Enhanced Defence Responses of Chickpea Plants Against Rhizoctonia solani by Pre-Inoculation with Rhizobia

Abstract

The aim of this work was to study the antagonist
effect of two Rhizobium strains Pch Azm and Pch
S.Nsir2 to Rhizoctonia solani and for an evaluation of
the relative impact of rhizobia on the expression of
the plant’s defence response against Rhizoctonia.
First, these strains reduced fungal growth observed in
vitro using the same or separately Petri dishes. More-
over, these isolates led to reduced chickpea infection
by R. solani, resulting from the direct effect of rhizo-
bia on pathogens and possible induced resistance in
chickpea. Concomitantly, reduction in infection was
accompanied by enhanced level of defence-related
enzymes, phenylalanine ammonia lyase (PAL) and
peroxidase (POX). An increased level of phenol con-
tent was recorded in the roots of bacterized plants
grown in the presence of pathogen. The results prom-
ise the use of rhizobia for protection of chickpea
against R. solani.

Full text

Unit
e d’Utilisation M
edicale et Agricole des Techniques Nucl
eaires, Centre National des Sciences et Technologies Nucl
eaires,
Ariana, Tunisia
Enhanced Defence Responses of Chickpea Plants Against Rhizoctonia solani by
Pre-Inoculation with Rhizobia
Imen Hemissi
1,2,3
, Yassine Mabrouk
2
, Sonia Mejri
2
, Mouldi Saidi
2
and Bouaziz SIFI
1
Author’s addresses:
1
Institut National de Recherche Agronomique de Tunisie (INRAT), Rue H
edi Karray, 2049 Ariana,
Tunisia;
2
Unit
e d’Utilisation M
edicale et Agricole des Techniques Nucl
eaires, Centre National des Sciences et Technologies
Nucl
eaires, 2020 Ariana, Tunisia;
3
Institut National d’Agronomie de Tunis (INAT), 43, Avenue Charles Nicolle, 1082 Tunis,
Mahraje
`ne, Tunisia (correspondence to Y. Mabrouk. E-mail: mabrouk.yassine@cnstn.rnrt.tn)
Received June 15, 2012; accepted December 6, 2012
Keywords: chickpea, Rhizoctonia solani, biological control, peroxidase, phenylalanine ammonia lyase, phenolic compounds
Abstract
The aim of this work was to study the antagonist
effect of two Rhizobium strains Pch Azm and Pch
S.Nsir2 to Rhizoctonia solani and for an evaluation of
the relative impact of rhizobia on the expression of
the plant’s defence response against Rhizoctonia.
First, these strains reduced fungal growth observed in
vitro using the same or separately Petri dishes. More-
over, these isolates led to reduced chickpea infection
by R. solani, resulting from the direct effect of rhizo-
bia on pathogens and possible induced resistance in
chickpea. Concomitantly, reduction in infection was
accompanied by enhanced level of defence-related
enzymes, phenylalanine ammonia lyase (PAL) and
peroxidase (POX). An increased level of phenol con-
tent was recorded in the roots of bacterized plants
grown in the presence of pathogen. The results prom-
ise the use of rhizobia for protection of chickpea
against R. solani.
Introduction
The importance of legumes in human nutrition is well
known. Chickpeas (Cicer arietinum L.) are one of the
most utilized legumes in the world because they are
considered to be an excellent source of dietary protein
(Frias et al. 2000). However, chickpea production in
most countries falls short of the demand due to dis-
eases that reduce yield and crop quality. Chickpeas
(Cicer arietinum L.) can be affected by more than 50
diseases of varied aetiology in different parts of the
world (Mazur et al. 2004). A few of them are devas-
tating to the crop on a worldwide basis, of which
some of the most important are induced by soilborne
fungi including Fusarium wilt, black collar and root
rot, dry root rot, Phytophthora root rot and Pythium
damping-off (Mazur et al. 2004). The widespread
soilborne pathogen Rhizoctonia solani is responsible
for serious damage to many economically important
agricultural and horticultural crops as well as trees
worldwide (Grosch et al. 2006). The first report of
R. solani AG2-3 causing root and collar rot on chick-
pea in Tunisia was in the Beja area (Ben Youssef
et al. 2010).
Strategies to control Rhizoctonia diseases are limited
because of its ecological behaviour, its extremely
broad host range and the high survival rate of sclero-
tia under various environmental conditions (Grosch
et al. 2006). Furthermore, cultivars with complete
resistance are not available at present (Li et al. 1995).
