Folia Microbioi. 44 (1), 77-84 (1999) w~a. b i o m e d , c a s .

c z / t a b u / f o l i a / i n d e x , htm

Fungal Utilization of Organophosphate Pesticides

and Their Degradation by Aspergillusflavus
and A. sydowii in Soil
H.A.H. HASAN

Botany Depanmeng Faculty of Science, Assiut University, Egypt

Received June 1s 1998

Revised version August 1~ 1998

ABSTRACT. Fungal species were isolated which utilize organophosphate pesticides, v/z. phosphorothioic (pirimiphos-methyl
and pyrazophos), phosphorodithioic (dimethoate and malathion), phosphonic (lancer) and phosphoric (profenfos) acid deriva-
tives. Pesticide degradation was studied/n vitro and in vivo (soil).Aspergillus flavus, A. fumigatus, A. niger, A. sydowii, A. terreus,
Emericella nidulans, Fusarium oxysporura and Penicillium chrysogenum were isolated from pesticide-treated wheat straw. The
number ofA. sydowii colonies was significantly promoted by I mmol/L pirimiphos-methyl, pyrazophos, lancer, dimethoate and
malathion when used as phosphorus sources and by pirimiphos-methyl and pyrazophos when used as carbon sources. The
number of A. flavus colonies increased with 0.5 mmol/L lancer and malathion used as the only carbon sources. A. sydow//,
A. niger, A. flavus, E. nidulans and F. oxysporum grew on, and utilized, 5 pesticides as phosphorus source and showed more than
50 % mass growth. A. sydowii, A. flavus and F. oxysporum phosphatase hydrolyzed the pesticides suggesting that these species
are important pesticide degraders. A. sydowii produced higher amounts of the phosphatase than A. flavus and F. oxysporum.
The enzyme was highly active against pyrazophos, lancer and malathion used as the only sources of organic phosphate. A. fla-
vus and A. sydowii phosphatases efficiently hydrolyzed pesticides at 300 ppm in soil, the degradation at 1000 ppm was lower.
Mineralization of 1000 ppm pesticides in soil amended with wheat straw was higher than in nonamended soil. All added pesti-
cides except profenfos were degraded within 3 weeks. Lyophilized adapted biomass ofA. flavus and A. sydow//could thus be
used for field biodegradation of these pesticides.

Successive applications of pesticides may result in combinations of pesticide residues in plants

or soils, which may cause premature inactivation of a pesticide, crop damage, or the formation of new
complex residues. Biodegradation of the residues in soil is therefore highly desirable.
Organophosphate residues in biosphere give rise to concern over their ultimate environmental
fate and possible recalcitrance. Cleavage of the inherently stable C - P bond is the central requirement
for complete organophosphate mineralization (Schowanek and Verstraete 1990).
Most studies of organophosphate fungicides, herbicides, and insecticides have been devoted to
their action on microorganisms (Abdel-Mallek 1984; Hasan 1988; (~erfi~ikovfiet al. 1992; Hasan and
Omar 1993; Ismail et al. 1995). A few studies on utilization of organophosphate and other pesticides by
bacteria have been conducted (Wanner 1994; ~erh~ikovfi 1995; McGrath et al. 1997) but no studies
exist on the biodegradation of organophosphate pesticides by fungi.
We isolated and screened fungi utilizing organophosphate pesticides. Efficient utilizers could
subsequently be used along with organophosphate pesticides in soil to serve as phosphate fertilizer for
crop production. Several experiments in this direction are running in vitro and in vivo.

M A T E R I A L S A N D METHODS

Organophosphate pesticides. Six pesticides belonging to 4 groups of organophosphate were

used:
(1) Phosphorothioic acid derivatives included the insecticide pirimiphos-methyl [O,O-di-
methyl-O-(2-diethylamino-6-methylpyrimidin-4-yl)phosphorothioate] (Plant Protection Division) and
the fungicide pyrazophos [O,O-diethyl-O-(5-methyl-6-ethoxycarbonyl-pyrazolo(1,5-a)pyrimidin-2-yl)-
phosphorothioate] (Hoechst Orient S,4.A).
(2) Phosphorodithioic acid derivatives included the insecticide dimethoate [O,O-dimethyl-
5-(N-methylcarbamoylmethyl)phosphorodithioate] (Kafr El-Zayat) and the insecticide malathion
[O,O-dimethyl-5-(1,2-dicarbethoxyethyl)phosphorodithioate] (EI-Naser Chemical Co).
(3) Phosphonic acid derivative was the herbicide lancer [N-phosphonomethylglycine] (Mon-
santo).
(4) Phosphoric acid derivative was the insecticide profenfos [O,O-diethyl-O-(2-chloro-4-bro-
mophenyl)phosphorie acid] ( Ciba Geigy).
78 H.A.H.HASAN Vol. 44

