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ANTIMICROBIAL EFFECT OF FERROCENYL DIARYL BUTENES AGAINST OLIVE PLANTLET DISEASES

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 utilisatio

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 utilisatio

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Journal of Plant Pathology (2011), 93 (3), 651-657 Edizioni ETS Pisa, 2011 651<br />

SUMMARY<br />

<strong>ANTIMICROBIAL</strong> <strong>EFFECT</strong> <strong>OF</strong> <strong>FERROCENYL</strong> <strong>DIARYL</strong> <strong>BUTENES</strong> <strong>AGAINST</strong><br />

<strong>OLIVE</strong> <strong>PLANTLET</strong> <strong>DISEASES</strong><br />

M. El Arbi 1,2,3 , P. Pigeon 2 , A. Chaari Rkhis 1 , S. Top 2 , A. Rhouma 1 , A. Rebai 3 , G. Jaouen 2 and S. Aifa 3<br />

1 Institut de l’Olivier. Route de l’Aéroport Km 1.5, 3003 Sfax, Tunisia<br />

2 Chimie ParisTech (Ecole Nationale Supérieure de Chimie de Paris), Laboratoire Charles Friedel,<br />

UMR CNRS 7223, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France<br />

3 Centre de Biotechnologie de Sfax (Université de Sfax). Route de Sidi Mansour Km 6, BP 1177, 3018 Sfax, Tunisia<br />

Many plant pathogenic bacteria and fungi are known<br />

to infect olive trees, causing considerable crop losses. The<br />

present work deals with targeting some olive tree<br />

pathogens by ferrocene derivatives [1,1-bis-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene]<br />

(P1); (Z+E)-1-(p-aminophenyl)-1-phenyl-2-ferrocenyl-but-1-ene<br />

(P2); 2-ferrocenyl-1-(4-N-acetylaminophenyl)-1-phenyl-but-1-ene<br />

(P3); tamoxifen analogous hydroxyferrocifen (P4); 1,1bis[4-(3-dimethylaminopropoxy)phenyl]-2-ferrocenyl-but-1-ene<br />

