ANTIMICROBIAL EFFECT OF FERROCENYL DIARYL BUTENES AGAINST OLIVE PLANTLET DISEASES

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).