Bio-Fungicides as an Alternative for Tomato Fusarium Crown

and Root Rot Control

Khaled Hibar, Laboratoire de Phytopathologie, ESHE Chott-Mariem, 4042 Sousse,
Tunisia, Mejda Daami-Remadi, INRAT/PRRDA-CE Chott-Mariem, 4042 Sousse,
Tunisia, Walid Hamada, INAT, 43 Avenue Charles Nicolle, 1082 Tunis, Tunisia,
and Mohamed El-Mahjoub, ESHE Chott-Mariem, 4042 Sousse, Tunisia

ABSTRACT
Hibar, K., Daami-Remadi, M., Hamada W., and El-Mahjoub M. 2006. Bio-fungicides as an
alternative for tomato Fusarium crown and root rot control. Tunisian Journal of Plant Protection
1: 19-29.
Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f.
sp. radicis-lycopersici (FORL) is a recent damaging disease of greenhouse crops in Tunisia. No or
some effective disease control methods are available. Therefore, alternative measures for disease
management are urgently required. In this study, the efficacy of some bio-fungicides to suppress FORL
was evaluated in vitro, in growth chamber as well as under greenhouse conditions. In in vitro tests, all
bio-fungicides inhibited mycelial growth of FORL at 50 to 73%. Under growth chamber trials, the
efficacy of all bio-fungicides was more significant when they were added to the substrate one week
before inoculation with the pathogen. Moreover, simultaneous addition of bio-fungicides and pathogen
spores to tomato plants has significantly reduced disease incidence. Under greenhouse conditions,
results were more encouraging. Indeed, the use of RootShield Drench reduces the percentage of dead
plants at 5.5%. Furthermore, tomato plants treated with this bio-fungicide produced more and have had
best fruits compared to those treated with fungicide, Hymexazol. This study demonstrated the efficacy
of some bio-fungicides in controlling FORL especially when they were applied early before pathogen
attack inoculation.
Keywords: Bio-fungicides, disease incidence, Bacillus spp., Trichoderma harzianum, Pythium
oligandrum

resistance have been developed, control

of FCRR is mainly restricted to
eliminating the pathogen in soil by
steaming or fumigating with chemicals
and by planting pathogen-free transplants
(39). However, complete eradication of
the fungus from soil has never been
achieved, due in part to the appearance of
fungicide- resistant strains in the
pathogen populations. The difficulties in
controlling FCRR have promoted
scientists to search for biological
alternatives that are efficient, reliable, and
safe for environment (7, 38, 39). Several
biocontrol strategies have been proposed
for controlling root pathogens, but
practical applications are still limited,

Fusarium crown and root rot of tomato

(FCRR) induced by Fusarium oxysporum
Schlect f. sp. radicis-lycopersici Jarvis
and Shomaker (FORL) is one of the most
damaging soil-borne diseases of tomato
causing heavy economic losses on plant
grown in sterilized soils (35). This disease
newly recorded in Tunisia, during 20002001 crop season (16, 19), caused heavy
losses reaching 90% of plants in some
geothermal greenhouses. Although some
cultivars with single dominant genes for
Corresponding author: K Hibar
Khaled_htn@yahoo.fr
Accepted for publication 23 January 2006.

Tunisian Journal of Plant Protection

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Vol. 1, No. 1, 2006

Table 1. Fusarium oxysporum f. sp. radicislycopersici isolates used in this study

Isolates
Host
plant Date
of
(Cultivar)
isolation

largely because of the lack of unequivocal

answers to key questions, including the
relationship the biocontrol agent may
establish with the plant and the exact
mechanisms by which it may directly
influence the pathogen or indirectly
influence the plant by inducing metabolic
changes (18).
Among the antagonists that show
satisfactory degrees of FORL control,
Trichoderma spp. (22, 39), Pseudomonas
spp. (5, 10, 14) and Bacillus spp. (24)
have been reported to reduce disease
incidence by inhibiting pathogen growth
and development in the rhizosphere.
In an attempt to validate this
assumption, the efficacy of some biofungicides for controlling FCRR was
investigated in vitro, in growth chamber
and under greenhouse conditions.
In this study we reported the effect of
some bio-fungicides on mycelial growth
and on disease incidence under growth
chamber and greenhouse conditions.