For these reasons, efficient strategies to control the
pathogen are urgently required. However, increasing
use of chemical inputs causes several negative effects
including development of pathogen resistance to the
applied agents and their non-target environmental
impacts (Gerhardson 2002). A growing awareness that
agricultural practices have a great impact on human
health and on the environment has spawned research
into the development of effective biocontrol agents to
protect crop plants against diseases. The uses of
antagonistic microorganisms against R. solani,Fusari-
um oxysporum,Fusarium solani and Sclerotinia sclero-
tiorum have been investigated as one of the alternative
control methods (Siddiqui et al. 1998). The antagonists
may act via competition for nutrients, antibiosis,
induced resistance, mycoparasitism, plant growth pro-
motion and rhizosphere colonization capability (Arfa-
oui et al. 2007; Siddiqui and Akhtar. 2007 and Bailey
et al. 2008).
The aim of this work was to evaluate the efficacy of
two Rhizobium strains to protect chickpea against root
rot and to assess the induction of defence enzymes
(PAL and POX) and phenolic compounds after chal-
lenge inoculation with R. solani.
J Phytopathol 161:412–418 (2013) doi: 10.1111/jph.12071
©2013 Blackwell Verlag GmbH

Materials and Methods
Fungal isolate
Rhizoctonia solani AG 2-3 (Ben Youssef et al. 2010)
was originally isolated from roots of infected chickpea
grown in a naturally infested field at Beja in the north
of Tunisia, according to the method described by
Gerhardson (2002). Fungal cultures of the pathogen
were stored in sterile sand tubes at 4°C. Active cultures
were obtained from small aliquots of a sand culture
plated on potato dextrose agar (PDA). Fungal cultures
were incubated at 22°C for 7 days.
Bacterial isolates
Rhizobium strains were isolated from the nodules of
chickpea as described by Beck et al. (1993). Bacterial
cells were stored in yeast extract mannitol agar at 4°C
(Vincent 1970). The two Rhizobium isolates, Pch Azm
and Pch SidNsir2, were selected after a nodulation test
(L’taief et al. 2007).
Antagonism test in dual culture
The in vitro inhibition of mycelial growth of R. solani
by the bacterial isolates was tested using the dual cul-
ture technique as described by Paulitz et al. (1992) and
Landa et al. (1997). Three 50-ll drops from the
10
8
cfu/ml bacterial suspension were equidistantly
placed on the margins of PDA plates and incubated at
25°C for 24 h. A 4-mm agar disc from fresh PDA cul-
tures of R. solani was placed at the centre of the PDA
plate for each bacterial isolate and incubated at 28°C
for 7 days. The radius of the fungal colony towards
and away from the bacterial colony was measured.
The percentage growth inhibition was calculated using
the following formula:
%inhibition ¼Rr
R100
where ris the radius of the fungal colony towards the
bacterial colony and R is the maximum radius of the
fungal colony away from the bacterial colony.
Volatile antifungal compounds
The production of volatile antifungal compounds by
the Rhizobium isolates was assayed by a sealed plate
method as described by Fiddman and Rossall (1993).
From 72-h-old cultures of Rhizobium strains in yeast
extract mannitol (YEM) liquid media (Vincent 1970),
200 ll was spread on YEMA medium in a Petri
dish. After incubation at 28°C for 24 h, a second
Petri dish containing PDA was inoculated with a
plug (Φ: 6 mm) of the test fungus in the centre of
the plate, inverted and placed over the bacterial cul-
ture. Each two plates, containing pathogen and bac-
teria, were sealed together with Parafilm and
incubated at 25°C. This ensured that both organisms
were growing in the same atmosphere though physi-
cally separated. As a control, a Petri dish containing
YEMA medium without bacteria was placed under
the PDA medium inoculated with the fungal
pathogen. Fungal growth was measured as increases
in the radial length after 5 days. Each test was repli-
cated three times.
In vitro assay for effect of rhizobia and R. solani on
germinated seeds
Chickpea seeds were surface sterilized with 0.5%
sodium hypochlorite and washed five times with dis-
tilled sterile water. The seeds were thoroughly soaked
in 5 ml of the bacterial suspension (10
8
cells/ml) to
ensure uniform coating of the surface. These seeds
were inoculated with 1 g of crushed fragment infected
by R. solani. The seeds were aseptically plated in PDA
medium. The plates were incubated at 28°C for 7 days.
After germination, the number of necrotic root was
measured.
