Vol.4, No.9, 516-520 (2013) Agricultural Sciences
http://dx.doi.org/10.4236/as.2013.49069
Fungi associated with wheat seed discolouration
and abnormalities in in-vitro study
Neha Pathak*, Razia. K. Zaidi
Section of Plant Pathology and Nematology, Department of Botany, Aligarh Muslim University , Aliga r h, India ;
*Corresponding Author: [e-mail]
Received 31 January 2013; revised 1 March 2013; accepted 15 April 2013
Copyright © 2013 Neha Pathak, Razia. K. Zaidi. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The main aim of present study is to ascert ain the
fungal species and their effect on germination
associated with wheat seeds. Seeds of three
varieties WH896, PBW-373 and HD264 of wheat
(Triticum aestivum) were collected from Quarsi
Agriculture Farm Aligarh. These three seed
samples of wheat showing different forms of
discouloration and abnormalities were screened
for associated fungi. Microscopic examination
of wheat seeds reveals that seeds of all the va-
rieties of wheat possess injuries to varying ex-
tent. Detailed examination of the seeds has
shown that the seeds can be classified on the
basis of extent of injury in the three categories
viz., seeds having minor cracks, cracks without
exposed embryo and cracks with exposed em-
bryo. Seed soaking and washing techniques
were also employed. Fusarium moniliforme and
Alternaria alternata were isolated from all the
categories of seed tested. Floating mycelial bits
and conidia of Alternaria, Fusarium, Drechslera,
Curvularia lunata, Mucor were found in all the
three varieties of wheat. The seeds were sub-
jected to visual observation and examination
under stereoscopic microscope. These findings
are to study and detect the phytopathogenic
mycoflora which causes damage and loss to our
seeds and crops.
Keywords: Wheat; Seed-Borne Fungi; Incidence;
Discolouration; Wrinkled Seeds
1. INTRODUCTION
Wheat (Triticum aestivum L) is an important cereal
crop belonging to Gramineae family .The production
rates of these crops have failed to keep pace with re-
quirements of the growing population [1]. During recent
studies, it has been reported that 100 g of edible portion
of wheat grains contain 11.50% proteins, 59.40% carbo-
hydrates, 9.70% fats, 10.60% crude fibers, and 1.80%
ash [2]. Along with some abiotic limitations, this protein
and starch rich cereal crop is also highly susceptible to
various pests and diseases and a number of storage grain
molds cause considerable losses [3]. Quality characters
of wheat seeds, such as seed germination, moisture con-
tent, seed discolouration and seed-borne fungal preva-
lence have long been known to be influenced by various
factors. The major mycotoxigenic fungi in rice are As-
pergillus spp. [4], Fusarium sp p. [5], an d Pen icilliu m spp
[6] in cereal. The type and severity of seed abnormality
may be dependent on the type and pathogenic potential
of the associated fungi as well as the prevailing weather
conditions [7]. The harmful effects of such fungal inva-
sion are glume or grain discolouration. Other abnormali-
ties such as deformation and damage in seeds are a major
constraint in crop production in most of the developing
countries. Seed abnormality due to the influence of seed-
borne fungi is very common and often accounts for a
large percentage of crop losses [8]. Seeds that showed
distinct symptoms were selected for study and catego-
rized in to three groups viz discoloured seeds, wrinkled
seeds, and seeds with discoloured embryo. Seed abnor-
mality could be in the form of shrunken seeds, seed ne-
crosis, seed rot, reduction in seed size or total seed dis-
colouration [9]. Discoloured seeds were further sub-
grouped, to isolate, identify and obtain information about
the incidence of various seed-borne fungi associated with
different types of discolouration and abnormalities of
seeds related to crop wheat in Aligarh district.
2. MATERIAL AND METHODS
2.1. Dry Inspection of Seeds
Seeds of three varieties of wheat viz WH896, PBW-

N. Pathak, R. K. Zaidi / Agricultural Sciences 4 (2013) 516-520 517
373 and HD264 were obtained from the Quarsi farm,
Aligarh. The seeds were subjected to visual observation
and examination under stereoscopic microscope. Seeds
that showed distinct symptoms and abnormalities were
selected and categorized in to different groups viz, dis-
coloured seeds, wrinkled seeds and seeds with discol-
oured embryo and blackened seeds. 100 seeds per culti-
var were examined under four replicate.
