American Journal of Plant Sciences, 2011, 2, 43-49
doi:10.4236/ajps.2011.21005 Published Online March 2011 (http://www.SciRP.org/journal/ajps)
43
Participation of Chitin-Binding Peroxidase
Isoforms in the Wilt Pathogenesis of Cotton
Egor Pshenichnov1, Nigora Khashimova1, Alik Akhunov1, Zamira Golubenko1, Robert D. Stipanovic2
1A.S. Sadykov Institute of Bioorganic Chemistry, Academy of Science, Tashkent, Uzbekistan; 2 USDA-Agricultural research Service,
Southern Plains Agricultural Research Center, College Station, USA.
Received December 17th, 2010; revised January 2nd, 2011; accepted January 19th, 2011.
ABSTRACT
Specific chitin-binding isozymes of peroxidase (POX) play an important role in pathogenesis of plant diseases caused
by fungi. We studied the dynamics of peroxidase activity in two varieties of cotton (Gossypium hirsutum L.); one was
susceptible and the other resistant to the plant pathogen Verticillium dahliae. After infection with strongly and weakly
virulent isolate of V. dahliae, we observed a correlation between the level of seedling tissue lesion and peroxidase ac-
tivity. Thus, the first POX activity was observed in all infected plants 2 hour s after inoculation, but POX activity of the
resistant variety rapidly increased and maximized 3 days after infection, while POX activity in the susceptible variety
showed a slow increase that continued to increase during the remaining 8 days of experimental observation. The in-
crease of POX activity in the susceptib le variety after infection may be explain ed by progressive fungal colon ization of
cotton tissues leading to irreversible senescence. Microscopic examination of plant tissues supports this hypothesis. The
more virulent isolate caused more necrosis and significantly more POX activity than the mildly virulent in both suscep-
tible and resistant plants. Control plants showed no changes in POX activity; however, the POX activity in the control
resistant varieties was higher than the control susceptible varieties. These findings indicate the potential utilization of
chitin binding POX as a biochemical tool to guide breeding programs to increase resistance to V. dahliae.
Keywords: Peroxidase, Plant Protection, Cotton, Gossypium Hirsutum, Verticillium Dahliae
1. Introduction
Recognition of a potential pathogen is required to trigger
a defense response in living organisms. Plants do not
produce immunospecific antibodies; however, some con-
stituents of pathogens can elicit the activation of plant
defense pathways. Among these biogenic inductors (eli-
citors) are proteins, glycoproteins, polyenic fatty acids,
and oligosaccharide fragments of fungi wall cells [chitin,
β-(1,3)-glucans] [1]. Among the numerous enzymes in-
volved in plant defense mechanisms, peroxidase (POX)
occupies a crucial position in an early plant response to
pathogens [2]. The pathogen related protein PR-9 POX
belongs to an enzyme involved in lignin formation [3].
The increase of lignification processes is connected with
active interactions between pathogen structures and POX.
Lignin formation on the surface of fungal mycelia in the
presence of this enzyme has been described for POX
isolated from cucumber [4]. Aver’yanov et al. [5] showed
an increase in POX activity in rice blast infected plants
compared to healthy plants, and researchers have shown
that cell wall bound POX produces H2O2 in response to
fungal pathogen elicitors [6,7]. Hammond-Kosak and
Jones [8] have published an extensive review of the in-
teraction between specific plant pathogen elicitors and
receptors that elicit plant resistant genes.
Hence, the race specificity of pathogens can be deter-
mined by their ability to activate the defense mechanisms
of host plants. The varieties of agricultural plants differ
in their range of resistance to phytopathogens. Recently,
the ability of individual POX isozymes to bind chitin was
discussed [9]. This specific sorption of POX on infec-
tious components of fungi containing chitin was ob-
served as abundant scurf of phenol polymers on the haus-
toria of Uromices vicia-fabae [10]. A polysaccharide
binding domain was observed in POX anionic isozymes
of Arabidopsis thaliana and Cucurbita pepo [11]. The
hypothetical role of POX in pathogenesis relates to per-
oxidation of chitin fragments of cell walls of the patho-
gen followed by production of oligosaccharide elicitors
[12]. Thus, the role of chitin-binding POX likely includes

Participation of Chitin-Binding Peroxidase Isoforms in the Wilt Pathogenesis of Cotton 44
initiation of plant defense mechanisms after pathogen
attack. Herein we report our investigation of the dynamic
activation of POX in seedlings from two cotton varieties
(Gossypium hirsutum L.) varieties triggered by the action
of two different strains of the fungal wilt pathogen Verti-
cillium dahliae and discuss its POX activation.
