Vol.4, No.11, 585-594 (2013) Agricultural Sciences
http://dx.doi.org/10.4236/as.2013.411079
A Kunitz trypsin inhibitor from chickpea
(Cicer arietinum L.) that exerts an antimicrobial
effect on Fusarium oxysporum f.s p. ciceris
Meera Nair*, Sardul Singh Sandhu
Fungal Biotechnology and Invertebrate Pathology Laboratory, Department of Biological Sciences, Rani Durgavati University,
Jabalpur, India; *Corresponding Author:
Received 31 August 2013; revised 29 September 2013; accepted 15 October 2013
Copyright © 2013 Meera Nair, Sardul Singh Sandhu. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
ABSTRACT
Fusarium oxysporum f.sp. ciceris (Foc) is one of
the most important fungal pathogens of chick-
pea and is re garded a s a c onstant threat in trop i-
cal and subtropical countries. In order to corre-
late Fusarium wilt resistance/susceptibility in
Cicer arietinum to the presence or absence of
trypsin inhibitor (TI) in the crude extract, trypsin
inhibitory assay (TIA) and in vitro activity of TI
against Foc were studied. In the present study, a
20 kDa trypsin inhibitor was purified from Fusa-
rium wilt resistant cultivar (viz. JG 2001-12) by
ammonium sulfate precipitation, dialysis and
chromatographies with Sephadex G-100 and
Die thyl ami noeth yl cellulose (DEAE-cellulose-52)
ion-exchange column. Results of pathogenecity
assay were found to be in correlation to the
trypsin inhibitor assay where the Fusarium wilt
resistant cultivar showed high trypsin inhibitory
activity (99%) in the presence of trypsin enzyme
using both natural and synthetic substrates. Pre-
li m in a ry s tu d i es using crude extra ct s o f JG 200 1-
12 showed a decrease in radial growth of Foc. A
45% - 82% reduction in conidium germination at
20 µg·mL−1 Cicer arietinum trypsin inhibitor (CaTI)
concentration was observed, thereby, indicating
the use of CaTI in suppression of pathogen and
in its deployment through transgenic plants for
the management of Fusarium wilt.
Keywords: Cicer arietinum; Fusarium Wilt; Kunitz;
Proteinase Inhibitor; Trypsin Inhibitor
1. INTRODUCTION
Cicer arietinum (L.) is a worldwide leguminous crop,
which ranks third in the world among pulse crops after
peas and beans. Chickpea accounts a substantial propor-
tion of human dietary nitrogen uptake and plays a crucial
role in food security in developing countries [1]. Fusa-
rium wilt caused by the Deuteromycetes fungal pathogen
Fusarium oxysporum f.sp. ciceris (Foc) Schlechtend.
Emend, Synder and Hansen, is a serious devastating dis-
ease of chickpea in India, Iran, Pakistan, Nepal, Burma,
Spain, Tunisia and Mexico. It is a major pathogen,
among 67 reported pathogens, to cause disease in Cicer
arietinum (L.). Annual yield losses due to this disease
estimate a 10% to 15% loss, but Fusarium wilt epidemics
can cause a 100% loss under favorable conditions [2].
Persistence of the pathogen in soil and its capacity to
survive there for years even in the absence of host [3]
renders its control difficult [4]. Soil applications of fun-
gicides are costly and lead to indiscriminate killing of
beneficial soil microflora, therefore alternative biocon-
trol agents or other eco-friendly control strategies are
necessary [4].
One of the eco-friendly control measures opted to pro-
vide resistance against fungal pathogens is using a pro-
teinase inhibitor (PI) proteins produced in plant tissues,
which, act as a defensive mechanism against microorgan-
isms [5]. Plant PIs rely on inhibition of proteases se-
creted by microorganisms, causing a reduction in the
availability of amino acids necessary for their growth
and development [6].
C. arietinum (L.) seeds are rich in serine proteinase
inhibitors (PIs) which show strong inhibitory activity
against trypsin, chymotrypsin or other proteolytic en-
zymes [7]. In our earlier study, trypsin inhibitor showed
antimetabolitic activity against Helicoverpa armigera [8].
