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Journal of Applied Sciences and Environmental Management
World Bank assisted National Agricultural Research Project (NARP) - University of Port Harcourt
ISSN: 1119-8362
Vol. 5, Num. 1, 2001, pp. 29-32
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Journal of Applied Sciences & Environmental Management, Vol. 5, No.
1, June, 2001, pp. 29-32
Antifungal Activities of Some Leaf Extracts on Seed-borne
Fungi of African Yam Bean Seeds, Seed Germination and Seedling Emergence
*Nwachukwu, E. O ; Umechuruba,C.I.
Department of Plant Science and Biotechnology, Faculty of Science, University
of Port Harcourt, Port Harcourt. Nigeria.
*Corresponding author
Code Number: ja01005
ABSTRACT
The efficacy of leaf extracts of basil (Ocimum basilicum), bitter
leaf (Vernonia amygdalina), lemon grass (Cymbopogen citratus),
neem (Azadirachta indica) and paw-paw (Carica papaya) on major
seed-borne fungi: Aspergillus niger, Aspergillus flavus, Botryodiplodia
theobromae and Fusarium moniliforme of African yam bean ( Sphenostylis
stenocarpa ) seeds, and on seed germination and seedling emergence were
studied in vitro and in vivo. All the plants leaf extracts (crude
and aqueous) significantly (P £ 0.05) reduced the
incidence of seed-borne fungi tested and increased seed germination and seedling
emergence except lemon grass leaf extract when compared with the untreated
control. Neem extract was the most effective while lemon grass extract was
the least. Crude extracts from all the plant leaves tested increased seed germination
and seedling emergence of African yam bean seeds and gave significant (P£ 0.05)
reduction of mycelial growth of all the fungi tested when compared with their
aqueous extracts. Leaf extracts of neem, basil, bitter leaf and paw-paw, which
are cheap and environmentally safe, are promising for protecting African yam
bean seeds against major seed-borne fungi and in the improvement of the crop. @
JASEM
African yam bean, Sphenostylis
stenocarpa (Hochst ex. Rich) Harms is an important grain legume in most
tropical African countries such as Nigeria, Ivory Coast, Ghana, Togo, Gabon,
Congo, Ethiopia and some parts of East Africa where it is used as food. The
crop also helps agriculturally to enrich the soil by its ability to fix nitrogen
from the atmosphere.
A survey of the seed-borne fungi associated with this crop grown in Eastern
Nigeria revealed high percentage incidence of Aspergillus niger, Aspergillus
flavus, Fusarium moniliforme and Botryodiplodia theobromae
(Nwachukwu and Umechuruba, 1991). Umechuruba and Nwachukwu (1994) also
reported significant reduction in seed germination, seedling emergence and
low seed and tuber yield of African yam bean caused by these important seed-borne
fungi, as well as reduction in nutritional qualities of the seeds (Umechuruba
and Nwachukwu 1997).
It is therefore necessary to search for control measures that are cheap,
ecologically sound and environmentally safe to eliminate or reduce the incidence
of these economic important pathogens, so as to increase seed germination
and yield of African yam bean.
In recent years much attention
has been given to non- chemical systems for seed treatment to protect them
against seed-borne pathogens. Plant extracts have played significant role
in the inhibition of seed-borne pathogens and in the improvement of seed
quality and field emergence of plant seeds. Shah et al, (1992), reported
that Argemone mexicana seed extract was effective in eliminating most
of the seed-borne fungi of cowpea but not against Alternaria alternata,
Curvularia lunata, Mucor sp. and Macrophomina phaseolina. Leaf
extracts of Delonix regia, Pongamia glabra and Acacia
nilotica significantly inhibited spore germination, mycelial growth and
spore production of A.. helianthi, M. phaseolina and F.
solani from sunflower seeds (Thiribhuvanamala and Narasimhan, 1998).
Parimelazhagan and Francis, (1999), in their own studies, reported reduction
in the radial growth of C. lunata associated with rice seeds when
treated with leaf extracts of Clerodendrum viscosum which also increased
seed germination and root and shoot lengths of rice.