Plant inoculations with bacteria and fungus and disease
assessment
The study was conducted to determine the efficacy of
two Rhizobium strains Pch Azm and Pch S.Nsir2 to
reduce the incidence of root rot on chickpea cultivar
Beja1. Seeds were surface disinfected in 2% NaOCl for
3 min, washed three times with sterile distilled water
and germinated on autoclaved layers of paper towels
in moist chamber at 25°C for 3 days. Germinated
seeds, selected for the uniformity (length of radicle),
were sown in R. solani–infested soil at a depth of
2 cm. The pathogen was cultured in a liquid medium
of potato dextrose for 7 days on a shaker at 200 rpm.
Mycelium was separated from the liquid medium by
filtration using Whatman paper No 1. Soil was inocu-
lated with 25 mg of prepared mycelium mixed with
1 kg of autoclaved soil. The soil moisture content was
maintained at field capacity by daily watering. No sup-
plementary fertilizer was added. After transplanting of
seedlings, each pot was drenched with 5 ml of each of
the bacterial suspension (10
8
cells/ml). The treatments
in the in vivo biocontrol experiment were as follows:
plants inoculated with R. solani and rhizobia, plants
inoculated only with R. solani (control a) and non-
inoculated chickpea (control b). The treatments were
arranged in a randomized block design in three repli-
cations. Seedling length, nodule number and shoot dry
weight were recorded at the end of the experiment.
Samples were kept for 72 h at (60 2) °Cinanoven
for measuring the dry weight of seedling. Development
of disease symptoms associated with Rhizoctonia root
rot infection and the virulence of pathogen was evalu-
ated at scale indices from 0 to 4 according to Tezcan
and Yildiz (1991).
The recorded data were based on Mass Disease
Index (MDI,%) according to the formula:
MDIð%Þ¼ P4
1ni
4N
!
100
n, number of plant with indices i.; N, total number of
plants.
Enhanced Defence Responses of Chickpea Plants 413

Analysis of some biochemical parameters
Peroxidase (Pox) and phenylalanine ammonia lyase
(PAL) assays
Procedures were carried out as described by Anderson
et al. (1995) and Lin and Kao (1999) for the peroxi-
dase assay and by Westcott and Henshaw (1976) for
the PAL assay. Chickpea roots were ground in liquid
nitrogen. The frozen powder was added to the extrac-
tion buffer (ratio 1 : 3, w/v) containing 100 mM
KH
2
PO
4
/K
2
HPO
4
(pH 7), 1% (V/V) Triton X-100 and
2% (w/v) insoluble polyvinylpyrrolidone. The mixture
was homogenized and centrifugated for 20 min at
10 000 g (4°C). The supernatant was used immediately
for total protein quantification (Bradford 1976) and
both enzyme assays.
Soluble peroxidase activity was assayed on a spec-
trophotometer at 470 nm in a reaction medium con-
taining 9 mM gaiacol, 1 mM hydrogen peroxide and
crude enzyme extract. Peroxidase activity was esti-
mated at 30°C and expressed as U/mg protein
(U: lmole tetragaiacol produced per minute). Phenyl-
alanine ammonia lyase activity was determined at
290 nm in a reaction mixture (2.3 ml) containing
1.4 ml of borate buffer (100 mM, pH 8.8), 0.6 ml of L-
phenylalanine (100 mM) and 0.3 ml of the crude
enzyme. Following 2 h of incubation at 40°C, the reac-
tion was stopped by the addition of 0.05 of 5 MHCl.
Activity was expressed as microgram cinnamic acid
produced per hour per milligram proteins.
Estimation of total phenol
Frozen roots from different treatments were extracted
three times with 80% aq. MeOH at 4°C under con-
tinuous stirring. The homogenate was centrifuged at
7000 g for 15 min, and the supernatant was stored at
20°C until use. To estimate the concentration of
total phenolics (milligram equivalent of catechin per
milligram of fresh weight (FW), Folin–Ciocalteu
reagent was used at 760 nm (Waterman and Mole
1994).
Data analysis
Five plants were grown per treatment in all the experi-
ments. The data are given as means confidence
interval (n=5, a=0.05, Student’s t-test). Data were
analysed by multifactorial analysis of variance (ANOVA,
SPSS 12.0 for Windows), and significant differences
among treatments were considered at the P <0.05
level.
Results
Fungal inhibition assays
We were interested in determining whether the two
Rhizobium strains Pch Azm and Pch S.Nsir2 were
directly inhibiting the growth of R. solani in vitro. The
results of in vitro dual culture revealed that the two
bacterial isolates reduced the mycelial growth of R. so-
lani by forming an inhibition zone (Fig. 1). The fungal
inhibition growth percentage was greater than 40%
(Table 1).
