SALICYLIC ACID

212 Pages·2009·1.19 MB·English

SALICYLIC ACID (ELICITOR) INDUCED HYPERSENSITIVE
RESPONSES IN Capsicum annum. L. INFECTED WITH
Colletotrichum capsici (Syd.) BUTLER & BISBY.

Thesis submitted to the Pondicherry University
for the award of the degree of

DOCTOR OF PHILOSOPHY
in

LIFE SCIENCES (BIOCHEMISTRY AND MOLECULAR
BIOLOGY)

Submitted by

S.N.RAJESWARI, M.Sc., M.Phil.,

DEPARTMENT OF BIOCHEMISTRY & MOLECULAR
BIOLOGY
PONDICHERRY UNIVERSITY
PONDICHERRY 605 014

March 2009
PONDICHERRY UNIVERSITY
DEPARTMENT OF BIOCHEMISTRY
AND MOLECULAR BIOLOGY
Dr. B. KANNABIRAN
Reader
CERTIFICATE
I hereby certify that this thesis entitled “SALICYLIC ACID (ELICITOR)
INDUCED HYPERSENSITIVE RESPONSES IN Capsicum annum L.
INFECTED with Colletotrichum capsici (Syd.) BUTLER & BISBY” submitted
for the award of degree of Doctor of Philosophy in Biochemistry and Molecular
Biology is a bonafide research work done by the candidate Ms.S.N.RAJESWARI
during the period of her study under my guidance at the Department of Biochemistry
and Molecular Biology, Pondicherry University, Pondicherry and the thesis has not
previously formed the basis for the award of any Degree or Diploma or other similar
titles.

I further certify that the thesis represents the independent work of the candidate.
Professor and Head (Dr. B. KANNABIRAN)
Department of Biological Sciences Research supervisor

Dean
School of Life Sciences
Place : Pondicherry
Date :
PONDICHERRY UNIVERSITY
DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY
DECLARATION
I hereby declare that this thesis entitled “SALICYLIC ACID (ELICITOR)
INDUCED HYPERSENSITIVE RESPONSES IN Capsicum annum L.
INFECTED WITH Colletotrichum capsici (Syd.) BUTLER & BISBY” is a record
of research work done by me under the guidance of Dr. B. Kannabiran, Reader in
Department of Biochemistry and Molecular Biology, Pondicherry University,
Pondicherry submitted for the award of degree of Doctor of Philosophy in Life
Sciences (Biochemistry and Molecular Biology) and has not previously formed the
basis for the award of any Degree or Diploma or other similar titles.

Puducherry S.N.Rajeswari

Date: Research Scholar
CONTENTS

List of Tables II
List of Figures IV
List of Plates V
List of Abbreviations VI
Introduction 01
Materials and Methods 29
Results 74
Discussion 89
Summary 130
Appendix 134
References 138
I
LIST OF TABLES
No. TITLE

Table 1 Effect of salicylic acid (Elicitor), culture filtrate of G.virens and bavistin
on the conidial germination and radial mycelial growth of Colletotrichum
capsici (in vitro).

Table 2 Effect of salicylic acid (Elicitor), culture filtrate of G.virens and bavistin
on the activity of polymethyl galacturonase (exo-PMG) of Colletotrichum
capsici (in vitro).

Table 3 Effect of salicylic acid, G. virens and bavistin on the activity of pectin
transeliminase (exo-PTE) of Colletotrichum capsici (in vitro).

Table 4 Effect of salicylic acid and formulation of G.virens, and bavistin on the
activity of 1, 4 β-exo-glucanase (C ) of Colletotrichum capsici (in vitro).
1

Table 5 Effect of salicylic acid and formulation of G. virens, and bavistin on the
activity of endo β-1,4-glucanase (exo C ) of Colletotrichum capsici. (in
x
vitro).

Table 6 Effect of salicylic acid and formulation G. virens, and bavistin on the
activity of cellobiase of Colletotrichum capsici (in vitro)

Table 7 Effect of the foliar spray of salicylic acid (10mM) on total chlorophyll and
carotenoid content of Capsicum annum leaves (in vivo).

Table 8 Effect of the foliar spray of salicylic acid (10mM) on DNA and RNA
content of Capsicum annum leaves (in vivo).

Table 9 Effect of the foliar spray of salicylic acid (10mM) on protein, amino acid
and proline content (mg/g f.w) in the leaves of Capsicum annum (in vivo).

II
Table 10 Effect of the foliar spray of salicylic acid (10mM) on carbohydrate
content in the leaves of Capsicum annum (in vivo).

Table 11 Effect of the foliar spray of salicylic acid (10mM) on sucrose and strach
content in the leaves of Capsicum annum (in vivo).

Table 12 Effect of the foliar spray of salicylic acid (10mM) on total nitrogen content
in the leaves of Capsicum annum (in vivo)

Table 13 Effect of the foliar spray of salicylic acid (10mM) on amino nitrogen
content in the leaves of Capsicum annum (in vivo).

