BIOLOGICAL AND MOLECULAR CHARACTERIZATION OF APPLE STEM PITTING VIRUS
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Date
2015
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Abstract
ABSTRACT
Surveys conducted during 2011 and 2012 in different apple growing areas of Himachal
Pradesh, Uttarakhand and Jammu and Kashmir revealed viral disease incidence ranging from 3 to 60
per cent. Chlorotic spots coupled with necrotic lesions on apple leaves were the predominant
symptoms. Orchards located at Regional Horticulture Research Station, Mashobra and Dhangvi
village of Kotkhai area were selected for conducting biological and serological detection of apple
stem pitting virus (ASPV). Serological detection through DAC and DAS-ELISA resulted in the
detection of apple stem pitting virus (ASPV) either alone or in mixed infection with apple chlorotic
leaf spot virus (ACLSV), apple mosaic virus (ApMV) and apple stem grooving virus (ASGV).
Biological detection of one of the ASPV seropositive isolate on herbaceous hosts resulted in the
production of symptoms on Chenopodium quinoa, C. amaranticolor, Nicotiana tabacum var. White
Burley and Phaseolus vulgaris. Detection on woody indicators (Malus pumila Spy 227 and Jay
Darling) under field conditions through double grafting of inoculators and indicator budwood resulted
in the production of typical viral symptoms on leaves in Jay Darling and Spy 227indicators. Graft
incompatibility and necrotic symptoms were produced at the graft union of Spy 227 indicator
followed by decline and dieback. Leaf samples drawn during March to May months were found
suitable for the ELISA of ASPV whereas petals were the best source in addition to seropositive
detection of ASPV in anthers and sepals. Association of ASPV with viral symptoms in apple was also
confirmed by RT-PCR assays. Internal control primers along with coat protein gene specific primers
were used to overcome the problem of false negative results. Molecular characterization of full coat
protein gene of 3 ASPV isolates, 5 ASGV isolates and partial characterization of coat protein gene of
10 ACLSV isolates was carried out and the resultant sequences were then submitted to NCBI.
Phylogenetic analysis of sequences showed the presence of variability among isolates and confirmed
that there is no correlation between the geographic origin and genetic diversity of these isolates,
which does not allow drawing conclusion on their origin and dispersion. Serological indexing resulted
in the selection of 13 trees of 7 cultivars free from infection of ASGV, ACLSV, ASPV and ApMV in
ELISA test of 36 symptomless trees of 12 cultivars
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Keywords
apples, diseases, biological phenomena, viruses, planting, fruits, elisa, storage structures, pcr, proteins, APPLE STEM PITTING VIRUS