FEMS Microbiology Letters 161 (1998) 29^35

Di¡erent toxic e¡ects of the sulfonylurea herbicides metsulfuron

methyl, chlorsulfuron and thifensulfuron methyl on £uorescent
pseudomonads isolated from an agricultural soil
a;b a;b;
Tina S. Boldt , Carsten S. Jacobsen *
a
Geological Survey of Denmark and Greenland, Department of Geochemistry, Thoravej 8, DK-2400 Copenhagen NV, Denmark
b
Microbiology Section, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Copenhagen F, Denmark

Received 21 November 1997; revised 18 January 1998; accepted 30 January 1998

Abstract

A total of 77 strains of genetically different fluorescent Pseudomonas strains were isolated from an agricultural soil. In pure
culture growth experiments the strains were screened for their ability to grow in various concentrations of the sulfonylurea
herbicides metsulfuron methyl, chlorsulfuron and thifensulfuron methyl. We found that the presence of the herbicides resulted
in a reduction of the growth of the fluorescent pseudomonads. Metsulfuron methyl was shown to be toxic to a major
proportion of the strains in low concentrations. Chlorsulfuron was found to be less toxic in low concentrations but toxic in high
concentrations. Thifensulfuron methyl was toxic only to a minority of the strains. Indirectly, the growth-reducing effect of the
sulfonylurea herbicides was shown to be caused by an inhibition of the enzyme acetolactate synthase. The enzyme is involved in
the synthesis of the branched amino acids valine, leucine and isoleucine, and we demonstrated that the toxic effects of the
sulfonylurea herbicides could be neutralized when the strains were grown in the presence of an excess amount of the three
amino acids. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

Keywords : Soil £uorescent pseudomonad; Toxicity; Sulfonylurea ; Metsulfuron methyl; Chlorsulfuron ; Thifensulfuron methyl

1. Introduction trum of weeds and have low toxicity to humans and

The sulfonylurea herbicides are a class of widely
used herbicides, and they are expected to get a larger
share of the market in the future. The sulfonylurea
herbicides have low application rates compared to
traditional herbicides, are e¡ective on a wide spec-
* Corresponding author. Tel.: +45 38 14 23 13;
Fax: +45 38 14 20 50; E-mail: csj@geus.dk
animals. The sulfonylurea herbicides exert toxic ac-
tivity by interfering with the enzyme acetolactate
synthase (ALS), which is speci¢c for plants and mi-
croorganisms [1]. The enzyme is involved in the syn-
thesis of the branched amino acids leucine, valine
and isoleucine [2].
The sulfonylurea herbicides have been developed
from the triazine herbicides and have the triazine
structure in common with other triazine herbicides,
e.g. atrazine [1] which has been used widely in corn
and spruce plantations.

