Romanian Biotechnological Letters
Copyright © 2014 University of Bucharest
Vol.19, No3, 2014
Printed in Romania. All rights reserved
ORIGINAL PAPER
Induction of plant antioxidant system by interaction with beneficial
and/or pathogenic microorganisms
Received for publication, July 05, 2013
Accepted, January 20, 2014
Florenţa Elena Helepciuca, Monica Elena Mitoia*, Anca Manole-P unescua,
Florentina Aldeaa, Aurelia Brezeanua, C lina Petruţa Corneab
a
Department of Plant and Animal Cytobiology, Institute of Biology Bucharest of
Romanian Academy, 296 Splaiul Independenţei, 060031 Bucharest, P.O. Box 5653, Romania
b
Department of Biotechnology, University of Agronomical Sciences and Veterinary
Medicine, Bucharest, Romania
Corresponding author: MONICA ELENA MITOI
E-mail:
[email protected]
Abstract
The interaction of plant with both pathogenic and non-pathogenic microorganisms can trigger active
defense reactions in plant. The defence response of Cucumis sativus L. plants treated with beneficial
bacteria and phytopathogenic fungi was evaluated. Cucumber plantlets treated with bacterial isolates
having antifungal activity (Bacillus licheniformis B40, Bacillus amyloliquefaciens Bw, Bacillus sp. Bw1,
Pseudomonas aeruginosa P14) and fungal (Pythium debaryanum Hesse) suspensions were examined for
antioxidant system activation. Potential alteration in superoxide dismutases, catalases, guaiacol
peroxidases and lignin production was approached using biochemical and histochemical methods.
Bacterial inoculation increased the number of superoxide dismutase and catalase isoforms, and also the
enzymatic activity of these antioxidants. Reinforcement of epidermal and cortical cell walls through lignin
deposition was observed using histochemical methods. These results showed that antioxidant enzymes
activity and lignin production were intensified in treated variants, indicating the activation of some
defense mechanisms, and a potential increase of plant resistance to pathogen attack.
Keywords: defense response, antioxidant enzymes, antagonistic bacteria, Cucumis
sativus, Pythium debaryanum.
Introduction
Plant rhizosphere is colonized by numerous microorganisms, which are attracted by plant
root exudates. Some of the microorganisms from the rhizosphere induce plant systemic
resistance, which is associated with activation of plant defense mechanisms. Some defense
mechanisms have a structural nature and block the microorganisms to enter and spread
through the plant, like the case of lignin deposition. Also, as a result of plant-microbe
interaction, in plants are activated molecular mechanisms which refer to rapid generation of
high amount of reactive oxygen species (ROS), production of lytic enzymes or other
chemicals with antimicrobial activity [1, 2]. ROS production is common in healthy plants,
and they were initially considered just toxic by-products of different metabolic pathways [3].
However, it is now shown their role in establishment of defense mechanisms (papillae
formation and the assembly of structural barriers, programmed cell death and hypersensitivity
response), but also in signaling by synergistic interaction between themselves and other
signaling molecules such as salicylic acid, jasmonate and ethylene [4]. An important role as
9366
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
Induction of plant antioxidant system by interaction with beneficial and/or pathogenic microorganisms
signals in other critical plant processes like growth and development is attributed to these
highly reactive molecules [2]. Despite these activities, ROS react easily with various
biomolecules, causing irreversible damage and leading to necrosis and plant death [5]. To
overcome ROS toxicity, plants produce enzymatic and nonenzymatic antioxidants that
scavenge oxygen species. The first line in antioxidant defence is enzymatic antioxidant
system, which neutralizes superoxid anion (O2· -) and hydrogen peroxide (H2O2). ROS
enzymatic neutralization is ensured by antioxidant enzymes, like superoxide dismutase
(SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and guaiacol peroxidase (GPX, EC
1.11.1.7). SOD catalyses O2· - dismutation and is considered the most effective intracellular
enzymatic antioxidant being present in all subcellular compartments prone to ROS mediated
oxidative stress [6]. The H2O2 resulted from this reaction is subsequently converted to H2O
and O2 by CAT, another indispensible enzyme for ROS neutralization. H2O2 can also be
degraded by GPX activity, which has an additional role in structural cell wall modifications
and production of secondary metabolites with antimicrobial activity. In this study we
evaluated the induction of structural and enzymatic defense response by new bacterial strains
with antifungal activities.