Fo2.01
Fo4.02
Fo1.03
Fo1.04

2001
2002
2003
2004

Plant material. Tomato seed

(Lycopersicon esculentum Mill. cv.
Riogrande, susceptible to FORL) were
sterilized by immersion in absolute
ethanol for 7 min, followed by extensive
rinsing in sterile distilled water (3). Seeds
were sown in alveolus plates filled with
previously sterilized peat. Seedlings were
grown in a growth chamber at 24 to 26 C
with 12-h photoperiod and 70% humidity.
They were watered daily and fertilized
twice a week with a standard nutrient
solution according to Pharand et al. (31).
Experiments were performed with 5week-old tomato plants carrying five or
six fully expanded leaves (2).
Bio-fungicides. Various fungi and
bacteria with known biocontrol activity
against soil-borne fungal pathogens
including
biocontrol
isolates
of
Trichoderma
harzianum,
Pythium
oligandrum, Bacillus subtilis, B. pumilus
and others were used in this study. T.
harzianun strain T22 (RootShield Drench,
BioWorks Inc. Geneva, NY), T.
harzianun (Biocont-T WP and Biocont-T
Gr, National Ammonia & Chemical
Industries, Amman), Bacillus subtilis
strain QST 713 (Serenade, AgraQuest,
Davis, CA), B. pumilus strain QST 2808
(Sonata, AgraQuest, Davis, CA), Pythium
oligandrrum (Polyversum, Biopreparty
Ltd., Czech Republic) and naturally
occurring micro-organisms (Agralan
Revive, Agralan Ltd., Swindon, UK)
were evaluated for their effects on
disease development caused by FORL.
Peat and perlite were used as the
transplanting medium in growth chamber
and greenhouse experiments respectively.
All bio-fungicides were applied at
recommended label rates as drenches:

MATERIALS AND METHODS

Fungal isolates. Four FORL isolates
were used in this study. They were
recovered from greenhouses tomato
plants showing typical crown and root rot
symptoms at 5th season exploitation in
Hammet Gabs in South Tunisia where
tomato culture heated with geothermal
water is practiced.
Fungal pathogen was isolated by
planting plant tissues (surface-disinfected
with 1% sodium hypochlorite for 2 min)
on PDA (Potato Dextrose Agar) and
incubating them at 25C for 5 days (23).
Isolates were identified as F. oxysporum
morphologically based on characteristics
of
the
macroconidia,
phialids,
microconidia,
chlamydospores,
and
colony growth traits (29). The forma
specialis of this pathogen was identified
using pathogenicity tests (19). Based on
pathogenicity tests, the more aggressive
isolates were selected for this study. The
four isolates used in in vitro and in vivo
tests are presented in table 1.
Tunisian Journal of Plant Protection

Durintha
Bochra
Sarnia
Olivette

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Vol. 1, No. 1, 2006

week-old tomato plants carrying five or

six fully expanded leaves (2). Plants were
carefully removed from alveolus plates
and transplanted into pots (12 cm
diameter) filled with previously sterilized
peat.
After the pathogen was cultured in the
PDB (Potato Dextrose Broth), a spore
suspension was obtained. The cultured
liquid medium was filtered and a
concentration of 107 spores/ml was
determined using a Malassez Blade. After
one week, plants with six to seven leaves
were inoculated with 10 ml of the spore
suspension of the pathogen applied as a
drench. The control plants were similarly
treated with sterile distilled water.
To test preventive and curative
effects of the bio-fungicides tested, three
experiments were performed: (i) biofungicides were added to the media (peat)
one week before the pathogen spores
were added; (ii) bio-fungicides and the
pathogen spores were applied at the same
time; and (iii) bio-fungicides were added
to the media one week after adding the
pathogen spores.
The disease severity was recorded on
0 to 3 visual scale, in which 0 = no
symptoms; 1 = light yellowing of leaves,
light or moderate rot on taproot and
secondary roots and crown rot; 2 =
moderate or severe yellowing of leaves
with or without wilting, stunting, severe
rot on taproot and secondary roots, crown
rot with or without hypocotyls rot, and
vascular discoloration in the stem; and 3
= dead seedlings (41).
Disease incidence percentage was
determined using the following formula
(40):

RootShield (0.9 g/liter) at 50 ml per plant,

Biocont-T WP (1 g/liter) at 50 ml per
plant, Biocont-T Gr (500 ml/m3) at 50 ml
per plant, Serenade (1ml/liter) at 50 ml
per plant, Sonata (1ml/liter) at 50 ml per
plant, Polyversum (1 g/liter) at 50 ml per
plant and Agralan Revive (1ml/liter) at 50
ml per plant.
Effect of bio-fungicides on mycelial
growth of FORL. The antagonistic
activities of the seven bio-fungicides on
mycelial radial growth of the pathogen
were determined by growing the fungus
on a PDA containing the different biofungicides in Petri plates (85-mm
diameter). Each bio-fungicide was added
at recommended label rates to 100 ml
sterilized PDA media at 60C, and then
poured equally into five Petri plates.
Control plates were made by replacing
the quantity of bio-fungicides with the
same quantity of sterile distilled water. A
disc (6mm diameter) of 6-day-old
pathogen mycelial culture was aseptically
transferred to the center of the solidified
PDA media in plates. The plates were
subsequently incubated for 6 days at 25C
(19).
Mycelial growth of the pathogen was
measured on each plate, and the growth in
PDA containing bio-fungicides was
compared with the growth of the
pathogen in plates containing water
(control). The experiment was replicated
three times for each treatment and the
mean values taken.
Effect of bio-fungicides on mycelial
growth of FORL was recorded in terms of
percentage
colony
inhibition
and
calculated according to Hmouni et al.
(21). Percentage growth inhibition was
determined as (1- Cn/Co) 100, where
Cn is the average diameter increase of
fungal colony with treatment, and Co is
the average diameter increase of fungal
colony with control.
Effect of bio-fungicides on disease
incidence. The effect of the seven biofungicides on FCRR disease incidence
was evaluated under growth chamber.
Experiments were performed with 5Tunisian Journal of Plant Protection