0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 4 5 - 7

FEMSLE 8055 19-3-98

30 T.S. Boldt, C.S. Jacobsen / FEMS Microbiology Letters 161 (1998) 29^35
The pseudomonads are a group of bacteria known
to play important ecological roles in the soil habitat
[3]. Among the roles of the £uorescent pseudomo-
nads of commercial interest are their ability to act
as antagonists against plant pathogens because of
their production of siderophores and antibiotics
[4,5].
An unintended consequence of the application of
the herbicides could be the in£uence on the microbial
ecological balance of the soil, leading to a signi¢cant
change of the quality of the population. This could
end up a¡ecting the productivity of crops negatively.
In assessing the in£uence of chemicals on the in-
digenous population of £uorescent pseudomonads
the isolation of identical clones would lead to tedious
repetition of the toxicological tests. An easy group-
ing of culturable soil bacteria can be done using
repetitive extragenic palindromic polymerase chain
reaction (REP-PCR) [6]. The genome of bacteria
consists of about 1% of conserved palindromic se-
quences at about 35 bp in transcribed but not trans-
lated areas of the genome [7]. By using these sequen-
ces in a PCR reaction the DNA between the
palindromes will become ampli¢ed. The DNA can
be separated by gel electrophoresis and create a
strain speci¢c pattern [8].
In this study we isolated 77 strains belonging to
the group of £uorescent pseudomonads from an
agricultural soil. The e¡ect of the sulfonylurea herbi-
cides metsulfuron methyl, chlorsulfuron and thifen-
sulfuron methyl on the growth of the £uorescent
pseudomonads was studied in pure culture growth
experiments. The e¡ect of the sulfonylurea herbi-
cides was studied further in a medium consisting of
the amino acids of which the synthesis is shown to
be blocked in plants by the sulfonylurea herbicides
[2].
2. Materials and methods
2.1. Isolation of bacteria and culture conditions
The £uorescent pseudomonads used in this experi-
ment were isolated from an agricultural soil located
at Drengsted, Denmark as described previously [6]
on Gould S1 agar [9], a medium selective for £uo-
rescent pseudomonads.
FEMSLE 8055 19-3-98
By the API 20 NE test system (BioMeèrieux SA,
Marcy L'Etoile, France) it was veri¢ed that the iso-
lated strains belonged to the group of £uorescent
pseudomonads. The manufacturer's instructions
were followed.
In the following experiments, single colonies of the
strains were obtained from the frozen culture by
streaking on Luria-Bertani agar supplemented with
0.1% (w/v) glucose. Unless otherwise stated, the cul-
tures were incubated at 25³C.
2.2. Toxicity tests on the isolated strains
Metsulfuron methyl, chlorsulfuron methyl (Riedel-
de-Haeën, Seelze, Germany) and thifensulfuron meth-
yl (Dupont, Nambsheim, France) were dissolved in
acetonitrile to ¢nal concentrations of 4 mg ml31 .
From these stock solutions, the herbicides were
transferred to MicroWell plates (Nunc, Naperville,
IL, USA), leading to concentrations of 0, 5, 50 or
300 ppm (0, 13.11 WM, 131.11 WM or 0.79 mM re-
spectively) in 300 Wl bacterial isolate suspension with
OD600 = 0.15 in Davis minimal medium. The tests
were run in triplicate. The acetonitrile was allowed to
evaporate before inoculation with the bacterial iso-
late suspension. Cell growth was measured spectro-
photometrically (600.0 nm) by a microplate reader
(Microplate bio-kinetics reader EL 312e, Bio-Tek In-
struments).
2.3. Neutralization of the toxic e¡ect of metsulfuron
methyl by the addition of valine, leucine and
isoleucine
Strains of the £uorescent pseudomonads belonging
to di¡erent REP-PCR groups (see below) were inves-
tigated for their ability to compensate for the
reduced production of valine, leucine and isoleucine
by assimilation of the amino acids from the me-
dium.
The amino acids valine, leucine and isoleucine
were added to a ¢nal concentration of 1 mM each
to MicroWell plates containing metsulfuron methyl
and bacterial isolate suspensions as described in Sec-
tion 2.2. The e¡ect of the addition of the three amino
acids on the cell growth was measured spectrophoto-
metrically as described in Section 2.2. The tests were
run in triplicate.
 T.S. Boldt, C.S. Jacobsen / FEMS Microbiology Letters 161 (1998) 29^35 31

2.4. Characterization by repetitive extragenic
palindromic polymerase chain reaction
REP-PCR [10] was used to verify that the isolated
strains of £uorescent pseudomonads were genetically
di¡erent. Template DNA was prepared, PCR reac-
tion performed and fragments visualized as described
previously [6].
2.5. Analysis of REP-PCR results
The photographs of the gels were scanned into the
program CREAM 1.0A (for Windows. Kem-En-Tec
A/S, Copenhagen, Denmark). The program calcu-
lated the size of the ampli¢ed sequences and the
results were veri¢ed manually. A binary matrix was
created in which 1 is the presence of the PCR prod-
uct and 0 is the absence of the PCR product.
NTSYSpc 1.80 (Applied Biostatistics Inc., Setauket,
NY) was used to calculate simple matching qualita-
tive coe¤cients and afterwards to create a dendro-
gram (complete clustering) in accordance with the
similarity of the di¡erent strains of REP-PCR prod-
ucts [11,12]. Dendrograms with single and un-
weighted pair-group method with arithmetic aver-
ages (UPGMA) clustering were calculated using
NTSYSpc 1.80 to evaluate dissimilarities between
cluster methods [13].
3. Results
3.1. Toxicity tests on the isolated strains
In a random and diverse group of soil bacteria, 77
strains belonging to the group of £uorescent pseudo-
monads were tested for their ability to grow in in-
creasing concentrations of the sulfonylurea herbi-
cides metsulfuron methyl, chlorsulfuron and
thifensulfuron methyl.
The e¡ect of the sulfonylurea herbicide metsulfur-
on methyl on the growth of one of the isolated
strains designated TBD7 is shown in Fig. 1. The
¢gure shows that the presence of 50 and 300 ppm
metsulfuron methyl reduced the growth of strain
TBD7, whereas the growth was not reduced in the
presence of 5 ppm. The strain was not a¡ected by
300 ppm chlorsulfuron or thifensulfuron methyl.

