Journal of Plant Pathology (2002), 84 (2), 83-93
Edizioni ETS Pisa, 2002
83
ULTRASTRUCTURE AND CYTOCHEMISTRY OF IN VITRO INTERACTIONS
OF THE ANTAGONISTIC BACTERIA BACILLUS CEREUS X16
AND B. THURINGIENSIS 55T WITH FUSARIUM ROSEUM VAR. SAMBUCINUM
M. Chérif1, N. Sadfi1, N. Benhamou2, A. Boudabbous3, A. Boubaker1, M.R. Hajlaoui4 and Y. Tirilly5
1 Laboratore
de Phytopathologie, Institut National Agronomique de Tunisie, 43 Avenue Charles Nicolle,
1082 Cité Mahrajène, Tunis, Tunisie
2 Recherches en Sciences de la Vie et de la Santé, Université Laval, Pav. Charles-Eugène Marchand, Quebec G1k 7P4, Canada.
3 Laboratore de Microbiologie, faculté des Sciences de Tunis, 1060 Campus Universitaire, Tunisie
4 Laboratoire de Cryptogamie-Bactériologie, Institut National de la Recherche Agronomique de Tunisie, Rue Hédi Karray,
2049 Ariana, Tunisie
5 Ecole Supérieure de Microbiologie et Sécurité Alimentaire de Brest, Technopôle Brest-Iroise, 29 280 Plouzané, France
SUMMARY
The present studies were undertaken to investigate
the interaction of the bacterial antagonists Bacillus
cereus X16 and B. thuringiensis 55T with Fusarium roseum var. sambucinum, the causal agent of potato dry rot. On wounded potato tubers, both bacilli effectively suppressed the development of Fusarium dry
rot. Nevertheless, confrontation of the fungal pathogen
with the antagonists on nutrient agar revealed that B.
cereus X16, induced a strong visible inhibition zone
but B. thuringiensis 55T did not. Light microscopy of
the interaction regions after confrontation with B.
cereus X16 on nutrient agar generally showed apparently intact fungal cells with densely stained protoplasm, indicating that the inhibition was due to stasis
rather than toxicity. By contrast, in presence of B.
thuringiensis, Fusarium cells appeared markedly damaged, with partial to complete cell wall disintegration
and disorganization and generally complete loss of protoplasm. These observations suggest that, contrary to
our expectations, antibiotic and chitinolytic activities
of B. thuringiensis 55T may be highly significant in the
parasitism of the pathogen. Confrontation in liquid
medium revealed that both bacteria completely inhibited the germination of Fusarium macroconidia and resulted in their destruction. This suggests that, to produce their fungitoxic and hydrolytic effects, the two
bacteria should make intimate contact with Fusarium
cells.
Key words: Bacillus, biocontrol, Fusarium dry rot,
microscopy, cytochemistry.
Corresponding author: M. Chérif
Fax: 216.-1.-799 391
E-mail: [email protected]
Abbreviations: B = bacterium; CY = cytoplasm;
F = fungus; N = nucleus; NA = nutrient agar; S = septum; TEM = transmission electron microscopy;
VA = vacuole; W = wall; WA = wall apposition;
WT = wall thickenings.
INTRODUCTION
Fusarium spp. are responsible for dry rot of potato
tubers and important crop losses under traditional
and cold storage conditions (Tivoli et al., 1985; Daami-Remadi and El Mahjoub, 1996; Carnegie et al.,
1998; Chérif et al., 2000). Of the different Fusarium
species studied, F. roseum var. sambucinum appears to
be the most dominant and the most destructive fungus
prevailing on stored potatoes (Boyd, 1972; Schisler et
al., 1997; Chérif et al., 2000). This pathogen is commonly reported to be responsible for losses higher
than 25%, especially under traditional storage, where
environmental conditions are particularly conducive
to dry rot development (Daami-Remadi and El
Mahjoub, 1996; Chérif et al., 2000). All commonly
grown potato cultivars are susceptible to Fusarium dry
rot (Pawlak et al., 1987; Schisler et al., 1997), and
management of the disease with chemical measures
has proved impractical, mainly due to the appearance
of fungicide-resistant strains, concern over the presence of chemical residues in the food chain, and problems of environmental pollution (Kawchak et al.,
1994; Secor et al., 1994).
