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Microbial Consortium-Induced Changes in Oxidative StressMarkers in Pea Plants Challenged with Sclerotinia sclerotiorum
Akansha Jain • Akanksha Singh • Surendra Singh •
Harikesh Bahadur Singh
Received: 19 April 2012 / Accepted: 2 October 2012 / Published online: 6 December 2012
� Springer Science+Business Media New York 2012
Abstract The ability for rhizobacteria and fungus to act as
bioprotectants via induced systemic resistance has been
demonstrated, and considerable progress has been made in
elucidating the mechanisms of plant–biocontrol agent–
pathogen interactions. Pseudomonas aeruginosa PJHU15,
Trichoderma harzianum TNHU27, and Bacillus subtilis
BHHU100 from rhizospheric soils were used singly and in
consortium and assessed on the basis of their ability to
provide disease protection by relating changes in ascorbic
acid and hydrogen peroxide (H2O2) production, lipid per-
oxidation, and antioxidant enzymes in pea under the chal-
lenge of Sclerotinia sclerotiorum. Increased production of
H2O2 24 h after pathogen challenge was observed and was
254.4 and 231.7–287.7 % higher in the triple consortium
and singly treated plants, respectively, when compared to
untreated challenged control plants. A similar increase in
ascorbic acid content and ascorbate peroxidase activity was
observed 24 and 48 h after pathogen challenge, respec-
tively, whereas increased activities of catalase, guaiacol
peroxidase, and glutathione peroxidase were observed 72 h
after pathogen challenge. Similarly, lipid peroxidation
reached a maximum at 72 h of pathogen challenge and was
61.2 and 11.2–32.1 % less in the triple consortium and
singly treated plants, respectively, when compared to
untreated challenged control plants. These findings suggest
that the interaction of microorganisms in the rhizosphere
enhanced protection from oxidative stress generated by
pathogen attack through induction of antioxidant enzymes
and improved reactive oxygen species management.
Keywords Biocontrol � Bacillus subtilis � Microbial
consortium � Pea � Pseudomonas aeruginosa � Trichoderma
harzianum � Sclerotinia rot
Introduction
The plant pathogen defense response in various plant–
pathogen interactions has often been associated with oxi-
dative stress (Apel and Hirt 2004), including Sclerotinia
sclerotiorum infection (Malencic and others 2010). Stem
rot or white mold caused by this fungal pathogen is one of
the most devastating and cosmopolitan soilborne plant
pathogens that infects more than 500 species of plants
worldwide (Willets and Wong 1980).The oxidative burst is
thought to be required for several subsequent defense
responses and is expressed in most, if not all, plant species
(Cessna and others 2000). The hypersensitive response
apparently does not protect plants against attack by nec-
rotrophic pathogens, which can utilize dead cells as a
nutrient source and may indeed promote fungal coloniza-
tion of the host tissues (Walz and others 2008).
Oxidative metabolism of normal cells and different
stress situations like pathogen invasion generate highly
reactive oxygen species (ROS). At low levels, ROS may
actually be beneficial, serving as secondary messengers in
intra- and intercellular signalling pathways (Williams and
others 2011). Increased ROS decrease seed viability and
root growth, stimulate leaf abscission and desiccation,
cause peroxidation of essential membrane lipids, and
damage proteins, carbohydrates, nucleic acids and
A. Jain � A. Singh � S. Singh
Department of Botany, Banaras Hindu University, Varanasi,
Uttar Pradesh, India
H. B. Singh (&)
Department of Mycology and Plant Pathology, Institute of
Agricultural Sciences, Banaras Hindu University, Varanasi,
Uttar Pradesh 221005, India
e-mail: hbs1@rediffmail.com
123
J Plant Growth Regul (2013) 32:388–398
DOI 10.1007/s00344-012-9307-3
pigments such as chloroplasts or carotenoids (Philosoph-
Hadas and others 1994). To counteract oxidative damage
from ROS, plants have acquired antioxidant protective
systems to maintain the lowest possible levels of ROS
inside the cell (Wojtaszek 1997). These systems include
antioxidant enzymes such as superoxide dismutase (SOD),
catalase (CAT), glutathione reductase (GR), glutathione-S-
transferase (GST), ascorbate peroxidase (APx), guaiacol
peroxidase (GPx), and different kinds of plant peroxidases
(POx), as well as some nonenzymatic plant antioxidants
such as glutathione, tocopherols, carotenoids, ascorbate,
flavonoids, proline, and other phenolic compounds (Kang
and Saltveit 2002; Nizamuddin 1987). A principal antiox-
idative system is the ascorbate–glutathione (GSH) cycle,
which detoxifies hydrogen peroxide (H2O2) in the chloro-
plast. These reactions seem to be general strategies to
improve stress tolerance (El-Zahaby and others 1995).
Susceptibility of a plant to pathogen-generated oxidative
stress may depend on the balance between biotic factors
that increase oxidant generation and those cellular com-
ponents that exhibit an antioxidant capability.
Defensive-related mechanisms associated with plant
growth-promoting microorganisms (PGPM) that mediate
induced systemic resistance (ISR) against incoming
pathogens have been thoroughly investigated in the past.
PGPM also showed the capability to activate antioxidants
that are associated with the ISR response and provide pro-
tection against diverse pathogens (Jetiyanon 2007; Xu and
others 2008; Singh and others 2011). An emerging trend in
this area is the use of a microbial consortium that can
enhance protection against pathogens through augmented
elicitation of host defence responses. Synergistic microbial
mixtures may be functional under various environmental
conditions and against various pathogens at a time. Inter-
estingly, several researchers have shown improved disease
management in different crops using microbial consortia
(de Boer and others 2003; Jetiyanon 2007; Akhtar and
Siddiqui 2008; Srivastava and others 2010).
