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15(1): 005-008, 2020
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IN-VITRO EVALUATION OF FUNGICIDES AND BIOAGENTS

AGAINST TOMATO EARLY BLIGHT PATHOGEN
ALTERNARIA SOLANI (L.)

V. SUMBULA* AND S.P KURIAN

1
Department of plant pathology,
College of Horticulture, Kerala Agricultural University, Thrissur - 680 656, Kerala, INDIA
e-mail: sumbulav101@gmail.com

KEYWORDS ABSTRACT
Alternaria solani Early blight disease caused by Alternaria solani (L.) is one of the most destructive diseases of tomato in the tropical
Bioagents and subtropical regions. In the present study, efficacy of different fungicides at different concentration and
Early blight bioagents against A. solani was assessed under in vitro conditions. A total of eight fungicides and seven bioagents
Fungicides were tested. It was found that, all the three concentrations of propineb (0.1%, 0.2% and 0.3%), hexaconazole
Tomato (0.05%, 0.1% and 0.15%) and iprodione 25% + carbendazim (0.1%, 0.2% and 0.3%) and difenoconazole at
0.075% concentration showed cent per cent inhibition of A.solani. It was followed by lower concentrations of
Received on : difenoconazole (0.05% and 0.025%) with 97.21 and 94.99 per cent inhibition of pathogen respectively. Among
08.01.2020 remaining fungicides, all the three concentrations of pyraclostrobin was significantly superior over copper
hydroxide, azoxystrobin and trifloxystrobin 25% + tebuconazole in inhibiting mycelial growth and it ranged
Accepted on : from 79.44 to 85.55 per cent. Among the bioagents tested, Trichoderma viride (KAU) and plant growth promoting
16.03.2020 microbial consortium (PGPM mix of KAU) were recorded cent per cent inhibition of pathogen. Among different
bacterial antagonists Bacillus subtilis 1; an endophyte isolated from cocoa recorded maximum growth inhibition
*Corresponding of 51.66 per cent.
author

INTRODUCTION copper hydroxide and iprodione were effective against A.

solani. Sahu et al. (2013) reported that, the fungicides like
Tomato (Solanum lycopersicum L.) is a vital solanaceous pristine, maccani, boscalid, pyraclostrobin and mancozeb
vegetable crop of global importance grown in an area of 4.8 can be used not only to manage early blight disease but to
million hectares (FAO, 2017). In India tomato occupies an increase the yield of tomato as well.
area of 0.797 million hectare, with a production of 20708 Although the use of chemicals to reduce or prevent losses
metric tons and productivity of 20.7 metric tons/ha in the year caused by this agent seems simple and successful, the damage
2017 (FAO, 2017). Varied climatic adaptability and high
inflicted by the residual effects of chemicals on humans and
nutritive value made the tomato cultivation more popular in
environment should certainly be taken into account. So,
the recent years. However, disease and pests pose serious
biological control is an alternative method which provides
threat to tomato cultivation among which, early blight is one
environment-friendly, sustainable and promising strategies
of the most destructive diseases, causing considerable loss to
against plant diseases. Biocontrol methods such as seedling
quality and quantity of fruits. Under favourable conditions
more than 80 per cent disease severity of early blight has dip and foliar applications of T. harzianum, T. viride and P.
been recorded in tomato crops (Kumar and Srivastava, 2013). fluorescens were found to decrease the early blight incidence
up to 62 per cent and increased tomato yield up to 37 per
Primary methods of controlling the disease include preventing cent (Ramanujam et al., 2015). Koley et al. (2015) studied
long periods of wetness on the leaf surface, cultural scouting, efficacy of B. subtilis and P. fluorescens against A. solani and
sanitation, and development of disease resistance varieties
showed 52.77% and 47.22% of growth inhibition respectively.
(Kirk et al., 2005 and Kumar and Srivastava, 2013). Moreover,
Basamma and Kulkarni (2016) also reported a significant
the ultimate control of this disease is achieve through
reduction in early blight incidence of tomato under
cultivation of resistant varieties, but it remains limited by the
greenhouse conditions by application of Bacillus subtilis.
evolution of new strains of the pathogens. Hence, farmers
mainly rely on fungicides for the control of pathogens viz. Unplanned, overdosed and wide use of fungicides often leads
Alternaria solani, the casual organism of early blight. Several to serious environmental problems besides affecting the health
research workers have reported that timely application of of users and consumers. Hence, application of fungicides at
fungicides is the best method to control early blight (Singh proper dose and time interval is mandatory. Moreover,
and Singh, 2006; Patel and Choudhary; 2010; Horsfield et innovative and safe methods like use of biocontrol agents
al., 2010). According to Tofoli et al. (2010) fungicides viz. need to be identified and evaluated for continuous search to
difenoconazole, tebuconazole, chlorothalonil, mancozeb, develop ecofriendly strategies to reduce the dependence on

