Agric. sci. dev., Vol(4), No (3), March, 2015. pp.

41-44

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Agriculture Science Developments

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The Influence of Pesticides and Herbicides on the Growth and Spore

Germination of Trichoderma Harzianum
Abbas Mohammadi*
Assistant Professor of Mycology, Dept. Of Plant Pathology, College of Agriculture, University of Birjand, Birjand, Iran

Yadegar Amini
MS student of Crop Protection, Dept. Of Plant Pathology, College of Agriculture, University of Birjand, Birjand, Iran
*Corresponding author: Amohammadi@birjand.ac.ir

Keywords

Abstract

Pesticide
Biocontrol
Trichoderma
Spore germination
Growth rate

In this paper, the effects of several pesticides on Trichoderma harzianum were studied using the growth rate
and spore germination test methods. The results suggested that the nine pesticides tested at different
concentrations showed different controlling effects on the mycelial growth and spore germination of T.
harzianum. Phosalone, Amitraz and Ethalfluralin showed the maximum inhibition of spore germination and the
lowest colony growth rate was observed in the presence of Ethalfluralin, Amitraz and Malathion pesticides.
The efficacy of the pesticides on the inhibition of the mycelial growth and spore germination showed that they
were both reduced with the increasing concentration of the insecticide. The results showed that the chemical
control of pests during the growing season may be greatly reducing the population of the biocontroler.

1. Introduction
In modern agriculture, pesticides are frequently used in the fields to increase crop production. Pesticides, when applied to crops, come into
contact with both the target and non-target organisms, and have been implicated to exert some effect, inhibitory or stimulatory, on the
development of the non-target organisms [15; 18; 17]. The growth of some organisms has been inhibited by the herbicides which are meant to
destroy the weeds, while some fungi have been affected by the application of insecticides [2; 4; 6]. T. harzianum is a fungus that is used as a
fungicide. The modes of action of Trichoderma are antibiosis, parasitism and competition. It is used for foliar application, seed and soil
treatment for the suppression of various disease-causing fungal pathogens [11; 14]. Commercial biotechnological products such as 3Tac have
been useful in the treatment of Botrytis, Fusarium and the Penicillium species. It is also used for manufacturing enzymes [19]. In the IPM
programs it is essential to know the influence of compatibility between the biocontrol agents and pesticides used in crop protection. The use of
incompatible pesticides may inhibit the development and reproduction of these pathogens. If the Trichoderma spp. has to be incorporated into a
pest and disease management program, it is essential to determine the effects of the pesticides on it [11; 3]. The aim of this research is to
investigate the in vitro effect of commonly used pesticides by farmers in Iran on the T. harzianum growth and spore germination.

2. Methodology
The pesticides selected were among those commonly used for pest management in Iran. The effect of these pesticides on the radial growth of the
colony and spore germination of T. harzianum was evaluated. The pesticide doses were calculated for three concentrations (field application rate
500) based on the ppm concentration of the commercial form of the pesticides. Nine pesticides were evaluated by poisoned food technique [9]
in Potato Dextrose Agar (PDA) medium. First, 20 ml of PDA medium was sterilized in individual boiling tubes and the insecticide emulsions of
the required concentration were incorporated into the melted sterile PDA aseptically, thoroughly mixed, poured into 9 cm diameter sterile Petri
dishes and allowed to solidify under laminar flow cabinet.
An agar disc along with the T. harzianum mycelium mat was cured from the periphery of 10-day-old colonies of Trichoderma by 10 mm
diameter cork borer and transferred into the center of the PDA plate. The growth medium (PDA) without insecticide, but inoculated with the
mycelial disc served as the untreated control. The plates were sealed with parafilm and incubated at room temperature for several days to enable
maximum growth. Each treatment was replicated thrice. The diameter of a growing culture in excess of the plugs in each Petri dish was
measured when the radial growth in the control plate fully covered the medium. The data were expressed as the percentage growth inhibition of
T. harzianum by pesticide treated PDA [8]. The effect of the pesticides on the germination of the spores was tested in vitro on 1% sterile water
agar (Difco-Bacto). The pesticides were added to the agar of each Petri dish at three concentrations A (field application rate - 500), B (field
application rate) and C (field application rate + 500) of Phosalone (1500, 2000, 2500 ppm), Amitraz (3000, 3500, 4000 ppm), Malathion (2000,
2500, 3000 ppm), Chlorpyrifos (2000, 2500, 3000 ppm), Fenpropathrin (2000, 2500, 3000 ppm), Thiodicarb (1500, 2000, 2500 ppm),
Haloxyfop-r-methyl (1000, 1500, 2000 ppm), Ethalfluralin (4000, 4500, 5000 ppm), Binapacryl (1000, 1500, 2000 ppm). Suspension of T.
harzianum spores were placed in each Petri dish. Three replicates of each fungicide dose and controls were used. The plates were incubated at
25C and spore germination was checked under a light microscope after three days, until six days of incubation [3].
The area, at the end of the experiment, was compared by ANOVA, and group separation was performed with the Duncan test ( 0.05). Data
were analyzed with the SPSS statistical software, Version 14.0.ll[7]