In this study, the effects of leaf extracts of neem (Azadirachta indica),
basil (Ocimum basilicum), bitter leaf (Vernonia amygdalina),
paw-paw (Carica papaya) and lemon grass (Cymbopogen citratus)
on major seed borne fungi of African yam bean seeds; and on seed germination
and seedling emergence were investigated.
MATERIALS AND METHODS
Extraction of leaf extracts:
The fresh leaves of neem, basil, bitter leaf, paw- paw and lemon grass were
carefully washed each alone under tap water and cut into tiny pieces. Aqueous
extract was prepared as follows: 1kg whole leaves of each plant was blended
separately in one litre of distilled water in a waring blender and sieved
through a Buchner funnel to remove debris. Similarly the crude extract was
prepared by blending 1kg of each of the plant leaves without water. The extract
was sieved through a Buchner funnel also to remove debris.
In vitro tests:
The seed-borne fungi (Aspergillus niger,
A. flavus, F. moniliforme and B. theobromae) used for this
investigation were isolated from naturally infected African yam bean seeds.
The in vitro tests were carried out to measure the effects of the
leaf extracts on radial growth of the seed-borne fungi. Potato dextrose
agar (PDA ) medium was used in the study. To every 15ml of sterile potato
dextrose agar medium in Petri dishes, 5ml of either crude or aqueous extract
of each plant were added. The solution in each Petri dish was gently swirled
and allowed to solidify. The extract-amended medium in the Petri dishes
were inoculated each alone at the centre with 5mm inoculum-disc of each
test fungus and incubated at 25 + 2°C for 14 days. The medium with
inoculum disc but without any extract served as control. Percentage inhibition
of mycelial growth by the leaf extracts was calculated using the formula:
Where: %MGI = %
Inhibition of mycelial growth
DC = diameter of control
DT = diameter of test
In vivo tests: African
yam bean seeds naturally infected with A. niger, A. flavus,
F. moniliforme and B. theobromae were used for the in vivo tests.
The efficacy of the leaf extracts was tested using the blotter method and
soil mix in buckets. Seeds were treated with the same crude and aqueous
extracts of the plant leaves as previously described. The seeds were soaked
in the crude and aqueous leaf extracts separately for one hour and then plated
on moist blotters as well as in sterilised soil mix in buckets. The untreated
seeds were soaked in distilled water for one hour and plated on moist blotters
and sterilised soil mix as control. A total of 400 seeds were soaked per
extract (crude or aqueous) and 10 seeds plated on blotter per Petri dish.
The extract- treated and untreated seeds were incubated in an incubator at
20 + 2°C for seven days. Seeds plated in blotter were examined for
fungal growth and percentage seed germination after 7 days of incubation
while percentage seedling emergence was recorded in seeds sown in sterilized
soil mix after 14 days. For soil method, 20 seeds were planted per bucket
equidistantly at the depth of 2.0cm and 5 buckets were used per extract treatments.
RESULTS
In vitro tests: The effects
of the various plant leaf extracts on mycelial growth of major seed-borne
fungi of African yam bean seeds are presented in Table 1. All the leaf extracts
significantly (P £ 0.05) inhibited the radial
mycelial growth of the fungi (Table 1). The percentage inhibition of mycelial
growth of the fungi varied with the type of leaf extracts, extract concentration,
as well as the type of fungus. Neem leaf extract gave the highest inhibition
while the lemon grass extract gave the lowest inhibition. The crude leaf
extracts of all the plants significantly (P £ 0.05)
inhibited growth of all the fungi more than their aqueous extracts (Table1).
All the leaf extracts (crude and aqueous) gave highest percentage inhibition
of A. flavus when compared with the results of other fungi. The control
did not inhibit mycelial growth of all the fungi (Table 1).
Table 1. Mean percentage inhibition
of mycelial growth of seed- borne fungi by various leaf extracts.
