Comparative studies on algal seaweed extracts JBiopest 7(2):167-176(2014)

Comparative studies on brown, red and green alga seaweed

JBiopest 5(1):
extracts for1-6
their antifungal activity against Fusarium oxysporum
f.sp. udum in Pigeon pea var. CO (Rg)7 (Cajanus cajan (L.) Mills.)
S. Ambika* and K. Sujatha1
ABSTRACT
In vitro studies was conducted to evaluate the effect of seaweed liquid extracts of
Caulerpa racemosa (green alga), Sargassum myricocystum (brown alga) and Gracilaria
edulis (red alga) at different concentrations of 10, 15, 20, 25 and 30% along with
control against the mycelial growth of Fusarium oxysporum f. sp. udum by poison food
technique. Result revealed that extract of S. myricocystum showed significant antifungal
activity against pathogen followed by G. edulis and C. racemosa. S. myricocystum
(30%) extract recorded the lowest mycelial growth (28.1, 33.9, 34.7, 39.4 and 44.3 mm)
at 24, 48, 72, 96 and 108 hrs after incubation. Among the antagonists tested against
Fusarium oxysporum f.sp. udum, the fungal antagonists Trichoderma viride was found to
be most effective in reducing the mycelial growth than the bacterial antagonist Pseudomonas
fluorescens. Both the antagonistic of fungi and bacteria has compatability with seaweed
extracts in all the concentrations.

MS History: .25.09.2014 (Received)-08.11.2014 (Revised)-18.11.2014 (Accepted)

Citation: S. Ambika and K. Sujatha. 2014. Comparative studies on brown, red and green alga seaweed extracts for
their antifungal activity against Fusarium oxysporum f.sp. udum in Pigeon pea var. CO (Rg)7 (Cajanus cajan (L.)
Mills.). Journal of Biopesticides, 7(2):167-176.
Key words: Seaweeds, soil borne pathogen, red gram, Fusarium oxysporum f.sp. udum.
INTRODUCTION 1981) and the annual crop losses due to wilt
Pigeonpea (Cajanus cajan (L.) Mills.) is one alone have been estimated about 36 million
of the major legume crops grown in the US dollars in India (Kannaiyan et al., 1984).
tropics and sub-tropics, and accounts for about Most of the soils borne pathogens are difficult
5% of world legume production. The largest to control by conventional strategies such as
producer is India, where the dried pea is the the use of resistant cultivars and synthetic
favoured choice for the preparation of dhal. fungicides (Weller et al., 2002). To avoid the
Due to cultivation of pigeonpea in diversified implication of yield losses due to plant
climatic situations in various geographical diseases, variety of control measures presently
areas it is attacked by more than 100 are in use. The chemical compounds are most
pathogens (Nene et al., 1989 and Raju et al., commonly used for the controlling of plant
2008). Soil borne diseases are the most diseases. No doubt the use of chemicals has
important in pulses causing heavy losses in been found very effective in controlling plant
seed yield. Fusarium wilt caused by Fusarium fungal diseases but some major problems
udum is the most important soil borne disease threaten to limit the continued use of
of pigeonpea capable of causing 30-100% loss fungicides.
in grain yield (Reddy et al., 1990) and the
incidence occurs at flowering and crop The toxic effect of synthetic chemicals can be
maturity stages (Kannaiyan and Nene, 1981). overcome, only by persistent search for new
The disease was first reported from Bihar state and safer pesticides accompanied by wide use
in India (Butler, 1906). Pigeonpea wilt is of pest control methods, which are ecofriendly
widely prevalent throughout the world and and effective (Mohana et al., 2011). In recent
more important in India (Kannaiyan and Nene years, there have been many reports of macro

