Nucleotide sequencing and an improved diagnostic for screening okra (Abelmoschus esculentus L.) genotypes for resistance to a newly described begomovirus in IndiaFull Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=thsb20
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Nucleotide sequencing and an improved diagnostic for screening okra (Abelmoschus esculentus L.) genotypes for resistance to a newly described begomovirus in India
V. Venkataravanappa, C. N. L. Reddy, N. S. Chauhan, B. Singh, S. K. Sanwal & M. Krishna Reddy
To cite this article: V. Venkataravanappa, C. N. L. Reddy, N. S. Chauhan, B. Singh, S. K. Sanwal & M. Krishna Reddy (2016): Nucleotide sequencing and an improved diagnostic for screening okra (Abelmoschus esculentus L.) genotypes for resistance to a newly described begomovirus in India, The Journal of Horticultural Science and Biotechnology
To link to this article: http://dx.doi.org/10.1080/14620316.2015.1123407
Published online: 17 Feb 2016.
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aCentral Horticultural Experimental Station, Regional Station of IIHR, Chettalli 571 248, Kodagu, Karnataka, India; bDivision of Crop Protection, Indian Vegetable Research Institute, Varanasi 221 305, Uttar Pradesh, India; cDepartment of Plant Pathology, College of Sericulture, University of Agricultural Sciences (Bangalore), Chintamani 563 125, Karnataka, India
ABSTRACT Ten okra (Abelmoschus esculentus L.) plants showing distinct yellow vein mosaic disease (YVMD) symptoms were collected from different fields in Karnataka State, India. The genomic DNA of the isolated viruses was amplified, cloned, and sequenced. Sequence analysis revealed that the DNA- A-like sequences of all ten isolates were identical. Sequence analysis of a representative virus isolate (OYSK2) with other begomovirus sequences available in GenBank showed ≥90% sequence identity with Bhendi yellow vein Maharashtra virus (BYVMaV; EU482411) and ≤89% homology with full-length Bhendi yellow vein mosaic virus (BYVMV) infecting okra on the Indian subcontinent. These results suggested that a new strain of BYVMaV was present in all ten samples collected from the field. A source of resistance to BYVMaV and naturally present virus isolates causing YVMD was identified by screening okra genotypes under artificial and natural inoculation condi- tions, respectively. None of the genotypes tested showed complete immunity to BYVMaV. However, the okra genotypes ‘Tulasi’ and ‘Trisha’ were only moderately susceptible under glass- house and field conditions. The new begomovirus strain could be detected by dot-blot hybridisa- tion using a non-radioactive DNA probe in the virus samples collected from both symptomless and symptomatic okra plants.
ARTICLE HISTORY Received 26 March 2015 Accepted 12 November 2015
KEYWORDS Abelmoschus exculentus; okra; Begomovirus; resistance; diagnostics
Okra (Abelmoschus esculentus L.) belongs to the family Malvaceae and is native to South Africa. It is one of the most important vegetable crops grown in tropical, subtropical, and warm temperate zones of the world, occupying total area of 0.78 million ha and producing an annual yield of 4.99 million metric tonnes (MT), with an average yield of 6.39 million MT ha–1 (Gopalakrishnan, 2007). India is a leading producer of okra, followed by Nigeria, with an area of 0.31 million ha producing 6.0 million MT with a maximum productivity of 9.8 MT ha–1 (FAO-STAT, 2012). Okra provides a source of vitamins and minerals for consumers in developing countries who otherwise depend on cereal crops which lack these essential nutrients (Pal, Singh, & Swarup, 1982).
