EFFICACY OF FOUR ESSENTIAL OILS ON FUSARIUM HEAD BLIGHT CAUSED BY FUSARIUM OXYSPORUM F. SP. RADICIS LYCOPERSICI (FORL), A FUNGAL TELLURIC PATHOGEN OF TOMATO GROWN UNDER COVER

Fusarium head blight caused by Fusarium oxysporum f. sp. Radicis-lycopersici (FORL) is a constraint to tomato cultivation in Korhogo, CÃƒÂ´te dIvoire. To control this disease, the antifungal activity of essential oils extracted from certain plants was evaluated in comparison with that of a synthetic fungicide for use. In vitro tests were performed on the different life stages of FORL. The in vivo evaluations consisted in carrying out two modes of treatment (curative and preventive) on plants of a susceptible variety of tomato inoculated with FORL. The results showed that the essential oils significantly reduced the different life stages of F.oxysporum f. sp. Radicis-lycopersici (FORL) as did the synthetic product. Thus, the essential oil Cymbopogon citratus at concentrations of 4000 and 6000 ppm strongly inhibited in vitro the different stages of FORL. In preventive and curative treatment, the essential oils not only improved the growth parameters of tomato plants but also reduced the incidence and severity of diseases. Thus, it was found that Cymbopogon citratus (4000 ppm) in preventive treatment, and Banko plus (250 ppm) in curative treatment reduced the mortality rate of FORL up to 6.66% and improved the growth parameters and reduced the flowering time of the tomato plant.The essential oil of C. citratus could be used for biological control of F. oxysporum f. sp. Radicis lycopersici.

Fusarium head blight caused by Fusarium oxysporum f. sp. Radicislycopersici (FORL) is a constraint to tomato cultivation in Korhogo, Côte d'Ivoire. To control this disease, the antifungal activity of essential oils extracted from certain plants was evaluated in comparison with that of a synthetic fungicide for use. In vitro tests were performed on the different life stages of FORL. The in vivo evaluations consisted in carrying out two modes of treatment (curative and preventive) on plants of a susceptible variety of tomato inoculated with FORL. The results showed that the essential oils significantly reduced the different life stages of F.oxysporum f. sp. Radicis-lycopersici (FORL) as did the synthetic product. Thus, the essential oil Cymbopogon citratus at concentrations of 4000 and 6000 ppm strongly inhibited in vitro the different stages of FORL. In preventive and curative treatment, the essential oils not only improved the growth parameters of tomato plants but also reduced the incidence and severity of diseases. Thus, it was found that Cymbopogon citratus (4000 ppm) in preventive treatment, and Banko plus (250 ppm) in curative treatment reduced the mortality rate of FORL up to 6.66% and improved the growth parameters and reduced the flowering time of the tomato plant.The essential oil of C. citratus could be used for biological control of F. oxysporum f. sp.

…………………………………………………………………………………………………….... Introduction:-
Obtaining the fungal strain Isolation was made from the crown and stem of the plant symptomatic of Fusarium head blight. These parts were cut into small fragments that, after being disinfected, in sodium hypochlorite solution (4%), rinsed with sterile distilled water and then dried in sterile blotting paper. The steilized explants were inoculated into Petri dishes containing PDA (Potato Dextrose Agar) culture medium. This culture medium was supplemented with an antibiotic, "Moxilfloxacin hydrochloride" (50 mg: 1000 ml) in order to avoid the proliferation of bacterial colonies. Petri dishes containing the explants were incubated in the dark at a temperature of 26±1°C for 3 to 4 days. Purification of the fungal colony from the explants was performed by removing the mycelial fragments from the growth front and subculturing onto new PDA medium. From this new colony, a monospore culture was performed. A spore suspension was spread on the PDA medium and incubated in the dark for 24 h at 26±1°C. A germinating spore was removed under a binocular magnifying glass and transplanted onto a new PDA medium. After two weeks of incubation in an oven at 28°C, the spore gave rise to a homogeneous clone of the fungus. Identification of the pathogen was carried out macroscopically and microscopically according to the determination key given by Nelson et al. (1983). Pathogenicity test on tomato seedlings was carried out according to the technique of Bachir(2017).

