Antifungal Activity of Psoralea corylifolia Hairy Root Extract against Sugarcane

Red Rot Pathogen under Controlled Condition Treatment Chamber

Keywords:
Hairy root, Antifungal activity, Sugarcane, Red rot
ABSTRACT:

Red rot disease is the major constraint for sugarcane production in India and
the pathogen has gained virulence in recent years. About 33 % reduction in yield was
observed and loss in sucrose and commercial cane sugar was estimated upto 32 to
50 % in average infections. The present investigation was carried out in sugarcane
breeding institute, Coimbatore to study the effect of Psoralea corylifolia hairy root
extract against high intensity Colletotrichum falcatum spore suspension (10
6
spores
ml
-1
) causing red rot disease reaction in canes under Controlled Condition Treatment
(CCT) Chamber. Nodal infection, green top, internodal discoloration and internal
discoloration of the canes in CCT chamber were taken as the parameters for fixing the
disease evaluation after 10 days of incubation. The results of CCT method
authenticated the results obtained under laboratory conditions. The study revealed
100 per cent effectiveness of two per cent P. corylifolia hairy root extract over red rot
pathogen infection when compared to canes treated only with spore suspension of
C. falcatum.
173-179 | JRA | 2013 | Vol 2 | No 2
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www.jagri.info
Journal of Research in
Agriculture
An International Scientific
Research Journal
Authors:
Rajkumar D* and
Murugesan R





Institution:
Department of Agricultural
Microbiology, Tamil Nadu
Agricultural University,
Coimbatore, 641 003,
Tamilnadu, India.





Corresponding author:
Rajkumar D
















Email:




Web Address:
http://www.jagri.info/
documents/AG0047.pdf.


Dates:
Received: 10 June 2013 Accepted: 01 July 2013 Published: 13 July 2013
Article Citation:
Rajkumar D and Murugesan R.
Antifungal Activity of Psoralea corylifolia Hairy Root Extract against Sugarcane Red Rot
Pathogen under Controlled Condition Treatment Chamber.
Journal of Research in Agriculture (2013) 2(2): 173-179
Original Research
Journal of Research in Agriculture
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An International Scientific Research Journal


INTRODUCTION
Sugarcane is one of the most important cash
crops grown in India, for its adaptability to be cultivated
under a wide range of climate, cultural and soil
conditions. This crop occupies 2.8 % of cultivated area
and contributes to the tune of 7.5 % of agricultural
production of the country. Sugarcane crop is one among
the important cash crops in India and a main source of
white crystal sugar and also provides gur and
khandasari (brown sugar)which are the main substitute
of sugar. In India about 35 million farmers involved in
sugarcane cultivation and about 50 million people
depend on depend on sugar factories and other related
industries for their employment. Different pathogens viz.,
fungi, bacteria, viruses and phytoplasmas infects
sugarcane crop (Agnihotri, 1990). In India, the red rot
disease caused by fungus Colletotrichum falcatum
(Went, 1893) is considered as very serious disease where
sugarcane is cultivated. The disease was responsible for
the elimination of many elite sugarcane varieties
(Beniwal et al., 1989). Various chemical fungicides are
available for the control of red rot in sugarcane. As
sugarcane is a long duration crop, the treatment of sett
with fungicides will not be sufficient to protect
sugarcane crop from red rot pathogen (Viswanathan,
2010). The use of synthetic fungicide leads to several
problems such as residue in food and feed, pathogen
resistance, toxicity to non target organism and
environmental pollution in different agricultural
ecosystems. In addition to these, elimination of soil born
inoculum through chemicals is difficult and costly; and
development of resistant varieties through breeding
methods is long term endeavour (Alexander and
Viswanathan, 1996). Therefore, with the increasing
public awareness of environmental safety and persistent
demand for ecofriendly products, we are forced to
produce quality products both for export and domestic
consumption. Hence, an alternative approach is the use
of botanicals in management of this disease which are
eco friendly in nature, in addressing the problem (Ahmad
et al., 1998). Botanicals are the rich sources of secondary
metabolites viz., triterpenes, glycosides, flavonoids,
tannins, alkaloids and other aromatic compounds (Singh
et al., 1976; Beniwal et al., 1988). Some of the plant
extracts were well known for their antifungal,
antibacterial and antiviral properties (Amoros et al.,
1992; Jayakumar et al., 2007). When compared to
synthetic pesticides botanicals have low mammalian
toxicity and they are also target specific. The biocidal
compounds of botanicals were highly degradable and its
activity extends to wide range of insect pests and
pathogens (Kalaycioglu et al., 1997). Botanical
fungicides contains numerous ingredients so that the
pathogens requires several mutations to develop
resistance to them (Das and Das, 1994; Naqvi et al.,
1991). In our earlier studies it was found that 15 %
aqueous extract of Psoralea corylifolia leaves and 2 %
methanol extract of P. corylifolia hairy roots showed 100
% inhibitory to C. falcatum under in vitro conditions. In
continuation of our work, in the present study an attempt
was made to evaluate the antifungal activity of methanol
extract of P. corylifolia hairy roots against C. falcatum
under Controlled condition treatment (CCT) Chamber.
The controlled condition testing method was found to be
an effective method when compared to other methods
available for identifying the resistant or susceptible
sugarcane variety against the infection of red rot
pathogen C. falcatum (Mohanraj et al., 1997).

