BSc - MSc (Int.)

The present research work entitled Identification of virus(es) and phytoplasma
associated with mixed infection of mosaic and yellowing disease in Elephant foot
yam was carried out at ICAR CTCRI, Sreekaryam during the year 2021-2022, with
the primary objective of identification and characterization of virus(es) /
phytoplasma causing mixed infection of mosaic and yellowing in elephant foot yam.
Elephant foot yam (EFY) plants having symptoms such as mosaic, yellowing, vein
yellowing, mosaic with yellowing, curling etc. were collected from different fields of
CTCRI as well as from KVK (Kottayam) and Erode (Tamil Nādu). Genomic DNA
and RNA were isolated from symptomatic leaf samples using CTAB method.
Detection of phytoplasma was carried out by PCR using universal primers P1/P7
followed by nested PCR using R16F2n/ R16R2. The expected band size of 1.2kb has
not been obtained from any of the infected leaf samples. Detection of DNA virus was
carried out using primers CLTY F / CLTY R which are begomovirus specific. The
expected band size of 500bp was obtained. RNA virus was detected using RT PCR.
The cDNA synthesized using Verso cDNA synthesis kit was amplified using
different sets of primers viz., GBNV F/GBNV R, MKTOSPOSGF/MKTOSPOSGR,
DsMV RPA1 F/ DsMV RPA1 R, MKGNNSF/WEICNR, PRSF8313/PRSR9420,
ChVMCPF/ChVMCPR, ChVMF2393/ChVMF3560 and BCWZ F/BCWZ R. An
amplicon size of 400bp was obtained from symptomatic leaf samples collected from
CTCRI, KVK Kottayam and Tamil Nadu using DsMV RPA primers. A specific
band of 1.2kb size and nonspecific band of size 500bp was obtained using potyvirus
genus specific primer MKGNNSF/WEICNR and nonspecific band of size 300bp was
obtained using Groundnut bud necrosis virus (GBNV) specific primer
GBNVF/GBNVR .
Positive amplicons were eluted from the gel and sequenced. Eluted amplicons were
also ligated to pTZ57R/T vector, cloned to competent DH5α E.coli cells and screened
through Blue-white screening on LB ampicillin/X-Gal plate. The white transformed
colonies that gave an expected band size in Colony PCR were chosen forsequencing.
87
BLASTn analysis revealed that the DNA virus sequences isolated from the
infected leaf samples from CTCRI had 91.77 per cent similarity with Sri
Lankan cassava mosaic virus (SLCMV). PCR analysis was carried out using
different sets of primers viz., SL RPA F/R, SLRP Rep F/R for further
confirmation of SLCMV and an amplicon of size 1000bp and 734 bp was
obtained respectively. BLASTn analysis of RNA viral sequences obtained by
using BCWZ F and BCWZ R primers revealed that the infected leaf samples
from CTCRI, Tamil Nadu and KVK had only 74 per cent identity with
Moroccan watermelon mosaic virus and those obtained by using potyvirus
genus specific primers MKGNNSF/WEICNR had only 74 per cent similarity
with Dasheen Mosaic Virus. Phytoplasma has not been detected in any of the
collected symptomatic samples.
In conclusion, the characterization of DNA and RNA viruses from EFY leaf
samples revealed that the Elephant foot yam plants were subjected to mixed
infection. The Sequence information of DNA virus isolate reveals the infection
of SLCMV in leaf samples and that of RNA virus isolates obtained from leaf
samples reveals the probability of infection with viruses that belong to families
other than potyviridae.