Materials and Methods
Plant and microbial cultures and growth conditions
Cucumber seeds (Marketer cultivar) were surface sterilized with a solution of 1% sodium
hypochlorite for one hour, rinsed and then aseptically transferred in culture vessels on sterile
filter paper with 10 ml of water. The seeds were germinated in glass jars at 25ºC, with a
photoperiod of 16 h light and eight hours dark. Three weeks old cucumber plantlets were
treated with five ml of 108 CFU ml -1 bacterial suspension and/or five ml of three days fungal
suspension. The microbial strains used in this study are presented in Table 1.
Table 1. Microbial strains used in the experiment
Strain
Bacillus
amyloliquefaciens
Bw
Bacillus sp.Bw1
Bacillus
licheniformis B40
Pseudomonas
aeruginosa P14
Pythium
debaryanum
Characteristics
antifungal activity
antifungal activity
α amylase, antifungal
activity, indole 3acetic acid production
antifungal activity,
indole 3-acetic acid
production
plant pathogen
Origin
Isolated from soil,
Faculty of Biotechnology,
UASVM, Bucharest
Isolated from soil,
Faculty of Biotechnology,
UASVM, Bucharest
Collection of Faculty of
Biotechnology, UASVM,
Bucharest
Isolated from soil,
Faculty of Biotechnology,
UASVM, Bucharest
Collection of RDIPP,
Bucharest
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
References
[7]
[8,9,10]
[11,9,10]
-
9367
Florenţa Elena Helepciuc, Monica Elena Mitoi, Anca Manole-P unescu,
Florentina Aldea, Aurelia Brezeanu, C lina Petruţa Cornea
Bacterial strains were grown on LB broth on a rotary shaker, 150 rpm at 28ºC, while fungal
suspension was obtained by inoculating 30 ml of PD (potato dextrose) broth with 3 x 1cm2 of
actively growing P. debaryanum mycelium. The treatment consisted of 5 ml of bacterial,
fungal or mixed suspension and was applied on plant root. The bacterial suspensions were
maintained on cucumber roots, removed after 24 hours, when the fungal suspension was
added. After 24 hours cucumber plantlets were grinded and total protein extract was obtained.
Isoenzymes detection
Total protein extract was obtained by grinding whole cucumber plantlets and adding 1:1 (w/v)
extraction buffer (0.05 M phosphate buffer, pH 7, 2 mM EDTANa2 and 4% (w/v) PVP). The
homogenate was maintained at 4°C overnight, centrifuged at 15000 rpm for 15 minutes, and
the supernatant was used for quantification and electrophoretic analyses. Isoenzymes
detection was performed using native polyacrylamide gel electrophoresis (PAGE) technique.
The activity of peroxidase isoforms was detected according to a modified method of Kavitha
& Umesha [12]. The gel was incubated in 0.5 M acetate buffer (pH 5) containing 0.08%
benzidine, 180 μl of glacial acid acetic and several drops of H2O2 (total volume 30 ml) until
brown bands appeared on violet background.
For catalase staining the method of Weydert & Cullen [13] was used, with some
modifications. The gel was first washed 3 times for 15 min in distilled water, incubated in
phosphate buffer 0.01 M, pH 7, supplemented with 0.003% hydrogen peroxide, and then
immersed in a mix of 2% FeCl3 and 2% K3Fe(CN)6 in equal proportions. After staining
yellow bands appear and the gel is colored in light green.
