Preservation of seeds against fungi using wood-ash of some tropical forest trees in Nigeria

Abstract

Fresh visually healthy seeds of Zea mays (maize), Cucumeropsis mannii (melon) and Phaseolus vulgaris (bean) were stored under three conditions, wood ash of some tropical forest trees in Nigeria namely; Khaya grandifoliola, Nauclea diderrichii, Piptadeniastrum africanum, Mangifera indica, Mansonia altissima, Triplochiton scleroxylon, Ceiba pentandra, Terminalias superba, Terminalia ivorensis). Seeds treated with benlate, an orthodox fungicide and seeds without any treatment to serve as the control of the experiment. These were set-up at two different locations on the campus (the laboratory and the botanic garden) for six months. The seeds stored with ashes of Nauclea diderrichii and Piptadeniastrum africanum were the most effective, stopping fungal growth and eliminating weevils compared to those seeds stored with benlate which is only effective against fungal growth. P. vulgaris (bean) seeds are the best stored of the three seeds probably due to the low moisture content of the seed. Four pathogenic fungi were isolated from the seeds (maize, melon and bean) at both locations and these include; Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus and Rhizopus racemosa. Preliminary phytochemical screening revealed that some of these ashes contained a number of active compounds which enable them (ashes extracts) to inhibit the growth of the pathogenic fungi.

Full text

African Journal of Microbiology Research Vol. 4 (4), pp. 279-288, 18 February, 2010
Available online http://www.academicjournals.org/ajmr
ISSN 1996-0808 ©2010 Academic Journals
Full Length Research Paper
Preservation of seeds against fungi using wood-ash of
some tropical forest trees in Nigeria
Temitope O. Oguntade* and Adedotun A. Adekunle
Department of Botany and Microbiology, Faculty of Science, University of Lagos, Akoka, Lagos, Nigeria.
Accepted 22 December, 2009
Fresh visually healthy seeds of Zea mays (maize), Cucumeropsis mannii (melon) and Phaseolus
vulgaris (bean) were stored under three conditions, wood ash of some tropical forest trees in Nigeria
namely; Khaya grandifoliola, Nauclea diderrichii, Piptadeniastrum africanum, Mangifera indica,
Mansonia altissima, Triplochiton scleroxylon, Ceiba pentandra, Terminalias superba, Terminalia
ivorensis). Seeds treated with benlate, an orthodox fungicide and seeds without any treatment to serve
as the control of the experiment. These were set-up at two different locations on the campus (the
laboratory and the botanic garden) for six months. The seeds stored with ashes of Nauclea diderrichii
and Piptadeniastrum africanum were the most effective, stopping fungal growth and eliminating weevils
compared to those seeds stored with benlate which is only effective against fungal growth. P. vulgaris
(bean) seeds are the best stored of the three seeds probably due to the low moisture content of the
seed. Four pathogenic fungi were isolated from the seeds (maize, melon and bean) at both locations and
these include; Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus and Rhizopus racemosa.
Preliminary phytochemical screening revealed that some of these ashes contained a number of active
compounds which enable them (ashes extracts) to inhibit the growth of the pathogenic fungi.
Key words: Seeds, storage fungi and wood-ash.
INTRODUCTION
Seeds like maize, beans and melons are of the world’s
staple food which provides energy and proteins for many
populations. Seeds are effectively preserved in order to
secure the availability of food all year round and to
ensure the supply of good seeds for planting programs
whenever needed (Oyeniran, 1980).
Seeds when improperly stored deteriorate and spoil
during preservation for two reasons, either as a result of
insufficient drying of the seeds before storage or as a
result of the presence of small quantities of spores of
storage fungi may be present on seeds going into storage
(Van Egmond et al., 2007).
The term “storage fungi” describe a group of fungi
adapted to growth in an environment of relatively low
moisture content and high osmotic pressure that are
involved in and responsible for deterioration of stored
seeds (Alexopoulos et al., 2007). Storage fungi include a
*Correspondence author. E-mail: temmitade@yahoo.com. Tel:
+2348023449102.
few species of Aspergillus, Penicillium and a few still
unidentified species of fungi imperfection. Some storage
fungi produce toxic compounds called mycotoxins which
are harmful to human and livestock (Agrios, 1997). Some
storage fungi invade seeds to such an extent as to render
the seeds unfit for human and animal consumption
(Dowd, 1994). The use of chemicals on seed in store is
an effective storage method is however complicated by
the fact that some of these chemicals have been found to
have serious limitation of one sort or another, such as
toxicity to animals, excessive cost, difficulty of appli-
cation, undesirable on the processing quality of seeds
and the odor and flavor of some chemical compounds on
the storage seeds. This probably makes foods processed
from it unacceptable to most people (Level, 1990). Wood-
ash is composed of the organic and inorganic residue
remaining after the combustion of wood, this is used as a
means of biological control which is generally favored as
a method of storing seeds because it does not have any
of those disadvantages of chemicals and tend to be more
durable in its effect (Adekunle and Uma, 2005).
This work investigated the efficacy and justifies

