J. Seetharamulu.pmd

NSave Nature to Survive
QUARTERLY
6(1&2): 93-97, 2012
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EFFECT OF MEDICINAL PLANTS AND BIOFUNGICIDES ON
DEFENSE ENZYME LEVELS AND DISEASE CONTROL IN
MULBERRY
J. SEETHARAMULU*, J. UMAMAHESHWARI 1 , A. SREERAMULU 1 , A. K. GOEL AND P. J. RAJU
Andhra Pradesh State Sericulture Research and Development Institute,
Kirikera - 515 211, Hindupur, Andhra Pradesh, INDIA
1 Department of Biochemistry, Sri Krishnadevaraya University,
Anantapur - 515 003, Andhra Pradesh, INDIA
e-mail: sithara_jolapuram@rediffmail.com
INTRODUCTION
Mulberry (Morus alba L.) is one of the most important commercial crops grown
extensively as food plant for the silkworm (Bombyx mori L.). Being a vegetatively
propagated perennial crop, initial establishment of mulberry is affected by many
soil borne fungal diseases namely stem canker and die-back caused by
Botryodiplodia theobromae, cutting rot caused by Fusarium solani and collar rot
caused by Phoma sorghina, Phoma mororum and Sclerotium rolfsii (Gupta et al.,
1997) and cutting die-back caused by Alternaria tenuissima (Seetha et al., 2004
& 2008). These diseases affect almost all the mulberry varieties cultivated in India.
Chemical fungicides have been suggested for the effective control of mulberry
diseases, but these chemicals could not get wide acceptance among sericulturists
due to their prohibitive cost, possible toxicity to silkworm and various hazards to
mankind and environment. The use of plant products and microbial agents for
controlling the plant diseases is generally regarded as a potential and safe alternative
to chemical fungicides. The plant kingdom represents an enormous reservoir of
potential chemical compounds and also leads to an increase in the enzyme
activity viz. peroxidase and polyphenol oxidase. Similarly the chemical
compounds like phenols, alkaloids and primary metabolites also increased which
it increases the resistance in plants. In the present study some efforts have been
made in controlling the pathogens at nursery stage with an integration of plant
extracts and bio-control agents.
MATERIALS AND METHODS
In vitro studies
Medicinal plant extracts were tested against the mycelial growth of the pathogenic
fungi viz. A. tenuissima and F. solani through poisoned food technique (McCallan,
1947) under in vitro conditions. The plant extracts were prepared according to
the method of Awuah, 1989. The results were expressed in terms of per cent
inhibition of mycelium over control.
The fungal antagonists viz, T. viride, T. harzianum and T. pseudokoningii and the
bacterial antagonist P. fluorescens were tested against the A. tenuissima and F.
solani by Dual culture technique (Hung and Hoes, 1976).
In vivo studies
To test the efficacy of promising plant extracts and bio-fungicides against the
pathogenic fungi, five treatments with three bio-fungicides {T. viride (T 1 ), T.
harzianum (T 2 ) and P. fluorescens (T 3 )}, two plants extracts {L. camara (T 4 ) P.
juliflora (T 5 )} and one chemical fungicide (T 6 ) (Mancozeb) were used for conducting
ABSTRACT
Medicinal plant extracts along with biofungicides
were tested under in vitro to control
the Fusarium solani and Alternaria tenuissima,
the incitants of root rot and die-back diseases
of mulberry. Plant extract of Prosopis juliflora
showed maximum inhibition on the mycelial
growth (above 80.0%) followed by L. camara
(above 66.0%). Among the antagonists
Pseudomonas fluorescens and Trichoderma
viride showed maximum inhibition on the
mycelial growth of both pathogenic fungi. The
promising plant extracts (P. juliflora and L.
camara) and antagonists (P. fluorescens and T.
viride) were then tested against both the
pathogenic fungi under in vivo along with
popular chemical Mancozeb. Maximum
survival percentage of mulberry cuttings was
recorded in the treatment with T. viride and P.
fluorescens (above 90%) followed by T.
harzianum (above 80%) against both the
pathogens. In case of plant extracts, P. juliflora
recorded higher survival (above 55%) followed
by L. camara (above 50%). Mancozeb recorded
55 % survival. Whereas in control the survival
was recorded 10 - 15% only. Alterations in
the enzymatic activities due to infection of
mulberry stem with A. tenuissima and F. solani
were also evident.
