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EFFECT OF SEED BORNE MYCOFLORA ON THE

QUALITY OF THREE VARIETIES OF ARACHIS
HYPOGEA

Article · March 2013

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International Journal of Agricultural
Science and Research (IJASR)
ISSN 2250-0057
Vol. 3, Issue 1, Mar 2013, 35-42
© TJPRC Pvt. Ltd.

EFFECT OF SEED BORNE MYCOFLORA ON THE QUALITY OF THREE VARIETIES OF

ARACHIS HYPOGEA

NAIKOO ABBAS1, WANI MUBASHIR2, NAZIR AHMAD BHAT3, WAHEED –U-ZAMEEN4,

MD. SULIMAN DAR4 & MAHMOOD AHMAD TAK4
1
Institute of Basic Science, Department of Botany, Bundelkhand University Jhansi, U.P, India
2
Department of Botany, University of Kashmir, India
3
Sam Higgenbottom Institute of Agriculture Technology and Sciences, Allahabad, U.P, India
4
Govertment Degree College Boys Anantanag Kashmir, India
ABSTRACT

Three genotypes of Arachis hypogea viz, Chandra, Indori, Rajasthani were collected from NBPGR New Delhi. 10
species of fungi were collected from these genotypes. Effect of one of the dominant fungi Aspergillus flavus was studied
on the quality of Arachis hypogea seeds. Fungal infection caused both quantitative and qualitative damage to the seed. It
resulted in characteristic decrease in sugar and oil quantity of Arachis hypogea seeds. There was marked reduction in
germination percentage and vigour index. The oil extracted from diseased kernel contain high amount of fatty acids as
compared to healthy seeds of all the genotypes.

KEYWORDS: Seed Borne Mycoflora, Sugar, Oil, Fatty Acids, Germination, Vigour Index, Arachis Hypogea

INTRODUCTION

Oilseed crops are the main source of raw materials for vegetable oils. The world is experiencing a shortage of
edible oil and proteins, Arachis hypogea plays a useful role in alleviating these deficiencies. Arachis hypogeae L. also
known as peanut or earthnut famous by its Indian name Mongphali is a native to its region in eastern South America
(Weiss, 1983). Arachis hypogeal is grown as an annual crop particular for its edible oil and protein rich kernels seeds,
borne in pods which develop and mature below the soil surface. The Arachis hypogea is a herbaceous plant that grows to a
height of 20-60cm. Peanut is grown world wide in the tropic and temperate zones primarly as an oilseed crop (Bensal et
al.,1993).Peanut usually requires a minimum of 100 to 150 days from planting to maturity depending on the variety
planted. Arachis hypogea is a major oil seed crop grown in about 100 countries covering 26.4 million hectares with a
total production of 36.1 million tons of nuts in shell.The major Arachis hypogeal producing countries are China, India,
Nigeria, U.S.A., Indonesia and Sudan (ICRISAT., 2007). Arachis hypogeal is important in the diets of rural people,
because it is rich in proteins (21-30%), fats 41-52%), and carbohydrates (11-27%). It has calcium, potassium, phosphorus,
magnesium, and vitamin E.

Arachis hypogeal haulms are nutritious and widely used for feeding livestock (Waliyar, 2006). Arachis hypogeal
kernels contain more protein than meat and two and half times more than eggs and ten times more than any other vegetable
food except for soybean (Gopalan et al., 1971). Like other high value crops Arachis hypogea is attacked by over 50 genera
of fungi and 1 bacterium, 15 viruses, 16 nematodes, and 2 phanerogamic parasites (Jackson and Bell, 1969). Many of these
agents are major constraints of groundnut production worldwide and reduce yields as well as quality of crop substantially.
Peanut seeds and seedlings are highly susceptible to several pathogens, several pathologists isolate the following seed
borne fungi from peanut, Rhizoctonia spp,Aspergillus spp, Fusarium spp, Alternaria spp, Pythium spp, Cladosporium spp,
penicillium spp. (Abou- talib et al.,1970). Deterioration of Arachis hypogea seeds due to fungal activity is normally
36 Naikoo Abbas, Wani Mubashir, Nazir Ahmad Bhat, Waheed –U-Zameen,
MD. Suliman Dar & Mahmood Ahmad Tak

associated with the production of off-colours and flavors, rancidity, discoloration effects on yield and quality of oil, loss of
seed viability and formation of mycotoxins (Twiddy, 1994).The purpose of this study is to evaluate the effect of seed
borne Aspergillus flavus on the quality of Arachis hypogeal genotype Chandra, Indori, Rajasthani by studying its effect
on sugar content oil content germination percentage vigour index and fatty acid content.

