Bioscience Methods 2015, Vol.6, No.3, 1-9
2
integration of biocontrol agents and commonly used
fungicides showed positive association by reducing
the seed infection compared to fungicide and the
fungal antagonists’ individually. Recently, biological
control combined with chemical fungicide at lower
concentration is applicable.
It is being a part of IPM
(Integrated Plant disease Management) strategy which
was applied since chemical controls have used
individually cause environmental pollution. Therefore the
present study,
Trichoderma
species
viz., T. viride, T.
harzianum, T. koningii, T. pseudokoningii
and
T. virens
were tested for compatibility with fungicides like
Mancozeb and Captan.
1 Materials And Methods
1.1 Source of
Trichoderma
spp
Rhizospheric soils of irrigated and non irrigated plants
were collected from different parts of Marathwada
region of Maharashtra, India. From the rhizosphere
soil samples,
Trichoderma
spp were isolated by using
PDA and
Trichoderma
selective medium (TSM) by dilution
plate technique (Johnson, 1957). The
isolated species
were
identified up to species level based on colony characters,
growth, structure of mycelium, conidiophores, phialides and
conidia (Kubicek and Harman, 2002). All
Trichoderma
spp
were purified by hyphal tip technique (Tuite, 1996). The
isolated
Trichoderma
spp were maintained throughout the
study by periodical transfers on PDA and TSM slants under
aseptic conditions to keep the culture fresh and viable.
1.2 Fungicides
a) Mancozeb 75% WP (1000 – 8000 μg/ml) is a broad
spectrum contact fungicide with a protective action
which belongs to the dithiocarbamates (Manganese
ethylene bisdithiocarbamte) family of chemicals,
which also includes maneb.
b) Captan 50% WP (100-700 μg/ml) (N-trichlorome-
thylthio-4-cyclohexene-1, 2-dicarboximide) were used
for
in vitro
.
1.3 Compatibility with fungicides
Fungicides like Mancozeb and Captan was incorporated
into the medium after sterilization. The fungicides in
proportionate dosage were incorporated in to the molten
Czapek Dox Agar (CZA) medium after sterilization and
dispersed thoroughly by continuous shaking. This was
poured in to 90 mm petridishes. Mycelial discs of 8 mm
cut from the growing margin of 7 days old culture of
Trichoderma
species was inoculated at the centre of the
petridish and incubated at 26 ± 2 C. The CZA plate
without fungicide served as control. The diameter of the
colony was measured after 7 days and compared with the
control (Tronosmo, 1989).
1.4 Source of pathogenic fungi
The test fungi were isolated from naturally infected
plants viz. leaf spot of brinjal (
Solanum melongana
L.)
caused by
Alternaria alternata
, Fruit rots of sapota
(
Manilkar zapota L.
) caused by
Rhizoctonia solani,
Aspergillus niger
and
Geotrichum candidum
, leaf spot
ofspinach (
Spinacea oleraea
L.) and fruit rot of ivy
guard (
Coccinia indica
Wight & Arn
.
) caused by
Macrophomina phaseolina.
1.5 Antagonistic Activity
Antagonistic efficacy of
Trichoderma
spp viz.,
T.
viride
,
T. harzianum
,
T. koningii
,
T. pseudokoningii
and
T. virens
were tested against the isolated
pathogenic fungi by dual culture experiment (Morton
and Stroube 1955).
Trichoderma
spp and test fungi
were inoculated 6 cm apart. Three replicates were
maintained for each treatment and incubated at 28 ±
2°C for 7 days. Monoculture plates of both served as
control. Seven days after incubation (DAI), radial
growth of test fungi and
Trichoderma
spp were
measured. Colony diameter of test fungi in dual
culture plate was observed and compared with control.
Percentage of radial growth inhibition (%RGI) was
calculated by using the formula: 100 X [C - T / C],
Where C = growth in control and T = growth in
treatment (Vincent, 1947).
The degree of antagonism between each of the
Trichodema
species and test pathogens in dual culture was scored on
scale of R
1
-R
5
(Bell et al., 1982).
2 Statistical Analyses
Statistics recitation
in vitro
compatibility was statistically
analysed using the main factor fungicide i.e. Mancozeb and
Capton and pathogenic fungi i.e.
Alternaria alternate,
Rhizoctonia solani, Aspergillus niger, Geotrichum candidum,
Fusariumoxysporum
f. sp.
spinacae,Macrophomina phaseolina
and the sub-factors were the
Trichoderma
species. Arcsine
transformation of biological control (
Trichoderma
species)
percentagewas calculatedbyusing the following formula:
Y = arcsine
= sin
-1
Where, p is the percentage of inhibition and Y is the
result of transformation
