vigna unguiculata (L.) Walp.] Germplasm using Serological and Molecular Diagnostics

Background: Bean common mosaic disease (BCMD) caused by Bean common mosaic virus (BCMV) has been regarded as one of the most important seed-borne and aphid vector transmitted destructive disease of cowpea not only affecting its yield but also reducing its grain quality globally. Since the dawn of crop improvement, germplasm served as the source of resistance for various biotic stresses. Currently, host plant resistance is the best, reliable, economic and environmental friendly practice of virus disease management. The objective of this study was to assess the response of diverse cowpea germplasm against BCMV under natural and controlled conditions followed by serological and molecular detection. Methods: In this study, 85 cowpea germplasm accessions and one known BCMV susceptible (C152) and one resistant (CP55) varieties are evaluated for their response to the BCMD in augmented block design (ABD). The reaction of cowpea accessions were assessed using percent disease incidence (PDI). Transmission Electron microscopy (TEM), Direct Antigen Coating-Enzyme-linked Immunosorbent Assay (DAC-ELISA) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were used for confirming the BCMV. Result: Natural and controlled condition screening data analysis revealed a significant difference among diverse cowpea germplasm for BCMV diseases incidence. Based on field screening at Ranchi and New Delhi (2019 and 2020) and artificial confirmation. 24 out of 85 accessions were found to be immune and confirmed through DAC-ELISA and RT-PCR. As a consequence these accessions could be a potential new source of disease resistance for BCMV in breeding programs.Further, DAC ELISA of seed coat and Embryo revealed the presence of virus in 5% and 15% of samples tested. Accession IC418505 recorded highest disease incidence (40.0%).

Development of Polyclonal Antibodies-Based Serological Method for the Detection of Calanthe Mild Mosaic Virus and Application in Virus Certification Programme.

Calanthe mild mosaic virus (CalMMV) infecting orchids is an important potyvirus which is known to cause mild leaf mosaic and flower colour-breaking symptoms in Calanthe and other orchid plants. The present study reports the production of polyclonal antibodies against CalMMV using bacterially expressed recombinant coat protein as immunogen, which in turn would be useful in routine indexing and screening of orchid germplasm. The coat protein (CP) gene (~ 807bp) of CalMMV isolated from infected orchid sample was cloned in expression vector, pET-28a ( +) that yielded ~ 31kDa fusion protein with Histidine tag (His6BP). The expression of fusion CP was confirmed through SDS-PAGE and Western blotting. The His6BP-CalMMV-CP obtained in soluble state after purification was used to immunize New Zealand white rabbit for the production of polyclonal antibodies (PAb). The PAb produced against the purified fusion protein successfully detected CAlMMV in the orchid samples at a dilution of 1:2000 in direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). This study presents the first report of Histidine tag (His6BP) fusion CalMMV-CP-based antibody production and its successful application in the identification of the virus in orchid plants. Outcome of this study will be helpful in routine certification programmes, screening of orchid germplasm and production of CalMMV-free planting materials of orchids.

Detecting Tomato Leaf Curl New Delhi Virus Causing Ridge Gourd Yellow Mosaic Disease, and Other Begomoviruses by Antibody-Based Methods.

The incidence and severity of begomovirus diseases have been increasing around the world recently, and the ridge gourd [Luffa acutangula (Roxb.) L.] is the latest example of a crop that has become highly susceptible to the outbreak of the tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in India. Accurate diagnosis of causal agents is important in designing disease management strategies. In this study the coat protein (CP) gene from a ToLCNDV-Rg ridge gourd isolate was used to produce polyclonal antibodies (ToLCNDV-Rg-CP-PAb) in a rabbit. The antibodies successfully detected a 30.5 kDa ToLCNDV-Rg-CP in extracts of symptomatic ridge gourd leaf samples by several assays, such as Western Blotting (WB), Dot Immuno Binding Assay (DIBA), Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA), Immuno Capture Polymerase Chain Reaction (IC-PCR), and Immuno Capture Loop-Mediated Isothermal Amplification (IC-LAMP) assays. However, none of the negative samples tested positive in either of the detection methods. Among all the methods tested, the immunocapture assay, IC-LAMP, was the most sensitive in detecting ToLCNDV-Rg. Furthermore, antibodies generated in this study also detected other commonly occurring begomoviruses in South India, such as tomato leaf curl Palampur virus and squash leaf curl China virus in cucurbits. Together, ToLCNDV-Rg-CP-PAb can be used for detecting at least three species of begomoviruses infecting cucurbits. The obtained antibodies will contribute to monitoring disease outbreaks in multiple crops.