In our previous studies we demonstrated that Tricho-
derma harzianum (NRRL 30596), Bacillus subtilis
(JN099686), and Pseudomonas aeruginosa (JN099685)
singly and in consortium mode, reduced plant mortality and
increased phenolic and proline contents and activities
of defence-related enzymes in pea after inoculation with
S. sclerotiorum under greenhouse conditions (Jain and
others 2012). However, it is unclear whether the observed
disease protection relates to changes in ascorbic acid and
H2O2 production, lipid peroxidation, and antioxidant
characteristics with reference to CAT, APx, GPx, and GR.
We aim to achieve an improvement in plant adaption to
Sclerotinia stress and to evaluate and select biocontrol
agent (BCA) treatments with higher tolerance levels to
biotic stress caused by S. sclerotiorum.
Materials and Methods
Inoculum Preparation of Biocontrol Agents
and Pathogen
Pseudomonas aeruginosa PJHU15 (GenBank accession
No. JN099685) and B. subtilis BHHU100 (GenBank
accession No. JN099686) were isolated from the rhizo-
sphere of Pisum sativum (Jaipur) and P. sativum (Hyder-
abad), respectively, as described previously (Jain and
others 2012). The Trichoderma isolate TNHU27 selected
for the experiments was previously identified as T. har-
zianum (ATCC No. PTA-3701) and was isolated from an
agricultural farm (Pantnagar). These isolates were selected
on the basis of their antagonistic potential against S. scle-
rotiorum and can be used in consortium mode as they
showed compatibility in vitro and in vivo by giving seed
treatment as described previously (Jain and others 2012).
The bacterial strains PJHU15 and BHHU100 were
grown on nutrient agar (Himedia MV001) for routine use
and maintained in nutrient broth (NB; Himedia M002) with
20 % glycerol at -80 �C for long-term storage. Single
colonies of B. subtilis and P. aeruginosa were transferred
to 500 ml flasks containing 200 ml of NB and were grown
on a rotary shaker (150 rpm) for 48 h at 27 ± 2 �C. The
bacterial culture was centrifuged at 6,0009g for 10 min at
4 �C and washed twice with sterile distilled water. The
final pellet was resuspended in a small quantity of sterile
distilled water, and the final concentration was adjusted to
4 9 108 CFU ml-1 using a Thermo Scientific UV 1
spectrophotometer (Thermo Fisher Scientific, Waltham,
MA, USA). Similarly, T. harzianum was grown on potato
dextrose agar (PDA; Himedia M096) for 6 days at
27 ± 2 �C, and the spores were harvested and brought to a
final concentration of 2 9 107 CFU ml-1. The pathogen
(S. sclerotiorum) was multiplied on bajra (Pennisetum ty-
phoides Pers.) seed meal–sand medium (bajra seeds, 250 g;
washed white sand, 750 g; distilled water, 250 ml) at
20 ± 2 �C for 15 days (Sarma and others 2007).
Greenhouse Experiment
A soil mixture of sandy soil, vermicompost, and farmyard
manure (2:1:1) was placed in polypropylene bags and
sterilized in an autoclave for three consecutive days at
15 lbs. of pressure for 30 min, then 1.5 kg of the mixture
was placed in plastic pots (15 9 10 cm). Seeds of pea (Cv.
Arkel) were surface sterilized with 1 % sodium hypo-
chlorite for 30 s, rinsed twice with sterile distilled water,
and dried under a sterile air stream. The seeds were coated
with B. subtilis, P. aeruginosa, and T. harzianum either
singly or two or three together, with the suspensions of the
organisms prepared in 1 % carboxymethyl cellulose used
J Plant Growth Regul (2013) 32:388–398 389
123
as adhesive. For coating, seeds were soaked in their
respective suspensions for 10 h [in the case of a consor-
tium, equal amounts of suspension (v/v) were mixed].
Then, the microbial suspension was drained off and the
seeds were dried overnight in sterile Petri dishes. The
following treatments were examined: (1) B. subtilis
(BHHU100), (2) T. harzianum (TNHU27), (3) P. aeru-
ginosa (PJHU15), (4) BHHU100 ? TNHU27, (5)
BHHU100 ? PJHU15, (6) TNHU27 ? PJHU15, and (7)
BHHU100 ? TNHU27 ? PJHU15. Two sets of noninoc-
ulated controls were also maintained. Five pots with six
seeds sown per pot were used for each treatment. The pots
were placed in the greenhouse and irrigation was provided
as needed or at 2 day intervals until partial saturation. A
cycle of 10:14 h dark:light and a temperature of 18 ± 2 �C
were maintained in the greenhouse. The design of the
experimental setup was completely randomized. Ten days
after germination three plants from each treatment were
harvested and dried in an oven at 60 �C for recording the
total biomass. Rhizosphere colonization of the three
microbes was also assessed in different treatment in terms
of log10 colony forming unit (CFU) g-1. Colonized path-
ogen culture was blended well prior to use as inoculum.
The collar region of the plants was inoculated in all the
treatments, except one set of controls that was left
unchallenged, at the rate of 50 g per pot 4 weeks after
sowing. Disease reduction was monitored 20 days after
infection by recording the number of infected plants in
untreated challenged control plants and BCA-treated
challenged plants.