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 Ambika and Sujatha
algae derived compounds that have a broad 168
o
range of biological activities, such as Long. 079 08.558’E), Tamil Nadu, were
antifungal, antibacterial, antiviral, antioxidant, washed with seawater initially to remove
anti-inflammatory, cytotoxic and antimitotic macroscopic epiphytes and sand particles and
activities (Demirel et al., 2009).The majority then with fresh water to remove adhering salt.
of these compounds are terpenes and The materials were shade dried for 2 weeks
polyphenols (Blunt et al., 2006). Marine followed by oven drying at 40oC for 24 h and
bioactive substances extracted from seaweeds powdered. A 150 ml of alcohol was added to
have been used for several decades to enhance 20 g powder and kept for overnight with
plant growth and productivity (Rathore et al., intermittent stirring and extracted through
2009). Brown seaweed extract is a well known rotary evaporator at 40oC and 45 rpm (Bhosle
plant growth stimulator, which improves et al., 1975 with slight modification). The
general plant health and enhances plant liquid fertilizer was collected and stored in air
resistance to nematodes, pests and fungal tight container. The different concentrations
diseases (Jayaraj et al., 2008). Present were prepared by taking 10, 15, 20, 25 and 30
investigation was undertaken to evaluate ml of the stock preparation and mixing with
different seaweed extracts (red, brown and distilled water to get 10, 15, 20, 25 and 30 %
green) for their antifungal activity against concentrations.
Fusarium oxysporum f.sp. udum and their Antifungal activity
compatibility with Trichoderma viride and Poisoned food technique (Schmitz, 1930) was
Pseudomonas fluorescens. employed to screen the antifungal efficacy of
seaweed extracts. Potato dextrose agar media
MATERIALS AND METHODS amended with seaweed extracts (10, 15, 20, 25
and 30%) were autoclaved and poured into
Isolation of pathogen sterile Petriplates. Fungal disc of 9mm
The pathogen was isolated from the diseased diameter were cut with the help of sterile cork
tissues of red gram by tissue segment method borer from the periphery of 5 days old culture
(Rangaswami, 1958). The infected portions of of Fusarium oxysporum f. sp. udum and the
diseased plants were cut into small pieces disc were transferred aseptically on PDA
using sterilized scalpel and these were surface plates poisoned with seaweed extracts. A plate
sterilized with 0.1 per cent mercuric chloride only with PDA and fungal disc was
for one minute and washed in three changes of considered as control (standard) and the
sterile water. The surface sterilized tissues diameter of growth of fungus in this plate was
were plated on PDA in sterile Petri plates and used as a control for the calculation of percent
incubated at room temperature (28  2˚C) for inhibition of test fungus.
14 days. The hyphal tips of fungi grown from Radial growth
the pieces were transferred aseptically to PDA Measurement of the radial growth in
slants for maintenance of the culture. The centimeters (cm) was done and the radial
fungus was further purified by single spore growth was determined by using the formula
isolation and maintained on PDA. The Kr according to Reeslev and Kjoller (1995).
pathogen was identified based on colony Radial growth (Kr) = (R1 – R0) /(t1 - t0)
character, conidial production and spore Where, R0 and R1 are the colony radial growth
morphology. at time t0 and t1 respectively, determined after
24, 48, 72, 96 and 108 hrs from inoculums.
Collection and preparation of extracts Inhibition percentage
The marine alga C. racemosa, The inhibition percentage was calculated
S.myricocystumand G. edulis were collected measuring the radial growth of the fungus
from Mandapam coast (Lat. 09o 17.417’N; grown on control and amended plates after 24,
48, 72, 96 and 108 hrs after incubation, using
the following formula (Harlapur et al., 2007).
 Comparative studies on algal seaweed extracts JBiopest 7(2):167-176(2014)
I% = 100 × (C – T) / C 169
Where,
JBiopestI%5(1):
= 1-6
inhibition percentage of Then the antagonistic bacterial isolates were
pathogen growth, C = average radial growth in inoculated in the poisoned media. The plates
control plates and T = average radial growth were incubated under room temperature for
in plates amended with seaweed extract. 48 h and the growth of bacteria was recorded
Efficacy of bacteria antagonist visually and scored either as highly
Culture of Pseudomonas fluorescens obtained compatible or moderately compatible or not
from the Department of Plant Pathology, compatible.
AC&RI, Madurai and tested for their Compatibility between antagonistic fungi
antagonistic effect on F.o. f.sp. udum by dual and seaweed extracts
culture plate technique (Dennis and Webster, PDA medium was amended with the
1971).Psuedomonas fluorescens was Caulerpa racemosa (green alga), Sargassum
multiplied on King’s B medium (King’s et al., myricocystum (brown alga) and Gracilaria
1954). A total of 9 mm culture disc of the edulis (red alga) with 10, 15, 20, 25, 30 per
pathogen was placed on the PDA medium in cent concentrations. Then the antagonistic
sterilized petri dish at one side 1.5 cm away fungi isolates were inoculated in the poisoned
from the edge of the plate and incubated at media. The plates were incubated under room
room temperature (282oC). Simultaneously temperature and the growth of fungi was
test bacteria were streaked on the medium at expressed in cm.
the opposite side of the plate, 1.5 cm away Data analysis
from the edge of the plate. Potato dextrose The data from various experiments were
agar medium inoculated with the pathogen analyzed statistically adopting the procedure
alone served as the control. The inoculated described by Panse and Sukhatme (1985).
plates were incubated at room temperature Wherever necessary, the percentage values
(28+2oC) with three replications. When the were transformed to arc sine values before
control plate showed full growth of the carrying out the statistical analysis.
pathogen, the radial growth of the mycelium RESULTS AND DISCUSSIONS
was measured. The results were expressed as Among the concentrations, 30% showed better
per cent growth inhibition over control. performance. Significant difference were
observed in the seaweed extarct of S.
Efficacy of the fungal antagonist myricocystum (30%) inhibited the mycelial
Culture of T. viride obtained from the growth of Fusarium oxysporum f. sp. udum
Department of Plant Pathology, AC&RI, which recorded lowest mycelial growth of
Madurai and tested for their antagonistic 28.1, 33.9, 34.7, 39.4 and 44.3 mm followed
effect on F.o. f.sp. udum by dual culture plate by G. edulis (30%) with 33.2, 38.1, 40.5, 47.6
technique (Dennis and Webster, 1971). A total and 53.1 mm where as in control the highest
of nine mm mycelial disc of F.o. f.sp. udum mycelial growth of 48.5, 62.4, 81.9, 85.2 and
and Trichoderma sp. were placed opposite to 90 mm after 24, 48, 72, 96 and 108 hrs was
each other near the periphery of the petri plate recorded (Table- 1). Cotton seeds soaked in
and incubated at room temperature (2820C). seaweed solution (1:500 S.wightii for 12 h)
After incubation, mycelial growth of the provided seedlings with considerable
pathogen and inhibition zone was measured as resistance against Xanthomonas campestris
well as in control plates. (Raghavendra et al., 2007).Seaweed extract of
Compatibility between antagonistic S. myricocystum showed inhibited radial
bacteria and seaweed extracts growth in the range of 19 to 42% after 24 hrs,
King’s B medium was amended with the C. 20 to 46% after 48 hrs, 34 to 58% after 72 hrs,
racemosa , S. myricocystum and G. edulis 27 to 54% after 96 hrs and 27 to 51 after 108
with 10, 15, 20, 25, 30 per cent concentration. hrs compared to control (Table 2). Jayaraj et
al. (2008) found that the seaweed in carrot
plants reduced leaf blights caused by