2. Materials and Methods
2.1. Biological Material
Cottonseed (Gossypium hirsutum L.) of AN-Bayaut-2
and C-4727 varieties were grown in an experimental
field at the Institute of Cotton Breeding and Seed Pro-
duction (Tashkent, Uzbekistan). AN-Bayaut-2 is consid-
ered to be highly resistant to V. dahliae and C-4727 is
considered to be susceptible [13]. Conidia of V. dahliae
Kleb. (weakly virulent T-4 and highly virulent AN-3
isolates) were originally isolated from the diseased cotton
and are from the collection housed at the Institute of Ge-
netics, Academy Sciences of Republic of Uzbekistan,
Tashkent, Uzbekistan. The pathogen was grown on
Czapek’s agar.
2.2. General
Chitin from crab shells (Fluka Chemical Company) was
used for chromatography. Isoelectric focusing of proteins
was carried out on an LKB Multiphor-2117 apparatus.
Sigma Chemical Company’s IEF mix 3.5-10.6 was used
as the isoelectric focusing marker. Isoelectrofocusing of
POX was carried out on a horizontal plate containing 7%
polyacrylamide gel, 0.016% N, N-methylene-bis-acryla-
mide, 10% glycerol, 1.5% ampholines pH 3.5-10 (LKB,
Sweden) and 0.033% ammonium persulfate in 8 M urea.
The anode was 0.5% HCI, and the cathode was 0.5%
NaOH. The POX activity was ascertained on the gel us-
ing a 0.1% solution of benzidine dihydrochloride. POX
isozymes were quantitated by reaction with a 0.01% ben-
zidine dihydrochloride solution (Reachim Company,
Russia) and 0.005% Н202 in a 0.1% sodium acetate
buffer (рН 4.7), and the absorption maxima at 620 nm
was measured after one minute of reaction time. The
POX activity of each isozyme was determined by meas-
uring the color intensity using a LKB Densitometer. The
total protein concentration was determined by the method
of Lowry [15]. Assays were performed using an optical
microscope [Neofot-2 (Carl Zeiss, Germany)] at 90 x
magnification. All experiments were repeated at least
three times. Data were subjected to analysis of variance
(ANOVA), and differences between treatments assessed
by Student’s two-sample t-test at P < 0.05.
2.3. Infection of Cotton Seedlings
Cottonseed were cleansed with H2SO4, rapidly washed
with running water and kept in water overnight. Seed-
lings were wrapped in paper towels for 7 days at 27˚C.
Infection of seedlings was initiated by placing the towels
in flasks containing a conidial suspension (107 conidia
per mL) of V. dahliae .
2.4. Lesion Assay
Cottonseeds were sterilized by treatment with a solution
containing 36 % H2O2 and 96 % ethanol (1:1). V. dahliae
(AN-3 and 4-T strains) conidia were incubated in sepa-
rate tubes (2.0x20 cm) with Czapek’s agar (1 × 106 per
tube; 2.0-2.5 cm columns of media) for 10 days. Then a
warm agar solution was added to the tubes and a single
sterilized seed was placed in this layer in each of six
tubes for each treatment. The lesion on seedlings was
evaluated by visual observation and plant damage was
assayed by measuring the height of the seedlings and the
size of the necrotic zones on the surfaces of damaged
seedlings.
2.5. Crude Enzyme Preparation
The enzyme preparation was a modification of that pre-
viously described [14]. That is, raw plant material was
ground in liquid nitrogen and stirred with 0.1M sodium
phosphate buffer (pH 6.6), and 0.5M NaCl (5 mL per 1 g
of plant tissue) with a magnetic mixer 1 h at 4˚C. The
precipitate was removed by centrifugation (6000 g, 20
min). The supernatant solution was used for further in-
vestigations.
