STUDIES ON VIRUS(ES) ASSOCIATED WITH GRAPEVINE LEAFROLL DISEASE IN INDIA
Loading...
Date
2013
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
IARI DIVISION OF PLANT PATHOLOGY INDIAN AGRICULTURAL RESEARCH INSTITUTE NEW DELHI
Abstract
Studies on virus(es) associated with grapevine leafroll disease in India
Grapevine leafroll disease (GLD) is a complex viral disease of grapevine accounting for
more than 60 % of yield losses due to viruses. It is caused by eleven distinct filamentous
viruses (recently proposed to be recognized as genetically five distinct viruses)
belonging to the family Closteroviridae. These viruses are named according to their
sequential discovery as Grapevine leafroll-associated virus 1, -2, -3 and so on (GLRaV-
1, -2, -3 and so on). Despite being known to be present since one decade in Indian
vineyards, no authentic study on GLD has been carried out. On the basis of serology,
electron microscopy, RT-PCR (reverse transcriptase polymerase chain reaction) and
sequencing, we report for the first time, the association of GLRaV-1 and -3 with GLD
symptomatic vines of the vineyards of Nashik and Pune regions of India. These two
viruses could not be detected from the vineyards of Koppal (Karnataka), Srinagar
(Jammu and Kashmir) and Delhi regions. The p24 gene of two isolates of GLRaV-1 was
characterized to be of 630 base pairs (bp) and based on p24 gene phylogeny the global
isolates of GLRaV-1 segregated into three distinct groups. Two Indian isolates of
GLRaV-1 clustered in group 1 with Claretvine and RRG isolates from USA. Partial
HSP70h (heat shock protein 70 homologue) and entire p19.7 genes of 546 bp and 540
bp, respectively were characterized from the eleven isolates of GLRaV-3. Based on
p19.7 and partial HSP70h phylogenies the global isolates of GLRaV-3 segregated into
six clusters irrespective of their geographic origins. Most of the Indian isolates clustered
in group 2 of the global isolates but isolates Revella-4/12, Revella-4/14, KS-B-7 and
Nashik showed discordant grouping behaviour based on different gene based
phylogenies. Globally, this was the first such report of incongruent grouping patterns of
different isolates of GLRaV-3 based on different genes. p19.7 gene based recombinant
analysis revealed Nashik isolate as a putative recombinant with parental sequences of 6-
18 from USA and Manjri-A2-38/36 from India. In recombination analysis of partial
HSP70h gene, none of the Indian isolates of GLRaV-3 was identified to be as
recombinant. An estimation of the normalized value for the ratio of nonsynonymous
160
substitution per nonsynonymous site to synonymous substitutions per synonymous site
revealed the different purifying selection pressures acting on both p19.7 and HSP70h
genes, with HSP70h being subjected to stronger functional constraint than p19.7 gene.
CP (coat protein) of Nashik isolate from cultivar Cabernet Souvignon was characterized
and found to be of 942 bp. In CP gene based clustering Nashik isolate clustered with the
group 2 of global isolates. The CP gene of Nashik isolate was over-expressed as
recombinant fusion protein at in vitro condition and used as immunogen for the
production of polyclonal antisera. The produced antisera was purified and conjugated
with enzyme alkaline phosphatase (ALP) to generate GLRaV-3 specific conjugate or
secondary antisera. These two immunoreagents could detect the presence of GLRaV-3
in infected sample (1:4 dilutions) at 1:8000 and 1:10,000 dilutions of primary and
secondary antisera, respectively. The primary antiserum was also used to develop a
modified immuno-capture RT-PCR (IC-RT-PCR) for the sensitive identification of
GLRaV-3. The generated immunoreagents can be used for indexing of producing
GLRaV-3 free grapevine propagating materials and in GLRaV-3 specific quarantine
certification programmes of the country. An attempt to trace out the exact route of entry
of GLD to India was not successful because of poor documentation and inefficient
quarantine procedures followed during and after the import of grapevine propagating
materials. These studies provide a base for the better understanding of biology and
epidemiology of grapevine leafroll disease across grape growing regions of India. The
findings also draw the attention for making Indian quarantine system more stringent
after revisiting the existing one so that the future introduction of viral diseases can be
prevented.
Description
Keywords
fruit crops, biological phenomena, proteins, genes, diseases, viruses, fruit products, fruits, productivity, land resources