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 Ambika and Sujatha

170
Table 1. Effects of seaweed extracts on mycelial growth (mm) of Fusarium oxysporum f. sp. in vitro.

Mycelial growth (mm)

Hours

24 48 72 96 108 Mean
Sargassum myricocystum (10%)
39.4 49.7 54.2 62.1 65.7 54.22
Sargassum myricocystum (15%)
38.2 45.7 49.4 57.5 61.6 50.48
Sargassum myricocystum (20%)
35.3 36.8 43.3 46.8 52.8 43.00
Sargassum myricocystum (25%)
30.1 34.7 38.3 42.4 45.8 38.26
Sargassum myricocystum (30%)
28.1 33.9 34.7 39.4 44.3 36.08
Gracilaria edulis (10%)
43.7 53.4 58.8 69.3 73.8 59.80
Gracilaria edulis (15%)
40.5 47.1 56.5 60.7 67.2 54.40
Gracilaria edulis (20%)
37.3 42.8 47.7 55.1 62.0 48.98
Gracilaria edulis (25%)
36.4 39.8 44.9 53.6 59.4 46.82
Gracilaria edulis (30%)
33.2 38.1 40.5 47.6 53.1 42.50
Caulerpa racemosa (10%)
43.0 58.1 62.7 72.6 79.2 63.12
Caulerpa racemosa (15%)
41.5 50.4 55.7 63.8 71.2 56.52
Caulerpa racemosa (20%)
40.2 44.7 51.3 58.7 68.3 52.64
Caulerpa racemosa (25%)
39.0 41.0 45.8 54.0 61.6 48.28
Caulerpa racemosa (30%)
37.7 39.7 44.7 50.7 58.3 46.22
Control
48.5 62.4 81.9 85.2 90.0 73.60
Mean
38.26 44.89 50.65 57.47 63.39 50.93
C T CX T
SEd 0.273 0.489 1.095
CD (0.05) 0.539** 0.964** 2.157**