Superoxide dismutases were stained according to Beauchamp & Fridovich [14], in a solution
of phosphate buffer 0.036M (pH 7.8), 2.4 mM NBT, 0.028 M TEMED, and 27.9 µM
riboflavin, until white bands appear.
Enzyme activity quantification
Quantification of superoxide dismutase activity was performed according to Beauchamp &
Fridovich [14]. The volume of enzyme extract required to inhibit NBT reduction by 50% was
defined as one unit of SOD activity.
Catalase activity was assayed at 240 nm by measuring the rate of H2O2 degradation from the
reaction mixture, using the method described by Garcia-Limones & al. [15]. One enzymatic
unit was defined as the volume of enzymatic extract that decomposes one μmol of H2O2 in
one minute at 25oC.
Guaiacol peroxidase enzymatic activity was assayed by measuring the absorbance at 470 nm
for 5 min. The method was described by Garcia-Limones & al. [15]. One unit of enzyme
activity represented the amount of enzyme catalyzing the oxidation of 1 μmol of guaiacol in 1
minute.
Statistical analysis was performed using SPSS (Statistical Package for Social Sciences). The
significance of the differences between the samples was tested by employing T-test
(independent samples two-tailed). The results with p<0.05 were considered statistically
significant.
Histochemical analyses
The treatment procedure for histochemical analyses was done as described before, except that
for lignin detection the plants were used 12 days after fungal inoculation. Histochemical
staining of peroxidase was performed using 3,3’-diaminobenzidine (DAB) as substrate,
following the producer recommendations (Carl Roth GmbH). Fresh handmade cross-sections
through hypocotyl were placed in an incubation solution (0.05M Tris-HCl buffer, pH 7,
0.01M imidazole and 0.1%DAB) for five minutes. The sections were then immersed in the
staining solution (0.05M Tris-HCl buffer, pH 7, 0.01M imidazole, 0.1% DAB and 0.003%
9368
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
Induction of plant antioxidant system by interaction with beneficial and/or pathogenic microorganisms
H2O2). The sections were maintained in this solution for two minutes, and the reaction was
stopped with distilled water. Brown colored tissues indicate enzymatic activity of POX.
Wiesner test was used for lignin localization employing the method described by FernandezGarcia & al. [16], with some modifications. Sections were immersed in 1% phluoroglucinol
dissolved in a mixture of 3:1 ethanol:HCl (95% ethanol and 20% HCl) for two minutes.
Positive lignin staining is indicated by areas colored in red-violet. All stained sections were
visualized using bright-field light microscopy under a Nikon Eclipse E200 microscope and
micrographs were recorded using a Nikon Coolpix 5400 digital camera.
Results
Superoxide dismutase activity increased in all treated variants, except for plants treated with
P14 strain (Figure1 a). The highest activity was detected in plants treated with B40 bacterial
strain. Differences in SOD electrophoretic patterns were observed in microbial treated plants
comparing to control, which presented only three isoforms with SOD activity (1, 2 and 6)
(Figure 1 b).
a
b
Figure 1. Enzymatic activity (a) and native PAGE analysis (b) of SOD from cucumber
non-infected plants (C), and plants treated with: B. amyloliquefaciens (Bw), B.
amyloliquefaciens – P. debaryanum (Bw-Py), Bacillus sp. (Bw1), Bacillus sp. – P.
debaryanum (Bw1-Py), B. licheniformis (B40), B. licheniformis – P. debaryanum (B40-Py),
P. aeruginosa (P14), P. aeruginosa - P. debaryanum (P14-Py). Asterisks indicate significant
differences between control and treated variants according to the t-test at level p<0.05 (*).
The results obtained with SOD isoforms allowed the detection of maximum six
isoforms/variant. The highest number of bands was detected in Bw-Py and Bw1-Py variants.
In all treated variants SOD 3 isoform was present, while in control was not expressed.