(P5); 1-[4-(3-dimethylamoniumpropoxy)<br />

phenyl]-1-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene<br />

citrate (P7) and 1,1-bis[4-(3-dimethylaminiumpropoxy)<br />

phenyl]-2-ferrocenyl-but-1-ene citrate (P8)] and an organic<br />

compound 1-[4-(3-dimethylamoniumpropoxy)<br />

phenyl]-1-(4-hydroxyphenyl)-2-phenyl-but-1-ene (P6),<br />

whose antimicrobial activity was tested in vitro against<br />

Pseudomonas savastanoi pv. savatanoi (the agent of olive<br />

knot), Agrobacterium tumefaciens (responsible for crown<br />

gall) and the fungus Fusarium solani (agent in nature of<br />

occasional root rot). Young olive plantlets inoculated<br />

with the three pathogens, developed symptoms when not<br />

treated with the ferrocene derivatives. These compounds<br />

efficiently inhibited the growth of these microorganisms<br />

in culture media and in inoculated plantlets which did<br />

not contract the corresponding diseases, especially when<br />

treated with P8. These findings shed light on the potential<br />

utilisation of ferrocene derivatives, analogues of<br />

hydroxytamoxifen, in curing microbe-mediated plant<br />

diseases.<br />

Key words: bioorganometallics, hydroxytamoxifen,<br />

olive tree, ferrocene, antimicrobial compounds, Pseudomonas<br />

savastanoi, Agrobacterium tumefaciens, Fusarium<br />

solani.<br />

Corresponding author: M. El Arbi, S.Top<br />

Fax: +216.74241033<br />

E-mail: mehdi.el_arbi@etu.upmc.fr<br />

E-mail: siden-top@chimie-paristech.fr<br />

INTRODUCTION<br />

Since the discovery of ferrocene (Kealy and Pauson,<br />

1951), many derivatives have been synthesized and used<br />

for theoretical studies and for various applications. In<br />

the last two decades, ferrocene derivatives were utilized<br />

for very interesting applications in biorganometallic<br />

chemistry (Jaouen, 2006). In fact, ferrocene is used as a<br />

precursor for the preparation of organometallic complexes<br />

serving as bioconjugates, such as ferroceneoligonucleotide<br />

conjugates for electrochemical probing<br />

of DNA (Ihara et al., 1996; Nakayama, 2002), or for<br />

electrochemical recognition of cations, anions and neutral<br />

guest species by redox-active receptors (Beer and<br />

Bayly, 2007). Many ferrocene-based biosensors have<br />

been developed, e.g. an amperometric immunoelectrode<br />

based on the ferricinium-ion-mediated oxidation<br />

of glucose by glucose oxidase (Di Gleria, 1986) and an<br />

electrocatalytic dopamine biosensor based on ferroceneencapsulated<br />

Pd-linked ormosil (Pandey et al., 2001).<br />

Ferrocene polymers have found also application in<br />

aerospace material development (Neuse, 2005), whereas<br />

non polymeric ferrocene derivatives proved useful in<br />

biomedicinal chemistry since they are stable, non toxic<br />

and can easily cross the cell membrane (Dombrowski et<br />

al., 1986), which potentially makes them appropriate<br />

drugs for disease therapy. Many ferrocene derivatives<br />

of known drugs have shown great potentialities to<br />

overcome resistance developed by cells against treatments.<br />

In fact, hydroxyferrocifen (P4), a hydroxytamoxifen<br />

analogue, was more effective on breast cancer<br />

cell lines than the parental tamoxifen (Top et al., 1996,<br />

2003) and a ferrocene chloroquine analogue (ferrochloroquine)<br />

was active against chloroquine-resistant<br />

strains of Plasmodium falciparum, a deadly agent of<br />

malaria (Domarle et al., 1998; Biot et al., 1997, 1999).<br />

Some ferrocenic derivatives of steroidal drugs were<br />

recently shown to be more effective against tumour cell<br />

lines compared to their parent compounds, and to acquire<br />

an anti-oxidative activity (Manosroi et al., 2010).<br />

The addition of a ferrocene moiety to some antibiotics,<br />

e.g. ferrocenyl-penicillin and ferrocenyl-cephalosporin,<br />

increased their activity (Edwards et al., 1979) and, similarly,<br />

the antifungal efficacy of flucanozole against Can-


652 Ferrocenyl diaryl butenes against olive plantlets diseases Journal of Plant Pathology (2011), 93 (3), 651-657<br />

dida was enhanced by the addition of ferrocene (Biot et<br />

al., 2000). Finally, new pyrazole derivatives containing a<br />

ferrocene unit have shown a wide range of activities<br />

against bacterial and fungal/yeast strains (Damljanovic<br />

et al., 2009).<br />

Ferrocenyl-substituted derivatives of antifungal drugs<br />

used in plant protection have also been developed. A ferrocene<br />

triadimefon derivative showed lower antifungal<br />

activity than the parent triadimefon, but exhibited an unexpectedly<br />

promising plant growth regulatory activity<br />

(Zhong et al., 2006). Ferrocene-containing di- and<br />

tetrahydroxylic acetylenic alcohols showed bactericidal<br />

activity against the phytopathogenic bacteria Pectobacterium<br />

aroideae, Xanthomonas campestris, Agrobacterium<br />

tumefaciens, and the actinomycetes Streptomyces sp. and<br />

Nocardiophsis sp. (Asatiani et al., 1984). The use of ferrocene<br />

compounds as fertilizers and for the prevention<br />

and cure of iron deficiency-induced disorders in plants<br />

has long been patented (Mues et al., 1977). In spite of<br />

these multiple applications, the use of ferrocene derivatives<br />

in agriculture is still little investigated.<br />

We have previously shown that some ferrocene derivatives,<br />

analogue of hydroxytamoxifen, exhibit a high antiproliferative<br />

effect on both hormone-dependent and<br />

Fig. 1. Chemical structure of the tested compounds.<br />

hormone-independent breast cancer cell lines, and possess<br />

also antimicrobial activity (El Arbi et al., 2011; Pigeon<br />

et al., 2011). We now report the successful protection<br />

of olive plantlets from bacteria and fungi by some<br />

ferrocene derivatives (Fig. 1) prior to planting in the<br />

field. For this study we have selected two bacterial<br />

pathogens, Pseudomonas savastanoi pv. savastanoi (CF-<br />

BP 5514) and Agrobacterium tumefaciens (CFBP 1903),<br />

and a strain of Fusarium solani isolated in our laboratory.<br />

Doxycycline and copper sulphate were chosen as<br />

standards for assessing the anti-microbial activity of the<br />

compounds under study.<br />

MATERIALS AND METHODS<br />

Chemical compounds. The compounds dihydroxy<br />

(P1) (Jaouen et al., 2000), amino (P2) (Buriez et al.,<br />

2008), acetamido (P3) (Pigeon et al., 2009), hydroxyferrocifen<br />

(P4) (Jaouen et al., 2000), 1-[4-(3-dimethylamoniumpropoxy)phenyl]-2-ferrocenyl-but-1-ene<br />