Identification of BCMV resistant germplasm in mungbean (Vigna radiata) using serological and molecular diagnostics

Diverse germplasm of mungbean serves as a source of resistant gene, screening of germplasm for BCMV resistance will provide resistance source. So, in 2018–2019 a total of 65 diverse mungbean accessions along with susceptible and resistant checks were screened against BCMV under natural conditions in the field and artificial inoculation under controlled conditions. Transmission electron microscopy, per cent disease incidence, serological Direct antigen coating- Enzyme-linked immunosorbent assay (DAC-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) diagnostics were used to detect and identify the BCMV resistant accessions. The study revealed that a total of 29 accessions were found to be immune to BCMV infections. However, these accessions need to be further evaluated in BCMV disease hotspots and under multilocation trials. Therefore, in the present study, accessions that were immune to BCMV may serve as valuable donors for the BCMV resistant mungbean breeding programme.

Thrips diversity of cotton ecosystem and the role of parthenium pollen grains in the transmission of tobacco streak virus (TSV) infection in cotton.

The online version contains supplementary material available at 10.1007/s13205-021-02967-6.

Graphene oxide based electrochemical immunosensor for rapid detection of groundnut bud necrosis orthotospovirus in agricultural crops

Groundnut bud necrosis orthotospovirus (GBNV) is one of the causative plant viruses responsible for the outbreak of many viral epidemics in food crops across India and other south-Asian countries. Its management is a major challenge due to fast vector transmission, and the non-availability of appropriate agrochemical treatment. The timely detection of GBNV becomes indispensable for the effective management of viral infection and the periodic monitoring of plant health. We report the fabrication of graphene oxide (GO) based electrochemical immunosensor for the rapid and sensitive detection of GBNV. The immunoelectrode is prepared by depositing GO onto indium-tin oxide (ITO) coated glass substrates and functionalized by anti-GBNV antibodies using N-ethyl-N′-(3- dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide (EDC-NHS) conjugation chemistry. The response measurements of the immunoelectrodes revealed a sensitivity of 221 ± 1 μA μg−1 mL−1(n = 3) and limit of detection (LOD) of 5.7 ± 0.7 ng mL−1(n = 3) for the standard concentrations of GBNV antigen. Further, the GBNV detection was carried out in infected leaf extracts of three different host plants i.e., Tomato, Cowpea, and N. benthamiana, and the results have been compared with the conventionally used direct antigen coated enzyme-linked immunosorbent assay (DAC-ELISA) technique. The comparable results obtained for the detection of GBNV in infected plants using electrochemical immunosensing and DAC-ELISA techniques advocated the immense potential of GO based immunosensor as a point-of-care sensing device that is poised to overcome the limitations of the traditional methods of virus detection in field conditions and may transform the diagnostics in agriculture.

Prevalence and Molecular Characterization Coat Protein Gene of Tobacco streak virus Causing Peanut Stem Necrosis Disease in Coastal and Rayalaseema Districts of Andhra Pradesh, South-India

Background: Peanut stem necrosis disease (PSND) caused by Tobacco streak virus (TSV) is a major constraint for groundnut production in Andhra Pradesh (A.P.). However, studies on prevalence and spread of the disease confined to only few districts of A.P. with this background current study focused on incidence and spread of the disease in entire state of A.P. Further an isolate of TSV occurring in A.P. characterized on the basis of genetic features by comparing with other TSV isolates originated from different hosts and locations from world.Methods: Roving survey was conducted during kharif 2017-18 in groundnut growing districts of Andhra Pradesh (A.P.) for peanut stem necrosis disease incidence. Groundnut plants showing PSND symptoms were collected and tested with direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). Groundnut samples found positive by ELISA once again tested by reverse transcription polymerase chain reaction (RT-PCR). The representative TSV-GN-INDVP groundnut isolate from Prakasham district was maintained on cowpea seedlings by standard sap inoculation method in glasshouse for further molecular characterization. The Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny.Result: Thirty-eight (52.7%) of seventy-two groundnut samples collected from different locations in A.P were given positive reaction to TSV by DAC-ELISA. For the first time, PSND incidence observed in coastal districts (Krishna, Guntur, Sri Pottisriramulu Nellore, Prakasham) of A.P. Maximum PSND incidence recorded from Bathalapalli (22.2%) and the minimum incidence in Mulakalacheruvu (4.1%). The coat protein (CP) gene of TSV-GN-INDVP groundnut isolate was amplified by RT-PCR and it shared maximum per cent nucleotide identity (97.51-98.62%) with TSV isolates from groundnut and other different crops reported in India. All Indian isolates cluster together irrespective of crop and location based on the phylogenetic analysis.