KEY WORDS
Antagonists, Alternaria tenuissima
Defence enzymes, Fusarium solani
Mulberry nursery, Plant extracts
Survivability
Received : 17.11.2011
Revised : 22.02.2012
Accepted : 24.04.2012
*Corresponding author

J. SEETHARAMULU et al.,
the experiment under controlled conditions. Control pots
were inoculated with the test fungi separately and left without
treatment. All the treatments were given before planting the
cutting in to the inoculated soil. The sterilized soil was used
for inoculation.
Inoculation
The test fungi were grown in corn meal - sand medium for
inoculation (Rangaswamy and Mahadevan, 2001). The 15
days old culture incubated at 29 ± 1ºC of A. tenuissima and
F. solani were used for inoculation of sterilized soil. The corn
meal-sand culture of the A. tenuissima and F. solani was
thoroughly mixed with sterilized soil separately at 10% level
(w/w - 90g sterilized soil + 10g inoculum). Each of the test
fungus was mixed with sterilized soil separately and poured
in to polythene bags (1kg inoculated soil/bag). Later twenty
cuttings were treated with each test bio-fungicide, plant
products and Mancozeb separately and planted in the
polythene bags (one cutting/bag). Twenty cuttings were
maintained for each treatment and control. All the bags were
watered on alternate days with sterilized water and kept in
open condition.
The cuttings were dipped in plant extracts of required
concentration (L. camara at 100% and P. juliflora at 10%
concentration) for half an hour and 100 mL of plant extract
was added to the inoculated soil in the bag and planted the
treated cuttings with plant extract. The talc based bio-fungicide
was mixed with distilled water to form fine slurry and the
cleaned mulberry cuttings were dipped in to the slurry for 30
minutes and 10 g of bio-fungicide was added to each bag
containing inoculated soil with test fungi and treated cuttings
with bio-fungicide were planted in the bags. In case of chemical
treatment the cuttings were dipped in 0.1% Mancozeb
solution for 30 minutes and 100 ml of 0.1% Mancozeb
solution is added to the inoculated bag and cuttings were
planted. The disease index was observed and the survival
percentage was calculated to each treatment and assessed
their efficacy.
Enzymatic studies
The mulberry stem tissues from the all treatments and control
bags were collected separately in to polythene bags at different
intervals i.e. 15 th , 30 th , 45 th and 60 th day of plantation (Stage I,
II, III and IV) and these samples were used for enzyme
Table 1: Efficacy of plant extracts against the mycelial growth of A. tenuissima and F. solani under in vitro by poisoned food technique
Botanical source A. tenuissima F. solani
Mycelialgrowth (cm) Inhibition (%) Mycelialgrowth (cm) Inhibition (%)
Prosopis juliflora, leaf 1.70 81.17 1.80 80.03
Calotropis procera, leaf 3.80 57.80 3.60 60.03
Annona squaimosa, seed 3.60 60.03 5.90 34.50
Cassia occidentalis, leaf 7.00 22.27 7.60 15.60
Ocimum sanctum, leaf 6.20 31.17 7.40 17.80
Azadiracta indica , seed 3.40 62.30 6.90 23.40
Eucalyptus globules, leaf 7.60 15.60 7.80 13.40
Aloe vera, leaf 7.73 14.13 8.60 4.50
Lantana camara, leaf 3.00 66.70 2.80 68.93
Vinca rosea, leaf 7.90 12.27 4.10 54.50
Mancozeb 0.40 95.56 0.60 93.33
Control 9.00 — 9.00 —
CD (P d” 0.05) 0.29 3.22 0.25 2.75
94
extraction. The small pieces (25g from each treatment and
control) of the tissues were transferred to warring blender and
added 100 mL of chilled Acetic acid-acetate buffer at pH 5.2
blended for 10 minutes, filtered through two layers of
cheesecloth and centrifuged at 2000 g at 35ºC for 20 minutes.
