Antifungal activity of psoralea corylifolia hairy root extract against sugarcane red rot pathogen...

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 (106 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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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

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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 6th, 7th and 8th 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.

T1 – 100 parts of spore suspension

(10 x 105 spores ml-1)

T2 – 50 parts of spore suspension + 50 parts of

P. corylifolia hairy root extract (2 %)

(5 x 105 spores ml-1)

T3 - 25 parts of spore suspension + 75 parts of


(2.5 x 105 spores ml-1)

T4 - 10 parts of spore suspension + 90 parts of


(1.0 x 105 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 32º 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, pathogen´s 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 106 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


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



S.S - spore suspension of C. falcatum (1 x 106 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 106 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



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



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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