280 Afr. J. Microbiol. Res.
scientifically the use of wood-ash use in seed storage.
MATERIALS AND METHODS
Sample collection
Fresh visually healthy seeds of Zea mays (maize), Phaseolus
vulgaris (Bean) and Cucumeropsis mannii (Melon) were collected
from the local markets in Bariga Lagos. Diseased seeds were
separated from the visually health ones. Seventy-two pieces of
plastic bowls were purchased from same market for the storage.
The woods (Khaya grandifoliola, Nauclea diderrichii,
Piptadeniastrum africanum, Mangifera Indica, Mansonia altissima,
Triplochiton scleroxylon, Ceiba pentandra, Terminalia superba and
Terminalia ivorensis) were brought from a wood merchant at
Okobaba, Ebute-meta, Lagos.
Wood-ash preparation
Two hundred grammes of each of wood was burnt into ash, the
quantity of ash produced varies, but was approximately five
hundred grams each. The cool dried ashes were parked into sterile
nylon bags with label containing the information on each ash
respectively.
Calculation of the seeds moisture content
The moisture content of the seeds was determined at the beginning
and the end of the storage period. Few seeds were in clean glass
Petri-dishes and weighed, the weight served as the initial weight
(g). The seeds, in glass Petri-dishes were put in an oven at 60oC for
48 h to allow the moisture to escape. The seeds were re-weighed to
deduce the final weight (Agrawal, 1980).
% moisture content = Initial weight – Final weight x 100
Initial weight
Seed preservation
Two storage locations on campus; The Botany laboratory and the
glass house in Botanical garden were used. At each storage
location, there were thirty-three disinfected plastic bowls which were
divided into three groups (eleven bowls for each type of seed).
Each bowl contained 1000 healthy seeds weighing about 200 g.
The seeds were mixed with the wood-ashes (in ratio 1:2), benlate
and one bowl of each type of seed serves as the control that is
without any treatment. The mixture of seeds and wood-ashes were
then stored in bowls with tight lids with labels containing information
like the storage date, seed name, preservative agent and the
location of storage. During storage period, seeds were collected
randomly from each storage bowl from both locations of storage
monthly. Also physical changes in each bowl were noted. The
collected seeds were then used in determining the fungi occurrence
in each storage bowl.
*Potato Dextrose Agar (PDA) was used in this work as the
culturing medium.
Isolation and identification of fungi
Preliminary survey of storage fungi invading seeds was carried out
in three different markets (Bariga, Oyingbo and Yaba) in Lagos
state for three consecutive months in order to get the frequency of
these fungi. Four of each diseased seeds collected from market
survey were placed on plates of PDA after been surface sterilized
with 40% of sodium hypochlorite. All the plates were incubated at
26oC for 3 to 5 days. Developing colonies of fungi were sub-
cultured to obtain pure culture. The fungi cultures were examined
both macroscopically and microscopically for colony, mycelia and
spore characteristics. The characteristics were compared with those
in a standard mycology text (Vashishta and Sinha, 2005). The fungi
were kept on PDA slants in the refrigerator prior use.
Extraction from the wood-ashes
Fifty grammes of each of the wood-ashes were soaked in 150 ml of
sterile distilled water and 70% ethanol for 24 h; the solutions were
then filtered into beaker with the aid of a Whatman No. 1 filter
paper. The extracts were concentrated by evaporating it in rotator
evaporator at a moderate high speed. The extracts were stored in
the refrigerator until use.
Control preparation
Three types of control were employed, that is, Griseofulvin, Benlate
and water. 125 mg of griseofulvin was dissolved in 10 ml of sterile
distilled water in a beaker; 5 g of benlate was dissolved in 10 ml of
sterile distilled water in another beaker and 10 ml of sterile distilled
water in a separate beaker.
Antifungal activity test
Four disc of sterilized perforated Whatman filter paper disc were
soaked in each extracts and the prepared controls for six hours.
The fungi obtained from the monthly market survey and those from
the monthly sampling from the storage seeds at both locations were
sub-cultured to get the “working cultures” unto which sterile distilled
water was added to produce the microbial suspension. Two drops
of each of the fungus suspension was spread on PDA plates and
four perforated disc soaked in the extracts and the controls were
carefully dropped on these PDA plates with the aid of sterilized
forceps.
The PDA plates were labeled and incubated at 29oC.
Measurement and recording of zone of inhibition were taken at
every 24 h for 5 days (Parker, 1979).
Phytochemical analysis
Phytochemical analysis studies were carried on each of the wood-
ash using the method described by Harborne (1998). Both alcohol
and water extracts of the nine wood-ashes were tested for the
presence of alkaloids, tannins, saponins, flavonoids, phlobatanins,
cardiac-glycosides and anthraquinone.
RESULTS
At the end of the first month of storage, no change
occurred. In the second, some changes were noted,
examples of such changes include weevils
(Callosobruchus maculatus) infestation and microbial
growth. And at the end of the storage period, more
changes had occurred (Tables 1 and 2).
The diversity of fungi isolated from the stored seeds

