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1. INTRODUCTION
Chickpea, an ancient crop of modern times, was first cultivated at least 9500 years ago
in the Fertile Crescent, from Turkey to Iran, at the beginning of agriculture. Chickpea
cultivation in the Indian subcontinent dates back at least 4000 years. Chickpea is
cultivated in nearly 50 countries around the world. Due to its high nutritional value, it is
an integral part of the daily dietary system for millions of people. Chickpea dominates
international markets over other legume crops and its trading is more than $8 billion
annually. The consumers’ preferences for extra-large seed size have provided an
excellent opportunity for its premium price and higher profitability. Therefore, this
ancient crop has been accepted as the crop of modern management.
Chick pea (Cicer arietinum L.) is, after soyabean and pea, the most important grain
legume crop in the world. This plant is native of Syria, South-Eastern-Turkey and
around the Mediterranean Basin, and constitutes a very important legume crop in
North Africa. In developing countries, it is an important source of high quality
protein for the proper inhabitants. Chick pea is able to grow under poor soil
conditions and can therefore be grown on marginal lands; where, owing to its
capacity to fix nitrogen, it improves soil fertility. The plant is erect and
freestanding, ranging in height from 15 to 60 cm although well- grown plants may
reach 80 cm. They have a fibrous tap root system, a number of woody stems
forming from the base, upper secondary branches and fine, froud like leaves.
Each leaflet has a thick covering of glandular hairs that secrete a strong acid
(malic) particularly during pod-set, and this provides some protection from
insects. The plant can derive more than 70% of its nitrogen from symbiotic
nitrogen fixation. Yields are best in areas with reliable seasonal rainfall and mild
spring conditions during seed filling. They are well suited to well-drained, non-
acidic soils of a medium to heavy texture. Gram husks, green or dried stems and
leaves are used for stock feed; whole seeds may be milled directly for feed. Gram
is one of the best legumes for human consumption as the seeds are very
nutritive. It furnishes an important food for lower classes and the flour is quite
nutritious. Among the food legumes, chick pea is most nutritive pulse extensively
used as protein adjunct to starchy diet (Sastri , 1950). Chickpea is traditionally a
low-input crop and is grown extensively in the moisture stress environments. The global
chickpea production has increased only marginally, unlike the manifold increase in
cereal production over the last 40 years. There are many constraints to production from
diseases, insects-pests, soil problems, environmental stresses and non-adoption of
modern management techniques. The high protein and low fat content of chick
peas makes them attractive to vegetarians, dieters and others interested in a
healthy lifestyle. When combined with other healthy foods, such as grains, nuts
or eggs, chick peas can be used to provide your body with the complete profile of
amino acids needed to make the protein that it needs for proper health. Chick pea
occupies more than 10 million hectares of the cultivated areas in the world, with a
total production of approximately 8 million tons and an average yield of 858
kg/ha. The average production of chick pea is 25-30 quintals per hectare which is
low inspite of high yielding varieties and new agronomic practices. Low yields are
attributed to different factors, among which pathogenic micro-organisms and

