Vol. 35 – 2009 - Ecologia Mediterranea - Université d'Avignon et des ...

KACEM MOURAD, KAZOUZ HAFIDA
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conservation à 4 o C, 0 o C et –20 o C pendant 60
jours, tandis qu’aucune activité n’a été détectée
après leur conservation à 37 o C pendant 80 à
120 jours. Le tween 80, le tween 20, le SDS ou
le Triton X-100 semblent avoir eu un effet marqué
sur FII24 ; inversement l’activité de FII83 n’a
pas été réduite par ces traitements. La substance
antibactérienne présente dans la fraction FII24
apparaît avoir des propriétés communément
associées aux « vraies » bactériocines et a montré
des effets inhibiteurs sur certaines souches
de Rhizobium sp. Sinorhizobium sp., de Propionibacterium,
de Lactobacillus et d’Erwinia chrysanthemi.
Pseudomonas savastanoi CFBP 2074
et Pseudomonas savastanoi 13519a, deux bactéries
phytopathogènes induisant des galles ou
des tuberculoses de l’olive, ont également été
inhibées par FII24. Ce travail suggère une utilisation
potentiellement efficace de la souche-
Rhizobium sp. ORN24, ou de ces substances
FII24, comme des agents de bio-contrôle pour la
prévention des maladies végétales causées par
les espèces de Pseudomonas savastanoi ou d’Erwinia
chrysanthemi.
Introduction
In nature, all organisms need to compete in
order to survive in their habitat. This biological
task can be achieved by the development
of competitive mechanisms such as the production
of toxins, enzymes and antimicrobial
agents (Roslycky 1967). Various bacterial
species can enter symbiosis with legume
plants (Spaink et al. 1998). These bacteria are
collectively called rhizobia and have the
capacity to induce the formation of root nodules
in the host plant (Lerouge et al. 1990;
Pedrosa et al. 2000).
Rhizobia have been shown to produce bacteriocins
(which cause antagonism between
closely related strains) characterized as small,
medium or large on the basis of their assumed
sizes, diffusion characteristics, and sensivity
to heat and proteolytic enzymes (Schwinghamer
& Brockwell 1978; Gross & Vidaver
1978; Schripsema et al. 1996).
The first description of bacteriocin production
by a number of species within the economically
important genus Rhizobium was published
by Roslycky (1967). Many rhizobial
species (bacteriogenic strains) produce bacteriocins,
designated as rhizobiocins (Hirsch
1979; Goel et al. 1999; Sridevi; Mallaiah
2008). The activity of new bacteriogenic
strains is of great importance, either when
strain is introduced from inoculants or as a factor
conditioning the persistence of the legume
year by year. Inability of the inoculant strains
to successfully compete with established Rhizobium
populations in soil has been frequently
observed (Van Rensburg & Strijdom 1985). As
concluded by Vargas & Graham (1989), the
success of an inoculant increases with bacteriogenic
rhizobia population in soil. Furthermore
the bacteriocin producing strains form
the highest number of nodules and could
increase nitrogen fixation by means of the suppression
of competition for nodulation sites in
the native population (Hodgson et al. 1985).
Venter et al. (2001) and Oresnik et al. (1999)
have reported that a mutant unable to produce
bacteriocin was unaltered in its competitive
properties, both in broth and in nodulation
assays, suggesting that the bacteriocin may not
play a major role in determining the ecological
success of this strain.
In this study we report a bacteriocin like substances
produced by Rhizobium sp. ORN 24
and ORN 83 strains previously isolated by
Merabet et al. (2006) and Merabet (2007)
from root nodules of Medicago ciliaris and
Medicago polymorpha, two legumes of forage
and ecological importance in Algeria,
especially in saline soil regions. Our investigation
includes partial purification and preliminary
characterization of the bacteriocins.
Materials & Methods
Bacterial cultures and media
Rhizobium sp. (ORN83, ORN24 and ORN26)
and Sinorhizobium sp. (ORN16, ORN88 and
ORN89) strains were previously isolated in
saline soils in the region of Oran (Algeria)
(Merabet et al. 2006; Merabet 2007). They
were maintained as a frozen stock at – 20 o C
in distilled water plus 20% (v/v) glycerol and
propagated twice in YEM broth (van Brussel
et al. 1977) at 28 o C before use.
Bacterial interaction and detection
of antimicrobial activity
Strains were cross tested for their antagonistic
activity by the well diffusion method as previously
described Kacem et al. (2005). Briefly,
each strain was propagated in 200 ml YEM
broth for 24 h at 30 o C. Culture was centrifuged
(10.000 rpm for 20 min. at 4 o C) and
the supernatant was adjusted to pH 7.0 with
3M NaOH to exclude the antimicrobial effect
of organic acid, followed by filtration of the
ecologia mediterranea – Vol. 35 – 2009