Effects of selected herbicides on soil microbial populations in oil palm plantation of Malaysia: A microcosm experiment

Abstract

Herbicides are commonly used in Malaysia to control weeds in oil palm plantation. In addition to their impact on weeds, these herbicides are also affecting soil microorganisms which are responsible for numerous biological processes essential for crop production. In the present study, we assessed the impact of four commonly used herbicides (paraquat, glyphosate, glufosinate-ammonium and metsulfuron-methyl) on soil microbial populations in oil palm plantation. Our study showed that the herbicide treatments significantly inhibited the development of microbial populations in the soil, and the degree of inhibition closely related to the rates of their applications and varied with the types of herbicide. Paraquat caused the highest inhibitory effect to bacteria and actinomycetes, whereas fungi were most affected by glyphosate. Metsulfuron-methyl had least inhibitory effects to all the microbial populations. The highest inhibition (59.3%) for fungal population was observed at 6 DAT (days after treatment), whereas for the bacteria and actinomycetes (82.0 and 70.6%, respectively) were at 4 DAT. Increasing trend of inhibition on growth of microbial populations was observed from the initial effect until 6 DAT, followed by a drastic decrease of the inhibition at 10 DAT. No inhibition was observed at 20 DAT. The study suggests that the herbicide application to soil of oil palm plantation cause transient impacts on microbial population growth, when applied at recommended or even as high as double (2x) of the recommended field application rate.

Full text

African Journal of Microbiology Research Vol. 7(5), pp. 367-374, 29 January, 2013
Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR12.1277
ISSN 1996 0808 ©2013 Academic Journals
Full Length Research Paper
Effects of selected herbicides on soil microbial
populations in oil palm plantation of Malaysia: A
microcosm experiment
Nur Masirah Mohd. Zain1, Rosli B. Mohamad1*, Kamaruzaman Sijam2,
Md. Mahbub Morshed1* and Yahya Awang1
1Department of Crop Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang,
Selangor D.E., Malaysia.
2Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang,
Selangor D.E., Malaysia.
Accepted 9 January, 2013
Herbicides are commonly used in Malaysia to control weeds in oil palm plantation. In addition to their
impact on weeds, these herbicides are also affecting soil microorganisms which are responsible for
numerous biological processes essential for crop production. In the present study, we assessed the
impact of four commonly used herbicides (paraquat, glyphosate, glufosinate-ammonium and
metsulfuron-methyl) on soil microbial populations in oil palm plantation. Our study showed that the
herbicide treatments significantly inhibited the development of microbial populations in the soil, and the
degree of inhibition closely related to the rates of their applications and varied with the types of
herbicide. Paraquat caused the highest inhibitory effect to bacteria and actinomycetes, whereas fungi
were most affected by glyphosate. Metsulfuron-methyl had least inhibitory effects to all the microbial
populations. The highest inhibition (59.3%) for fungal population was observed at 6 DAT (days after
treatment), whereas for the bacteria and actinomycetes (82.0 and 70.6%, respectively) were at 4 DAT.
Increasing trend of inhibition on growth of microbial populations was observed from the initial effect
until 6 DAT, followed by a drastic decrease of the inhibition at 10 DAT. No inhibition was observed at 20
DAT. The study suggests that the herbicide application to soil of oil palm plantation cause transient
impacts on microbial population growth, when applied at recommended or even as high as double (2x)
of the recommended field application rate.
Key words: Herbicides, soil microbes, soil microcosm, field application rate, oil palm plantation.
INTRODUCTION
Soil, an important component of the ecosystem, serves
as a medium for plant growth through the activity of
microbial communities. This soil microbial communities
(like bacteria, fungi and actinomycetes) play critical role
in litter decomposition and nutrient cycling, which in turn,
affect soil fertility and plant growth (Singh et al., 1999;
Chauhan et al., 2006; Tripathi et al., 2006; Pandey et al.,
*Corresponding authors. E-mail: rosli@agri.upm.edu.my;
mmorshed_bd@yahoo.com. Tel: 603- 89474831. Fax: 603-
89474918.