Alternaria and Botrytis as effectively as the B-1, 3-glucanase, peroxidase, polyphenol

fungicide chlorothalonil. In carrot application oxidase, phenylalanine ammonia lyase, and
of SLF enhanced activities of chitinase, B-1-3 lipoxygenase due to SLF application
flucanase, polyphenol oxidase and lipoxynase (Jayaraman et al., 2011). The commercial
which are factors regulating plant disease. extract from the brown seaweed Ascophyllum
Similar results were found in cucumber which nodosum was found to reduce fungal diseases
showed enhanced activities of various in cucumber (Jayaraman et al., 2011). Brown
defence-related enzymes including chitinase, algae have shown effectiveness in controlling
 Comparative studies on algal seaweed extracts JBiopest 7(2):167-176(2014)
171
JBiopest 5(1): 1-6 Fig 1.Spores of Fusarium oxysporum f.sp. udum

Table 2. Effects of seaweed extracts on inhibition over control of Fusarium oxysporum f. sp.
udum in vitro.

Inhibition over control (%)

Seaweed Hours
24 48 72 96 hr 108 Mean
Sargassum myricocystum (10%) 18.76 20.35 33.82 27.11 27.00 25.40
Sargassum myricocystum (15%) 21.24 26.76 39.68 32.51 31.56 30.35
Sargassum myricocystum (20%) 27.22 41.03 47.13 45.07 41.33 40.35
Sargassum myricocystum (25%) 37.94 44.39 53.24 50.23 49.11 46.98
Sargassum myricocystum 30%) 42.06 45.67 57.63 53.76 50.78 49.98
Gracilaria edulis (10%) 9.90 14.42 28.21 18.66 18.00 17.83
Gracilaria edulis (15%) 16.49 24.52 31.01 28.76 25.33 25.22
Gracilaria edulis (20%) 23.09 31.41 41.76 35.33 31.11 32.54
Gracilaria edulis (25%) 24.95 36.22 45.18 37.09 34.00 35.48
Gracilaria edulis (30%) 31.55 38.94 50.55 44.13 41.00 41.23
Caulerpa racemosa (10%) 11.34 6.89 23.44 14.79 12.00 13.69
Caulerpa racemosa (15%) 14.43 19.23 31.99 25.12 20.89 22.33
Caulerpa racemosa (20%) 17.11 28.37 37.36 31.10 24.11 27.61
Caulerpa racemosa (25%) 19.59 34.29 44.08 36.62 31.56 33.22
Caulerpa racemosa (30%) 22.27 36.38 45.42 40.49 35.22 35.95
Mean 22.53 29.92 40.70 34.72 31.53 31.88

C T CX T
SEd
0.313 0.181 0.701
CD (0.05)
0.618** 0.357** 1.383**

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 Ambika and Sujatha
plant diseases. The laminarin polysaccharide 172
isolated from Laminaria digitata is able to extracts, compared to water extracts (Hanaa et
elicit host defense responses in plants al., 2008). The water extract of Padina
(Klarzynski et al., 2000). The extract of the tetrastromatica and ethanolic extract of
seaweed Ascophyllum nodosum stimulates the Padina tetrastromatica and Sargassum
activity of peroxidases and phytoalexin tennerrimum showed activity against
synthesis in some plants of commercial value, Fusarium solani and F. oxysporum by well
increasing their resistance. The Ulva fasciata diffusion and disc diffusion method,
extract is able to effectively reduce the respectively (Asnad and Tanver, 2014).
number of colonies in powdery mildew. The Table 3. Effect of antagonists on the mycelial
brown seaweeds shows high antifungal growth of Fusarium oxysporum f.sp.udum in vitro.
activity as compared to red and green algae. (Dual culture technique)
The brown seaweeds contain high amount of Per cent
*Mycelial
Fungi reduction
flavanoid and phenolic compounds could be growth (mm)
over control
the reason for antifungal activity (Cowan et Pseudomonas 68.5 23.8
al., 1999). Using organic solvents which are fluorescens (29.20)
able to extract a large quantity of lipophilic Trichoderma 63.8
32.5
compounds (glycolipid, phenolic- viride (53.01)
Control 90.0 -
terpenoidsds, unsaturated-fatty acids and
SEd 1.41
hydroxylated unsaturated-fatty acids), the CD (0.05) 3.19**
higher antifungal activity found in ethanol

Fig 2.Efficacy of seaweed extracts against the mycelial growth of Fusarium oxysporum f. sp. udum in vitro
condition.