The cultivation of okra in India is susceptible to many insect pests and diseases from seed germination to harvest. Among these, yellow vein mosaic disease (YVMD) caused by many mono- and bi-partite bego- moviruses and their satellite DNA (subgenomic
components consisting of ssDNA) pose a major threat to the cultivation and production of okra. The impor- tant monopartite begomoviruses associated with YVMD are Bhendi yellow vein mosaic virus (BYVMV), Okra yellow vein mosaic virus (Fauquet et al., 2008), Cotton leaf curl Alabad virus, Cotton leaf curl Bangaluru virus, Bhendi yellow vein Bhubaneswar virus (Venkataravanappa, Reddy, Jalali, & Krishna Reddy, 2013a), Bhendi yellow vein Maharashtra virus (BYVMaV), and the bipartite Bhendi yellow vein Delhi virus (Venkataravanappa, Reddy, Jalali, & Krishna Reddy, 2012). These diverse begomoviruses have been associated with YVMD under natural field infection conditions and cannot be differentiated based on the symptoms expressed by infected okra plants. Depending on the stage of crop growth at which infec- tion occurred, total yield losses range from 50% to 94% (Sastry & Singh, 1974).
The identification of sources of resistance against the viruses that cause YVMD is the best method to control YVMD. Different hybrids and varieties of A.
CONTACT V. Venkataravanappa venkatrajani@gmail.com
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Another approach to control YVMD in okra would be to reduce whitefly vector populations using insecti- cides, physical barriers, or to remove symptomatic virus-infected plants. The use of insecticides has proved effective only when whitefly populations were low, but became a problem when insect populations were high (Rashida, Sultan, Khan, Noor-Ul-Islam, 2005). Due to repeated applications of all commonly used insecticides under natural conditions, virus vector whiteflies have developed moderate to strong resistance to these chemicals, which supports claims that many insecticides are losing their effectiveness against white- fly (Rashida et al., 2005).
The present study aimed to characterise a new strain of a begomovirus associated with YVMD, and to iden- tify potential sources of resistance in a collection of okra germplasm.
Materials and methods
Isolate
Leaf samples from ten okra (Abelmoschus esculentus L.) plants showing prominent YVMD symptoms were col- lected from fields at Srinivaspur in Karnataka State, India. Two samples of non-symptomatic okra plants were also collected. As field-collected plants may har- bour more than one virus, insect transmission was repeated three times under controlled conditions and YVMD symptoms were exhibited following each trans- mission of each sample to the susceptible okra cultivar ‘1685’. This ensured the absence of any virus not trans- mitted by Bemisia tabaci. The newly isolated virus was maintained in an insect-proof greenhouse on the sus- ceptible okra genotype ‘1685’ for later screening of okra genotypes and for characterisation of the virus.
DNA isolation
Total DNA was extracted separately from leaf samples of each of the ten symptomatic okra plants, from two non- symptomatic plants collected from the field, and from plants maintained in a glasshouse after insect transmission with each sample, using the cetyl trimethyl ammonium bromide (CTAB) method (Doyle & Doyle, 1990). Each extract of DNA was adjusted to 50 ng ml–1 and diluted tenfold in sterile distilled deionised water before being subjected to PCR amplification and stored at –20°C.
PCR amplification, cloning, and sequencing
The complete genome of the new virus isolate causing YVMD was amplified as described by Venkataravanappa et al. (2012). To test for the presence of a second DNA component (DNA-B) and/or a β-satellite (DNA-β), degen- erate primers specific to begomovirus DNA-B (Rojas, Gilbertson, Russel, & Maxwell, 1993; Venkataravanappa et al., 2012) and β-satellite DNA (Briddon, Bull, Mansoor, Amin, & Markham, 2002) were used.
The PCR-amplified products of DNA-A were pur- ified and cloned into the vector PTZ57R (MBI- Fermentas GmbH, St. Leon-Rot, Germany) according to the manufacturer’s instructions, then transformed into competent cells of Escherichia coli DH5α (Invitrogen Bioservices India Pvt. Ltd, Bengaluru, India). The complete nucleotide sequence of three clones of each of the ten DNA samples were deter- mined using an ABI PRISM 3730 automated DNA sequencer (Applied Biosystems – Invitrogen Bioservices India Pvt. Ltd) by Anshul Biotechnologies DNA Sequencing (Hyderabad, India).