Among alternatives being explored, use of microbial
agents has shown significant potential (Benhamou et
al., 1997; El-Ghaouth et al., 1998). Currently different
species of microorganism are registered for commercial
use against fungal plant pathogens in Europe and in
the United States. These include fungi (Gliocladium
virens G.21; Trichoderma harzianum KRL-AG2; Candi-
84
Potato dry rot: antagonism of Bacillus spp. in vitro
da oleophila 182), gram-negative bacteria
(Pseudomonas fluorescens EG1053, P. syringea ESC-10
and ESC-11, Burkolderia cepacia) and gram-positive
bacteria such as Bacillus subtilis GB03 and MBI 600
(Kim et al., 1997; El-Ghaouth et al., 1998). Bacillus
species, as a group offer several advantages over other
gram-negative bacteria, including longer shelf life because of their ability to form endospores and the
broad-spectrum activity of their antibiotics (Fiddman
and Rossall, 1995; Kim et al., 1997). Bacillus species
are generally soil-inhabitating or exist as epiphytes and
endophytes in the spermosphere and rhizosphere
(Walker et al., 1998). They are also found in many environments as their survival is aided by their ability to
form endospores resistant to UV irradiation, dessication, heat and organic solvents. Some Bacillus species,
such as B. thuringiensis and B. sphaericus, are entomopathogenic as they produce toxins effective against
the larvae of a wide range of insects. The success of B.
thuringiensis as an insecticide has promoted research
and development for additional Bacillus-based products. There is a growing list of reports of postharvest
fungal disease control with Bacillus species, including
B. subtilis for control of green mold, sour rot, and stem
end rot on citrus (Singh and Deverall, 1984), brown
rot on stone fruit (Utkhede and Sholberg, 1986), and
several diseases of apple (Sholberg et al., 1995) and
avocado (Korsten et al., 1997).
Recently, 83 Bacillus isolates, obtained from salty
soils, and five B. thuringiensis strains were screened in
our laboratory for ability to suppress growth of F. roseum var. sambucinum in vitro and to control dry rot
on potato tubers (Sadfi et al., 2001). Among these isolates, strains X16 of B. cereus and 55T of B.
thuringiensis showed high levels of control in vivo.
Nevertheless, while B. cereus inhibited the growth of
the fungal pathogen in vitro by forming inhibition
zones in dual cultures, B. thuringiensis (55T) failed to
produce such inhibition zones and seemed to be ineffective on agar plates. According to the literature,
Bacillus spp. may cause their antagonistic effects
against fungal pathogens by antibiosis, nutrient competition, site exclusion, parasitism and/or induced resistance (Kehlenbeck et al., 1994; Muninbazi and
Bullerman, 1998; Walker et al., 1998). The current
study was undertaken to investigate more closely the
interaction of B. cereus (X16) and B. thuringiensis
(55T) with F. roseum var. sambucinum in vitro by
means of light and transmission electron microscopy
with gold cytochemistry.
Journal of Plant Pathology (2002), 84 (2), 83-93
MATERIALS AND METHODS
Fungal and bacterial isolates and growth conditions.
The isolate of F. roseum var. sambucinum used was obtained from infected potato tubers with typical symptoms of Fusarium dry rot and reported to be virulent on
potato tubers (Chérif et al., 2000). The fungal pathogen
was grown on potato-dextrose agar (PDA) medium at
25°C.
Isolate X16 of B. cereus was selected among a collection of 83 Bacillus spp. isolated from samples of salty
soils collected from different locations in the south of
Tunisia (Sadfi et al., 2001). Strain 55T of B. thuringiensis was selected among six strains kindly provided by
Dr. A. Boudabbous from the laboratory of Microbiology of the Faculté des Sciences de Tunis. Bacterial isolates were maintained on slants of nutrient agar (NA,
Oxoid) at 4°C and subcultured at two-month intervals.
