ARTICLE IN PRESS

Physiological and Molecular Plant Pathology 71 (2007) 201–209

www.elsevier.com/locate/pmpp

Reinforcement of cell wall in roots of Lycopersicon esculentum through

induction of phenolic compounds and lignin by elicitors
Sudhamoy Mandal,1, Adinpunya Mitra
Natural Product Biotechnology Group, Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur 721302, India
Accepted 4 February 2008

Abstract

Changes in phenolic metabolism and lignin deposition have been studied in roots of tomato plants after elicitation with four elicitors
which are Fusarium mycelium extract (FME), chitosan (CHT), Fusarium culture filtrate (FCF) and Trichoderma mycelium extract
(TME). Most profound effect of elicitors was observed on ferulic acid among the phenolic compounds. After 24 h elicitation, the increase
in ferulic acid content of root cell wall was 3.71 and 3.30 times by FME and CHT, respectively. The increase of 4-hydroxybenzoic acid
was 2.71 and 2.16 times by these two elicitors. The level of 4-coumaric acid was little more than double by these two elicitors after 24 h
elicitation. Most pronounced increase in lignin synthesis was also effected by FME followed by CHT. Lignin deposition in the root cell
wall was increased 3.6, 5.4 and 7.1 times by FME during 12, 24 and 36 h after elicitation, respectively. Similarly, CHT increased lignin
deposition by 2.8, 5.1 and 6.8 times at 12, 24 and 36 h after elicitation, respectively. FCF and TME also increased lignin deposition
significantly in the cell walls of tomato roots during the above time periods of elicitation. Activity of phenylalanine ammonia lyase
reached highest level at 24 h post elicitation under the influence of the elicitors. Peroxidase activity registered a sharp increase at 24 h post
elicitation. Markedly increased level of polyphenol oxidase activity was found at 12 h post elicitation. Cinnamyl alcohol dehydrogenase
activity was observed to reach highest level at 48 h post elicitation. Cell wall strengthening, through the deposition of lignin, preceded by
the induction of the synthesizing enzymes appears to play an important role in the defense response of Lycopersicon esculentum in
reaction to elicitors, including one derived from Fusarium oxysporum f. sp. lycopersici, the causal organism of Fusarium wilt of tomato.
r 2008 Elsevier Ltd. All rights reserved.

Keywords: Cell wall-bound phenolics; Chitosan; Cinnamyl alcohol dehydrogenase; Ferulic acid; Fusarium mycelium extract; Fusarium oxysporum f. sp.
lycopersici; Lignin; Lycopersicon esculentum; Phenylalanine ammonia lyase; Peroxidase; Polyphenol oxidase

1. Introduction production is lost to plant diseases [1]. Fusarium oxysporum

f. sp. lycopersici, causal agent of Fusarium wilt of tomato, is
Tomato (Lycopersicon esculentum Mill.) is one of the responsible for important crop losses in the field and
most widely grown vegetable crops in the world. Tomato, commercial greenhouses [2]. Although some tomato
like any other crops, suffers from many destructive diseases cultivars with single dominant genes for resistance have
caused by fungi, bacteria and viruses. It has aptly been been developed, control of Fusarium wilt in tomato is
stated in a recent review that plant diseases pose a threat to mainly restricted to eliminating the pathogen in soil by
crop production, because at least 10% of global food steaming or fumigating with chemicals and planting
pathogen-free stocks [3]. Emergence of new races of the
Abbreviations: CAD, cinnamyl alcohol dehydrogenase; CHT, chitosan; pathogen has drastically reduced the effectiveness of the
CWB, cell wall-bound; FCF, Fusarium culture filtrate; FME, Fusarium classical control measures. Besides, application of pesti-
mycelium extract; PAL, phenylalanine ammonia lyase; POD, peroxidase; cides has raised wide spread concern for damaging the
PPO, polyphenol oxidase; TME, Trichoderma mycelium extract. sustainability of the nature. Hence, there is a multitude of
Corresponding author. Tel.: +91 674 2471712; fax: +91 674 2471867.
E-mail address: sudhamoy.blitz@gmail.com (S. Mandal).
efforts for developing alternative disease management
1
Present address: Central Horticultural Experiment Station (ICAR), methods which are practically effective and environmen-
Aiginia, Bhubaneswar, PIN 751019, India. tally safe.