First Report of Natural Infection by Capsicum Chlorosis Virus on Amaryllis (Hippeastrum hybridum) Plants from India

First Report of Natural Infection by Capsicum Chlorosis Virus on Amaryllis (<i>Hippeastrum hybridum</i>) Plants from India

Heterologous expression of pepper mild mottle virus coat protein encoding region and its application in immuno-diagnostics

Pepper mild mottle virus (PMMoV), a tobamovirus of family Virgaviridae affects the quality and quantity of Capsicum. PMMoV is highly contagious, capable of transmitting through infected seeds and soil. Symptoms are more severe when crop is infected at young stage but remain unnoticed when infection takes place at maturity. Therefore, cost effective diagnostic techniques are required for timely and accurate detection of virus. In present study, coat protein encoding region of PMMoV-HP1 isolate was cloned into expression vector system, pET28a and expressed in BL21, a protease deficient strain of Escherichia coli. The PMMoV-HP1 pathotype was identified as PMMoV-P12 on the basis of coat protein amino acid sequence analysis in our previous study. The overexpression of recombinant coat protein of 26kDa, corresponding to the expected 6X Histidine tag fused recombinant protein was purified using Ni-NTA columns from insoluble fraction. For antisera production, the purified recombinant protein was dialyzed ~ 24h to remove urea and then used for raising polyclonal antisera. The specificity and sensitivity of antiserum obtained was demonstrated using different dilutions of antiserum for western blot assay and direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). In Western blot assay, the test antiserum reacted strongly both with PMMoV-CP in purified protein and native CP in crude sap from PMMoV infected pepper plants, whereas no reaction was observed with healthy plant sap. In DAC-ELISA antiserum dilution up to 1:1000 was capable of detecting the virus in infected sample. The absence of any cross reactivity of test antiserum was confirmed against tobacco mosaic virus, cucumber mosaic virus, tomato spotted wilt virus, pepper veinal mottle virus, potato virus Y and tomato yellow leaf curl virus antigen, known to infect capsicum.

Retracted: Verification of single nucleotide polymorphism (SNP) markers associated with maize (Zea mays. L) streak virus resistance in early generation maize lines

Maize streak virus (MSV) is a devastating viral disease of maize in sub-Saharan Africa. The disease may cause up to 100% yield loss in susceptible crops. The use of molecular markers can facilitate the development of varieties resistant to the virus. The objective of this study is to assess the usefulness of Single Nucleotide Polymorphism (SNP) markers linked to MSV resistance in selecting for resistance at early generations of inbred line development in maize. A total of 160 maize lines were genotyped with three SNP markers that are linked to MSV resistance. These lines were tested for their reaction to MSV through artificial inoculation using viruliferous Cicadulina triangular at the three leaf stage; maize streak virus symptom was scored from 7 days after inoculation for six weeks at weekly interval on a scale of 1 to 5. MSV titer on the upper and lower leaves was determined using Direct Antigen Coating Enzyme linked Immunosorbent Assay (DAC-ELISA). One hundred and forty-two (142) of the 160 maize lines had the favourable marker allele for MSV resistance while 18 maize lines did not have the allele. Differences among the 160 maize lines for MSV symptom on upper leaves at six week after inoculation were significant (P< 0.01). Favourable allele of the SNP markers was significantly associated with MSV symptom score at 6 week after inoculation and MSV titer status. The percentage of maize lines with desirable marker allele with resistance based on symptoms score and ELISA were 97.9 and 93%, respectively. The three SNP markers showed high efficiency in the identification of MSV resistant maize lines and therefore have potential for use in marker-assisted selection. The SNP markers were not effective in detecting MSV resistance in few genotypes, indicating a need to develop other markers for resistance. Key words: ELISA, genotyping, inoculation, maize streak virus, SNPs. See: Notice of Retraction