Antagonistic activity on solid medium. Two-day-old
cultures of B. cereus X16 and B. thuringiensis 55T were
studied in their interaction with F. roseum var. sambucinum on NA in 9-cm Petri plates using a dual culture technique. Bacteria were streaked across the center
of each agar plate with a loopful of pure bacterial culture. Two 5-mm disks cut from a 7-day-old culture of F.
roseum var. sambucinum, were placed 2.5 cm apart on
each side of the bacteria. The plates were then incubated at 25°C. Mycelial samples were collected from the
interaction region 2 to 6 days after inoculation and
processed for light and transmission electron microscopy (TEM).
Antagonistic activity in liquid medium. These tests
were performed in 250 ml-Erlenmeyer flasks containing
100 ml of sterile nutrient broth (NB). Fungal spore suspensions and bacterial suspensions were obtained by
flooding 10-day-old PDA cultures of F. roseum var.
sambucinum and 2-day-old NA cultures of Bacillus spp.,
respectively with sterile distilled water. The protagonists were added to the flasks containing NB and adjusted to obtain a final concentration of 106 CFU ml-1 of
the bacteria and 105 spores ml-1 of the fungus. Bacterial
and fungal concentrations were determined, respectively by dilution plating and counting with a heamocytometer. The flasks were then incubated at 25°C and
100 rev min-1 on a rotary shaker. Samples were collected from 2 to 6 days after incubation. Fungal and bacterial cells were pelleted by centrifugation at 7000 g for 15
min. The pellets were embedded in 2% water agar and
processed for light microscopy and TEM.
In order to determine the effect of the bacterial antagonists on the fungal mycelium, the same procedure
Journal of Plant Pathology (2002), 84 (2), 83-93
was adopted with the only exception that bacteria were
added to the liquid medium 5 days after inoculation
with fungal macroconidia. By that time, fungal spores
germinated and developed a dense mycelium.
Antagonistic activity on potato tubers. Potato tubers
cv. ‘Spunta’ were surface-sterilized by soaking in 2%
aqueous sodium hypochlorite for 10 min. They were
then thoroughly rinsed, dried by using sterile filter papers, and then wounded by removing a plug 3 mm in
diameter and 3 mm in depth with a sterile cork-borer.
B. cereus X16 and B. thuringiensis 55T isolates were
used after, respectively, 24 h and 48 h of culturing at
106 CFU ml-1, and bacterized potato wounds received
20 µl of bacterial suspension. Potato wounds were challenged with the pathogen immediately after bacterial inoculation and received 20 µl of a conidial suspension
adjusted to 105 spores ml-1. The treated wounds were
sealed with scotch tape and potato tubers were placed
in plastic bags to maintain a high humidity and then incubated at 20°C for one week. For each treatment 20
potato tubers were assayed and the experiment was repeated at least twice.
Light microscopy and TEM. Before fixation and
processing for TEM, samples were always examined by
light microscopy and photographed with a Zeiss Axioskop microscope (Carl Zeiss, Thorwood, NY). Samples collected from the interaction regions of solid dual
cultures, from liquid cultures, as well as samples from
pure cultures of the fungus and each bacterium, were
fixed in 2% (v/v) glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2, for 2 h at room temperature and
overnight at 4°C. Samples were rinsed in three changes
of cacodylate buffer and post-fixed in 1% (w/v) osmium tetroxide in the same buffer. They were then dehydrated in a graded series of ethanol and embedded in
Epon 812 resin. For light microscopy, thin sections
(0.25 to 0.5 µm) were collected on microscope slides
and stained with ethylene blue. For TEM, ultrathin sections were collected on 200-mesh nickel grids coated
with Formvar and stained with uranyl acetate and lead
citrate. Grids were examined with a JEOL 1200 EX
transmission microscope (JEOL, Tokyo) operating at
80 kV.
For each treatment, samples were collected from at
least four replicates to obtain a representative sampling
of the interactions. For each sample, more than 10 thin
or ultrathin sections were examined by light microscopy and TEM, respectively.
Cytochemical labeling. Colloidal gold suspension
was prepared as described by Grandmaison et al.
Chérif et al.
85
(1988). A lectin with N-acetylglucosamine binding
specificity, was used for localizing fungal chitin according to a previously described procedure (Benhamou,
1989; Chérif and Benhamou, 1990). Because of its low
molecular weight, this lectin could not be directly complexed to colloidal gold. It was used in a two-step procedure, using ovomucoid as a second step reagent.