2.6. Adsorption of POX Isozymes on Chitin and
V. dahliae Conidia
All procedures were carried out at 4˚C. The supernatant
obtained as described above was treated with ammonium
sulfate to 30% saturation. The supernatant solution was
collected and brought to 70% (NH4)2SO4 saturation. The
residue was collected by centrifugation (6000 g, 20 min),
resuspended in a minimal volume of twice-distilled water
and desalted in 0.01M sodium phosphate buffer (pH 6.6)
using “Vivaspin 20” tubes (Sartorius, Germany) at 6000
g. The chitin adsorption assay was performed on a chro-
matographic column (2 cm × 6 cm) packed with chitin
previously equilibrated with 0.1M sodium phosphate
buffer (pH 6.6). The crude protein preparation was ap-
plied to the top of the column and the column was
washed with 0.1 M phosphate buffer with a flow of 20
mL/h to remove the unbound protein. Fractions were
collected and protein fractions were detected by moni-
toring the UV absorption at 280 nm using a flow cell on
an Uvicord system (LKB, Sweden). When all of the un-
bound protein had been eluted, the bound protein was
eluted with 1 M NaCl in 0.01 M phosphate buffer (pH
6.6). The V. dahliae conidia adsorption assay was carried

Participation of Chitin-Binding Peroxidase Isoforms in the Wilt Pathogenesis of Cotton 45
out in “volume”. Conidia (500 mg) were previously
treated with 1N NaOH (5 min) and spun down (6000 g,
20 min.) and then washed with 0.1M sodium phosphate
buffer (pH 6.6). After a second spin (6000 g, 20 min.) the
desalinated total protein preparation was suspended with
conidia in a minimal volume of the same buffer (30 min),
and conidia were eluted with some portions of buffer.
The column was eluted with 1M NaCl in 0.01M sodium
phosphate buffer (pH 6.6). Fractions were monitored at
280 nm as indicated above.
3. Results and Discussion
3.1. Peroxidase Activity in Seedlings
Segments of the two varieties of 7-day-old cotton seed-
lings were harvested each hour after infection with V.
dahliae conidia (AN-3 strain) up to 6 hour (Figure 1(a))
and then daily for 8 days (Figure 1(b)). POX activity in
control samples was not statically different at any period
compared to time zero. In the case of the AN-Bayaut-2
cotton variety, we observed an increase in POX activity
at 2 hours after infection in all segments (Figure 1(a)).
At 3 hours the POX activity had slightly decreased and
further significant increase was observed after 3 days
(Figure 1(b)) with a gradual lost of activity up to 8 days.
The POX activity of the C-4727 was not significantly
different than the control after 2 hour and 8 days in any
of the time segments; there was a slight increase in POX
activity beginning 3 days after infection in C-4727 (Fig-
ure 1(a), (b)) and continuing until 8 days. This is proba-
bly due to the additional tissue that is infected with time.
In the case of AN-Bayaut-2, the pathogen infection is
quickly contained and additional tissues are not infected;
thus, the level of POX decreases.
3.2. Lesion of Seedlings
The effect of the V. dahliae isolates on the two cotton
cultivars are shown in Table 1. The C-4727 cotton seed-
lings were 1.8 cm shorter than the control seedlings when
they were treated V. dahliae isolate 4-T, while those for
AN-Bayaut-2 were only 0.5 cm shorter than the control
when treated with the same isolate. The V. dahlia e AN-3
isolate had a greater effect on these cotton varieties. That
is, C-4727 was 2.6 cm shorter than the control and
AN-Bayaut-2 was 2.2 cm shorter.
Surface symptoms of infection appeared for C-4727
cotton seedlings after 4-5 days as small dark necrotic
lesions on stems, cotyledon leaves, and roots. These le-
sions were about 1 cm in length on stem and cotyledon
tissues with desiccation of tissues. Fifteen to eighteen
days after infection of the C-4727 cultivar with the
highly virulent V. dahliae isolate AN-3 the seedlings
were dead. Thus, the C-4727 cultivar was highly suscep-
(a)
(b)
Figure 1. Dynamics of POX activity in 7-days seedlings of
two cotton varieties susceptible (C-4727) and resistant (AN-
Bayaut-2) after infection (a – in hours, and b – in days) with
V. dahliae (AN-3 isolate) conidia.
Table 1. Growth inhibition of cotton seedlings after infec-
tion with different strains of V. dahliae (AN-3, highly viru-
lent; 4-T weakly virulent).
