Fungi associated with wheat seed discolouration and abnormalities in <i>in-vitro</i> study

doi:10.4236/as.2013.49069

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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: [email protected]

Received 31 January 2013; revised 1 March 2013; accepted 15 April 2013

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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