New Phytol.

(1999), 141, 497–510

Process of tomato root colonization by a

pathogenic strain of Fusarium oxysporum
f. sp. lycopersici in comparison with a
non-pathogenic strain

C H A N T A L O L I V A I N    C L A U D E A L A B O U V E T T E*
Laboratoire de Recherches sur la Flore pathogeZ ne dans le sol, INRA-CMSE, BV 1540,
17 rue Sully, 21034 DIJON Cedex, France
Received 26 June 1998 ; accepted 26 November 1998


A pathogenic strain of Fusarium oxysporum f. sp. lycopersici transformed with the glucuronidase (GUS) reporter
gene was used to study the colonization process of tomato (Lycopersicon esculentum) roots in hydroponic culture.
The plants were treated exactly as those used in a different study with a non-pathogenic strain of F. oxysporum
in order to compare the two types of colonization process. The pathogenic strain rapidly colonized the root surface,
forming a dense network of hyphae – shown by the GUS staining assay – as early as 48 h after inoculation. The
first images of the pathogen penetrating into the epidermis of the root were observed 24 h after inoculation. The
plant showed defence reactions mainly in the hypodermis, but also in the cortex. Generally these barriers failed
to prevent the centripetal growth of the pathogen towards the stele. The GUS activity moved with the actively
growing hyphae into the tissues ; the stele appeared intensely stained 7 d after inoculation, whereas by this time
the hyphae at the root surface did not show any more staining. The hyphae formed a dense network at the apex
of the root, but direct contact between hyphae and living cells was prevented by several layers of sloughed cap cells.
Although rarely observed, penetration of the pathogen into the apex was possible and led to a rapid destruction
of apical cells. The only differences between this pattern of root colonization and that observed for a non-
pathogenic strain of F. oxysporum appear to concern the frequency of dead apices and the intensity of fungal
colonization in the cortex. When the pathogen passed around the barrier formed in the hypodermis it always
reached the xylem, although this barrier and other defence reactions induced at different levels in the cortex always
prevented the non-pathogenic strain from reaching the stele. These observations suggest that the main differences
between the two types of interaction – between the plant and either the pathogenic or the non-pathogenic
strain – are quantitative rather than qualitative.

Key words : light microscopy, electron microscopy, GUS gene marker, ultrastructural study, penetration, infection
process, tomato (Lycopersicon esculentum), Fusarium oxysporum.

important question as to what makes the difference


The fungus Fusarium oxysporum has a worldwide
distribution in soil and occurs in the rhizosphere of
many plant species. Some strains that induce root
rots or vascular wilts are responsible for diseases on
crops of economical importance. However, most of
the strains are termed non-pathogenic because they
are unable to induce disease on a given plant species.
Nevertheless, they are parasitic because they colonize
the root tissues of the plant to some extent (Nagao et
al., 1990 ; Mandeel & Baker, 1991 ; Postma &
Luttikholt, 1996 ; Olivain & Alabouvette, 1997). The
*Author for correspondence (fax j33 3 80 69 32 26 ; e-mail
alabouvette!dijon.inra.fr).
between a pathogenic and a non-pathogenic strain on
a given species remains unanswered. Comparison of
the process of root colonization of a plant species by
a pathogenic and a non-pathogenic strain might
enable this problem to be addressed.
Having established that Fo5a4, a non-pathogenic
strain effective in controlling fusarium wilt of tomato
(Olivain et al., 1995), is able to colonize the surface
and to some extent the cortex of tomato roots
(Olivain & Alabouvette, 1997), the process of root
colonization by a pathogenic strain of F. oxysporum
belonging to forma specialis lycopersici was studied
under the same experimental conditions. The ob-
jective of this study was to determine whether