2. insect attacks are considered the most serious. The reasons of low yield are so
many apart from other reasons; the main cause of low yield of this crop is the
incidence of diseases. India is the world leader in chick pea production followed
by Pakistan. The chick pea crop is attacked by 172 pathogens (67 fungi, 22
viruses, 3 bacteria, 80 nematodes and Mycoplasma) from all over the world.
Among all, only a few of them have the potential to devastate the crops. Some of
the serious disease in order of their importance are dry root rot (Rhizoctonia
bataticola), wilt (Fusarium oxysporum f. sp. Ciceri), wet root rot (Rhizoctonia
solani), Ascochyta blight (Ascochyta rabiei) and collar rot (Sclerotium rolfsie).
The fungus Rhizoctonia bataticola is the major biotic constraint in the successful
cultivation of this important crop. It is most important and widespread soil borne
disease of chick pea grown between latitudes 20 N and 20 S, Where the climate is
relatively dry and warm.
COLLECTION OF DISEASED MATERIAL:-
Naturally infected Chick pea plants, showing characteristic symptoms of dry root rot
were collected from the field of pulse section, Bihar Agriculture University (BAU),
Sabour, Bhagalpur which were surveyed. Such Rhizoctonia bataticola affected plants
were brought to the laboratory. Dry Root Rot disease caused by Rhizoctonia bataticola
was observed on variety PUSA-256/PG-256 in Sabour, Bhagalpur during March-
April 2013.Considering the importance of the disease and crop, the present
investigation were undertaken to know the nutritional quality in terms of a
protein, carbohydrate of a normal and infected chick pea cultivar , to understand
the carbohydrate metabolism of normal and infected chick pea , to identify the
proteins responsible for the nutritional deficiency caused by Rhizoctonia
bataticola and to evaluate the changes in enzymatic activities of normal and
infected chick pea cultivar from germination to growth phase.
PUSA-209: It was developed at I.A.R.I, New Delhi, from a cross P-827 and C-235 and
was released for general cultivation in 1980 for Rajasthan, Delhi, Uttar Pradesh,
Haryana and Punjab. The crop matures in about 145-165 days. Plant height is about 65
cm. Seeds are attractive and medium bold (135 g/1000 seeds) and light brown in color.
Its yield potential is about 25-30 quintals per hectare.

3. Total Soluble Sugars
The dried plant materials stem, root, leaf, and callus (50 mg each) were homogenized
separately in a mortar and pestle with 20 ml of 80% ethanol and left overnight. Each of
the sample was centrifuged at 1200 rpm for 15 min, the supernatants were collected
separately and concentrated on a water bath using method of Loomis and Shull (1973).
Distilled water was added to make up the volume up to 50 ml and processed further for
quantitative analysis.
Starch
The residual mass obtained after extraction of total soluble sugars of each of the
test samples was suspended in 5.0 ml of 52% perchloric acid (Mc Cready et al,
1950). Later, 6.5 ml of water was added to each sample and the mixture was
shaken vigorously for 5 min.
Quantitative Estimation of sugar
1 ml aliquot of each sample was used for the estimation of carbohydrates using the
phenol-sulphuric acid method of Dubois et al, (1951). A standard regression curve of
standard sugar (glucose) was prepared. A stock solution of glucose 100 μg / ml was
prepared in distilled water. From this solution, 0.1 to 0.8 ml was pipette out into eight
separate test tubes and volume was made up to 1 ml with distilled water. These tubes
were kept on ice; 1 ml of 5% phenol was added in each tube and shaken gently. 5 ml of
conc. sulphuric acid was rapidly poured so that the steam hits the liquid and tubes were
gently shaken during the addition of the acid. Finally the mixture was allowed to stand
on a water bath at 26-30o
C for 20 min. The characteristic yellow orange colour was
developed. The optical density was measured at 490 nm using spectrometer (Carlzeiss,
Jena DDR, VSU 2 P) after setting for 100% transmission against a blank (distilled
water). Standard regression curve was computed between the known concentrations of
glucose and their respective optical density, which followed Beer’s Law.