Name of plant
|
Extract
|
Seed borne fungi
|
|
Conc.
|
*Fm
|
Bt
|
An
|
Af
|
Azadirachta indica
(Neem)
|
Crude
Aqueous
|
68.0g
56.0f
|
78.5h
53.3f
|
88.3h
70.1f
|
91.5h
80.5f
|
Vernonia amygdalina
(Bitter leaf )
|
Crude
Aqueous
|
55.3F
30.5d
|
68.29
49.0e
|
76.1g
61.5e
|
88.3g
70.0e
|
Ocimum basilicum
( Basil )
|
Crude
Aqueous
|
44.0e
28.3d
|
50.5e
37.6d
|
62.0e
50.3d
|
71.0e
61.8d
|
Carica papaya
( Paw-paw )
|
Crude
Aqueous
|
48.7e
32.1d
|
55.3f
39.8g
|
70.0f
60.3e
|
78.0f
60.3d
|
Cymbopogen citratus
( Lemon grass )
|
Crude
Aqueous
|
14.5b
10.3b
|
24.5c
15.0b
|
30.5c
19.7b
|
36.7c
21.8b
|
Control
|
Distilled water
|
0.0a
|
0.0a
|
0.0a
|
0.0a
|
|
|
|
|
|
|
|
Mean of 6 replicates. Means followed by the
same letter(s) do not differ significantly at 5% (DMRT)
*Fm = Fusarium moniliforme, Bt = Botryodiplodia
theobromae, An = Aspergillus niger, Af = Aspergillus flavus
In vivo test:
The effects of the plants leaves
extracts on the incidence of seed-borne fungi, seed germination and seedling
emergence are presented in Table 2. All the leaf extracts (crude and aqueous)
significantly (P £ 0.05) reduced the incidence
of all the seed-borne fungi, increased seed germination and seedling emergence
except leaf extract from lemon grass which was ineffective when compared
with the untreated control seeds (Table 2).
The crude leaf extracts of all
the plants were more effective than the aqueous leaf extracts in reducing
seed-borne fungi and in increasing seed germination and seedling emergence.
The extract from neem leaf was the most effective in the reduction of seed-borne
fungi and increase in seed germination and seedling emergence(Table 2).The
crude leaf extract of this plant also gave complete control of A. niger and A.
flavus (Table 2). Seed germination and seedling emergence were
higher in seeds treated with leaf extracts of neem (89.0% and 81.3%) respectively
followed by leaf extracts of bitterleaf (82.5% and 75.0%), paw-paw (80.1%
and 68.8%) and basil (66.0% and 56.8% ) respectively. The extracts from lemon
grass did not reduce the incidence of seed- borne fungi. Also seed germination
was not enhanced by this extract.
Table 2. Effect of different
leaf extracts on the incidence of seed- borne fungi, seed germination and
seedling emergence of African yam bean seeds.
Name of plant
|
Extract
Conc.
|
Mean % seed- borne fungi
*Fm Bt An Af
|
Mean % seed germination
|
Mean % seedling emergence
|
A.. indica
(Neem)
|
Crude
Aqueous
|
5.3a
8.8b
|
4.0a
0.0a
|
0.0a
5.3b
|
0.0a
4.0b
|
|
89.0g
75.0e
|
81.3g
69.5de
|
V. amygdalina
|
Crude
Aqueous
|
2o.5a
3o.1c
|
18.4a
24.8b
|
16.5d
25.0b
|
15.3a
20.5b
|
|
82.5e
61.3c
|
75.0f
60.0c
|
O. bacilicum
(Basil)
|
Crude
Aqueous
|
28.0b
34.3cd
|
22.0a
26.8b
|
24.2b
28.3c
|
22.0b
28.3c
|
|
66.0d
56.8b
|
60.1c
53.5b
|
C. papaya
( Paw- paw )
|
Crude
Aqueous
|
29.3b
32.5c
|
20.3a
27.0bc
|
28.0c
35.0d
|
21.5b
30.1d
|
|
80.1e
68.8d
|
70.5e
67.0d
|
C. citratus
( Lemon grass )
|
Crude
Aqueous
|
39.5d
41.3e
|
28.2c
30.3d
|
55.5e
60.0f
|
60.0e
66.8f
|
|
50.0a
53.3a
|
46.3a
48.5a
|
Untreated control
|
Distilled water
|
42.5e
|
31.0d
|
62.8f
|
68.5f
|
|
55.0a
|
48.0a
|
*Fm= Fusarium moniliforme, Bt= Botryodiplodia
theobromae An = Aspergillus niger and Af = Aspergillus
flavus
Figures followed by different letters in columns differ significantly
when subjected to DMRT (P £ 0.05).