2007). However, soil micro-organisms are greatly
influenced by factors including the application of
herbicides (Pampulha et al., 2007), which are applied in
modern agricultural practices to attain optimum crop
yields (Zabaloy et al., 2008). If, microorganisms are
sensitive to particular herbicide, its application will
interfere with vital metabolic activities of microbes
(Oliveira and Pampulha, 2006), thus affect the availability
of nutrients in the soil (Nautiyal, 2006). Numerous studies
have shown the effect of herbicides on soil micro-
organism populations that ultimately affect the rates of
decomposing labile, celluloses and recalcitrant like lignin,
respectively, in a variety of ecosystems (Taylor and

368 Afr. J. Microbiol. Res.
Parkinson, 1988; Tripathi and Singh, 1992a,b; Pandey et
al., 2007; Osono et al., 2003; Osono and Takeda, 2007;
Osono et al., 2008). Although, their accurate numbers are
still not very clear mainly because of rapid changes in the
populations (Chauhan et al., 2006; Das et al., 2006), but
a healthy population of microorganisms can stabilize the
ecological system in soil (Chauhan et al., 2006). Thus,
the changes in the population of these micro-organisms
will affect the ability of the soil to regenerate nutrients to
support plant growth.
Malaysia is the world’s largest producer and exporter of
palm oil that covers over 5 million hectares of land
(MPOB, 2011). Weed management is a major problem in
the oil palm plantation during the immature phase to
avoid suppression of growth and late yield of the oil palm
(Chee et al., 1992), so the herbicides are frequently used
to manage weeds. Most commonly used herbicides are
paraquat, glufosinate-ammonium, glyphosate and
metsulfuron-methyl (Chuah et al., 2005; Kuntom et al.,
2007). The presence of herbicide residues in soil could
have direct impacts on soil microorganisms is matter of
great concern. At normal field recommended rates,
herbicides are considered to have no major or long-term
effect on microbial populations (Audus, 1964; Bollen,
1961; Fletcher, 1960). It has been reported that some
microorganisms were able to degrade the herbicide,
while some others were adversely affected depending on
the application rates and the type of herbicide used
(Wilkinson and Lucas, 1969; Sebiomo et al., 2011).
Therefore, effects of herbicides on microbial growth,
either stimulating or depressive, depend on the chemicals
(type and concentration), microbial species and
environmental conditions (Bollen, 1961; Hattori, 1973).
Studies on pesticide residual effects on soil
microorganisms are often done in soil microcosm small-
scale experiment which can be interpreted accurately at
larger scales (Benton et al., 2007). Microcosms
containing soil microfauna of field communities offer
higher resolution of ecotoxicological effects of chemicals
in soil environments (Parmelee et al., 1993). As the
precise assessment of the potential non-target effects of
herbicides on soil microorganisms in oil palm plantation
are of growing interest, therefore, soil microcosm can
provide better understanding of possible response of soil
microbes to herbicides. The study was aimed to evaluate
the effect of commonly used herbicide on bacterial,
fungal and actinomycetes populations in soil microcosms
from oil palm plantation.
MATERIALS AND METHODS
Herbicide treatments
The herbicide treatments consisted of paraquat (Gramoxone®
PP910), glyphosate (Roundup®), glufosinate-ammonium (Basta
15®) and metsulfuron-methyl (Ally® 20 DF). Three different
concentrations (rates) of each herbicide treatment: paraquat and
glufosinate-ammonium at 0.44, 0.88 and 1.76 mg a.i./g soil each;
glyphosate at 0.88, 1.76 and 3.52 mg a.i./g soil; and metsulfuron-
methyl at 0.015, 0.03 and 0.06 mg a.i./g soil were applied in this
study. These treatment rates represented 0.5, 1 and 2 times (x)
their recommended field rates (paraquat: 400 g a.i./ha; glufosinate-
ammonium: 400 g a.i./ha; glyphosate: 800 g a.i./ha; metsulfuron-
methyl: 15 g a.i./ha). The treatments were calculated using the
formula:
Soil sampling and preparation of microcosm
Soils were collected from a young oil palm (3 years old) area at
Universiti Putra Malaysia (UPM), Serdang, Selangor, Malaysia. The
site has a history of herbicide application at 6- months interval, and
the herbicide used is glyphosate (Roundup®). Eighty soil cores
(approximately 40 kg) were sampled to a depth of 15 cm using
auger, collected randomly from underneath the surrounding palms
and betw een the palm rows. The samples w ere mixed thoroughly to
form a composite sample and taken back to Microbiology
Laboratory, Department of Plant Pr otection, Faculty of Agriculture,
UPM, and processed accordingly. The pH value of sampled soil
was determined as 4.1 ± 0.01. Soil chemical properties were
determined w hich were as follows: 1.94% C, 0.32% N; 219 ppm P,
104 ppm K, 119 ppm Ca and 32 ppm Mg, and the soil was
classified as sandy clay (40% clay, 10% silt and 50% sand).