Sample Collection for Biochemical Analysis
For each treatment, plants were carefully uprooted without
causing any damage at 24 h intervals after pathogen
inoculation up to 96 h. The plants were washed in running
tap water and stored in a deep freezer (-80 �C) until used
for biochemical analysis.
Hydrogen Peroxide Production
Histochemical detection of H2O2 was done by 3,30-diam-
inobenzidine (DAB) staining as described by Thordal-
Christensen and others (1997). H2O2 reacts with DAB to
form a reddish-brown stain. Leaf disks were incubated in
1 mg ml-1 DAB solution (pH 7.5). After incubation in the
dark at room temperature for 20 h, leaf tissues were boiled
in a solution containing alcohol and lactophenol (2:1) for
5 min and rinsed twice with 50 % ethanol.
For H2O2 quantification, a 0.1 g leaf sample from each
of the treatments was homogenized in an ice bath with
2.0 ml of 0.1 % (w/v) of trichloroacetic acid (TCA). The
homogenate was centrifuged at 12,0009g for 15 min and
0.5 ml of the supernatant was mixed with 10 mM potas-
sium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium
iodide solution and incubated for 5 min. The oxidation
product formed was measured at 390 nm (Velikova 2000).
The amount of H2O2 formed was determined from the
standard curve made with known concentrations of H2O2
and expressed as nmol H2O2 g-1 fresh weight (FW).
Ascorbic Acid Content (AAC)
A 0.1 g leaf sample was homogenized in 2 ml of oxalic
acid and Na EDTA extraction solution. To 1 ml of super-
natant, 5 ml of 20 lg ml-1 of 2,6-dichlorophenol-indo-
phenol (DCPIP) dye was added to develop color, and the
absorbance was recorded at 520 nm against a reagent
blank. Ascorbic acid content was quantified using the
method of Keller and Schwager (1977).
Lipid Peroxidation (LPO)
Lipid peroxidation was measured in terms of the amount of
malondialdehyde (MDA), which is a secondary end product
of polyunsaturated fatty acid oxidation, determined by the
thiobarbituric acid (TBA) reaction. The assay was carried out
by the method described by Ohkawa and others (1979). A
leaf sample (0.1 g) from each of the treatments was
homogenized and incubated with 2.0 ml of 20 % TCA (w/v)
containing 1 % TBA (w/v) for 30 min at 95 �C. The reaction
was stopped by cooling on ice for 10 min and the product was
centrifuged at 10,0009g for 15 min. The absorbance of the
reaction product was measured at 532 nm. The amount of
MDA was computed using the extinction coefficient of
155 mM-1 cm-1 and expressed as nmol MDA g-1 FW.
Antioxidant Enzyme Activities
Ascorbate peroxidase (APx) (E.C. 1.11.1.11) activity was
measured as per the method described by Nakano and
Asada (1981).The reaction mixture consisted of 25 mM
phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.25 mM
ascorbic acid, 1.0 mM H2O2, and 0.2 ml enzyme extract. A
decrease in absorbance was noted 60 s after addition of
enzyme extract. The oxidation of ascorbic acid was
recorded at 290 nm and the enzyme activity was expressed
as nmol ascorbate oxidized min-1 mg-1 protein.
Catalase (CAT) (E.C. 1.11.1.6) activity was assayed
according to the method described by Aebi (1984). Leaf
samples (0.1 g) were homogenized in 50 mM Tris–HCl
buffer (pH 8.0) containing 0.5 mM EDTA, 2 % w/v pol-
yvinylpyrrolidone, and 0.5 % (v/v) Triton X100 using a
chilled mortar and pestle. The homogenate was centrifuged
at 15,0009g for 10 min at 4 �C, and the supernatant was
used for the enzyme assay. The reaction mixture consisted
390 J Plant Growth Regul (2013) 32:388–398
123
of 300 lM phosphate buffer (pH 7.2) and 100 lM H2O2 in
1 ml enzyme extract. Activity was determined by recording
O2 released from enzymatic dissociation of H2O2 in dark-
ness for 1 min. O2 produced by the enzymatic reaction was
estimated by measuring the decrease in H2O2 absorption at
240 nm (extinction coefficient of H2O2 is
0.036 mM-1 cm-1) and enzyme activity was expressed as
lM H2O2 oxidized min-1 g-1 FW.
Glutathione reductase (GR) (E.C. 1.6.4.2) activity was
determined by using the method described by Anderson
(1996). A leaf sample (0.1 g) was homogenized using a
chilled mortar and pestle in 5 ml of 50 mM Tris–HCl
buffer (pH 7.6). The homogenate was centrifuged at
15,0009g for 30 min at 4 �C, and the supernatant was used
for enzyme assay. The reaction mixture contained 50 mM
Tris–HCl buffer (pH 7.6), 10 ml NADPH (0.15 mM),
100 ll oxidized glutathione (1 mM GSSG), 3 mM MgCl2,
and 0.3 ml enzyme extract. GR activity was recorded by
measuring the decrease in absorbance of NADPH at
340 nm and the activity of enzyme was expressed as nmol
NADPH oxidized min-1 mg-1 protein.