(P5) (El Arbi<br />

et al., 2011), 1-[4-(3-dimethylamoniumpropoxy) phenyl]-<br />

1-(4-hydroxyphenyl)-2-phenyl-but-1-ene (P6) (Top et al.,<br />

2006), 1-[4-(3-dimethylaminopropoxy) phenyl]-1-(4-hy-


Journal of Plant Pathology (2011), 93 (3), 651-657 El Arbi et al. 653<br />

droxyphenyl)-2-ferrocenyl-but-1-ene citrate (P7) (El Arbi<br />

et al., 2011) and 1,1-bis[4-(3-dimethylamoniumpropoxy)<br />

phenyl]-2-ferrocenyl-but-1-ene citrate (P8) (El Arbi et al.,<br />

2011) were synthesized as described in the above references<br />

and reported for their antiproliferative activity on<br />

breast cancer cells.<br />

Materials for biological assays. Plantlets of cv. Chemlali<br />

were produced from in vitro-grown explants at the Institut<br />

de l’Olivier de Sfax using standard procedures<br />

(Rugini, 1984). P. savastanoi pv. savastanoi (CFBP 5514)<br />

was a gift of Dr. M.M. López (Instituto Valenciano de<br />

Investigaciones Agrarias, Valencia, Spain), A. tumefaciens<br />

(CFBP 1903) was obtained from Dr. X. Nesme<br />

(Université Lyon 1, France), whereas F. solani was isolated<br />

at the Institut de l’Olivier de Sfax.<br />

Plate count agar tests. The antimicrobial effect of ferrocene<br />

compounds, used at a dose of 1 mg ml –1 , was assessed<br />

using the well method (Tagg and Mc Given, 1971).<br />

The different pathogens (Fig. 2) were plated on plate<br />

count agar (PCA) medium (5 g l –1 peptone, 2.5 g l –1 yeast<br />

extract, 1 g l –1 glucose and 15 g l –1 agar) and the effect<br />

was evaluated measuring the size of inhibition zones.<br />

Fig. 2. Antimicrobial effect of P4 and P5 (1 mg ml –1 ) against<br />

Agrobacterium tumefaciens (CFBP 1903).<br />

Determination of the minimal inhibitory and minimal<br />

bactericide concentrations. Antimicrobial activity<br />

was determined by the tube dilution method. Twofold<br />

dilutions of each compound under trial were prepared<br />

in PCA liquid medium. A suspension of the standard<br />

microorganisms, prepared from 24 h cultures of bacteria<br />

in PCA liquid medium at a concentration of 10 6<br />

CFU ml –1 , was added to each dilution in a 1:1 ratio.<br />

The same procedure was followed for fungi by using a<br />

spore suspension. Growth (or lack thereof) of the microorganisms<br />

was determined visually after incubation<br />

for 24 h at 37°C. The lowest concentration at which<br />

there was no visible growth was taken as the minimal<br />

inhibitory concentration (MIC). Then, one loopful<br />

from each tube was cultured on PCA and incubated<br />

for 24 h at 37°C. The lowest concentration of the compound<br />

that showed no colony formation was defined as<br />

the minimal bactericide concenration (MBC). Doxycycline<br />

and CuSO 4 were used as standards. Solutions of<br />

the test compounds and Doxycycline were prepared in<br />

ethanol at a concentration of 2 mg ml –1 . The twofold<br />

dilutions of the compounds were prepared (200, 100,<br />

50, 25, 12.5 µg ml –1 ). Microorganism suspensions at<br />

10 6 CFU ml –1 were inoculated into the corresponding<br />

test tube.<br />

Phytotoxicity test. The method of Zucconi et al. (1981)<br />

was used, determining the germination index of tomato<br />

seeds according to the following formula:<br />

IG = Number of germinated seeds in the assay ×<br />

Number of germinated seeds in the control<br />

Average of root length in the assay × 100<br />

Average of root length in the control<br />

In vivo antimicrobial tests. One-year-old and 4-monthold<br />

plantlets of cv. Chemlali from in vitro culture were<br />

wounded and infected with 10 µl of 10 8 CFU ml –1 inocula<br />

of P. savastanoi pv. savastanoi and A. tumefaciens, respectively.<br />

Three days after inoculation, the wounds were<br />

treated with a P8 solution. Inoculation with F. solani was<br />

performed by spraying the wounds (in leaves and stems) with<br />

a suspension of 10 6 spores ml –1 and treated by spraying with<br />

P8 solution after 1 h. A. tumefaciens-inoculated plantlets<br />

were maintained in a growth chamber at 25±1°C, under<br />