The microcosms were prepared according to Oliveira and
Pampulha (2006) with minor modifications. The soils w ere air-dried
slowly in laboratory environment (25°C; 50% RH) for 24 h before
sieving through a 2 mm mesh. The sieved soils were then analyzed
to estimate the moisture content and the Water Holding Capacity
(WHC). The laboratory determination of the moisture content of soil
samples was done by placing 10 g of soil sample in a weighing
glass beaker w as initially w eighed, followed by oven drying at 70°C
for 24 h. Glass beaker containing the dried soil w as then w eighed
again to get the final w eight of the soil. The moisture content w as
calculated as percentage using the formula:
Water Holding Capacity (WHC) of the soil w as determined by
placing 3 g of soil sample on a piece of Whatman filter paper w hich
had been initially w eighed, follow ed by oven drying at 70 °C for 24 h.
Oven-dried soil on the w eighed Whatman filter paper was weighed
before dipping into w ater until the soil w as saturated. The soil w as
then placed in humid enclosure to drain off the water before
weighing again, and calculated using the formula (ASTM, 2010):
The bulked soils w ith determined moisture content of 13% w ere
then mixed together, and 56 ml sterile distilled w ater was added to
achieve the moisture level of 18.5%, w hich was 50% of its
maximum MHC. The soil was then placed in 39 sterile glass bottles,
each containing 1 kg of soil. Each bottle w as loosely fit w ith cap to
allow gas exchange. The soil-containing glass bottles w ere then
incubated in dark, in a 25°C incubator, for 10 days to allow time for
adaptation of microorganisms before treatment with the herbicides.
The herbicide treatments w ere applied w ith the following
procedures, conducted aseptically under laminar flow unless stated

otherwise. 50 ml of each herbicide treatments were sprayed to 36
out of 39 glass bottle accordingly, using hand sprayer. The
herbicide w as mixed thoroughly by constant shaking for 5 min. The
remaining 3 glass bottle soils were served as control, and sprayed
with 50 ml sterile distilled w ater. The soil microcosms w ere then
formed by transferring the treated soils into each sterile square
plastic container (15 cm x 15 c m x 7.4 cm) w ith lids loosely fitted.
The soil microcosms were then incubated in darkness at 25°C.
Sterile distilled w ater was added on weekly basis to restore the
initial w eight of each microcosm, maintaining the constant moisture
content.
Enumeration of microbial population
Enumeration of the microbial populations was done using specific
media for each microorganism. Three different grow th media
supplemented with inhibitors w ere prepared: Potatoe Dextrose Agar
(PDA, Difco) supplemented w ith 30 mg/L streptomycin sulphate
(Sigma-Aldrich) for enumeration of fungi; Nutrient Agar (NA, Oxoid)
supplemented w ith 0.1g/L cyclohexamide ( Merck) for enumeration
of bacteria; and Actinomycetes Isolation Agar (AIA, Difco)
supplemented w ith 0.5 g/L cyclohexamide (Merck) for enumeration
of actinomycetes (Araujo et al., 2003). The inhibitors were added
into sterilized media (121°C, 15 min) accordingly, and mixed
thoroughly on hotplate and stirrer (Jenway) before pouring into
each Petri dish, marked at the bottom dividing it into three sections.
Soil w as collected from each microcosm at 2, 4, 6, 10 and 20
DAT (days after treatment) to assess the herbicidal effect on the
microbial populations present in the soil. Five sub-samples w ere
collected randomly from each microcosm treatment using sterile
cork borer (10 mm diameter). Sub-samples from each microcosm
were mixed together, and 1 g of the soil was taken to make a serial
dilution. Serial dilutions were made aseptically under laminar flow
by suspending the soil in 9 ml of sterile distilled w ater in a test tube
and vortexed us ing vortex mixer (Vision Scientific) for 30 s to
thoroughly mix them. This pr ocess w as repeated until the dilutions
were made up to 10-5 to complete the serial dilutions.