4. All samples were analyzed in the same way as described above and contents of the
total soluble sugars and starch were calculated by computing optical density of each of
the samples with standard curve.
Proteins
Extraction
The test samples (50 mg each) were separately homogenized in 10 ml of cold 10%
trichloroacetic acid (TCA) for 30 min and kept at 4o
C for 24 hr. These mixtures were
centrifuged separately and supernatants were discarded. Each of the residues was
again suspended in 10 ml of 5% TCA and heated at 80o
C on a water bath for 30 min.
The samples were cooled, centrifuged and the supernatants discarded. The residue
was then washed with distilled water, dissolved in 10 ml of 1N NaOH, and left overnight
at room temperature (Osborne, 1962).
Quantitative Estimation
Each of the above samples (1ml) was taken and the total protein content was estimated
using the spectrophotometer and method of Lowry et al, (1951). A regression curve of
the standard protein (bovine serum albumin, BSA) was prepared. A stock solution of
BSA (Sigma Chem. Co., St. Louis, USA) was prepared in 1N NaOH (1mg/ml). Eight
concentrations (ranging from 0.1 to 0.8 mg/ml) were separately measured in test tubes
and the volume of each was made up to 1ml by adding distilled water. To each, 5ml of
freshly prepared alkaline solution (Prepared by mixing 50 ml of 2% Na2CO3 in 0.1 N
NaOH and 1 ml of 0.5% CuSO4.5H2O in 1% Sodium potassium tartarate) was added
and kept at room temperature for 10 min. In each sample 0.5 ml of Folin-Ciocalteau
reagent (commercially available reagent was diluted with equal volume of distilled water
just before use) was added rapidly with immediate mixing and optical density of each
sample was measured after 30 min at 750 nm using spectrophotometer against the
blank (Lowry et al, 1951). Five replicates of each concentration were taken and the
average value was plotted against their respective concentrations to compute a
regression curve.

5. All samples were processed in the same manner and the concentration of the total
protein content in each sample was calculated by referring the optical density of each
sample with standard curve. Five replicate samples were taken in each case and mean
value was calculated.
Results of primary metabolites of normal Cicer arietinum L. Seeds
S. No. Plant parts Carbohydrates
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Seed 8.65 9.12 45.41

6. 8.65 9.12
45.41
0
5
10
15
20
25
30
35
40
45
50
Carbohydrates mg/gdw Starch mg/gdw Protein mg/gdw
SEED
Primary metabolites of normal Cicer arietinum L. Seeds
Results of primary metabolites of infected Cicer arietinum L. Seeds
S. No. Plant
parts
Carbohydrates
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Seed 8.20 8.89 44.88

7. 8.2 8.89
44.88
0
5
10
15
20
25
30
35
40
45
50
Carbohydrates
mg/gdw
Starch mg/gdw Protein mg/gdw
SEED
Results of primary metabolites of infected Cicer arietinum L. Seeds
Primary metabolites of normal Cicer arietinum L. Seedlings (Germinated seeds
15 days old)
S.
No.
Plant parts Carbohydrate
s
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 7.65 5.12 37.41
2 Stem 4.61 4.94 29.13
3 Roots 4.17 5.67 33.73

8. 7.65
4.61 4.175.12 4.94 5.67
37.41
29.13
33.73
0
5
10
15
20
25
30
35
40
Leaf Stem Roots
Carbohydrate
Starch
Protein
Primary metabolites of normal Cicer arietinum L. Seedlings (Germinated seeds
15 days old)
Primary metabolites of infected Cicer arietinum L. Seedlings (Germinated seeds
15 days old)
S. No. Plant
parts
Carbohydrates
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 7.13 5.00 36.90
2 Stem 4.12 4.60 29.03
3 Roots 4.00 5.18 32.96

9. 7.13
4.12
4
5.00
4.60
5.18
36.9
29.03
32.96
0
5
10
15
20
25
30
35
40
Leaf Stem Roots
Carbohydrate
Starch
Protein
Primary metabolites of infected Cicer arietinum L. Seedlings (Germinated seeds
15 days old)
Results of primary metabolites of normal Cicer arietinum L. Seeds Seedlings (30
days old)
S. No. Plant
parts
Carbohydrates
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 8.65 7.12 38.41
2 Stem 4.98 5.49 27.13
3 Roots 4.23 6.39 36.73

10. 8.65
4.98
4.23
7.12
5.49
6.39
38.41
27.13
36.73
0
5
10
15
20
25
30
35
40
45
LEAF STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites of normal Cicer arietinum L. Seeds Seedlings (30 days old)
Results of primary metabolites Cicer arietinum L. infected Seeds Seedlings (30
days old)
S. No. Plant
parts
Carbohydrates
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 8.46 7.02 38.10
2 Stem 4.80 5.40 27.00
3 Roots 4.10 6.10 36.40

