Immunological and molecular detection of banana viruses and production of disease free planting materials
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Date
2014
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Department of Plant Pathology, College of Agriculture, Vellayani
Abstract
The study entitled "Immunological and molecular detection of banana viruses
and production of disease free planting materials" was conducted in College of
. Agriculture, Vellayani, and Thiruvananthapuram during !he period of2011-2014.
Symptomatological studies showed that the characteristics symptoms caused
by BBTV were small, brittle leaves with thickened veins which remained bunched at
the top of the pseudostem. Plants with early infection did not produce fruits, where
plants with later infection produce bunch with reduced size, weight and mishapen
fingers. The characteristic symptoms caused by BBrMV were reddish spindle shaped
lesion in the pseudostem, flag leaf sheath, leaf petiole, and bract. Leaves of infected
plants showed characteristic chlorotic spindle shaped lesion on the leaf lamina. The
characteristic symptoms of BSV were chlorotic streaks in the leaf lamina. Later the
chlorotic streaks became necrotic. The characteristic symptom of CMV was mosaic
pattern in the leaf lamina.
The pathophysiological studies conducted in cultivar Nendran revealed that
there was significant difference in carbohydrate, chlorophyll, protein and phenol
content in infected plant when compared to healthy ones .. The activity of defence
related enzymes like peroxidase, polyphenol oxidase and phenylalanine
ammonialyase were found to be more in infected plants. Electrophoretic analysis of
protein in virus infected samples through SDS-PAGE revealed the presence of an
additional protein in the protein profile. The protein profile of BBTV infected sample
showed one extra band with molecular weight of 20 kDa, BBrMV infected sample
showed three additional protein band with molecular weight of 38 kDa, 29 kDa and
22 kDa, BSV infected sample showed three additional proteins with molecular weight
of 25 kDa, 19 kDa, and 12 kDa, CMV infected sample showed one extra band with
molecular weight of 25 kDa. Electrophoretic analysis of isozyme though native gel
revealed the increased action of peroxidase enzyme in infected sample.
Detection of VIruS infecting banana was carried out using varIOUS
immunological techniques such as DAC-ELISA and DIBA using polyclonal
antiserum (Agdia) and monoclonal antiserum. Both the techniques were found to be
efficient in detecting virus infecting banana. Molecular diagnosis of the BBTV was
carried out using CP gene and replicase gene specific primers. PCR product with
amplicon size of about 530 bp was observed for coat protein gene specific primer
where 237 bp was observed for replicase gene specific primer. Molecular diagnosis
of BSV was carried out using two CP gene specific primers resulted in PCR product
with amplicon size of 664 bp and 730 bp. Molecular diagnosis of CMV was canied
out using CP gene specific primer resulted in PCR product with an amplicon size of
687 bp. CP gene specific primer for BBrMV did not give positive result. Cluster
dendrogram analysis revealed that the BBTV isolate was mostly related to BBTV
coat protein gene of Burundi isolate, BSV isolate was mostly related to banana streak
virus isolate Trichi, CMV isolate was mostly related to cucumber mosaic virus isolate
Trichi coat protein gene.
The meristematic region of the virus infected banana suckers were excised
and inoculated to MS media with BAP and NAA. The regeneration of plants from
meristematic region was difficult because of high phenol production and
contamination by endogenous bacteria. Meristem culture eliminated BBTV, CMV
and BBrMV but not the BSV.
Based on the research result, the banana VIruses can be detected usmg
immunological and molecular technique and the meristem culture can eliminate all
the banana viruses except BSV.
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173373