Page 21 - Volume 6, Issue 2, July 2013

1

Laboratory of Pesticide Science, Agricultural Universi-

ty of Athens, Iera Odos 75, GR-118 55 Athens, Greece.

2

Faculty of Applied Biology and Biotechnology, De-

partment of Biology, University of Crete, GR-714 09

Heraklion, Greece

.

3

Pesticides Residues Laboratory, Department of Pes-

ticides Control and Phytopharmacy, Benaki Phyto-

pathological Institute, 8 St. Delta str., GR-145 61 Kifis-

sia (Athens) Greece.

Corresponding author:

e.bempelou@bpi.gr

Hellenic Plant Protection Journal

6:

69-82, 2013

Biodegradation of diazinon by the epiphytic yeasts

Rhodotorula glutinis

and

Rhodotorula rubra

E.D. Bempelou

1,3

, J.G. Vontas

2

, K.S. Liapis

3

and V.N. Ziogas

1

Summary

The capability of the epiphytic yeasts

Rhodotorula glutinis

and

Rhodotorula rubra

to biode-

grade diazinon in pure cultures and tomato fruits was investigated. Higher biodegradation rates were

observed as the concentration of diazinon and the inoculum of the microorganisms were increased,

and the yeasts proved to be more active at 25

o

C and 15

o

C. The presence of glucose in the mineral nu-

trient medium, as an extra source of carbon, delayed the biodegradation by

Rh. glutinis

, while

Rh. ru-

bra

biodegrade diazinon faster in the presence of glucose. The detection and quantification of diazi-

non and its degradation product 2-isopropyo-6-methyl-4-pyrimidinol (IMP) were achieved successful-

ly in the LC-MS/MS chromatographic system. The in vitro enzymatic assays applied suggested that glu-

tathione-S-transferases may be involved in the biodegradation of diazinon, a fact that was further en-

hanced after the addition of the synergists triphenyl phosphate, diethyl maleate and piperonyl butox-

ide in the biodegradation trials. The decrease of diazinon residues on tomato fruits confirmed the cor-

responding on pure cultures, resulting in the suggestion that the yeasts

Rh. glutinis

and

Rh. rubra

can

possibly be used successfully for the removal or detoxification of diazinon residues ontomatoes.

Additional keywords:

biodegradation, diazinon, epiphytic yeasts, glutathione-S-transferases, synergists

Introduction

The excessive and frequent application of

pesticides in agricultural practice has result-

ed in high levels of pesticide residues accu-

mulated on vegetables. This matter poses a

potential health risk to consumers (Bologne-

si and Morasso, 2000), particularly in coun-

tries where highly toxic insecticides are still

in use. An increased interest in the develop-

ment of means for removing pesticide resi-

dues from vegetables, using environmental

friendly approaches, such as microorgan-

isms or recombinant enzyme systems has

been observed.

Diazinon,

[Ο, O-diethyl O-[6-methyl-2-(1-

methylethyl)-4-pyrimidinyl] ester,

has mainly

been used extensively as an insecticide or

nematicide for the control of a broad range

of insects and pests, playing a distinguished

role in agriculture and veterinary. It is a non

systemic pesticide with action through

stomach or suffocation. The main paths of

its degradation in the environment are ox-

idation in air (Schomburg

et al.

, 1991; Seiber

et al.

, 1993) and hydrolysis, photolysis and

biodegradation in water and soil (Domagal-

ski and Kuivila, 1993; Frank

et al.

, 1991; How-

ard, 1991). The half-life of diazinon in plants

has been measured from 1 to 14 days while

in tissues with high fat concentration has

been found to be more persistent (Bartch,

1974). The corresponding time in animals is

too short with a maximum of 12 hours half-

life. The pesticide is rapidly excreted though

urine and feces, with the 70% of the total ex-

cretion to be consisted of metabolites (US

Public Health Service, 1995). Moreover, di-

azinon has an intermediate toxicity to mam-

mals and may affect the central nervous

system, the cardiovascular system and the