0885-5765/$ - see front matter r 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.pmpp.2008.02.003
 ARTICLE IN PRESS
202 S. Mandal, A. Mitra / Physiological and Molecular Plant Pathology 71 (2007) 201–209

Higher plants have the ability to initiate various defense to yield a complex net of crosslinks among monolignols,
reactions such as hypersensitive responses, production of proteins and polysaccharides. Cell wall associated PODs
phytoalexins and antimicrobial proteins, and reinforce- (E.C. 1.11.1.7) have been implicated in these crosslinking
ment of cell walls when they are infected by various reactions [15]. PPO (E.C. 1.14.18.1) has been associated
pathogens [4,5]. Soluble and cell wall-bound (CWB) with many vascular diseases [16].
phenolic compounds accumulate in plant tissues challenged Though induced resistance is a widely studied subject,
by fungal pathogens [6]. These phenolics play an important very little is known about the inducible defense responses
role in the resistance of plants to pathogen attack as they resulting from interactions between L. esculentum–F.
belong to the antimicrobial defense arsenal [7]. Plants can oxysporum f. sp. lycopersici. In the present investigation,
detect specific pathogens through the perception of signal we have attempted to explore aspects of induced resistance
molecules (elicitors) mostly generated/secreted from patho- in tomato in respect to cell wall strengthening effected by
gens. Fragments of cell surface macromolecules typical of four elicitors which are Fusarium mycelium extract (FME),
microorganisms such as cell wall polysaccharides and CHT, Fusarium culture filtrate (FCF) and Trichoderma
secreted proteins often serve as a potent elicitor to induce mycelium extract (TME). Of the four elicitors used, FME
defense reactions. One strategy for crop protection, which and FCF were derived from the tomato wilt pathogen
also takes into account environmental considerations, is to F. oxysporum f. sp. lycopersici. With this approach, we
mimic the pathogen aggression in order to trigger defense have also attempted to simplify plant–pathogen interaction
responses in plants prior to a possible pathogen infection. study in roots of tomato through the use of pathogen-
Elicitors are molecules that are capable of doing this by derived elicitors by comparing their respective efficacies
mimicking the perception of a pathogen by the plant. They with CHT and TME in enhancing the defense responses
are generally defined as molecules that stimulate any of a against the pathogen.
number of defense responses of plants, including the
formation of phytoalexins [8]. As the signal transduction 2. Materials and methods
pathways responsible for triggering plant defense responses
become clearer, the possibility of sensitizing a plant by 2.1. Plant material and chemicals
prior application of elicitors would become a promising
option for effective management of plant diseases [9]. Tomato (L. esculentum cv. Arka Saurabh) was grown in
Treatment of parsley cell suspensions with Phytophthora a fertile and irrigated plot in open ambient climate. Roots
megasperma f. sp. glycinea elicitor results in the accumula- were collected from 60-day-old uniformly developed plants
tion of substantial concentrations of coumarin phytoalex- for use in all elicitation experiments. Analytical grade
ins as well as esterification of phenylpropanoids, in chemicals were used in sample preparation. Deionized
particular ferulic acid, to the cell walls [10]. Chitosan water for all procedures was obtained from a Barnstead/
(CHT), a deacetylated form of chitin, has been shown to Thermolyne (Dubuque, USA) Diamond-NanopureTM
have the capacity to induce resistance against Fusarium water purification system. All authentic standards were
wilt in susceptible tomato plants [2]. CHT treatment procured from Sigma-Aldrich Chemical Co. Ltd (New
induced a significant increase in the activities of polyphenol Delhi, India).
oxidase (PPO), peroxidase (POD), and enhanced the
content of phenolic compounds in tomato fruits; thus, 2.2. Elicitor preparation
providing protection against gray mould and blue mould
diseases [11]. Four elicitors used in the elicitation experiment were
The incorporation of phenylpropanoid-derived material CHT, FME, FCF and TME. CHT was prepared essentially
in lignin and lignin-like polymers following infection or as described by Villegus and Brodelius [17]. Crude fungal
elicitation, results in reinforcement of cell walls and elicitors were prepared from F. oxysporum f. sp. lycopersici
structural rigidity which can lead to resistance against (causal organism of Fusarium wilt of tomato) and
enzymic degradation and the restriction of diffusion of Trichoderma viride (a biocontrol agent) according to the
enzymes and toxins from the fungus to the host [12]. method of Eilert et al. [18]. Both fungi were obtained from
Lignification makes the cell wall more resistant to Indian Type Culture Collection, IARI, New Delhi.
mechanical pressure applied during penetration by fungal
appressoria [13]. Lignin is an aromatic polymer composed 2.3. Time course elicitation of tomato roots
mainly of cinnamyl alcohols which originates from
t-cinnamic acid, the product of phenylalanine ammonia This was carried out according to cotton leaf disk
lyase (PAL, E.C. 4.3.1.5). Cinnamyl alcohol dehydrogenase elicitation experiment of Dubery and Slater [19]. Root
(CAD, E.C. 1.1.1.95) catalyses the second reductive step of fragments (approx. 20 mm) were cut from 60-day-old
the lignin-committed branch, leading to hydroxycinnamyl tomato plants. The root fragments were washed several
alcohols, the monomeric precursors of lignin [14]. These times with deionized water to remove dirt and wound
precursors are enzymically dehydrogenated in the cell walls metabolites and then immersed in 20 ml of elicitor solution
to phenoxy radicals, which then polymerize spontaneously under sterile conditions in Petri dishes. The elicitor solution
 ARTICLE IN PRESS
S. Mandal, A. Mitra / Physiological and Molecular Plant Pathology 71 (2007) 201–209 203