Fig. 1. Bacterial growth of the £uorescent pseudomonad TBD7 measured as optical density (OD = 600.0 nm) in media containing various
concentrations of the sulfonylurea herbicide metsulfuron methyl. The bars indicate standard deviations of three replicates.

FEMSLE 8055 19-3-98

32 T.S. Boldt, C.S. Jacobsen / FEMS Microbiology Letters 161 (1998) 29^35

Fig. 2. The concentrations of the sulfonylurea herbicides metsulfuron methyl, chlorsulfuron and thifensulfuron methyl needed to cause a
reduction in growth of the 77 strains of £uorescent pseudomonads isolated from soil. N.T.E. : no toxic e¡ect observed at 300 ppm herbi-
cide.

The results of the toxicity tests revealed di¡erences
in sensitivity among the strains and di¡erences in
toxicity of the sulfonylurea herbicides. This is out-
lined in Fig. 2. Metsulfuron methyl had a reducing
e¡ect on the growth of 76 out of the 77 isolated
strains of £uorescent pseudomonads. The growth
of 15 strains was a¡ected by metsulfuron methyl at
a concentration of 5 ppm (19.5%). Further, 48
strains were a¡ected by a concentration of 50 ppm
(62.3%).
Thifensulfuron methyl was shown to be toxic to
only a minor proportion of the strains. Seven strains
were shown to be a¡ected by thifensulfuron methyl
at concentrations up to 300 ppm (10%). Of these
seven strains, three were a¡ected by 50 ppm, and
one strain was a¡ected by 5 ppm thifensulfuron
methyl.
Chlorsulfuron was shown to a¡ect the growth of
the strains only in high concentrations. Altogether,
54 strains showed reduced growth in the presence of
up to 300 ppm chlorsulfuron. Three strains were af-
fected by 5 ppm, and 11 strains were a¡ected by 50
ppm chlorsulfuron.
No correlation was found between the strain and
its general sensitivity or tolerance towards all three
herbicides.
The dendrogram revealed that the e¡ect of the
sulfonylurea herbicides was not restricted to a par-
ticular REP-PCR group of the £uorescent pseudo-
monads, in that the sensitivity towards the three her-
bicides was not dependent on the strains' placement
in the di¡erent clusters (data not shown).
3.2. Neutralization of the toxic e¡ect of metsulfuron
methyl by the addition of valine, leucine and
isoleucine
Four randomly chosen strains belonging to di¡er-
ent REP-PCR groups, which had been shown to be
a¡ected by the presence of metsulfuron methyl, were
grown in the presence of both metsulfuron methyl
and the amino acids leucine, valine and isoleucine.
For all of the four strains we showed that the met-
sulfuron methyl mediated reduction in growth was
neutralized by the further addition of valine, leucine
and isoleucine. The neutralization of the toxic e¡ect
of metsulfuron methyl for strain TBD7 is shown in
Fig. 3. Fig. 3 shows that the expected growth reduc-

FEMSLE 8055 19-3-98

 T.S. Boldt, C.S. Jacobsen / FEMS Microbiology Letters 161 (1998) 29^35 33

Fig. 3. Bacterial growth of the £uorescent pseudomonad TBD7 measured as optical density (OD = 600.0 nm) in media containing various
concentrations of the sulfonylurea herbicide metsulfuron methyl and the amino acids valine, leucine and isoleucine. The bars indicate
standard deviations of three replicates.