MATERIALS AND METHODS

Seeds of three genotype of Arachis hypogea namely Chandra, Indori and Rajasthani were collected from
germplasm collection of National Beureu of Plant Genetic Resource, New Delhi. The seeds were then placed in B.O.D for
24 hrs at 250C. A large number of seed borne mycoflora were obtained from seeds placed in petri plates in incubator. The
rest study was carried out on the most dominant isolated species of fungi. The identification of fungi was made according
to (Ainsworth and Bisbys, 1973).The dominant seed borne mycoflora Aspergillus flavus was cultured on PDA for 7 days at
room temperature to get large number of spores. The spores were washed off with sterile distilled water. Spore suspension
was prepared by gently scraping the culture and transferring it to culture tube containing 100 ml distilled water.

The spore suspension obtained was adjusted to a concentration of 1×10¯7spores ml-1. The seeds of all the three
genotypes of Arachis hypogea were taken in equal quantity (100 seed/each) these seed were surface sterilized by 1%
NaOCl and then rinsed with distilled water and kept a side Seeds were soaked in spore suspension of Aspergillus flavus
and shake by hand for about 10 minutes and kept for 1 hour After words seeds were collected and redried for about 5 hours
under laminar flow.

The number of conidia were counted by a haemocytometer and approximated at 5×104 conidia seeds-1.Both
inoculated and un-inoculated seeds were used for subsequent studies. Estimation of Sugar was done by method evolved by
(Dey, 1990). Sugar was extracted from grinded seeds (1gm) both infested and healthy in ethanol (90%). It was kept in oven
at 600C for 1 hour and final volume was made to 25ml. To 1 ml aliquot of 5% phenol was followed by addition of 5ml
sulphuric acid. Optical density was read at 485 nm, the concentration were determined against curve by using glucose.100
grams of each genotype of Arachis hypogeal both infested and control were grinded with the help of grinder and oil of
each genotype was obtained through Soxlet apparatus for 9 hours per sample using Hexane as solvent (Jhamet al., 1982).
The oil obtained was put in specific voils that were already sterilized. The oil obtained through Soxlet apparatus was then
subjected to GLC analysis to study variation in free fatty amino acid (Uppstromet al., 1978).

Materials of the experiment are ethylated reagent Petroleum ether/0.02M NAOH in ethanol (2/3) and a salt
solution (80g Nacl and 3grams Sodium hydrogen sulphate in liter water). About 5 grams of oil or equivalent amount of oil
seed was taken. The sample was extracted and transesterified at the same time with N 5ml ethylated reagent and shake. The
sample was kept overnight at room temperature, 10ml of salt solution was added and shaked. As soon as the two layers are
separated the benzene phase was transferred to small test tube. A Philips PU ( ) 4500 chromatographic instrument was used
with flame ionization detector (FID). With this column the injection post, column and detector temperature was set at
2200C, 1850C and 2400C.

Nitrogen flow (used as carrier gas) rate was 22 ml/minute; the injection volume was 2HL. Peak areas were
measured with an electronic digital integrator. The percentage of seed germination was observed at room temperature after
3rd, 5th and 7th day of plating. The room temperature fluctuate form 32-350C.With the help of fine thread the root length
and shoot length of the emerging seedlings was measure and recorded for each seed in the plate. Average of each plate was
calculated then vigour index was calculated using the formula: Vigour Index = Germinating seed percentage × Radicle
length.
Effect of Seed Borne Mycoflora on the Quality of three Varieties of Arachis Hypogea 37

RESULTS AND DISCUSSIONS

Effect on Sugar Content

The present study investigated that there is reduction in sugar content in genotypes of Arachis hypogeal Chandra,
Indori, Rajasthani by fungal infection. The sugar content in variety Chandra healthy seeds were found to be 14.19±1.25
mg/gmfw as compared to 7.13 ±0.89 mg/gm.fw in infested seeds In case of healthy seeds of variety Indori the sugar was
observed as 32.86 ±1.91 mg/gm.fwand it was reduced to 31.22 ±1.86 mg/gm.fw in indori infested. The sugar content of
healthy seeds of genotype Rajasthani was observed as 30.69 ±1.85 mg/gm.fw and in the same variety infested the sugar
content was reduced to 17.92 ±1.42 mg/gm.fw.(Catherine et al.,1987) demonstrated decrease in sugar content with the
infection of Aspergillus flavus, Fusarium oxysoporium in seeds of some autoclaved oil palm kernels. Mathur and
Sinha1978 reported that fungi association with seeds bring certain biochemical changes in seeds by decreasing sugar
content. Decreased in sugar may be due to fungi utilized sugar as a substrate for its growth.