DISCUSSIONS
The efficacy of different plant
leaf extracts (neem, basil, pawpaw, bitter leaf and lemon grass) against
major seed borne fungi of African yam bean seeds was tested in vitro and in
vivo. The results showed that the five plant leaf extracts significantly
(P £ 0.05) inhibited the radial growth of all
the test fungi with inhibition varying from one extract to another (Table
1). Percentage inhibition of radial growth of all the fungi was highest in
neem extract and lowest in extract from lemon grass (Table 1). Parimelazhagan
and Francis (1999), reported a significant reduction of radial growth and
mycelial dry weight of C. lunata in extract of Clerodendrum viscosum.
Nidiry (1999), also obtained a similar result with tomato seed extract, which
reduced the mycelial growth of Colletotrichum gloeosporioides and
spore germination of Cladosporum cucumerinum.
All the leaf extracts were
effective in the reduction of the incidence of all the seed borne fungi tested
except extracts from lemon grass that did not give significant(P £ 0.05)
reduction of the fungi when compared with untreated control seeds (Table
2). This result indicates that the leaf extracts of neem, paw paw, basil
and bitter leaf probably have some fungicidal properties that inhibit the
growth of the seed borne fungi. The crude extracts were more effective in
reducing the incidence of fungi than the aqueous extracts. This is an indication
that dilution of the extracts reduced toxic effects of the leaf extracts
on the seed-borne fungi. This result agrees with the findings of Zaman et
al (1997), that the efficacy of garlic, neem, ginger and onion extracts
on seed borne fungi of mustard declined with increase dilution.
The extracts were not phytotoxic
to African yam bean seeds rather they improved seed germination and seedling
emergence significantly (P£ 0.05) more than the
untreated control seeds (Table 2). The extracts from lemon grass were ineffective
in improving seed germination and seedling emergence (Table 2).
The ability of the extracts to
increase seed germination and seedling emergence could be attributed to the
suppression of the incidence of the seed borne fungi that could have killed
the embryo of the seeds. This result is consistent with that of Parimelazhagan
and Francis, (1999) who established that leaf extracts of C. viscosum increased
seed germination and improved seedling development of rice seeds.
This present study has therefore
shown that leaf extracts of neem, paw-paw, basil and bitter leaf can be used
as fungicidal seed treatments for the control of seed-borne fungi of African
yam bean seeds and for increasing seed germination and seedling emergence.
REFERENCES
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Nidiry, E. S. J. (1999).
Antifungal activity of tomato seed extracts.Fitoterapia, 70 (2):181-183.
-
Nwachukwu, E.O. and Umechuruba,
C.I. (1991). Seed-borne fungi associated with African yam bean seeds
in four Eastern States of Nigeria. Seed Research, 19 (1):
13-20.
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Nwachukwu, E.O. and Umechuruba, C. I. (1997). Changes
in nutritional values of African yam beanseeds due to seed- borne fungi. Global
Journal of Pure and Applied Sciences, 3 (2):141-147.
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Parimelazhagan, T. and Francis, K. (1999). Antifungal
activity of Clerodendrum viscosum against Curvularia lunata in
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Shah, N.H., Khan, M.I.and Azam, M.F. (1992). Seed microflora
of cowpea and its control with extract of Argemone mexicana.Bioved 3 (2):
176-168.
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Thiribhuvanamala, G. and Narasimhan, V. (1998). Efficacy
of plant extracts on seed- borne pathogens of sunflower . Madras Agricultural
Journal, 85 (5/6): 227-230.
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Umechuruba, C.I. and Nwachukwu, E.O. (1994). Efficacy
of certain fungicides against seed-borne fungi of African yam bean seeds. International
Journal of Pest Management, 4 (2): 126-131.
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Zaman,M.A, Saleh, A.K.M., Rahman, G.M.M, and Islam, M.T.
(1997). Seed-borne fungi of mustard and their control with indigenous plant
extracts. Bangladesh Journal Plant Pathology, 13 (1/2): 25-28.
Copyright 2001 - Journal of Applied Sciences & Environmental Management
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