Treatment with B40 and P14 bacterial strains induced the expression of a supplementary
isoform (SOD 4) comparing with Bw and Bw1 variants. Colonization with both bacteria and
fungi induced the expression of some characteristic bands, SOD 4 and 5, except for B40-Py
variant.
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
9369
Florenţa Elena Helepciuc, Monica Elena Mitoi, Anca Manole-P unescu,
Florentina Aldea, Aurelia Brezeanu, C lina Petruţa Cornea
Catalase activity was higher in treated variants comparing with the control (Figure 2 a).
Generally, mixed treatment induced a slight increase of enzymatic activity comparing with
bacterial treatment. However, the highest CAT activity was detected in plants treated with
P14 strain. The electrophoretic pattern of CAT isoenzymes (Figure 2 b) showed the presence
of supplementary isoforms in variants that received mixed treatment (CAT5 for Bw1-Py,
CAT5 and CAT6 for Bw-Py and B40-Py, CAT4 for P14-Py). CAT3 was present only in P14,
while the treatment with Bw bacterial strain induced the expression of CAT7.
a
b
Figure 2. Enzymatic activity (a) and native PAGE analysis (b) of CAT from cucumber
non-infected plants (C), and plants treated with: B. amyloliquefaciens (Bw), B.
amyloliquefaciens – P. debaryanum (Bw-Py), Bacillus sp. (Bw1), Bacillus sp. – P.
debaryanum (Bw1-Py), B. licheniformis (B40), B. licheniformis – P. debaryanum (B40-Py),
P. aeruginosa (P14), P. aeruginosa - P. debaryanum (P14-Py). Asterisks indicate significant
differences between control and treated variants according to the t-test at level p<0.05 (*).
Guaiacol peroxidase activity was detected in all variants, with the highest values in plants
treated with Bw1 and P14 strains (Figure 3). Mixed treatment (bacteria and fungal pathogen)
allowed the detection of increased GPX activity only in Bw-Py and P14-Py variants,
comparing with control and Pythium debaryanum treatment. No significant qualitative
differences between variants in GPX isoforms electrophoretic pattern were observed.
However, more intensified bands were detected in some samples (data not shown).
9370
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
Induction of plant antioxidant system by interaction with beneficial and/or pathogenic microorganisms
Figure 3. Enzymatic activity of GPX in cucumber non-infected plants (C), and plants treated
with: B. amyloliquefaciens (Bw), B. amyloliquefaciens – P. debaryanum (Bw-Py), Bacillus sp. (Bw1),
Bacillus sp. – P. debaryanum (Bw1-Py), B. licheniformis (B40), B. licheniformis – P. debaryanum
(B40-Py), P. aeruginosa (P14), P. aeruginosa - P. debaryanum (P14-Py). Asterisks indicate
significant differences between control and treated variants according to the t-test at level p<0.05 (*).
Histochemical analyses were performed in order to detect possible cellular differences in plants
subjected to treatments with beneficial bacteria, pathogen or combined treatments. The peroxidase
activity (Figure 4 b, c) increased compared with control variant (Figure 4 a) in epidermal and
subjacent cortex but also in vascular bundles especially in cambial and xylem tissues, and in fiber cap
(sclerenchyma).
Lignin deposition
Comparing with control (Figure 4 d), in variants treated with bacteria it was observed an increased
lignin accumulation in epidermal cell walls of and also in xylem vessels (Figure 4 e, f). An irregular
lignin deposition was also obvious in some cortical cells, prefiguring an angular collenchyma.
Figure 4. Histochemical staining of POX (A, B, C – brown coloration; ep – epidermis,
phl v – phloem vessels, cb – cambium, xv – xylem vessels) and lignin deposition (D, E, F –
red-violet coloration, see arrows) in cucumber hypocotil sections: A – control variant POX; B
- plants treated with Bw strain; C – plants treated with Bw1 strain; D – control variant lignin;
E – plants treated with Bw strain; F – plants treated with Bw1 strain. Scale bars = 100μm.