MATERIALS AND METHODS
Evaluation by controlled condition testing
method was done essentially as per the method
developed by Mohanraj et al., (1997).The experiment
was done at Controlled condition treatment (CCT)
chamber in Sugarcane Breeding Institute, Coimbatore.
The testing chamber is of 3m x 3m x 3.6m dimensions
which was fabricated with steel frames covered with
high density polythene sheets. The inside of the chamber
174 Journal of Research in Agriculture (2013) 2(2): 173-179
Rajkumar and Murugesan, 2013
was illuminated each day for 8 h by fluorescent lamps
with a total light energy output of 320 watts. A timer
controlled humidifier (make - L and TEM 1000) was
operated inside the chamber so as to maintain 90 per cent
relative humidity throughout experimentation period
(Figure 1). Eight months old sugarcanes of variety CoC
671 were collected from the field, and they were placed
inside the chamber such that the lowest node of the
sugarcane stalk was 10 to 15 cm below the surface of the
wet sand bed in trays. Nodes of 6
th
, 7
th
and 8
th
position
were selected for inoculation from which leaf sheaths
were removed using a fine knife, without injuring the
nodal regions (Mohanraj et al., 1997). Red rot inoculum
C. falcatum (Cf671) was prepared as a spore suspension
(one million spores/ ml). In order to check the efficacy of
P. corylifolia suspension extract against the test
pathogen, the spore suspension was mixed with different
combinations of 2.0 % P. corylifolia (effective
percentage which exerted 100 per cent inhibition under
in vitro condition) suspension extract as given below.
T
1
100 parts of spore suspension
(10 x 10
5
spores ml
-1
)
T
2
50 parts of spore suspension + 50 parts of
P. corylifolia hairy root extract (2 %)
(5 x 10
5
spores ml
-1
)
T
3
- 25 parts of spore suspension + 75 parts of
P. corylifolia hairy root extract (2 %)
(2.5 x 10
5
spores ml
-1
)
T
4
- 10 parts of spore suspension + 90 parts of
P. corylifolia hairy root extract (2 %)
(1.0 x 10
5
spores ml
-1
)
Six canes were taken for each treatment. Two ml
from the above treatments was swabbed on the selected
nodes and covered with thick cotton pads and tied with
polythene strips. Inoculated canes were incubated under
90 % relative humidity (RH) and the temperature was
maintained at 32 C (Figure 2 and Figure 3). Inoculated
canes were evaluated for disease reaction after 10 days.
Criteria for disease evaluation was based on the nature of
nodal and internodal lesions, spread of the lesions, colour
of the lesions, pathogens growth and sporulation on
nodal and internodal regions, bud necrosis, internal
symptoms and histological examinations.

RESULTS AND DISCUSSION
Two per cent hairy root extract of P. corylifolia
which exhibited 100 per cent control over mycelial
growth and spore germination of C. falcatum under in
vitro condition in our earlier study was evaluated for its
effectiveness on red rot reaction under Controlled
Condition Treatment Chamber. The canes treated with
the spore suspension (1 x 10
6
spores/ ml) with a
combination of 2 % P. corylifolia hairy root extract
showed a significant reduction in disease symptoms,
when compared to canes treated only with spore
suspension. The effect of hairy root extract on disease
reaction was clearly observed on evaluation as control.
The significant reduction in nodal infection (91.77 %)
was observed in the canes treated with 90 parts of hairy
root extract and 10 parts of spore suspension, where the
nodal infection rate was only 8.33 %. The canes treated
only with spore suspension recorded 77.77 % nodal
Journal of Research in Agriculture (2013) 2(2): 173-179 175
Rajkumar and Murugesan, 2013
Table 1. Effect of 2.0 per cent hairy root extract of P. corylifolia on red rot disease reaction.
S.No Treatments
% of nodal
infection
% of internodal
discoloration
% of internal
discoloration
% of green top
1 100 parts S.S 77.77 91.60 88.88 16.66
2 50 parts S.S + 50 parts HR.E 36.11 0.00 8.33 33.33
3 25 parts S.S + 75 parts HR.E 19.44 0.00 5.55 100.00
4 10 parts S.S + 90 parts HR.E 8.33 0.00 0.00 100.00
S.S - spore suspension of C. falcatum (1 x 10
6
spores/ ml)
HR.E hairy root extract of P. corylifolia