Oguntade and Adekunle 281
Table 1. Physical observation and occurrence of fungi on the three seed stored with various ashes, benlate, and the control at two different locations of storage (garden).
Fungi species isolated from the seed
Physical observation Aspergillus flavus
Aspergillus niger Aspergillus
fumigatus
Rhizopus
Seed type Storage means
1st
Month
2nd
Month
3rd
Month
4th
Month 1st 2nd 3rd 4th
1st 2nd 3rd 4th 1st 2nd 3rd 4th 1st 2nd 3rd 4th
Khaya grandifoliola Fewer
weevils
Few
weevils Many
weevils /
color
changed
Many
weevils /
color
changed
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
Nauclea diderrichii No
weevil No
weevil Fewer
weevils Fewer
weevils - - - - - - - - - - - - - - - +
Piptadeniastrum africanum No
weevil No
weevil Fewer
weevils Fewer
weevils - - - - - - - - - - - - - - - -
Mangifera Indica No
weevil Fewer
weevils Fewer
weevils Fewer
weevils - - + + - - - - - - - - - - + +
Mansonia altissima Fewer
weevils Few
weevils Many
weevils Many
weevils - - + + - + - - - - - - - - + +
Triplochiton scleroxylon Fewer
weevils Few
weevils Many
weevils Many
weevils - - + + - - - - - - - - - - + +
Ceiba pentandra Fewer
weevils Few
weevils Many
weevils Many
weevils - - + + - - - - - - - - - - +
Terminalia superba Fewer
weevils Few
weevils Many
weevils /
color
changed
Many
weevils /
color
changed
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
Terminalia ivorensis Fewer
weevils Few
weevils Many
weevils /
color
changed
Many
weevils /
color
changed
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
Benlate Fewer
weevils Few
weevils Many
weevils Many
weevils - - - - - - - - - - - - - - - -
Maize
Control Few
weevils Many
weevils Much
weevils Much
weevils - - + + - + - - - - + + - - + +
Khaya grandifoliola No
weevil No
weevil No
weevil No
weevil - - - - - - - - - - - - - - - - Beans
Nauclea diderrichii No
weevil No
weevil No
weevil No
weevil - - - - - - - - - - - - - - - -

282 Afr. J. Microbiol. Res.
Table 1. Contd.
Piptadeniastrum africanum No
weevil No weevil No weevil No weevil - - - - -
- - - - - - - - - - -
Mangifera Indica No
weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Mansonia altissima No
weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Triplochiton scleroxylon No
weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Ceiba pentandra No
weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Terminalia superba No
weevil No weevil No weevil No weevil - - + + - - - - - - - - - - - -
Terminalia ivorensis No
weevil No weevil No weevil No weevil - - + + - - - - - - - - - - - -
Benlate No
weevil Few weevils Few weevils Few weevils - - - - - - - - - - - - - - - -
Control few
weevils Few weevils Many weevils Many weevils - + + + - - + + - - - - - + - -
Khaya grandifoliola No
weevil No weevil No weevil No weevil - - - - - + + + - - - - - - - -
Nauclea diderrichii No
weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Piptadeniastrum africanum No
weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Mangifera Indica No
weevil No weevil No weevil No weevil - + + + - - - - - - - - - - - -
Mansonia altissima No
weevil No weevil No weevil No weevil - + - + - - + + - - + + - - - -
Melon
Triplochiton scleroxylon No
weevil No weevil No weevil No weevil - - + + - + - - - - + + - - - -
varied each month with Aspergillus niger as the
most frequent and Aspergillus flavus as the least
(Figure 1).
The wood-ashes of Nauclea diderrichii and
Piptadeniastrum africanum proved to be effective
in preserving the seeds, eliminating the fungi and
preventing the manifestation of weevils (C.
maculatus). Benlate, an orthodox fungicide proved
to be effective against the fungi attack but does
not prevent the weevils (C. maculatus) invasion.
Monthly market survey carried out at the three
different markets in Lagos for three consecutive
months showed five pathogenic fungi to be
involved with seed storage. These are
Macrophomina species, Sclerotium rolfsii,
Aspergillus niger, Aspergillus fumigatus, and
Rhizopus racemosa. Rhizopus racemosa was the
most frequent; S. rolfsii was the least frequent