(scale number of plants infected)

Disease Ten
incidenceplants
(%) = per elementary treatment 100
(highest scale total number of plants)

Ten plants per elementary treatment

were used and variance analysis of the
treatment effect on measured data was
performed by using the general linear
21

Vol. 1, No. 1, 2006

Inhibition percentage (%)

model procedure of SPSS (SPSS 10.0).

Experiments were analyzed using
standard analysis of variance (ANOVA)
with factorial treatment structure and
interactions. When F values were
significant at p>0.05, differences among
the treatments were determined by S-N-K
(Student-Newman-Keuls) test.
Greenhouse experiment. Greenhouse experiment for controlling FCRR
was performed using only one product
(RootShield Drench).
The greenhouse experiment was
carried out in 2003-04 crop season at the
5th Season exploitation, situated in
Hammet Gabs in Southern Tunisia.
RootShield Drench was applied twice
before transplanting in the breedingground to tomato plants cv. Bochra.
These latest, with 3-4 true leaves, were
transferred from breeding-ground to the
greenhouse, heated with geothermal
water. Soilless culture was adopted in
sausage bags filled with perlite infested
with FORL and using a drip irrigation
system. In the greenhouse, the biofungicide was applied once a month from
the date of transplantation (1st September
2003) to the end of the crop season (30
May 2004). This experiment was
performed with a total number of 18000
plants and the number of dead plants
along the crop season was recorded.
Notes concerning fruit yield and fruit
size were taken and compared to those
obtained with the same number of plants
treated with fungicide, Hymexazol.

Serenade

70

Sonata
Polyversum

60

Biocint-T W-P

50

Biocont-T Gr

40

Agralan Revive

30

RootShield

20
10
0
FO2-01

FO4-02

FO1-03

FO1-04

F.oxysporum f. sp.radicis lycopersici isolates

Fig. 1. Effect of various bio-fungicides on mycelial

growth inhibition of F. oxysporum f. sp. radicislycopersici after an incubation period of 6 days at
25C.

than 73%. With the exception of Sonata

which entailed the lowest growth
inhibition (about 50%), the other products
have significantly inhibited mycelial
growth of FORL and the growth
reduction recorded was more than 60%.
Effect of bio-fungicides on disease
incidence under growth chamber
experiments. By applying bio-fungicides
one week before inoculation with the
pathogen, disease incidence was low for
all bio-fungicides treatments, and
statistical analysis did not separate out
any biological treatment from healthy
plants (control). Results obtained show
that disease incidence has never exceeded
13.3% and it reached 3% with the biofungicides Serenade and Agralan Revive
(Table 2). The other bio-fungicides have
also significantly reduced disease
incidence when compared with control.
Moreover, tomato plants treated one week
before inoculation showed healthy
appearance and an optimal vegetative
growth compared to uninoculated plants.
For example, Fig. 2 illustrates the
comparison between a plant treated with
Serenade and an uninoculated control
plant.
Applying bio-fungicides one week
before inoculation has also a beneficial
effect on root system development
compared to untreated plants (Fig. 3).

RESULTS
In vitro inhibition of FORL by biofungicides. Adding bio-fungicides to the
PDA media has inhibited mycelial growth
of FORL isolates. Results obtained (Fig.
1) show that all bio-fungicides have
inhibited the development of FORL by
more than 50% and the highest values
were obtained with the two bio-fungicides
(Serenade and RootShield). Indeed, the
use of these two bio-fungicides halted
mycelial growth of FORL by more
Tunisian Journal of Plant Protection

80

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Vol. 1, No. 1, 2006

Fig. 2. Comparison between an uninoculated control

plant (A) and a plant treated with Serenade one
week before its inoculation with F. oxysporum f. sp.
radicis-lycopersici (B), one month after inoculation
at 25C.

Fig. 3. Comparison between the root system of a plant

treated with Serenade one week before inoculation with
F. oxysporum f. sp. radicis-lycopersici (A) and the root
system of an
uninoculated control plant (B), one
month after inoculation at 25C.