infection. In case of internodal discoloration, except the
canes treated with 100 parts of spore suspension, all
other canes treated with both spore suspension and hairy
root extract showed 100 % free from internodal
discoloration. For internal discoloration observation, the
canes from all the treatments were cut opened
longitudinally with a sharp knife inorder to observe the
internal symptoms of disease infection. A significant
reduction of internal discoloration was observed in the
canes treated with P. corylifolia hairy root extract when
compared to canes treated with spore suspension alone.
The leaves of the canes treated with hairy root extract
remained green throughout the experimental period,
whereas the leaves of the canes treated only with fungal
spore suspension dried up completely (Table 1)
(Figure 4). The solute transport might be affected by
severe discoloration in the internodal region and resulted
in drying up of leaves. The study clearly authenticates
the outcome of the results obtained under in vitro
conditions. It was a clear-cut demonstration that supports
the principle antimicrobial compound responsible for
controlling the dreadful pathogen.
Controlled condition testing method is a precise
method to evaluate the sugarcane clones for disease
resistance, where the results can be observed within
10 days (Mohanraj et al., 1997). In this method the
intensity of the spore load taken in the cotton swab was
much higher (1 x 10
6
spores ml
-1
) and it cannot replicate
the true natural condition of the soil. Such severe
condition is well throttled by the presence of antifungal
compound in 2 % P. corylifolia hairy root extract and
successfully controlled and blocked the infection of
pathogen into the cane. When compared with the results
obtained in the pot culture studies, the effect of hairy root
extract on control of pathogen was higher in controlled
condition testing method. The reason might be due to
controlled condition testing method, where the spores are
in direct contact with the antimicrobial compound.
Whereas, under pot culture conditions there were no
direct contact between the pathogen and the compound.
Also the experimental period was very short (10 days) in
case of controlled condition testing method than in the
pot culture experiments. The antimicrobial compound
might be attributed to natural degradation in pot culture
176 Journal of Research in Agriculture (2013) 2(2): 173-179
Rajkumar and Murugesan, 2013
Figure 1.Control Condition Treatment Chamber
Figure 2. Cotton Swabbing on nodes
Figure 3. Incubation of canes inside the chamber
conditions and only high percentage of compound might
work in the pot culture conditions. Previously a 0-9 scale
screening methodology was universally accepted as red
rot resistance variety screening method, but the method
was time consuming and also influenced by
environmental factors. Whereas controlled condition
testing (CCT) method is a rapid, precise and less
influenced by environment factors while screening
sugarcane genotypes for red rot resistance (Srinivasan
and Bhat, 1961). Among the methods used to evaluate
clones for disease resistance against red rot pathogen
such as nodal method, plug method and controlled
condition testing method, the results obtained in the
controlled condition testing method showed very precise
results and also very much suitable to identify field
tolerant clones with more reliability in a short time
(Viswanathan et al., 1998; Kalaimani, 2002). Ramesh
Sundar et al., (2002) studied the induction of systemic
resistance to C. falcatum in sugarcane by acibenzolar-s-
methyl (CGA- 245704) a noval synthetic signal molecule
Journal of Research in Agriculture (2013) 2(2): 173-179 177
Rajkumar and Murugesan, 2013
Figure 4. Disease reaction in canes upon various treatments in CCT chamber
a. Nodal infection
b. Inter Nodal infection
c. Green top
d. Internal discoloration


by controlled condition testing method and reported that
this method was less injurious and evaluation can be
done in a more natural way.

CONCLUSION
Sugarcane red rot disease was responsible for the
elimination of many elite sugarcane varieties and upto
100 % yield loss has been reported in severe
epiphytotics. The use of synthetic fungicides for disease
control leads to the environmental pollution in different
agricultural ecosystems. This study revealed that 2 %
hairy root extract of P. corylifolia was fully effective
against red rot pathogen under controlled condition
testing chamber. Further studies are needed to test the
fungitoxic effect under field conditions, their
thermostability, stability to storage and also their
phytotoxicity towards the host plant.

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Journal of Research in Agriculture (2013) 2(2): 173-179 179
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