Isolation and identification of wheat straw-borne fungi utilizing organophosphate pesticides as

phosphorus and carbon sources. The dilution-plate method (Johnson and Curl 1972) was used. Cza-
pek-Dox agar medium (g/L: sodium nitrate 3.0, magnesium sulfate 0.5, potassium chloride 0.5, agar
15.0) was used for isolation of fungi. Potassium dihydrogen phosphate (5 mmol/L) served as a phos-
phorus source and glucose (50 mmol/L) served as carbon source. When organophosphates were used
as sole phosphorus source, inorganic phosphate was replaced by the pesticides at 0.5, 1, 3 and
5 mmol/L. When the pesticides were used as the sole source of carbon, 5 mmol/L glucose was used as
control and replaced by pesticides at 0.5, 1, 3 and 5 mmol/L. Rose-bengal was added to the medium as
a bacteriostatic agent. Five plates were used for each concentration. The plates were incubated at 28 ~
for 1 - 5 weeks and the growing fungi were counted and identified according to Raper and Fennell
(1965) for Aspergillus, Booth (1977) for Fusadum, Christensen and Raper (1978) for Emericella and
Pitt (1985) for Penicillium species. The average number of colonies per dish was multiplied by the dilu-
tion factor to obtain the number of colonies per g of wheat straw.
Screening of fungal isolates for the ability to utilize organophosphate pesticides in enrichment liq-
uid medium. The culture medium (Czapek-Dox broth) contained (g/L): glucose 30, NaNO3 3, MgSO4
0.5 and KC1 0.5. KH2PO4 was omitted from the medium and replaced by the organophosphate pesti-
cides in a final concentration of 0.5 mmol/L. The pH of the media was adjusted to 7. 250-mL Eden-
meyer flasks containing 50 mL of a sterilized medium were inoculated with I mL of spore suspension
ofA. flavus, A. niger, A. sydowii, A. fumigatus, A. terreus, E. nidulans, F. oxysporum and P. chrysogenum.
Ammonium sulfate was used in culture media of A. niger and P. chrysogenum due to the NaNO3 toxic-
ity. The flasks were then incubated at 28 ~ on a shaking platform at a frequency of 1.7 Hz. After 7 d,
the cultures were filtered and dry mycelial mass was determined. Phosphate release into the culture
supernatant was monitored by the method of Olsen et al. (1954). Inoculated medium with KH2PO4
(0.5 retool/L) served as a standard against which fungal growth on pesticides was scored. Three flasks
were used for each treatment and control.
Activity ofphosphatases against different pesticides. Three isolates from a pesticide-treated liq-
uid medium (tt. flavus, A. sydowii and F. oxysporum) were tested further for their ability to produce
phosphatase. The optimum conditions of Famurewa and Olutiola (1994) were used. Maximum phos-
phatase production was determined after 4 d of fungal growth in a medium containing 70 mmol/L
phosphate, 5 % glucose and 0.5 % ammonium sulfate. Mycelial mats were extracted by acetate-acetic
acid buffer (pH 4.5) or glycine-NaOH (pH 9.6). The homogenate was centrifuged at 10000g for
30 min at 4 *C. Phosphatase activity in pesticide hydrolysis was determined using the organophosphates
as sole substrates. The reaction mixture consisted of 2.5 mL of the substrate (1 mmol in 0.1 mol/L
sodium acetate-acetic acid buffer, pH 4.5, or glycine-NaOH buffer, pH 9.6) and 0.5 mL of enzyme
preparation. After 1 h at 25 ~ the released phosphates were determined as mentioned above. One
unit of phosphatase activity (1 nkat) is the amount of the enzyme which produced 1 nmol of phospho-
rus per second. Specific activity is given in nkat per mg dry mass.
Pesticide degradation in soil. Clay soil was obtained from the Botanical Garden, Faculty of Sci-
ence, Assiut University. It has the following properties: organic matter 1.64 %, total N 0.12 %, soluble
salts 0.42 %, pH 7.5. The soil was air-dried, passed through a 4 mm sieve, and remoistened with sterile
distilled water to 20 % to permit good aeration. Amounts of 100 g of nonsterile soil were packed in
polythene bags. The following variants were set up in triplicate: soil + pesticides (control I), soil +
pesticides + inoculum (test I). Each pesticide was added at 300 and 1000 ppm. The soil was also
amended with wheat straw in triplicate: soil + wheat straw + 1000 ppm pesticides (control II), soil +
wheat straw + pesticides + inoculum (test II). Inoculations were done with 2 mL of spore suspension
(A. flavus and A. sydowii). The bags were incubated at 25 ~ Weekly, the available phosphorus was
extracted with NaHCO3 (0.5 mol/L). Phosphorus in the filtrate was determined by the colorimetric
molybdate blue method of Olsen et al. (1954). The phosphorus produced by the fungi in test I was
compared to the phosphorus produced in control I and that produced in test II was compared to
control II.
Statistical analysis of the results. Triplicate data of each experiment were analyzed statistically
using one-way analysis of variance (PC program).
1999 FUNGAL UTILIZATIONOF ORGANOPHOSPHATEPEA'TICIDES "19