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
9371
Florenţa Elena Helepciuc, Monica Elena Mitoi, Anca Manole-P unescu,
Florentina Aldea, Aurelia Brezeanu, C lina Petruţa Cornea
Discussions
It was shown by numerous authors that microorganisms induce various plant defense
mechanisms [17, 18, 19, 20, 21] or promote plant growth and development [22]. However, in
case of studies regarding the impact of beneficial bacteria on plant antioxidant system
differences were recorded among both plant species and microorganisms used. In this order,
the aim of this study was the evaluation of plant response, mainly the activation of cucumber
antioxidant system as a result of interaction with pathogenic and/or beneficial
microorganisms. Three types of antioxidant enzymes were examined: superoxide dismutases,
catalases and guaiacol peroxidases.
Within a cell, the superoxide dismutase is considered a key enzyme in regulation of
intracellular levels of ROS [23], as it constitutes the first line of antioxidant system defence
against ROS [24, 6], and is capable to scavenge the superoxide radical with the formation of
hydrogen peroxide. The hydrogen peroxide molecules formed as a result of superoxide
dismutases activity are subsequently degraded by catalases and peroxidases to H2O and O2.
The isosuperoxide dismutase expression was different between treated variants and the
control (SOD 3), and also according to the treatment applied (SOD 4 is present in B40 and
P14, but not in Bw and Bw1 variants). Mixed treatment generally conduced to expression of
SOD 4 and SOD 5 in case of Bw-Py and Bw1-Py and SOD 5 in case of P14-Py. Only in case
of B40-Py it was observed the absence of both SOD 4 and 5 isoforms, although treatment
with B40 triggers the expression of SOD 4. From the total of six isosuperoxid dismutases,
only three are present in control (SOD 1, SOD 2 and SOD 6), suggesting that the interaction
with beneficial bacteria and phytopathogenic fungi activate the production of other three
isoforms (3, 4 and 5). An early response in plant-microbe interaction is the formation of
superoxide radicals and plants have to eliminate these radicals using superoxide dismutases or
other antioxidants. The presence of SOD 3, SOD 4 and SOD 5 can be attributed to the plant
necessity to activate new isoforms in order to counteract the excessive accumulation of this
free radical. As a result of SOD activity, superoxide radicals are transformed in hydrogen
peroxide, which is also a highly reactive molecule and can cause important damages.
Hydrogen peroxide can be degraded both by the action of catalases and peroxidases. Our
results showed that catalases presented a significant increased activity in treated variants
comparing with the control. Similar to isosuperoxide dismutase electrophoretic pattern, some
new catalase isoforms appear as a result of interaction between the microorganisms used and
cucumber plants. Moreover, treatment-characteristic bands appear in three situations: CAT 3
for P14, CAT 4 for P14-Py and CAT 7 for Bw.
Although a significant increase in peroxidase activity in plant-microbe interactions have been
previously reported [25, 26, 18, 19], we did not observed significant modifications in GPX
enzymatic activity except for three variants. Also, unlike SOD and CAT, the electrophoretic
spectrum of guaiacol POX did not showed qualitative differences among the samples treated
with bacteria or with mixtures of beneficial bacteria and phytopathogenic fungi. The relatively
uniform presence of the guaiacol POX isoforms can be attributed to the implication of these
enzymes in different plant processes like formation of diferulic bonds or the cross bonds
between cell wall proteins, in oxidizing cinnamic alcohols during the process of lignification
and suberization, but also in processes of oxidation of phenolic compounds during the
hypersensitive response [27]. Moreover, maintaining of peroxidase activity at the level of the
control samples could be explained by the involvement of catalases in H2O2 degradation.