Table 2. Disease incidence when tomato plants were treated with bio-fungicides one week before the Fusarium
oxysporum f. sp. radicis-lycopersici spores were inoculated to plants
Disease incidence (%)
FO2-01z

FO4-02z

FO1-03z

FO1-04z

Serenade

6,67 a

3,33 a

6,67 a

3,33 a

Sonata

10 a

6,67 a

10 a

6,67 a

Polyversum

6,67 a

13,33 a

10 a

10 a

Biocont-T W-P

6,67 a

13,33 a

13,33 a

6,67 a

Biont-T Gr

13,33 a

6,67 a

10 a

10 a

Agralan Revive

6,67 a

3,33 a

6,67 a

10 a

RootShield

6,67 a

6,67 a

10 a

6,67 a

83,3 b

90 b

93,33 b

0a

0 a

0a

Inoculated and untreated control 80 b

Uninoculated control
0a
z

Within columns, means followed by the same letters are not significantly different (P=0.05) according to S.N.K.
test.

with Polyversum, Biocont-T W-P, BiontT Gr, Agralan Revive and RootShield
were
statistically
classified
with
uninoculated plants when they were
inoculated with three FORL isolates
(FO2-01, FO4-02, FO1-03). Also in this
experiment, disease incidence did not
exceed 40% and it ranged from 13.3% for
Polyversum, Biocont-T W-P and Agralan
Revive with FO2-01and FO4-02 isolates
to 40% for Biocont-T W-P with FO1-03
isolate, (Table 4).

Treating plants one week after the

inoculation with the pathogen did not
significantly reduce disease incidence
compared to untreated plants. Results
obtained showed that with all biofungicides, disease incidence was always
more than 50% and it reached 66.67%
with the bio-fungicide Sonata (Table 3).
Simultaneous application of biofungicides and pathogen spores to tomato
plants has significantly reduced disease
incidence. Indeed, tomato plants treated
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Vol. 1, No. 1, 2006

Table 3. Disease incidence when tomato plants were treated with bio-fungicides one week after the Fusarium
oxysporum f. sp. radicis-lycopersici spores were inoculated to plants
Disease incidence (%)
Serenade

FO2-01z
60 b

FO4-02z
60 bc

FO1-03z
60 b

FO1-04z
53,33 b

Sonata

63,33 b

60 bc

66,67 b

63,33 b

Polyversum

56,67 b

53,33 b

46,67 b

60 b

Biocont-T W-P

50b

60 bc

53,33 b

60 b

Biont-T Gr

53,33 b

56,67 b

56,67 b

63,33 b

Agralan Revive

53,33 b

63,33 bc

60 b

63,33 b

RootShield

56,67 b

63,33 bc

56,67 b

56,67 b

Inoculated and untreated plants

80 c

83,33 c

90 c

93,333 c

0a
0a
0a
0a
Healthy plants
z
Within columns, means followed by the same letters are not significantly different (P=0.05) according to S.N.K.
test.
Table 4. Disease incidence when bio-fungicides treatment and Fusarium oxysporum f. sp. radicis-lycopersici spore
inoculation to tomato plants were performed at the same time
Disease incidence (%)
Serenade

FO2-01z
26,67 b

FO4-02z
26,67 b

FO1-03z
33,33 b

FO1-04z
36,67 b

Sonata

30 b

36,67 b

36,67 b

36,67 b

Polyversum

16,67 ab

13,33 ab

16,67 ab

20 b

Biocont-T W-P

20 ab

13,33 ab

20 ab

40 b

Biont-T Gr

20 ab

23,33 ab

20 ab

36,67 b

Agralan Revive

13,33 ab

16,67 ab

20 ab

23,33 b

RootShield

23,33 ab

26,67 b

20 ab

26,67 b

Inoculated and untreated plants

80 c

83,33 c

90 c

93,33 c

0a
0a
0a
0a
Healthy plants
z
Within columns, means followed by the same letters are not significantly different (P=0.05) according to S.N.K.
test.

controlling Fusarium crown and root rot of

tomato (19). This surplus in fruit quantity
compensated and even exceeded the loss
caused by dead plants which was about
11.88 tons (an average of 12kg/plant).
Also, plants treated with the bio-fungicide
presented a good quality and fruit size
compared
to
those
treated
with
Hymexazol.

Control of Fusarium crown and root

rot under greenhouse conditions.
Based on its efficacy in vitro and in vivo
and on its availability, only one biofungicide (RootShield Drench) was used to
control
FORL
under
greenhouse
conditions.
From 1st September 2003 to 30 May
2004, only 990 plants were dead on a total
number of 18000 plants representing 5.5%.
Moreover, plants treated with this biofungicide showed better vegetative growth
and produced more than tomato plants
treated with Hymexazol (Fig. 4).
Plants treated
with RootShield
produced 18 tons (an average of 1kg/plant)
more than the same number of plants
treated with Hymexazol, also effective in
Tunisian Journal of Plant Protection

DISCUSSION
Because high disease pressure and high
crop value require frequent applications of
chemical
pesticides,
significant
environmental pollution and selection of
resistant pathogen strains are among the
main
problems
encountered
in
greenhouses.
24

Vol. 1, No. 1, 2006

Fig. 4. Comparison between plants treated with fungicide Hymexazol (A) or bio-fungicide RootShield Drench (B)
under greenhouse conditions: plants treated with Hymexazol exhibit symptoms of yellowing and wilting (arrows)
whereas plants treated with RootShield Drench exhibit a good vegetative growth without yellowing and wilting
symptoms.