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Obtaining the different concentrations of essential oils and synthetic product for the tests The amendment of the PDA culture medium with the essential oils or the synthetic fungicide was performed according to the technique described by Soroet al. (2011). The choice of concentrations was made according to previous studies (Doumbouyaet al., 2012; Diagne, 2015 and Tiendrebeogo et al., 2017). Thus, the concentrations of products (synthetic fungicide and essential oils) selected to be tested against the pathogen were 250 ppm, 500 ppm, 1000 ppm, 2000 ppm, 4000 ppm and 6000 ppm.The preparation of the stock solutions of each essential oil was carried out using DMSO. Thus, 1ml of DMSO is mixed with 5 ml of essential oil and then homogenized by manual shaking for 5 minutes. This step makes the essential oils miscible in the culture medium. Each dose taken from the stock solution was added to 100 ml of PDA just before its distribution in the 90 mm diameter Petri dishes. For the preparation of the stock solution of the synthetic fungicide (Banko plus), a dilution is made with sterilized distilled water. Thus 1.4 ml of Banko plus is dissolved in 84.5 ml of sterilized distilled water. From this Banko plus stock solution, each volume taken, in the objective of the retained concentrations, was added to 100 ml of culture medium (PDA) then homogenized and distributed in Petri dishes.

Mycelial growth inhibition test and mycelial disc recovery
A 5 mm diameter mycelial disc was collected and placed in the centre of new Petri dishes containing 15 ml of PDA with added essential oil or Banko plus. For each product, six (6) concentrations were tested, with three replicates per concentration. The controls were carried out under the same conditions and consisted only of PDA medium with added DMSO. The test was performed three times. The seeded plates were incubated in a photoperiod of 12 h at room temperature. Mycelial growth was measured every 24 h. For this purpose, two perpendicular diameters passing through the middle of the mycelial disc were traced on the reverse side of the Petri dish. The daily growth of the mycelium was measured along the axis of the two diameters. These measurements were taken for seven (7) days. From the measurements taken, the rate of inhibition of mycelial growth of the two pathogens compared to the control was calculated using the formula below. At the end of the mycelial growth inhibition test, all explants that had not grown on the culture media amended with the different products were transplanted onto new PDA medium. The aim is to determine whether the product is fungicidal if the explant does not grow or fungistatic if it does grow.
Evaluation of the activity of essential oils and synthetic products on the production of spores of FORL After seven (7) days of incubation, ten (10) 5 mm diameter discs were removed from the Petri dishes used for mycelial growth measurements and placed in a test tube containing 2 ml of sterile distilled water. The tubes were shaken in 15-second sequences for 2 min to detach the spores from the conidiophores. The resulting suspensions were filtered through wattman paper to remove mycelial fragments. The number of spores was counted using a Malassez slide. The sporulation inhibition rate was thus determined by the formula: NSo: Average number of spores in the control, NStc: Average number of spores in the tubes at concentration C Evaluation of the activity of the products on the germination of spores of FORL From a 6-day culture on PDA medium, 10 mycelial washers were collected and placed in a tube containing 2 ml of sterile distilled water. The spores released after shaking were counted on a Malassez slide in order to calibrate the spore suspension to 105 spores. A volume of 100 µL of this suspension was spread in petri dishes containing PDA medium only (control) or with the addition of different concentrations of essential oils and synthesis product. Three replicates per concentration were performed. After 48 h of incubation in 12 h photoperiod, the number of germinated 1021 spores was counted on a total of 500 spores. The percentage of germination inhibition compared to the control was calculated.

In vivo evaluation of the activity of essential oils and synthetic fungicide
The essential oils C. citratus and O. basilicumwere used in addition to the synthetic fungicide Banko plus for testing under semi-controlled conditions. This choice was based on their in vitro efficacy. For each product tested, the lowest concentration that gave total inhibition of mycelial growth or MIC was used for the in vivo tests.

Setting up the nursery
Seeds of the tomato cultivar to be tested were superficially disinfected by soaking in a sodium hypochlorite solution (4%) for 3 minutes and then rinsed thoroughly with sterile distilled water to remove chlorine and residues of pesticides used in seed treatment. After drying, the seeds were sown in three honeycomb plates containing sterile commercial potting soil. Watering was done with tap water daily and the plants were transplanted one month after sowing.

Preparation of the inoculum
The FORL inoculum was prepared from a six (6) day culture on PDA culture medium. Dipping ten (10) mycelial washers in a tube containing 2 ml of sterile distilled water obtained it. After manual shaking to loosen the spores, the resulting solution was filtered through 4-wattman paper to remove residues. The concentration was assessed using the Malassezhematimeter and adjusted to 10 6 spores/ml.

Transplanting substrate and soil inoculation
The transplanting substrate is a mixture of sand and sterilized soil. The sand was taken from a lowland market garden plot in the 'disaster' district of Korhogo (Côte d'Ivoire). The sampling was done randomly, in different places on the plot. The proportion of the sand/soil mixture is 2/1. The mixed substrate is distributed in the transplanting pots pierced at the base to allow drainage.
The inoculation technique consisted in spraying 1ml of the inoculum in each transplanting hole made in the pots, whichwill be closed by a thin layer of substrate. A light watering was carried out, in order to favour the development of the mushrooms. After three days of incubation, the 21-day-old nursery plants were transplanted into the pots of inoculated or uninoculated substrate (control).