The drop plate method, conducted under sterile condition, w as
used for enumeration of the colonies. The test tubes of the serial
dilutions w ere vortexed before five drops (10 µL drop-1) of the
suspension w ere pipetted out onto each particular section of the
media ( marked by dividing lines) according to dilution value of the
suspensions. Dilutions selected for plating on PDA were 10-2 to 10-4
(for culturing fungi), whereas, NA and AIA were plated with the
dilutions of the 10-3 to 10-5 (for culturing bacteria and actinomycetes,
respectively). The plates w ere prepared in triplicates, covered and
allowed to dry. After 1 h, the plates were inverted, sealed w ith
parafilm to avoid contamination and incubated in darkness at 25°C.
Enumeration of colonies for bacteria, fungi and actinomycetes
were done using the Colony Counter (Rocker) after 24 h, 7 and 10
days, respectively. The total up of the colonies was used to
calculate the Colony-forming unit ( CFU)/g dry weight of soil. Dry
weight of soil was determined after oven drying at 70°C for 24 h
using the formula:
Dry weight of soil = (w eight of moist soil) X (1% moisture soil
sample/100), and the CFU was calculated using the formula:
Data analysis
The experiment w as conducted by Complete Randomized Design
Zain et al. 369
(CRD) w ith three replicates. Data w ere expressed as inhibition
percentages relative to the control, and analyzed follow ing 2-way
Analysis of Variance (ANOVA) betw een herbicides and each
exposure dates. Mea ns w ere compared using Duncan’s Multiple
Range Test (DMRT) at P<0.05 using Statistical Analysis System
(SAS).
RESULTS
The effect of herbicide treatments on soil microbial
population was determined based on the inhibition
percentages of the growth of fungal, bacterial and
actinomycetes colonies in each treatment media. The
growth inhibition showed an increasing trend with
increased herbicide concentrations, and the microbial
population showed different degree of sensitivity to the
herbicide compounds at different sampling dates
(exposure periods). The inhibition percentages of fungal
colony development by the herbicides relative to the
control (without herbicide treatment) were shown in Table
1. The inhibition percentage of fungi increased with
higher application rates of each herbicide. Highest
inhibitions of 63.1 to 81.4% were observed at 2x the
recommended field application rate. At 0.5x the
recommended field application rate, the herbicides
inhibited fungal development by 42.2 to 54.1%. At
recommended field application rate, these herbicides
could be considered as only moderately toxic to the
fungal colony development, causing moderate inhibition
of 54 to 59.3%. This indicated that applications of the
herbicides even at lower than the recommended field
rates could be moderately detrimental to the fungal
development in soil.
Moderately high inhibition percentages of the fungal
colony development of more than 44% were observed
within 2 DAT for the herbicides, except for paraquat.
Paraquat, however, caused significantly lower inhibition
(25.8%) at 2 DAT. The highest inhibition for paraquat of
54.3% was observed at 6 DAT, but was statistically
insignificant compared with the inhibition rate of
glyphosate and glufosinate-ammonium. Inhibition
observed for glyphosate and glufosinate-ammonium were
comparable at specific rates of application and times of
sampling. Subsequently, the inhibition percentages of the
fungal colony development at recommended field rate
were insignificant among the herbicides from 6 DAT
onwards. Inhibition of the fungal colony development was
abruptly low for all the treatments at 10 DAT, ranging
from 2.3 to 10.6%. The fungal colonies, therefore,
showed their ability to recover from the toxic effect by 10
DAT, and at 20 DAT, no further inhibition or full colony
recovery was observed.
Bacterial population development in soil was also
affected significantly until 10 DAT by paraquat,
glyphosate, glufosinate-ammonium and metsulfuron-
methyl. The percentages of inhibition of the bacterial

370 Afr. J. Microbiol. Res.
Table 1. Effect of herbicide treatments on soil fungal population at five exposure periods in soil
microcosm.