Ovomucoid was conjugated to gold at pH 5.4.
Sections were first incubated on a drop of WGA [25
µg ml-1 in phosphate- buffered saline (PBS), pH 7.4]
for 60 min at room temperature, rinsed with PBS, and
transferred to a drop of ovomucoid gold-complex for
30 min at room temperature. After washing with PBS
and rinsing with double-distilled water, sections were
contrasted with uranyl acetate and lead citrate, and examined by TEM.
Specificity of labeling was assessed by the following
controls: (i) incubation with WGA to which was previously added N,N’,N’’ triacetyl-chitotriose (1 mg ml-1 in
PBS); (ii) incubation with WGA, followed by unlabeled
ovomucoid and finally by ovomucoid gold-complex;
and (iii) direct incubation with the ovomucoid goldcomplex, the lectin step being omitted.
Reagents. Tetrachloroauric acid was purchased from
BDH chemicals, Montreal. All other reagents for electron microscopy were obtained from JBEM company,
Pointe-Claire, QB, Canada.
RESULTS
Macroscopic observations. Coinoculation of F. roseum var. sambucinum with bacterial antagonists on NA
revealed that B. thuringiensis 55T was apparently unable
to inhibit growth of the fungus after 5 days of incubation
(Fig. 1A). By that time, however, the fungus was strongly
inhibited by B. cereus X16, with the appearance of inhibition zones (Fig. 1B). While the first contact between B.
thuringiensis 55T and the pathogen was observed by 3
days after coinoculation, intimate contact between F. roseum var. sambucinum and B. cereus X16 was never observed even after many weeks of incubation.
Potato wounds inoculated with F. roseum var. sambucinum alone started to show typical dry rot symptoms
by the third day of incubation at 20°C. By the seventh
day, all potato tubers infected with the pathogen were
diseased and showed lesions of more than 20 mm in diameter (Fig. 1C).
Application of both Bacillus isolates to potato
wounds before challenge with the pathogen, significantly reduced development of dry rot (Fig. 1D and E). Tubers treated with B. cereus X16 and inoculated with the
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Potato dry rot: antagonism of Bacillus spp. in vitro
Journal of Plant Pathology (2002), 84 (2), 83-93
Fig. 1. Effect of the bacterial antagonists
B. thuringiensis 55T and B. cereus X16
on (A,B) mycelial growth of the
pathogen F. roseum var. sambucinum in
dual cultures, after 5 days of incubation
on nutrient agar medium, and on (C-F)
dry rot development on wounded potato
tubers cv. ‘Spunta’, after 7 days of inoculation. (A) B. thuringiensis 55T and the
fungal pathogen in dual culture. (B) B.
cereus X16 and the fungal pathogen in
dual culture. (C) Control potato tuber
inoculated with the pathogen F. roseum.
var. sambucinum alone. (D) Coinoculation with B. cereus X16. (E) Coinoculation with B. thuringiensis 55T. (F) Inoculation with B. cereus X16 alone.
pathogen showed no symptoms of infection after 7 days
at 20°C (Fig. 1D). Although some small, brownish lesions could be seen in potatoes bacterized with B.
thuringiensis 55T and inoculated with the pathogen after 7 days of incubation (Fig. 1E), their frequency and
diameter never reached those in the infected controls.
Tubers inoculated with either B. cereus X16 (Fig. 1F) or
B. thuringiensis 55T alone were free of disease symptoms.
Light microscopy observations. Mycelial samples
from pure cultures of F. roseum var. sambucinum revealed densely stained hyphal cells (Fig. 2A). After 3
days of confrontation, examination of sections from the
edge of Fusarium colonies in contact with the inhibition
zones caused by B. cereus X16 on NA generally showed
apparently intact fungal cells with densely stained protoplasm (Fig. 2B). A relatively low percentage of Fusarium cells showed signs of cell damage, even after a
much longer period of exposure to B. cereus X16. By
contrast, in presence of B. thuringiensis 55T, where
contact between the two protagonists was macroscopically observed, Fusarium cells appeared markedly damaged, as evidenced by disorganization of the cytoplasm
and generally complete loss of protoplasm (Fig. 2C, D).
