Download Effect of nitrogen fixation, nitrogen fertilization and viral infection on

Plant Foods for Human Nutrition 47: 257-263, 1995.
© 1995 Kluwer Academic Publishers. Printed in the Netherlands.
Effect of nitrogen fixation, nitrogen fertilization and
viral infection on yield, tannin and protein contents and
in vitro protein digestibility of faba bean
E.E. BABIKER, E.A.E. EL SHEIKH, A.J. OSMAN & A.H. EL TINAY
Department of Biochemistry and Soil Science, Faculty of Agriculture, Shambat, Sudan
Received 1 April 1994; accepted in revised form 10 December 1994
Key words: Faba bean, Protein digestibility, Rhizobium, Tannin, Viruses
Abstract. A field investigation of two faba bean cultivars (cv.), Agabat and Silaim, showed that
bean yellow mosaic virus (BYMV) infection reduced (p ~<0.001) yield (Kg/ha), protein content and
in vitro protein digestibility (IVPD) but increased (p ~<0.05) tannin content (rag/100 ml). Nitrogen
fertilization with viral infection significantly reduced yield and IVPD for cv. Silaim and increased
( p ~<0.05) protein and tannin contents, Nitrogen fertilization alone was found to increase ( p ~<0.05)
yield, protein and tannin contents but slightly reduced IVPD. Rhizobium inoculation with viral
infection significantly decreased yield per unit area, protein content and IVPD, but increased
(p ~<0.05) tannin content. Rhizobium inoculation alone significantly increased (p ~<0.001) yield and
tannin content and slightly increased protein content but decreased IVPD. The results indicated
that nitrogen fertilization or nitrogen fixation increased yield, protein and tannin contents and
decreased IVPD. Viral infection had an adverse effect on yield, protein content and IVPD but had
no effect on tannin content.
Introduction
In different parts of the world, vigorous efforts are directed to improve yield,
protein quality and quantity in sorghum and faba bean as field crops with
methods for overcoming the antinutritional factors such as tannin, by application of fertilizers and genetic manipulation. In field beans (Viciafaba) protein
content is raised by sulphur applications with or without additional nitrogen (N)
[1]. Higher levels of N fertilization was found to enhance grain protein content
and digestibility of ryegrass [2]. In contrast, Witman et al. [3] reported that
protein digestibility was not affected by N application, however, N tended to
reduce digestibility and increase yield and protein content of ryegrass. Reduction
in digestibility was found to be accompanied by high levels of tannins which were
found to have an adverse effect on proteolytic enzymes such as pepsin and
trypsin [4]. In the Middle East, where the faba bean is widely grown, the crop is
usually fertilized and sometimes inoculated for N fixation, however, infestation
with viruses is common. The aim of this study is to determine the effect of N
fixation, N fertilization and viral infection on yield, tannin and protein contents
and in vitro protein digestibility (IVPD) of faba bean cv. Silaim and Agabat.
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Materials and methods
Faba bean cuttivars. Seeds of Agabat and Silaim were ground to pass a 0.4 mm
screen for determination of tannins and protein content, and ground to pass a
0.16 mm screen for determination of digestibility.
Field experiment. The field experiment was conducted in the Demonstration
Farm of the University of Khartoum at latitude 15 ° 40'N and longitude
32 ° 32'E during 1992/1993 cropping season. Seeds of both cultivars were
either inoculated with Rhizobium leguminosarum biovar viceae strain TAL 1397
or fertilized with N (50 Kg/ha). In addition, there was an untreated control for
both cultivars. All treatments were either sap inoculated or uninoculated with
BYMV after ten days from sowing. Treatments were:
(1)
(2)
(3)
(4)
(5)
(6)
Untreated control plants (Silaim)
Rhizobium inoculated plants (Silaim)
Nitrogen fertilized plants (Silaim)
Untreated control plants (Sitaim) + BYMV
Rhizobium inoculated plants (Silaim) + BYMV
Nitrogen fertilized plants (Silaim) + BYMV
Similar treatments were carried out for Agabat cultivar. The experiment was
arranged in a factorial design with four replicates per treatment. Plants were
irrigated every ten days. At maturity, seeds were collected and the yield of each
treatment was expressed on per hectare basis.
Protein and tannin analysis. Protein (N x 6.25) and tannin as tannic acid
(rag/100 ml) were determined by the method of AOAC [5]. For determination
of tannin content five grams of sample were extracted with 400 ml of water at
100 °C for 30 min, left to cool and transferred to a 500 ml volumetric flask and
diluted to the mark, the mixture was then gently shaken and filtered. 5 ml
Folin-Denis reagent and 10 ml saturated Na2CO3 solution were added to the
extract (10m l) and the volume was increased to 100ml with distilled water,
mixed well. The absorbance of the color developed after 30 rain was read at
760 nm. A standard curve was prepared expressing the results as tannic acid
(mg/ml) after correcting for blank.
