TY  - BOOK
AU  - Geethu Gokul G
AU  - Vimi Louis (Guide)
TI  - Molecular characterization of erwinia species causing rhizome rot in banana
U1  - 660.6 
PY  - 2017///
CY  - Vellanikkara
PB  - Centre for Plant Biotechnology and Molecular Biology, College of Horticulture
KW  - Plant Biotechnology and Molecular Biology
N1  - MSc
N2  - The rhizome rot or tip over disease affecting the rhizome caused by Erwinia spp.
is a major and emerging issue which leads to a substantial economic loss in banana. The
widely cultivatedbanana varieties like Grand Naine, Rasthali and Nendran are very
susceptible to the disease. Different Erwinia species are reported to cause the disease.
Understanding the correct etiology of the disease and variability of the pathogen based on
molecular characterization is very much important in crop improvement programmes.
Isolation of pathogen from infected rhizomes from different locations yielded 18
bacterial isolates. In nutrient agar medium, the bacterial colonies were small, cream to
yellow in colour, slightly raised and mucoid.The bacterium when streaked on the crystal
violet pectate medium produced fissures or pits characteristic of Pectobacterium spp.
The pathogenicity tests were carried out by inoculation in rhizome bits (in-vitro),
rhizome of three months old plants (in-vivo) and pseudostem of two months old tissue
culture plants (in-vivo) of banana var. Nendran. Inoculation of rhizome bits and
inoculation at collar region of three months old banana plants produced rotting symptoms
after eight and 37 days respectively. Pseudostem inoculation of two months old tissue
culture plants var. Nendran also produced characteristic rotting symptoms after six days.
After proving pathogenicity, the 18 bacterial isolates were characterized by
cultural, morphological, biochemical, physiological and molecular methods. Cultural
characterization was carried out by using nutrient agar (NA), yeast extract glucose
calcium carbonate (YGC), Logan’s medium and nutrient broth. On nutrient agar, cream to
slightly yellowish, mucoid, slightly raised convex colonies with entire margin varying in
size were formed. The bacterial growth in the nutrient broth was recorded at different
intervals and variation in OD values was observed between isolates. On YGC medium,
cream to yellow coloured semi mucoid colonies were formed with a clear zone around
each colony after 24 h of inoculation. Small bubbles were observed on the top of each
bacterial colony due to the emission of CO2 which is the characteristic feature of
Pectobacterium spp. In Logan’s medium small to medium colonies of 1.6 to 2 mm
diameter with purple colour colonies were formed, which is the characteristic feature of
Erwinia carotovora/Pectobacteriumcarotovorum.
Morphological characterization of the bacterium was carried out by staining viz.,
Gram staining, capsule staining, flagellar staining. The bacteria appeared as red coloured
short rods indicating the Gram negative nature with 3.10 μm-0.95 μm x 1.10 μm-0.57 μm
in size. The bacterium was non-capsular in nature with peritrichous flagella.
Biochemical characterization of the bacterial isolates were carried out by
conducting different test viz., solubility in KOH, potato soft rot test, carrot soft rot test,
intrinsic antibiotic resistance, growth in three and four per cent NaCl, growth in CVP
medium and catalase test. The bacteria, in three per cent KOH produced viscous filament.
The potato and carrot slices inoculated with bacterial culture produced rotting symptoms
with the emission of characteristic foul smell in three to four days and five to seven days
respectively and variation was observed between isolates. The bacterial isolates showed
resistance to erythromycin and susceptibility to streptomycin. Bacterial growth was
observed in the inoculated peptone broth containing three and four percent NaCl. The
bacterial culture inoculated on CVP medium produced characteristic fissures or
depressions due to pectate degradation. Catalase test was positive for all the isolates.
Physiological characterization of the bacterium was carried out by growing at
different temperature and pH. The maximum growth was recorded at 27oC and at pH 7.0.
Based on the results of cultural, morphological, biochemical and physiological
characterization of the 18 bacterial isolates, dendrogram was constructed which classified
the isolates into six groups. The molecular characterization of bacterial isolates was
carried out by total genomic DNA isolation, PCR amplification of 16S rRNA gene and
sequencing. Colony PCR of 18 bacterial isolates were also carried out. The sequences
obtained for six isolates, representative of each group were used for homology analysis,
phylogenetic analysis and phylogenetic tree construction. All the six groups of bacterial
isolates were belonged to Pectobacterium carotovorum but variation was observed at
subspecies level. Out of the six groups, there were two subspecies viz., Pectobacterium
carotovorum ssp. carotovorum and Pectobacterium carotovorum ssp. brasiliense. The
Barcode
gap
of
Pectobacterium
carotovorumandPectobacteriumcarotovorumssp.brasiliense
sequences were deposited in the NCBI GenBank.
carotovorum
was
assessed
ssp.
and
the

UR  - http://krishikosh.egranth.ac.in/handle/1/5810132644
ER  -