Guaiacol peroxidase (GPx) (E.C. 1.11.1.7) was assayed
by measuring the increase in absorbance at 470 nm due to
oxidation of guaiacol to tetraguaiacol (Zheng and Van
Huystee 1992). The reaction mixture contained 10 mM
sodium phosphate (pH 6.0), 0.3 % (v/v) H2O2, 1 % (v/v)
tetraguaiacol, and 0.3 ml enzyme extract. The reaction was
initiated by the addition of H2O2. The absorbance was
recorded at 470 nm and the linear portion of the activity
curve was used to express enzyme activity (expressed as
U mg-1 protein). One unit of enzyme activity represented
the amount of enzyme catalysing the oxidation of 1 lmol
of guaiacol min-1. Protein was estimated following the
method of Lowry and others (1951).
Statistical Analysis
Values from different experiments shown in the figures are
mean ± standard deviation (SD) of at least three replica-
tions of each of the experiments. All the data collected in
this study were subjected to analysis of variance
(ANOVA). The treatment mean values were compared by
Duncan’s multiple range test at the P B 0.05 significance
level. The software used for analysis was SPSS ver. 16
(SPSS, Inc., Chicago, IL, USA).
Results
Total Biomass
Plants treated with beneficial microbes had increased plant
growth in terms of dry weight in comparison to untreated
control plants (Fig. 1). Plants treated with a consortium of
B. subtilis ? P. aeruginosa ? T. harzianum showed an
increase in growth of 261.9 % in comparison to control
plants not treated with any microbial agents. Significant
increase in colonization in plants treated B. subtilis ?
T. harzianum ? P. aeruginosa was observed in compari-
son to single microbe treated plants (Table 1).
Disease Reduction
Challenge inoculation with S. sclerotiorum resulted in soft
brown lesion development on the collar region. All the
BCA-treated plants challenged with the pathogen showed
significant reduction in plant mortality compared with
BCA-untreated challenged plants (Fig. 2). The maximum
disease reduction was observed in plants treated with a
consortium of B. subtilis ? P. aeruginosa ? T. harzianum
(85.7 %), followed by B. subtilis ? T. harzianum, and
P. aeruginosa ? T. harzianum (71.4 %) in comparison to
control challenged plants.
H2O2 Production
H2O2 production was visualized as a reddish brown stain
by DAB, which was more prominent in BCA-treated
challenged plants (Fig. 3). Quantitative measurement of
H2O2 content was studied in BCA-treated plants both
before and after pathogen challenge (Fig. 4a, b). A sig-
nificant change was observed in the BCA-treated pea plants
both before and after challenge, but the response was
stronger and more visible upon Sclerotinia stress. Pathogen
stress caused a sharp increase in H2O2 at 24 h after chal-
lenge in all the BCA-treated plants. Maximum induction
was observed in pathogen-challenged T. harzianum-treated
Fig. 1 Dry weight of pea plants raised from seeds treated with B.subtilis BHHU100, T. harzianum TNHU27, and P. aeruginosaPJHU15 either individually or in combination at 10 days after
germination. Results are expressed as the mean of three replicates and
vertical bars indicate SD of the mean. Different letters indicate
significant differences among treatments within the results taken at
the same time interval according to Duncan’s multiple range test at
p B 0.05
J Plant Growth Regul (2013) 32:388–398 391
123
plants, which showed a 287.8 % increase in comparison to
noninoculated challenged control plants. However, H2O2
content was nearly equal in challenged plants treated with a
consortium of B. subtilis ? P. aeruginosa ? T. harzianum,
where it was 254.4 and 593.5 % higher than that in chal-
lenged and unchallenged noninoculated control plants,
respectively. After 48 h of pathogen stress, H2O2 content
decreased gradually in all the BCA-treated plants.
Although a slight increase was also observed in noninoc-
ulated challenged controls, it was insignificantly low in
comparison to other treatments. No change was observed in
untreated nonchallenged plants.
Ascorbic Acid Content
In healthy leaves the ascorbic acid content did not change
with time (Fig. 5). In contrast, after 24 h of pathogen
challenge, the AAC in P. aeruginosa ? T. harzianum-
inoculated plants increased sharply and was 67.9 and
171.0 % higher when compared with noninoculated chal-
lenged and unchallenged controls, respectively; thereafter,
a declining trend was observed in BCA-treated plants.
Although the AAC in the noninoculated challenged plants
increased gradually up to 48 h after pathogen challenge, its
content was maximized in plants treated with P. aerugin-
osa ? T. harzianum followed by a consortium of B. sub-
tilis ? P. aeruginosa ? T. harzianum and was 60 and
156.6 % higher than that in noninoculated challenged and
unchallenged plants after 24 h of pathogen stress,
respectively.
Lipid Peroxidation
The MDA content was significantly higher in noninocu-
lated challenged plants and was greatly reduced in the
plants inoculated with PGPMs and challenged with path-
ogen (Fig. 6). In the noninoculated challenged plants, the
MDA content increased and rose sharply 96 h after path-
ogen challenge. It was 61.2 % less and 19.0 % higher in
the plants inoculated with the consortium of B. subtil-
is ? P. aeruginosa ? T. harzianum in comparison to
noninoculated challenged and unchallenged plants,
respectively. However, during the whole period of the
study, the content of MDA increased slowly with all the
treatments but at a particularly lower level in consortium-
treated plants when compared with the singly inoculated
and noninoculated challenged plants.
