IN VITRO INTEGRATED CONTROL OF COLLETOTRICHUM GLOEOSPORIOIDES WITH BIOLOGICAL AND CHEMICAL AGENTS

Abstract

To study the effect of different biological and chemical agents on the growth of Colletotrichum gloeosporioides, in vitro experiments were conducted with Trichoderma spp. and two fungicides alone and in integration, at the Department of Plant Pathology, NWFP Agricultural University, Peshawar, in 2006. The results indicated that all the treatments had a significant inhibitory effect on the culture of C. gloeosporioides and reduced its colony diameter. Treatments with any of the species of Trichoderma integrated with 300 mg mancozeb/L PDA were effective and did not allow any growth of the pathogen. When mancozeb was used alone at 100 mg/L PDA or 300 mg/L PDA, it reduced the growth of the pathogen by 49.21% and 100%, respectively, as compared to the control. The cultures of T. viride, T. harzianum and T. hamatum reduced the growth of C. gloeosporioides by 28.00 %, 50.40 % and 58.41 %, respectively, when used alone. The dose of 300 mg captan/L PDA reduced the colony diameter by 31.74 %, while its integration with T. hamatum reduced the growth of the pathogen by 75.07 %. It is concluded that mancozeb is more effective than captan against the pathogen. Trichoderma spp. are also effective in controlling the growth of C. gloeosporioides whether used alone or in integration with the fungicides. However, further studies are needed to evaluate their potential under field conditions.

Full text

Sarhad J. Agric. Vol.24, No.1, 2008
IN VITRO INTEGRATED CONTROL OF COLLETOTRICHUM GLOEOSPORIOIDES
WITH BIOLOGICAL AND CHEMICAL AGENTS
Adil Hussain, Fazli Raziq and Hakim Khan
ABSTRACT
To study the effect of different biological and chemical agents on the growth of Colletotrichum gloeosporioides, in vitro
experiments were conducted with Trichoderma spp. and two fungicides alone and in integration, at the Department of Plant
Pathology, NWFP Agricultural University, Peshawar, in 2006. The results indicated that all the treatments had a significant
inhibitory effect on the culture of C. gloeosporioides and reduced its colony diameter. Treatments with any of the species of
Trichoderma integrated with 300 mg mancozeb/L PDA were effective and did not allow any growth of the pathogen. When
mancozeb was used alone at 100 mg/L PDA or 300 mg/L PDA, it reduced the growth of the pathogen by 49.21% and 100%,
respectively, as compared to the control. The cultures of T. viride, T. harzianum and T. hamatum reduced the growth of C.
gloeosporioides by 28.00 %, 50.40 % and 58.41 %, respectively, when used alone. The dose of 300 mg captan/L PDA reduced
the colony diameter by 31.74 %, while its integration with T. hamatum reduced the growth of the pathogen by 75.07 %. It is
concluded that mancozeb is more effective than captan against the pathogen. Trichoderma spp. are also effective in controlling
the growth of C. gloeosporioides whether used alone or in integration with the fungicides. However, further studies are needed to
evaluate their potential under field conditions.
Keywords: Colletotrichum gloeosporioides, Trichoderma spp., Integrated Control, Mancozeb
INTRODUCTION
Colletotrichum gloeosporioides is a destructive
pathogen of many crop species. It causes diseases in
many annual, biennial and perennial plants. The
fungus has got a large host range and infects a variety
of economically important plants such as cucurbits,
blue berries, citrus, guava, strawberry, turf grasses,
cereals and some other fruit crops both in nursery and
field. This pathogen exists in different forms in
different hosts. It is found both in sexual (Glomerella
cingulata) and asexual (C. gloeosporioides) forms
(Agrios, 1997).
It is very difficult to control the pathogen. Chemical
control may not be effective because the pathogen
often forms hard overwintering structures (acervuli)
which enable the pathogen to survive as dormant
under a wide range of unfavorable environmental
conditions. Moreover, problems like leaching,
degradation, environmental pollution and killing the
non-target organisms are also associated with
chemical control. Host resistance is broken down
easily by the frequent appearance of new virulent
strains. Mainly due to these reasons, integrated
control strategies have been adopted extensively.
Different species of Trichoderma have been used
widely against many plant pathogens. Trichoderma is
a filamentous fungus distributed in species like
Trichoderma viride, T. harzianum, T. hamatum and
T. asperelum (Khuls et al. 1999). Trichoderma has
multiple mechanisms for control of pathogens
(Benitez et al. 2004). Trichoderma spp. have a wide
host range and so are strongly antagonistic to fungal
pathogens like Pythium, Rhizoctonia, Fusarium,
Botrytis, Sclerotium, Colletotrichum, Alternaria,
nematodes and many other plant pathogens (Harman,
1996). Winidham et al. (1986) reported that
Trichoderma increased plant growth by the
production of a growth-stimulating factor. Leinhos
and Buchenauer (1992) concluded that fungi release
toxic metabolites and enzymes into the medium in
which they grow. Benitez et al. (2004) isolated
chemicals like harzianic acid, tricholin and viridine,
which play a very important role in antagonistic
behavior of Trichoderma.
The use of fungicides sometimes becomes
unavoidable. However, their dose and frequency of
application can be minimized by integration with
other methods for effective control of plant
pathogens. Solano and Arauz, (1995) applied
mancozeb, captan, tricyclazole, chlorothalonil and
prochloraz against papaya anthracnose
(Colletotrichum gloeosporioides) and found that
mancozeb and prochloraz resulted in lowest disease
incidence. Freeman et al. (1997) assessed various
fungicides like folpet, captan and propaconazole for
their ability to control C. gloeosporioides. They
found that captan was effective in 50 % and 70 %
concentrations. Legard (2000) tested captan, thiram,
Benlate, Topsin-M and mancozeb against
Colletotrichum crown rot. He found Benlate, Topsin-
M and mancozeb more effective in controlling the
pathogen, as compared to captan and thiram. This
study was aimed at finding out the effect of
Trichoderma spp. alone and in integration with
fungicides on the growth of C. gloeosporioides.
Department of Plant Pathology, NWFP Agricultural University, Peshawar
–
Pakistan.