Sequence analysis
The viral sequences obtained were first analysed using Vector NTI Advance TM 9 software (Invitrogen, Foster City, CA, USA) to remove any vector sequences, then the ORF Finder Programme (http://www.ncbi.Nlm.nih.Gov/projects/gorf/) was used to identify putative coding regions. Sequence similarity searches were performed by comparing these sequences to all available sequences in GenBank using BLASTN (Altschul, Gish, Miller, Myers, & Lipman, 1990; Supplementary Table SI). Sequences showing the highest identity scores with the present isolate were aligned using the SEAVIEW Programme (Galtier, Gouy, & Galtier, 1996). Sequence identity matrices for begomoviruses were generated using the Bioedit Sequence Alignment Editor (Version 5.0.9; Hall, 1999). A phylogenetic
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Plant material
The 30 okra genotypes used to screen for YVMD- associated virus resistance or tolerance in this study were popularly grown resistant varieties or hybrids from different locations in India: ‘Arka Anamika’, ‘Arka Abhay’, ‘Bhanupriya’, ‘Bio-8066’, ‘Mahalaxmi’, ‘Majuka’, ‘NS-531’, ‘NS-532’, ‘NS-502’, ‘OA47’, ‘Pusa Sawani’, ‘S-51’, ‘Solani’, ‘S71’, ‘1685’, ‘Sulkeerthi’, ‘Trisha’, ‘Tulasi’, ‘US-7109’, ‘US-7112’, ‘US-7111’, ‘US-136’, ‘US-5’, ‘US-7003’, ‘Vishal’, ‘Varsha Improved’, ‘VRO-5’, ‘No. 223646’, ‘Pusa Makhmali’, and ‘Hyb.218’. All were obtained from various pri- vate or government organisations.
Whitefly maintenance and plant inoculation
Whitefly collection, maintenance, and virus transmis- sion were carried out as described by Venkataravanappa et al. (2012) and Venkataravanappa, Reddy, and Krishna Reddy (2013b). After transmission, all inoculated plants were sprayed with a systemic insec- ticide and maintained in an insect-proof glasshouse for YVMD symptom expression. Leaf samples from symp- tomatic and non-symptomatic plants were used for analysis.
Artificial and natural screening of okra genotypes
All 30 genotypes of okra were screened for resistance to the new YVMD virus isolate under artificial and nat- ural conditions. Virus transmission was carried out under artificial conditions as described by Venkataravanappa et al. (2012, 2013b). The virus iso- lated characterised and maintained in the present study was used for artificial inoculations.
Natural screening of the same 30 genotypes for resistance to the natural YVMD-causing virus popu- lation was carried out at the Experimental Field of the Indian Institute of Horticultural Research Farm, Bangalore, Karnataka. The experiment was laid out in four rows, with 100 plants of each resistant genotype in three replications, with 60 cm between rows and 20 cm between plants. Two rows of 100 plants of the susceptible okra cultivar ‘1685’ were sown to provide an adequate supply of virus for the
whiteflies. All recommended cultural practices to raise a healthy okra crop were followed and no plant protection measures were applied. The responses of the okra genotypes were recorded based on the incidence of YVMD symptoms using the modified scale described by Venkataravanappa et al. (2013b).
Labelling the DNA probe
To prepare a non-radioactive DNA probe for the new virus isolate, a nucleic acid-labelling kit was used, as described by the manufacturer (Roche Diagnostics, Mannheim, Germany). A highly conserved, 770-bp region of the BYVMaV genome coding for the coat protein (GenBank Accession No. GU112012) was selected to develop the DNA probe. All DNA extrac- tion, pre-hybridisation, hybridisation, and detection procedures were carried out as described by Venkataravanappa et al. (2013b).