cool white fluorescent lamps (45 µmol m -2 s -1 ) with a<br />

16 h photoperiod, whereas plantlets inoculated with P.<br />

savastanoi pv. savatanoi and F. solani were maintained in<br />

glasshouse at room temperature.<br />

RESULTS AND DISCUSSION<br />

Bioassays. When tested on PCA, products P4 and<br />

P5 inhibited the growth of the three microorganisms<br />

under trial. Compared to controls the highest inhibition<br />

was given by 1-bis[4-(3-dimethylaminopropoxy)phenyl]<br />

-2-ferrocenyl-but-1-ene (P5) (Fig. 2). The conversion to<br />

citric acid salts of products P4 and P5 allowed the total<br />

hydrosolubility of bis[4-(3-dimethylamoniumpropoxy)<br />

phenyl]-2-ferrocenyl-but-1-ene citrate (P8) and the partial<br />

hydrosolubility of 1-[4-(3-dimethylamoniumpropoxy)<br />

phenyl]-1-(4-hydroxyphenyl)-2-ferrocenyl-but-1-ene<br />

citrate (P7).


654 Ferrocenyl diaryl butenes against olive plantlets diseases Journal of Plant Pathology (2011), 93 (3), 651-657<br />

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

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


Journal of Plant Pathology (2011), 93 (3), 651-657 El Arbi et al. 655<br />

Table 1. MIC and MBC determination.<br />

P3 P2 P1 Products<br />

50<br />

100<br />

100<br />

200<br />

50<br />

100<br />

– 1<br />

CMI µg m l<br />

CMB µg m l – 1 Agrobacterium tumefaciens<br />

50<br />

200<br />

25<br />

200<br />

50<br />

100<br />

– 1<br />

CMI µg m l<br />

CMB µg m l – 1 Pseudomonas savastanoi<br />

50<br />

100<br />

100<br />

200<br />


656 Ferrocenyl diaryl butenes against olive plantlets diseases Journal of Plant Pathology (2011), 93 (3), 651-657<br />

Fig. 5. In vivo effect of P8 against the crown gall of olive plantlets.<br />

A : Non treated, B : Treated (250 µg ml –1 ).<br />

(not treated) and only 30% of the plants treated with 250 µg<br />

ml –1 of P8 developed the symptoms of crown gall (Fig. 5).<br />

We have previously shown that compounds P4, P5<br />

and P6 are active against both hormone-dependent and<br />

hormone-independent breast cancer cell lines and have<br />

also antimicrobial activity (Vessières et al., 2005; Pigeon<br />

et al., 2009, 2011; El Arbi et al., 2011). Now evidence is<br />

provided that the same chemicals are also active in vitro<br />

against microorganisms pathogenic to young olive trees.<br />

The antimicrobial activity of P6 confirms the action<br />

of tamoxifen against Candida albicans (Dolan et al.,<br />

2009). Phenyl substitution by ferrocene, leading to compound<br />

P4, did not improve its efficacy, meanwhile the<br />

substitution of OH function by a dimethylaminopropoxy<br />

chain led to a more active compound P5.<br />

Another advantage of these ferrocenyl compounds<br />

such as P5 is their decreased cytotoxicity due to the low<br />

water-octanol partition coefficient (log P o/w ) (El Arbi et<br />

al., 2011), that restrains their persistence in the soil<br />

(Barceló et al., 1997). These features are compatible<br />

with agricultural applications and help preserving the<br />

environment.<br />

Since the action of P5 mimics that of Doxycycline,<br />

which is allowed in certain countries such as the USA<br />

(Kuldip et al., 2005), this compound could be a good alternative<br />

to antibiotics used in agriculture.<br />

In conclusion, the main objectives of our work were the<br />

synthesis and study of new products that could be useful in<br />

the chemical control of olive tree pathogens. P5 displayed a<br />

significant antimicrobial activity in vitro that could compete<br />

with antibiotics and performed better than copper sulphate.<br />

To facilitate field applications, P5 was converted into citrate<br />

salt (P8) which is hydrosoluble, maintained the antimicrobial<br />

activity, was not phytotoxic and was effective both in vivo<br />

and in vitro against the tested pathogens. Thus, ferrocenic<br />

derivatives of hydroxytamoxifen, especially P5, protect<br />

olive plantlets prior to their culture in the field and seem be<br />

promising potential pesticides for plant protection.<br />

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