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498 C. Olivain and C. Alabouvette
surface colonization, penetration sites and internal
colonization are similar for the pathogenic and the
non-pathogenic strain. Indeed, in contrast to the
numerous studies reporting the colonization process
in plant stems (Baayen & Elgersma, 1985 ; Beckman,
1987) and in induced resistance (Benhamou et al.,
1994a,b ; Benhamou et al., 1996a,b), the ‘ primary
determinative phase ’ (Beckman, 1987) – including
root-surface colonization, penetration of the fungus
through the epidermis and the colonization of cortex
and endodermis to the vascular system – has rarely
been studied (Rodriguez-Galvez & Mendgen, 1995).
In the case of the pathogenic F. oxysporum, the
question as to whether the pathogen is penetrating
the root only at the apex or through differentiated
tissues of the root is still controversial. Observations
on flax (Turlier et al., 1994) seem to establish that F.
oxysporum f. sp. lini penetrates the root only at the
apex, through undifferentiated tissues, although
studies on tomato (Bishops & Cooper, 1983) reported
penetration both at the apex and through differen-
tiated tissues of the root. Localizing the colonization
and penetration zones is important for establishing
whether the pathogen and the non-pathogenic strain
are competing for infection sites and whether this
competition takes place all along the root or only at
the apices.
  
Preparation of the fungal strain and inoculum
A pathogenic strain of F. oxysporum f. sp. lycopersici
(Fol32) isolated in 1989 from a wilted tomato plant
in French Brittany was used. This strain was
transformed with the Escherichia coli β-D-glucuro-
nidase (GUS) gene according to the procedure
described by Couteaudier et al. (1993). In the
presence of 5-bromo-4-chloro-3-indolyl β-D-glu-
curonide, the GUS activity of the fungus enables its
histochemical localization in plant tissues and cells.
Because the GUS gene is under the control of the
promoter of glyceraldehyde-3-phosphate dehydro-
genase, an enzyme that catalyses the second step of
glycolysis, the intensity of the blue staining resulting
from the GUS activity gives an indication of the
metabolic activity of the transformed strain (Epar-
vier & Alabouvette, 1994). After transformation, the
pathogenicity of the GUS-marked strain was com-
pared to that of the wild strain by inoculation of
tomato in a standardized biotest and found not to be
significantly different. Until use, the fungus was
stored at k80mC as microconidia in 25% glycerol.
The fungus was grown in 10 g l−" malt broth
(Biokar Diagnostics, 60000 Beauvais, France) at
25mC on a rotary shaker (250 rpm). After 6 d of
culture, the mycelial mats were removed by filtration
through a 40 µm mesh. The microconidia in the
filtrate were washed three times by centrifugation
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(11 000 g, 20 min) and resuspended in sterile distilled
water. The density of the conidial suspension was
determined with a haemocytometer.
Conditions of plant growth and inoculation
Tomato seeds (Lycopersicon esculentum cv. Mar-
mande Verte), provided by INRA (Station d’Ame! li-
oration des Plantes, Montfavet, France), were sur-
face sterilized in 1.25% sodium hypochlorite for 20
min and rinsed three times in sterile distilled water.
Seeds were germinated on nutrient agar, prepared by
adding 10 g l−" agar in the nutrient solution used to
grow tomatoes commercially in rockwool (Hydro-
kani nutrient solution, Hydro-Azote, 92200 Neuilly,
France). Some 5–10 seeds were deposited at the
surface of the nutrient agar, which had been poured
into Petri dishes that were kept inclined at 60m and
incubated in the dark at 25mC for 3 d. Seedlings with
a 1.5 cm-long radicle were gently removed from the
agar surface to be inoculated. Inoculation involved
dipping the radicles in a conidial suspension (10'
microconidia ml−") for 1 h. The control plants were
similarly dipped in sterile distilled water. The
seedlings were then aseptically transferred into tubes
containing a sterilized nutrient solution adapted for
tomato crops in soil-less culture (Hydrokani nutrient
solution) without oxygen supply. The plants were
cultivated in a growth chamber at 25mC with a
day–night cycle of 14 h–10 h under 8000 lux.
Uninoculated control plants were cultivated simi-
larly. Radicles were sampled for observation at
different time intervals from 17 h to 10 d after
inoculation. Six inoculated plants and three control
plants were taken at each sampling time. All plants
were observed under a stereomicroscope, and three
inoculated plants and one control, chosen at random,
were processed for microscopic observations.
Light microscopy on whole roots
The roots were transferred into a 1 mM solution of
5-bromo-4-chloro-3-indolyl β-D-glucuronide (Sig-
ma-Aldrich, Paris, France Ref. B.6650) in 0.1 M
phosphate buffer, pH 7.2, at 25mC for 1 h. Each
radicle was layed on a ‘ deep ’ glass slide in distilled
water, beneath a cover slip and observed under a
stereomicroscope or a bright-field light microscope.
Observations were recorded on Kodak Gold 100
film.
Microscopy on root sections
Three zones were characterized for each radicle, and
transverse sections were taken from these : the apex
and the elongation zone ; the intermediate zone
without secondary roots ; and the distal part of the
root where secondary roots were present. The
secondary roots were removed before processing the
tap root. One-cm-long pieces were cut in these three
 Tomato root colonization by a pathogenic Fusarium sp.
zones and fixed with 1% glutaraldehyde in phosphate
buffer (0.1 M, pH 7.2) at room temperature for 4 h.
After washing in phosphate buffer, the samples were
postfixed with 1% osmium tetroxide in the same
buffer for 1 h at room temperature. Samples were
dehydrated in a graded series of ethanol followed by
propylene oxide and embedded in an Epon–Araldite
mixture (Glauert & Hall, 1991). Semi-thin sections
(0.5 µm) and ultra-thin sections (0.1 µm) were cut
using an ultramicrotome (Reichert Ultracut E, Lyon,
France). Semi-thin sections were stained with 1%
aqueous toluidine blue O and methylene blue O in
1% sodium tetraborate and examined under a
bright-field light microscope (Leica Sarl, Rueil-
Malmaison, France). Ultra-thin sections were given
contrast using a methanolic solution of uranyl acetate
(2.5%) followed by Reynold’s lead citrate. Micro-
scopic observations were made with a Hitachi 600
electron microscope (Verrieres-le-Buisson, France)
operating at 75 kV.