Catalase is unique among H2O2–degrading enzymes, as it not requires a reductant in order to
catalyze H2O2 dismutation, the reaction of H2O2 removal being performed using a very
energy-efficient mechanism [28, 29]. Although they have low substrate affinities at low
substrate concentrations [30], catalases have a very fast turnover rate and a very high
9372
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
Induction of plant antioxidant system by interaction with beneficial and/or pathogenic microorganisms
specificity for H2O2, characteristics that distinguish them from other peroxide-metabolizing
enzymes [29].
The different affinities of peroxidases and catalases for H2O2 suggest that the function of
these two different classes of H2O2-scavenging enzymes differs, catalases being involved in
the removal of H2O2 excess, whereas peroxidase would be mainly involved in fine modulation
of ROS [30, 31].
Thus, catalase appears to be the first enzyme involved in H2O2 detoxification, and only in
systems where catalase is no longer sufficient, H2O2 or its derivatives such as organic
peroxides must primarily be metabolized by peroxidases, which all require reductants [32,
29].
Histochemical peroxidase staining also indicate that in the system used by us peroxidase
activity increased in plants subjected to bacterial treatment, mostly in metabolic active tissues
(cambium and xylem). In case of lignin, an increased lignification of protective tissues
(epiderma and collenchima) of treated plants was observed, that could prevent the microbial
spread through the plant. The accumulation of lignin could also limit the diffusion of
microbial enzymes and toxins, and also the uptake of water and nutrients by the
microorganisms [33]. Lignification represents thus an important structural defense mechanism
used by plants against microorganisms.
Conclusions
Plants treatment with beneficial bacteria leads to expression of new SOD and CAT isoforms,
while mixed treatment increases even more the number of isoforms. This shows that the
treatments with the selected beneficial bacteria activate plant enzymatic antioxidant system,
and presumably increases plant capacity to resist pathogen attack. Moreover, the fortification
of protective tissues shows an improved structural defense capacity of treated plants. We can
conclude that prior treatment with non-pathogenic bacteria activates some components of the
structural and biochemical defense systems in cucumber, this indicating an improved
resistance to potential phytopathogen attack. Induction of plant antioxidant system by
antagonistic bacteria is of great interest because plant treatment with bioproducts based on
such bacteria could enhance plant protection against fungal pathogens.
References
1. S. EBRAHIM, K. USHA, B. SINGH, Pathogenesis related (PR) proteins in plant defense mechanism. In A.
Méndez-Vilas, editor. Science against microbial pathogens: communicating current research and technological
advances. Badajoz: Formatex; pp. 1043–1054 (2011).
2. P. SHARMA, A. B. JHA, R. S. DUBEY, M. PESSARAKLI, Reactive Oxygen Species, Oxidative Damage,
and Antioxidative Defense Mechanism in Plants under Stressful Conditions. Journal of Botany, 2012, 1,26
(2012).
3. J. BAILEY-SERRES, R. MITTLER, Editorial, The Roles of Reactive Oxygen Species in Plant Cells. Plant
Physiol., 141, 311 (2006).
4. G.P. BOLWELL, and A. DAUDI, Reactive oxygen species in plant-pathogen interactions. In: Reactive
oxygen species in plant signaling, Signaling and communication in plants, L.A. RIO, and A. PUPPO, eds.,
Berlin Heidelberg: Springer, 2009, pp. 113-133.
5. A. PITZSCHKE, C.FORZANI, H. HIRT, Reactive Oxygen Species Signaling in Plants. Antiox. Redox
Signaling, 8, 1757, 1764 (2006).
6. S.S. GILL, N. TUTEJA, Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop
plants. Plant Physiol.Bioch. 48, 909, 930 (2010).
7. O.A. SICUIA, F. CONSTANTINESCU, S. DINU, Bacterial biocontrol strains that reduce Rhizoctonia
damping-off in tomato seedlings. Scientific Pap, Series B, Horticulture. LVI, 175, 180 (2012).