Oguntade and Adekunle 283
Table 2. Physical observation and occurrence of fungi on the three seed stored with various ashes, benlate and the control at two different location od storage (laboratory).
Physical observation Fungi species isolated from the seed
Aspergillus flavus Aspergillus
niger Aspergillus fumigatus Rhizopus
Seed
type Storage means 1st
Month 2nd
Month 3rd
Month
4th
Month 1st 2nd 3rd 4th 1st 2nd 3rd 4th 1st 2nd 3rd 4th 1st
2nd 3rd 4th
Khaya grandifoliola
Fewer
weevils
Few
weevils
Many
weevils
Many
weevils /
color
changed
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
Nauclea diderrichii No
weevil No
weevil Fewer
weevils Fewer
weevils - - - - - - - - - - - - - - - +
Piptadeniastrum africanum No
weevil No
weevil Fewer
weevils Fewer
weevils - - - - - - - - - - - - - - - -
Mangifera Indica No
weevil Fewer
weevils Fewer
weevils Fewer
weevils - - + + - - - - - - - - - - + +
Mansonia altissima Fewer
weevils Few
weevils Many
weevils Many
weevils - - + + - + - - - - - - - - + +
Triplochiton scleroxylon Fewer
weevils Few
weevils Many
weevils Many
weevils - - + + - - - - - - - - - - + +
Ceiba pentandra Fewer
weevils Few
weevils Many
weevils Many
weevils - - + + - - - - - - - - - - + +
Terminalia superba
Fewer
weevils
Few
weevils
Many
weevils
Many
weevils /
color
changed
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
Terminalia ivorensis
Fewer
weevils
Few
weevils
Many
weevils
Many
weevils /
color
changed
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+
Benlate Fewer
weevils Few
weevils Many
weevils Many
weevils - - - - - - - - - - - - - - - -
Maize
Control Few
weevils Many
weevils Much
weevils Much
weevils - - + + - + - - - - + + - - + +

284 Afr. J. Microbiol. Res.
Table 2. Contd.
Khaya grandifoliola No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - -
Nauclea diderrichii No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Piptadeniastrum africanum No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Mangifera Indica No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Mansonia altissima No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Triplochiton scleroxylon No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Ceiba pentandra No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Terminalia superba No weevil No weevil No weevil No weevil - - + + - - - - - - - - - - - -
Terminalia ivorensis No weevil No weevil No weevil No weevil - - + + - - - - - - - - - - - -
Benlate No weevil Few weevils Few weevils Few weevils - - - - - - - - - - - - - - - -
Beans
'
Control No weevil Few weevils Many weevils Many weevils - + + + - - + + - - - - - + - -
Khaya grandifoliola No weevil No weevil No weevil No weevil - - - - - + + + - - - - - - - -
Nauclea diderrichii No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Piptadeniastrum africanum No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Mangifera Indica No weevil No weevil No weevil No weevil - + + + - - - - - - - - - - - -
Mansonia altissima No weevil No weevil No weevil No weevil - + - + - - + + - - + + - - - -
Triplochiton scleroxylon No weevil No weevil No weevil No weevil - - + + - + - - - - + + - - - -
Ceiba pentandra No weevil No weevil No weevil No weevil - - + + - - - - - - - - - - - -
Terminalia superba No weevil No weevil No weevil No weevil - - + + - - + + - - - - - - - -
Terminalia ivorensis No weevil No weevil No weevil No weevil - + + + - + + + - - - - - - - -
Benlate No weevil No weevil No weevil No weevil - - - - - - - - - - - - - - - -
Melon
Control No weevil No weevil No weevil No weevil - + + + - + + + - + + + - - - -