consisted of 1 ml of elicitor in 19 ml of deionized water. by the increase in absorbance at 470 nm in a Systronics

Control root fragments were immersed in 20 ml sterile UV-visible scanning spectrophotometer (Ahmedabad,
deionized water. Petri dishes with root fragments were India). One unit of enzyme activity represented the amount
incubated for 0, 12, 24, 36, 48 and 72 h at 25 1C in the dark. of enzyme catalyzing the oxidation of 1 mmol of guaiacol in
All experiments were performed in triplicate. 1 min.

2.4. Extraction of CWB phenolic compounds 2.9. PPO assay

CWB phenolics were extracted according to the method Root segments (elicited and control) were homogenized
described by Parr et al. [20] with slight modification. (1:2 w/v) in 0.1 M potassium phosphate buffer (ice cold
Approximately 25–30 mg of dry cell wall material was extraction buffer pH 6.8). The homogenate was centrifuged
obtained per gram of root tissue. The dry residue was at 20 000 rpm for 30 min at 4 1C. The supernatant was
subjected to alkaline hydrolysis using 2 N sodium hydro- used directly in the enzyme assay. The reaction mixture
xide (NaOH) for 24 h. contained 1 mM catechol in 0.05 M sodium phosphate
buffer (pH 6.5) and 500 ml enzyme extract. The reference
2.5. HPLC analysis of phenylpropanoid derivatives contained only substrate. PPO activity was determined
using catechol as substrate and monitoring the increase
The samples of cell wall extracts of tomato roots were in absorbance at 405 nm [26]. The linear portion of the
prepared for HPLC by dissolving in 50% (v/v) methanol. activity curve was used to express enzyme activity
HPLC analyses were performed on a PhenomenexTM (nkat mg1 protein). One unit was defined as a change in
(Torrance, USA) C18 column (RP-Hydro, 4 mm, 250  absorbance of 0.001 under the assay conditions.
4.6 mm) using a Waters HPLC system (Milford, USA)
equipped with a dual absorbance UV-detector. Chromato- 2.10. CAD assay
grams were monitored simultaneously at 254 and 310 nm
and analyzed on a Windows XPTM platform with a CAD was extracted in 0.1 M Tris–HCl (pH 7.5) contain-
BREEZETM software ver. 3.2 (Waters). An isocratic linear ing 15 mM b-mercaptoethanol, polyethylene glycol (10%
solvent system of aqueous trifluoroacetic acid (68%) and v/v) and 5% polyclar T. Root segments were homogenized
methanol (32%) with a flow rate of 1 ml/min for 30 min (1:2 w/v) and centrifuged at 20 000 rpm for 20 min at 4 1C.
was used to elute the phenolic compounds [21]. Identifica- The supernatants were directly used in the enzyme assay.
tion of the phenolic acids was achieved by comparing their The reaction mixture consisted of 2.5 ml 0.1 M Tris–HCl
retention times with those from authentic standards. (pH 8.8), 200 ml 0.1 M Tris–HCl (pH 8.8) containing 3 mM
The phenolic compound contents were expressed as mg g1 coniferyl alcohol, 200 ml 0.1 M Tris–HCl (pH 8.8) contain-
biomass dry wt. ing 6 mM NADP and 100 ml enzyme extract. CAD activity
was measured following the oxidation of the appropriate
2.6. Determination of lignin-like polymers hydroxycinnamyl alcohol at 30 1C [27]. Assays with
coniferyl alcohol as substrate were monitored by following
