AbstractStrains of bacterial and fungal bicontrol agents were

tested for their effectiveness on the sugarbeet root rot pathogen,

S. rolfsii. Out of 12 strains of Pseudomonas fluorescens tested,
maximum per cent inhibition of 48.9 was exhibited by Pf1 in dual
plate technique. The B. subtilis strain, EPCO 16 showed greatest per
cent inhibition (44.4) than other two strains. Among 12 different
Trichoderma sp. tested, Trichoderma asperellum, TTH 1 exhibited
better inhibition of mycelial growth of the pathogen (64.4 per cent).
The Farm yard manure based bioformulations of these effective
biocontrol agents were tested individually and in combination against
root rot of sugarbeet under pot culture (glasshouse) conditions. Next
to the difenoconazole treatment, significant reduction in root rot
incidence was observed in the combination of P. fluorescens (Pf1)
and T. asperellum (TTH1) than individual and control treatments.
Similarly, increased yield was observed in the combination of Pf1
and TTH1 treated sugarbeet plants under pot culture conditions.

KeywordsIn vitro antagonism, P. fluorescens, B. subtilis,
Trichoderma sp., pot culture, root rot, S. rolfsii,
I. INTRODUCTION
UGARBEET (Beta vulgaris L. ssp. vulgaris var. altissima
Doll. Chenopodiaceae) is an important sugar producing
tuber crop as it accumulates high concentration of sucrose in
its white roots of conical shape. The conditions suitable for
growth and development of the crop are also favourable for
the quick development, proliferation and spread of disease,
among them root rot caused by S. rolfsii is the most serious
one resulting in significant yield loss. Control of root rot
diseases using fungicides are normally recommended which
are not economical and causing environmental hazards. In this
context, biological control of plant diseases is advocated
instead of chemical pesticides. Application of single bioagent
Thilagavathi Rasu. Senior Research Fellow, Department of Plant
Pathology, Tamil Nadu Agricultural University, Coimbatore, India (phone:
+91 9629442611; e-mail: rthilagaphd@gmail.com).
Nakkeeran Sevugapperumal. Associate Professor, Department of Plant
Pathology, Tamil Nadu Agricultural University, Coimbatore, India. (e-mail::
nakkeeransingai@yahoo.com)
Raguchander Thiruvengadam. Professor, Department of Plant Pathology,
Tamil Nadu Agricultural University, Coimbatore, India. (e-mail:
raguchander@rediffmail.com)
Samiyappan Ramasamy Former Director, Centre for Plant Molecular
Biology, Tamil Nadu Agricultural Univeristy, Coimbatore, India (e-mail:
rsamiyappan@rediffmail.com)


can have limitations with regard to the consistency of the
product and efficacy in different environments [1]. Cocktails
of biocontrol agents may have advantages of broad spectrum
activity, enhancing the efficacy and reliability of the
biological control and they communicate with each other to
maximize biocontrol efficacy. The present study was
undertaken to study the combination of effictive biocontrol
agents against root rot of sugarbeet and to develop ecofriendly
management practices to control the disease.

II. MATERIALS AND METHODS
A. Pathogen and biocontrol agents
The root rot pathogen Sclerotium rolfsii was isolated from
infected sugarbeet plant. Trichoderma asperellum (TTH1),
T. viride (TNAU TV1), strains of P. fluorescens and Bacillus
subtilis were obtained from culture collection section,
Department of Plant Pathology, Centre for Plant Protection
Studies, Tamil Nadu Agricultural University, India. Other
Trichoderma sp. isolates were obtained from rhizosphere soil
of different crops grown in different locations of Tamil Nadu.

B. In vitro screening
To study the antagonism of bacterial antagonists viz.,
P. fluorescens and B. subtilis strains on S. rolfsii, a nine mm
mycelial disc from actively growing colony of S. rolfsii was
placed at the centre of the Petri plate containing PDA medium.
After 12 h of incubation, a sterile Whatman No. 40 filter paper
discs with six mm dia were placed one cm away from the
edges of the Petriplate at four sides centering around the
fungal disc. About 25l of bacterial cell suspension from 48 h
old broth cultures was dropped over the filter paper discs.
Sterile water was used as a check. Four replications were
maintained for each bacterial antagonist [2]. To study the
antagonism of fungal antagonists on S. rolfsii, three days old
culture from Trichoderma sp. isolates was placed five mm
away from the periphery of the PDA plate and opposite to the
culture disc of S. rolfsii [3]. Control plate was maintained with
S. rolfsii alone and incubated at room temperature (272C).
Four replications were maintained. Observations were taken
after the S. rolfsii reached full growth in the control plate. The
radial mycelial growth of the S. rolfsii and per cent reduction
over control was calculated by using the formula, Per cent
inhibition over control
={(C-T)/C} x 100 (1)
Where, C- mycelial growth of S. rolfsii in control;
T- mycelial growth of S. rolfsii in dual plate.
Biological Control of Sugarbeet Root Rot
Caused by Sclerotium rolfsii