Oguntade and Adekunle 285
0
5
10
15
20
25
30
35
40
45
50
1
Fungi
Frequency (%)
Aspergillus flavus
Aspergilus niger
Aspergillus fu
igatus Rhizopus
Figure 1. Frequency of pathogenic fungi over six months period of seed preservation.
0
5
10
15
20
25
30
35
40
45
1
Fungi
Frequency (%)
Macrophomina
sp
ecies
Sclerotium rofsii
Aspergillus niger
Aspergillus fumigatus
Rhizopus
Figure 2. Frequency of pathogenic fungi obtained from monthly markets survey for three consecutive months.
(Figure 2).
There was reduction in the moisture contents of the
three seeds stored with wood-ashes at both locations at
the end of the storage period except for the control
samples which tend to increased a little.
Activity test using Benlate extract had the highest zone
of inhibition against all the isolated pathogenic fungi. Both
ethanol and water extracts of N. diderrichii and

286 Afr. J. Microbiol. Res.
Table 3. Antifungal activity of the nine ashes.
Sample Zone of inhibition (Mean + S. E. (mm)) fungi
Ethanol Extract of:
Macrophomina
species
Sclerotium
rofsii
Aspergillus
niger
Aspergillus
fumigatus
Rhizopus
Khaya grandifoliola 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Nauclea diderrichii 14.75 ± 0.54 14.50 ± 0.25 14.00 ± 0.25 14.00 ± 0.25 12.00 ± 0.00
Piptadeniastrum
africanum
15.25 ± 0.22 12.00 ± 0.00 11.00 ± 0.00 13.00 ± 0.00 12.00 ± 0.00
Mangifera Indica 0.00 ± 0.00 13.50 ± 0.25 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Mansonia altissima 0.00 ± 0.00 0.00 ±0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Triplochiton scleroxylon 0.00 ±0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Ceiba pentandra 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Terminalia superba 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Terminalia ivorensis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Water Extract of:
Khaya grandifoliola 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Nauclea diderrichii 15.50 ± 0.25 13.50 ± 0.75 13.50 ± 0.75 14.00 ± 0.00 13.00 ± 0.00
Piptadeniastrum
africanum
15.00 ± 0.00 12.00 ± 0.00 11.50± 0.25 11.00 ± 0.00 10.00 ± 0.00
Mangifera Indica 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Mansonia altissima 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Triplochiton scleroxylon 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Ceiba pentandra 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Terminalia superba 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Terminalia ivorensis 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Control (Distilled water) 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00
Benlate 15.50 ± 0.25 16.5 ± 0.25 26.00 ± 0.00 21.00 ± 0.00 13.50 ± 0.25
Fulcin 0.00 ± 0.00 0.00 ± 0.00 12.75 ± 0.22 7.75 ± 0.22 09.00 ± 0.00
P. africanum also showed inhibition against all the
pathogenic fungi. Only ethanol extract of Mangifera indica
inhibited the growth of S. rolfsii. Fulcin an orthodox
animal fungicide showed inhibition against A. niger, A.
fumigatus and R. racemosa only. Other extracts showed
zero inhibition (Table 3).
Preliminary photochemical screening for active com-
pounds (alkaloids, flavonoids, tannins, cardiacglyco-
sides, saponins and phlobatanins) in the extracts of the
nine ashes showed that both water and ethanol extracts
of N. diderrichii contained flavonoids and tannins, how-
ever, flavonoids was found to be in higher concentration
in the ethanol extract (Table 4). No alkaloid, cardiacglyco-
sides, saponins or phlobatanins was found in both the
water and ethanol extracts of N. diderrichii. Water and
ethanol extracts of P. africanum showed the presence of
alkaloids, flavonoids and tannins with flavonoids
concentrated in its ethanol extract. Cardiacglycosides,
saponins and phlobatanins were absent in the extraction
of P. africanum. Both extracts water and ethanol of M.
indica were found to contain alkaloids and flavonoids,
with alkaloids concentrated in the ethanol extract of it.
Alkaloids were also present in both extracts (water and
ethanol) of Triplochiton scleroxylon. The extracts (water
and ethanol) of Terminalia superba and Terminalia
ivorensis showed the presence of tannins (Table 4).
DISCUSSION AND CONCLUSION
This work showed that the wood-ash of N. diderrichii and
P. africanum were capable of preventing a number of
pathogenic fungi due to the presence of some active
compounds in them. Six pathogenic fungi were isolated
from storage seeds over the period of six months, which
included S. rolfsii, A. niger, A. flavus, A. fumigatus,
Rhizopus and Macrophomina species The various
experiments conducted in this work showed that wood-
ash have fungicidal effects which probably explain why
the ashes of N. diderrichii and P. africanum were able to
prevent the invasion of pests and microbial growth
effectively. Nigerian natives use wood ash to preserve
seeds against pests and microbes. They claim that the
wood-ash was able to preserve the seeds by absorbing