Lignin was extracted according to the method of Bruce the formation coniferaldehyde at 400 nm. CAD activity
and West [22] and assayed quantitatively in the alcohol was expressed as nkat mg1 protein.
insoluble residue (AIR) as thioglycolic acid derivatives
(TGA-derivatives) following alkali hydrolysis [23]. Results 3. Results
were expressed as the increase in A280 nm g1 of AIR fresh wt.
3.1. Effect of elicitors on phenolics profile in roots of tomato
2.7. PAL assay
Six CWB phenolic compounds were detected and
Enzyme extraction steps were carried out at 4 1C. PAL identified by HPLC analysis in the tomato roots from
was assayed directly in the supernatant after concentration control samples (non-elicited roots). They were 4-hydroxy-
through AmiconRTM Ultra-4 CFU membrane (Millipore, benzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanil-
Bedford, USA) by detecting the formation of t-cinnamic lin, 4-coumaric acid and ferulic acid. When elicited roots
acid at 280 nm with HPLC [24]. were analyzed by HPLC, these six CWB phenolic
compounds were detected in case of all four elicitors.
2.8. POD assay Though no qualitative differences were observed between
CWB phenolic profiles of elicited and non-elicited tomato
POD activity was determined from the crude enzyme roots, quantitative differences could be clearly discerned in
extract using an assay system consisting of 20 mM guaiacol the amount of the phenolic compounds. All elicitors could
(0.5 ml), 0.1 mM acetate buffer (pH 5.0) (2.1 ml), 40 mM increase the level of phenolic compounds in varying degrees
H2O2 (0.2 ml) and the enzyme extract (0.2 ml) with a final right from 12 h of elicitation compared to the correspond-
volume of 3 ml [25]. Oxidation of guaiacol was measured ing control. However, most effective elicitors were FME
 ARTICLE IN PRESS
204 S. Mandal, A. Mitra / Physiological and Molecular Plant Pathology 71 (2007) 201–209

and CHT among the four elicitors, and most profound returned to the level of control in respect to elicitation by
induction effect was observed on ferulic acid among FME and CHT.
the phenolic compounds. After 24 h elicitation, the increase
in ferulic acid content of root cell wall was 3.71 and 3.30 3.2. Effect of elicitors on lignin-like polymers in roots of
times by FME and CHT, respectively. The increase of tomato
4-hydroxybenzoic acid was 2.71 and 2.16 times by these
two elicitors. The level of 4-coumaric acid was little more It was observed that all elicitors increased lignin deposi-
than double by these two elicitors after 24 h elicitation tion in varying degrees starting from 12 h of elicitation
(Fig. 1). The contents of vanillic acid, 4-hydroxybenzalde- compared to the corresponding control (Fig. 2). Most
hyde and vanillin were also fairly higher than the level pronounced increase in lignin synthesis was effected by
found in control (data not shown). There was a gradual FME followed by CHT. Lignin deposition in the root cell
decrease in the levels of CWB phenolic compounds wall was increased 3.6, 5.4 and 7.1 times by FME during 12,
with passage of time, but the level of ferulic acid never 24 and 36 h after elicitation, respectively. Similarly, CHT

1000
4-HBA 4-CA FA
900
Concentration of phenolics (µg g-1d.wt)