Control S. myricocystum (30%)

Fig 3.Effect of antagonists on the mycelial growth of Fusarium oxysporum f. sp. udum in
vitro (Dual culture technique) 3
Trichoderma viride Control
 Comparative studies on algal seaweed extracts JBiopest 7(2):167-176(2014)
173
Fig 1-6
JBiopest 5(1): 4. Compatibility between antagonistic fungi and seaweed extracts.
Pseudomonas fluorescens

S. myricocystum (30%)

Control
Control S. myricocystum (30%)

Fig 5. Compatibility between antagonistic bacteria and seaweed extracts.

S. myricocystum 30%) Control

S. myricocystum 30%) Control
The effect of bacterial antagonists and fungal results revealed that the growth of T. viride
antagonist were tested against the growth of was found to be not sensitive to the extracts of
Fusarium oxysporum f. sp.udum by following seaweeds S. myricocystum,Caulerpa racemosa
dual culture technique in vitro. Among these and Gracilaria edulis. Among the
antagonists the fungal antagonist viz., concentrations 30% in all species recorded
Trichoderma viride was found to be most highest growth compared to other
effective by recording 63.8 per cent reduction concentrations. Mycelial growth of T. viride is
over control. Pseudomonas fluorescens 87, 82 and 80 mm for Sargassum
recorded the mycelial growth reduction of myricocystum (30%), Caulerpa racemosa
23.8 per cent over control (Table 3, Fig. 2). (30%) and Gracilaria edulis (30%),
The result was supported by Naik et al. (2010) respectively (Table 4). Rajendrandran and
in Fusarium oxysporum f. sp. vanillae. Ranganathan, (1996) reported that T. viride, T.
The effect of different concentrations of harzianum, T. hamatum, T. koningii and T.
seaweed extracts was tested for their pseudokoningii were antagonistic to Fusarium
compatibility with fungal antagonist of oxysporum f. sp. cepae which causes basal rot
T.viride and bacterial antagonist of P. in onion. Trichoderma harzianum was
fluorescens under in vitro condition. The

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 Ambika and Sujatha
174
Table 4. Compatibility between antagonistic fungi Trichoderma viride and antagonistic
bacteria Pseudomonas fluorescens with seaweed extracts
Trichoderma viride Pseudomonas fluorescens
Mycelial growth (mm) Compatibility level
+++
Sargassum myricocystum (10%) 66.2
+++
Sargassum myricocystum (15%) 72.4
Sargassum myricocystum (20%) +++
76.7
Sargassum myricocystum (25%) 82.4 +++
Sargassum myricocystum (30%) 86.9 +++
Gracilaria edulis (10%) 65.0 +++
Gracilaria edulis (15%) 69.6 +++
Gracilaria edulis (20%) 75.4 +++
Gracilaria edulis (25%) 77.9 +++
Gracilaria edulis (30%) 81.5 +++
Caulerpa racemosa (10%) 61.3 ++
Caulerpa racemosa (15%) 65.7 ++
Caulerpa racemosa (20%) 71.8 ++
Caulerpa racemosa (25%) 76.3 ++
Caulerpa racemosa (30%) 80.2 ++
Control 90.0 ++
Mean 74.9
SEd 1.56**
CD (0.05) 3.15 **
+++: Highly compatible, ++: moderately compatible, +: slightly compatible -: No compatible

antagonistic to F. oxysporum in plates and at 30% concentration effectively controlled

pots by reducing the population of F. and inhibited the mycelial growth of F.
oxysporum (Goodwin-Egein and Arinzae, oxysporum f.sp. udum in red gram under in
2001). Chandel (2011) reported that T. vitro studies.
harzianum exhibited 68 per cent reduction in
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Rathore, S.S., Chaudhary, D.R., Boricha, ___________________________________
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Reddy, M.V., Nene, Y.L., Kannaiyan, J., *Communication author
Raju, T.N. Saka, V.N., Davor, A.T., Phone no: 08489174468
Email: [email protected]