5
V. SUMBULA AND S.P KURIAN

harmful chemicals. Therefore, the present study was aimed to In vitro evaluation of antagonists
assess the efficacy of different doses of fungicides and bioagents Three endophytic Bacillus sp (B. subtilis 1, B. subtilis 2 and B.
against Alternaria leaf blight of tomato under in vitro conditions. subtilis 3) isolated from cocoa leaves, three reference cultures
viz. Trichoderma viride (KAU), Pseudomonas fluorescens
MATERIALS AND METHODS (KAU) and B. subtilis (KAU) and one plant growth promoting
microbial consortium (PGPM mix of KAU) were screened for
Isolation, purification and maintance of the pathogen their antagonistic activity against the pathogen, A. solani by
A. solani was isolated from infected tomato leaves and purified adopting dual culture technique (Johnson and Curl, 1972).
by following single spore isolation method (Ho and Ko, 1997). Antagonistic activity of T. viride was tested by employing
Pure cultures of different isolates of A. solani were tested for deferred antagonism and bacterial antagonists by
the pathogencity on tomato leaves, reisolated and maintained simultaneous antagonism methods. Monoculture of the
on potato dextrose agar slants for further investigations. pathogen served as control. Experiment was laid out in
Completely Randomized Design (CRD) and three replications
In vitro evaluation of fungicides
were kept for each antagonist. Observations were recorded
In vitro evaluation of eight fungicides in three different dosses daily till the pathogen attained full growth in control and per
viz. propineb 70% WP (0.1%, 0.2% and 0.3% ), copper cent inhibition was calculated.
hydroxide 77 WP (0.15%, 0.2% and 0.25%), pyraclostrobin
20% EC (0.025%, 0.05% and 0.075%), azoxystrobin 23%
SC (0.1%, 0.15 and 0.2%), hexaconazole 5% EC (0.05%, RESULTS AND DISCUSSION
0.1% and 0.15%), difenconazole 25% EC (0.025%, 0.05%
In vitro evaluation of fungicides
and 0.075%), iprodione 25% + carbendazim 25% WP (0.1%,
0.2% and 0.3%) and trifloxystrobin 25% + tebuconazole The efficacy of eight fungicides against A. solani at different
55% (0.025%, 0.05% and 0.075%) was carried out by concentrations is given in Fig. I. All the fungicides tested were
poisoned food technique (Zentmyer, 1955). The fungicides found to be effective against the pathogen; however, the
were mixed separately with sterilized potato dextrose medium efficiency varied with the chemical. There was a positive
in suitable proportion to get the desired concentrations and correlation between the concentration and per cent inhibition
poured to sterilized Petri dishes @ 20 ml/plate. Eight mm sized of growth of mycelium except propineb, hexaconazole
disc from five day old culture of the pathogen was placed at iprodione 25% + carbendazim. All the three concentrations
the center of each Petri dish containing poisoned medium. of propineb (0.1%, 0.2% and 0.3%), hexaconazole (0.05%,
Experiment was in Completely Randomized Design (CRD) with 0.1% and 0.15%) and %), iprodione 25% + carbendazim
three replications for each fungicide. Observations were (0.1%, 0.2% and 0.3%) recorded cent per cent inhibition of
recorded, till pathogen attained full growth in control. Per the pathogen. Hence, it revealed that, even the lower
cent inhibition of the pathogen was calculated using the concentration of these fungicides were effective against the
formula suggested by Vincent (1927). pathogen. Difenconazole was significantly superior over
copper hydroxide, pyraclostrobin, azoxystrobin and
trifloxystrobin 25% + tebuconazole in inhibiting mycelial
growth and recorded 94.99-100 per cent inhibition. The
combination fungicide, trifloxystrobin 25% + tebuconazole,
C = Growth of the pathogen in control was the least effective and recorded only 73.33 per cent
T = Growth of the pathogen in treatment inhibition even at the concentration of 0.075 per cent.

