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Detection of Virus Particles and Double-stranded Rna in Dieback Affected Dalbergia Sissoo Roxb. from Bangladesh

Stephanie Vogel
Molecular Phytopathology and Genetics, University of Hamburg, Biocentre Klein Flottbek, Ohnhorststrasse 18, D-22609 Hamburg, Germany

Hanny Tantau
Molecular Phytopathology and Genetics, University of Hamburg, Biocentre Klein Flottbek, Ohnhorststrasse 18, D-22609 Hamburg, Germany

Nicole Mielke-Ehret
Molecular Phytopathology and Genetics, University of Hamburg, Biocentre Klein Flottbek, Ohnhorststrasse 18, D-22609 Hamburg, Germany

MI Hoque
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

RH Sarker
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

ML Saha
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh.

Sk Shamimul Alam
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

M Salim Khan
Tissue Culture Laboratories, BCSIR, Dhaka-1205, Bangladesh.

Hans-Peter Muhlbach
Molecular Phytopathology and Genetics, University of Hamburg, Biocentre Klein Flottbek, Ohnhorststrasse 18, D-22609 Hamburg, Germany

Abstract/ Executive Summary:

Dieback of Dalbergia sissoo Roxb. (sissoo) is a disastrous disease, which has destroyed millions of forest trees in South Asia. Plant pathogenic fungi and bacteria were found associated with diseased trees, but the causative disease agent has not yet been identified unequivocally. In order to examine whether plant viruses could be detected in diseased trees, present author applied bioassays, transmission electron microscopy and double-stranded RNA (dsRNA) isolation, followed by cDNA cloning. Unknown virus particles and a complex pattern of dsRNA could be detected in leaf homogenates from dieback affected sissoo trees, in contrast to an uninfected tree. Disease associated dsRNA bands ranged in size from 0.6 to 3.5 kbp, and a cDNA clone derived from dsRNA showed partial sequence homology to a plant viral RNA polymerase. Our data indicated for the first time the presence of unknown virus(es) in dieback-affected Dalbergia sissoo in Bangladesh.

Keywords: Dieback disease, Sissoo, Double stranded RNA, Reverse transcription, DOP-PCR

Location: Various sites in Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Sisso

Objectives:

To find out the presence of plant viruses in diseased trees of Sissoo Roxb.

Methodology:

Leaves of dieback affected Dalbergia sissoo trees were collected from various sites in Bangladesh. Mixed leaf samples from various parts of each individual tree were used in this investigation. Samples P6 and P9 were collected from Sirajganj in the Eastern Rajshahi division, while the others were sampled at sites in the Dhaka division, P8 at Tangail, P20 and P22 at Mirzapur, and P29 - P33 at Pubail/Tangail. Severity of sissoo dieback disease was assessed. ‘No’ disease means no typical symptoms of dieback, ‘mild’ disease with chlorosis and necrosis on leaves as well as initial crown transparency, ‘medium’ disease with strong leaf necrosis, advanced crown transparency, gummosis and necrosis (black spots) at the bottom parts of the trunk, while ‘severe’ disease means almost all foliage and most of twigs and branches of higher order lost (stagheadedness), and black spots at the trunk up to at least 2 m height. Collected leaves were kept cool and wet in sealed plastic bags for maximum 48 hours during transport and finally stored at 70⁰ C. For bioassays the following herbaceous indicator plants were used: Nicotiana tabacum var. ‘Xanthi’ and ‘Samsun’, Chenopodium quinoa and Phaseolus vulgaris. Seeds were provided by Dr. C. Heinze (Department of Biology, University of Hamburg) or were obtained from the seed collection of the Botanical Garden, University of Hamburg. Plants were grown in the greenhouse under natural light at temperatures ranging from 16 to 30° C. Bioassays were performed with homogenates made from dieback affected sissoo leaves in the following homogenisation buffers: 0.1 M KH2PO4, pH 7.0; 0.1 M sodium phosphate buffer (pH 7.0); 0.01 M potassium phosphate buffer (pH 7.0), containing 0.01 M Na₂SO₃. Sissoo leaves (1 g) were first ground in liquid nitrogen to a fine powder. Subsequently 2 or 20 ml of the corresponding buffer were added, homogenized further, and used for inoculation of leaves of indicator plants dusted with carborundum (P320). Aliquots of 30 μl of the original homogenate or in a dilution of 1/10 with the corresponding homogenization buffer were used per leaf. Alternatively, leaves of indicator plants were inoculated using the dry homogenized powder of sissoo leaves after grinding in liquid nitrogen without any buffer added, called dry inoculation according to Dr. Anan Kadri (unpublished, University of Stuttgart, Institute of Biology, Pfaffenwaldring 57, 70569 Stuttgart, Germany). For dry inoculation corresponding quantities of dry homogenates were used. The inocula were softly distributed on the leaves using sterile, frosted microscopic slides. Plants were kept for five weeks in the greenhouse under the same conditions as described above and visually inspected every third day. For TEM studies aliquots of 0.5 g of sissoo leaves were homogenized in 100 ml of 0.1 M sodium phosphate buffer, pH 7.0, or in 0.1 M potassium phosphate, pH 7.0. Hexagonal nickel grids (300 mesh) coated with 0.5% pioloform (Plano GmBH Wetzlar, Germany) were placed onto 15 μl drops of homogenate and incubated 5 min at 20° C. The grids were washed with 40 μl water and treated with 2% uranyl acetate solution for 2 min. The grids were inspected using TEM Leo 906 E.

Source of Information: Bangladesh J. Bot. 40(1): 57-65, 2011 (June)

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

It is still unknown to what extent a putative virus infection could contribute to the dieback disease of D. sissoo. There are very limited reports on the association of plant viruses with the dieback syndrome of woody plants.

Knowledge Management: Journal