Antioxidant Enzyme Activities
Pathogen stress resulted in a general increase in the anti-
oxidant enzyme activities throughout the experiment in
both inoculated and noninoculated plants. However, it was
much more pronounced in the plants inoculated with a
consortium of B. subtilis, P. aeruginosa, and T. harzianum
(Figs. 7, 8, 9, 10). In the noninoculated challenged plants,
Fig. 2 Disease reduction in pea plants raised from seeds treated with
B. subtilis BHHU100, T. harzianum TNHU27, and P. aeruginosaPJHU15 either individually or in combination 20 days after challenge
inoculation with S. sclerotiorum. Results are expressed as the mean of
three replicates and vertical bars indicate SD of the mean. Differentletters indicate significant differences among treatments within the
results taken at the same time interval according to Duncan’s multiple
range test at p B 0.05
Table 1 Effect of Bacillus subtilis BHHU100, Trichoderma harzianum TNHU27 and Pseudomonas aeruginosa PJHU15 either singly or in
combination on population density of each other in pea (log10 CFU g-1)
Treatments B. subtilis T. harzianum P. aeruginosa
Untreated control – – –
BHHU100 5.28 ? 0.03b – –
TNHU27 – 5.51 ? 0.32b –
PJHU15 – – 6.0 ? 0.2b
BHHU100 ? TNHU27 6.95 ? 0.99a 5.0 ? 0.46b –
BHHU100 ? PJHU15 6.0 ? 0.46b – 6.3 ? 0.46ab
TNHU27 ? PJHU15 – 6.9 ? 0.92a 6.85 ? 0.69a
BHHU100 ? TNHU27 ? PJHU15 6.3 ? 0.46a 7.45 ? 0.23a 6.9 ? 0.08a
Results are expressed as means of three replicates ±SD of the means. Different letters indicate significant differences among treatment results
taken at the same time interval according to Duncan’s multiple range test at p B 0.05
392 J Plant Growth Regul (2013) 32:388–398
123
the activities of APx, CAT, GR, and GPx increased after
pathogen challenge, peaking after 72 h, and decreased
slightly thereafter. In contrast, in the B. subtilis ? P. aeru-
ginosa ? T. harzianum-treated plants, the activities of APx
and CAT increased significantly, reaching maximum values
after 48 and 72 h of pathogen challenge, respectively, and
GR and GPx activities peaked after 72 h of pathogen chal-
lenge. The activities of APx, CAT, GR, and GPx were 110.3,
45.4, 28.54, and 107.3 % greater than those of the nonin-
oculated challenged control plants, respectively, and there-
after a declining trend was observed. During our study
period, the enzyme activities in general were always higher
in the B. subtilis ? P. aeruginosa ? T. harzianum-treated
plants, followed by the two-species consortium- and single-
species-treated plants when compared with the noninocu-
lated challenged and unchallenged controls.
Discussion
The mechanisms involved in microbial induced systemic
resistance (ISR) vary depending on the host–pathogen
system and the microbes chosen to control the disease. ISR
in plants is a physiological state of enhanced defensive
capacity elicited by specific stimuli, whereby the plant’s
innate defenses are potentiated against subsequent chal-
lenges (Vallad and Goodman 2004). The results obtained
from the present study indicate that the microbes in con-
sortium mode play a positive role in protecting against
S. sclerotiorum-induced oxidative stress and enhance tol-
erance by partially increasing the H2O2 content and the
activities of antioxidant enzymes.
An oxidative burst is one of the earliest and most uni-
versal resistance responses mounted by plant tissues
against an invading microbe: a controlled release of O2-
and H2O2 at the point of pathogen challenge is believed to
be required for pathogen defence (Bestwick and others
1997). Plant cells are relatively tolerant of H2O2 in com-
parison to pathogens (Lu and Higgins 1999). H2O2 not only
directly inhibits and reduces the growth of plant pathogens,
but in the early plant–pathogen interaction it contributes to
cell wall strengthening by papillae formation, lignifica-
tions, cross-linking of hydroxyproline/proline-rich pro-
teins, and other cell wall polymers (Kuzniak and Urbanek
2000). In the present investigation, it was found that H2O2
content initially increased significantly in the BCA-chal-
lenged plants, indicating that plants treated with microbes
were better equipped to mitigate stress generated by
Fig. 3 H2O2 production (arrows) in pea leaves as visualized by 3,30-diaminobenzidine (DAB) staining. H2O2 production (revealed by
reddish-brown stain) was detected in (a) unchallenged controls, and
24 h after Sclerotinia sclerotiorum challenge in (b–i), where
b BHHU100, c TNHU27, d PJHU15, e BHHU100 ? TNHU27,
f BHHU100 ? PJHU15, g TNHU27 ? PJHU15,
h BHHU100 ? TNHU27 ? PJHU15, and i challenged control
J Plant Growth Regul (2013) 32:388–398 393
123
Sclerotinia, which is known to suppress H2O2 production.
However, a downward trend appeared thereafter in the
BCA-treated plants after a temporary increase, with a
maximum decrease in plants treated with a consortium of
B. subtilis ? P. aeruginosa ? T. harzianum. In noninoc-
ulated challenged plants, although an increase in H2O2
Fig. 4 H2O2 content at
different time intervals in pea
plants raised from seeds treated
with B subtilis BHHU100, Tharzianum TNHU27, and Paeruginosa PJHU15 either
singly or in combination before
(a) and after (b) challenge with
S. sclerotiorum. Results are
expressed as the mean of three
replicates and vertical barsindicate the SD of the mean.
Different letters indicate
significant differences among
treatments within the results
taken at the same time interval
according to Duncan’s multiple
range test at p B 0.05
Fig. 5 Ascorbic acid content at
different time intervals in pea
plants raised from seeds treated
with B. subtilis BHHU100, T.harzianum TNHU27, and P.aeruginosa PJHU15 either
individually or in combination
and challenged with S.sclerotiorum. Results are
expressed as the mean of three
replicates and vertical barsindicate the SD of the mean.