RESULTS A N D DISCUSSION

Pesticide utilization as phosphorus source

The utilization by wheat straw mycoflora of different pesticides containing the carbon-phos-
phorus bond as sole phosphorus sources is shown in Table IA. Aspergillus flavus, A. fumigatus, A. niger,
A. sydowii, A. terreus, Emericella nidulans, Fusatium oxysporum and Penicillium chrysogenum were able
to utilize 0.5 mmol/L organophosphate pesticides as phosphorus source.
A. flavus and A. sydowii utilized 1 mmol/L phosphorothioic (pirimiphos-methyl and pyra-
zophos), phosphorodithioic (dimethoate and malathion) and phosphouic (lancer) acid derivatives after
1 week, and 1 mmol/L phosphoric (profenfos) acid derivative after 5 weeks of incubation. P. chrysoge-
hum utilized 1, 3 and 5 mmol/L pirimiphos-methyl, dimethoate, and malathion. A. fumigatus utilized
pirimiphos-methyl and lancer, F. oxysporum used malathion and lancer, and E. nidulans used lancer in
concentration of 1, 3 and 5 mmol/L as sole phosphorus sources.
Screening of fungal species utilizing pesticides showed that more than 75 % species utilized
pirimiphos-methyl, dimethoate, malathion and lancer at 1 mmol/L and more than 50 % utilized these
compounds at 3 mmol/L (Table IC). More than 50 % utilized pirimiphos-methyl, malathion and lancer
at 5 mmol/L. Pyrazophos was utilized by 50 % at 1 mmol/L and less than 25 % at 3 and 5 mmol/L.
Profenfos at 1 mmol/L was utilized by 25 % of fungal species after 5 weeks and not utilized before thi~
period. More than 60 % ofA.flavus andA. sydowii isolates utilized 5 mmol/L malathion and lancer as
phosphorus sources. A. sydowii also utilized all pesticides at 1 mmol/L.

Pesticide utilization as carbon source

The ability of wheat straw mycoflora to use organophosphate pesticides as a sole carbon
source is shown in Table lB. A. sydowii utilized pirimiphos-methyl and pyrazophos (phosphorothioie
acid derivatives) at 0.5, 1 and 3 mmol/L, dimethoate (phosphorodithioic acid derivative) at 0.5 and
1 mmol/L after 1 week of incubation. Malathion and lancer (0.5, 1 and 3 retool/L) were used as carbon
source by A. flavus. Profenfos (0.5 mmol/L) preserved the growth of A. sydowii and A. flavus after
5 weeks of incubation.
16-33 % of fungal species utilized the pesticides as a sole carbon source (Table IC). More
than 85 % ofA. flavus isolates utilized 3 mmol/L lancer and more than 75 % ofA. sydowii isolates uti-
lized pirimiphos-methyl, and their colony number increased over the control at 1 mmol/L. Also, A. sy-
dowii colony count increased 3-fold compared to control with 1 mmol/L pyrazophos.

Pesticide utilization in enrichment culture

In liquid enrichment cultures of eight fungal species supplied with 0.5 mmol/L pesticides
(Table II), seven species grew on pirimiphos-methyl and pyrazophos and gave 58-81 and 50-101% of
dry mass, respectively, compared to the control (0.5 mmol/L KH2PO4). Five species utilized malathion
and gave 60-115 % dry mass. Also, four species utilized lancer and dimethoate and gave more than
50 % of dry mass. A. sydowii followed by F. oxysporum,A, niger, E. nidulans andA. flavus were the best
degrading species. A. fumigatus, A. terreus and P. chrysogenum also utilized the pesticides but produced
less than 50 % growth mass. Zboinska et al. (1992) found that P. citrinum in liquid media did not use
the herbicide lancer in 0.5 mmol/L. This may be explained by the inhibitory effect of sodium nitrate
which was used as a nitrogen source. In our experiments A. niger and P. chrysogenum grew in a medium
containing ammonium sulfate as nitrogen source instead of sodium nitrate.
The enrichment cultures thus utilized organophosphates as a sole source of phosphorus but
their metabolism was not accompanied by a detectable release of inorganic phosphate. This may be
due to the incorporation of soluble phosphorus into the fungal biomass. Shishkina and Trotsenko
(1991) found that the phosphorus residue participates in the synthesis of ATP from ADP as well as in
the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate.

