antimicrobial effect of ferrocenyl diaryl butenes against olive ... - SIPaV

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Journal of Plant Pathology (2011), 93 (3), 651-657

Edizioni ETS Pisa, 2011

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ANTIMICROBIAL EFFECT OF FERROCENYL DIARYL BUTENES AGAINST OLIVE PLANTLET DISEASES M. El Arbi1,2,3, P. Pigeon2, A. Chaari Rkhis1, S. Top2, A. Rhouma1, A. Rebai3, G. Jaouen2 and S. Aifa3 1 Institut

de l’Olivier. Route de l’Aéroport Km 1.5, 3003 Sfax, Tunisia ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel, UMR CNRS 7223, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France 3 Centre de Biotechnologie de Sfax (Université de Sfax). Route de Sidi Mansour Km 6, BP 1177, 3018 Sfax, Tunisia 2 Chimie

SUMMARY

INTRODUCTION

Many plant pathogenic bacteria and fungi are known to infect olive trees, causing considerable crop losses. The present work deals with targeting some olive tree pathogens by ferrocene derivatives [1,1-bis-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene] (P1); (Z+E)-1-(p-aminophenyl)-1-phenyl-2-ferrocenyl-but-1-ene (P2); 2-ferrocenyl-1-(4-N-acetylaminophenyl)-1-phenyl-but-1-ene (P3); tamoxifen analogous hydroxyferrocifen (P4); 1,1bis[4-(3-dimethylaminopropoxy)phenyl]-2-ferrocenyl-but-1-ene (P5); 1-[4-(3-dimethylamoniumpropoxy) phenyl]-1-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene citrate (P7) and 1,1-bis[4-(3-dimethylaminiumpropoxy) phenyl]-2-ferrocenyl-but-1-ene citrate (P8)] and an organic compound 1-[4-(3-dimethylamoniumpropoxy) phenyl]-1-(4-hydroxyphenyl)-2-phenyl-but-1-ene (P6), whose antimicrobial activity was tested in vitro against Pseudomonas savastanoi pv. savatanoi (the agent of olive knot), Agrobacterium tumefaciens (responsible for crown gall) and the fungus Fusarium solani (agent in nature of occasional root rot). Young olive plantlets inoculated with the three pathogens, developed symptoms when not treated with the ferrocene derivatives. These compounds efficiently inhibited the growth of these microorganisms in culture media and in inoculated plantlets which did not contract the corresponding diseases, especially when treated with P8. These findings shed light on the potential utilisation of ferrocene derivatives, analogues of hydroxytamoxifen, in curing microbe-mediated plant diseases.

Since the discovery of ferrocene (Kealy and Pauson, 1951), many derivatives have been synthesized and used for theoretical studies and for various applications. In the last two decades, ferrocene derivatives were utilized for very interesting applications in biorganometallic chemistry (Jaouen, 2006). In fact, ferrocene is used as a precursor for the preparation of organometallic complexes serving as bioconjugates, such as ferroceneoligonucleotide conjugates for electrochemical probing of DNA (Ihara et al., 1996; Nakayama, 2002), or for electrochemical recognition of cations, anions and neutral guest species by redox-active receptors (Beer and Bayly, 2007). Many ferrocene-based biosensors have been developed, e.g. an amperometric immunoelectrode based on the ferricinium-ion-mediated oxidation of glucose by glucose oxidase (Di Gleria, 1986) and an electrocatalytic dopamine biosensor based on ferroceneencapsulated Pd-linked ormosil (Pandey et al., 2001). Ferrocene polymers have found also application in aerospace material development (Neuse, 2005), whereas non polymeric ferrocene derivatives proved useful in biomedicinal chemistry since they are stable, non toxic and can easily cross the cell membrane (Dombrowski et al., 1986), which potentially makes them appropriate drugs for disease therapy. Many ferrocene derivatives of known drugs have shown great potentialities to overcome resistance developed by cells against treatments. In fact, hydroxyferrocifen (P4), a hydroxytamoxifen analogue, was more effective on breast cancer cell lines than the parental tamoxifen (Top et al., 1996, 2003) and a ferrocene chloroquine analogue (ferrochloroquine) was active against chloroquine-resistant strains of Plasmodium falciparum, a deadly agent of malaria (Domarle et al., 1998; Biot et al., 1997, 1999). Some ferrocenic derivatives of steroidal drugs were recently shown to be more effective against tumour cell lines compared to their parent compounds, and to acquire an anti-oxidative activity (Manosroi et al., 2010). The addition of a ferrocene moiety to some antibiotics, e.g. ferrocenyl-penicillin and ferrocenyl-cephalosporin, increased their activity (Edwards et al., 1979) and, similarly, the antifungal efficacy of flucanozole against Can-

Key words: bioorganometallics, hydroxytamoxifen, olive tree, ferrocene, antimicrobial compounds, Pseudomonas savastanoi, Agrobacterium tumefaciens, Fusarium solani.

Corresponding author: M. El Arbi, S.Top Fax: +216.74241033 E-mail: [email protected] E-mail: [email protected]


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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 (CFBP 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-


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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.

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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 CuSO4 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 106 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 108 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 106 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.

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 106 CFU ml–1, was added to each dilution in a 1:1 ratio.

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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Fig. 3. In vivo effect of P8 against Pseudomonas savastanoi pv. savastanoi. A. Control, B. Untreated, C. Treated (100 µg ml–1).

Fig. 4. In vivo effect of P8 against Fusarium solani infecting olive plantlets. A and B, Non treated. C, Treated (100 µg ml–1).


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Table 1. MIC and MBC determination. P3 50 100 50 200 50 100 P6 12.5 25 50 100 12.5 25 P9 (Doxycycline)