2.2. Plating and Incubation of Seeds
Infected seeds of each cultivar were surface sterilized
in 2% HgCl2 for 15 min and then rinsed for 2 min each in
three changes of sterile distilled water prior to plating.
Ten seeds were plated in each petri dish containing 10 ml
of potato dextrose agar (PDA) medium and moist blotter
papers. For each of the categories and subgroupings, a
total of 400 seed were plated in four replicates of 100
seeds per variety. These petri dishes were incubated at
22˚C ± 2˚C under alternating cycles of 12 h light and 12
h darkness. On the 8th day, incubated seeds were ob-
served for fungal growth and identification under stereo
binocular and compound microscopes. Identification was
based on presence of physical characteristics of the
structures such as conidia and hypha [10-12]. The num-
ber of seeds infected with each kind of fungus was
counted and more than one fungal growth on the same
seeds, was regarded as multiple infection.
2.3. Statistical Analysis
The data obtained from the seed health testing me-
thods were subjected to statistical analysis to find out the
significance of the results. The data collected were trans-
formed prior to analysis using square root transformation
method and analysis of variance and mean separation
were performed using Statistical Analysis Software (SAS,
1994).Germinated seeds were counted and expressed as a
measure of seed viability using the formula.
100Svn N
where Sv is % seed viability, n is the number of seeds
germinated from each normal or abnormal seed type and
N is the total number of seeds plated on blotter and PDA.
3. RESULTS
Inspection of dry seeds of wheat varieties WH896,
PBW373, and HD264 indicated that certain seeds were
damaged, deformed, discoloured and contaminated with
inert matter and infected with mycelial fragments. In all
the three varieties, colour of pericarp was found Golden,
Golden Brown and Light Golden Brown respectively.
Inspection of seeds revealed that normal seeds were
12.57% - 28.7% while among the different abnormal
seed types. Deformed seeds were (21.25%), Broken
seeds (7.25%), and Malformed seeds (32.8%) were
found across all the three varieties tested. (Tab l e 1 , Fig-
ure 1) show the percentage of normal and abnormal
seeds after dry inspection of seeds. There were more ab-
normal than normal seeds. Wrinkled seed type was the
most common form of abnormality with 64.4% - 72.2%.
Incidence of entirely discoloured seed was 12.50 and
8.50 on variety PBW-373 and HD264 respectively (Ta-
ble 1). Incidence of seeds with discoloured embryo and
brush end was 1.25 to 1.80% and 1.75% - 6.0%. Alter -
naria alternata and A. clamydophor and Fusarium
moniliforme were isolated from both normal and abnor-
mal seeds (Table 2). However, with a higher incidence in
abnormal than normal seeds (Table 2). Rizopus spp.
(12.5%) and Penicillium spp. (4.2%) were also isolated
additionally from entirely discoloured seeds. Incidence
of Alternaria alternata was 2.2% on normal seeds while
it was 2.25% - 78.0% on the abnormal seeds. Incidence
of Fusarium moniliforme (30.2%) on seed with discol-
oured embryo end (Tab le 2). Seed abnormality that are
associated with seed infection and discoloration had a
higher incidence of fungal infection of seeds (Ta b le 2 ).
Germination percentage of normal seeds was 80% and
this was higher than that (2.2% - 42.0%) of the abnormal
seeds (Table 2).
Fusarium moniliforme, Alternaria alternata, and As-
pergillus flavus were associated with white streaks from
the all three varieties of wheat. However th e incidence of
F. moniliforme (94.0) was significantly higher than of
any of the other fungi in all the three varieties tested
Figure 1. Seed discoloration and isolation of fungus on Blotter
Method; Indicates the infection of Fusarium spp. on Blot-
ter.

N. Pathak, R. K. Zaidi / Agricultural Sciences 4 (2013) 516-520
518
(Ta b le 3 ). On variety WH896, F. moniliforme was iden-
tify from 94% of seeds that showed white streaks. The
incidence of F. moniliforme on seeds that showed pur-
ple/pink discolouration was significantly higher than any
other fungi isolated on variety PBW373 (56%) and
HD264 (42%). Fusarium moniliforme, Drechslera aus-
traliensis and Curvularia lunata were observed on seeds
with brown spots. The incidence of D. australiensis
(62.8%) on variety HD264 was significantly higher than
that of other fungi. Aspergillus flavus. Cladosporium,
Drechslera were mostly associated with blackened seeds
of three varieties tested. The percentage incidence of
Aspergillus flavus on the blackened seed of the three
varieties incubated (31%, 28%, and 25%) on WH896,
HD264 and PBW372 respectively were significantly
higher than any o ther observed fungi. Table 2 also show s
that the incidences of Alternaria alternata and Fusarium
moniliforme were significantly high on seeds of all the
var ie ti es r eg ar dless of the type of discolouration (Table 3,
Figure 2).