Invitroproteindigestibility(IVPD). IVPD was determined according to Saunder
et al. I-6]. A 0.2 g sample was placed in a 50 ml centrifuge tube, 15 ml of 0.1 M HC1
containing 1.5 mg pepsin was added, and the tube was incubated at 37 °C for
three hours. After incubation, the sample was centrifuged at 5000 x g for 20 min
at room temperature. Nitrogen in the supernatant was estimated using the
Kjeldahl method. Digestibility was calculated using the formula:
protein digestibility (%) -
N in supernatant - enzyme N
x 100
N in sample
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Statistical analysis. Each sample was analyzed in triplicate and the figures were
then averaged. Data were assessed by analysis of variance (ANOVA) [7] and
by the Duncan multiple range test with a probability of p ~<0.05 [8].
Results
Effect of N fertilization, N fixation and viral infection on yield. Figures 1 and 2
show the effect of N fertilization, N fixation and viral infection on yield of cv.
Agabat and Silaim, respectively. For both cultivars, viral infection (BYMV)
caused a significant (p...<0.001) reduction in yield per unit area of all treatments. Nitrogen fertilization and fixation significantly increased yield per unit
area for all treatments for both cultivars. The yield of cv. Agabat (Fig 1) was
significantly lower than that of cv. Silaim (Fig. 2) for all treatments.
Effect of N fertilization, N fixation and viral infection on protein content. Table I
shows the effect of N fertilization, N fixation and viral infection on protein
content of the two faba bean cultivars. For both cultivars viral infection caused
slight reduction in protein content. For cv. Agabat when both N fertilization
and virus were applied, protein content was significantly (p ~ 0.05) increased
while in the case of Silaim, protein content was significantly (p~<0.05) increased when both N fixation and virus were applied. For both cultivars
(Table 1), N fertilization alone significantly increased protein content but N
fixation alone was found to have no effect.
4200
3500
ConDo/
V/ral Infect~on(V)
2800
N Fefti/iza-
2100
.~
P
14oo
~
i
[------~
N FTxation
(AF2
,~ + v
7O0
0
Fig. I.
''
',
'
. . . . . .
Effect of N fixation and fertilization and viral infection on yield of, faba bean, cv. Agbat.
260
42003500
"
2 8 0 0
"
Control
~?al I n l e t Non (V~
21 O0 "
~
N Ferti/ization
[N)
N+V
.g' 1400
)-
i
~
I
At F/xatfon
i ~--+
v
7 0 0 "
0
Fig. 2. Effect of N fixation and fertilization and viral infection on yield of, faba bean, cv. Silaim.
Table 1. Effect of N fixation, N fertilization and viral infection on
protein content (%) of two faba bean cv. Agabat and Silaim
Treatments
cv. Agabat
cv. Silaim
36.0 a (_+ 0.86)
39.08 (_+ 0.70)
36.3 a (_+ 0.86)
37.1" (_+ 0.72)
39.08 (_+ 0.24)
37.9 a (_+ 0.80)
34.8 c (+ 0.82)
40.0 a (_+ 0.53)
34.6 ~ (± 1.1)
36.1 ~ (_+ 1.30)
38.2 b~ (_+ 0.82)
35.6 c (_ 0.93)
No virus
Control
N fertilization
N fixation
BYMV
Control
N fertilization
N fixation
Values are means (+ SD). Means not sharing a common superscript(s)
in a column are significantly different at p~<0.05 as assessed by
Duncan's multiple range test.
Effect of N fertilization, N fixation and viral infection on tannin content. F i g u r e s 3 a n d 4 s h o w the effect of N fertilization, N f i x a t i o n a n d viral
i n f e c t i o n o n t a n n i n c o n t e n t of cv. A g a b a t a n d Silaim, respectively. F o r b o t h
c u l t i v a r s a n d in all t r e a t m e n t s , viral i n f e c t i o n significantly i n c r e a s e d t a n n i n
c o n t e n t ( m g / 1 0 0 ml). B o t h N f e r t i l i z a t i o n a n d f i x a t i o n w e r e f o u n d to c a u s e a n
i n c r e a s e in t a n n i n c o n t e n t of cv. A g a b a t (Fig. 3) a n d cv. S i l a i m (Fig. 4).
Effect of N fertilization, N fixation and viral infection on in vitro protein
digestibility. T a b l e 2 s h o w s the effect of N fertilization, N f i x a t i o n a n d viral
261
80
Control
70
Viral infect/on ~ )
0
0
hi F e r t i l i z a Non 07)
60
-o
c~
50"
.2
}-
~/-/77-/-/~
N Fixation
(NF)
[--1
NF
+
V
40"
33
Fig. 3. Effect of N fixation and fertilization and viral infection on tannin content of, faba
bean, cv. Agbat.