1st concentration 2nd concentration 3rd concentration

120
% inhibition over control

100
120
80
% inhibition over control

100
60
80
60 40

40 20
20 0
Trichoderma viride (KAU)

Bacillus subtilis (KAU)

Bacillus subtilis 1 (Endophyte from

Bacillus subtilis 2 (Endophyte from

PGPM mix (KAU)

Pseudomonas fluorescens (KAU)

cocoa)

cocoa)

cocoa)
Bacillus subtilis 3 (Endophyte from

0
Propineb 70% WP

Copper hydroxide 77% WP

Pyraclostrobin 20% EC

Iprodione 25% + Carbendazim

25% WP

Trifloxystrobin 25% +
Tebuconazole 55%
Azoxystrobin 23% SC

Hexaconazole 5% EC

Difenconazole 25% EC

Figure1: In vitro evaluation of fungicides against Alternaria solani Figure 2: In vitro evaluation of bioagents against Alternaria solani

6
 IN-VITRO EVALUATION OF FUNGICIDES AND BIOAGENTS

Azole fungicides like hexaconazole and difenconazole can In the present attempt we tried to find out suitable fungicides
inhibit the pathogen either by destroying their cell membrane with its effective dose and bio-control agents against A. solani,
or its permeability or by inhibiting metabolic processes of the the casual organism of early blight of tomato. Though, all the
pathogen and hence are extremely effective (Fisher et al., 2004). fungicides and bioagents evaluated in vitro were found effective
Similarly, propineb, a contact fungicide which interferes at against the pathogen, among different fungicides tested all the
different locations in the metabolism of the pathogen; on several three concentrations of propineb (0.1%, 0.2% and 0.3%),
points of the respiration chain, in the metabolism of hexaconazole (0.05%, 0.1% and 0.15%) and %), iprodione
carbohydrates and proteins, in the cell membranes and this 25% + carbendazim (0.1%, 0.2% and 0.3%) were found to
multi-site mode of action of propineb also prevents be most effective and among the bioagents, T.viride (KAU)
development of resistance in the pathogen. Strobilurin group was most efficient. Hence, the present study revealed that,
fungicides like azoxystrobin, pyraclostrobin and trifloxystrobin even the lower concentration of these fungicides were effective
are broad spectrum in action and excellent inhibitors of spore against the pathogen. However, further studies are needed to
germination and comparatively less effective for direct mycelial evaluate their potential under field conditions and that will
inhibition than above chemicals. Moreover, strobilurins help the farmers to dissuading the use of over doses of
inhibits mitochondrial respiration by blocking electron transfer chemicals to avoid residual toxicity in the tomato fruits, in
at the cytochrome bc1 complex (Jiang et al., 2009). The addition of reducing the cost of chemical.
induction of the alternative oxidase respiratory pathway at the
cytochrome bc1 target site has been proposed as the likely ACKNOWLEDGEMENTS
reason for the low mycelial sensitivity to strobilurins displayed
by the pathogen. Complete inhibition of mycelial growth of A. The present work was supported by Kerala Agricultural
solani by hexaconazole at 0.025 per cent has been reported University, College of Horticulture, Vellanikkara, Thrissur by
earlier by Singh and Singh (2006). Efficacy of fungicides like giving infrastructure and technical facilities.
mancozeb, copper oxychloride, copper hydroxide,
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