that, in dual culture, a strain of T.

harzianum was able to produce chitinases
and inhibit growth of FORL. Mycelial
growth of this pathogen was also inhibited
by using a local strain of T. harzianum
which limited FORL development by more
than 65% (20). The same strain of T.
harzianum was found to reduce mycelial
growth of Pythium spp. and Fusarium spp.
isolates causing potato tuber rot in Tunisia
(12, 13).
In vivo application of these biofungicides,
has
limited
FCRR
development. The disease incidence
reduction was greater when bio-fungicides
were added one week before inoculation
with the pathogen. By treating plants one
week before inoculation, disease incidence
did not exceed 10% compared to untreated
plants for which disease incidence ranged
from 80 to 93%. In this case, antagonists
may induce systemic resistance against
FORL; indeed, several studies demonstrate
that T. harzianum (1, 42), Pythium
oligandrum (3), B. subtilis (33), B. pumilus
(25) and soil microorganisms (27) earlier
applied to roots can effectively protect
plants against soilborne pathogens.
Moreover, application of bio-fungicides

This situation has prompted the search

for biological alternatives that could be
efficient either for conventional disease
management programs or for integration
with other methods (4).
Various bio-fungicides were tested
for biological control of FCRR. Data
obtained in this study demonstrated that
bio-fungicides
Serenade,
Sonata,
Polyversum, Biocont-T W-P, Biont-T Gr,
Agralan Revive and RootShield Drench
were consistently effective in reducing
mycelial growth of FORL. Indeed,
addition of these bio-fungicides to the
culture media has inhibited mycelial
growth by more than 70%.
Similar studies have been conducted
for other pathogens using the same
antagonists to try to demonstrate if these
ones can inhibit mycelial growth of some
pathogens. For example Cavaglieri et al.
(8) found that 10 Bacillus strains have
significantly
inhibited
Fusarium
verticillioides growth and the greatest
antifungal activity was obtained with B.
subtilis strain CE1.
Inhibitory activity of T. harzianum has
been reported in several studies; for
example Chrif and Benhamou (9) found
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Vol. 1, No. 1, 2006

Application of bacteria to seeds has

been used for biological control of soilborne plant pathogens affecting many host
plants (17).
In our study we have used two bacteria
(B. subtilis and B. pumilus) to control
FCRR. Both antagonists tested have
significantly reduced disease incidence
caused by FORL. These results are
relatively consistent with several studies
showing the effect of these bacteria in
reducing disease severity throughout the
experiments. For example Collins and
Jacobsen (11) found that an application of
the biocontrol agent B. subtilis isolate,
BacB, 3 days before inoculation has
provided significantly better control of
sugar beet Cercospora leaf spot, caused by
Cercospora beticola. Cavaglieri et al. (8)
also reported that B. subtilis CE1 at 108
and 107 CFU ml-1 inocula was able to
reduce rhizoplane and endorhizosphere
colonization of F. verticillioides, one of
the most commonly reported soil-borne
fungal
pathogens
infecting
maize.
Similarly, Kilian et al. (24) found that this
antagonist formed a 0.4-0.8mm thick film
around roots of tomato plants when seeds
were previously treated with FZB24 B.
subtilis. The efficacy of B. subtilis in
controlling soil-borne pathogen was
reported by Estevez de Jensen et al. (15)
who found that this antagonist strain
GBO3 has limited the development of
bean root rot caused by F. solani f. sp.
phaseoli.
B. pumilis was also reported to be
efficient in controlling various pathogens.
For example, Bottone and Peluso (2003)
found that B. pumilus (MSH) is able to
produce an antifungal compound that is
active against Mucoraceae and Aspergillus
species. Similarly, Reynaldi et al. (36)
reported that B. pumilus (m435) has
inhibited the growth of the fungus
Ascosphaera apis, the causal organism of
chalkbrood disease in honeybee larvae. In
the same way, Raupach and Kloepper (32)
demonstrated that B. pumilus strain INR7,
used alone or in a mixture with
Curtobacterium flaccumfaciens strain