Colonization of the root surface
At 24 h after inoculation, numerous hyphae were
observed at the surface, especially in the root-hair
zone (Fig. 1a), the intensity of colonization decreas-
ing progressively towards the root tip (Fig. 1b). The
mycelium at the root surface and the root hairs
exhibited the blue staining resulting from the
glucuronidase activity (Fig. 1a).
At 48 h after inoculation, the colonization of the
root surface was intense in the mature zone where
secondary roots were emerging, and hyphae of the
pathogen had also colonized the root tip (Fig. 1c).
The glucuronidase activity was obvious but localized
at the root tip (Fig. 1c) and some spots in the mature
zone of the root. It affected mainly the hyphae at the
surface of the root, but was seldomly observed in the
superficial layers of the root tissues (Fig. 1d).
At 4 d after inoculation, hyphae formed a
continuous network all over the root surface (Fig. 2a)
and, with the exception of some of their tips,
secondary roots were also colonized. Globally, the
intensity of the blue staining corresponding to the
glucuronidase activity was clearly decreased but still
noticeable preferentially in hyphae localized at the
base of the secondary roots (Fig. 2b) and in some
cases at the apex.
At 7 d after inoculation, the mature part of roots
was still well colonized by the fungus, but the
growing parts of both the secondary and the tap
roots exhibited a less intense colonization. At that
stage, the glucuronidase activity was not detected at
the root surface any more, and at the same time
chlamydospores were observed (Fig. 2c). However,
on thick sections of root, the glucuronidase activity
was very intense in the stele (Fig. 2d).
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499
Observations made 10 and 14 d after inoculation
confirmed this pattern of colonization by the patho-
gen. The vascular colonization of the fungus was
confirmed by microbial isolation made from the
hypocotyl. The few plants left showed typical
symptoms of fusarium wilt 24 d after inoculation.
Penetration into the root
Observations by electron microscopy of ultra-thin
sections revealed attachment of hyphae to the
external wall of epidermal cells in the root-hair zone
as soon as 17 h after inoculation (Fig. 3a). At the
same time, the first images of hyphae penetrating
into epidermal cells and root hairs were observed
(Fig. 3b). There was no specialized penetration
structure, but the penetrating hyphae appeared
constricted and often formed a wall on a level with
the penetration point (Fig. 3b). There was apparently
no direct relationship between the intensity of the
colonization of the root surface and the frequency of
penetrating hyphae.
Colonization of root tissues
Colonization pattern in the differentiated part of the
root. After penetration of the pathogen into an
epidermal cell, the adjacent hypodermal cells reacted
intensively. These ‘ sensitive cells ’ (Beswetherick &
Bishop, 1993) showed an aggregated cytoplasm with
an electron-dense layer attached to the plasmalemma
(Fig. 3c), and formed ‘ buckles ’ with thick walls that
tended to entrap the fungus (Fig. 3d). In a very few
cases, several misshapen hypodermal cells formed a
discontinuous barrier between the epidermis and the
cortex (Fig. 4a). Such images were not observed in
control plants, where slightly misshapen hypodermal
cells never formed thick-walled buckles (Fig. 4b).
Sometimes, another barrier was observed deeper in
the cortex ; this was formed by hypertrophied cells
with thick walls, and the fungus was limited to the
outer part of the cortex where it was observed in
sloughed dead cells (Fig. 5a). These situations of
limited colonization of the root by the pathogen were
still occasionlly observed 10 d after inoculation ; they
were similar to those described when the plants were
colonized by the non-pathogenic strain (Olivain &
Alabouvette, 1997).
In general, the pathogen reached the stele after
invasion of the cortex. Fungal colonization of the
cortex was usually not obvious ; in these cases, the
pathogen was mainly observed in the intercellular
spaces (Fig. 5b) and growing between cells (Fig. 6a).
However, the pathogen could also be present in cells
and a few pictures showed it growing from one cell
to another by digestion of the cell wall (Fig. 6b).
Although difficult to show on transverse sections,
continuity of fungal growth was evident through the
cortex towards the stele where the vessels of the
500 C. Olivain and C. Alabouvette