11. 8.46
4.8
4.1
7.02
5.40
6.10
38.1
27
36.4
0
5
10
15
20
25
30
35
40
45
LEAF STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites Cicer arietinum L. infected Seeds Seedlings (30 days old)
Results of primary metabolites of normal Cicer arietinum L. plant (60 days old)
S. No. Plant
parts
Carbohydrates
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 9.02 8.00 37.20
2 Stem 6.20 5.40 27.00
3 Roots 5.22 6.10 36.40

12. 9.02
6.2
5.22
8.00
5.40
6.10
37.2
27
36.4
0
5
10
15
20
25
30
35
40
LEAF STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites of normal Cicer arietinum L. plant (60 days old)
Results of primary metabolites Cicer arietinum L. infected plant (60 days old)
S. No. Plant
parts
Carbohydrate
s
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 8.94 7.90 36.70
2 Stem 5.60 5.08 26.80
3 Roots 5.03 6.00 33.80

13. 8.94
5.6
5.03
7.90
5.08
6.00
36.7
26.8
33.8
0
5
10
15
20
25
30
35
40
LEAF STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites Cicer arietinum L. infected plant (60 days old)
Results of primary metabolites of normal Cicer arietinum L. plant (90 days old)
S. No. Plant
parts
Carbohydrate
s
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 9.32 8.24 37.64
2 Immature
pod
6.18 5.16 33.22
2 Stem 6.22 5.55 27.36
3 Roots 5.52 6.42 36.86

14. 9.32
6.18
6.22
5.52
8.24
5.16
5.55
6.42
37.64
33.22
27.36
36.86
0
5
10
15
20
25
30
35
40
LEAF IMMATURE
POD
STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites of normal Cicer arietinum L. plant (90 days old)
Results of primary metabolites Cicer arietinum L. infected plant (90 days old)
S. No. Plant
parts
Carbohydrate
s
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 8.00 6.55 33.40
2 Immature
pod
5.18 5.06 30.22
2 Stem 5.40 4.12 22.16
3 Roots 5.32 5.82 32.44

15. 8
5.18
5.4
5.32
6.55
5.06
4.12
5.82
33.4
30.22
22.16
32.44
0
5
10
15
20
25
30
35
40
LEAF IMMATURE
POD
STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites Cicer arietinum L. infected plant (90 days old)
Results of primary metabolites of normal Cicer arietinum L. plant (120 days old)
S. No. Plant
parts
Carbohydrate
s
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 8.00 6.55 33.40
2 Seeds 8.68 9.26 45.46
2 Stem 5.40 4.12 22.16
3 Roots 6.60 7.82 32.44

16. 8
8.68
5.4
6.6
6.55
9.26
4.12
7.82
33.4
45.46
22.16
32.44
0
10
20
30
40
50
LEAF SEED STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites of normal Cicer arietinum L. plant (120 days old)
Results of primary metabolites Cicer arietinum L. infected plant (120 days old)
S. No. Plant
parts
Carbohydrate
s
mg/gdw
Starch
mg/gdw
Protein
mg/gdw
1 Leaf 6.09 6.20 32.16
2 Seeds 8.19 8.68 44.60
2 Stem 4.96 4.00 19.06
3 Roots 5.96 6.92 28.64

17. 6.09
8.19
4.96
5.96
6.20
8.68
4.00
6.92
32.16
44.6
19.06
28.64
0
5
10
15
20
25
30
35
40
45
50
LEAF SEED STEM ROOTS
Carbohydrates mg/gdw
Starch mg/gdw
Protein mg/gdw
primary metabolites Cicer arietinum L. infected plant (120 days old)