The supernatant was collected in conical flask. The crude
enzyme in the flask was used for the measurement of
endoglucanase (β-1, 3-glucanase) activity. This enzyme extract
was stored at 2-4ºC by adding a few drops of tolune for later
use (Aneja, 2003). The activity of endoglucanase was
quantified by CMC method (Ghosh, 1987) and the activities
of polyphenol oxidase (PPO) and peroxidase (PEO) was
determined by Fehrmann and Dimond (1967).
RESULTS AND DISCUSSION
In vitro studies
Among all the plant extracts P. juliflora showed highest
inhibition on both fungi (81.2 % over A. tenuissima and 80.0
% over F. solani) and followed by L. camara (66.7 % over A.
tenuissima and 68.9 % over F. solani). In the present study
significant differences in inhibition of mycelial growth was
observed in A. tenuissima and F. solani by the interaction with
T. viride and P. fluorescens. These two antagonists showed
high levels of inhibition (80-88 %) as shown in table 2, where
as T. pseudokoningii and T. harzianum showed moderate
level of inhibition against both pathogenic fungi.
In vivo studies
All the tested plant products and bio-fungicides showed
inhibitory effect on the growth of F. solani and A. tenuissima.
The maximum survival percentage was recorded in T. viride
and P. fluorescens (above 90 %) followed by T. harzianum
(80 %). Incase of the treatments with plant extracts, P. juliflora
(above 55 %) showed higher survival percentage and followed
by L. camara (above 50 %). Mancozeb recorded 55 %
survivability. Percent improvement of survivability over control
under disease stress conditions of F. solani was recorded 85.0
%, 70.0 %, 80.0 %, 45.0 %, 50.0 % and 45.0 % in the
treatments with T. viride, T. harzianum, P. fluorescens, L.
camara, P. juliflora and Mancozeb respectively. In case of A.
tenuissima inoculated bags the percent improvement of
survivability over control was recorded higher with 75.0 %,
70.0 %, 75.0 %, 35.0 %, 40.0 % and 40.0 %.

Units/mL
Specific activity/mg protein
Specific activity/mg protein
120
100
80
60
40
20
0
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
180
160
140
120
100
80
60
40
20
0
0
I II III IV
C I T.V P.F L.C P.J M
Treatments
I II III IV
C I T.V P.F L.C P.J M
Treatments
I II III IV
Endoglucanase
Peroxidase
Polyphenol oxidase
C I T.V P.F L.C P.J M
Treatments
Figure 1: Effect of F. solani infection on the activity of defense
enzymes of mulberry stem tissues at different stages of disease
development under different treatments
The present study disclose that, the plant extracts of P. juliflora
and L. camara were found to be effective in inhibiting the
growth of pathogenic fungi. The present findings are agreeing
with the earlier reports of many researchers. Raghavendra et
al., (2002) reported the fungi toxic properties of P. juliflora
against eight species of Fusarium and one species of Alternaria.