800

700

600

500

400

300

200

100

0
CHT

CHT

CHT

CHT

CHT

CHT
CON

CON

CON

CON

CON

CON
FME

TME

FME

TME

FME

TME

FME

TME

FME

TME

FME

TME
FCF

FCF

FCF

FCF

FCF

FCF
0 12 24 36 48 72

Treatment duration (h)

Fig. 1. Time course formation of 4-hydroxybenzoic acid (4-HBA), 4-coumaric acid (4-CA) and ferulic acid (FA) in elicited and non-elicited (control) roots
of tomato. Values are mean7SD of triplicate analysis. Key to the elicitors: CHT, Chitosan; FME, Fusarium mycelium extract; FCF, Fusarium culture
filtrate; TME, Trichoderma mycelium extract; CON, Control.

8
TGA-derivatives at 280 nm g-1AIR fresh wt

Control FME CHT

7
FCF TME
6

0
0 12 24 36 48 72
Treatment duration (h)

Fig. 2. Deposition of lignin (expressed as thioglycolic acid derivatives at 280 nm g1 alcohol insoluble residue) in the cell wall on a time course after
elicitation of the roots of tomato with different elicitors. Values are mean7SD of triplicate analysis. Key to the elicitors: CHT, chitosan; FME, Fusarium
mycelium extract; FCF, Fusarium culture filtrate; TME, Trichoderma mycelium extract.
 ARTICLE IN PRESS
S. Mandal, A. Mitra / Physiological and Molecular Plant Pathology 71 (2007) 201–209 205

increased lignin deposition by 2.8, 5.1 and 6.8 times at 12, 24 increase of 3.7 and 5.9 times by FME at 12 and 24 h post
and 36 h after elicitation, respectively. FCF and TME also elicitation. In the same pattern, the enzyme activity was
increased lignin deposition significantly in the cell walls of increased 2.4 and 4.8 times by CHT during the same
tomato roots during the above time periods of elicitation. elicitation periods. The enhancement of PAL activity was
However, as time progressed during the experiment, there 3.7 and 4.1 times by FCF and TME, respectively, at 24 h
was marginal increase in lignin deposition in the root cell post elicitation. The rise in PAL activity under the
walls and this was evident after 36 h of elicitation. influence of elicitors was transient and showed a sharp
decreasing trend immediately after 24 h of elicitation of the
roots.
3.3. PAL activity in elicited roots of tomato

The activity of PAL, a key enzyme of the phenylpropa- 3.4. POD activity in elicited roots of tomato
noid metabolism, was studied after elicitation of the
tomato roots on a time course. Activity of this important In this elicitation experiment, it was observed that a slow
defense enzyme was found to increase right from 12 h of increase of POD activity at 12 h post elicitation was
elicitation and peaked at 24 h post elicitation under the followed by a sharp increase at 24 h post elicitation (Fig. 4).
influence of the elicitors (Fig. 3). PAL activity registered an POD activity registered a sharp increase by 3.2 times at

0.8
Control FME
0.7 CHT FCF
PAL activity (nkat mg-1 protein)

TME
0.6

0.5

0.4

0.3

0.2

0.1

0
0 12 24 36 48 72
Treatment duration (h)

Fig. 3. Phenylalanine ammonia lyase (PAL) activity (nkat mg1 protein) on a time course after elicitation of the roots of tomato with different elicitors.
Values are mean7SD of triplicate analysis. Key to the elicitors: CHT, chitosan; FME, Fusarium mycelium extract; FCF, Fusarium culture filtrate; TME,
Trichoderma mycelium extract.

120
Control FME
CHT FCF
100
POD activity (nkat mg-1 protein)

TME

80

60

40

20

0
0 12 24 36 48 72
Treatment duration (h)

Fig. 4. Peroxidase (POD) activity (nkat mg1 protein) on a time course after elicitation of the roots of tomato with different elicitors. Values are
mean7SD of triplicate analysis. Key to the elicitors: CHT, chitosan; FME, Fusarium mycelium extract; FCF, Fusarium culture filtrate; TME, Trichoderma
mycelium extract.
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206 S. Mandal, A. Mitra / Physiological and Molecular Plant Pathology 71 (2007) 201–209