Thilagavathi Rasu, Nakkeeran Sevugapperumal, Raguchander Thiruvengadam
and Samiyappan Ramasamy
S

International Journal of Biological, Ecological and Environmental Sciences (IJBEES) Vol. 2, No. 1, 2013 ISSN 2277 4394
7
C. Preparation of bioformulation
Biocontrol agents selected from in vitro antagonism were
used for the preparation of bioformulation. Talc based
bioformulation was prepared for P. fluorescens (Pf1), B.
subtilis (EPCO16) and Trichoderma asperellum (TTH1) as
follows. A loopful of Pf1 and EPCO16 strains were inoculated
into the Kings B and nutrient broths respectively, incubated in
a rotary shaker for three days at 150 rpm at 282
o
C. The 400
ml of bacterial suspensions containing 9 x 10
8
cfu/ml, one kg
of the carrier material (talc powder), 15 g calcium carbonate
(to adjust the pH to neutral) and 5 g CMC (adhesive) were
mixed under sterile conditions [4]. For fungus, an actively
growing mycelial disc of TTH1 was inoculated into yeast
molasses broth (30 ml molasses; 5 g yeast; made up to 1000
ml), incubated for 15 days at 282
o
C. The fungal biomass
(containing 3x10
8
cfu/ml) along with the spent broth was
incorporated into the sterilized talc powder carrier material @
50 ml suspension per 100g and thoroughly mixed with
addition of 500 mg CMC [5]. The freshly prepared talc based
bioformulations were mixed with autoclaved farm yard
manure at 1:1 ratio. Incubated for two to seven days to obtain
an enriched farm yard manure based bioformulation and kept
at room temperature for further experiments.

D. Biocontrol of root rot on potted sugarbeet plants
Farm yard manure based bioformulation of each biocontrol
agent was tested individually or in combination against
sugarbeet root rot under glass house conditions. Pots were
filled with autoclaved pot mixture (Red soil: sand: garden
soil), upper 5 cm soil was challenge inoculated with 100
sclerotia by mixing with 100g of soil. Treatments receiving
single biocontrol agent were applied with one gram of
bioformulation per sowing hole, while the treatments with
more than one bioagent, one gram of each bioformulation was
applied. The susceptible sugarbeet cv. Indus was sown @ 10
seeds per pot, after one week three plants were maintained.
Soil applications were done at 30, 60 and 90 days after sowing
@ five gram per pot or five gram of each for the treatments
with individual or combination of biocontrol agents
respectively.
The experiment was conducted in a completely randomized
block design with four replications. The top yield and tuber
yield were recorded at 120 days after sowing. The per cent
disease incidence (PDI) was assessed using the following
formula.
=(Number of infected plants) /(Total number of plants) x
100 (2)

III. RESULTS AND DISCUSSION
In the present study, the result from in vitro antagonistic
activity of biocontrol agents against S. rolfsii revealed that
among strains of P. fluorescens used, maximum per cent
inhibition of 48.9 was exhibited by Pf1 when compared to all
other strains. The B. subtilis strain, EPCO 16 showed highest
per cent inhibition (44.4) than others. The result showed that
among different Trichoderma sp. tested, T. asperellum
(TTH1) exhibited better inhibition of mycelial growth of the
pathogen (64.4 per cent) (Table I; Fig. 1,2,3). Similarly,
Pseudomonas cf. monteilii inhibited the growth of S. rolfsii up
to 94 per cent under in vitro conditions by the production of
diffusible antibiotic, volatile metabolites, hydrogen cyanide
and siderophore [6]. An inhibitory effect of Bacillus on wide
range of fungi including S. rolfsii was documented by several
researchers [7]-[8]-[9]-[10]. It has been reported to produce
an array of compounds that demonstrated antifungal
activity [1]-[11]. Toxic metabolites produced by the
Trichoderma sp. might be responsible for the inhibitory effect
on different plant pathogens [12].
Efficacy of biological control of plant pathogens can be
increased by employing effective biocontrol agents with
diverse modes of action [13]-[14]. In the present study, farm
yard manure based bioformulations of effective biocontrol
agents were tested individually and in combination against
root rot of sugarbeet under pot culture conditions. The results
showed that among different treatments tested, next to the
chemical (100 per cent reduction), the combination treatment
Pf1+TTH1 recorded least root rot incidence of 25 per cent
than individual treatments at 120 days after sowing. The
control treatment recorded 83.3 per cent disease incidence.
Similarly. combined application of P. fluorescens and
T. viride has an improved biocontrol activity against stem rot
(Sclerotium rolfsii) in groundnut [15], root rot
(Macrophomina phaseolina) in green gram [16].
The results of biometrical observations revealed that the
application of farm yard manure based bioformulation
increased the top and tuber yield of sugar beet compared to
untreated control. They were significantly higher in the
combination of Pf1+TTH1 challenged with S. rolfsii than
individual treatments. The same combination treatment
recorded maximum top yield of 173 g and tuber yield of
294 g as against control treatment which recorded least yield
of 128 g of top and 240 g of tuber per plant (Table II). The
combined application of T. viride and P. fluorescens increased
the root length and shoot length in tomato [17] and chilli [18]
and maximum shoot length, root length and vigour index were
observed in black gram [19] and greengram [16]. The present
study clearly indicates that the combination of effective
biocontrol agents significantly reduced the disease incidence
and increased the yield than they were applied alone. The
work has to be intensified to study the mechanisms involved
in disease control by the combination of biocontrol agents.