Antifungal properties of extracts of P. juliflora have also been
reported by Muthulakshmi (1990) and Narasimhan et al.,
(1991) against A. tenuissima. Ram and Thakore (2005) reported
the extracts from L. camara were effective in inhibiting the
growth of F. solani. In case of bio-fungicides P. fluorescens
and T. viride were found to be effective in controlling F. solani
95
Table 2: Efficacy of antagonists against the mycelial growth of A.
tenuissima and F. solani under in vitro conditions
Name of the antagonist Inhibition of mycelial growth (%)
A. tenuissima F. solani
Trichoderma viride 87.7 88.9
Trichoderma harzianum 64.4 68.9
Trichoderma pseudokoningii 73.3 80.0
Pseudomonas fluorescens 83.3 78.9
Control 0.0 0.0
C D (P d” 0.05) 2.61 2.66
Units/mL
Specific activity/mg protein
Specific activity/mg protein
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
180
160
140
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
EFFECT OF MEDICINAL PLANTS AND BIOFUNGICIDES
I II III IV
C I T.V P.F L.C P.J M
Treatments
I II III IV
C I T.V P.F L.C P.J M
Treatments
I II III IV
Endoglucanase
Peroxidase
Polyphenol oxidase
C I T.V P.F L.C P.J M
Treatments
Figure 2: Effect of A. tenuissima infection on the activity of defense
enzymes of mulberry stem tissues at different stages of disease development
under different treatments
and A. tenuissima. Philip et al., (1996) reported the
antagonistic effect of a local isolate of T. harzianum against
the mulberry root rot pathogen with 74 % protection to the
mulberry plants. Dhoke (2005) reported the efficacy of
fluorescent Pseudomonas isolates against some Fusarium
species. Such effective inhibition of mycelial growth of F. solani

J. SEETHARAMULU et al.,
Table 3: Efficacy of plant extracts and antagonistic fungi against the A. tenuissima and F. solani under simulated conditions (In vivo)
Sl.No. Treatments No. of cuttings Soil inoculated with F. solani Soil inoculated with A. tenuissima
planted No. of cuttings Survival Percent over No. of cuttings Survival Percent over
survived % survived % control
1 Trichoderma viride 20 19 95 + 85 18 90 + 75
2 Trichoderma harzianum 20 16 80 + 70 17 85 + 70
3 Pseudomonas fluorescens 20 18 90 + 80 18 90 + 75
4 Lantana camara 20 11 55 + 45 10 50 + 35
5 Prosopis juliflora 20 12 60 + 50 11 55 + 40
6 Mancozeb 20 11 55 + 45 11 55 + 40
7 Control 20 2 10 0 3 15 0
+ = Increase over control ; Each value is an average of three replications
by T. viride has been reported by Jha and Jalali (2006). Bohra
et al., (2005) reported the treatment with Pseudomonas
significantly reduces the disease severity of Fusarium wilt in
the nursery. Mostapha et al., (2006) reported P. fluorescens
have an excellent potential to be used as biocontol agents
against mulberry root rot pathogens viz. B. theobromae, F.
solani, F. oxysporum and R. solani. All these findings were
agreeing with the present findings.
It is finally reveals from the present research findings, the biofungicides
viz, Trichoderma viride and Pseudomonas
fluorescens and Prosopis juliflora (plant extract) were suggested
as pre-plantation treatment for effective control of cutting rot
and die-back diseases of mulberry nursery.
Defense enzymes
The endoglucanase (β-1, 3- glucanase) activity under disease
stress conditions of F. solani was high in T (15.7 %, 39.5 %,
1
30.5 % and 21.9 % at 1, 2, 3 and 4 stages respectively than
the control) and T (20.6%, 42.8%, 25.2% and 27.1% at stage
2
1, 2, 3 and 4 respectively than the control) treated stems than
all other treatments and control. Infection had a pronounced
effect on endoglucanase activity and steep increase up to 2nd
stage of infection, later slight decrease in the enzyme activity
was noticed (Fig. 1). In case of the disease stress conditions of
A. tenuissima, endoglucanase activity in T and T was
1 2
recorded very high than the control and infected stem.