(a) (b)

(c) (d)

Figure 1. Light microscopy of the whole root, showing colonization of tomato roots by the GUS-marked
Fusarium oxysporum f. sp. lycopersici. (a,b) 24 h after inoculation, showing colonization of the root-hair zone
(a) ; and very few hyphae (arrow) in the elongation zone (b). (c,d) 48 h after inoculation, showing a root tip with
active hyphae and blue-stained, colonized cap cells (arrow) (c) ; and colonization of the elongation zone, blue
staining of hyphae (arrow) at the surface of the root and blue staining of superficial tissues (between
arrowheads) (d). Scale bars represent 40 µm.

xylem were colonized (Fig. 5b). Less frequently, the (Fig. 5c). In the cells, many hyphae appeared as
fungal invasion was massive, and all cortical cell longitudinal sections indicating their centripetal
layers were colonized by numerous hyphae present growth towards the stele. Whatever the intensity of
both inside the cells and in the intercellular spaces fungal colonization of the cortex, many images of

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 Tomato root colonization by a pathogenic Fusarium sp. 501

(a) (b)

(c) (d)

Figure 2. Light microscopy of the whole root, showing colonization of tomato roots by the GUS-marked
Fusarium oxysporum f. sp. lycopersici. (a,b) 4 d after inoculation, showing a continuous network of unstained
hyphae at the root surface (a) ; and hyphae stained blue at the base of a secondary root (arrows) (b). (c,d) 7 d
after inoculation, showing a dense network of unstained hyphae with chlamydospores (arrows) at the root
surface (c) ; and blue staining as a result of glucuronidase activity in the stele of the root (arrow) (d). Scale bars
represent 40 µm (a–c) and 200 µm (d).

defence reactions were observed : osmiophilic globu-
lar deposits attached to hyphae in the intercellular
spaces (Fig. 7a) ; occlusion papillae limiting the
penetration of the fungus into cells (Fig. 7b) ; and
extensive production of electron-opaque material in
cells presenting degenerated hyphae (Fig. 7c). De-
fence reactions were also observed, with xylem
vessels appearing occluded with electron-dense gran-
ular material (Fig. 7d). In the case of massive
invasion, very intense reactions were observed at the
endodermal level (Fig. 5c). The cells appeared
misshapen, distorted and collapsed, creating a physi-
cal barrier difficult for the fungus to cross. This
barrier was not continuous around the stele, but
formed only in contact with infected cortical tissue
(Fig. 5c). The vessels were always colonized, indi-
cating that, in contrast to the non-pathogenic strain,
the pathogen was always able to reach the stele. The