Seed transmission of a distinct soybean yellow mottle mosaic virus strain identified from India in natural and experimental hosts

Soybean yellow mottle mosaic virus (SYMMV) is a newly identified member of the genus Gammacarmovirus from grain legumes in India. As the modes of transmission of this virus have not been described, we assessed the possibility of SYMMV to be transmitted through seed collected from field infected mungbean plants and mechanically sap inoculated French bean plants using serological and molecular techniques followed by progeny assays. Direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) and reverse transcription polymerase chain reaction (RT-PCR) results are inconsistent with field infected mungbean seed tissues to ensure seed transmissibility irrespective of seed number used. Seed from mechanical sap inoculated French bean showed higher absorbance values in DAC-ELISA and amplification corresponding to replicase, movement and coat protein regions of SYMMV genome. The relative accumulation of SYMMV was higher in pod walls, immature seed and stamens and stigma of mechanical sap inoculated French bean. Progeny assays with infected seed revealed the seed transmissibility of SYMMV at the rate of 63.33% in mungbeanand 73.33% in French bean. Mechanical sap inoculation of mungbean progeny seedlings on French bean cv. Pusa Parvati produced characteristic symptoms of SYMMV. The results obtained from this study demonstrate that SYMMV is seed borne in nature and can be transmitted to next generation seedlings. This is the first report of seed transmission of SYMMV in mungbean and French bean.

Detection of mixed infection of potyvirus and tobamovirus in Bitter Gourd (Momordica charantia) in Karnataka State, India

Bitter gourd, (Momordica charantia) is one of the most popular vegetables in India. However, the major limiting factor is the virus disease for poor productivity of this crop. In this study, virus infection in bitter gourd plant at Experimental fields of ICAR-Indian Institute of Horticultural Research, Bengaluru was studied. Leaf dip preparations of infected sample under transmission electron microscope (TEM), direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) using specific antisera and reverse transcription polymerase chain reaction (RT-PCR) using primers targeting coat protein gene of potyvirus and tobamovirus revealed the presence of two types of viruses potyvirus and tobamovirus in the infected bitter gourd plants; to our knowledge, this is the rare report of mixed infection of potyvirus and a tobamovirus in bitter gourds in Karnataka state.

Xanthomonas campestris pv. musacearum: a major constraint to banana, plantain and enset production in central and east Africa over the past decade.

Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Order Xanthomonadales; Family Xanthomonadaceae; Genus Xanthomonas; currently classified as X. campestris pv. musacearum (Xcm). However, fatty acid methyl ester analysis and genetic and genomic evidence suggest that this pathogen is X. vasicola and resides in a separate pathovar. Xcm can be isolated on yeast extract peptone glucose agar (YPGA), cellobiose cephalexin agar and yeast extract tryptone sucrose agar (YTSA) complemented with 5-fluorouracil, cephalexin and cycloheximide to confer semi-selectivity. Xcm can also be identified using direct antigen coating enzyme-linked immunosorbent assay (DAC-ELISA), species-specific polymerase chain reaction (PCR) using GspDm primers and lateral flow devices that detect latent infections. Causes Xanthomonas wilt on plants belonging to the Musaceae, primarily banana (Musa acuminata), plantain (M. acuminata × balbisiana) and enset (Ensete ventricosum). There is a high level of genetic homogeneity within Xcm, although genome sequencing has revealed two major sublineages. Yellowing and wilting of leaves, premature fruit ripening and dry rot, bacterial exudate from cut stems. Xcm has only been found in African countries, namely Burundi, Ethiopia, Democratic Republic of the Congo, Kenya, Rwanda, Tanzania and Uganda. Xcm is transmitted by insects, bats, birds and farming implements. Long-distance dispersal of the pathogen is by the transportation of latently infected plants into new areas. The management of Xcm has relied on cultural practices that keep the pathogen population at tolerable levels. Biotechnology programmes have been successful in producing resistant banana plants. However, the deployment of such genetic material has not as yet been achieved in farmers' fields, and the sustainability of transgenic resistance remains to be addressed.