Accordingly, an attempt has been made to analyze anti-
fungal activity of trypsin inhibitor towards the growth of
Foc and its influence on hyphal growth. To the best of

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594
586
our knowledge, this is the first study focusing on the use
of C. arietinum (L.) trypsin inhibitor (CaTI) on Fusarium
wilt control.
2. MATERIALS AND METHODS
2.1. Fungal Cultures
Standard isolates of Foc (MTCC # 2087) was procured
from Microbial Type Culture Collection and Gene Bank
(MTCC), IMTECH (Institute of Microbial Technology),
Chandigarh where they were already characterized and
classified for their race specificity using conventional
method of race identification. The cultures were main-
tained on Potato Dextrose Agar (PDA) slants with timely
sub-culturing and infection to a susceptible cultivar, JG
62.
2.2. Plant Material
Cicer arietinum (L.) cultivar seeds viz. JG 2001-12, JG
16 and JG 62 used in this study were obtained from De-
partment of Genetics and Plant Breeding, Jawaharlal
Nehru Krishi Vishwavidhyalaya (J.N.K.V.V.), Jabalpur,
India. Each plant were grown from single seed, planted
in a rectangular trough and maintained under green
house conditions. Cultivar JG 62 (pedigree-selection
from germplasm), is susceptible to Fusarium wilt.
2.3. Pathogenecity Assay
Seeds of C. arietinum (L.) cultivar viz. JG 62, JG
2001-12 and JG 16 were pre-germinated and grown in 20
× 10 × 2.25 cm trays filled with peat soil. Freshly pre-
pared spore suspension (1 × 106 spores·mL−1) of Foc #
2087 was added individually to the sterile trays
containing 7 day old chickpea plants. Seedlings grown in
trays with no pathogen (un-inoculated plants) served as
control. The pathogenecity assays were conducted in
triplicates. Seedlings showing typical disease symptoms
such as wilt, xylem discoloration and stunting were re-
corded for 21 days. Final disease severity data was re-
corded on 8th week after inoculation. Pathogen was
re-isolated from representative diseased plants so to
prove Koch’s postulates.
The seed extract of resistant cultivar viz. JG 2001-12
was prepared as per the method described by Nair et al.
[8]. Soluble proteins were extracted from milled, defatted
and depigmented seeds of chickpea in 10 mM phosphate
buffer, pH 7.2 with constant stirring at 4˚C for 4 h. In-
hibitor proteins were fractionated with ammonium sul-
phate (40% - 60% saturation), recovered by centrifuga-
tion at 12000 × g for 30 min at 4˚C and later dissolved in
a minimal amount of deionized water. The protein was
concentrated against 50 mM Tris/HCl buffer, pH 7.5 us-
ing dialysis membrane (Sigma Aldrich USA, 12 kDa cut
off) and loaded on a Sephadex G-100 (50 cm × 1 cm)
equilibrated with chilled 10 mM Tris/HCl buffer (pH 7.5)
and eluted with the same buffer. Active fraction showing
trypsin inhibitor activity was pooled and concentrated via
acetone precipitation. Acetone treated aliquots (500 µl)
was loaded and left undisturbed for 30 min. The inhibitor
was eluted using 0 to 2 M NaCl linear gradients at 4˚C.
Total protein was assayed by Lowry et al. [10] using
bovine serum albumin as a standard.
2.4. Preliminary Proteinase (Trypsin)
Inhibitor Assay
Trypsin inhibitor activity (TIA) of crude extract was
determined using both natural (casein) and synthetic oli-
gopeptides substrate (BApNA) by pre-incubating 0.25 µg
trypsin (10 mg·mL−1 of bovine trypsin in 1 mM HCl) and
50 µl of crude extract (12.5 mg protein) for 15 min in a
water bath (D’sco, India) at 37˚C. Natural or synthetic
oligopeptide substrate was then added and the reaction
was carried out for 30 min at 37˚C. For caseinolytic as-
say, reaction was stopped by adding 50 µl of 50% (w/v)
TCA which was allowed to stand for 15 min at 4˚C.
