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2007 Plant Management Network.
Accepted for publication 25 January 2007. Published 8 May 2007.


Occurrence of Begomovirus Associated with Yellow Vein Mosaic Disease of Kenaf (Hibiscus cannabinus) in Northern India


Raju Ghosh, Sujay Paul, Anirban Roy, Javid Iqbal Mir, and S. K. Ghosh, Plant Virus Laboratory, Division of Crop Protection, Central Research Institute for Jute and Allied Fibres, Barrackpore, Kolkata-700120, West Bengal, India; and R. K. Srivastava and U. S. Yadav, Crop Research Station, Bahraich, Uttar Pradesh, India


Corresponding author: Anirban Roy. anirbanroy75@yahoo.com


Ghosh, R., Paul, S., Roy, A., Mir, J. I., Ghosh, S. K., Srivastava, R. K., and Yadav, U. S. 2007. Occurrence of begomovirus associated with yellow vein mosaic disease of kenaf (Hibiscus cannabinus) in northern India. Online. Plant Health Progress doi:10.1094/PHP-2007-0508-01-RS.


Abstract

The occurrence, distribution, and disease incidence of yellow vein mosaic (YVM) disease of kenaf (Hibiscus cannabinus) in northern India are reported. The disease was found in both commercial and experimental farms in the Bahraich district, Uttar Pradesh, India and caused significant reduction in plant height and crop yield. Southern hybridization with begomovirus-specific probe and PCR amplification with DNA β and coat protein primers confirmed the association of begomovirus with the disease.


Introduction

Kenaf (Hibiscus cannabinus L.), an annual herbaceous crop of the Malvaceae family, is cultivated primarily for the production of bast fibers in more than 20 countries, particularly in China, India, Thailand, and Vietnam. Kenaf is also used in paper making, oil absorption, potting media, board making, filtration media, and animal feed. USDA recognizes kenaf as the best non-woody paper-making plant. In India, this crop is widely cultivated in Andhra Pradesh, Orissa, and West Bengal and to some extent in Uttar Pradesh, where it is grown as a mixed crop with maize, pigeon-pea, sorghum, and sugarcane. A large number of viral diseases have been reported to cause serious damage in this crop at different countries but no detailed data have yet been reported from India.

Begomoviruses (family Geminiviridae) an economically important group of plant viruses, are reported to cause serious losses to many important crops (e.g., bean, cucurbits, tomato, etc.) in tropical and subtropical regions (3,4). Recently, a begomovirus containing a satellite DNA β was found to be associated with yellow vein mosaic disease (YVMD) in kenaf in the eastern part of India (5). With an aim to record the distribution pattern of this disease, an investigation was undertaken at different commercial and experimental farms of the Bahraich region at Uttar Pradesh located in the northern part of India.


Survey of Virus Diseases and Sample Collection

In order to study the occurrence of YVMD in kenaf a survey was carried out at three different villages, namely Susrauli (4 plots), Kalpipara (3 plots), and Chichdi (3 plots), of the Bahraich district of Uttar Pradesh. Seventeen accessions including varieties and germplasm of kenaf that were planted in 2--2-m plots in an experimental field of the Crop Research Station, Narendranath Dev Agricultural University, Bahraich, Uttar Pradesh, India were also screened for this viral disease. Data were collected 100 days after planting. Disease incidence (percent of plants showing symptoms over total number of plants screened), disease severity (number of leaves showing symptoms over total number of leaves on a plant), and plant height reduction were recorded. Leaves from symptomatic plants were collected for molecular analysis.


DNA Isolation

Total plant DNA was isolated from the symptomatic leaves of the collected samples using the CTAB method (10). After electrophoresis on 0.8% agarose gels the DNA was quantified by comparing the intensity of bands with known concentration of uncut λ-DNA using Quantity One 1-D Analysis Software (Bio-Rad, Hercules, CA).


Southern Hybridization with Begomovirus Specific Probe

DNA obtained from symptomatic leaf samples, collected from three villages and also from the Crop Research Station, was electrophoresed in a 0.8% agarose gel and then transferred to a nylon membrane. DNA isolated from virus-free kenaf samples, grown under controlled glasshouse conditions, served as the healthy control. Hybridization was carried out following standard procedures (7) with a α-P radiolabeled probe to DNA A of the Cotton leaf curl Rajasthan virus (the probe was supplied by Dr. V. G. Malathi, Indian Agricultural Research Institute, New Delhi). The cotton leaf curl virus probe was used because kenaf, like cotton, is a member of Malvaceae family and the begomoviruses of this family are very similar (1).