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502 C. Olivain and C. Alabouvette

(a) (b)

(c) (d)

Figure 3. Electron micrographs of transverse ultra-thin sections, showing the colonization of tomato root
tissues by Fusarium oxysporum f. sp. lycopersici. (a) Attachment of a hypha to the wall of an epidermal cell
(arrow), 17 h after inoculation. (b) A hypha penetrating the cell wall of a root hair, 89 h after inoculation. (c)
Colonization of an epidermal cell with an adjacent misshapen hypodermal cell, showing an aggregated
cytoplasm (arrowhead) and an electron-dense layer (arrow). (d) A hypha entrapped in a buckle (arrow) formed
by the thick wall of a hypodermal cell. Abbreviations : EC, epidermal cell ; H, hypha ; HC, hypodermal cell ;
RH, root hair. Scale bars represent 1 µm (a,b,d) and 2 µm (c).

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 Tomato root colonization by a pathogenic Fusarium sp. 503

(a) (b)

Figure 4. Light micrographs of transverse sections showing colonization of tomato root tissues by Fusarium
oxysporum f. sp. lycopersici (toluidine blue O and methylene blue O staining). (a) A barrier (between arrows)
formed by a layer of misshapen hypodermal cells between the epidermis and the cortex. (b) Uninoculated
control plant : the hypodermal cells are slightly misshapen, and are not exhibiting thick-walled buckles.
Abbreviations : C, cortex ; H, hypodermis ; XV, xylem vessel. Scale bars represent 10 µm.

acropetal growth of the pathogen in the vessels of the
roots towards the hypocotyl and the stem of the plant
was indicated by sections showing infected vessels in
the upper part of the root where no colonization was
detected in the cortex.
When the xylem was abundantly colonized, the
hyphae multiplied in the vascular parenchyma
causing cell disorganization and cell wall alteration –
these became less electron dense (Fig. 6c). Cell
disintegration was observed in the stele and in the
cortex, indicating centrifugal, necrotrophic growth
of the pathogen. At the final stage, the whole inner
part of the root appeared decayed, and only the cell
walls of the lignified vessels and of the suberized
hypodermis subsisted (Fig. 8a). Where there was
early, intense colonization, this root decay was
observed just 8 d after inoculation.
Colonization pattern of the apical zone. As already
indicated, there was always intense colonization at
the surface of the root tip (Fig. 1c). Semi-thin
sections showed numerous hyphae around the cap
cells that were being exfoliated (Fig. 8c). Cell
exfoliation, which was much more intense in the
inoculated plants (Fig. 8c) than in the control (Fig.
8d), maintained fungal hyphae at a distance from the
meristematic cells. Defence reactions limited to a
few subapical cells have also seldomly been observed.
In this study, direct penetration of the pathogen in
the meristematic zone has only been observed twice
(Fig. 6d). The necrosis of apical cells was visible
(Fig. 6d) and ultimately led to the total destruction
of the apex by disruption of the cell walls as soon as
89 h after inoculation (Fig. 8b).
In contrast with the non-pathogenic strain, dead
apices have frequently been observed after inocu-
lation with the pathogenic F. oxysporum f. sp.
lycopersici. These dead apices, which appeared either
non-colonized (Fig. 8e) or heavily colonized (Fig. 8f)
by the fungus, were observed on short tap roots,
arrested in their development by an early infection in
the upper part. The invasion of the apex by the
fungus occurred either from the external coloniz-
ation at the root surface or from the internal growth

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504 C. Olivain and C. Alabouvette

(a)

(b)

(c)

Figure 5. Light micrographs of transverse sections showing colonization of tomato root tissues by Fusarium
oxysporum f. sp. lycopersici (toluidine blue O and methylene blue O staining). (a) A barrier (between arrows)
formed in the cortex by a layer of hypertrophied cells with thick walls. (b) Discrete colonization of the cortex
with hyphae (arrowheads) in the intercellular spaces and colonization of the vessels. (c) Intense colonization of
all the cortical cell layers and of the stele in spite of defence reactions at the endodermal level (between arrows).
Abbreviations : C, cortex ; DCC, dead cortical cells ; HCC, hypertrophied cortical cells ; ICC, infected cortical
cells ; XV, xylem vessel. Scale bars represent 40 µm (a) and 10 µm (b,c).