TCA-soluble material was collected by centrifuging the
reaction mixture at 2000 × g for 20 min. In BApNA, the
reaction was terminated by adding 0.5 mL of 10% acetic
acid and absorbance at 410 nm was determined. The
percentage at which casein was digested by trypsin was
calculated by measuring the absorption of trichloroacetic
acid (TCA) filtrate at 280 nm by using the tyrosine
standard curve. Similarly, pNA (p-nitroaniline) was used
as a standard for BApNA. Trypsin inhibition (%) was
calculated from the difference between untreated (with-
out crude extract) and treated (with crude extract) sam-
ples divided by untreated sample reading, multiplied by
100 [9].
2.5. Extraction of Chickpea PIs
2.6. Polyacrylamide Gel Electrophoresis
Analysis
Sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis (SDS-PAGE) was performed according to the
protocol of Laemmli [11] with 4% stacking gel and 12%
separating gel using a vertical gel electrophoresis system
(GeNei, Bangalore, India), where anion exchange frac-
tion along with standard protein molecular weight mark-
er (Bangalore Genei, India) was loaded to the wells. The
gel was run at a constant voltage of 50 V at 4˚C in elec-
trophoresis buffer (25 mM Tris/HCl pH 8.8, 192 mM
glycine and 0.1% (w/v) SDS) following which, the entire
gel was washed with distilled water and stained with
Commassie brilliant blue stain (Calbiochem®) for 30 min
with constant shaking. The gel was later destained with

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594 587
methanol:glacial acetic acid:distilled water (v/v) (3:2:15)
for one hour giving three changes of destain.
2.7. In-Gel Assay of Proteinase Inhibitor
Gelatin zymography were performed according to Le
and Katunuma [12] with resolving 10% acrylamide solu-
tion containing 0.1% gelatin. Ion exchange chromatog-
raphy fraction equivalent to 40 μg protein were mixed
with equal volume of sample buffer containing 25 mM
Tris/HCl (pH 6.8), 20% glycerol, 4% SDS and 0.02%
bromophenol blue was loaded along with 80 µg of soy-
bean trypsin inhibitor (Calbiochem®; 10 mg·mL−1 stock
solution in Tris/HCl pH 8.2) which was used as a marker.
Electrophoresis was performed at a constant voltage of
50 V at 4˚C in electrophoresis buffer (25 mM Tris/HCl,
pH 8.8, 192 mM glycine and 0.1% SDS). The gel was
washed for 30 min at room temperature with 0.1 M Tris-
HCl at pH 7.2 containing 2.5% Triton X-100, followed
by distilled water for 20 min. Subsequent incubation at
37˚C for 3 h with reaction buffer containing 10 mg·mL−1
trypsin solution (10 mg trypsin in 0.1 M Tris/HCl, pH 7.5)
and 0.02% NaN3 followed by Coomassie Brilliant Blue
R250 (CBB) staining for 1 h and destaining produced
blue bands against a clear background.
2.8. Determination of Proteinase (Trypsin)
Inhibitor Activity at Different Stages of
Purification Steps
The proteinase inhibitor activity on natural and syn-
thetic chromo(non-chromo)genic substrates was deter-
mined in a reaction mixture containing 50 mM Tris/HCl
buffer (pH 7.5), 0.25 µg trypsin (EC 3.4.21.4) (10
mg· mL −1 of bovine trypsin in 1 mM HCl), different con-
centration of all three ammonium sulphate cut off (100
μg), gel filtrate fraction (100 μg) and ion exchange frac-
tions. The anion exchange fractions (43 μg, 86 μg, 430
μg protein) with 100 µl casein, 40 µl BApNA (10
mg· mL −1 in dimethyl sulfoxide); 0.23 mM BAEE in 67
mM potassium phosphate buffer (pH 7.5), 12.5% azoca-
sein (w/v) in 50 mM Tris, 5 mM CaCl2 (pH 8.0). Con-
trols with a trypsin enzyme (100˚C, 5 min) were run in
parallel. The amount of p-nitroaniline (pNA) released
was determined by measuring the change in absorbance
at 410 nm using pNA calibration curve. Proteinase in-
hibitor (TI) activity was also determined using a non-
chromogenic substrate N-benzoyl-L-arg ethyl ester
(BAEE) according to Schwert and Takenaka [13].
In azocasein, the reaction was terminated by adding
200 µl of ice-cold 5% TCA to 500 µl of reaction mixture.