Table 14 Effect of the foliar spray of salicylic acid (10mM) on O.D and Total
Phenol in the leaves of Capsicum annum. (in vivo)

Molecular Biology

Table 1 Summary of the Purification of Polygalaturonase inhibiting protein (PGIP)
from Capsicum annum leaves.

III
LIST OF FIGURES
No. TITLE

Fig. 1 Effect of salicylic acid (Elicitor), culture filtrate of G.virens and bavistin
on the activity of pectinolytic enzyme-pectin methyl esterase (PME) of
Colletotrichum capsici. (in vitro)

Fig. 2 Effect of salicylic acid (Elicitor), culture filtrate of G.virens and bavistin
on the activity of polymethyl galacturonase (endo-PMG) of Colletotrichum
capsici. (in vitro)

Fig. 3 Effect of salicylic acid G. virens and bavistin on the activity of pectin
transeliminase (endo-PTE) of Colletotrichum capsici (in vitro)

Fig. 4 Effect of salicylic acid G. virens and bavistin on the activity of endo B-1,
4-glucanase (endo Cx) of Colletotrichum capsici (in vitro)

Fig. 5 Effect of the foliar spray of salicylic acid (10mM) on the electrolytic
leakage from Capsicum annum leaves (in vivo)

Fig. 6 Effect of the foliar spray of salicylic acid (10mM) on superoxide
dismutase (SOD) activity in Capsicum annum leaves (in vivo)

Fig. 7 Effect of the foliar spray of salicylic acid (10mM) on catalase activity
in Capsicum annum leaves (in vivo)

Fig. 8 Effect of the foliar spray of salicylic acid (10mM) on peroxidase activity in
Capsicum annum leaves (in vivo)

Fig. 9 Effect of the foliar spray of salicylic acid (10mM) on polyphenol oxidase
activity in Capsicum annum leaves (in vivo)

Fig. 10 Effect of the foliar spray of salicylic acid (10mM) on nitrate reductase
(NR) activity in Capsicum annum leaves (in vivo)

Fig. 11 Effect of the foliar spray of salicylic acid (10mM) on the activity of ß 1,3
glucanse (PR-protein) (in vivo)
IV
Fig. 12 Effect of the foliar spray of salicylic acid (10mM) on phenyl alanine
ammonia-lyase (PAL) activity in Capsicum annum leaves (in vivo)

Fig. 13 Effect of foliar spray of salicylic acid on total concentration of endogenous
salicylic acid in Capsicum annum leaves (in vivo)

Molecular Biology

Fig. 1 Ion exchange column chromatography (Carboxyl methyl-cellulose) of
Polygalacturonase inhibting protein activity (PGIP)

Fig. 2A Gel permeation column chromatography of PGIP from fractions 7-18 of
the ion exchange separation. (Fig.1)

Fig. 2B Gel permeation column chromatography of PGIP from fractions 27-40 of
the ion exchange (Fig.1)

Fig. 3 Inhibition of C.capsici PGs by increasing amounts of chilli PGIP purified
from capsicum annum leaves

LIST OF PLATES
No TITLE

Plate 1 SDS-PAGE gel of proteins in fractions from the gel permeation
chromatography separations of chilli PGIP

Plate 2 PG agarose cup plate assay

V
LIST OF ABBREVIATIONS
µg : microgram
0C : degree centigrade
ANOVA : analysis of variance
APS : ammonium persulphate
ASA : acetylsalicylic acid
BSA : Bovine serum albumin
CAT : Catalase
CBB R-250 : coomassie brilliant blue
Da : Dalton
DMF : N,N-Dimethyl formamide
DMF : (N,N-Dimethyl formamide)
DTT : Dithiothreitol
EDTA : Ethylene diamine tetra acetic acid
et al. : and others
Fig. : figure
H O hydrogen peroxide
2 2 :
HCl : hydrochloric acid
HEPES : (N-2-hydroxyethyl piperazine-N′-2-ethanesulphonic acid)
HR : hypersensitive response
M : molar
mg : milligram
mM : millimolar
MW : molecular weight
NADPH : β-Nicotinamide adenine dinucleotide phosphate reduced tetrasodium
salt
nm : nanometer
NRA : Nitrate reductase activity
NTB : Nitroblue tetrazolium
O.D : optical density
PAL : phenylalanine ammonia-lyase
PCA : Perchloric acid
VI
pH : hydrogen ion concentration
PIRMG : Percent inhibition of radial mycelial growth.
PMSF : Phenyl methyl sulphonyl fluoride
PO : Peroxidase
PPO : Polyphenol oxidase
PR : pathogenesis related
PVP : Polyvinyl pyrrolidone phosphate
SA : salicylic acid
SAR : systemic acquired resistance
SAU : Specific activity units
SDS-PAGE : Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
SIR : systemic induced resistance
SOD : Superoxide dismutase
sp. : species (single)
spp. : species (plural)
TMRT : Tukey’s HSD multiple range test
TBA : Thiobarbituric acid
TCA : Trichloro acetic acid
TEMED : tetramethyl ethylene diamine
Tris : tris (hydroxyl methyl) amino methane
v/v : volume per volume
Ve : elution volume
Vo : void volume
w/v : weight per volume

VII