Herbicide Treatments
(mg a.i./gm)
% Population inhibition relative to control
(Mean ± SE)
RFR
2 DAT
4 DAT
6 DAT
10 DAT
20 DAT
Paraquat
0.44
0.5x
14.8ef ± 2.7
23.3g ± 5.1
45.2cd ± 3.3
0.2e ± 4.2
0.0a ± 0.0
0.88
1x
25.8e ± 1.8
33.0fg ± 1.4
54.3bcd ± 5.0
2.3cde ± 0.7
0.0a ± 0.0
1.76
2x
44.2cd ± 1.4
59.1bc ± 6.0
63.1abc ± 2.9
12.4bcd ± 1.5
0.0a ± 0.0
Glyphosate
0.88
0.5x
37.9d ± 4.9
40.6def ± 4.9
54.1bcd ± 6.4
9.7bcde ± 1.2
0.0a ± 0.0
1.76
1x
44.0cd ± 0.9
46.1cdef ± 2.1
59.3abc ± 4.5
10.6bcde ± 1.3
0.0a ± 0.0
3.52
2x
57.8b ± 5.0
60.8bc ± 5.9
75.5a ± 4.0
14.4b ± 2.0
0.0a ± 0.0
Glufosinate-ammonium
0.44
0.5x
10.3fg ± 1.1
46.2cdef ± 3.9
44.1cd ± 8.2
0.6de ± 0.3
0.0a ± 0.0
0.88
1x
40.2d ± 5.6
55.6bcd ± 5.8
53.3bcd ± 8.6
5.1bcde ± 0.6
0.0a ± 0.0
1.76
2x
58.3b ± 4.3
81.4a ± 8.2
59.3abc ± 6.9
33.7a ± 6.5
0.0a ± 0.0
Metsulfuron-methyl
0.015
0.5x
42.2cd ± 5.0
36.7efg ± 6.4
35.2d ± 7.9
5.3bcde ± 8.5
0.0a ± 0.0
0.03
1x
54.0bc ± 7.0
53.7bcde ± 8.9
48.2cd ± 5.0
6.1bcde ± 1.3
0.0a ± 0.0
0.06
2x
71.1a ± 3.2
70.3ab ± 3.6
68.5ab ± 6.4
12.7bc ± 4.3
0.0a ± 0.0
Control
0.0g ± 0.0
0.0h ± 0.0
0.0e ± 0.0
0.0e ± 0.0
0.0a ± 0.0
Values in the same column follow ed by superscript similar letter(s) are not significantly different by DMRT
(P<0.05). Data are presented as mean values (standard error) of three replicates at each exposure period.
RFR, Recommended field rate; the rate which is recommended in the product label to apply in the field.
colony development relative to the control are shown in
Table 2. The herbicides caused higher inhibition to
bacterial population development compared with that of
the fungi. At all sampling times and treatment rates of the
herbicides, the inhibition percentages of bacterial
colonies were higher than those observed for the fungal
colony development, except for the glufosinate-
ammonium treatment at 4 DAT and 6 DAT.
The highest inhibitions of the bacterial population were
from 77.9 to 87.9%. These highest inhibitions, however,
were observed from the 2 times recommended field rate
for all herbicides. Treatment of herbicides at 0.5, 1 and 2
times their recommended field rate also indicated
increased inhibition percentages with the increased in the
herbicide rates, when sampled at 2 days after treatment
until 10 DAT. However, the lowest treatment at 0.5 times
the field recommended rate had also caused significantly
high inhibition of the colony development compared with
the control, and comparable with those of treatments at
recommended field rate.
At the recommended field rate, the herbicides could be
considered as moderately to highly toxic to bacterial
population. Highest inhibition of bacterial growth was
recorded at 68.7, 74 and 82% at 4 DAT for metsulfuron-
methyl, glyphosate and paraquat, respectively, and 73%
for glufosinate-ammonium at 2 DAT. However,
glufosinate-ammonium caused the maximum
suppression through growth inhibition of the bacterial
colony development (73%) at faster rate (2 DAT) than
paraquat, glyphosate and metsulfuron-methyl with 45.5,
55 and 67.3%, respectively. The inhibition percentages of
bacterial population for all treatments reduced
significantly by 10 DAT with a range of 8 to 22.8%. The
observations were comparable with that observed for the
fungal colony development discussed earlier. No
inhibition at 20 DAT indicates that the bacterial population
recovers from the earlier effects, similar to the fungal
population.