In pure liquid cultures (NB), fungal macroconidia
germinated after 24 h of incubation (Fig. 3A) and gave
a very dense mycelium with numerous newly formed
macroconidia 5 days later (Fig. 3B). Coinoculation with
fungal spores and either B. cereus X16 or B. thuringiensis 55T completely inhibited the germination of macroconidia after 24h (Fig. 3C, and D) and 6 days (Fig. 3EG) of incubation. By the end of the experiment, the
Bacillus spp. had abundantly colonized the medium and
fungal macroconidia, resulting in destruction of a high
percentage of macroconidia, which appeared brightly
stained (Fig. 3E-G). In some instances, bacterial cells of
B. thuringiensis 55T seemed to be inside macroconidia
(Fig. 3G, arrow). This was never observed with B.
cereus X16. Nevertheless, this bacterial isolate caused
severe damage to the macroconidia in liquid medium
(Fig. 3F).
Journal of Plant Pathology (2002), 84 (2), 83-93
Chérif et al.
87
Fig. 2. Light microscope observations of F.
roseum var. sambucinum cells from pure cultures (A), from the edge of Fusarium colonies
in contact with the inhibition zone caused by
B. cereus X16 (B), or from fungal colonies in
contact with B. thuringiensis 55T (C, D), after
3 days of incubation on nutrient agar plates.
F = Fungus.
Ultrastructural and cytochemical observations
Interaction of B. cereus X16 with the pathogen. The
time course study of fungal growth in dual cultures in
presence of B. cereus X16 revealed that colonization of
the NA medium by Fusarium ceased 48-72h after confrontation. Up to that time, the majority of Fusarium hyphae were free of any sign of alteration, as judged by the
integrity of the cytoplasm and cell wall (Fig. 4A). From 3
to 6 days after inoculation, some hyphae showed various
degrees of cytoplasm disintegration, leading in some cases to complete depletion of their cytoplasm (Fig. 4B).
Nevertheless, hyphae showing such severe alteration
were very rarely observed and most cells of F. roseum
var. sambucinum displayed apparently well-preserved organelles and cytoplasm. Labeling with the WGA/ovomucoid-gold complex revealed that even hyphae with
advanced cytoplasm alteration preserved the integrity of
their cell walls, which were intensely and regularly labeled with gold particles (Fig. 4C). In many instances,
Fusarium cells developed cell wall thickenings, which
were also intensively labeled (Fig. 4D). By contrast,
coinoculation of Fusarium and B. cereus X16 in liquid
medium (NB), resulted in much more damage to fungal
cells (Fig. 4E, F). Under these conditions, the contact
between the pathogen and B. cereus X16 was more intimate and direct (Fig. 4E). Six days after inoculation,
most fungal cells appeared severely damaged and the
main characteristic of these cells was partial to complete
wall disintegration associated with depletion of cytoplasm contents (Fig. 4E). This observation was confirmed by labeling with the WGA/ovomucoid-gold complex, which showed that some fungal cells were reduced
to traces that could only be identified by the presence of
gold particles in remaining wall debris (Fig. 4F).
Interaction of B. thuringiensis 55T with the pathogen.
Mycelial samples from the edge of growing colonies of
F. roseum var. sambucinum examined two days after
coinoculation with B. thuringiensis 55T on NA, revealed generally dense hyphal cells with apparently preserved cell walls, nucleus and organelles (Fig. 5A).
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Potato dry rot: antagonism of Bacillus spp. in vitro
Journal of Plant Pathology (2002), 84 (2), 83-93
Fig. 3. Light microscope observations of F.
roseum var. sambucinum cells after incubation
of macroconidia in liquid medium (nutrient
broth) in pure cultures (A, B) or in presence of
the bacterial antagonists B. cereus X16 (C, E,
F) and B. thuringiensis 55T (D, G). (A) Pure
culture Fusarium after 24 h of incubation. (B)
Pure culture of Fusarium after 7 days of incubation. (C) Coinoculation with B. cereus X16
after 24h of incubation. (D) Coinoculation
with B. thuringiensis 55T after 24h of incubation. (E, F) Coinoculation with B. cereus X16
after 6 days of incubation. (G) Coinoculation
with B. thuringiensis 55T after 6 days of incubation. A and C-F: Bar = 20 µm; B: Bar = 60
µm; G: Bar = 10 µm.