Results and discussion
PCR amplification
Ten samples of okra leaves expressing YVMD symp- toms were collected from three locations in India. All resulted in PCR amplification using the three pairs of primers designed to amplify DNA-A-like sequences of begomoviruses. Multiple alignments of the full- length genomic sequences (three clones per DNA sample) were identical, suggesting that the same virus was present in all ten samples from different locations. All DNA samples failed to amplify a sec- ond genomic component (DNA-B), but gave positive PCR signals for β-satellite DNA (data not shown), suggesting that the new virus infecting okra was monopartite. No PCR amplification occurred from the two non-symptomatic okra leaf samples.
Genome organisation and sequence analysis
The full-length DNA-A-like sequence of the new begomovirus isolate, designated OYSK2, was deter- mined in both orientations and was found to be 2741 nucleotides in length and was deposited in the NCBI-GenBank Database (Accession No. GU112013).
The sequence of OYSK2 contained features typical of ‘Old World’ monopartite begomoviruses, with seven predicted ORFs (AV1, AV2, AC1, AC2, AC3, AC4, and AC5). Sequence alignments showed that OYSK2 had the highest nucleotide sequence identity
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(91.9%) with Bhendi yellow vein Maharashtra virus (BYVMaV; EU482411) and the lowest sequence iden- tity (75.0–87.6%) with BYVMVs present in India. This result was supported by phylogenetic analysis showing that OYSK2 clustered with BYVMaV (Figure 1). The threshold cut-off value for strain demarcation in begomoviruses is currently 89% (Fauquet et al., 2008). This suggests that OYSK2 is a new strain of BYVMaV-IN (India:Karnataka:okra). The intergenic
region (IR) of OYSK2 shared 85.8% identity with the IR of BYVMV-Pakistan, for which a full-length sequence is available in GenBank (Accession No. AJ002453). The length of the IR was 273 nucleotides and encompassed a highly conserved hairpin structure containing the nona-nucleotide sequence (TAATATTAC) which is the origin of virion-strand DNA replication, and repeated sequences known as ‘iterons’ (GGTACC) adjacent to the TATA box, which
Figure 1. Phylogenetic tree based on nucleotide sequences of the DNA-A component of the new begomovirus virus isolate OYSK2 (identified with ♦) with 41 other begomoviruses (see Supplementary Table SI) using the Neighbour-Joining method in MEGA6.01 software. Horizontal distances are proportional to sequence distances. Vertical distances are arbitrary. The tree is unrooted. Bootstrap analysis was performed with 1000 replicates and only values >50% are shown at the branch points. Each branch has the GenBank Accession Number of the virus isolate followed by the abbreviated virus name, the country in which it was reported, the location from which it was collected, the isolate number, and the year of collection, in all parentheses. Bhendi yellow vein mosaic virus (BYVMV), Okra yellow vein mosaic virus (OYVMV), Bhendi yellow vein Maharashtra virus (BYVMaV), Bhendi yellow vein Haryana virus (BYVHV), Bhendi yellow vein Bhubaneswar virus (BhVBhV).
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is a recognition sequence for binding to the replicase (Heyraud et al., 1993).
Evaluation of okra genotypes
Screening of 30 okra genotypes following artificial whitefly inoculation showed variations in their sus- ceptibility to BYVMaV OYSK2 infection. YVMD symptoms appeared 8–10 days after inoculation (DAI) and increased gradually in all 30 okra geno- types (Table I; Figure 2A) except ‘Tulasi’ and ‘Trisha’, which started to show symptoms of virus infection 25–30 DAI (data not shown) that later developed fully and the plants died.
Under field conditions, the incidence of YVMD symp- toms among the 30 genotypes screened varied from 26% to 100% (Table II). Symptoms of YVMD appeared 30 days after sowing (DAS) in all genotypes except ‘Tulasi’ and ‘Trisha’, in which symptoms appeared at 60 DAS. The incidence of YVMD increased steadily in all 30 genotypes by 30 DAS, and ranged from 60% to 100%, except in ‘Tulasi’ (28%) and ‘Trisha’ (26%) at 105 DAS (Figure 2B). Variations in the incidence of YVMD in the different okra genotypes at different growth stages might be due to host preference by the vector whitefly. Similar results have been reported in earlier studies on screening different okra genotypes for resistance to BYVMV (Dhankhar, Dhankar, & Saharan, 1996; Sangar, 1997; Venkataravanappa et al., 2013b).