Table 1: Effect of Aspergillus Flavus on Sugar Content of Arachis Hypogeal Genotype

Sugar Content mg/gm F.W
Variety
Control(Mean±S.E) Infested(Mean±S.E)
Chandra 14.19±1.25 17.13±0.89
Indori 32.86±1.91 31.22±1.86
Rajasthani 30.69 ± 1.85 17.92±1.42
Data given are mean of three replicates ± standard error

Change in Oil Content

The present study observed that fungi Aspergillus flavus change the amount of oil in infested seeds compared to
healthy seeds. The oil quantity of healthy seeds of genotype Chandra was observed to be 21.95 ±1.56 gm and it was
reduced to 5.10 ±0.75 gm in infested seeds of the same genotype Chandra. There was also decrease in oil quantity of
infected indori and Rajasthani genotype, the oil content of healthy seeds of genotype Indori was observed as 29.74 ±1.81
gm and the oil content was decreased to 25.32 ±1.67gm in infested seeds of the same genotype. The oil quantity of
genotype Rajasthani healthy seeds was observed as 50.55 ±2.36 gm and it was reduced to 16.20 ±1.34 gm in infested
seeds.(Umechruba et al., 1992) also observed decreased in oil content of inoculated seeds of Arachis hypogea. A.flavus
caused loss of 2.3% oil; A.niger caused oil loss of 3.6% and M. phaseolina 1.7% loss of oil. (Angelo and Ory, 1983)
investigated decrease in oil content may be due to fatty acids hydrolyzed indiscriminately by the lipase enzyme of the
fungus. The structural damage caused by fungal growth in the seed coat and the internal tissues of cotyledon will expose
the lipid to oxidation, which is responsible for deteriorating oil quality (Dhingra et al., 1998).According to our
consideration the reduction in oil content might be due to lipolytic activity of seed borne fungi. The colour of the oil of all
infested seeds was changed as compared to healthy seeds, in all the three genotypes of Arachis hypogea. The oil from the
infested seeds has unpleasant odour. (Ward and Diener, 1961) suggested that changes in colour may be due to pigments
synthesized by invading fungi. The role of seed borne fungi in rancid odour development in oil has been reported in
peanut(Wilson, 1947).

Table 2: Effect of Aspergillus Flavus on Oil Content of Arachis Hypogeal Genotype

Oil Content in Grams
Genotype Control(Mean±S.E) Infested(Mean±S.E)
Chandra 21.95±1.56 5.1±0.75
Indori 29.74±1.81 25.32±1.67
Rajasthani 50.55±2.36 16.2±1.34
Data given are mean of three replicates ± standard error
38 Naikoo Abbas, Wani Mubashir, Nazir Ahmad Bhat, Waheed –U-Zameen,
MD. Suliman Dar & Mahmood Ahmad Tak

Variation in Fatty Acid Content

The present study revealed that there is increase in fatty acid content of all the infested seeds as compared to
healthy seeds. The fatty acid content of genotype Chandra healthy were observed as palmitic acid, 11.28% oleic acid,
38.65% stearic acid, 3.46% linoleic, 38.46% and arachidic acid 1.43% and it was increased in infested seeds as palmitic
acid, 12.10%,oleic acid,41.32% stearic acid 3.69% linoleic acid 39.15% and arachidic acid 1.63%. The fatty acid content
of oil obtained from healthy seeds of genotype Indori was observed as palmitic acid, 12.31% oleic acid, 36.30% stearic
acid, 3.40% linoleic acid, 35.30% and arachidic acid, 0.80%,the fatty acid content increased in infested seeds of the same
variety indori up to extend of palmitic acid,14.22% oleic acid,39.47% stearic acid, 3.59% linoleic acid, 36.55% and
arachidic acid 1.51%.There is also increase in fatty acids of oil of infested seeds as compared to healthy seeds of genotype
Rajasthani palmitic acid,9.94% oleic acid 49.19% stearic acid,2.40% linoleic acid 28.45% arachidic acid 2.31% in healthy
seeds. The fatty acids content of infested seeds was observed as palmitic acid, 12.13% oleic acid,51.32% stearic acid,
2.95% linoleic acid, 29.01% and arachidic acid, 3.21%. Bhattacharya and Raha, (2002) found increase in oleic acid content
of maize,groundnut and soybean seeds infected by different fungi species including Aspergillus,Peicillium, Fusarium,
Curvularia, Alternaria and Rhizopus. Sharma et al(1987) observed higher quanties of palmetic,oleic,linoleic and arachidic
acids in diseased kernels of Arachis hypogea seeds infected by M.phaseolina. Mckevith, (2005) stated that after processing
oxidation is the main problem to affect oil and lipid leading to aldehyde production that may result in high fatty acid
content. The present study revealed that lipids present in seeds are primarily neutral triglycerides and their hydrolysis to
free fatty acids and glycerol are catalyzed by seed infecting fungi.