Detection, diagnosis of orchid virus and inactivation of cymbidium mosaic virus (CYMV) on plants

Floriculture is one of the disciplines of Horticulture which is dealing with growing of ornamental plants flowering plants and garden maintenance etc. orchids are one of the floriculture plant. It is a member of Orchidaceae family consisting of more than 25,000 species, which are distributed almost all over the world. Vanda Orchid plant is collected from different nurseries showing chlorotic and mosaic symptoms were observed and it was suspected to infect with virus. So the symptomatic plants were tested with Direct Antigen Coating- Enzyme Linked Immunosorbent Assay (DAC-ELISA) for Cymbidium Mosaic Virus (CYMV), Odontoglossum ring spot virus (ORSV), Poty virus and Tomato Spotted Wilt Virus (TSWV) and further confirmed by Transmission Electron Microscopy (TEM). With the two methods CYMV were detected positively from the samples and low positive results were observed for ORSV, Potex, Poty virus and Tomato Spotted Wilt Virus (TSWV). High incidence of Cymbidium Mosaic Virus (CYMV) was observed. Chemicals were evaluated for inactivation of CYMV on pruning tools for disease control. Skimmed milk was found to be effective, non caustic and inexpensive for inactivation of CYMV inoculated on local lesion indicator host plants. However, systemic host plants (Orchids) were used in evaluation.

Response of some wild species of tomato againstPeanut bud necrosis virusunder open-field conditions

Thrips-borne Tospovirus pathogens adversely affect many globally important crops. Among 16 distinct virus species in the Tospovirus genus, four species including Peanut bud necrosis virus (PBNV) causing necrosis disease in tomato have been reported in India. Identification of stable sources and further utilization of wild relatives as gene sources to increase levels and diversify the bases of resistance may offer good management for the disease. A total of 13 wild species of tomato (Solanum peruvianum), two S. pimpinellifolium, one S. chilense, one S. pennellii and three check cultivars (S. lycopersicum) along with two cultivars (S. lycopersicum) having the Sw-5 and Sw-7 genes were evaluated under field conditions during three consecutive seasons (June to October 2008, July to December 2009, August 2010 to February 2011). Among all, a high degree of field resistance (>80%) was detected in seven lines of S. peruvianum (L00735, L00671, L00887, L06138), S. chilense (TL02226) and S. pimpinellifolium (L03708, TL02213) lines. The field data was also supported by negative reaction against a polyclonal antiserum of the nucleocapsid protein (N) of PBNV in direct antigen coating-enzyme linked immunosorbent assay (DAC-ELISA). The cultivars with Sw-5 and Sw-7 genes were highly susceptible to PBNV.

COAT PROTEIN GENE OF NEW ISOLATE OF CUCUMBER MOSAIC VIRUS INFECTING BANANA IN EGYPT

Banana plants showing typical mosaic and yellow stripes on leaves as symptoms were collected from Assiut Governorate in Egypt. The causal agent was identified as Cucumber mosaic virus (CMV) on the basis of symptoms, transmission, serology, transmission electron microscopy (TEM) and reverse transcription polymerase chain reaction (RT-PCR). Coat protein (CP) gene was amplified using gene specific primer, followed by cloning into desired cloning vector and sequencing. In this study the CMV was transmitted into propagation host either by aphid or mechanically. The transmission was confirmed through direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). Analysis of the 120 deduced amino acid sequence of the coat protein gene revealed that the Eg-A strain of CMV shared from 97.50 to 98.33% with those strains belonging to subgroup IA. The cluster analysis grouped the Egyptian isolate with strains Fny and Ri8 belonging sub-group IA. It appears that there occurs a high incidence of CMV infecting banana belonging to IA subgroup in most parts of Egypt.

Production of virus-free garlic (Allium sativum L.) through meristem tip culture after solar or hot air treatment of cloves