Table I. Responses of 30 genotypes of okra to inoculation with BYVMaV OYSK2 strain under artificial conditions#.
Okra cultivar/ hybrid
Rate of infection*
Incubation period (days)
‘1685’ (Susceptible control)
20/20 8–10 100.0 YVM, vein twisting, downward curling HS NT NT
‘Arka Abhay’ 20/15 9–10 75.0 Vein clearing, veinal chlorosis and petiole bending
HS +++ ++
‘Arka Anamika’ 20/20 8–10 100.0 Vein clearing, veinal chlorosis, complete yellowing
HS NT NT
‘Bhanupriya’ 20/13 8–10 66.6 YVM, minute enation S +++ ++ ‘Bio 8066’ 20/17 8–10 85.0 YVM HS +++ +++ ‘Hyb.219’ 20/20 8–10 100.0 YVM HS NT NT ‘Majuka’ 20/13 8–10 66.6 Vein netting, yellow mosaic, stunted
growth S +++ ++
‘No.223646’ 20/18 8–10 90.0 YVM HS +++ +++ ‘NS 531’ 20/19 8–10 95.0 YVM, complete yellowing S +++ +++ ‘NS 532’ 20/20 8–10 100.0 YVM, complete yellowing, enation HS NT NT ‘NS502’ 20/20 8–10 100.0 YVM and vein thickening HS NT NT ‘OA 47’ 20/18 8–10 90.0 Vein netting, YVM HS +++ +++ ‘Pusa Makhmali’ 20/16 8–10 70.0 YVM, complete yellowing, stunted
growth HS +++ ++
‘Pusa Sawani’ 20/13 8–10 66.6 YVM, complete yellowing S +++ ++ ‘S 71’ 20/13 12–15 76.0 Chlorotic spots, stunted of plants HS +++ ++ ‘S-51’ 20/8 12–14 76.0 Petiole bending, yellow vein mosaic HS +++ ++ ‘Solani’ 20/12 8–10 60.3 Vein netting, yellow vein mosaic S +++ ++ ‘Sulkeerthi’ 20/20 8–10 100.0 YVM and enation, upward curling HS NT NT ‘Trisha’ 20/7 23–25 25.0 Initially small chlorotic spot, later turn
into yellow vein mosaic MS ++ +
‘Tulasi’ 20/6 27–10 26.6 Initially small chlorotic spot, later turn into yellow vein mosaic
MS ++ +
‘US 136’ 20/17 8–10 85.0 Vein clearing, veinal chlorosis, stunted growth
HS +++ ++
‘US 5’ 20/20 8–10 100.0 YVM, petiole bending, vein netting HS NT NT ‘US 7003’ 20/18 8–10 90.0 YVM, petiole bending, vein netting HS +++ +++ ‘US 7109’ 20/12 8–10 70.0 Complete yellowing, petiole bending,
vein thickening S +++ ++
‘US 7111’ 20/16 8–10 70.3 Petiole bending and yellow vein mosaic HS +++ ++ ‘US 7112’ 20/10 8–10 65.0 YVM, petiole bending S +++ ++ ‘US-419’ 20/20 8–10 100.0 YVM and vein netting HS NT NT ‘Varsha improved’
20/11 18–20 70.0 YVM and vein thickening MS +++ ++
‘Vishal’ 20/18 8–10 60.0 YVM, vein thickening S +++ ++ ‘VRO-5’ 20/17 8–10 85.0 YVM HS +++ ++
#Inoculation was carried out using ten whiteflies per plant that had a 24 h acquisition feeding period on infected okra plants and a 24 h inoculation feeding period.