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 Tomato root colonization by a pathogenic Fusarium sp. 505

(a) (b)

(c) (d)

Figure 6. Electron micrographs of transverse ultra-thin sections showing the colonization of tomato root
tissues by Fusarium oxysporum f. sp. lycopersici. (a) A hypha growing between cells. (b) Hyphae growing from
one cell to another (arrowhead) by digestion of the cell wall (arrow). (c) Disorganization of the vascular
parenchyma with cell wall alterations (arrows). (d) Penetration of hyphae and necrosis of a cell in the
meristematic zone. Abbreviations : H, hypha ; XV, xylem vessel. Scale bars represent 2 µm (a,c,d) and 1 µm (b).

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506 C. Olivain and C. Alabouvette

(a) (b)

(c) (d)

Figures 7a–d. Electron micrographs of transverse ultra-thin sections, showing the defence reactions of tomato
against Fusarium oxysporum f. sp. lycopersici.(a) Osmiophilic globular deposit (arrowhead) attached to a hypha
in an intercellular space. (b) An occlusion papilla (arrow) formed around a hypha penetrating a cell wall. (c) A
cell with degenerated hyphae and abundant osmiophilic deposits (arrowheads). (d) Xylem vessel with hyphae
and occluded with electron-dense material. Abbreviations : DH, degenerated hypha ; H, hypha ; IS, intercellular
space ; OXV, occluded xylem vessel ; XV, xylem vessel. Scale bars represent 1 µm (a–c) and 2 µm (d).

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 Tomato root colonization by a pathogenic Fusarium sp. 507

(a) (b)

(c) (d )

(e) (f )

Figure 8. Light micrographs of transverse ultra-thin sections, showing the colonization of tomato root tissues
by Fusarium oxysporum f. sp. lycopersici (toluidine blue O and methylene blue O staining). (a) 8 d after
inoculation, showing decay of the cortex ; only walls of the hypodermal cells (arrowheads) and of the vessels are
conserved. (b) 89 h after inoculation, showing destruction (arrow) of an apex after penetration of the pathogen.
(c) Apical zone of an inoculated plant, showing hyphae (arrowheads) around numerous exfoliated cells (arrows).
(d) Uninoculated control plant with limited exfoliation of cap cells. (e) A dead apex without hyphae. (f) Hyphae
(arrows) both outside and inside a dead apex. Abbreviation : XV, xylem vessel. Scale bars represent 40 µm (a,b)
and 10 µm (c–f).

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508 C. Olivain and C. Alabouvette
of the fungus down from the upper colonized part of
the root.