Paraquat, glyphosate, glufosinate-ammonium and
metsulfuron-methyl treatment to soil also affected the
development of actinomycetes population (Table 3). The
growth inhibition of actinomycetes colonies caused by the
herbicides was similar to those recorded for the fungi and
bacteria, which increased with the increased of the
herbicides application rates. However, treatments at 0.5x
and 1x the recommended field rates were significantly

Zain et al. 371
Table 2. Effect of herbicide treatments on soil bacterial population at five exposure periods in soil microcos ms.
Herbicide Treatments
(mg a.i./g)
% Population inhibition relative to control
(Mean ± SE)
RFR
2 DAT
4 DAT
6 DAT
10 DAT
20 DAT
Paraquat
0.44
0.5x
30.1e ± 1.0
73.6bc ± 2.5
62.2b ± 5.2
17.8bc ± 3.1
0.0a ± 0.0
0.88
1x
45.5d ± 3.0
82.0ab ± 3.5
67.9b ± 4.9
22.8ab ± 6.5
0.0a ± 0.0
1.76
2x
46.7d ± 3.7
87.9a ± 4.5
82.9a ± 2.6
32.9a ± 1.4
0.0a ± 0.0
Glyphosate
0.88
0.5x
52.8d ± 0.9
73.3bc± 4.7
63.5b ± 2.2
13.3bcd ± 4.9
0.0a ± 0.0
1.76
1x
55.0cd± 8.3
74.0bc ± 5.8
67.9b ± 4.4
14.6bc ± 2.7
0.0a ± 0.0
3.52
2x
67.0bc ± 0.8
81.0ab ± 2.6
83.7a ± 2.4
18.3bc ± 6.2
0.0a ± 0.0
Glufosinate-ammonium
0.44
0.5x
69.5ab ± 6.1
39.0d ± 3.4
27.1c ± 6.1
0.6d ± 0.6
0.0a ± 0.0
0.88
1x
73.0ab ± 3.7
48.0d ± 3.7
30.9c ± 2.3
8.0cd± 5.3
0.0a ± 0.0
1.76
2x
82.1a± 2.3
66.6c ± 4.4
35.7c ± 1.1
17.0bc± 2.1
0.0a ± 0.0
Metsulfuron-methyl
0.015
0.5x
53.8d ± 5.3
68.1c ± 3.5
58.9b ± 2.3
10.4bcd ± 6.2
0.0a ± 0.0
0.03
1x
67.3bc ± 3.9
68.7c ± 4.8
59.7b± 1.0
17.3bc ± 5.0
0.0a ± 0.0
0.06
2x
69.6ab ± 5.5
77.9abc ± 3.2
67.2b ± 7.4
21.1abc ± 1.8
0.0a ± 0.0
Control
0.0f ± 0.0
0.0e ± 0.0
0.0d ± 0.0
0.0d ± 0.0
0.0a ± 0.0
Values in the same column follow ed by superscript similar letter(s) are not significantly different by DMRT (P<0.05). Data are presented as
mean values (standard error) of three replicates at each exposure period. RFR, Recommended field rate; the rate which is recommended in
the product label to apply in the field.
lower than that at 2x the recommended field rate.
Herbicides, at rates recommended for use in the field,
were considered as moderately toxic to actinomycetes
population in soil. Highest inhibition at the recommended
field rate for all herbicides were 70.6, 47.0, 64.3 and
59.4% for paraquat, glyphosate, glufosinate-ammonium
and metsulfuron-methyl, respectively. These inhibition
percentages were observed by 4 DAT for paraquat,
glyphosate and metsulfuron-methyl, whereas it was
slower for glufosinate-ammonium, observed at 6 DAT. By
10 DAT, however, the inhibition rate for actinomycetes
were still relatively high, in comparison with that of the
earlier sampling period, and also to that of the fungal and
bacterial populations. This could indicate slower recovery
period of actinomycetes after the initial effect of the
herbicides. However, by 20 DAT, no further inhibition to
the actinomycetes population was observed for all
treatments, which indicate full recovery from the
treatment.
