PhD

Sugarcane (Saccharum officinarum) is a monocotyledonous perennial cash
crop cultivated worldwide both under tropical and sub tropical conditions. It is being
cultivated in more than 120 countries in the world. Like any other crops, it is also
susceptible to biotic stress. Of which, diseases caused by viruses not only pose
serious threat to sugarcane cultivation but also result in deterioration and exclusion of
elite varieties of the germplasm. One of the major viral disease which affects global
exchange of sugarcane germplasm is leaf fleck disease caused by Sugarcane
bacilliform virus (SCBV).
The research project entitled ‘Characterization and management of Sugarcane
bacilliform virus causing leaf fleck in sugarcane’ was initiated with purposive
sampling surveys in selected sugarcane fields in districts of Kerala and Tamil Nadu in
order to document the symptoms under natural conditions, to assess the disease
incidence, severity and to collect infected samples for further studies. The per cent
disease incidence of the leaf fleck disease in Kerala ranged from 12 to 51 per cent
whereas severity ranged from 10 to 36.5%. In Tamil Nadu the per cent disease
incidence ranged from 28 to 56 per cent while severity ranged from 28 to 50.41%.
Major symptoms observed on leaves were mottling, chlorotic flecks, chlorotic
patches streaks and stripes with general yellowing of the canopy. In the case of
severely affected clones, there was reduction in tillering, internodal length, number of
internodes and appearance of deep longitudinal cracks. In highly susceptible clones,
stunted growth with bunchy top appearance was noticed. On the basis of phenotypic
variability of symptom expression, genotypes were classified into five groups. The
development of the symptoms was also studied under artificial condition through
insect transmission of the virus using pink mealy bug, Saccharicoccussacchari.
Morphological characterisation of the virus done using electron microscopy revealed
the presence of bacilliform virus particles of size 30 X 130–150 nm which indicated
that the virus belongs to genus BADNA and family Caulimoviridae and the etiology
of the disease was confirmed as Sugarcane bacilliform virus. The molecular detection
of SCBV was also standardized through polymerase chain reaction (PCR). PCR
amplification of RNaseH/RT gene was done using BADNA specific and SCBV129
specific primers. The amplicons were sequenced and in silco analysis of sequences
showed sequence homology of 99 to 100 percent identity to SCBV.
Widespread occurrence of the disease was observed even in the early
generation of varietal development and in newly developed varieties. The
transmission of the virus was suspected through true seed (fluff) developed by
biparental crossing during sugarcane varietal development programme. Hence, the
study was conducted to establish possible transmission of the virus from sugarcane
parents to their progenies and the role of maternal and paternal parents in disease
transmission through true seeds to the progenies. Samples from eight months old
seedlings, three months old seedlings and parental clones were tested positive to the
virus in PCR assays. Real time PCR was also standardized to assay these clones.
Immunodiagnostic technique was validated using DAC ELISA. The technique of
immunocapture PCR was also standardized. Minimal dilution of antisera with which
SCBV could be detected was 2:1000 (V/V). Plant extract (antigen) at a dilution of 1:5
was found to be optimal for the detection of SCBV. Molecular detection of SCBV
from mealy bug vector was also standardized. Both phenotypic and molecular
methods were utilized to identify potential sources of natural resistance against
SCBV. Based on the severity of symptom expression and PCR assays these were
further classified as highly susceptible (HS), moderately susceptible (MS) moderately
resistant (MR) and resistant (R).
For generation of RNAi hair pin construct, initially forward (SF) and reverse
primer (SR) were used to amplify 700 bp fragment of RT/RNase H gene to be cloned
in sense orientation of the vector, pHANNIBAL. The linearized vector and the insert
were ligated, and the ligation mixture was used to transform competent cells of
Escherichia coli and the transformants were selected. Later antisense forward (AF)
and reverse (AR) primer pairswere used to amplify 700 bp fragment of RT/RNase H
gene to be cloned in antisense orientation. PCR product ligated into antisense
direction of the vector and transformed into competent cells of E. coli. The
recombinant pHANNIBAL vector was digested with restriction enzymes. The
recombinant pHANNIBAL vector harbouringRNase H /RT gene was released from
the vector through Not I site and subcloned into plant expression binary vector. Thus,
cassette for RNA silencing was prepared.130
Meristem tip culture was also standardized with antiviral chemical tenofovir.
Recovery percentage of meristem varied from 70 to 75 per cent and the viral load was
quantified using real time PCR.
The outcome of the study would facilitate early detection and elimination of
the source of infection and prevent the spread of the disease in the field. Information
generated in the study could be utilized while planning biparental crossing and reduce
the spread of the virus in varietal development programmes. The hair pin construct
developed in this study could be further utilized to generate transgenic disease
resistant plants.