The reaction mixture was then placed at room tempera-
ture for 10 min and centrifuged at 3,000 × g for 10 min,
the supernatant (500 µl) was later aspirated and 1500 µl
of 0.5M NaOH was added. The absorbance was recorded
at A428 [14].
2.9. Screening of Fungitoxic Activity
The in vitro fungitoxic activity of crude extract of
fusarium wilt resistant cultivar (viz. JG 2001-12) and
fusarium wilt susceptible cultivar (viz. JG 16) was per-
formed by mycelial disc method as described by Trema-
coldi and Pascholati [15]. The mycelial disc was placed
onto PDA medium, previously flooded with filtered (0.2
µm, Millipore) crude extracts in 25 µl, 500 µl, 1000 µl
and 2000 µl volumes. The experiment was performed in
triplicate for each concentrations and controls (with no
crude extract added) under strict aseptic conditions. The
fungitoxic activity of each concentration was expressed
in terms of the mean of colony diameter in cm (mean
value ± standard deviation) produced by respective ex-
tract at the end of the incubation period (7 d). Gel filtra-
tion fraction (Fraction A) was also tested against mycelia
growth of Foc isolates at 10, 100 and 200 µg of protein
(as described above).
2.10. Effect of Anion Exchange Fractions on
the Hyphal and Conidial Growth of
Fusarium oxysporum f.sp. ciceris
The effect of purified CaTI protein (anion exchange
fractions) on hyphal growth and conidial germination
was assayed. Conidial suspension was prepared by flood-
ing the cultures with sterile distilled water containing
0.05% (v/v) Tween-80 which was later filtered through
four layers of sterilized cheesecloth to remove adhering
mycelia. The spore concentration was adjusted to 105
conidia per mL with the aid of Neubauer hemocytometer.
Conidia were allowed to germinate and grown in the
presence of CaTI at 0, 5, 10, 15, 20 µg·mL−1 concentra-
tions in 6-well plate at 25˚C in darkness with a positive
control and negative control plates containing heat inac-
tivated CaTI and phosphate buffer. For each treatment,
hyphal length was measured from 40 randomly selected
hyphae and the mean hyphal length was used for com-
parison using a light microscope equipped with an ocular
micrometer after 12 h and 24 h of incubation. As Foc is
multicelled therefore; a conidium was considered germi-
nated, if hyphae were visible for at least one of the cells.
Each treatment contained three replicates and the ex-
periment was repeated twice.
2.11. Statistical Analysis
The results were subjected to analysis of variance
(ANOVA) and the significance of differences among
means was determined by using t-test using the MS Ex-
cel program where p values <0.05 were considered sig-
nificant. All experiments were performed in triplicate
unless stated otherwise. Comparisons were made for all

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594
588
parameters within the amount of inhibitor used as well as
the hours of incubation during antimicrobial activity on
Foc.
3. RESULTS AND DISCUSSION
3.1. Pathogenecity Assays
Chickpea plants viz. JG 62, JG 2001-12 and JG 16
were inoculated with fungal spores of Foc individually
where un-inoculated plants served as control. Upon in-
fection, JG 62 and JG 16 showed wilting symptoms by
10 days after inoculation (dai), while no wilting was ob-
served in JG 2001-12 cultivar even by the 8th week.
Figure 1 shows the chickpea cultivars, where JG 16 ex-
hibit wilting symptoms whereas JG 2001-12, display no
wilting symptoms.
3.2. Preliminary Proteinase (Trypsin)
Inhibitor Activity
The inhibitory activity of the crude extract (viz., JG
2001-12 and JG 16) against bovine trypsin was deter-
mined using natural and synthetic substrate viz. casein,
BApNA respectively.
Caseinolytic activity: Crude extract of JG 2001-12
and JG 16 was used to determine the proteinase inhibi-
tory activity where the specific activity of trypsin was
found to be 227 ± 0.5 nmol·tyr·min−1·mg−1 protein in
absence of crude extract (trypsin inhibitor). The crude
extract of Fusarium wilt resistant cultivar (viz. JG 2001-
12) displayed a 99% trypsin inhibition with 0.7 ± 0.4
nmol·tyr·min−1·mg−1 protein. The caseinolytic activity of
Fusarium wilt susceptible cultivar (viz. JG 16) showed a
12% inhibition with 200 ± 0.5 nmol·tyr·min−1·mg−1 pro-
tein TIA (Table 1).