This study examines the colonization of tomato roots
by a strain of pathogenic F. oxysporum f. sp.
lycopersici (Fol32) for comparison with that pre-
viously described for a non-pathogenic F. oxyporum
(Fo5a4) (Olivain & Alabouvette, 1997). Although
several papers have already been dedicated to the
penetration and colonization processes of tomato
root by strains of F. oxysporum f. sp. lycopersici
(Bishop & Cooper, 1983) and F. oxysporum f. sp.
radicis lycopersici (Charest et al., 1984 ; Brammall &
Higgins, 1988 ; Benhamou et al., 1990a,b), here we
describe the colonization process under the same
experimental conditions as those used for the non-
pathogenic strain in order to compare the two
patterns of colonization.
The pathogenic F. oxysporum f. sp. lycopersici
rapidly colonized the root surface and, 7 d after
inoculation, the first chlamydospores were formed,
indicating a decrease of the fungal activity. The
pattern of root colonization by the pathogen was very
similar to that observed for the non-pathogenic
strain, indicating that both fungi have the same
ability actively to colonize the tomato root surface.
These results are in agreement with those of
Beswetherick & Bishop (1993), who described ran-
dom growth of several fungal species on the tomato
root surface. However, the techniques used in this
study did not allow the quantification of root
colonization by the fungus, and the same pattern of
colonization might result in a different intensity of
colonization. Indeed, Recorbet et al. (1998), using an
indirect technique of isolation, showed that the
intensity of root colonization by this pathogen and
another non-pathogenic strain was similar until the
second day after inoculation ; later, colonization by
the pathogen continued to increase, although it
started to diminish for the non-pathogenic strain.
The blue staining as a result of the glucuronidase
activity was intense 48 h after inoculation. It affected
both the external hyphae and the superficial tissues
of the root, indicating their colonization by the
fungus. In comparison with the non-pathogenic
strain, the extension of the blue patches was more
limited and the blue staining of the root tissues was
detectable for a shorter time (indeed, it was always
lower 4 d after inoculation). This difference in the
persistence of the glucuronidase activity in the
superficial tissues of the root might be explained by
the differential behaviour of the two fungi. Whereas
the colonization of the non-pathogenic strain was
restricted to the superficial layers of cells by the
defence reactions of the plant, the pathogen grew
rapidly, centripetally towards the stele, which ap-
peared intensely blue stained 7 d after inoculation.
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The penetration of the pathogenic F. oxysporum
did not require a specialized structure, and hyphae
penetrating into the root were observed as early as 24
h. These results are in agreement with others already
published and concerning several formae speciales of
F. oxysporum (Bishop & Cooper, 1983 ; Smith &
Peterson, 1983 ; Bhalla et al., 1992). The number of
penetrating hyphae was very limited in comparison
with the intensity of the colonization of the root
surface – this was in contrast to the observations
reported by Rodriguez-Galvez & Mendgen (1995),
who used pieces of colonized agar as inoculum. The
pathogenic and non-pathogenic F. oxysporum were
able to penetrate the root into the well differentiated
and apical zones. The penetration process was
similar for both the pathogen and the non-pathogenic
strain. Therefore, preferential sites of infection did
not exist, and competition between the two types
occurred at the root surface, but not for specific sites
of penetration.
The penetration of the pathogen into the root, at a
level where the tissues are differentiated, did gen-
erally result in a vascular infection. However,
sometimes the colonization was limited by intense
defence reactions occurring in the superficial cell
layers, mainly at the hypodermis level. As soon as the
hypodermis was invaded, the pathogen grew rapidly
into the cortex towards the stele and induced a
vascular infection. The defence reactions at the
hypodermis level were similar to those observed with
the non-pathogenic strain ; however, in response to
the colonization by the non-pathogenic strain, the
barrier was formed more rapidly and the defence
reactions were more intense than with the pathogen.
The barrier was generally effective in limiting the
extension of the non-pathogenic strain, which was
restricted to the superficial layers of cells. This
interpretation of our observations is in agreement
with that of Brammall & Higgins (1988), who suggest
that the hypodermis might be an important barrier
to colonization of the inner cortex of tomato roots by
F. oxysporum f. sp. radicis lycopersici. On the
contrary, the pathogen passed around this barrier
and grew rapidly, intercellularly towards the stele.
Although mostly intercellular, the pathogen has also
been observed in the cells of the cortex, where it was
able to dissolve the cell walls and to progress from
cell to cell. The same but less intense and numerous
defence reactions as described for the non-patho-
genic strain have been observed in the root invaded
by the pathogen : wall appositions and thickenings ;
intracellular and intercellular deposits attached on
hyphae ; and accumulation of granular plugs in the
vessels. These defence reactions have been observed