DISCUSSION
Herbicide treatments of paraquat, glyphosate,
glufosinate-ammonium and metsulfuron-methyl showed
significant effects on microbial growth and development
in soil environment. Significant increased of fungal,
bacterial and actinomycetes growth inhibition were
observed from 0.5x to 2x their recommended field
application rates, indicating a positive correlation
between growth inhibition and treatment rates. Bacterial
and actinomycetes populations were severely affected by
Paraquat which inhibited their population growth by 70 to
82% at recommended field rate. However, the fungal
population in soil was moderately inhibited (54.3%).
Paraquat has also been reported to inhibit several
microorganisms in soil by Smith and Mayfield (1977).
They reported that paraquat could inhibit a great number
of cellulolytic microflora and that might cause injurious
effects to symbiotic, anaerobic and nitrogen fixing
microorganisms. Paraquat is also known to be bounded
strongly and coherently to soil components, including clay
minerals and organic matter, therefore limits the access
of microorganisms to paraquat in soil water (Bromilow,
2003; Isenring, 2006). Thus, adsorption of paraquat to
soil rapidly decreases the bioavailability of the herbicide
in the soil environment and demonstrated the capability
of adsorption process to deactivate hundreds or even
thousands of paraquat application over many soil types
(Roberts et al., 2002). The sandy clay classification of the

372 Afr. J. Microbiol. Res.
Table 3. Effect of herbicide treatments on soil actinomycete population at five exposure periods in soil
microcosms.
Herbicide Treatments
(mg a.i./g)
% Population inhibition relative to control
(Mean ± SE)
RFR
2 DAT
4 DAT
6 DAT
10 DAT
20 DAT
Paraquat
0.44
0.5x
29.7cd ± 7.4
68.7ab ± 5.8
36.5d ± 6.5
26.3bc ± 3.1
0.0a ± 0.0
0.88
1x
31.1cd ± 8.5
70.6ab ± 7.2
45.1cd ± 4.4
31.4bc ± 2.5
0.0a ± 0.0
1.76
2x
40.6bc ± 4.3
82.5a ± 0.8
60.6b ± 8.9
47.4a ± 3.3
0.0a ± 0.0
Glyphosate
0.88
0.5x
11.9ef ± 6.0
38.2d ± 6.7
20.1e ± 3.0
11.2de ± 5.1
0.0a ± 0.0
1.76
1x
19.4de ± 7.2
47.0cd ± 3.4
22.7e ± 3.8
20.9cd ± 8.4
0.0a ± 0.0
3.52
2x
28.5cde ± 1.8
55.8bc ± 7.6
38.4d ± 4.1
25.6bc ± 7.9
0.0a ± 0.0
Glufosinate-ammonium
0.44
0.5x
0.0f ± 0.0
0.0e ± 0.0
60.9b ± 0.0
0.0e ± 0.0
0.0a ± 0.0
0.88
1x
0.0f ± 0.0
10.4e ± 2.0
64.3b ± 0.7
0.0e ± 0.0
0.0a ± 0.0
1.76
2x
11.9ef ± 6.0
46.5cd ± 6.8
79.2a ± 0.0
26.5bc ± 5.0
0.0a ± 0.0
Metsulfuron-methyl
0.015
0.5x
23.9cde ± 5.6
57.0bc ± 6.6
54.2bc ± 3.5
26.9bc ± 4.0
0.0a ± 0.0
0.03
1x
48.2b ± 4.6
59.4bc ± 6.3
57.5bc ± 6.4
38.8ab ± 1.7
0.0a ± 0.0
0.06
2x
69.1a ± 7.0
79.9a ± 1.8
63.3b ± 3.4
51.3a ± 4.8
0.0a ± 0.0
Control
0.0f ± 0.0
0.0e ± 0.0
0.0f ± 0.0
0.0e ± 0.0
0.0a ± 0.0
Values in the same column follow ed by superscript similar letter(s) are not significantly different by DMRT (P<0.05).
Data are presented as mean values (standard error) of three replicates at each exposure period. RFR,
Recommended field rate; the rate which is recommended in the product label to apply in the field.
experimental soils might have reduced the binding of
paraquat to soil components and thus increasing the
availability of paraquat in soil water, and hence affecting
the soil microorganisms significantly.
Glyphosate was observed to be less toxic than
paraquat to bacterial and actinomycetes populations. At
recommended field rate, it inhibited the bacterial
population by 74%. The inhibition of actinomycetes and
fungal populations were moderate with 47 to 59.3%.