ing e¡ect (Fig. 1) was neutralized by the further ad-
dition of valine, leucine and isoleucine. In the pres-
ence of both herbicide and amino acids the growth
did not depend on the concentration of the herbi-
cide.
4. Discussion
We showed that the presence of the sulfonylurea
herbicides metsulfuron methyl, chlorsulfuron and
thifensulfuron methyl had a growth reducing e¡ect
on a number of strains belonging to the group of
£uorescent pseudomonads. Metsulfuron methyl was
generally shown to be toxic to most of the strains at
low concentrations, whereas an e¡ect of chlorsulfur-
on was seen generally at higher concentrations only.
Thifensulfuron methyl was generally toxic only to a
minority of the strains. It was found that the sensi-
tivity to inhibition of the herbicides varied greatly
among strains. Previously, it has been shown that
sulfonylurea herbicides can have a toxic e¡ect on
microorganisms [14,15]. Chlorsulfuron has been re-
ported to be toxic in concentrations ranging from
3 WM [16] to 2.8 mM [14] to di¡erent microorgan-
isms. The results of Forlani et al. [15] show that
di¡erent strains of pseudomonads had di¡erent tol-
erances to chlorsulfuron. Burnet and Hodgson [14]
showed a di¡erence in toxicity of the sulfonylurea
herbicides sulfometuron methyl and chlorsulfuron
in that sulfometuron methyl reduced the growth of
a larger number of strains than did chlorsulfuron.
The di¡erence in strain sensitivity and herbicide
toxicology might be due to di¡erent genetic and
physiological strategies of the bacteria. The bacteria
might contain genes encoding di¡erent isoenzymes of
ALS [14,16]. Furthermore, the physical interactions
between enzyme and herbicide might be di¡erent.
Results of Burnet and Hodgson [14] indicate that
the membrane acts as a barrier in keeping the sulfo-
nylurea herbicides out of the cell. The authors found
that although chlorsulfuron and sulfometuron meth-
yl often a¡ected the growth of the bacteria di¡er-
ently, their e¡ects on ALS activity of toluenized cells
were similar. The greater toxicity of sulfometuron
methyl might be due to a greater hydrophobicity,
and thereby greater permeability, of this herbicide
than of chlorsulfuron [1]. Furthermore, Forlani et

FEMSLE 8055 19-3-98

34 T.S. Boldt, C.S. Jacobsen / FEMS Microbiology Letters 161 (1998) 29^35
al. [15] found a discrepancy between the in vivo tol-
erance of the cell and a high sensitivity of the enzyme
in that the addition of a detergent increased the tox-
icity of some sulfonylurea herbicides.
The e¡ects of sulfonylurea herbicides on microor-
ganisms and their activity in soil habitats have been
elucidated in several investigations. In general, the
observed e¡ects have been minor and temporary.
The e¡ects on nitri¢cation and dehydrogenase activ-
ity are often reported to be small or absent with a
stimulation shortly afterwards [16]. Ismail et al. [17]
found that the microbiological populations decreased
and later increased. This observed stimulation of the
enzymatic activity and the increase in the popula-
tions might be due to an increased supply of nu-
trients in the form of weeds and microorganisms
killed by the herbicide. The quick recovery of the
enzymatic activities after the application of the her-
bicides is not necessarily due to the activity of the
indigenous soil bacteria normally active. Forlani et
al. [15] showed that the application of a sulfonylurea
herbicide changed the composition and colonization
of introduced strains to the rhizosphere. Repeated
applications of these herbicides could result in a
change in the microbiological structure, leading to
a change in the quality of the soil if the a¡ected
bacteria are bene¢cial to plant growth. Rovira and
McDonald [18] showed that application of chlorsul-
furon increased root damage in wheat and barley by
Rhizoctonia solani. This could be the result of the
ousting of antagonistic microorganisms. We showed
that the e¡ect of the sulfonylurea herbicides was not
restricted to a particular biovar or REP-PCR group
of the £uorescent pseudomonads but was rather a
broad e¡ect.
We showed indirectly that the reduction in growth
as a result of the presence of the herbicides was due
to the inhibition of the enzyme ALS. This is because
the addition of the amino acids the synthesis of
which is blocked was shown to neutralize the growth
reducing e¡ect. We showed that the microbiological
growth inhibiting e¡ect of sulfonylurea herbicides
was a simple inhibition of the enzyme ALS. The
further addition of the amino acids valine, leucine
and isoleucine was shown not to neutralize the her-
bicides' toxic e¡ect on plant cells [19], indicating a
more complex toxic e¡ect of the sulfonylurea herbi-
cides in plants than in bacteria.
FEMSLE 8055 19-3-98
Acknowledgments
The authors thank farmer H.P. Jacobsen,
Sk×rb×k, Denmark, for providing the soil, and Du-
Pont for providing technically pure thifensulfuron
methyl. We also thank Janne HÖybye for correcting
the manuscript. This work was supported by the
Danish Research Council Grant J. 9400352 under
the Danish Interministries Research Programme on
Pesticides.
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