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
9373
Florenţa Elena Helepciuc, Monica Elena Mitoi, Anca Manole-P unescu,
Florentina Aldea, Aurelia Brezeanu, C lina Petruţa Cornea
8. R. MATEESCU, C.P. CORNEA, I. GREBENIŞAN, O. SIREAN, Improvement by mutagenesis and
electroporation of antifungal properties of different Bacillus spp. strains. Rom. J. Gen. 1, 39, 51 (2005).
9. F.E. HELEPCIUC, E.M. CARASAN, A. BREZEANU, C.P. CORNEA, C.VOAIDEŞ, The evaluation of
stimulative and protective effect induced by treatments with biocontrol agents on plant development, Biotechnol,
„Proc Int Symp New Res Biotechnol”, Serie F, Special volume: 562, 568 (2008).
10. M. E. MITOI, F. E. HELEPCIUC, A. BREZEANU, C. P. CORNEA, Characterization of the impact of
Bacillus licheniformis and Pseudomonas aeruginosa against Alternaria alternata by phase contrast microscopy
and transmission electron microscopy. Sci Ann “Alexandru Ioan Cuza” Univ Iaşi – Veg Biol, 58 (2), 5, 18
(2012).
11. C.P.CORNEA, C. VOAIDEŞ, M. CIUC , S. DINU, M. COSTACHE, M. DRAGANOIU, V. SEVERIN, F.
OANCEA, In vitro inhibition of Erwinia amylovora Romanian isolates bz new antagonistic bacterial strains
Genetic diversity of some bacterial strains with biotechnological importance. Rom. Biotech. Lett., 13 (3), 3737,
3746 (2008).
12. R.KAVITHA AND S. UMESHA, Regulation of defense-related enzymes associated with bacterial spot
resistance in tomato. Phytoparasitica 36 (2), 144, 159 (2008).
13. C. J. WEYDERT AND J. J.CULLEN, Measurement of superoxide dismutase, catalase, and glutathione
peroxidase in cultured cells and tissue. Nat Protoc. 5 (1), 51, 66 (2010).
14. C. BEAUCHAMP, I. FRIDOVICH, Superoxide dismutase: improved assays and an assay applicable to
acrylamide gels. Anal. Biochem., 44, 276, 287 (1971).
15. C. GARCIA-LIMONES, A. HERVÁS , J.A. NAVAS-CORTÉS , R.M. JIMÉNEZ-DÍAZ, M.TENA.
Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and
incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris .Physiol
Mol Plant Pathol., 61 (6), 325, 337 (2002).
16. N. FERNANDEZ-GARCIA, M. CARVAJAL, E.OLMOS, Graft Union Formation in Tomato Plants:
Peroxidase and Catalase Involvement. Ann Bot , 93, 53, 60 (2004).
17. N. BENHAMOU, C. GARAND, A.GOULET. Ability of nonpathogenic Fusarium oxysporum strain Fo47 to
induce resistance against Pythium ultimum infection in cucumber. Appl Environ Microbiol. 68 (8), 4044, 4060
(2002).
18. S. MANDAL, A. MITRA, N. MALLICK, Biochemical characterization of oxidative burst during interaction
between Solanum lycopersicum and Fusarium oxysporum f. sp.lycopersici. Physiol Mol Plant Pathol., 72, 56, 61
(2008).
19. M.C. SILVA, L. GUERRA-GUIMARAES, A. LOUREIRO, M.R. NICOLE, Involvement of peroxidases in
the coffee resistance to orange rust (Hemileia vastatrix). Physiol. Mol. Plant. Pathol, 72, 29, 38 (2008).
20. J. NAWROCKA, M. SNOCHOWSKA, E. GAJEWSKA, P. E. IETROWSKA, M. SZCZECH, U.
MALOLEPSZA. Activation of defense responses in cucumber and tomato plants by selected polish Trichoderma
strains. Veg Crops Res Bull. 75 (1), 105, 116 (2011).