Alterations in wall labeling with the WGA/ovomucoidgold complex started to appear by 3 days after inoculation, at which time the two protagonists were very close
to each other (Fig. 5B). These wall-labeling alterations
were always associated with abundant presence of gold
particles in NA medium (Fig. 5B, arrows). At this stage,
fungal nuclei and vacuoles often showed obvious signs
of malformation, as exemplified by contortion of their
membranes (Fig. 5B, and C).
In many instances, fungal cells responded to B.
thuringiensis 55T attack by the accumulation of cell
wall appositions and thickenings (Fig. 5D). Such appositions were intensely labeled with the WGA/ovomucoid-gold complex indicating high accumulation of
chitin at these sites (Fig. 5D). As early as 3 days after inoculation, significant alterations of the fungal cytoplasm
were readily discernable, such as retraction, aggregation
and organelle disintegration (Fig. 5C). By that time,
fungal cell walls generally showed good structural
preservation. Past this stage, from 4 to 6 days after inoculation, most Fusarium cells became affected (Fig. 6A),
with pronounced damage, as judged by total loss of
protoplasm (Fig. 6A-C) and partial or complete wall
disintegration (Fig. 6B), leading to empty hyphal shells
with many holes, corresponding to locally digested areas in the cell walls (Fig. 6C1, and C2). Such cell wall
degradation was generally accompanied by the release
of gold particles, which were scattered over the surrounding agar medium (Fig. 6C2)
Examination of ultrathin sections from samples of
Fusarium macroconidia and mycelium confronted with
B. thuringiensis 55T in liquid medium (NB) revealed
the same events of fungal cytoplasm disintegration and
cell wall degradation (Fig. 6D), with the exception that
alterations started to appear by 24 h after inoculation,
thus earlier than for solid cultures, where the first contact between the two protagonists was not observed before 3 days of incubation.
Journal of Plant Pathology (2002), 84 (2), 83-93
Chérif et al.
89
Fig. 4. Transmission electron micrographs of
F. roseum var. sambucinum cells grown on nutrient agar (A-D) or in liquid medium (nutrient broth, E, F) in presence of the antagonistic
bacterium Bacillus cereus X16. (A) After 48 h
of incubation. (B) After 3 days of incubation.
(C) Labeling with the WGA/ ovomucoid-gold
complex of fungal hyphae incubated for 6
days in presence of the antagonist. (D) Fungal
cell with a cell wall thickening, intensively labeled WGA/ ovomucoid-gold complex. (E) In
liquid medium, bacterial cells are in close contact with the pathogen by the sixth day of
incubation. (F) Fusarium hyphae showing
highly degraded Cell walls after labeling
with the WGA/ovomucoid-gold complex.
Bar = 1 µm. B = Bacterium; CY = Cytoplasm;
F = Fungus; S= Septum; W = Wall; WT =
Wall thickenings.
Specificity of labeling with the WGA/ovomucoidgold complex was assessed by the negative results obtained with all control tests including the previous adsorption of the WGA with N,N’,N’’-triacetylchitotriose
(data not shown).
DISCUSSION
Our results based on ultrastructural observations
and cytochemical localization of N-acetylglucosamine
residues showed that contrary to our expectations, antibiotic and chitinolytic activities of B. thuringiensis 55T
may be highly significant in the parasitism of F. roseum
var. sambucinum. In fact, although in vitro antagonism
tests performed on NA showed no apparent inhibition
of Fusarium growth in presence of B. thuringiensis 55T,
microscopic observations revealed that this antagonist
was very destructive to fungal hyphae, on both solid
and liquid media. Moreover, although apparently ineffective in vitro on NA, B. thuringiensis 55T effectively
inhibited dry rot in vivo on wounded tubers. This confirms the idea that Bacillus isolates unable to form inhibition zones on solid medium are not necessarily incapable of killing the pathogen in vitro and inhibiting disease development in vivo.