immediately prior to the pathogen

penetration can also significantly reduce
disease incidence. In fact, disease
incidence for treated plants ranged from 13
to 40% and it was about 90% for untreated
plants.
More promising results were obtained
under greenhouse conditions by using
RootShield Drench to control FCRR. By
treating tomato plants twice in the seed
bed before transplanting and once a month
during the crop season, percentage of
completely wilted plants was about 5.5%.
Moreover, plants treated with RootShield
Drench produced more and showed best
fruit size compared to those treated with
Hymexazol. Larkin and Fravel (26) also
reported that RootShield Drench applied at
0.2% has significantly reduced the
development of Fusarium wilt of tomato;
however, when applied at 0.05 or 0.1%
this bio-fungicide did not reduce pathogen
development. The use of this bio-fungicide
(RootShield) in soil with asparagus
seedlings has increased root weight and
decreased root disease compared with the
infested control when a low level of F.
proliferatum and F. oxysporum f. sp.
asparagi was used (34). However, used
with cucumber seedlings, RootShield
Drench did not provide significant control
of Fusarium root and stem rot on
cucumber (37). Several other studies
demonstrate the efficacy of biocontrol
agents in reducing disease severity. In fact,
Marois et al. (28) reported the successful
biological control of FCRR with conidial
suspension consisting of three isolates of
T. harzianum, one of Aspergillus
ochraceus and one of Penicillium
fumiculosum in infested soil with the
pathogen at low concentration.
The present study demonstrates that a
single bio-fungicide alone significantly
reduced disease severity. Similar results
have been reported by Muslim et al. (30)
showing that biological control agent,
hypovirulent binucleate Rhizoctonia, alone
has significantly reduced disease severity
of FCRR even under high pathogen
inoculum pressure.
Tunisian Journal of Plant Protection

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ME1 and B. subtilis strain GBO3, showed

a higher level of protection against
Pseudomonas syringae pv. lachrymans the
causal organism of angular leaf spot
disease in cucumber.
The efficacy of biocontrol agents, in
particular T. harzianum (RootShield
Drench) against FCRR grown under
growth
chamber
and
greenhouse

conditions was well illustrated in this

study. However, to be more affirmative,
the other bio-fungicides (Serenade, Sonata,
Polyversum, Biocont-T W-P, Biont-T Gr
and Agralan Revive) must be tested under
greenhouse and field conditions to be used
as alternatives to control FCRR and other
soil-borne pathogens.

RESUME
Hibar K., Daami-Remadi M., Hamada W. et El-Mahjoub M. 2006. Les bio-fungicides comme une
alternative dans la lutte contre la pourriture Fusarium des racines et du collet de la tomate.
Tunisian Journal of Plant Protection 1: 19-29.
La fusariose des racines et du collet de la tomate (Lycopersicon esculentum) cause par Fusarium
oxysporum f. sp. radicis-lycopersici (FORL) est une grave maladie rcemment signale dans les serres
gothermales du sud tunisien. Labsence de mthodes de lutte efficaces contre ce pathogne nous a
pouss chercher dautres alternatives de lutte pour contrler cette maladie. Dans cette tude, l'efficacit
de quelques bio-fongicides a t value in vitro, dans une chambre de culture et sous serres chauffes
par les eaux gothermales. Dans les essais le lutte in vitro, tous les bio-fongicides ont limit la croissance
myclienne de FORL et le pourcentage dinhibition tait compris entre 50 et 73%. Test in vivo,
l'efficacit de tous les bio-fongicides tait plus significative surtout quand ils ont t ajouts aux substrats
de culture une semaine avant inoculation. De plus, l'addition simultane de ces bio-fongicides et du
pathogne aux plants de tomate a significativement rduit l'incidence de cette maladie. Sous serres, les
rsultats obtenus taient plus encourageants. En effet, par l'utilisation de RootShield Drench le
pourcentage de plants fltris tait seulement de 5.5%. De plus, les plants de tomate traits par ce biofongicide ont eu une production et une qualit de fruits meilleures compare au fongicide, lHymexazol.
Cette tude a dmontr l'efficacit de quelques bio-fongicides dans la lutte contre FORL particulirement
quand il ont t appliqus tt avant inoculation.
Mots cls : Bio-fungicides, incidence de la maladie, Bacillus spp., Trichoderma harzianum, Pythium
oligandrum


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Tunisian Journal of Plant Protection