4. DISCUSSION
Seed-borne pathogens affect directly and indirectly the
quality of seeds in commerce. The germination of seed
infected with seed-borne pathogens may be reduced be-
cause the pathogens attack and kill the seedling.
Seed-borne diseases have been found to affect the growth
and productivity of crop plants [13-15]. Seeds are re-
garded as highly effective means for transferring plant
pathogens over the years. Seed-borne diseases have been
found to affect the growth and productivity of cr op plan ts.
Some of the fungi infect the seed and cause discolour-
ation of the seed and also some other seed borne patho-
gens are known to be associated with wheat seed which
are responsible for deterioration of seed quality during
storage [16]. Detecting seed-borne fungi on seeds is ac-
complished by a number of methods. Examination of dry
seeds with the naked eye and at magnifications of 10 to
30 times reveals a number of plant pathogens that occur
mixed with the seeds as fungus bodies or have converted
the seed into fungus structure. The spores of some path o-
gens may be associated in such great numbers with indi-
viduals seeds that merely mo unting the seeds in drops of
water, tearing the seeds apart, in such conditio n and then
examining the exuding spores microscopically are suffi-
cient for fungal detection. The dry inspection of seeds
Table 1. Incidence of normal and abnormal wheat seeds from dry inspection of seeds.
Variety No Normal seeds (%) Wrinkled seeds (%)Seeds with discoloured
embryo end (%) Entirely discoloured seeds (%) Seeds with discoloured brush end (%)
WH896 15.25 72.20 1.80 4.0 6.0
HD264 12.57 64.4 0.00 8.50 0.25
PBW373 26.57 38.2 1.25 12.50 1.75
Table 2. Viability and mycoflora of normal and abnormal seeds of all three wheat varieties.
Seed type Germination % Associated fungi Seed infections % Incidence of fungi %
A.alternata, 2.25 ± 0.25
Normal seeds 80.0 ± 0.15 F. moniliforme, 0.50 ± 0.10 0.60%
Alternaria, 20.5 ± 1.50
Fusarium, 42.0 ± 4. 5 0
Wrinkled seed 42.0 ± 2.4
Aspergillus 34.0 ± 5.50
6.25%
A. clamydophore, 57.5 ± 0.30
A. flavus, 30.0 ± 1.0
Entirely discoloured seed 8.0 ± 6.0
D. australiensis 18.40 ± 2.24
30.6%
A. alternata, 18.25 ± 0.50
D. australiensis, 78.0 ± 1.52
C. lunata 60.50 ± 0.20
Seed with discoloured embryo end 2.5 ± 10.3
Fusarium 30.20 ± 1.20
40.2%
A. clamydophore, 10.25 ± 2.10
Penicillium spp., 24.0 ± 4.10 Seed with discoloured brush end 4.7 ± 3.0
Cladosporium spp. 15.4 ± 2.50
28.18%
Values are means from four varieties of wheat. *±S.E.—Standard error.

N. Pathak, R. K. Zaidi / Agricultural Sciences 4 (2013) 516-520 519
Table 3. Seed borne fungi associated with different types of discolouration on seed of three wheat varieties.