International Journal of Biological, Ecological and Environmental Sciences (IJBEES) Vol. 2, No. 1, 2013 ISSN 2277 4394
8

Fig. 1 S. rolfsii & Pf1 Fig. 2 S. rolfsii & EPCO16

Fig. 3 S. rolfsii S. rolfsii & TTH1


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TABLE I










TABLE I
IN VITRO ANTAGONISM OF BIOCONTROL AGENTS ON S. ROLFSII
Biocontrol
agents
Growth of S. rolfsii
Per cent Inhibition of S.rolfsii
over control
Trichoderma sp.
TNAU TV1 3.6
b
60.0
b
(50.8)
TTH 1 3.2
a
64.4
a
(53.4)
TED 1 3.6
b
60.0
b
(50.8)
TSBO 1 3.8
c
58.1
c
(49.7)
TSBO 2 4.1
d
54.4
d
(47.5)
TSBO 3 4.9
e
45.9
e
(42.6)
TSFCE 1 5.5
f
39.2
f
(38.8)
TED 2 6.5
h
28.1
h
(32.0)
TED 3 5.9
g
34.1
g
(35.8)
TDH 1 3.8
c
58.1
c
(49.7)
TCOO 1 3.8
c
58.1
c
(49.7)
TTNJ 1 4.1
d
54.4
d
(47.5)
P. fluorecens
Pf1 4.6
a
48.9
a
(44.4)
PFBV 22 5.3
b
41.4
b
(40.0)
PFKO 1 7.0
e
21.9
e
(27.9)
TDK 1 9.0
i
00.0
i
(00.0)
FP 7 8.2
g
09.4
g
(17.8)
PFRA 1 7.6
f
15.8
f
(23.4)
PFATR 1 8.5
h
05.6
h
(13.7)
PFBS 1 9.0
i
00.0
i
(00.0)
PFNL 1 6.4
d
29.4
d
(32.8)
PRA 4 5.7
c
37.2
c
(37.6)
PYR 6 6.4
d
29.4
d
(32.8)
PFNA 1 7.0
e
21.9
e
(27.9)
B. subtilis

EPC 5 5.4
b
39.7
b
(38.9)
EPC 8 5.6
c
37.8
b
(37.9)
EPCO16 5.0
a
44.4
a
(41.8)
Control 9.0
d
00.00


In both tables values are mean of four replications. Means followed by a
common letter are not significantly different at 5% level by DMRT. Data in
parentheses are arcsine transformed values.
TABLE II
EFFICACY OF BIOCONTROL AGENTS ON YIELD AND DISEASE INCIDENCE OF
POTTED SUGARBEET PLANTS
Treatments Yield (g plant
-1
)
Top Tuber
Per cent disease
incidence
Pf1 135
ef
258
cde
50.0
e
(45.0)
EPCO16 131
f
246
de
41.7
d
(40.2)
TTH 1 148
cd
264
bcd
45.9
e
(45.0)
Pf1+EPCO16 162
b
277
b
33.3
c
(35.4)
Pf1+TTH 1 173
a
294
a
25.0
b
(30.0)
EPCO 16+TTH 1 144
de
253
cde
50.0
e
(45.0)
Pf1+EPCO16+TTH 1 157
bc
271
bc
37.5
d
(40.2)
Difenoconazole 130
f
252
de
00.0
a
(0.0)
Carbendazim 136
ef
246
de
41.7
d
(40.2)
Inoculated control 117
g
227
f
83.3
f
(65.9)
Uninoculated control 128
f
240
ef
00.0
Healthy control 116
g
223
f
00.0


International Journal of Biological, Ecological and Environmental Sciences (IJBEES) Vol. 2, No. 1, 2013 ISSN 2277 4394
9
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