Polymerase Chain Reaction (PCR) for Virus Detection

PCR was done using viral coat protein primers designed for the Bhendi YVMV (8) and universal DNA β primers (2). In the previous study (5), DNA of the East Indian isolate of this virus amplified with these two primer sets but not with the DNA A-specific primers of Cotton leaf curl virus, Tomato leaf curl virus, and Mungbean yellow mosaic virus. Hence, these two primers were used for PCR amplification in this study. In all PCR reactions (50 l), 100 ng of total plant DNA and 0.2 M of primers were used. Each PCR reaction mixture contains 1X PCR buffer, 0.2 M dNTPs, 1.5 M MgCl2, and 0.6 units Taq DNA polymerase (Fermentus). DNA fragments were amplified in a PX2 thermal cycler (Thermo, Applied Biosystem, Foster City, CA) with an initial denaturation at 94C for 2 min followed by 30 cycles of 94C for 1 min, 55C (with DNA β primer set) or 52C (with coat protein specific primer set) for 2 min, and 72C for 3 min followed by a final extension at 72C for 10 min. The PCR amplified fragments were resolved after electrophoresis in 0.8% agarose gel and visualized in a UV gel documentation system (Bio-Rad, Hercules, CA) after staining in ethidium bromide.

Initial symptoms of YVMD were veinal chlorosis of the leaves (Fig. 1), progressing to a conspicuous yellow network of veins. The plants became stunted. In symptomatic plants, the fibre yield was greatly reduced when compared with healthy plants (data not shown).


 

Fig. 1. Symptoms of yellow vein mosaic disease on leaves of Kenaf.

 

The average disease incidence, disease severity, and height reduction were recorded to be approximately 59%, 78% and 22%, respectively, from a total of 1500 plants surveyed from three villages (Table 1). Several germplasm accessions of kenaf showed YVMD symptoms in the experimental fields of Crop Research Station, Bahraich (UP) (Table 2). Average disease incidence among different accessions varied from 29% to 74%. The accessions MT 150, KIN 061, and KIN 151 showed low disease incidence (<50%), while KIN 108, KIN 013, AMC 108, and Bahraich Local showed relatively high incidence (>50%) of the disease.

DNA obtained from symptomatic leaves of different accessions of kenaf showed strong hybridization signal with the cotton leaf curl DNA A probe (Fig. 2) while no such signal was observed with DNA from glasshouse-grown virus-free healthy samples. PCR amplification with specific primers revealed an amplicon of approximately 1.3 kb with DNA β primers and ca. 0.77 kb with coat protein specific primers (Fig. 3).


 

Fig. 2. Autoradiogram showing hybridization of DNA from symptomatic kenaf samples with the α-P radio-labeled Cotton leaf curl virus DNA A probe. Symptomatic samples were collected from: Susrauli (Lanes 1 and 2), Kalpipara (Lane 3), Chichdi (Lane 5), and germplasm accessions from our experimental field (Lanes 6 and 7). DNA isolated from virus-free kenaf samples, grown under controlled glasshouse conditions, served as healthy control (Lane 8).

 

Fig. 3. PCR products obtained using (A) the Bhendi YVMV coat protein primers and (B) the universal DNA β primers with symptomatic (of YVM disease) samples obtained from: Susrauli (Lanes 1 and 2), Kalpipara (Lanes 3 and 4), Chichdi (Lanes 5 and 6), and germplasm accessions from experimental field (Lanes 7 through 11). A positive control for the PCR, using the East Indian isolate of this virus, was included in Lane 12.


The present investigation has thus revealed the occurrence and distribution of yellow vein mosaic disease of kenaf in different areas of northern India. The disease produced marked height reduction and directly affected fibre production. Germplasm accession KIN 108 was found to be highly susceptible to YVMD. The DNA β satellite molecule and coat protein gene have been found associated with different begomoviruses such as Ageratum yellow vein virus (11), Cotton leaf curl virus (9), and Tomato yellow leaf curl virus (6). These viruses are spreading rapidly in a devastating manner throughout different crops and weed species in various parts of the world. Hybridization with the DNA A probe and positive PCR amplification with primers for DNA β and the Bhendi YVMV coat protein gene, as evident in the present investigation, confirmed the association of begomovirus with this disease.


Acknowledgments

Authors are grateful to the Director of CRIJAF for his keen interest during the present investigation and Dr. V.G. Malathi for providing the DNA A probe. The first two authors are also grateful to ICAR for providing financial assistance during the tenure of which this work was carried out.


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