3. Results and Discussion

Commercial formulations of Ethalfluralin and Amitraz reduced the mycelial growth and spore germination of Trichoderma even at the lowest
concentration (Table 1; Fig. 1, 2). Ethalfluralin effectively controls foxtail, barnyardgrass, fall panicum, crabgrass, pigweed, kochia, and black
nightshade when applied prior to planting or to the plant seedlings (20). In the presence of Ethalfluralin, spore germination zeroed and the
growth rate of the Trichoderma colony was less than 1cm per week. We found that the Ethalfluralin not only controlled the weeds, it also

Abbas Mohammadi *, Yadegar Amini

42

Agriculture Science Developments Vol(4), No (3), March, 2015.

suppressed the Trichoderma growth in the fields (Table 1; Figs. 1, 2). Amitraz is an insect repellent, insecticide and a pesticide synergist. Its
effectiveness is traced back to the alpha-adrenergic agonist activity, interaction with octopamine receptors of the central nervous system and
inhibition of the monoamine oxidases and prostaglandin synthesis [13]. Amitraz inhibited spore germination completely at the all of the
concentrations tested and the growth rate of the Trichoderma colonies was reduced to below two mm per day (Table 1; Figs. 1, 2). Based on the
results of this experiment, the application of Amitraz reduces the Trichoderma populations in the agricultural ecosystems and acts as a biocontrol
inhibitor. Phosalone, a phosphorodithioate acaricide and insecticide, is used to control the various insect species in/on almonds, apples, apricots,
cherries, grapes, peaches, pears and plums[5]. Phosalone inhibited spore germination in all the concentrations tested and significantly (P<0.05)
reduced the T. harzianum growth when pesticide-amended treatments were compared with the control (Table 1; Figs. 1, 2). Thiodicarb acts as an
insecticide against the major Lepidopterous pests, and suppresses the Coleopterous and some Hemipterous insect pests. Thiodicarb is used
primarily on cotton, sweet corn and soybeans [1]. Thiodicarb significantly (P<0.05) reduced spore germination in T. harzianum at 2000 and
2500ppm concentrations when compared with the control treatment (Table 1; Figs. 1, 2). No significant differences were observed in the T.
harzianum mycelial growth between concentrations of 1500, 2000 ppm of Thiodicarb (Table 1; Figs. 1, 2).
Haloxyfop is in the pyridine chemical family and is used as a pre- and post-emergent selective herbicide. Haloxyfop controls the annual and
perennial grasses in sugar beet, oilseed, potatoes, leaf vegetables, onions, sunflowers, strawberries and other crops [22]. Haloxyfop inhibited
spore germination at 2000 ppm completely and significantly (P<0.05) the control spore germination and colony growth of T. harzianum at other
concentrations (Table 1; Figs. 1, 2).
Fenpropathrin is a highly effective, broad-spectrum insecticide and acaricide of synthetic pyrethroids. The widely used insecticide fenpropathrin
in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals [12]. No inhibition
of T. harzianum spore germination and mycelial growth was observed with 2000-2500 ppm of fenpropathrin and there was no significant
difference between the control and fenpropathrin pesticide treatment (Table 1; Figs. 1, 2). Chlorpyrifos is a broad spectrum organophosphate
insecticide and acts by interfering with the activities of cholinesterase. It is used as an insecticide on grain, cotton, field, fruit, nut and vegetable
crops, and well as on lawns and ornamental plants [10]. Chlorpyrifos significantly (P<0.05) reduced the spore germination and growth of T.
harzianum mycelia at different concentrations when compared with the control treatment and conidial germination was inhibited 100% at
3000ppm concentration of this pesticide (Table 1; Figs. 1, 2). Malathion is an organophosphate pesticide that is used to kill insects on
agricultural crops, stored products, in home gardens as well as to kill mosquitoes and Mediterranean fruit flies in large outdoor areas [16].
Trichoderma growth in media amended with Malathion decreased for all test rates and spore germination was reduced at 30-85 when Malathion
was used in the 2500-3000 ppm concentration (Table 1; Figs. 1, 2). Binapacryl, a member of the dinitrophenol family, acts as a contact miticide
with ovicidal action and as a fungicide against powdery mildews [21]. Binapacryl did not show significant statistical differences, regarding the
control of the Trichoderma growth germination throughout the experiment, but a reduction in the spore germination of Trichoderma was
observed at 2000mg/lit of the fungicide rate (Table 1; Figs. 1, 2).
Table 1. Effect of pesticide on the growth and spore germination of Trichoderma harzianum in vitro
Pesticide