24 h post elicitation under the influence of FME compared levels of PPO activity (3.5 times higher than control) were
to the corresponding control. At the same time point, there found with FME at 12 h post elicitation. At 24 h post
was 2.9 times increase in POD activity under the influence elicitation, the activity was 4.4 times with the above elicitor.
of CHT. The other two elicitors, FCF and TME, increased In case of CHT, the activity was 2.6 times higher at 12 h post
POD activity by 2.4 and 2.7 times, respectively, at 24 h post elicitation and 3.7 times higher at 24 h post elicitation than
elicitation. After 24 h of elicitation, there was a gradual the corresponding controls. In a similar way, FCF and TME
decrease in activity of the enzyme up to 48 h of elicitation, increased PPO activity by 2.1 and 1.7 times after 12 h of
but still higher than the level of 12 h of elicitation. At 72 h elicitation and 3.5 and 2.7 times after 24 h of elicitation,
post elicitation, the activity of POD returned almost to the respectively. After 24 h of elicitation, there was a sharp fall
level of control. of PPO activity in case of all the elicitors up to 36 h of
elicitation and then a gradual decline in activity followed.

3.5. PPO activity in elicited roots of tomato

3.6. CAD activity in elicited roots of tomato
All the four elicitors could increase PPO activity in
varying degrees beginning with 12 h of elicitation compared In this elicitation study of defense enzymes, CAD
to the corresponding control (Fig. 5). Markedly increased activity showed gradual increase from 12 h of elicitation

16
Control FME
14 CHIT FCF
TME
PPO activity (nkat mg-1 protein)

12

10

0
0 12 24 36 48 72
Treatment duration (h)

Fig. 5. Polyphenol oxidase (PPO) activity (nkat mg1 protein) on a time course in elicited and non-elicited (control) roots of tomato. Values are
mean7SD of triplicate analysis. Key to the elicitors: CHT, chitosan; FME, Fusarium mycelium extract; FCF, Fusarium culture filtrate; TME, Trichoderma
mycelium extract.

0.6
Control FME
CHT FCF
CAD activity (nkat mg-1 protein)

0.5 TME

0.4

0.3

0.2

0.1

0
0 12 24 36 48 72
Treatment duration (h)

Fig. 6. Cinnamyl alcohol dehydrogenase (CAD) activity (nkat mg1 protein) on a time course after elicitation of the roots of tomato with different
elicitors. Values are mean7SD of triplicate analysis. Key to the elicitors: CHT, chitosan; FME, Fusarium mycelium extract; FCF, Fusarium culture
filtrate; TME, Trichoderma mycelium extract.
 ARTICLE IN PRESS
S. Mandal, A. Mitra / Physiological and Molecular Plant Pathology 71 (2007) 201–209 207