Adil Hussain, et al. In vitro integrated control of colletotrichum cloeosporioides …… 80
MATERIALS AND METHODS
The culture of C. gloeosporioides was obtained from
the Department of Plant Pathology, N.W.F.P.
Agricultural University, Peshawar. The culture was
then multiplied on Potato Dextrose Agar (PDA)
medium. The cultures of Trichoderma spp. were also
obtained from the Department of Plant Pathology,
N.W.F.P, Agricultural University, Peshawar. These
antagonists were grown on PDA medium at 25
o
C for
1 week for mass culturing. The two fungicides,
captan and mancozeb, were purchased from the local
market.
Setting up of the Experiment
The experiment was carried out at the Department of
Plant Pathology, NWFP Agricultural University,
Peshawar to test the relative efficacy of the three
species of Trichoderma (T. viride, T. harzianum and
T. hamatum) against C. gloeosporioides alone and in
integration with different doses of the two fungicides
(captan and mancozeb). Two doses, 100 mg/L and
300 mg/L, of both the fungicides were used in PDA
medium. The medium was prepared and autoclaved
at 121
o
C for 15 minutes. Both the fungicides were
added after sterilization and thoroughly mixed before
pouring into the Petri dishes. Petri dishes were
inoculated in the centre with blocks of 5 mm
diameter of a 15 days old culture of C
gloeosporioides. Each of the Trichoderma spp. was
applied individually as 5 mm diameter inoculum
plugs at four equidistant sites around the pathogen.
The Petri dishes were sealed with parafilm and
incubated for 2 weeks at 25
o
C. One treatment was
also left untreated that served as a control. All these
treatments were replicated seven times. The
experiment was laid out in a randomized complete
block design. Data were taken as the mean colony
diameter of C. gloeosporioides, twice at weekly
interval.
RESULTS AND DISCUSSION
Both the doses of the two fungicides and each of the
three species of Trichoderma as well as their
integration reduced the growth of Colletotrichum
gloeosporioides significantly as compared to the
control (Tables I and II). The high dose of 300 mg
mancozeb/L PDA when used alone or in integration
with any of the species of Trichoderma was the most
effective and did not allow any growth of the
pathogen. The low dose of 100 mg mancozeb/L PDA
reduced the growth of the pathogen by 49.21% after 7
days, which remained the same after 14 days. Captan,
on the other hand, reduced the colony diameter of the
pathogen by 21.71% and 31.74% when used in the
low and high doses of 100 and 300 mg/L PDA,
respectively. When integrated with T. hamatum, the
low dose of mancozeb reduced the growth of the
pathogen by 85.77% during the first 7 days of
incubation, but the growth inhibition was reduced to
76.98% after 14 days. T. hamatum also reduced the
colony diameter of the pathogen by 80.95% in
integration with 100 mg captan/L PDA in the first
week, which was reduced to 76.18% after the second
week. Among the Trichoderma spp., T. hamatum was
also the most effective alone and reduced the colony
diameter of the pathogen by 58.41%. T. harzianum
and T. viride reduced the colony diameters by
50.40% and 28.00% after 7 days and 50.40% and
25.40 % after 14 days, respectively.
Several workers have used different species of
Trichoderma against Colletotrichum, both in the field
and laboratory (Freeman et al. 1997). The present
study proved that among the fungicides, mancozeb as
compared to captan, was more effective in controlling
the growth of the pathogen whether used alone or in
integration with the antagonists. This result compares
favorably with the finding of Solano and Arauz
(19995), Haddad et al. (2003) and Legard (2000).
There may be several reasons for the greater efficacy
of this fungicide. Mancozeb is a contact protective
fungicide with a high molecular weight as compared
to captan. It is a derivative of dithiocarbamic acid.
These compounds are toxic to fungi because they are
metabolized to isothiocynate radicals (N = S = C)
inside pathogen cells, inactivating the –SH group of
amino acids and enzymes showing their effect. On
the other hand, captan is a low molecular weight
contact fungicide used mostly against oomycetes
(Haddad et al. 2003). Mancozeb also showed good
results when it was integrated with the species of
Trichoderma.
Among the species of Trichoderma, T. viride was not
as efficient as T. harzianum and T. hamatum, which
caused greater growth inhibition. These results are
similar to the findings of Malathie et al. (2002),
Marco (2003) and Verma et al. (2006). All the
species of Trichoderma were inoculated to the Petri
dishes at the same time but after 3 days of inoculating
C. gloeosporioides. Still the hyphae of T. hamatum
and T. harzianum grew vigorously over the culture of
the pathogen (Fig. 1). The growth of T. viride, on the
other hand, was much restricted. The culture was
light green in colour right from the beginning, as
compared to white colour of the other two species.
Absence of direct parasitism and less competition
may also be one of the reasons due to which T. viride
was not as successful in controlling C.
gloeosporioides.

