Bio fungicid FOXL.pdf

Tunisian Journal of Plant Protection Vol. 1, No. 1, 2006

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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

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 2000-2001 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 [email protected]

Accepted for publication 23 January 2006.

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,


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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 bio-fungicides 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.

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.

Table 1. Fusarium oxysporum f. sp. radicis-lycopersici isolates used in this study

Isolates Host plant (Cultivar)

Date of isolation

Fo2.01 Durintha 2001 Fo4.02 Bochra 2002 Fo1.03 Sarnia 2003 Fo1.04 Olivette 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 5-week-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:


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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 bio-fungicides 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 bio-fungicides on FCRR disease incidence was evaluated under growth chamber. Experiments were performed with 5-

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) bio-fungicides 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):

Ten plants per elementary treatment

Ten plants per elementary treatment were used and variance analysis of the treatment effect on measured data was performed by using the general linear

(scale number of plants infected) = 100

(highest scale total number of plants) Disease incidence (%)


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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. Green-house 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 breeding-ground 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 bio-fungicide 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.

RESULTS In vitro inhibition of FORL by bio-

fungicides. 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

Fig. 1. Effect of various bio-fungicides on mycelial growth inhibition of F. oxysporum f. sp. radicis-lycopersici 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 bio-fungicides 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).

0

10

20

30

40

50

60

70

80

FO2-01 FO4-02 FO1-03 FO1-04 F.oxysporum f. sp.radicis lycopersici isolates

Inhi

bitio

n pe

rce

nta

ge (%

)

Serenade

Sonata

Polyversum

Biocint-T W-P

Biocont-T Gr

Agralan Revive

RootShield


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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

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

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 bio-fungicides, disease incidence was always more than 50% and it reached 66.67% with the bio-fungicide Sonata (Table 3).

Simultaneous application of bio-fungicides and pathogen spores to tomato plants has significantly reduced disease incidence. Indeed, tomato plants treated

with Polyversum, Biocont-T W-P, Biont-T 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).

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

Inoculated and untreated control 80 b 83,3 b 90 b 93,33 b Uninoculated control 0 a 0 a 0 a 0 a

A B

A B


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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 (%) FO2-01z FO4-02z FO1-03z FO1-04z Serenade 60 b 60 bc 60 b 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

Healthy plants 0 a 0 a 0 a 0 a 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 (%) FO2-01z FO4-02z FO1-03z FO1-04z Serenade 26,67 b 26,67 b 33,33 b 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

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

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 bio-fungicide (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 bio-fungicide 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

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.

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.


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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.

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

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 bio-fungicides, 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

A B


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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.

Application of bacteria to seeds has been used for biological control of soil-borne 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


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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 bio-fongicide 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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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 86:405-416.

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 87:108-121.

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.

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