*Number of plants showing symptoms/total number of plants tested. $HS, highly susceptible; S, susceptible; MS, moderately susceptible. £NT, not tested because all plants showed symptoms; +++, strong; ++, moderate; +, weak hybridisation signal.
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Twenty-two of 30 genotypes screened under arti- ficial and field conditions differed in their suscept- ibility to BYVMaV OYSK2 and viruses causing YVMD present in the natural ecosystem of the experimental field. A DNA probe was used to con- firm the presence of BYVMaV OYSK2 and BYVMV isolates in plants of the 22 genotypes which did not show any symptoms after artificial inoculation and field screen- ing. The DNAprobe detected viral DNA in all okra plants showing YVMD symptoms and also in non-sympto- matic plants (Table II; Figure 3A). Based on the inten- sity of the signal on the nylon membrane, detection levels were catogorised from weak to strong. A low titre and a slow rate of virus accumulation in leaf tissues indicated the presence of a resistance mechanism (Rom, Antignus, Gidoni, Pilowsky, & Cohen, 1993; Aidawati, Hidayat, Hidayat, Suseno, & Sujiprihati, 2007). The titre of a virus in a genotype is an indicator of the resistance or susceptibility of the plant to the virus. However, in the present study, the titre of BYVMaV OYSK2 in symptomless okra plants was almost the same as that in okra plants showing YVMD symptoms, except for ‘Trisha’ and ‘Tulasi’ (Figure 3B). Similar approaches have been used to follow the accumulation of tomato leaf curl viral DNA in plant tissues and to correlate these with
symptom intensity and the level of viral DNA in tomato genotypes (Rom et al., 1993; Aidawati et al., 2007). Although the sensitivity of non-radioactive DNA probes is lower than radioactive probes, our results indicate the potential application of non-radio- active DNA probes to determine the responses of 30 okra genotypes to virus infection under artificial or field conditions.
In conclusion, this study has added further infor- mation on the diversity of begomoviruses infecting okra in India and on the development of DNA-based diagnostics to screen germplasm for virus resistance and to use sources of resistance identified in okra germplasm against viruses associated with YVMD.
This research was supported under the Project ‘Establishment of the association of begomovirus species with yellow vein mosaic disease (YVMD) in wild and cultivated species of okra and identification of source of resistance to the most predominant virus’, funded by the National Fund for Basic and Strategic Research in Agricultural Sciences (NFBSFARA), Indian Council of Agricultural Research, Government of India, New Delhi, India. We thank Dr M. Pitchaimuthu, Principal Scientist, Division of Vegetable Crops, IIHR, Bangalore for providing the 30 okra genotypes.
Figure 2. Incidence of YVMD in 30 okra genotypes at different time points following artificial inoculation with BYVMaV OYSK2 strain 5–35 days after sowing (DAS; Panel A) or under natural field conditions (15–105 DAS; Panel B). Cultivar ‘1685’ is the susceptible (control) variety of okra.
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Figure 3. Detection of BYVMaV OYSK2 in okra plants by dot-blot hybridisation using a digoxigenin-labelled DNA probe following inoculation with the new virus isolate and showing symptoms or no symptoms. Panel A, dots 1a–1c are susceptible controls (okra cultivar ‘1685’) showing symptoms; dots 2a–2c to 5a–5c represent leaf sap samples from okra plants not showing symptoms. Panel B, symptoms expressed in the susceptible okra cultivar, ‘1685’ and in the moderately susceptible cultivars ‘Trisha’ and ‘Tulasi’ during screening for resistance and subsequent detection by dot-blot hybridisation (dots 1–3, respectively).
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THE JOURNAL OF HORTICULTURAL SCIENCE AND BIOTECHNOLOGY 9
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Sequence analysis
Plant material
Artificial and natural screening of okra genotypes
Labelling the DNA probe
Evaluation of okra genotypes