Confrontation of B. cereus X16 with the pathogen on
solid medium (NA) demonstrated that Fusarium hyphae appeared generally intact with a very low percentage of damaged cells, indicating that the inhibition zone
observed exerted a fungistatic rather than a fungitoxic
role. These conclusions were confirmed by ultrastructural observations, which generally revealed fungal hyphae with preserved cytoplasm, organelles and cell
walls. Even the few pathogen cells showing damaged
protoplasm preserved the integrity of their cell walls as
evidenced by the intense and regular labeling for chitin.
By contrast, confrontation of the pathogen with B.
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Journal of Plant Pathology (2002), 84 (2), 83-93
Fig. 5 A-D. Transmission electron micrographs of F. roseum var. sambucinum hyphae
grown on nutrient agar medium in presence of
the antagonistic bacterium B. thuringiensis
55T. Fusarium cells after 2 days (A) and 3 days
(C) of incubation. (B, D) Fusarium cells after 3
days of incubation labeled with WGA/ ovomucoid-gold complex. In D, a fungal cell responding to bacterial attack by the formation
of a cell wall apposition. Bar = 0.5 µm. CY =
Cytoplasm; N = nucleus; VA = vacuole; W =
Wall; WA = Wall apposition.
thuringiensis 55T resulted in serious damage to a high
percentage of hyphae, associated with a series of degradation events, including alteration of chitin macromolecules and visible cell wall disruption, retraction of the
plasmalemma, distortion of the nucleus, generalized
disorganization of the cytoplasm, and, ultimately, complete loss of protoplasm. Fungal cell wall disintegration
and cytoplasm disorganization were observed as soon as
3 days after confrontation, just before first contact between the two protagonists was established. This suggests that fungitoxic compounds and hydrolytic enzymes produced by B. thuringiensis 55T diffuse a short
distance into the agar to cause the observed disturbances in advance of physical contact.
Cell wall appositions and thickenings in Fusarium
cells were observed in presence of both bacteria. Such
deposits contained large amounts of chitin and are
similar to wall appositions observed in the cells of different fungal pathogens submitted to the activity of antagonistic fungi (Benyagoub et al., 1998; Benhamou et
al., 1999) and to treatment with fungicides (Robertson
and Fuller, 1990), chitosan (Benhamou, 1992) or 2-deoxy-D-glucose (El-Ghaouth et al., 1997). The mechanisms that control the process of chitin deposition at
such sites and the exact biological function played by
these deposits are still unclear. Nevertheless, from the
literature the most accepted explanations indicate that
the deposited material may be laid down as newly synthesized molecules via deregulation of fungal membrane-bound enzymes involved in the synthesis of
structural compounds (Benhamou et al., 1999). Accordingly, the massive accumulation of these structural
compounds as abnormal wall-like deposits reflects a
defense strategy elaborated by the fungal pathogen for
preventing penetration of mycoparasites and fungitoxic compounds. Whatever the role played by the deposited material, our Bacillus antagonists, when in direct contact with the pathogen, i.e. in liquid medium,
were able to circumvent such barriers and cause severe
damage to fungal cells, which were rapidly reduced to
empty shells.
The differences observed between B. cereus X16 and
B. thuringiensis 55T on NA, relative to the percentage
of fungal cells affected, cell wall degradation and the
extent of cytoplasm disintegration, were not observed
in liquid medium. In fact, B. cereus X16 was as effective
as B. thuringiensis 55T and caused extensive cell wall
disruption and cytoplasm disorganization of the
Journal of Plant Pathology (2002), 84 (2), 83-93
Chérif et al.
91
Fig. 6. Transmission electron micrographs of
F. roseum var. sambucinum hyphae grown on
nutrient agar (A-C) or in liquid medium (D) in
presence of the antagonistic bacterium B.
thuringiensis 55T. (A, B) Fusarium hyphae
showing pronounced damage after 4 days of
incubation. (C1, C2) Fusarium hyphae labeled
with the WGA/ovomucoid-gold complex.