Phosphatase activity against pesticides

The metabolism of organophosphate pesticides includes oxidation of dithiophosphates to thio-
phosphates ( S - P = S ---* O - P = S ) and thiophosphates to phosphates ( O - P = S ~ O - P = O ) by oxid-
ase. Enzymic hydrolysis is probably the most important mechanism for the conversion of organic phos-
phate to inorganic phosphate in the soil (Brannon and Sommers 1985). The ability of phosphatases to
80 H.A.H. HASAN Vol. 44

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 1999 FUNGAL UTILIZATION OF ORGANOPHOSPHATE PF_SI'ICIDES 83

participate in the hydrolytic detoxification of pesticides was assessed. A. flavus, A. sydowii and F. oxy-
sporum were investigated for the production of cellular and extracellular phosphatase and their activity
in hydrolyzing the organophosphate pesticides was determined (Table III), The ftmgal species were
able to produce both acid (EC 3.1.3.2) and alkaline (EC 3.1.3.1) phosphatases, the production of alkal-
ine phosphatases being higher. They produced also both extra- and intraceUular phosphatases, the
intraceUular activity being higher.

Table I l L Alkaline phosphatase activity of the fungal species hydrolyzing pesticides at 25 ~

A, flavus A, sydowii F, oxysporum

Pesticides P specific P specific P specific

produced EAa activity produced EA activity produced EA activity
lamol/h nkat/mg lamol/h nkat/mg lamol/h nkat/mg

Pirimiphos-methyl 890 _+ 10 247 25 1150 • 12 319 32 820 • 8 231 23

Pyrazophos 1270 • 14 353 35 2170 ~ 21 603 60 1240 ~- 11 344 34
Dimethoate 900 +_ 16 250 25 1170 *_ 18 325 32 780 • 7 217 22
Malathion 1090 • 30 303 30 1670 ~ 17 464 46 920 • 13 256 26
Lancer 1060 • 18 294 29 1820 • 14 506 51 1060 • 9 294 29
Profenfos 600 • 11 167 17 920 _* 12 256 26 600 • 14 167 17

aEnzyme activity, p h o s p h o r u s (in nkat) released by 1 m L extract,

The enzymes hydrolyzed

the pesticides suggesting that these Table IV. Biodegradation of pesticides in soil (20 % moisture content) by
A. flavus and A. sydowii after 1 week at 25 ~
species may play an important role
in the degradation of pesticides.
A. sydowii phosphatase was highly Net change in p h o s p h o r u s
active against pyrazophos followed Pesticides Conc. produced, lag,/g soila
by lancer and malathion. Profenfos ppm
A. flavus A. sydowii
was more resistant to phosphatase
degradation. This may be explained Pirimiphos-methyl 300 36.8 • 4.5 43.3 • 3.9
by the chlorobromophenol pro- 1000 13-5 • 1A 7.5 -+ 0.9
ducts which may inhibit phos- Pyrazophos 300 29.3 • 2.3 25.3 • 3.0
phatase activity. Famurewa and 1000 14-5 • 0.8 12.5 • 1.5
Olutiola (1994) suggest that the Dimethoate 300 25.0 • 1,2 25.0 -+ 2.0
phosphatase may possess cysteine 1000 5.0 • 0.6 10.0 • 1,0
Malathion 300 31.5 • 3.3 26.5 • 2.2
residues or disulfide bridges.
1000 11.3 • 1,1 8.8 • 1.2
Lancer 300 45.0 • 2.2 65.0 • 4.2
Pesticide degradation in soil 1000 7.0 • 0.3 7.5 • 0.8
Profenfos 300 41-5 • 3.2 39.5 • 3.1
Since the conditions in soil
1000 1.8 • 0.4 4.3 -+ 0.6
are much more complex than those
in synthetic media, the ability for aNet phosphorus produced = phosphorus produced in test (nonsterile soil
pesticide degradation in nonsterile + pesticides + inoculum) - phosphorus produced in control (nonsterile
soil was investigated. Two isolates soil + pesticides).
of phosphatase-producing fungi
were tested further for their ability to hydroly-ze the organophosphates in soil.
Soluble phosphorus increased distinctly under the action of A. flavus and A. sydowii (Tab-
le IV). Even in nonsterile soil these two species were effective in hydrolyzing pesticides. Phosphatases
produced byA. flavus andA. sydowii hydrolyzed 300 ppm pesticides more than 1000 ppm. The mineral-
ization of 1000 ppm pesticides in amended soil with wheat straw was higher than in nonamended soil
(Table V). All added pesticides except profenfos were degraded by the end of week 3 (data not shown).
A. flavus and A. sydowii are to our knowledge the first fungi isolated from wheat straw capable
of degrading organophosphate pesticides and utilizing these compounds as sole phosphorus and carbon
sources by releasing phosphorus from these pesticides through the action of their phosphatases. These
strains could be bencficial as a fungal inocutum for efficient hydrolysis of pesticides.
84 H.A.H. HASAN Vol. 44

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