and its activity was observed to reach highest level at 48 h PAL activity is an extremely sensitive indicator of stress
post elicitation (Fig. 6). CAD activity increased 2.4, 3.2 and conditions and fungal challenge, and elicitor treatment
4.0 times higher than the corresponding controls at 24, 36 elevates the levels of the flux through the general
and 48 h post elicitation under the influence of FME. phenylpropanoid pathway, thereby supplying the carbon
Similarly, the increase of CAD activity was 1.6, 2.3 and 3.2 skeletons for secondary products such as phenolics which
times higher during the same post elicitation periods under are the precursor molecules of lignin [32]. Rapid and
the influence of CHT. Remaining two elicitors, FCF and transient increase in PAL activity was observed in sugar
CHT also increased CAD activity by 2.1 and 1.9 times at beet seedlings treated with cell wall protein fraction
36 h post elicitation and 2.6 and 2.1 times at 48 h post obtained from mycelial mat of Pythium oligandrum [33].
elicitation, respectively. After 48 h of elicitation, a fall in A rapid and strong induction of PAL transcripts was also
the activity of CAD was noticed in respect to all four observed in Medicago truncatula cell suspension cultures
elicitors. But the effect of FME and CHT could maintain a elicited with yeast extract [34]. Similarly, a recent study has
relatively much higher level of CAD activity than the shown that elicitation of grapevine leaves by CHT led
control level. marked induction of PAL activity [35]. It has been also
reported that application of CHT to soybean leaf tissues
4. Discussion caused increased activity of PAL [36]. In our study, the
rapid and transient increase in PAL activity in elicited
For investigating the biochemical defense responses in roots is in agreement with earlier reports. The induction of
tomato against Fusarium wilt, the plant–pathogen interac- PAL activity in our study also correlates well with the
tion in roots was simplified through the use of pathogen- accumulation of lignin in the cell walls of the roots.
derived elicitors that elicit a quantitatively similar response The last step in the synthesis of lignin and suberin has
of the live pathogen [28]. FME and FCF, derived from been proposed to be catalyzed by PODs, although other
F. oxysporum f. sp. lycopersici, were compared with two proteins may also be involved [37]. Lignin is highly
other elicitors CHT and TME for their effectiveness in resistant to attack by microorganisms, and lignified cell
inducing resistance. FME stood out prominently among walls are an effective barrier to pathogen entrance and
the four elicitors as the best one followed by CHT. Several- spread. The enhanced resistance of ASM-treated tomato
fold increase of ferulic acid in response to FME and CHT plants against Clavibacter michiganensis ssp. michiganensis
after 24 h of elicitation is particularly significant in respect (causal agent of bacterial canker of tomato) was associated
to crosslinking of ferulic acid to cell wall materials. with significant increases in the activities of POD [38]. Cell
Elicitation of plant tissues can result in accumulation of suspension cultures of Capsicum annuum responded to
substantial amounts of antimicrobial compounds as well as elicitation by both lyophilized mycelium and fungus filtrate
increased esterification of phenylpropanoids, particularly of Phytophthora capsici by synthesizing or accumulating
ferulic acid to the cell walls [10]. A common host response PR-proteins with POD activity and lignin-like polymer
is the esterification of ferulic acid to the host cell wall, and [39]. Elicitors evoke similar responses as that of pathogen
it has been suggested that crosslinking of such phenylpro- infection in plants. It was found that Rhizoctonia infection
panoid esters leads to the formation of lignin-like polymers resulted in early local and systemic increase in POD activity
[29]. The rapid enhancement of phenolic profile may be a in Picea abies [40]. Rapid induction of POD and PPO
result of sensing of elicitors at informational level by the activities was detected in cell wall reinforcement in banana
plant roots. This is because the elicitors are recognized as roots in response to elicitors from F. oxysporum f. sp.
signal compounds by appropriate perception systems cubense [32]. In addition to its antifungal activity, CHT has
displaying high sensitivity in several plants [4]. The increase the potential for inducing defense-related enzymes [41]. It
in CWB phenolics correlated with the deposition of has been demonstrated that POD activity was elicited by
lignin in FME-treated as well as other elicitor-treated CHT in cucumber, resulting in an increase in resistance
tomato roots. These observations resembled eminently against Botrytis cinerea [42]. A recent report indicated that
with the finding in Musa acuminata roots elicited with CHT, when injected in date palm roots, induced a
F. oxysporum extracts [30]. Pine cell cultures when elicited significant increase in the activities of POD and PPO,
with a fungal extract also accumulated a significantly and proved antifungal against F. oxysporum f. sp. albedinis
higher amount of lignin and CWB ferulic acid [23]. [43]. Results obtained in this study on POD activity
Similarly, elicitation of flax cell cultures with mycelium indicated that the tomato plants responded actively to
extracts of F. oxysporum f. sp. lini triggered a strong elicitation through the rapid induction or activation of
incorporation of monolignols in the lignin fraction. Flax specific isoforms. The importance of PPO activity in
cell cultures showed a specific response to elicitation with disease resistance stems from its property to oxidize
mycelium extracts of F. oxysporum f. sp lini [31]. In our phenolic compounds to quinones, which are often more
study also, strong and rapid lignification in tomato roots toxic to microorganisms than the original phenols [44]. It is
was observed in response to FME. This strong increase in reasonable to assume that an increased activity of PPO
lignin deposition in the cell wall could be a specific response would result in higher concentrations of toxic products of
to the wilt pathogen F. oxysporum f. sp. lycopersici. oxidation and therefore greater degrees of resistance to
 ARTICLE IN PRESS
208 S. Mandal, A. Mitra / Physiological and Molecular Plant Pathology 71 (2007) 201–209

pathogen infection. The possible involvement of PPO in References

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