Different letters indicate
significant differences among
treatments within the results
taken at the same time interval
according to Duncan’s multiple
range test at p B 0.05
394 J Plant Growth Regul (2013) 32:388–398
123
content was observed, it was insignificant in comparison to
plants treated with BCAs. It has been demonstrated that the
oxidative burst is indeed suppressed in the presence of
oxalate generated by virulent Sclerotinia, reducing H2O2
production which blocks a signalling event in the oxidative
burst pathway, even at the optimal pH of the pathway,
thereby compromising the defence responses generated by
the host plant (Cessna and others 2000).
ROS activate ethylene, salicylic acid, and jasmonic acid
signalling pathways. These pathways induce defence-gene
expression to counteract the invading pathogen (Conklin
and Barth 2004). An initial increase in AAC was observed
in our study, which was significantly higher in consortium-
treated and pathogen-challenged plants when compared
with noninoculated challenged and unchallenged controls.
Thereafter, a marked decline in AAC was observed in all
BCA-treated pathogen-challenged plants. An initial
increase in AAC in the present study can be explained by
the fact that ascorbic acid along with glutathione are the
major redox buffers of the plant cells and are present at
high concentrations in chloroplasts and other cellular
compartments (5–20 mM ascorbic acid and 1–5 mM glu-
tathione) where they play a crucial role in plant defence
against oxidative stress (Noctor and Foyer 1998).
The MDA content can be an indicator of oxidative
damage to membranes (Salin 1988). In general, the ele-
vated levels of H2O2 can be positively correlated to the
increased MDA content in plants exposed to a pathogen,
indicating that its accumulation leads to oxidative damage.
However, this damage was also observed in B. subtilis ?P.
aeruginosa ? T. harzianum-treated pea plants, followed
by P. aeruginosa ? T. harzianum-treated pea plants, but
Fig. 6 Lipid peroxidation at different time intervals in pea plants
raised from seeds treated with B. subtilis BHHU100, T. harzianumTNHU27, and P. aeruginosa PJHU15 either individually or in
combination and challenged with S. sclerotiorum. Results are
expressed as the mean of three replicates and vertical bars indicate
the SD of the mean. Different letters indicate significant differences
among treatments within the results taken at the same time interval
according to Duncan’s multiple range test at p B 0.05
Fig. 7 Ascorbate peroxidase activity at different time intervals in pea
plants raised from seeds treated with B. subtilis BHHU100, T.harzianum TNHU27, and P. aeruginosa PJHU15 either individually
or in combination and challenged with S. sclerotiorum. Results are
expressed as the mean of three replicates and vertical bars indicate
the SD of the mean. Different letters indicate significant differences
among treatments within the results taken at the same time interval
according to Duncan’s multiple range test at p B 0.05
J Plant Growth Regul (2013) 32:388–398 395
123
was less in comparison to noninoculated challenged control
plants. In untreated challenged plants, the rate of lipid
peroxidation was greatly elevated in which more cells were
stressed. The product of lipid peroxidation may act as a
signal for activation of defence reactions (Kuzniak and
Urbanek 2000). Similarly, T. harzianum-pathogen-cotreat-
ed sunflower plants were shown to have significantly lower
levels of MDA than pathogen-inoculated plants, implying
that the Trichoderma had potent efficacy in alleviating
pathogen-induced oxidative damage (Singh and others
2011).
An increase in antioxidant enzyme activity is often
linked to improvement in stress tolerance (Lee and others
2007; Murgia and others 2004). Earlier studies showed that
Erysiphe graminis f. sp. hordei infection leads to signifi-
cant changes in the antioxidant metabolism of barley (El-
Zahaby and others 1995). The increases of antioxidant
enzyme activities in the BCA-treated plants, especially in
consortium-treated plants, were more pronounced and
higher levels were observed, indicating that the BCA-
inoculated plants were highly efficient in scavenging from
the deleterious effects of ROS. In particular, APx activity
in the B. subtilis ? P. aeruginosa ? T. harzianum-treated
plants peaked after 48 h of pathogen challenge, when the
AAC and H2O2 content dropped down to the lowest levels.
