ANTIMICROBIAL EFFECT OF FERROCENYL DIARYL BUTENES AGAINST OLIVE PLANTLET DISEASES

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652 Ferrocenyl diaryl butenes against olive plantlets diseases Journal of Plant Pathology (2011), 93 (3), 651-657 dida was enhanced by the addition of ferrocene (Biot et al., 2000). Finally, new pyrazole derivatives containing a ferrocene unit have shown a wide range of activities against bacterial and fungal/yeast strains (Damljanovic et al., 2009). Ferrocenyl-substituted derivatives of antifungal drugs used in plant protection have also been developed. A ferrocene triadimefon derivative showed lower antifungal activity than the parent triadimefon, but exhibited an unexpectedly promising plant growth regulatory activity (Zhong et al., 2006). Ferrocene-containing di- and tetrahydroxylic acetylenic alcohols showed bactericidal activity against the phytopathogenic bacteria Pectobacterium aroideae, Xanthomonas campestris, Agrobacterium tumefaciens, and the actinomycetes Streptomyces sp. and Nocardiophsis sp. (Asatiani et al., 1984). The use of ferrocene compounds as fertilizers and for the prevention and cure of iron deficiency-induced disorders in plants has long been patented (Mues et al., 1977). In spite of these multiple applications, the use of ferrocene derivatives in agriculture is still little investigated. We have previously shown that some ferrocene derivatives, analogue of hydroxytamoxifen, exhibit a high antiproliferative effect on both hormone-dependent and Fig. 1. Chemical structure of the tested compounds. hormone-independent breast cancer cell lines, and possess also antimicrobial activity (El Arbi et al., 2011; Pigeon et al., 2011). We now report the successful protection of olive plantlets from bacteria and fungi by some ferrocene derivatives (Fig. 1) prior to planting in the field. For this study we have selected two bacterial pathogens, Pseudomonas savastanoi pv. savastanoi (CF- BP 5514) and Agrobacterium tumefaciens (CFBP 1903), and a strain of Fusarium solani isolated in our laboratory. Doxycycline and copper sulphate were chosen as standards for assessing the anti-microbial activity of the compounds under study. MATERIALS AND METHODS Chemical compounds. The compounds dihydroxy (P1) (Jaouen et al., 2000), amino (P2) (Buriez et al., 2008), acetamido (P3) (Pigeon et al., 2009), hydroxyferrocifen (P4) (Jaouen et al., 2000), 1-[4-(3-dimethylamoniumpropoxy)phenyl]-2-ferrocenyl-but-1-ene (P5) (El Arbi et al., 2011), 1-[4-(3-dimethylamoniumpropoxy) phenyl]- 1-(4-hydroxyphenyl)-2-phenyl-but-1-ene (P6) (Top et al., 2006), 1-[4-(3-dimethylaminopropoxy) phenyl]-1-(4-hy-
Journal of Plant Pathology (2011), 93 (3), 651-657 El Arbi et al. 653 droxyphenyl)-2-ferrocenyl-but-1-ene citrate (P7) (El Arbi et al., 2011) and 1,1-bis[4-(3-dimethylamoniumpropoxy) phenyl]-2-ferrocenyl-but-1-ene citrate (P8) (El Arbi et al., 2011) were synthesized as described in the above references and reported for their antiproliferative activity on breast cancer cells. Materials for biological assays. Plantlets of cv. Chemlali were produced from in vitro-grown explants at the Institut de l’Olivier de Sfax using standard procedures (Rugini, 1984). P. savastanoi pv. savastanoi (CFBP 5514) was a gift of Dr. M.M. López (Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain), A. tumefaciens (CFBP 1903) was obtained from Dr. X. Nesme (Université Lyon 1, France), whereas F. solani was isolated at the Institut de l’Olivier de Sfax. Plate count agar tests. The antimicrobial effect of ferrocene compounds, used at a dose of 1 mg ml –1 , was assessed using the well method (Tagg and Mc Given, 1971). The different pathogens (Fig. 2) were plated on plate count agar (PCA) medium (5 g l –1 peptone, 2.5 g l –1 yeast extract, 1 g l –1 glucose and 15 g l –1 agar) and the effect was evaluated measuring the size of inhibition zones. Fig. 2. Antimicrobial effect of P4 and P5 (1 mg ml –1 ) against Agrobacterium tumefaciens (CFBP 1903). Determination of the minimal inhibitory and minimal bactericide concentrations. Antimicrobial activity was determined by the tube dilution method. Twofold dilutions of each compound under trial were prepared in PCA liquid medium. A suspension of the standard microorganisms, prepared from 24 h cultures of bacteria in PCA liquid medium at a concentration of 10 6 CFU ml –1 , was added to each dilution in a 1:1 ratio. The same procedure was followed for fungi by using a spore suspension. Growth (or lack thereof) of the microorganisms was determined visually after incubation for 24 h at 37°C. The lowest concentration at which there was no visible growth was taken as the minimal inhibitory concentration (MIC). Then, one loopful from each tube was cultured on PCA and incubated for 24 h at 37°C. The lowest concentration of the compound that showed no colony formation was defined as the minimal bactericide concenration (MBC). Doxycycline and CuSO 4 were used as standards. Solutions of the test compounds and Doxycycline were prepared in ethanol at a concentration of 2 mg ml –1 . The twofold dilutions of the compounds were prepared (200, 100, 50, 25, 12.5 µg ml –1 ). Microorganism suspensions at 10 6 CFU ml –1 were inoculated into the corresponding test tube. Phytotoxicity test. The method of Zucconi et al. (1981) was used, determining the germination index of tomato seeds according to the following formula: IG = Number of germinated seeds in the assay × Number of germinated seeds in the control Average of root length in the assay × 100 Average of root length in the control In vivo antimicrobial tests. One-year-old and 4-monthold plantlets of cv. Chemlali from in vitro culture were wounded and infected with 10 µl of 10 8 CFU ml –1 inocula of P. savastanoi pv. savastanoi and A. tumefaciens, respectively. Three days after inoculation, the wounds were treated with a P8 solution. Inoculation with F. solani was performed by spraying the wounds (in leaves and stems) with a suspension of 10 6 spores ml –1 and treated by spraying with P8 solution after 1 h. A. tumefaciens-inoculated plantlets were maintained in a growth chamber at 25±1°C, under cool white fluorescent lamps (45 µmol m -2 s -1 ) with a 16 h photoperiod, whereas plantlets inoculated with P. savastanoi pv. savatanoi and F. solani were maintained in glasshouse at room temperature. RESULTS AND DISCUSSION Bioassays. When tested on PCA, products P4 and P5 inhibited the growth of the three microorganisms under trial. Compared to controls the highest inhibition was given by 1-bis[4-(3-dimethylaminopropoxy)phenyl] -2-ferrocenyl-but-1-ene (P5) (Fig. 2). The conversion to citric acid salts of products P4 and P5 allowed the total hydrosolubility of bis[4-(3-dimethylamoniumpropoxy) phenyl]-2-ferrocenyl-but-1-ene citrate (P8) and the partial hydrosolubility of 1-[4-(3-dimethylamoniumpropoxy) phenyl]-1-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene citrate (P7).
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ANTIMICROBIAL EFFECT OF FERROCENYL DIARYL BUTENES AGAINST OLIVE PLANTLET DISEASES

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