27

Vol. 1, No. 1, 2006

LITERATURE CITED
1. Benhamou, N. and Chet, I. 1996. Parasitism of
sclerotia of Sclerotium rolfsii by Trichoderma
harzianum: Ultrastructural and cytochemical
aspects of the interaction. Phytopathology 86:405416.
2. Benhamou, N. and Blanger, R. 1998.
Benzothiadiazole-Mediated induced resistance to
Fusarium oxysporum f. sp. radicis-lycopersici in
tomato. Plant physiology 118:1203-1212.
3. Benhamou, N., Rey, P., Cherif, M., Hockenhull, J.,
and Tirilly, Y. 1997. Treatment with the
mycoparasite Pythium oligandrum triggers
induction of defence-related reactions in tomato
roots when challenged with Fusarium oxysporum
f. sp. radicis-lycopersici. Phytopathology 87:108121.
4. Benhamou, N., Gagn, S., Le Qur, D., and
Dehbi, L. 2000. Bacterial-mediated induced
resistance in cucumber: Beneficial effect of the
endophytic bacterium Serratia plymuthica on the
protection against infection by Pythium ultimum.
Phytopathology 90:45-56.
5. Bolwerk, A., Lagopodi, A. L., Wijfjes, A. H. M.,
Lamers, G. E. M., Chin-A-Woeng, T. F. C.,
Lugtenberg B. J. J., and Bloemberg, G. V. 2003.
Interaction in the tomato rhizosphere of two
Pseudomonas biocontrol strains with the
phytopathogenic fungus Fusarium oxysporum f.
sp. radicis-lycopersici. Molecular Plant Microbe
Interaction 16:983-993.
6. Bottone, E. J. and Peluso R. W. 2003. Production
by Bacillus pumilus (MSH) of an antifungal
compound that is active against Mucoraceae and
Aspergillus species: preliminary report. Journal of
Medical Microbiology 52:69-74.
7. Caron, M., Fortin, J. A., and Richard, C. 1986.
Effect of phosphorus concentration and Glomus
intraradices on Fusarium crown and root rot of
tomatoes. Phytopathology 76:942-946.
8. Cavaglieri, L., Orlando, J., Rodriguez, M. I.,
Chulze, S., and Etcheverry, M. 2005. Biocontrol of
Bacillus subtilis against Fusarium verticillioides in
vitro and at the maize root level. Research in
Microbiology 156:748-754.
9. Chrif, M. and 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.
10. Chin-A-Woeng, T. F. C., Bloemberg, G. V., van
der Bij, A. J., van der Drift, K. M. G. M.,
Schripsema, J., Kroon, B., Scheffer, R. J., Keel, C.,
Bakker, P. A. H. M., Tichy, H. V., de Bruijn, F. J.,
Thomas-Oates, J. E., and Lugtenberg, B. J. J.
1998. Biocontol by phenazine-1-carbox-amideproducing Pseudomonas chlororaphis PCL139 of
tomato root rot caused by Fusarium oxysporum f.
sp. radicis-lycopersici. Molecular Plant Microbe
Interaction 11:1069-1077.
11. Collins, D. P. and Jacobsen, B. 2002. Optimizing
a Bacillus subtilis isolate for biological control of

Tunisian Journal of Plant Protection

sugar beet cercospora leaf spot. Biological Control

26:153-161.
12. Daami-Remadi, M. 2001 a. Activit antagoniste
de Trichoderma harzianum vis--vis de Pythium
aphanidermatum et de Pythium ultimum agents
responsables de la pourriture aqueuse des
tubercules de pomme de terre. Annales de lInstitut
National de la Recherche Agronomique de Tunisie
74: 167-186.
13. Daami-Remadi, M. 2001 b. Lutte biologique
contre les Fusarium spp. agents pathognes
responsables de la pourriture sche des tubercules
de pomme de terre. Mmoire de Diplme dEtudes
Approfondies en Protection des Plantes et
Environnement. Ecole Suprieure dHorticulture et
dElevage de Chott Mariem, Tunisia, 72 pp.
14. Dekkers, L. C., Mulders, I. H. M., Phoelich, C.
C., Chin-A-Woeng, T. F. C., Wijfjes, A. H. M.,
and Lugtenberg, B. J. J. 2000. The sss colonization
gene of the tomato-Fusarium oxysporum f. sp.
radicis lycopersici biocontrol strain Pseudomonas
fluorescens spp. bacteria. Molecular Plant Microbe
Interaction 13:1177-1183.
15. Estevez de Jensen, C., Percich, J. A., and Graham,
P. H. 2002. Integrated management strategies of
bean root rot with Bacillus subtilis and Rhizobium
in Minnesota. Field Crops Research 74:107-115.
16. Hajlaoui, M. R., Hamza, N., Gargouri, S., &
Guermech, A. 2001. Apparition en Tunisie de
Fusarium oxysporum f. sp. radicis lycopersici,
agent de la pourriture des racines et du collet de la
tomate. Bulletin OEPP 31: 505-507.
17. Hebbar, K. P., Martel, M. H., and Heulin, T.
1998. Suppression of pre- and post-emergence
damping-off in corn by Burkholderia cepacia.
European Journal of Plant Pathology 104:29-36.
18. Hervas, A., Trapero-Casas, J. L., and JimenezDiaz, R. M. 1995. Induced resistance against
Fusarium wilt of chickpea by non-pathogenic
races of Fusarium oxysporum f. sp. ciceris and
non-pathogenic isolates of F. oxysporum. Plant
Disease 79:1110-1116.
19. Hibar, K. 2002. La fusariose du collet et des
racines de la tomate : Pathognicit et moyens de
lutte. Mmoire de Diplme dEtudes Approfondies
en Protection des Plantes et Environnement. Ecole
Suprieure dHorticulture et dElevage de Chott
Mariem, Tunisia, 54 pp.
20. Hibar, K., Daami-Remadi, M., Khiareddine, H.,
& El Mahjoub, M. 2005. Effet inhibiteur in vitro et
in vivo du Trichoderma harzianum sur Fusarium
oxysporum
f.
sp.
radicis-lycopersici.
Biotechnologie
Agronomie
Socit
et
Environnement 9:163-171.
21. Hmouni, A., Hajlaoui, M. R., & Mlaiki, A. 1996.
Rsistance de Botrytis cinerea aux benzimidazoles
et aux dicarboximides dans les cultures abrites de
tomate en Tunisie. Bulletin OEPP 26: 697-705.
22. Howell, C. R. 2003. Mechanisms employed by
Trichoderma species in the biological control of
plant diseases: The history and evolution of current
concepts. Plant Disease 87: 4-10.