SummaryGarlic (Allium sativum L.) is a clonally propagated crop that can be infected by many virus species which tend to accumulate year-after-year, leading to yield reductions and varietal degeneration. Therefore, the production and use of virus-free planting material is vital to sustain productivity. In this study, Onion yellow dwarf virus (OYDV; Potyvirus), Garlic common latent virus (GarCLV; Carlavirus), Shallot latent virus (SLV; Carlavirus), and Garlic virus X (GarV-X; Allexivirus) were detected in cloves of the commercial garlic cultivar, ‘G-1’ (‘Jamuna Safed’) by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA). DAC-ELISA was performed using specific antisera against each virus species, and confirmed through reverse transcription-polymerase chain reactions (RT-PCR) using virus or genus-specific oligonucleotide primers. To obtain virus-free plants of ‘G-1’, cloves were subjected to solar heat treatment for 5, 10, or 15 d, or to hot air treatment (at 37°C, 40°C, or 42°C for 7, 14, or 21 d) combined with meristem isolation and in vitro culture, then indexed for all viruses using DAC-ELISA and RT-PCR. Meristems from solar heat-treated or hot air-treated cloves regenerated after 90 d and 45 d in culture, respectively. Solar heat treatment for 15 d resulted in 17 - 18% regeneration, and all were free from GarCLV, SLV, and OYDV. However, when solar heat-treated for 10 d, only 66% of the 17 - 18% regenerated plants were free from all viruses, including GarV-X. Hot air treatment at 37°C or 40°C eliminated GarCLV, SLV, and OYDV in 20 - 38% and 42 - 62% of regenerated plants, respectively, for the different durations of exposure. However, GarV-X could not be eliminated from any of the regenerated plants by hot air treatment, irrespective of the duration of exposure. Meristems from cloves that had been hot air-treated at 42°C resulted in 50 - 66% regeneration, of which 53 - 57% were free from GarCLV, SLV, and OYDV when treated for 7 d or 14 d, and 29% were free from all viruses when treated for 21 d. For most treatments, an increase in the duration of exposure to heat resulted in reduced regeneration, but increased the rate of recovery of virus-free plants. To our knowledge, this is the first study to report the use of solar treatment for virus elimination in garlic and the development of an efficient tissue culture protocol for the production of virus-free garlic plants using different thermotherapy treatments coupled with meristem tip culture.

Current status of Citrus tristeza virus incidence and its spatial distribution in citrus growing geographical zones of India

Citrus tristeza virus (CTV), a member of genus Closterovirus, is an important pathogen which has destroyed more than one million citrus trees in India till date. Survey was conducted in commercial Citrus sp. covering all the citrus growing geographical zones of India. For detection of CTV and estimation of disease incidence, direct antigen coated- enzyme linked immuno-sorbent assay (DAC-ELISA), reverse transcription- polymerase chain reaction and nucleotide sequence analyses were employed. The overall CTV incidence of 26.3% in Vidarbha region, 47.1-56.0% in Northeast (Asom, Meghalaya, Sikkim and the Darjeeling hills), 36-50% in South (Andhra Pradesh and Karnataka) and 16-60% in North-Northwest (Uttarakhand, Delhi and Punjab) were reported. CTV titre in infected tree samples of different locations was measured by ELISA . The high titre was observed in Kagzilime (C. aurantifolia) and sweet orange (C. sinensis) but low in mandarin (C. reticulata). Nucleotide sequence of 5’ORF1a fragment of CTV genome of nine representative CTV isolates, Kat-8, RA-3, B-6, Dh-1, An-9, TP-4, Pant-3, MU-2 and As-2 from all the citrus growing regions surveyed were analysed. The present Indian isolates showed 78-98% identities among them, and placed in different clusters in phylogenetic analysis. This is for the first time an overall incidence of CTV and its spatial distribution in citrus growing geographical zones of India were reported. Findings presented in this study will help in understanding epidemiology and formulating molecular based management strategy.

Sequence Diversity of the Nucleoprotein Gene of Peanut bud necrosis virus Isolates from South India

AbstractPeanut bud necrosis virus (PBNV), genus Tospovirus (family Bunyaviridae), is an important virus infecting peanut and other crops in South India. PBNV isolates naturally infecting groundnut, brinjal, tomato, black gram, field bean, cowpea, cotton, jute, taro and Calotropis plants were collected from different regions of South India and characterized. Infection was confirmed by direct antigen‐coating enzyme‐linked immunosorbent assay (DAC‐ELISA) using PBNV‐specific antiserum. The coat protein gene was further amplified using PBNV coat protein‐specific primers. The amplicon (830 bp) was cloned and sequenced; sequence analysis revealed that the N gene shared 93–100% and 95–100% sequence identity with PBNV at the nucleotide and amino acid levels, respectively.

Simultaneous detection of potato viruses Y and X by DAC-ELISA using polyclonal antibodies raised against fused coat proteins expressed in Escherichia coli

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).