Rhizobium volatile substance effect on R. solani growth
in vitro
The results of the physically speculated coculture indi-
cate that Pch Azm and Pch Rhizobium strains produce
volatile substance able to reduce the growth of the
pathogen. Inhibition can reach 38.28 and 35.75%,
respectively, after 5 days of incubation (Table 1).
Effect of Rhizobium strains and R. solani on chickpea
germinated seeds
The effect of symbiont and pathogen was first studied
at an early stage of plant development by assessing
root rot of germinated seedling. Rhizobium isolates
have a variable and significant effect on germination
of chickpea seeds inoculated with R. solani. The
healthy status of germinated seeds inoculated with the
pathogen was significantly improved with the Rhizo-
bium strains used in this study. These rhizobia reduce
the effect of the pathogen as seen by the% of necrotic
radicles. The highest values of chickpea seed germina-
tion were obtained with Rhizobium strain Pch S.Nsir2;
moreover, the amount of necrotic radicles was reduced
by 76.67% (Tables 1 and 2).
Effect of treatments on yield parameters
Infection of chickpea plants with the pathogen led to a
significant reduction in plant growth as measured by
shoot length and shoot dry weight (Table 3). In con-
trast, a significant increase was achieved following
the inoculation with Rhizobium strains of uninfected
Fig. 1 Dual culture of bacterial isolate Azm and Rhizoctonia solani
in vitro showing inhibition of mycelium growth and formation of a
visible clearly halo on the plate
Table 1
Effect of Rhizobium isolates on Rhizoctonia solani growth inhibition
in (a) dual coculture and (b) separate plate
Treatments
% growth inhibition
of fungus (a)
% growth inhibition
of fungus (b)
R. solani 0.0 0.0
R. solani +Azm 50.52*38.25*
R. solani +SidNsir2 76.15*35.75*
Values with * are significant different with the control values.
414 Hemissi et al.

chickpea plants. In the case of inoculation of infected
plants with one of the two Rhizobium strains, the
growth was increased compared to infected plants with-
out Rhizobium, but does not reach the rates observed
with inoculated and uninfected plants. It was checked
that plants not inoculated with rhizobia showed no
nodulation (Table 3). Inoculations with two Rhizobium
strains induce nodule formation in chickpea cultivar
Beja1. Inoculation of plants with Rhizobium strains
without the pathogen significantly increased plant
growth compared to uninoculated control (Table 3).
Inoculation with Pch Azm strain increased plant growth
more than Pch S.Nsir2 strain although the number of
nodule observed on roots inoculated by this stain is
higher.
Disease assessment
Inoculation with the fungus resulted in a pronounced
decrease in the dry weight of shoots compared to unin-
oculated control plants and to treatments with the bac-
teria isolates. Shoot dry weight reduction by
rhizoctonia was approximately 58, 25% of control.
Rhizobium treatment enhanced the resistance of
chickpea to R. solani. Two weeks after inoculation
with the fungal pathogen, 82.5% of the plants were
completely infected. However, pre-inoculation of pre-
germinated seeds of chickpea with Rhizobium reduced
significantly the percentage of infected plants to 12.24
and 12.35% with strains Pch Azm and Pch S.Nsir2,
respectively (Table 3). Seedlings inoculated only with
Rhizobium strains showed non-susceptible reaction.
Impact of Rhizobium inoculation on peroxidase and PAL
activities in chickpea roots
To assess whether defence reactions were stimulated
by the symbiont or pathogen, the levels of peroxidase
and PAL activities were analysed in root tissues at dif-
ferent times after inoculation in both infected and
inoculated chickpea (Figs 2 and 3).
Peroxidase activity remained at low level during the
35 days of culture in root of uninoculated chickpea
plants. Infection by the pathogen induced an increase
in peroxidase activity 7 days after inoculation (DAI),
which was maximal at 14 DAI and maintained during
35 days of culture. In roots co-inoculated with R. so-
lani and Pch Azm or Pch S.Nsir2 isolates, peroxidise
activity increased an obvious threefold rise fasted to
higher levels, with a maximum at 28 DAI and a 5-fold
stimulation.
Phenylalanine ammonia lyase activity in chickpea
roots was transiently increased at 7 DAI by R. solani
infection. When plants were co-inoculated with R. so-
lani and Rhizobium strains, enzyme activity was
increased at 7 DAI and maintained at slightly higher
levels during the whole period of the experiment.