Height of seedlings after 8 days (cm)
V. dahliae
isolate C-4727 AN-Bayaut-2
Control 14.5 ± 0.6 13.5 ± 1.2
4-T 12.7 ± 0.8 13.0 ± 1.6
AN-3 11.9 ± 0.5 11.3 ± 1.3
tible to the AN-3 isolate. In comparison, when the
AN-Bayaut-2 cotton seedlings were infected with the
AN-3 isolate, some growth inhibition was observed as
well as the appearance of necrotic zones in hypocotyl
tissue (about 2 mm length) 15 to 18 days after infection.
The isolate 4-T did not cause any surface symptoms ex-
cept a small inhibition in growth for AN-Bayaut, and
only a 1.8 cm inhibition in growth in the C-4727.
3.3. Optical Microscopic Assay of Cotton
Seedling Tissues after Infection with
V. dahliae
Pathogen penetration sites on root rhizoderma of AN-

Participation of Chitin-Binding Peroxidase Isoforms in the Wilt Pathogenesis of Cotton 46
Bayaut-2 cotton seedlings appeared under the micro-
scope as zones of necrosis around live cells. Figure 2(a)
shows the cytoplasm of root cortex cells of plants inocu-
lated with isolate 4-T. These cells were filled with opti-
cally dense material. In the case of AN-3 isolate, the
mycelia extended to the root cortex parenchyma (Figure
2(b)).
After infection of C-4727 cotton seedlings with both
4-T and AN-3 the mycelia of V. dahliae isolates had
more thoroughly permeated the root parenchyma. Hypo-
cotyl microscopic sections of AN-Bayaut-2 and C-4727
cotton seedlings had clear differences in pathogen pene-
tration. Hyphae penetrated both the inter- and intracellu-
lar C-4727 cotton seedlings. The pathogen freely pene-
trated the vascular tissues of susceptible C-4727 and can
be easily visualized. In the fourth day after infection of
C-4727 seedlings we observed colonization of intercel-
lular spaces with AN-3 fungal hyphae (Figure 3(a)). In
comparison, vascular cells of AN-Bayaut-2 cotton seed-
lings were thickened and separated from this pathogen
(Figure 3(b)), and pathogen penetration was not observed.
Tissues of C-4727 vascular system also exhibited cell
plasmolysis, i.e. plasmolemma discharge from vascular
cell wall (Figure 4(a)). A histochemical analysis of a
non fixed preparation of AN-Bayaut-2 vascular cells
shows that POX activity is localized, in external and in-
ternal surfaces of cell wall and in the zones of necrosis
(Figures 3(b) and 4(b)).
(a) (b)
Figure 2. V. dahliae (4-T, a and AN-3, b isolates) penetration of the root rhizoderma of AN-Bayaut-2 cotton seed-
lings. Arrows indicate nodules in cell wall and necrosis.
(a) (b)
Figure 3. Verticillium dahliae (AN-3 isolate) penetration sites of hypocotyls of C-4727 (a) and cotton seedlings
(AN-Bayaut-2) (b). Arrows indicate hyphae between cells of C-4727 (a) and cell wall and necrotic zone of
AN-Bayaut-2 (b).

Participation of Chitin-Binding Peroxidase Isoforms in the Wilt Pathogenesis of Cotton 47
(a) (b)
Figure 4. Tissues of C-4727 vascular system after infection with AN-3 isolate of Verticillium dahliae (a). Histo-
chemical analysis of POX localization in AN-Bayaut-2 vascular cells infected with AN-3 isolate of V. dahliae (b).
3.4. Isozymes with POX Activity in 7-Day-Old
Cotton Seedlings
Anionic isozymes of POX accumulate during cell wall
lignifications. Zheng [16] and Passardi [17] showed that
resistant plants rapidly accumulated lignin after infection
with fungal pathogens. The pathogen penetrates through
intercellular space by hyphal growth. Chitin biosynthesis
is localized in the apical zone of hyphae and its frag-
ments may penetrate to the intercellular space inducing
activity of extracellular anionic isozymes of POX. Thus,
these POXs may bind with and localize the fungal
pathogens. We designed an experiment which modeled
the interaction between the fungal pathogen and cotton
POX enzymes using V. dahliae conidia as a chroma-
tographic matrix. Isoelectrofocusing shows that 7-days
after cotton seedlings of AN-Bayaut-2 had two isozymes
that bind to both chitin and V. dahlia e conidia (Figure 5,
lane 3) while C-4727 had only one of these types of
isozyme (Figure 5, lane 6). This data suggest that chitin
binding isozymes of POX are signaling molecules in
plant defense mechanisms which identify the oligosac-
charide containing phytopathogens.