Sarhad J. Agric. Vol.24, No.1, 2008 81
CONCLUSION AND RECOMMENDATION
It is concluded that mancozeb is more effective than
captan against the pathogen. Trichoderma spp. are
also effective in controlling the growth of C.
gloeosporioides, whether used alone or in integration
with the fungicides. However, further studies are
needed to evaluate their potential under field
conditions.
Table I. Mean colony diameter (cm) of Colletotrichum gloeosporioides after 7 days of incubation at 25
º
C as
affected by fungicides and Trichoderma spp.
Treatment No. Treatments Colony diameter % reduction than
control (T1).
T1 Control (C. gloeosporioides alone) 9.00 A -
T2 Trichoderma viride 6.429 BC 28.00
T3 T. harzianum 4.457 EF 50.40
T4 T. hamatum 3.743 FG 58.41
T5 Captan (100 mg/L PDA) 7.029 BCD 21.71
T6 Captan (300 mg/L PDA) 5.857 B 31.74
T7 Mancozeb (100 mg/L PDA) 4.571 DEF 49.21
T8 Mancozeb (300 mg/L PDA) 0.0 K 100
T9 T. viride + Captan (100 mg/L PDA) 5.214 CDE 42.06
T10 T. harzianum + Captan (100 mg/L PDA) 3.429 FGH 62.0
T11 T. hamatum + Captan (100 mg/LPDA) 1.714 IJ 80.95
T12 T. viride + Captan (300 mg/L PDA) 4.386 EF 51.26
T13 T. harzianum + Captan (300 mg/L PDA) 3.014 GHI 66.51
T14 T. hamatum + Captan (300 mg/L PDA) 2.243 HIJ 75.07
T15 T. viride + Mancozeb (100 mg/L PDA) 3.429 FGH 61.90
T16 T. harzianum + Mancozeb (100 mg/L PDA) 2.571 GHIJ 71.43
T17 T. hamatum + Mancozeb (100 mg/L PDA) 1.286 JK 85.77
T18 T. viride + Mancozeb (300 mg/L PDA) 0.0 K 100
T19 T. harzianum + Mancozeb (300 mg/L PDA) 0.0 K 100
T20 T. hamatum + Mancozeb (300 mg/L PDA) 0.0 K 100
LSD
(0.05)
1.319
CV (%) 36.00
Means followed by different letters are significantly different from each other at 5 % level of significance.