21. D.J. SEO, D.M. NGUYEN, Y.S. SONG, W.J. JUNG, Induction of defense response against Rhizoctonia
solani in cucumber plants by endophytic bacterium Bacillus thuringiensis GS1. J Microbiol Biotechnol. 22 (3),
407, 415 (2012).
22. Y. AN, S. KANG, K.D KIM, B.K HWANG, Y.JEUN, Enhanced defense responses of tomato plants against
late blight pathogen Phytophthora infestans by pre-inoculation with rhizobacteria. Crop Prot. 29, 1406, 1412
(2010).
23. S. GAO, R. YAN, M. CAO, W. YANG, S. WANG, F. CHEN, Effects of copper on growth, antioxidant
enzymes and phenylalanine ammonia-lyase activities in Jatropha curcas L. seedling. Plant Soil Environ. 54 (3),
117, 122 (2008).
24. R. G.ALSCHER, N. ERTURK, L. S. HEATH, Role of superoxide dismutases (SODs) in controlling
oxidative stress in plants. J. Exp. Bot., Antioxidants and Reactive Oxygen Species in Plants Special Issue, 53
(372), 1331, 1341 (2002).
25. T. JABS, A.J. SLUSARENKO, The hypersensitive response, In: Mechanisms of Resistance to Plant
Diseases, A.J. SLUSARENKO, R.S.S. FRASER, L.C. van LOON, eds, Kluwer Academic Press, Dordrecht,
2000, pp 279-323.
26. C. HE, T. HSIANG, D.J.WOLYN, Activation of defense responses to Fusarium infection in Asparagus
densiflorus. Eur J Plant Pathol. 107 (5), 473, 483 (2001).
27. G. COG LNICEANU, M. MITOI, F. HELEPCIUC, M. MATEI, S. MATEI, Biochemical changes induced
in regenerants of Fragaria X Ananassa Duch. by the in vitro treatment with fungal elicitors. Rom. Biotech.
Lett., 15 (4), 5512, 5518 (2010).
28. J. G. SCANDALIOS, L. GUAN, A. N. POLIDOROS, Catalases in Plants: Gene Structure, Properties,
Regulation, and Expression, In: Oxidative Stress and the Molecular Biology of Antioxidant Defenses, J. G.
SCANDALIOS, ed., New York: Cold Spring Harbor Laboratory Press, 1997, pp. 343-406.
9374
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
Induction of plant antioxidant system by interaction with beneficial and/or pathogenic microorganisms
29. A. MHAMDI, G. QUEVAL, S. CHAOUCH, S. VANDERAUWERA, F. VAN BREUSEGEM, G.
NOCTOR, Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models. J. Exp. Bot., 61
(15), 4197, 4220 (2010).
30. G. NOCTOR, C. H. FOYER, Ascorbate and Glutathione: Keeping Active Oxygen Under Control. Annu.
Rev. Plant Physiol. Plant Mol. Biol,. 49, 249, 279 (1998).
31. A.A.ALI, F. ALQURAINY, Activities of antioxidants in plants under environmental stress, In: The
Lutein: Prevention and Treatment for Age-related Diseases, N. MOTOHASHI, ed., Transworld Research
Network, India, 2006, pp 187-256.
32.H. WILLEKENS, S. CHAMNONGPOL, M. DAVEY, M. SCHRAUDNER, C. LANGEBARTELS, M. VAN
MONTAGU, D. INZE, W.VAN CAMP, Catalase is a sink for H2O2 and is indispensable for stress defence in
C3 plants. EMBO J. 16 (16), 4806, 4816 (1997).
33. D. GUEST and J. BROWN, Plant defence against pathogens, In: Plant pathogens and plant disease, J. F.
BROWN, H. J. OGLE, eds., Australasian Plant Pathology Society, inc., Armidale, Australia, 1997 pp. 262-286.
Romanian Biotechnological Letters, Vol. 19, No. 3, 2014
9375