Remaining treatments also showed higher enzyme activity than
the control at all the stages. Infection had a pronounced effect
on peroxidase activity under the disease stress conditions of
F. solani and resulted in steep increase at various stages of
disease development. The higher enzyme activity was noticed
in the T and T treatments. The enzyme activity was recorded
1 2
high by 31.1 %, 38.7 %, 64.5 % and 63.5 % in T and 30.9 %,
1
69.0 %, 73.3 % and 68.9 % in T at 1 2 st , 2nd , 3rd and 4th stage
respectively than the control. Whereas the enzyme activity
under the disease stress conditions of A. tenuissima all the
treatments showed increased activity over control. In the
treatments with bio-fungicide (T & T ) the enzyme activity was
1 2
found very high at all stages than the control.
The activity of polyphenol oxidase under the disease stress of
both the fungi showed gradual increase in all the treatments.
T & T treatments recorded high enzyme activity. In the
1 2
treatments T and T also showed increased enzyme activity
3 4
over control. In case of A. tenuissima infection the enzyme
activity in all the treated tissues was increased according to
the stage than the control (Fig. 2). In case of T and T treatments
1 2
the enzyme activity was recorded higher than the infected
stem tissues.
96
In the present study it was noticed that the activities of β-1, 3glucanase,
PEO and PPO were increased in the plants treated
with biofungicides and plant products than the control. Similar
results were also found by many other researchers. According
to Karthikeyan et al. (2005) the enzymes viz, chitinase, β-1, 3glucanase,
peroxidase and polyphenol oxidase were induced
in the A. palandui inoculated plants, whereas an additional
increase in the synthesis of these enzymes was observed in
the P. fluorescens Pf pretreated plants challenge-inoculated
1
with A. palandui.
Increased PEO and PPO activity is a general phenomenon in
the inoculated plants. According to Van Loon et al., 1998
induction of defense enzymes makes the plant resistant to
pathogen invasion. These results agreeing with the findings of
Gupta et al. (2007) found higher amount of oxidative enzymes
viz, catalase, poly phenol oxydase and peroxidase were
present in anthracnose diseased leaves of French bean than
healthy ones and resistant varieties showed higher enzyme
activity than their susceptible counterparts. According to
Kloepper et al. (1996) induction of systemic resistance may
involve activation of multiple potential defense mechanisms,
including increased activity of β- 1, 3- glucanases, peroxidase.
Induced disease resistance has been demonstrated in a number
of plants – pathogen interactions by Sticher et al. (1997).
Alterations in the enzymatic activities due to infection of
mulberry stem with Alternaria tenuissima and Fusarium solani
were also evident. The activities of defence enzymes viz.
endoglucanase, peroxidase and polyphenol oxidase were
increased in the plants treated with bio-fungicides and plant
products under disease stress conditions of both the
pathogenic fungi at various stages of disease development.
Further, the increased enzyme activities were observed in the
mulberry stem tissues treated with Trichoderma viride and
Pseudomonas fluorescens as compared to control and also in
other treatments. Whereas in chemical treatment not much
difference in the enzyme activity was observed when compared
with corresponding healthy stem tissues. Prabhu et al. (2004)
also reported that, there was an increase in the enzyme activity
viz., peroxidase and polyphenol oxidase, when the plants
were treated with plant products. Similarly the chemical
compounds like phenols, alkaloids and primary metabolites
also increased which it increases the resistance in plants. In
case of diseases, the plant extracts may lock the susceptible
sites that prevent the entry of pathogen into the plant. Perez et
al. (2002) reported T. harzianum produce some fungal cell
wall hydrolyzing enzymes within their biocontrol mechanism.
According to Karthikeyan et al. (2005) the activity of β-1, 3glucanase
was greatly induced in Pf treated plants as
1

compared with controls and also showed significant increase
in the levels of the defense enzymes in comparison to the
plants challenged with the pathogen. According to
Schlumbaum et al. (1986) and Velazhahan et al. (2003)
chitinase and β-1, 3-glucanase have the potential to hydrolyze
chitin and β-1, 3-glucan respectively, the major components
of fungal cell walls, leading to direct inhibition growth of several
fungi. The studies indicate that, the plant products and biofungicides
are very much effective in controlling the disease
and also facilitating as plant growth promoter by triggering the
defense mechanism.
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