Oguntade and Adekunle 287
Table 4. Result of phytochemical test for active compounds in the nine ashes.
Extracts Alkaloids Flavonoid Tannis Cardiac glycosides Saponins Phlobatanins
Khaya grandifoliola - - - - - -
(Ethanol extract)
Khaya grandifoliola - - - - - -
(Water extract)
Nauclea diderrichii - ++ + - - -
(Ethanol extract)
Nauclea diderrichii - + + - - -
(Water extract)
Piptadeniastrum africanum + ++ + - - -
(Ethanol extract)
Piptadeniastrum africanum + + + - - -
(Water extract)
Mangifera Indica ++ ++ - - - -
(Ethanol extract)
Mangifera Indica + + - - - -
(Water extract)
Mansonia altissima - - - - - -
(Ethanol extract
Mansonia altissima
(Water extract) - - - - - -
Triplochiton scleroxylon + - - - - -
(Ethanol extract)
Triplochiton scleroxylon + - - - - -
(Water extract)
Ceiba pentandra - - - - - -
(Ethanol extract)
Ceiba pentandra - - - - - -
(Water extract)
Terminalia superba - - + - - -
(Ethanol extract)
Terminalia superba - - + - - -
(Water extract)
Terminalia ivorensis - - + - - -
(Ethanol extract)
Terminalia ivorensis - - + - - -
(Water Extract)
Control (distilled water) - - - - - -
+ = Present. + + = concentrated when present. = Absent.
the moisture in the seeds. Environmental conditions like
temperature, humidity and the moisture in the seeds
might have a role to play due to the slight differences in
the occurrence of the weevils in the seeds located at the
garden and that of the laboratory (Hagstrum and Flinn,
1993). Seeds of P. vulgaris had the lowest moisture

288 Afr. J. Microbiol. Res.
content and tend to be the best preserved seeds
probably due to the low moisture content of the seeds
Adekunle and Uma (1997).
Based on sensitivity test, it was obvious that both
extracts (water and ethanol extracts) of N. diderrichii, P.
africanum and M. indica contain substance with fungicidal
effect Adekunle (2001).
The preliminary screening of some extracts showed
some ashes to contain more active compounds than
others. The potency of the extracts may be a function of
the number and concentration of substances they
contain.
N. diderrichii contained flavonoids and tannins with
ethanol extract showing concentration of flavonoids. P.
africanum contained alkaloids, flavonoids and tannins.
The ethanol extract showed concentration of flavonoids.
M. indica contained alkaloids and flavonoids while the
ethanol extract showed alkaloids. The extracts of other
ashes contained only one active compound except for
Mansonia altissima and Ceiba pentrandra with all which
substances absent in their extracts. The inhibitory effects
of N. diderrichi and P. africanum against all the
pathogenic fungi may be due to the presence of
flavonoids and tannins in their extracts while only the
ethanol extract of M. indica showed inhibitory effect
against S. rolfsii and this was found to contain flavonoids
and alkaloids. The fact that ethanol extracts were found
to be more active or more potent than their aqueous
counterpart may be as a result of the fact ethanol diffuse
faster than water due to its volatility.
Benlate an orthodox plant fungicide showed the highest
inhibitory effect against all the pathogenic fungi. The
difference between the extracts of the ashes and the
benlate might be due to the fact that the active
ingredients in the ashes were not purified while benlate is
already a purified bioactive substance. Benlate tends to
be a better preservative agent against microbial growth
but not effective enough to prevent the insects invasion. It
was also noticed that the benlate tightly bound to the
seeds unlike the ashes that could be easily sieved away
from the seeds. Fulcin (a synthetic human fungicide drug)
showed inhibitory effect against A. niger, A. fumigatus
and R. racemosas only.
ACKNOWLEDGMENTS
I immensely thank Dr. A. A. Adekunle for the personal
research guidance and supervisory effort, and the entire
staff of the Department of Botany and Microbiology,
University of Lagos, for their assistance and
incomparable support towards the success of this
research work.
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