BApNA: Trypsin activity was rapidly inhibited
(99.4% inhibition) by the crude extract of C. arietinum
fusarium wilt resistant cultivar (viz. JG 2001-12),
indicating the presence of trypsin inhibitor with 1.0 ± 0.3
µmol pNA min−1mg−1 protein TIA against bovine trypsin
(Table 1).
3.3. Purification of Cicer Arietinum Trypsin
Inhibitor (CaTI)
The trypsin inhibitor from JG 2001-12 seed crude ex-
tract was identified by trypsin inhibitory activity (TIA)
assays using trypsin as the enzyme at preliminary identi-
fication and purification steps. The purification results
are presented in Figure 2 and Table 2. The proteinase
inhibitor activity was revealed in 40% - 60% ammonium
sulphate cut off (Figure 3). Chickpea proteinase (trypsin)
inhibitor was isolated by dialysis, desalting in Sephadex
G-100 and purified using DEAE-cellulose column eluted
with 0.1 M NaCl. Upon size-exclusion chromatography
Table 1. Trypsin Inhibitory activity (TIA) of crude extracts of
fusarium wilt resistant (JG 2001-12) and susceptible (JG 16)
cultivars.
Casein BApNA
TIA* TI# TIA* TI#
Control 227.6 ± 0.50 172.01 ± 0.50
Crude extract of cultivar
1)JG 16 200b ± 0.5 12 145.2a ± 0.315.5
2)JG 2001-120.7a ± 0.4 99 1.0b ± 0.3 99.4
a,bMeans with different superscripts in a column differs significantly (p <
0.05). *TIA = nmol tyr min−1mg−1 protein; **TIA = µmol pNA min−1mg−1
protein; TI# = Trypsin Inhibition (%).
Table 2. Purification performance of Trypsin Inhibitor from JG
2001-12 cultivar.
Sample
Total
protein
(mg)
Specific
Activity
(µM pNA
min−1mg −1)
TIA* Yield
(%)
Purification
Fold
Crude Extract 258 2.32 44.5 100 1
Sephadex G-1009 72.54 86.7 25 57.1
DEAE-cellulose-52
column 0.1 140 94.4 10 65.4
*TIA = Trypsin Inhibitory activity.
JG 2001-12 JG 16
Figure 1. Fusarium wilt susceptible cultivar (JG 16)
and resistant (JG 2001-12) showing wilting and
chlorosis.
on Sephadex G-100 column, two protein peaks were ob-
tained where proteinase inhibitor was detected in the first
peak (Figure 2(a)) which consisted of three fractions
(fraction 9, 10, 11 called as Fraction A). Fraction A
showed maximum trypsin inhibitory activity against
trypsin enzyme (Figures 4(a), (b)). Further, the trypsin
inhibitor was retained by anion exchange column-
DEAE-cellulose column and eluted in fractions of 0.1 M
NaCl (Figure 2(b)). Taken together, approximately 10%
recovery and 65.4 fold increase in specific activity re-
sulted (Table 2). Fractions of size exclusion chromatog-
raphy, anion exchange chromatography were employed
on SDS-PAGE and reverse zymography (using gelatin as
ubstrate) to verify the purity of trypsin inhibitor and s

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594 589
(a)
(b)
(c) (d)
Figure 2. Purification of Trypsin inhibitor from JG 2001-12 seeds. (a) Separation using Sephadex G-100
column chromatography of trypsin inhibitor from seeds of Fusarium wilt resistant Cicer arietinum L. cultivar
viz., JG 2001-12. (b) Purification of higher trypsin inhibitory region (fraction 9, 10, 11) identified after
Sephadex G-100 chromatography using DEAE-cellulose ion exchange chromatography column. Line across
the chromatography represents the linear 0-1 M NaCl gradient employed to elute adsorbed proteins. (c)
SDS-PAGE gel of trypsin inhibitor purified using ion exchange chromatography with standard molecular
weight marker (M). (d) In gel assay detection of trypsin inhibitory activity. Standard soybean kunitz trypsin
inhibitor (SKTI) and Purified trypsin inhibitor from JG 2001-12 seeds.
estimate its molecular mass. Trypsin inhibitor was re-
solved by SDS-PAGE and in-gel digestion of gelatin in
zymograms where both gels showed one band corre-
sponding to Mr ~20 kDa (Figures 2(c) and (d)).