in other plant species infected by other formae
speciales of F. oxysporum (Charest et al., 1984 ;
Jordan et al., 1988 ; Tessier et al., 1990 ; Benhamou
et al., 1990a). Based on our observations with F.
oxysporum f. sp. lycopersici, it seems that the decisive
 Tomato root colonization by a pathogenic Fusarium sp.
phenomenon for the success or failure of a vascular
infection is the kinetics at which these defence
reactions create barriers that are efficient or not in
preventing the fungus from reaching the stele.
At the apex of the root, hyphae formed a dense
network, but contact between hyphae and living cells
was rare. Indeed, before reaching apical cells, the
fungus had to cross several layers of exfoliated cap
cells. Comparison with the healthy control showed
that there was more exfoliation in the presence of the
fungus : it is possible that exfoliation constitutes a
defence mechanism. Vascular infection after pen-
etration through these undifferentiated cells at the
apex of the root is possible, but has rarely been
observed under our experimental conditions. This
apical infection led to a rapid destruction of the apex,
with many hyhae in disorganized cells. Although
typical defence reactions have been observed, they
did not limit the growth of the fungus as observed in
plants inoculated with the non-pathogenic strains.
The pathogen colonized up the vascular bundles
from these heavily infected apices.
This study demonstrates that the pathogen F.
oxysporum f. sp. lycopersici is able to perform a
vascular infection of tomato after penetration
through either the differentiated tissues of the
mature root or the young tissues of the apical zone.
By contrast with the results obtained with flax and its
pathogen F. oxysporum f. sp. lini (Turlier et al.,
1994), penetration into the meristematic zone did
not result in a biotrophic colonization of the cells but
led to a rapid destruction of the apex. However,
these results do not totally exclude the possible
existence of a biotrophic colonization phase for a
very short time. Indeed, Rodrigez-Galvez &
Mendgen (1995) described a biotrophic phase for F.
oxysporum f. sp. vasinfectum in the apex of cotton
root, but their observations were made only 24 h
after inoculation. In this study, the intercellular
growth of the pathogen towards the stele, when it did
not induce intense colonization of the cortex, could
be compared with a biotrophic phase described by
others. In contrast to this early phase of root
infection, the pathogen behaved as a necrotrophic
fungus when the root became senescent after total
invasion of the vessels. Images showing the inner
cortex invaded by hyphae coming from the vascular
parenchyma have been observed.
Our observations show that the patterns of tomato
root colonization by a pathogenic and a non-
pathogenic strain of F. oxysporum are quite similar,
especially the root-surface colonization and the
penetration into the epidermis. The main detectable
differences concern the intensity of the plant defence
reactions, and the rapidity at which they are formed.
In the case of the non-pathogenic strain, the barrier
raised in the hypodermis was generally efficient in
limiting the colonization of the root. On the contrary,
in the case of the pathogen, this barrier seemed
Printed from the C JO service for personal use only by...
509
ineffective, because it was discontinuous and formed
too late. The same type of reactions were observed in
the endodermis : the barrier was effective in pre-
venting the colonization of the stele by the non-
pathogenic form, although the pathogen was able to
pass around this discontinuous barrier. Thus, the
differences appear to be of a quantitative rather than
of a qualitative nature.
This study does not show whether the penetration
of a pathogenic F. oxysporum occurs preferentially
through the apical or the mature root zone. Indeed,
under these experimental conditions both means of
penetration have been detected, the second being
more frequent. This leads to the conclusion that
both possibilities exist, and that depending on the
strain studied, on the forma specialis and on the
experimental conditions, one or the other possibility
is predominant. This might explain why the research
published to date is so confusing (Alconero, 1968 ;
Smith & Peterson, 1983 ; Jordan et al., 1988 ;
Farquhar & Peterson, 1989 ; Bhalla et al., 1992).
Finally, this study supports the hypothesis that the
pathogenic and the non-pathogenic F. oxysporum
might compete for colonization of the root surface
and the root tissues. They can be present everywhere
at the root surface, but both are more abundant
where root exudation is important, indicating po-
tential competition for nutrients. However, as there
are no specific penetration sites, and as the surface
colonization by the non-pathogenic strain is never
totally complete, the pathogen always has a small
chance of finding a spot where it can penetrate the
root. Biological control provided by the non-
pathogenic strain needs a large amount of inoculum
and never achieves full protection (Alabouvette et
al., 1993).
              
This study has been partly supported by a grant from
Conseil Re! gional de Bourgogne, France. The observations
using electron microscopy were made at the Centre de
Microscopie Applique! e a' la Biologie, Universite! de
Bourgogne, France. We thank Paul Bremeersch for
technical help and Josette Relot for photographic work.

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