Microbial Consortium-Induced Changes in Oxidative Stress Markers in Pea Plants Challenged with...

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: [email protected]

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


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


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


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


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



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






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






Fig. 9 Glutathione reductase at different time intervals in pea plants








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.

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