Note that cell wall degradation is accompanied
with the release of gold particles (C2, a higher
magnification of C1, arrow). (D) Confrontation of the pathogen with B. thuringiensis 55T
in liquid medium. Bar = 1 µm. B = Bacterium;
F = Fungus; W = Wall.
pathogen. This suggests that in order to exert its fungitoxic and hydrolytic effects, B. cereus X16 must be in
intimate contact with the Fusarium cells. The absence
of inhibition zones on solid medium after application of
B. thuringiensis 55T may be explained by the fact that
the fungal pathogen can counteract fungistatic effects
and therefore reach intimate but deadly interaction
with the bacterium. In contrast, fungistatic compounds
produced by B. cereus X16 may halt fungal spread but
avoid the lethal effects of close contact.
When intimate contact between the protagonists is
achieved, fungal cell wall degradation by hydrolytic enzymes produced by both antagonists seems to play a
major role in the outcome of their interaction with the
pathogen. In a recent study, we have reported that B.
cereus X16 and B. thuringiensis 55T exhibited strong
chitinolytic activity as determined by the formation of
clearing zones on chitin agar, the release of reducing
sugars from colloidal chitin and by the release of p-nitrophenol (pNP) from dimeric, trimeric and tetrameric
chromogenic chito-oligosaccharides, thus showing that
these antagonists are able to produce N-acetyl-βD-glucosaminidases, chitobiosidases and endochitinases (Sadfi et al., 2001). Our TEM observations revealed
that while the alteration of chitin first occurred locally,
as illustrated by the release of N-acetylglucosamine
residues in the growing medium, it appeared to be
more generalized later on and at more advanced stages
of the interaction of the pathogen with the antagonistic
bacilli. Fusarium cell walls were completely digested.
From these observations, it can be speculated that the
synergistic and coordinated action of chitinases with
other polysaccharidases, such as β-1,3-glucanases, lipases and proteases may be an important determinant
in the antagonistic process (Sivan and Chet, 1989;
Chérif and Benhamou, 1990; Benhamou and Chet,
1996).
Confrontation of conidia and hyphae of F. roseum
var. sambucinum to the bacilli generally resulted in severe alteration of their protoplasm. Such alteration may
directly correlate with the toxic action of antifungal substances (i.e. antibiotics) produced by the antagonists. In
92
Potato dry rot: antagonism of Bacillus spp. in vitro
Journal of Plant Pathology (2002), 84 (2), 83-93
fact, fungal protoplasmic alterations have been reported
in other antagonistic interactions involving antibiosis as
the main mechanism of action (Hajlaoui et al., 1993;
Bélanger et al., 1995). For instance, Benyagoub et al.
(1996), showed that antibiotics from Sporothrix flocculosa caused protoplasm alteration and depletion in treated pathogenic fungi by promoting modification of the
lipid composition of the plasmalemma. This raises the
question as to what extent weakening of the cell wall of
pathogenic fungi through the action of hydrolytic enzymes of the antagonist may facilitate the diffusion of
toxic substances toward membrane receptors by increasing the wall permeability. A growing body of evidence indicates that the synergistic action of wall hydrolases, especially chitinases, and antibiotics is required in
the antagonistic process and that alteration of host cell
walls are essential prerequisites for further antibiotic
diffusion (Di Pietro et al., 1993). These findings are in
line with our observations, which have demonstrated a
high percentage of affected fungal cells with severely altered cytoplasm in liquid medium, where close contact
between the fungus and B. cereus X16 was well established and cell wall alterations of the pathogen were evident. Such results were not observed on solid medium,
where the fungal cell walls appeared intact, and chitin
breakdown was not detected by cytochemical labeling.
Benhamou N., Chet I., 1996. Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum: ultrastructural
and cytochemical aspects of the interaction. Phytopathology 86: 405- 416.
ACKNOWLEDGEMENTS
Chérif M., Benhamou N., 1990. Cytochemical aspects of
chitin breakdown during the parasitic action of a Trichoderma sp. on Fusarium oxysporum f.sp. radicis lycopersici.
Phytopathology 80: 1406-1414.
This research was supported by funds from the International Foundation for Science (C/2600-2), AUPELF-UREF (Fond International de Coopération Universitaire 2000/PAS/44) and IRESA, Tunisia (Projet
Fédérateur Pomme de Terre). We thank Alain Goulet
for his technical assistance.
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