Phenolic compounds
In control roots, the level of total soluble phenolic
compounds increased very slowly, reaching a maxi-
mum at 21 DAI (Fig. 4). These levels did not change
significantly when the roots were infected by the path-
ogen. Co-inoculation of the roots with R. solani and
one of the bacterial isolates, Pch Azm and Pch
S.Nsir2, increased total phenolic levels (Fig. 4), highest
phenol levels were recorded 14 and 21 days after inoc-
ulation in roots co-inoculated.
Table 2
Impact of the selected Rhizobium isolates on percentage of necrotic
radicles of chickpea seeds inoculated with R. Solani in vitro
Treatments Necrotic radicles (%)
Control 0
R. solani 96,67 3,79
S.Nsir2 23,33 2,59
Azm 40,00 2,72
Table 3
Effect of Rhizobium isolates on mass disease index (%) and growth
performance of chickpea seedling based on nodule number, shoot
length and shoot dry weight after 6 weeks of sowing in healthy and
R. solani–infested soil
Treatments
Mass
Disease Index
(MDI, %)
Nodule
number
Shoot
length (cm)
Shoot
dry
weight
(mg)
Water/
Water
0.00* 0.00 41.00* 867.00
Azm/Water 0.00* 41.33* 65.05* 2079.33*
SidNsir2/
Water
0.00* 63.33* 60.20* 1260.33*
Water/
R. solani
82.50 0.00 22.67 362.00
Azm/
R. solani
12.24* 25.00* 45.33* 1153.00*
SidNsir2/
R. solani
12.34* 22.00* 43.00* 1046.00*
The assays were repeated three times, and each treatment was con-
ducted with 20 plants. Mean values followed by *are significant
(P =0.05) by Duncan’s multiple range test as compared to the inoc-
ulated control with R. solani.
0
5
10
15
20
25
30
0 7 14 21 28 35
Peroxidase activity (UE/mg of proteins)
Days after inoculation
Fig. 2 Evolution of peroxidase in chickpea root inoculated with
Rhizobium isolates and infected by R. solani. Activities were mea-
sured on 7, 14, 21, 28 and 35 days after inoculation (DAI). Control
was performed with inoculated and healthy chickpeas (○) and non-
inoculated and infested chickpeas (□)(Δ, Pch Azm isolate; ■, Pch
S.Nsir2 isolate)
Enhanced Defence Responses of Chickpea Plants 415

Discussion
The ability of rhizobia to inhibit certain soilborne
plant pathogens and plant parasites (Siddiqui et al.
1998; Arfaoui et al. 2005; Mabrouk et al. 2007a) has
increased the importance of rhizobia besides their use
in nitrogen fixation. In this study, an experiment was
therefore carried out to study the antagonist effect of
2Rhizobium strains to R. solani and for an evaluation
of the relative impact of rhizobia on the expression of
the plant’s defence response against the pathogen.
The reduction in fungal growth observed in vitro,by
the two selected Rhizobium isolates, was presumably
due to metabolites being released from bacteria into
the culture medium. These metabolites could be antibi-
otics or cell wall degrading enzymes, as previously dis-
cussed by several authors (Charkrabotry et al. 1984
and Perdomo et al. 1995). Moreover, production of
volatile compounds that inhibit R. solani growth was
observed during in vitro culture using separate Petri
dishes. Volatile compounds from biological control
agents have an important part of the inhibitory mecha-
nism, especially under closed storage conditions. Pro-
duction of volatile ammonia has been implicated as a
possible mechanism to control soilborne pathogens
(Paulitz et al. 2000).
These results could explain the reduction in necrotic
lesions observed on germinated seeds infected by patho-
gen and inoculated by rhizobia. Before interaction with
its host plant and nodule formation, the rhizobia pres-
ent in the rhizosphere of plants presumably prevent the
contact of pathogenic fungi on roots by covering the
hyphal tip of the fungus and by parasitizing it.
Our results show that inoculation with some Rhizo-
bium strains significantly reduced the percentage of dis-
ease severity in infected chickpea plants. These
findings are in agreement with those of Siddiqui et al.
(1998) who found that pre-inoculation with Bradyrhiz-
obium and Rhizobium trifoli led to a significant reduc-
tion in disease severity caused by R. solani in
economically important crops (chickpea and sun-
flower). These rhizobia also increased nitrogen content
and dry weight of nodules, roots and shoots. Several
other workers have noticed the beneficial effects of rhi-
zobia on plant growth and reduction in disease inci-
dence (Smiley et al. 1986; Hussain and Ghaffar. 1990;
Arfaoui et al. 2006a).