Table 3: Impact of Aspergillus Flavus on Fatty Acid Composition of Arachis Hypogeal Genotype in Percentage

Genotype Chandra Indori Rajasthani

Fatty Acids Control Infested Control Infested Control Infested
Palmitic acid 11.28 12.10 12.31 14.22 9.94 12.13
Oleic acid 38.65 41.32 36.30 39.37 49.19 51.32
Stearic acid 3.46 3.69 3.40 3.59 2.90 2.95
Linoleic acid 38.46 39.15 35.30 36.55 28.45 29.01
Arachidic acid 1.43 1.63 0.80 1.51 2.31 3.21

Effect on Germination Percentage and Vigour Index

The germination percent of genotypes Chandra, Indori, and Rajasthani infested seeds was reduced as compared to
healthy seeds of all the three varieties. The reduction in germination percentage was observed after 3rd, 5th and 7th day of
both infested and healthy seeds. Germination percentage was reduced 20% after 3rd day 20% after 5th and 7th day
respectively by Aspergillus flavus in genotype Chandra compared to healthy seeds. In infested seeds of variety Indori
germination percentage was reduced to 30%, 40% and 40% as compared to healthy seeds after 3rd,5th and 7th day
respectively. In genotype Rajasthani the germination percentage was reduced to 30%, 20% and 10% after 3rd, 5th and 7th
day respectively as compared to healthy seeds. The observation of Chandra seeds depict that there was no impact of
A.flavus on the germination percentage of these seeds with regards to number of days. From the observations we can
conclude that in Indori seeds the effect of Aspergillus flavus was remarkable. Initially the Germination percentage was 100
% in healthy and 70 % in infested seeds which increased to 90 % on final observation where as healthy remained the same
The decrease in germination ability by fungal infection may be purpose due to damaging of the embryo by fungal infection
or by depletion of nutrient reserves reported by Christensen, 1973.Harman et al.,(1967) reported that the decrease in
germination percentage may be due to production of toxic metabolites. Harrington, (1967) suggested that depletion of
available oxidisable materials in meristematic cells might cause deterioration. Basavasaju et al., (2004 revealed that
Effect of Seed Borne Mycoflora on the Quality of three Varieties of Arachis Hypogea 39

Plasmopora halstedii resulted in lower germination percentage (47%) with less vigour index (470) in sunflower.
Aspergillus flavus, A. niger, Rhizoctonia spp. Fusarium spp. and Sclerotiumrolfsii reduced germination percentage and
vigour index in all genotype of Arachis hypogea (Nargund et al., 2003). The impact of Aspergillus flavus on seed produce
could be well studied by vigour index.Although the difference in germination percentage of healthy and infested seeds of
all the three genotypes was not very much significant, but there was marked difference in vigour index of healthy and
infested seeds. The vigour index of healthy seeds was probably thrice of that of infested seeds. In case of Chandra vigour
index of infested seeds was 240 and 756 of healthy seeds, Indori vigour index of healthy seeds was 399 and infested 162
and in Rajasthani the vigour index was 568 in healthy seeds and 174 of infested seeds after 7th day.

From these observations we can conclude that the infestation on culture filtrate of A.flavus was unable to check
germination as quality of seed would have been very viable but with time it could be observed that the healthy of emerging
seedling root lengh was affected and thus there was a noticiable difference in vigour index of healthy and infested seeds of
all the three genotypes of Arachis hypogea. The importance of production of toxic metabolites is obvious, when the
pathogen is seed borne. The seed borne fungi are known to affect adversely seed germination and seedling vigour possibly
due to production of toxic metabolites.

Table 4: Effect of Aspergillus Flavus on Germination Percentage and Vigour index of three Genotypes of Arachis
Hypogeal after 3rd Day, 5th Day and 7th Day

Genotype Chandra Indori Rajasthani

Germination Control 90 90 100
percentage after 3rd day Infested 70 60 70
Vigour index after 3rd Control 609 285 403
day Infested 133 138 107
Germination Control 100 100 100
percentage after 5rd day Infested 80 60 80
Vigour index after 5rd Control 743 346 483
day Infested 208 158 146
Germination Control 100 100 100
percentage after 7rd day Infested 80 60 90
Vigour index after 7rd Control 756 399 568
day Infested 240 162 174

CONCULSIONS

From present investigation it can be concluded that more studies are needed to explore a more safe and economic
method to check seed borne fungi, so that the quality and quantity of oil of the oilseeds may be safeguarded.

ACKNOWLEDGEMENTS

The Authors are grateful to Dr. Gazala Rizivi Head, Department of Botany, Bundelkhand University, Jhansi, U.P.
India for providing necessary laboratory.

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