White streaks Pink/Purple Brown spots Blackened seeds
Fungus *1 2 3 1 2 3 1 2 3 1 2 3
Alternaria alternata 49.2 a82.0 a 56.0 a 10.6 c15.0 b120 b8.5 c 2 6 .0 b18.5 b 0.2 c 4.6 c 8.0 c
Aspergillus niger 21.0 b42.5 a 45.2 a 13.3 c16.5 b30. 5 b81.1 a3 1 .2 b17.0 b 8.5 c 0.0 c 2.6 c
Alternaria clamydophor 42.6 b27.0 b 36.8 a 32.2 b28.2 b10.5 b36.2 b56.0 b45.2 b 24.2 b 0.0 c 8.2 c
Aspergillus flavus 32.1 b14.6 b 10.3 b 18.5 b26.2 b13.6 b28.6 b38.0 b26.0 b 31.0 b 28.5 b25.6 b
Fusarium moniliforme 94.0 b74.2 a 52.0 a 38.5 b42.5 b56.0 a36.2 b78.5 b45.0 c 8.6 c 17.0 b15.4 b
Mucor 0.0 c 0.2 c 4.0 c 2.0 c 8.2 c 6.5 c 10.2 b20.2 b8.4 c 0.0 c 0.0 c 0.0 c
Rhizopus oryzae 1.5 c 0.0 c 6.2 c 0.0 c 0.0 c 0.0 c 13.8 b18.1 b5.0 c 4.0 10 .0 c2.8 c
Drechsleria australiensis 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 0.0 c 46.6 b62.8 b26.2 b 16.0 b 24.o b20.4 b
Rhizopus spp. 0.0 c 0.1 c 2.1 c 0.2 c 4.0 c 10.4 b0.0 c 13.2 b5.5 c 5.6 c 8.0 c 10.4 c
Penicillium spp. 0.5 c 1.9 c 4.5 c 0.0 c 0.0 c 0.0 c 10.1 c26.4 b30.2 b 0.0 c 0.0 c 0.0 c
Curvularia lunata 0.0 c 0.0 c 0.0 c 8.0 c 6.0 c 10.0 c25.8 b37.4 b28.4 b 0.0 c 0.0 c 0.0 c
Cladosporium 0.0 c 0.0 c 0.0 c 2.0 c 1.0 c 11.7 b1 8.5 b15.2 b12.6 b 24.6 b 20.0 b16.8 b
*1) Variety WH896, 2) HD264, 3) PBW373; Each value is a mean of four rep li cates.
Figure 2. Culture of Alternaria alternata on PDA Method.
revealed a higher incidence of wrinkled and discoloured
than normal seeds. Grain weathering manifests as dis-
colouration, rough appearance, shriveling, loss of texture
or reduced size [17]. The association of Alternaria al-
ternata and Fusarium moniliforme with all the different
types of seed discolouration and abnormalities, and its
high incidence may be due to the susceptibility of the
wheat crop to attack by both fungi. The isolation of dif-
ferent microorganism associated with seeds of wheat as
detected by different techniques compared in terms of
percentage frequencies indicated that the Standard Blot-
ter method yielded quantitatively high percent frequency
of Aspergillus spp. and Alternaria spp. [18]. [19] Thomas
and Buddenhagen (1980) and [20] Zummo and Scott
(1992) observed higher incidence of F. moniliforne than
other fungi isolated from maize seeds. [21] Adlkha and
Joshi (1974) reported that severe infection of wheat
heads caused discolouration and shriveling of the seeds.
The association of Alternaria alternata and Fusarium
moniliforme with all forms of seed abnormalities and its
relatively high incidence on discoloured seeds indicates
the susceptibility of the wheat crop to these fungal spe-
cies, similar to the reports of [8] Varshney (1990). [22]
Parry et al. (1995) had listed F. grammearum as one of
the several Fusarium spp. associated with the head blight
of wheat. Fusarium mon iliforme was found to be associ-
ated with pink/purple seeds. Similar observation was
reported by [23] Neergaard (1979). Brown spots on
wheat seeds were associated with F. moniliforme, Drech-
slera australiensis and Curvularia lunata. Blackened
wheat seeds were infected with Aspergillus flavus, Al-
ternaria clamydophore, and Cladosporium. Alternaria
tenuis isolated from entirely discoloration seeds is also a
reputed black-point pathogen [8,24]. The reduced viabil-
ity of abnormal seeds is due to the influence of f u ngi . [ 2 5]
Fakir (1988) reported significant reduction in the germi-
nation of infected seeds while [26] Rena and Gupta
(1982) reported that localized discoloured areas, usually
around the embr yo end of seeds are often responsible for
reduced germ inability. The isolation of some economi-
cally important seed-borne fungi of wheat from the dis-
coloured embryo ends may be due to the fact that the
embryo contains abundant protein materials. The find-

N. Pathak, R. K. Zaidi / Agricultural Sciences 4 (2013) 516-520
520
ings in this investigation suggest that wheat seeds with
various types of discolouration and abnormalities have
the potential to cause seed deterior ation in storage and in
the field too.
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