80
-.
Control
70
~
V;rol / n f e c t/on (V)
~
iX~ F e r l ih~a-t [on (hi)
~
,v+v
~
N FzXation
r - - t
~ + v
0
0
'-'
60
u
50
•2
40
.
30
Fig. 4. Effect of N fixation and fertilization and viral infection on tannin content of, faba
bean, cv. Silaim.
infection on I V P D of cv. Agabat and cv. Silaim. Viral infection was found to
cause a reduction in I V P D for cv. Agabat while for cv. Silaim, I V P D was not
affected. Nitrogen fertilization with viral infection significantly increased I V P D
for cv. Agabat while for cv. Silaim it was significantly decreased. For both
cultivars N fixation with viral infection significantly decreased IVPD. Nitrogen
fertilization alone slightly decreased (p ~<0.05) I V P D for both cultivars, while
N fixation slightly increased (p~<0.05) I V P D for cv. Agabat but slightly
decreased it for cv. Silaim.
262
Table 2. Effect of N fixation, N fertilization and viral infection on in
vitro protein digestibility (IVPD) (%) of faba bean cv, Agabat and
Silaim
Treatments
cv. Agabat
cv. Silaim
62.0 a (_+ 3.30)
61.0 b ( ± 1.25)
63.0 ~ ( ± 2.05)
61,0" (+_ 2,50)
60.0 b ( ± 1.60)
60.0 b ( ± 2.05)
60.0 a ( ± 2.40)
67.0 ~ ( ± 2.16)
58.0 f (_+ 1.63)
60.7 b ( + 3.10)
58.0 ~ ( ± 0.82)
58.0 ~ ( ± 1.60)
No virus
Control
N fertilization
N fixation
BYMV
Control
N fertilization
N fixation
Values are means (_+ SD). Means not sharing a common superscript(s)
in a column are significantly different at p~<0.05 as assessed by
Duncan's multiple range test.
Discussion
For both cultivars, results showed that viral infection (BYMV) reduced yield
(kg/ha) and protein content. This could be due to the fact that viral infection
affected plant growth and development in critical stages, especially the flowering stage, and consequently affected seed production and composition. When
plants were fertilized with N, viral infection was found to have no effect on
protein content but significantly affected yield. The explanation for this is not
clear, but may lie in physiological processes (as well as the period between N
application and viral infection). When they were inoculated with Rhizobium
(N fixation), viral infection was found to have an adverse effect on yield and
protein content. This is because viral infection reduced the ability of Rhizobium
to fix N [9], which was reflected on plant growth and development and
consequently on seed production and composition. Nitrogen fertilization alone
or N fixation significantly improved both yield and protein content, because
plants make use of N added or fixed for growth and development and for seed
production and composition. This is in agreement with results obtained by
Mahdi [10].
For both cultivars, results showed that tannin content was increased for all
treatments. The explanation for this could be that viral infection caused an
impairment in plant growth and development; accordingly, seeds produced
were small, wrinkled and dark in color. As a result of these factors, tannins
were increased and this was found to have an adverse effect on proteolytic
enzymes such as pepsin and trypsin, and consequently in vitro protein
digestibility decreased for all treatments. However, N application and viral
infection were found to increase tannin content and IVPD for cv. Agabat
(Table 2 and Fig. 3). This observation is a departure from an other-wise good
correlation between tannin content and IVPD. The explanation for this
263
deviation is n o t clear, b u t m a y lie in chemical (as well as quantitative)
differences between the t a n n i n s of the two cultivars.
T h e adverse effect of N fertilization o n I V P D was p r o b a b l y n o t due to N as
such b u t due to a n increase in the t a n n i n c o n t e n t of the N-treated cuttivars.
The overall results indicate that N fertilization increased yield, p r o t e i n a n d
t a n n i n content, a n d decreased I V P D . N i t r o g e n fixation yielded results similar
to those o b t a i n e d by N fertilization. Viral infection had a n adverse effect o n
yield, p r o t e i n c o n t e n t a n d I V P D b u t had n o effect o n t a n n i n content.
Acknowledgement
A u t h o r s are grateful to Agricultural Research C o r p o r a t i o n , Shambat, Sudan,
N i t T A L Project, USA, D e p a r t m e n t of C r o p P r o t e c t i o n University of K h a r toum, S u d a n for p r o v i d i n g faba b e a n cv. A g a b a t a n d Silaim, Rhizobium
Ieguminosarum b i o v a r viceae strain T A L 1397, a n d pure freeze-dried isolates of
b e a n yellow mosaic virus, respectively.
References
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