OPEN A CCESS

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594
590
Figure 3. Trypsin Inhibitory Activity (TIA) in different ammo-
nium sulphate cut offs of protein homogenate of JG 2001-12
cultivar in comparison to control (with no crude extract) using
BApNA substrate.
*
*
**
*
***
*
0
50
100
150
200
TIA
ControlFraction 9Fraction 10Fraction 11
Treatments
Trypsin Inhibition (%)TIA
*
**
**
0
20
40
60
80
100
120
140
160
180
200
ControlJG 2001-12Gel Filtration Fraction
(Fraction A)
Anion Exchange
Fraction
Treatments
TIA (µmol pNA min
-1
mg
-1
protein)
Figure 4. Trypsin Inhibitory Activity (TIA) Assay.
3.4. Trypsin Inhibitor Activity Assay
Trypsin inhibitor activity assay (TIA) of purified tryp-
sin inhibitor at different purification steps using synthetic
substrates (BApNA, azocasein, BAEE) were carried
where the reliability of the reaction conditions was de-
termined in triplicate sets. The TIA using BApNA sub-
strate, at different concentration of CaTI protein [(5 μL)
(43 μg), 10 (86 μg), 15 μL (430 μg)] showed a progres-
sive reduction in trypsin activity, with increase in con-
centration of CaTI protein. The inhibitory activity was
increased from 86.2%, 97.2% to 98.61% with protein
concentration (Figure 5). Similarly, in azocasein and
BAEE substrate, absorbance at 428 nm and 253 nm was
found to be high in control (with no CaTI) with respect
to the proteinase activity in the presence of trypsin in-
hibitor in fusarium wilt resistant cultivar crude extract viz.
JG 2001-12. The trypsin activity was calculated in units
where, one unit of trypsin was defined as the amount that
increased A428 by 0.01/min under the assay conditions.
Figure 5. Effect of different concentration of trypsin inhibitor
on activity of trypsin using synthetic substrates (BApNA,
Azocasein, BAEE) as described in Materials and Methods.
Error bars represent standard deviations. Inset, showing the
protein concentration at different volume of trypsin inhibitor.
Trypsin inhibitory units were calculated from the number
of trypsin units under similar conditions in the presence
of trypsin inhibitor. 50 µl of inhibitor (320 µg equivalent
protein) was used as the optimum concentration so to
inhibit trypsin activity. It was found that after 30 min of
incubation, there was no increase in the absorbance at
428 nm and 253 nm, suggesting an inhibition in trypsin
activity (Figure 5). Our results demonstrated no varia-
tion in proteolytic activity of trypsin on natural and syn-
thetic substrates in presence and absence of trypsin in-
hibitor. The results indicated that the crude extract of
fusarium wilt resistant cultivar (viz. JG 2001-12) inhibit
proteolysis of natural and synthetic substrates in the
presence of bovine trypsin. The method we adopted here

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594 591
does not require ampholytes, so it is a cheaper and more
convenient method. In the present study, a comparison on
the trypsin inhibitor activity was carried out using dif-
ferent natural and synthetic substrates where, the TIA
results were in conformity to Spelbrink et al. [16]. Ear-
lier reports also suggest the use of both natural and syn-
thetic substrates in determination of antitryptic activity as
natural substrates are more difficult to displace from the
active site to form the enzyme inhibitor complex while
synthetic substrate are accurate in determining the in-
hibitor content of the materials [17] thereby providing a
value with physiological relevance [16].