The marked decline of AAC in BCA-treated and pathogen-
infected plants may be explained by the strong induction of
APx activity. The apparent decrease in ascorbic acid and
concomitant increase in antioxidative enzymes in BCA-
treated plants show that ascorbic acid plays a dominant role
in the antioxidative reaction against S. sclerotiorum. The
activities of CAT, GR, and GPx reached their maximum
Fig. 8 Catalase activity at different time intervals in pea plants raised
from seeds treated with B. subtilis BHHU100, T. harzianumTNHU27, and P. aeruginosa PJHU15 either individually or in
combination and challenged with S. sclerotiorum. Results are
expressed as the mean of three replicates and vertical bars indicate
the SD of the mean. Different letters indicate significant differences
among treatments within the results taken at the same time interval
according to Duncan’s multiple range test at p B 0.05
Fig. 9 Glutathione reductase at different time intervals in pea plants
raised from seeds treated with B. subtilis BHHU100, T. harzianumTNHU27, and P. aeruginosa PJHU15 either individually or in
combination and challenged with S. sclerotiorum. Results are
expressed as the mean of three replicates and vertical bars indicate
the SD of the mean. Different letters indicate significant differences
among treatments within the results taken at the same time interval
according to Duncan’s multiple range test at p B 0.05
396 J Plant Growth Regul (2013) 32:388–398
123
levels after 72 h of pathogen challenge in all the treat-
ments. The different affinities of APX (lM range) and
CAT (mM range) for H2O2 and their induction at 48 and
72 h, respectively, after pathogen challenge in the present
study also confirm that they belong to two different classes
of H2O2-scavenging enzymes: APX might be responsible
for the fine modulation of reactive oxygen intermediates
for signalling, whereas CAT might be responsible for the
removal of excess reactive oxygen intermediates during
stress (Mittler and others 2004). The cytosol, with its
ascorbate–glutathione cycle, and the peroxisomes, with
CAT, might therefore act as a buffer zone to control the
overall level of reactive oxygen intermediates reaching
different cellular compartments during stress and normal
metabolism (Mittler and others 2004). GR is believed to
maintain a high reduced peroxidised ratio of ascorbic acid
and glutathione which is essential for the proper scaveng-
ing of ROS in cells (Asada and Takahashi 1987; Noctor
and Foyer 1998). Increased activity of CAT, APx, SOD,
and GPx was also observed in a similar study involving T.
harzianum–Rhizoctonia solani and sunflower interaction
(Singh and others 2011). In our previous study, increased
activity of the antioxidant enzymes SOD and peroxidase
was observed in pea plants treated with the same microbial
consortium and challenged by S. sclerotiorum (Jain and
others 2012).
Our results show that involvement of BCAs leads to
significant changes in H2O2 and antioxidant metabolism of
pea, challenged by S. sclerotiorum. Thus, the stronger
induction of antioxidative enzymes in plants treated with a
consortium of B. subtilis, P. aeruginosa, and T. harzianum
may be explained by the fact that these microbes in con-
sortium mode equipped the plants with better elicitation of
stress response from greater numbers of cells than in singly
treated and untreated control plants. This study also sug-
gests that BCAs induced stronger antioxidative pathways
which are probably not only defensive reactions, but also
contribute to suppression of lipid peroxidation and necrotic
symptom expression.
Conclusion
In the present study enhanced protection from oxidative
stress is the most reasonable explanation of the beneficial
effects that plant growth-promoting rhizospheric microbes
have on host plants. Enhanced ROS management in con-
sortium-treated plants could explain the protection from
pathogen stress. It also explains resistance to many addi-
tional oxidative stresses generated by pathogen attack and
may account for the overall enhanced tolerance. The anti-
oxidant enzymatic activities offer an alternative mecha-
nism that is observed in plant–microbe defensive
mutualism. Further investigations will be needed to eval-
uate, ascertain, and support the details of the mechanisms.
Acknowledgments Akansha Jain is grateful to Department of Sci-
ence and Technology, Government of India, New Delhi, for financial
assistance under AORC scheme as INSPIRE-JRF.
References
Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126
Akhtar MS, Siddiqui ZA (2008) Biocontrol of root-rot disease
complex of chickpea by Glomus intraradices, Rhizobium sp. and
Pseudomonas striata. Crop Prot 27:410–417
Anderson ME (1996) Glutathione. In: Punchard NA, Kelly FJ (eds)
Free radicals: a practical approach. Oxford University Press,
Oxford, pp 213–226
Fig. 10 Guaiacol peroxidase at different time intervals in pea plants
raised from seeds treated with B. subtilis BHHU100, T. harzianumTNHU27, and P. aeruginosa PJHU15 either individually or in
combination and challenged with S. sclerotiorum. Results are
expressed as the mean of three replicates and vertical bars indicate
the SD of the mean. Different letters indicate significant differences
among treatments within the results taken at the same time interval
according to Duncan’s multiple range test at p B 0.05
J Plant Growth Regul (2013) 32:388–398 397
123
Apel K, Hirt H (2004) Reactive oxygen species: metabolism,
oxidative stress, and signal transduction. Annu Rev Plant Biol
55:373–399
Asada K, Takahashi M (1987) Production and scavenging of active
oxygen in photosynthesis. In: Kyle DJ et al (eds) Photoinhibi-
tion. Elsevier, Amsterdam, pp 227–287
Bestwick CS, Brown IR, Benneth MHR, Mansfield JW (1997)
Localization of hydrogen peroxide accumulation during the
hypersensitive reaction of lettuce cells to Pseudomonas syringaepv phaseolicola. Plant Cell 9:209–221
Cessna SG, Sears VE, Dickman MB, Low PS (2000) Oxalic acid, a
pathogenicity factor for Sclerotinia sclerotiorum suppresses the
oxidative burst of the host plant. Plant Cell 12:2191–2200
Conklin PL, Barth C (2004) Ascorbic acid, a familiar small molecule
intertwined in the response of plants to ozone, pathogens, and the
onset of senescence. Plant Cell Environ 27:959–970
de Boer M, Bom P, Kindt F, Keurentjes JJB, van der Sluis I, van Loon
LC, Bakker PAHM (2003) Control of Fusarium wilt of radish by
combining Pseudomonas putida strains that have different
disease-suppressive mechanisms. Biol Control 93:626–632
El-Zahaby HB, Gullner G, Kiraly Z (1995) Effects of powdery
mildew infection of barley on the ascorbate–glutathione cycle
and other antioxidants in different host-pathogen interactions.
