Page 28 - Volume 6, Issue 2, July 2013

© Benaki Phytopathological Institute
Bempelou et al. 76
glucose in the cultivation medium. We have
to mention that the above results are attrib-
uted to biodegradation of diazinon with a
maximum concentration of glucose in the
nutrient medium of 5 g/L, since Muncnero-
va and Augustin (1995) reported that in the
concentration of glucose of 10 g/L Rh. rubra
also started the degradation only after the
depletion of the saccharide.
Effect of diazinon concentration on biodeg-
radation
To investigate the effect of diazinon con-
centration on degradation, degradation tri-
als of diazinon with different concentrations
were conducted in the mineral salts medi-
um at 25o C. Rh. glutinis and Rh. rubra proved
to be capable degraders of diazinon. Both
yeasts degrade fully 25 μg/mL diazinon in
12 to 14 days (Figure 2). As it was observed
the higher the concentration it was, the
more active the yeasts were. In particular, al-
though Rh. glutinis maintained the delayed
action under the presence of glucose, it pre-
sented higher biodegradation rates when
it was incubated with 25 μg/mL diazinon
in both media. In flasks supplemented with
glucose the rate of 0.57 μg/mL/day (10 μg/
mL diazinon) increased to 1.14 μg/mL/day
(25 μg/mL diazinon). The corresponding val-
ues in the flasks without glucose in the min-
eral salts medium were 0.83 μg/mL/day and
2.5 μg/mL/day (Figure 2). Rhodotorula ru-
bra depleted diazinon in all cases present-
ing higher biodegradation rates in the con-
centration of 25 μg/mL, 2.5 μg/mL/day and
2.08μg/mL/day in comparison with 1.67 μg/
mL/day and 1.25 μg/mL/day in media with
or without glucose, respectively. The hydro-
lysis percentages of diazinon were less than
5% in all controls.
The metabolite 2-isopropyl-6-methyl-4-
-pyrimidinol (IMP) was produced as a deg-
radation product from both yeasts (Figure
3) in increasing levels up to the depletion
of the parent compound. Both yeasts pro-
duced bigger concentrations of IMP after
their incubation with 25 μg/mL diazinon.
As we can observe in Figures 2 and 3, af-
ter the consumption of diazinon the levels
of IMP remained almost stable and did not
decrease until the end of the experimental.
Having in mind that at that time IMP was the
only source of carbon available for the mi-
croorganisms, we can suspect that the two
yeasts can not mineralize this compound.
Similar studies have been conducted.
Yang et al., 2005 reported that Alcaligenes
faecalis DSP3 degraded 90% of 100μg/mL
diazinon in 10 days. Moreover, the bacte-
rium Enterobacter strain B-14 degraded 25
μg/mL diazinon in 2 days (Singh et al ., 2004).
Singh has also reported (2006) the degra-
dation of high concentration of diazinon
by soil microorganisms. Barik and Mun-
necke (1982) reported the depletion of diaz-
inon in 24 hours. Arthrobacter and Flavobac-
terium transformed 14-15 ppm diazinon
in rice fields (soil, water, rhizosphere) in 10
days (Sethunathan and Pathak, 1972). Final-
ly Cycon et al. (2009) described the degra-
dation (80-90%) of 50 μg/mL insecticide in
14 days by Pseudomonas sp., Serratia lique-
faciens and Serratia marcescens. The major-
ity of the microorganisms reported to be
able to degrade diazinon are soil bacteria.
No reference has been found describing the
biodegradation of diazinon by yeasts and
moreover by the epiphytic yeasts Rh. glu-
tinis and Rh. rubra.
Effect of temperature on biodegradation of
diazinon
The effect of different temperatures,
15o C, 25oC and 35oC, on diazinon biodegra-
dation in mineral salts medium is present-
ed in Figure 4. As shown after 14 days of in-
cubation no degradation took place at 35oC,
while the degradation rates at 25 oC and 15o C
were 0.67 μg/mL/day and 0.4 μg/mL/day for
Rh. glutinis and 0.83 μg/mL/day and 0.56 μg/
mL/day for Rh. rubra, respectively. The re-
sults are considered acceptable taking into
account that Rhodotorula yeasts belong to
psychrophyllic microorganisms.
Enzymatic assays
Inhibition studies were conducted by in-
cubation of homogenates of the two yeasts
with diazinon as described above. As it can