3.5. Effect of Crude Extract of Seeds on the
Mycelial Growth of Fusarium
oxysporum f.sp. ciceris
Studies have shown that an increasing level of pro-
teinase inhibitor correlates to an increase in resistance
against pathogens [18]. In many plant species, response
to infection by pathogenic bacteria, viruses and fungi or
to various abiotic stresses is accompanied by the synthe-
sis of a variety of proteins termed as Pathogenesis related
(PR) proteins [19], which have been isolated from both
dicots and monocots [20]. Preliminary short report by
Niderman et al. [20] has described a direct fungicidal
activity of PR-1 protein from tomato. Similar results with
PR-2, protein P14 against fungal pathogens have also
been elucidated. In the present communication, crude
extract of C. arietinum (L.) seed was preliminarily
screened so to observe the effect of trypsin inhibitor on
mycelial growth of Foc . The amount of proteins in the
crude extract was equivalent to 3.5% in relation to the
fresh weight of the seeds. The categorization of crude
extracts as per TIA assay was also in accordance to the
pathogenicity assay. Test plates containing crude extract
of JG 2001-12, exhibited a colony diameter of 4.3 ± 0.04
cm whereas control plates exhibited a colony diameter of
9.0 ± 0.05 cm (Figure 6(b)). Mycelial growth was found
to be static with a decrease in colony diameter in pres-
ence of JG 2001-12 crude extract whereas susceptible
cultivar, JG-16, and control plates where no crude extract
was added showed no inhibition. Observations suggest
CaTI to be antifungal, as JG 2000-12 showed significant
antifungal activity w.r.t JG 16 cultivar, whose growth
was analogous to control plates.
3.6. Effect of gel filtration fractions
(showing trypsin inhibitory activity) on
the mycelial growth of F. oxysporum
f.sp. ciceris
Results showed a decrease in the growth of mycelia as
the fraction showing trypsin inhibitor challenged the
growth of Foc isolate # 2087 (3.0 ± 0.04 cm) as com-
Control Test
(a)
0
1
2
3
4
5
6
7
8
9
10
Control25 µl500 µl1000 µl2000 µl
Treatments
Colony Diameter (cm
)
(b)
Figure 6. Effect of crude extract on the growth of Fusarium
oxysporum f.sp. ciceris. (a) In vitro plate assay of crude extract
from JG 2001-12 (T) on growth of Foc with respect to the con-
trol (C). (b) Effect of crude extract from JG 2001-12 on the
growth of Foc
pared to the control where the growth of the isolate was
found to be normal (9.0 ± 0.04 cm). Gel filtrate fractions
(fraction “A”) showed a significant inhibition in fungal
growth, indicating presence of trypsin inhibitor which
led to inhibition in radial growth at protein concentration
of 200 µg (Table 3). Thus, demonstrating trypsin inhibi-
tor’s defensive role in inhibiting fungal growth. Using
gel filtrate fraction, a 100-fold increase in inhibitory ac-
tivity was observed over the crude inhibitor preparation
against Foc (Table 3).
3.7. Effect of Anion Exchange Fractions
(Showing Trypsin Inhibitory Activity) on
the Conidial Growth and Hyphal Length
of F. oxysporum f.sp. ciceris
As the volume of fractions achieved after anion ex-
change was small therefore, the purified trypsin inhibitor
effect on conidial growth and hyphal length of Foc was
observed. For most isolates, conidium germination de-
creased dramatically with increasing trypsin inhibitor
(CaTI) concentration. Conidium germination was re-
duced to 50% at a CaTI concentration of 20 µg·mL−1.
After 12 h of incubation, a significant difference was ob-
served both visually (Figure 7) and quantitatively (Fig-
ure 8) in fungal growth; as condium germination was
found to be less as compared to the control (10% PDB
un-amended with CaTI). In 10% PDB amended with

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594
592
Table 3. Effect of gel filtrate fraction “A” on growth of
Fusarium oxysporum f. sp. ciceris at different concentration of
protein (µg).
Treatment Colony diameter (cm)
1) Control 9.0 ± 0.04
2) 10 µg 5.0 ± 0.2
3) 100 µg 4.0 ± 0.25
4) 200 µg 3.0 ± 0.04
(
a)
(b)
(c)
(d)
Figure 7. Inhibition of Fusarium oxysporum f.sp. ciceris co-
nidia germination. Ion exchange chromatography fractions are
mixed with PDB and incubated at 37˚C. Bar = 10 μm. (a) 10%
potato dextrose broth (PDB) alone at 0 h; (b) PDB amended
with trypsin inhibitor (20 μg·mL–1) at 12 h; (c) PDB amended
with trypsin inhibitor (20 μg·mL–1) after 24 h; (d) Control, with
no trypsin inhibitor.
CaTI (15 µg·mL−1), hyphal growth was observed to be
25 µm as compared with 200 µm in 10% PDB (control).