Antifungal treatments of plants
MICs of 4000 ppm, 4000 ppm and 250 ppm for O. basilicum essential oil, C. citratus essential oil and Banko plus fungicide were used.). For a final volume of 125 ml, 0.5 ml of C. citratus and O. basilicum essential oils were diluted with 4.5 ml of tween 20 and added to 120 ml of sterile distilled water. With the Banko plus, a dilution was made using a volume of 0.03 ml added to 125 ml of sterile distilled water. The effect of essential oils was compared to that of the synthetic fungicide (Banko plus) through two treatments.
As a preventive treatment, the products were applied before transplanting the plants. It consisted in spraying 2 ml of the product in question into the inoculated transplanting hole. The seedlings were then transplanted into the pots under glass. Two types of control were made. The first type consists of plants transplanted into inoculated holes and sprayed with 2 ml of an aqueous solution of tween 20 (0.5 ml of tween for 12 ml of sterile distilled water). The second type consists of plants transplanted into uninoculated holes and sprayed with 2 ml of sterile distilled water.
The curative treatment procedure consisted in spraying a volume of 2 ml of the different products from the stem to the leaves 3 days after transplanting the plants into the inoculated holes. Two types of control were also carried out. The first type of plants were transplanted into holes inoculated with the fungus and sprayed with 2 ml of an aqueous solution of tween 20 (0.5 ml per 12 ml of sterile distilled water). The second type consisted of seedlings transplanted into holes not inoculated with the fungus and sprayed with 2 ml of sterile distilled water.

Experimental device
The transplanted plants were watered twice a day throughout the experiment. The pots were placed under the greenhouse in a split-plot design with two (2) factors: treatments (product-dose), mode of application (preventivecurative). Ten (10) plants per treatment were used. The experiment was repeated three times. 1022 Monitoring the development of the disease Monitoring of growth parameters and disease symptoms was doneevery two days starting from the first week after transplanting.The growth parameters measured were plant height, stem neck diameter, number of leaves and flowering time. These parameters were measured until flowering. The flowering time variable was assessed in number of days after sowing (Djidjiet al., 2010). For each parameter, the mean was calculated.
Concerning the indicators of disease symptom expression, the incidence and severity of the disease were assessed. The severity was assessed using a symptom rating scale proposed by Vakalounakis and Fragakiadakis(1999). 0: healthy plant; 1: slight yellowing, slight pivot and secondary root rot and crown rot; 2: yellowing of leaves and stems with or without wilting or stunting of plants; 3: death of the plant. The disease severity index (SI) is obtained according to the following formula (Song et al.,2004): With Ni being the number of plants having received the same score; Zi: score (0; 1; 2; 3); Nt: total number of plants used for each treatment; Z: highest score (3).

Statistical analysis
These data were analyzed using Statistica version 7.1 software. An Analysis of Variance (ANOVA I) was performed and in case of significant difference between the means, the separation of the means was done by the NewmanKeuls test at the 5% threshold.

Results:-
Effect of the different products on the mycelial growth of FORL Inhibition of FORL was product and concentration dependent (Figure 1).The synthetic fungicide (Banko plus) completely inhibited the pathogen at all concentrations used. As for the essential oils, total inhibition was observed at concentrations of 4000 ppm for the oils of C. citratus, O. basilicumand 6000 ppm for the essential oil of L. multiflora, respectively. The essential oil of E. camaldulensis showed the lowest inhibition rates. However, its significant antifungal activity is around 85% from 4000 ppm. The highest inhibitory activity was observed with the essential oil of C. citratus. The differences between the inhibition rates of mycelial growth of FORLare significant (P<0.001) at the 5% threshold.

Effect of products on F. oxysporumf. sp.Radicis-lycopersici sporulation
Total inhibition of FORL sporulation was achieved with the fungicide Banko plus. As for the essential oils, E. camaldulensisoil showed the highest inhibition rates (above 70%), from 500 ppm. Conversely, the essential oil of L. multifora, with the highest sporulation inhibition rate (37.29%) obtained at 4000 ppm, showed the lowest activity overall ( Table 1).