Thiodicarb

Holoxyfop-r-methyl

Ethalfluralin

Binapacryl

Fenpropathrin

Phosalone

Amitraz

Malathion

Chlorpyrifos

Control

Concentration
(ppm)
1500
2000
2500
1000
1500
2000
4000
4500
5000
1000
1500
2000
2000
2500
3000
1500
2000
2500
3000
3500
4000
2000
2500
3000
2000
2500
3000
0
0
0

Mean of Spore Germination

Percent
Subset =.05
F
70
AB
2
A
0
DE
31.33
ABC
12
A
0
A
0
A
0
A
0
F
66
F
66
D
30
F
80
F
72
BCD
16.66
.66
A
A
0
A
0
A
0
A
0
A
0
F
64.66
CD
58
A
0
E
41.33
ABC
14
A
0
85
F
85
F
85
F

Mean of Growth
cm
Subset = .05
NO
8.03
N
7.95
NO
8.05
J
4.65
G
3.416
F
2.416
B
1.15
B
0.916
A
0.516
M
6.98
L
6.416
L
6.23
O
8.283
NO
8.08
M
7.13
5.48333
K
G
3.4
B
0.8833
EF
2.3333
DE
2.083
B
1
F
2.4333
D
1.9666
C
1.483
I
4.3
H
3.933
GH
3.666
9
P
P
9
P
9

43

The Influence of Pesticides and Herbicides on the Growth and Spore Germination of Trichoderma Harzianum
Agriculture Science Developments Vol(4), No (3), March, 2015.

Fig. 1. Effect of pesticide on growth of Trichoderma harzianum colonies

Fig. 2. Effect of pesticide on spore germination of Trichoderma harzianum

4. Conclusion
Overall our results suggest that various pesticides used to protect the plants against weeds and insects could exert an effect on the growth and
spore germination of the Trichoderma species. Moreover, this research study was conducted under laboratory conditions and does not represent
what could occur under field conditions where environmental factors influence the interactions between the pesticide and Trichoderma species.
Further research work regarding this with more herbicides should be initiated to confirm the present findings both in vitro and in vivo in greater
detail.

Abbas Mohammadi *, Yadegar Amini

44

Agriculture Science Developments Vol(4), No (3), March, 2015.

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