At the end of 24 h of incubation, PDB amended with
CaTI (15 µg·mL−1) showed an increase in hyphal growth
from 25 µm to 52 µm, whereas there was a drastic in-
crease in germination and hyphal growth of the mycelia
(340 µm) in 10% PDB (control).
At the end of 24 h of incubation, the hyphal length in
the PDB amended with CaTI at 20 μg·mL−1 remained
***
**
**
***
0
50
100
150
200
250
300
350
400
Control5 µg/mL10 µg/mL15 µg/mL 20 µg/mL
Treatments
Hyphal growth
(
µ
m
12 h24 h
(
Figure 8. Fungal hyphal growth (μm) with different treat-
ments of trypsin inhibitor fractions of anion exchange
column chromatography as described in Materials and
Method.
nearly the same as that at 12 h with little variation (i.e. at
12 h the hyphal growth was found to be 40 μm and after
24 h the hyphal growth was 42 μm). The results obtained
using conidial germination and hyphal growth was in
correlation to each other as the results showed inhibition
in the presence of trypsin inhibitor were consistent
(Figures 7 and 8). These studies were similar to Chen et
al. [21] reports, which showed that the resistance of cer-
tain corn genotypes to fungal infection is related to the
action of trypsin inhibitor, which is due to the lowering
in the production and activity of fungal alpha-amylase,
which in turn reduce the availability of simple sugars for
fungal growth. Similar studies on the inhibition of spore
germination and mycelium growth of Alternaria alter-
nata by buckwheat trypsin/chymotrypsin was observed
[22]. Likewise, cysteine proteinase inhibitors from pearl
millet also inhibited the growth of many pathogenic
fungi including Trichoderma reesei [23]. In addition, the
antifungal effect of trypsin inhibitor was observed in
wheat kernel [24], corn [21], barley [25] and cabbage
[26].
Our results were in corroborance with Chen et al. [21],
Huang et al. [27], Revina et al. [28] who described the
use of resistant TI in inhibiting germination and hyphal
growth of plant pathogenic fungi. Collating the present
and previously reported data, it appears that Cicer
arietinum trypsin inhibitor (CaTI) act as an antifungal
agent since in vitro activity of CaTI showed a 45% - 82%
suppression of conidial germination and hyphal growth,
thereby showing its effectiveness against Foc.
As Foc survives in soil, therefore use of TI containing
Cicer arietinum L. (CaTI) would be an easy approach in
order to reduce the use of chemicals/fungicides to com-
bat Fusarium wilt. In the present study, a sustainable ap-
proach in controlling Fusarium wilt was utilized via the
use of trypsin inhibitor as implementation of cultural,
physical, biological and chemical measures minimize
economic risks to consumers and the environment. To
sum up, CaTI provide a defensive property to host plant

M. Nair, S. S. Sandhu / Agricultural Sciences 4 (2013) 585-594 593
as a cultivar containing trypsin inhibitor showed resis-
tance against Fusarium wilt. In this context, future re-
search to identify eco-friendly tools for the biological
control of pathogens and further studies are warranted to
confirm its biotechnological potential against other
pathogens inflicting Cicer arietinum (L.).
3.8. Abbreviations and Acronyms
BAEE, N-benzoyl-L-Arg ethyl ester; BApNA, N-α-
Benzoyl-D, L-arginine p-nitroanilide hydrochloride; CLA,
carnation leaf agar; CaTI, Cicer arietinum Trypsin In-
hibitor; Foc, Fusarium oxysporum f.sp. ciceris; PDA,
Potato Dextrose Agar; PDB, Potato Dextrose Broth;
p-NA, para-nitroaniline; TI, Trypsin Inhibitor; TIA,
Trypsin Inhibitor Activity; TCA, Trichloroacetic acid.
4. ACKNOWLEDGEMENTS
The authors would like to thank the Head of the Department of Bio-
logical Sciences, Rani Durgavati University, Jabalpur (India) for labo-
ratory facilities and University Grant Commission Project (Govt. of
India), New Delhi for financial assistance vide project no. UGC
(32-370/2006) (SR) dated. 24/04/07. Authors also acknowledge
JNKVV, Jabalpur, India for providing the seed material.
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