Biochem Cell Biol 85(10):1225–1230
Jain A, Singh S, Sarma BK, Singh HB (2012) Microbial consortium
mediated reprogramming of defense network in pea to enhance
tolerance against Sclerotinia sclerotiorum. J Appl Microbiol
112(3):537–550
Jetiyanon K (2007) Defensive-related enzyme response in plants
treated with a mixture of Bacillus strains (IN937a and IN937b)
against different pathogens. Biol Control 42:178–185
Kang KM, Saltveit ME (2002) Chilling tolerance of corn, cucumber
and rice seedling leaves and roots are differentially affected by
salicylic acid. Physiol Plant 115:571–576
Keller T, Schwager H (1977) Air pollution and ascorbic acid. Eur J
Plant Pathol 7:338–350
Kuzniak E, Urbanek H (2000) The involvement of hydrogen peroxide
in plant responses to stresses. Acta Physiol Plant 22(2):195–203
Lee KP, Kim C, Landgraf F, Apel K (2007) EXECUTER1- and
EXECUTER2-dependent transfer of stress-related signals from
the plastid to the nucleus of Arabidopsis thaliana. Proc Natl
Acad Sci USA 104:10270–10275
Lowry OH, Rosenbrough JJ, Farr AL, Randall RJ (1951) Estimation of
protein with the folin phenol reagent. J Biol Chem 193:265–275
Lu H, Higgins VJ (1999) The effect of hydrogen peroxide on the
viability of tomato cells and of the pathogen Cladosporiumfulvum. Physiol Mol Plant Pathol 54:131–143
Malencic D, Kiprovski B, Popovic M, Prvulovic D, Miladinovic J,
Djordjevic V (2010) Changes in antioxidant systems in soybean
as affected by Sclerotinia sclerotiorum (Lib.) de Bary. Plant
Physiol Biochem 48(10–11):903–908
Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004)
Reactive oxygen gene network of plants. Trends Plant Sci
9:490–498
Murgia I, Tarantino D, Vannini C, Bracale M, Carrabvieri S, Soave C
(2004) Arabidopsis thaliana plants overexpressing thylakoidal
ascorbate peroxidase show resistance to paraquat-induced pho-
tooxidative stress and to nitric oxide-induced cell death. Plant J
38:940–995
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by
ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell
Physiol 22:867–880
Nizamuddin A (1987) NADPH-dependent and O2-dependent lipid
peroxidation. Biochem Educ 15(2):58–62
Noctor G, Foyer C (1998) Ascorbate and glutathione: keeping active
oxygen under control. Annu Rev Plant Physiol Plant Mol Biol
49:249–279
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxidation in
animal tissues by thiobarbituric acid reaction. Anal Biochem
95:51–58
Philosoph-Hadas S, Meir S, Akiri B, Kanner J (1994) Oxidative
defense systems in leaves of three edible herb species in relation
to their senescence rates. J Agric Food Chem 42(11):2376–2381
Salin ML (1988) Toxic oxygen species and protective systems of the
chloroplast. Physiol Plant 72:681–689
Sarma BK, Ameer Basha S, Singh DP, Singh UP (2007) Use of
nonconventional chemicals as an alternative approach to protect
chickpea (Cicer arietinum) from Sclerotinia stem rot. Crop Prot
26:1042–1048
Singh BN, Singh A, Singh SP, Singh HB (2011) Trichodermaharzianum-mediated reprogramming of oxidative stress response
in root apoplast of sunflower enhances defence against Rhizoc-tonia solani. Eur J Plant Pathol 131:121–134
Srivastava R, Khalid A, Singh US, Sharma AK (2010) Evaluation of
arbuscular mycorrhizal fungus, fluorescent Pseudomonas and
Trichoderma harzianum formulation against Fusarium oxyspo-rum f. sp. lycopersici for the management of tomato wilt. Biol
Control 53:24–31
Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997)
Subcellular localization of H2O2 in plants. H2O2 accumulation in
papillae and hypersensitive response during the barley-powdery
mildew interaction. Plant J 11:1187–1194
Vallad GE, Goodman RM (2004) Systemic acquired resistance and
induced systemic resistance in conventional agriculture. Crop
Sci 44:1920–1934
Velikova V (2000) Oxidative stress and some antioxidant systems in
acid rain-treated bean plants: protective role of exogenous
polyamines. Plant Sci 151:59–66
Walz A, Sell IZ, Theisen S, Kortekamp A (2008) Reactive oxygen
intermediates and oxalic acid in the pathogenesis of the
necrotrophic fungus Sclerotinia sclerotiorum. Eur J Plant Pathol
120(4):317–330
Willets HJ, Wong JAL (1980) The biology of Sclerotinia sclerotio-rum, S. trifoliorum and S. minor with emphasis on species
nomenclature. Bot Rev 46:101–165
Williams R, Rohr AM, Wang WT, Choi IY, Lee P, Berman NEJ,
Lynch SG, LeVine SM (2011) Iron deposition is independent of
cellular inflammation in a cerebral model of multiple sclerosis.
BMC Neurosci 12:59
Wojtaszek P (1997) Oxidative burst: an early plant response to
pathogen infection. Biochem J 322:681–692
Xu X, Qin G, Tian S (2008) Effect of microbial biocontrol agents on
alleviating oxidative damage of peach fruit subjected to fungal
pathogen. Int J Food Microbiol 126:153–158
Zheng X, Van Huystee RB (1992) Peroxide-regulated elongation of
segments from peanuts hypocotyls. Plant Sci 81:47–56
398 J Plant Growth Regul (2013) 32:388–398
123