Plants are constantly exposed to soilborne pathogens
and have evolved defence mechanisms for combating
infection by microbial disease organisms. Inducing the
plant’s defence mechanism by application of a biologi-
cal antagonist is thought to be a potential sustainable
crop production strategy. A number of biochemical
and physiological changes have been associated
with rhizobia inoculation such as the production of
antifungal or oxidative enzymes (Arfaoui et al. 2006b;
Mabrouk et al. 2007b).
Our results indicated that Rhizobium inoculation led
to a significant increase in the phenolic content and
the activities of the defence enzymes PAL and POX of
infected chickpea plants, suggesting that these parame-
ters are implicated in the disease resistance. The accu-
mulation of phenolic compounds is the most
commonly observed defence reaction in chickpea in
response to fungal infections (Vogelsang et al. 1994).
These compounds possess biological activity against a
wide range of pathogens and are considered as bio-
markers for the plant degree of resistance/tolerance
(Vogelsang et al. 1994). Phenolics produced in
response to infection by the pathogen such as phytoal-
exins constitute an active defence response (Ch
erif
et al. 2007). Defence responses may also include the
elaboration of cell wall thickenings usually accompa-
nied by the deposition of lignin, a polymer of aromatic
phenolics. This thickening limited the infection process
and played an important role as a physical barrier to
stop the pathogen invasion. In addition, phenolics
seem to inhibit disease development through different
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 7 14 21 28 35
Soluble phenolics (mg/g FW)
Days after inoculation
Fig. 4 Effect of Rhizobium isolates’ chickpea inoculation on phenolic
compounds accumulation in chickpea roots. Contents were measured
on 7, 14, 21, 28 and 35 DAI. Control was performed with inoculated
and healthy chickpeas (○) and non-inoculated and infested chickpeas
(□)(Δ, Pch Azm isolate; ■, Pch S.Nsir2 isolate)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0 7 14 21 28 35
PAL activity
(µg cinnamic acid/h/mg de MF)
Days after inoculation
Fig. 3 Evolution of phenylalanine ammonia lyase (PAL) activities in
chickpea root inoculated with the some Rhizobium isolates and
infected by R. solani. Activities were measured on 7, 14, 21, 28 and
35 DAI. Control was performed with inoculated and healthy chick-
peas (○) and non-inoculated and infested chickpeas (□)(Δ, Pch Azm
isolate; ■, Pch S.Nsir2 isolate)
416 Hemissi et al.

mechanisms involving the inhibition of extracellular
fungal enzymes and inhibition of fungal oxidative
phosphorylation (Hammerschmidt 2005).
Peroxidase (POX) is involved in the oxidation of
several compounds particularly phenolics increasing
their toxicity (Yedidia and Benhamou 1999). It is also
known to be important in symptom expression, a close
relationship between increased activity of POX, and
appearance of symptoms in infected tissues was
reported in several works (Mayer 1987). They are usu-
ally associated with induced resistance response (Ras-
mussen et al. 1995), and they are also implicated in
several plant defence mechanisms such as lignin syn-
thesis, oxidative cross-linking of different plant cell
wall components or generation of oxygen reactive spe-
cies (Mehdy 1994). Also, PAL is the first committed
enzyme in the phenylpropanoid pathway leading to the
conversion of L-phenylalanine into cinnamic acid with
elimination of ammonia (Da Cunha 1987). Many
plant-specific phenylpropanoid pathways and their cor-
responding functional diversity basically originate from
the core phenylpropanoid metabolism initiated by
PAL (Hahlbrock and Scheel 1989). Consequently, both
enzymes could be implicated in Rhizoctonia avoidance
of inoculated chickpea by increasing accumulation of
phenolic compounds and toxicity to pathogen.
We conclude that the selected rhizobia could be used
effectively as biocontrol agents of chickpea against
R. solani. Nevertheless, further studies are needed to
characterize the mechanisms involved in Rhizobium-
inoculated chickpea resistance to R. solani to improve
Rhizobium strains uses as biofertilizer biocontrol
agents in chickpea fields.
Acknowledgments
This work was supported by funds from the Ministry of Higher Edu-
cation and Research of Tunisia. The authors thank Prof. Martina
Rickauer (INP-ENSAT Toulouse, France) for critical review of the
manuscript.
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