28

Vol. 1, No. 1, 2006

23. Katan, T., Zamir, D., Sarfati, M., and Katan, J.

1991. Vegetative compatibility groups and
subgroups in Fusarium oxysporum f. sp. radicislycopersici. Phytopathology 81: 255-262.
24. Kilian, M., Steiner, U., Krebs, H., Junge, G.,
Schmiedeknecht, G., and Hain, R. 2000. FZB24
Bacillus subtilis-mode of action of a microbial
agent enhancing plant vitality. PflanzenschutzNachrichten Bayer 1:72-93.
25. Kloepper, J. W., Ryu, C. M., and Zhang, S. 2004.
Induced systemic resistance and promotion of
plant growth by Bacillus spp. Phytopathology
94:1259-1266.
26. Larkin, R.P. and Fravel, D.R. 1998. Efficacy of
various fungal and bacterial biocontrol organisms
for control of Fusarium wilt of tomato. Plant
Disease 82:1022-1028.
27. Liu, L., Kloepper, J. W., and Tuzun, S. 1995.
Introduction of systemic resistance in cucumber
against Fusarium wilt by plant growth-promoting
rhizobacteria. Phytopathology 85:695-698.
28. Marois, J. J., Mitchell, D. J., and Sanada, R. M.
1981. Biological control of Fusarium crown and
root rot of tomato under field conditions.
Phytopathology 71:1257-1260.
29. Messiaen, C. M. & Cassini, R. 1968.
Systmatique des Fusarium. Annales de
Phytopathologie 3: 386-454.
30. Muslim, A., Horinouchi, H., and Hykumachi, M.
2003. Control of Fusarium crown and root rot of
tomato with hypovirulent binucleate Rhizoctonia
in soil and rock wool systems. Plant Disease
87:739-747.
31. Pharand, B., Carisse, O., and Benhamou, N. 2002.
Cytological aspects of compost-mediated induced
resistance against Fusarium crown and root rot in
tomato. Phytopathology 92:424-438.
32. Raupach, G. S. and Kloepper, J. W. 2000.
Biocontrol of cucumber diseases in the field by
plant growth-promoting rhizobacteria with and
without methyl bromide fumigation. Plant Disease
84:1073-1075.
33. Raupach, G. S., Liu, L., Murphy, J. F., Tuzum, S.,
and Kloepper, J. W. 1996. Induced systemic
resistance in cucumber and tomato against mosaic
cucumovirus using plant growth-promoting
rhizobacteria (PGPR). Plant Disease 80:891-894.

Tunisian Journal of Plant Protection

34. Reid, T. C., Hausbeck, M. K., and Kizilkaya, K.

2002. Use of fungicides and biological controls in
the suppression of Fusarium crown and root rot of
asparagus under greenhouse and growth chamber
conditions. Plant Disease 86:493-498.
35. Rekah, Y., Shteinberg, D., and Katan, J. 1999.
Spatial distribution and temporal development of
Fusarium crown and root rot of tomato and
pathogen
dissemination
in
field
soil.
Phytopathology 89:831-839.
36. Reynaldi, F. J., De Giusti, M. R., and Alippi, A.
M. 2004. Inhibition of the growth of Ascosphaera
apis by Bacillus and Paenibacillus strains isolated
from honey. Revista Argentina de Microbiologia
36:52-55.
37. Rose, S., Parker, M., and Punja, Z.K. 2003,
Efficacy of biological and chemical treatments for
control of Fusarium root and stem rot on
greenhouse cucumber. Plant Disease 87: 14621470.
38. Sivan, A. and Chet, I. 1993. Integrated control of
Fusarium crown and root rot of tomato with
Trichoderma harzianum in combination with
methyl bromide or soil solarization. Crop
Protection 12:380-386.
39. Sivan A., Ucko, O., and Chet, I. 1987. Biological
control of Fusarium crown rot of tomato by
Trichoderma harzianum under field conditions.
Plant Disease 71:587-592.
40. Song, W., Zhou, L., Yang, C., Cao, X., Zhang, L.,
and Liu, X. 2004. Tomato Fusarium wilt and its
chemical control strategies in a hydroponic system.
Crop Protection 23:243-247
41. Vakalounakis, D. J. and Fragkiadakis, G. A.
1999. Genetic diversity of Fusarium oxysporum
isolates from cucumber: differentiation by
pathogenicity, vegetative compatibility and RAPD
fingerprinting. Phytopathology 89:161-168.
42. Yedidia, I., Benhamou, N., and Chet, I. 1999.
Induction of defense responses in cucumber plants
(Cucumis sativus L.) by biocontrol agent
Trichoderma
harzianum.
Applied
and
Environmental Microbiology 65:1061-1070.

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