Adil Hussain, et al. In vitro integrated control of colletotrichum cloeosporioides …… 82
Table II. Mean colony diameter (cm) of Colletotrichum gloeosporioides after 14 days of incubation at 25 ºC as
affected by fungicides and Trichoderma spp.
Treatment No. Treatments Colony diameter % reduction than
control (T1).
T1 Control (C. gloeosporioides alone) 9.00 A -
T2 Trichoderma viride 6.714 B 25.40
T3 T. harzianum 4.457 DE 50.40
T4 T. hamatum 3.743 EF 58.41
T5 Captan (100 mg/L PDA) 7.029 BC 21.71
T6 Captan (300 mg/L PDA) 6.143 B 31.74
T7 Mancozeb (100 mg/L PDA) 4.571 DE 49.21
T8 Mancozeb (300 mg/L PDA) 0.0 H 100
T9 T. viride + Captan (100 mg/L PDA) 5.214 42.06
T10 T. harzianum + Captan (100 mg/L PDA) 3.857 EF 57.14
T11 T. hamatum + Captan (100 mg/LPDA) 2.143 G 76.18
T12 T. viride + Captan (300 mg/L PDA) 4.386 DE 51.26
T13 T. harzianum + Captan (300 mg/L PDA) 3.014 FG 66.51
T14 T. hamatum + Captan (300 mg/L PDA) 2.243 G 75.07
T15 T. viride + Mancozeb (100 mg/L PDA) 3.857 EF 57.14
T16 T. harzianum + Mancozeb (100 mg/L PDA) 2.786 FG 69.04
T17 T. hamatum + Mancozeb (100 mg/L PDA) 2.071 G 76.98
T18 T. viride + Mancozeb (300 mg/L PDA) 0.0 H 100
T19 T. harzianum + Mancozeb (300 mg/L PDA) 0.0 H 100
T20 T. hamatum + Mancozeb (300 mg/L PDA) 0.0 H 100
LSD
(0.05)
1.279
CV (%) 33.94
Means followed by different letters are significantly different from each other at 5 % level of significance.

Sarhad J. Agric. Vol.24, No.1, 2008 83
Fig. 1: Growth of Trichoderma harzianum hyphae and spores over the colony of Colletotrichum gloeosporioides.

Adil Hussain, et al. In vitro integrated control of colletotrichum cloeosporioides …… 84
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