Effect of products on germination of F. oxysporum f. sp. Radicis-lycopersici
All oils induced a strong inhibitory activity on spore germination (more than 85%) from 1000 ppm ( Table 2). However, the essential oil of L. multiflora and the synthetic product Banko plus completely inhibited FORL spore germination from 1000 ppm. Complete inhibition (100%) was obtained at 4000 ppm with the essential oils of C. citratus and E. camaldulensis. Figure 2shows that the percentages of severity index were higher with inoculated and untreated plants (41.66%) compared to all treated plants. The severity index varied according to the treatment and the mode of application. It was noticed that curative treatments recorded the lowest severity indices except for the synthetic product Banko plus. Among the applied products, the highest index was obtained with the essential oil of O. basilicum in preventive treatment (21.73%) and the lowest value with that of C. citratus in curative treatment (P<0.001).

Effects of products on the incidence of F. oxysporum f. sp. Radicis-lycopersici
Sixty (60) days after transplanting, seedlings transplanted to soil inoculated with FORL and untreated (T+ Fus) showed high mortality rates (25%) (Figure 3). Depending on the product and the method of application, incidences  were low, less than or equal to 10%. The synthetic fungicide Banko plus as a preventive treatment induced the lowest mortality rate (3.84%).   The figures with the same letter are not significantly different at the 5% threshold using the Newman and Keuls test for the same parameter and for the same product associated with the application method.

Effects of products on growth parameters of inoculated tomato plants
Following the treatments, the highest average heights were obtained with C. citratus essential oil in preventive (18.35 cm) and curative (16.47 cm) treatments. Plant height was significantly better in curative treatment (15.27 cm) than in preventive treatment (11.56 cm) with the synthetic fungicide Banko plus ( Table 3).
The highest diameter was obtained in preventive treatment with C. citratus essential oil compared to the two types of control. The results also revealed that the mean diameters of plants in preventive and curative treatment of Banko plus  Thenumbers with the same letter are not significantly different at the 5% threshold.

Effect of essential oils and synthetic product on the average flowering time of inoculated tomato plants
The analysis of variance showed that there was no significant difference between the flowering time of the treated plants and that of the non-inoculated control plants. However, among the treatments carried out, the shortest flowering time was obtained following the preventive treatment with O. basilicumessential oil ( Table 4).

Discussion:-
This study showed that all the products tested at different concentrations have an effect on F. oxysporum f. sp. Radicis-lycopersici. This activity of the products, especially the essential oils would be due to the fact that these extracts possess natural organic compounds with antimicrobial activities recognized in several aromatic plants.  1028 Chlorothalonil) and Ocimum gratissimumessential oil completely inhibited mycelial growth at 600 ppm and 150 ppm concentrations, respectively, reflecting a better efficacy of this essential oil. The relative efficiency obtained in this work with Ocimum basilicumessential oil on FORL mycelial growth (total inhibition at 4000 ppm) would be due to a difference in chemotype but also in sensitivity of the fungal strains.
The FORL strain on the other hand was susceptible to C. citratus compared to the other essential oils. This could be explained by the quality, quantity and chemical structure of the antifungal molecules contained in each essential oil (Zarithet al., 2018). Radicis-lycopersici, treatment with the products at concentrations inhibiting the mycelial growth of the fungus resulted in a low attack of the fungus. However, the degree of attack varied depending on whether the products were applied as a preventive or curative measure.
As a curative treatment, the synthetic product improved plant height, number of live leaves and stem collar diameter. The essential oils of Ocimum basilicumand Cymbopogon citratus, when applied to plants inoculated with FORL, showed some efficacy on disease expression and on plant growth and development parameters. The essential oil C. citratus showed the best activity in preventive treatment. This essential oil stimulated the growth in height of the variety UC 82 B, induced the greatest diameter and number of functional leaves. The inhibition of FORL by this oil in controlled and semi-controlled conditions would be due to the fact that aromatic vegetable species would possess in a natural way compounds being able to inhibit the development of the microorganisms. Thus, a study conducted by Cabral et al. (2013) in relation to the effectiveness of the essential oil Cymbopogon citratus on the agent Fusarium oxysporum f. sp. Radicis-lycopersici, in vitro and in vivo, showed that it has bioactive compounds such as phytoalexins. These phytoalexinsare known to have antimicrobial activity for plant protection including alkaloids, flavonoids, isoflavonoids, tannins, cumarins, glycosides, terpenes, phenylpropanes and organic acids.

Conclusion:-
At the end of this study, it was found that, like the synthetic product, the essential oils significantly reduced the different life stages of Fusarium oxysporum f. sp. Radicis-lycopersici (FORL). Thus, the essential oil C. citratus at concentrations of 4000 and 6000 ppm strongly inhibited in vitro the different stages of FORL. In preventive and 1029 curative treatment, the essential oils not only improved the growth parameters of tomato plants but also reduced the incidence and severity of diseases. Thus, for management of Fusarium root and crown blight,C. citratus essential oil in preventive treatment and Banko plus in curative treatment could be used.