Findings from this study were supported by other studies
(Anderson and Kolmer, 2005; Franz et al., 1997;
Mekwatanakarn and Sivasithamparam, 1987; Toubia-
Rahme et al., 1995; Turkington et al., 2001; Wong et al.,
1993; Wyss and Muller-Scharer, 2001). However, few
studies contradict this result (Busse et al., 2001; Muller et
al., 1981; Stratton and Stewart, 1992; Wardle and
Parkinson, 1990; Weaver et al., 2007). As a weed killer,
glyphosate targets a single enzyme called 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS)
(Franz et al., 1997) which plays important role in the
shikimic acid pathway responsible for biosynthesis of
aromatic amino acids, and this enzyme is widely present
in plants and microorganisms, including bacteria and
fungi (Kishore and Shah, 1998; CaJacob et al., 2004).
The presence of EPSPS proteins in bacteria and fungi,
therefore, made the microorganisms vulnerable to
glyphosate. CaJacob et al. (2004) also reported that
EPSPS proteins have been isolated and characterized
from microorganisms, which some can tolerate
glyphosate while others were sensitive to the herbicide.
Glufosinate-ammonium was considered to be more
toxic than glyphosate to the actinomycetes population.
The inhibition of bacterial population by glufosinate-
ammonium (73%) was considered as being equally toxic
compared with glyphosate (74%). The growth-inhibition
by glufosinate-ammonium could be due to negative
effects on the dehydrogenase activity of soil
microorganisms as explained by Pampulha et al. (2007),
and subsequent decline of growth-inhibition likely due to
the compound’s rapid degradation process in soil ( Ismail
and Ahmed, 1994). A study done by Ahmad and Malloch
(1995) reported that bacterial growth was reduced only
about 40% by glufosinate-ammonium herbicide in
agricultural soils. Similarly, the herbicide at
recommended field rate reduced the bacterial population
temporarily, as they recovered after 7 days (Ismail et al.,

1995). Pampulha et al. (2007) reported significant
inhibition in growth of actinomycetes, Streptomyces spp.,
within six days after application of the herbicide to soil
microcosms. In contrary, Ahmad and Malloch (1995)
obtained insignificant result for the effects of glufosinate-
ammonium towards soil actinomycetes.
Metsulfuron-methyl was observed to be the least toxic
to fungal and bacterial populations compared to
paraquat, glyphosate and glufosinate-ammonium.
However, the toxicity of metsulfuron-methyl to
actinomycetes population was higher than glyphosate,
but similar to paraquat and glufosinate-ammonium.
Metsulfuron-methyl could be considered as being
moderately toxic to bacterial, actinomycetes and fungal
populations at recommended field rate. Ismail et al.
(1996) showed that bacterial population decreased when
the concentrations of metsulfuron-methyl increased
during the first 3 to 9 days after application, depending on
soil types. However, Ismail et al. (1996) also
demonstrated increase in fungal population with
increasing metsulfuron-methyl concentrations, which may
be influenced by the soil type. El-Ghamry et al. (2000)
reflected the toxicity effect of metsulfuron-methyl when
the soil microbial biomass significantly decreased with
increasing concentrations of the herbicide, which could
either be due to toxicity effect and the adsorption of the
herbicide in soil or because the soil microorganisms were
not adapted to the herbicide itself.
In this study, the herbicide treatments to soil indicated
short term growth-inhibitory effects on soil microbial
population. The treatment effects on soil microbial
population growth over the five exposure periods
exhibited rapid decreasing trends after 6 DAT, and the
effects were zero at 20 DAT which indicate full recovery
of the microbial populations from the initial herbicidal
effects. This was to be expected because the amounts of
herbicides molecules present in the soil were negligible to
have any influence on fungal population that ultimately
lead to zero inhibition of fungal growth.
CONCLUSION
Paraquat, glyphosate, glufosinate-ammonium and
metsulfuron-methyl caused significant inhibitory effects
on growth of fungal, bacterial and actinomycetes
populations in soil microcosms. However, the exposures
of the microorganisms upon herbicide applications cause
short term changes on the growth and development of
the microbial community in oil palm plantation soil.
ACKNOWLEDGEMENTS
Sincere thank is due to Universiti Putra Malaysia (UPM)
for funding this study under the Research University
Grant Scheme (RUGS).
Zain et al. 373
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