4. Conclusions
Expression of resistance genes to phytopathogens is best
observed during the infection process when plant cells
are exposed to the pathogen. The riposte reaction pre-
supposes induction of such resistance factors. In our
study we examined the dynamics of POX activity in
seedlings of two different cotton varieties infected with
weak (T-4) and highly virulent (AN-3) isolates of V.
dahliae. When infected with the highly virulent isolate
AN-3, we observed higher POX activity in the infected
AN-Bayaut-2 variety as compared to the susceptible
C-4727. An increase of POX activity of cotton seedlings
infected with V. dahliae had a biphasic characteristic
during 6 to 8 days.
The first enzyme activation of both varieties was noted
2 h after infection. This activation may be described as
Figure 5. Isozyme spectrum of POX from 7-day-old cotton
seedlings (guaiacol/H2O2 stained): a. AN-Bayaut-2 cotton
seedlings: 1 – total POX fraction; 2 – POX fraction not ad-
sorbed by chitin or V. dahliae conidia; 3 – chitin-specific
isozymes; b. C-4727 cotton seedlings: 4 – total POX fraction;
5 – POX fraction not adsorbed on chitin and V. dahliae co-
nidia; 6 – chitin specific isozyme; M – pI markers (Coo-
massie BB G-250 stained).

Participation of Chitin-Binding Peroxidase Isoforms in the Wilt Pathogenesis of Cotton 48
super sensitivity to the pathogen. The second activation
of POX was observed during the next few days, and ex-
hibited significant differences in activity. That is, POX
activity of AN-Bayaut-2 rapidly increased and maxi-
mized 3 days after infection. This was followed by a
gradual decreased in activity. POX activity in the C-4727
variety after infection showed a slow increase that con-
tinued to increase during the remaining days. The control
samples showed no changes in POX activity during the
experiment period, however, the POX activity was higher
in the resistant AN-Bayaut-2 plant compared to the sus-
ceptible C-4727. The increase of POX activity in the
C-4727 variety after infection with V. dahliae may be
explained by progressive fungal colonization of cotton
tissues leading to irreversible senescence. Microscopic
examination supports this hypothesis.
Thus, our data correlates well with the level of resis-
tance of cotton varieties to fungi pathogens. In addition,
the amount of tissue exhibiting necrosis also correlated
with the dynamics of POX activity in different parts of
the seedlings. The difference of these parameters was
dependent on the isolate of V. dahliae which was used
for infection. That is, the AN-3 isolate, which is the more
virulent, caused more necrosis and significantly more
POX activity than the 4-T strain.
The hypersensitivity plant reaction to pathogen attack
causes POX gene expression and a biphasic accumula-
tion of mRNA transcripts which encode biosynthesis of
anionic POX [18]. The first phase occurs 2 to 9 hours
after infection with the pathogen. The second phase of
POX transcript accumulation differs for susceptible and
resistant genotype. This phase in susceptible plants oc-
curs slower and in resistant plants occurs in 24 to 48
hours after the first phase. Thus, susceptible plants have a
slower response allowing greater cell damage. Pathogen
penetration and penetration inside the root tissues is ob-
served for both susceptible and resistant cotton varieties,
but in the resistant variety the surface symptoms do not
appear. However, the depth of pathogen penetration de-
pends on the resistance of cotton variety. Observation of
surface symptoms of wilt infection depends on over-
growth of fungal hyphae in plant cells. POX localization
around cell wall shows active lignifications which blocks
fungal penetration. In this case, chitin, the component of
cell walls of V. dahliae, may induce lignifications. Free
radicals produced by the action of POX are highly reac-
tive and form covalent bounds with proteins and carbo-
hydrates of fungi cell walls. Thus, POX may be consid-
ered as a defense factor in wilt pathogenesis [11].
These findings offer the potential utilization of chitin
binding POX as a biochemical tool to guide breeding
programs to increase resistance to V. dahliae.
5. Acknowledgements
This work was supported by a grant from the Department
of Science and Technologies Development, Republic of
Uzbekistan.
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