Faba bean, Vicia faba L. (Fabaceae), is considered as one of the most important
food legumes in Egypt. The faba bean is a major staple food crop in Africa.
Farmers plant around four million hectares of beans which correspond to 20%
of total crop area planted (Bond, 1987). Broad beans
can get better soil productiveness and decline the incident of infections, under
certain environmental conditions (Mwanamwenge et al.,
1998). A number of viruses may cause severe diseases in faba bean crops.
There are 15 different viruses in the genus Comovirus of the family Comoviridae,
such as BBTMV. Vicia faba L. one of the most important host plant to
BBTMV, including other legume plants like peas, (Quantz,
1953). Symptoms of BBTMV infection can be severe in cool includes mild mosaic,
malformation and necrosis (Paul and Quantz, 1959).
Salicylic Acid (SA) is a natural signaling molecule involved in plant defense
response against pathogen infection. Its function in plant disease resistance
is well recognized for plants, where it is required for basal resistance against
pathogens as well as for the inducible defense mechanism, Systemic Acquired
Resistance (SAR) which confers resistance against a broad-spectrum of pathogens.
(Chaturvedi and Shah, 2007). Our study appraised the
special effects of exogenous treatment SA on the disease severity, BBTMV infection
percentage, protein patterns in cultivars, protein bands numerals, molecular
weight for each band and percentage of bands in susceptible and tolerance broad
MATERIAL AND METHODS
The DAS-ELISA technique was applied as described by Clark
and Adams (1977) to measure BBTMV concentrations in the infected and (SA+V)-treated
leaves using three antibodies BBTMV, BBSMV and BYMV were obtained from International
center for Agricultural Research in the Dry Areas (ICARDA), Plant virology,
P.O. Box 5466, Aleppo, Syria.
Four cultivars of faba bean, cv. Tribe White, Giza 3, Giza 717 and Sakha l)
were planting in natural and favorable conditions suitable for broad bean growth.
BBTMV used in these experiments was prepared from fresh severely infected leaves
of faba bean cv. Giza 3. After 21 days of growth, plants with similar size were
selected and divided into five groups. Each group consists of four replicates
(a replicate is one pot containing three healthy plants). The namely of the
groups was as follows, group 1: Control (sprayed with water), group 2: Inoculated
with virus at the same time with the other groups, group 3: 50 μM (SA and
BBTMV), group 4: 100 μM (SA and BBTMV), sprayed by SA and inoculated with
virus 3 days later (SA and BBTMV) and group 5: 100 μM SA, sprayed without
followed virus inoculation.
Every part of plant leaves were sprayed by SA treated. All leaves were mechanically
inoculated by BBTMV inoculation after three days from SA spraying (Radwan
et al., 2008). Disease severity and percentage of infection were
calculated after 21 days from seedling inoculation and were recorded according
to the subsequent balance (0) No external symptoms, (1) Light mottling and crinkling,
(2) Mottling and crinkling, (3) Strict mosaic, size diminution and (4) Deformation.
Disease Severity (DS) values were calculated using the following formula according
to Yang et al. (1996):
The youngest developed leaves from both control and treated plants from four
cultivars broad bean were collected after three weeks from inoculation for analysis
of changes, 50 mg dry tissues of each cultivar from four broad bean leave cultivars
infected and uninfected by BBTMV were treated with different salicylic acid
concentrations and compared with control. the leaves cultivars were crocheted
and extracted with SDS reducing buffer to determine the total soluble protein
SDS-PAGE procedure was portrayed by Laemmli (1970).
Gels were photographed and scanned by Gel Doc Bio-Rad System (Gel-Pro analyzer
V3) t determine the percentage of similarity of four broad bean leaves cultivars
of each treatment group and electrophoretic profile of total soluble protein
extracted from treated and untreated four broad bean leaved cultivars.
ANOVA type one-way were used to calculated the noteworthy diversity in the
averages of the experimental treatments. A probability at level of 0.05 or less
was measured considerable.
RESULTS AND DISUSSION
The leaf cultivars of broad bean (Tribe White, Giza 3, Giza 717 and Sakha l)
were exhibited systemic rigorous symptom under BBTMV inoculation compared with
non-inoculated. Novel symptoms appeared including mosaic and crinkling, size
diminution and malformation in susceptible cultivars cv. Giza 3 and Tribe White
(Fig. 1), other than, mild mosaic, size reduction and light
mottling in tolerant cultivars CV. Giza 717 and Sakha l (Fig.
2). On the other hand, SA treatments diminished the exterior of damaging
symptoms reasoned by virus progress, principally when the leaves were treated
with 100 μM SA under BBTMV infection while 100 μM (SA+V) treated leaves
had no celebrate symptoms. On the other hand, mild BBTMV symptoms were observed
by 50 μM (SA+V) application (Radwan et al.,
2007;Radwan et al., 2008). This type of the
systemic resistance might be initiated by SA is spraying in broad bean plants
against BYMV Elbadry et al. (2006).
The products of several disobedience genes were triggered appearance and connected
with plant tolerance, possibly will playing significant responsibility in the
constraint of pathogen development and dispersal. Typical characteristic indicator
genes of Systemic Acquired Resistance (SAR) are for instance encoding pathogenesis-related
protein PR1 (Van Loon and Van Kammen, 1970; Fraser,
1982; Van Loon et al., 1994; Van
Loon and Van Strien, 1999).
Results showing in Fig. 3a-c, revealed that BBTMV infection
inoculated plants have higher percentage of infection, severity of disease and
concentration of virus while lower in case of SA treated plants and control.
This regular lessen of infection percentage, severity of disease and concentration
of virus with an increase level of SA play role important for resistance against
BBTMV infection in all cultivars of faba bean compared with untreated one. Radwan
et al. (2008) also recorded that SA treatments reduced the concentration
of BYMV, infection percentage and severity of disease.
The results tabulated in (Table1and 2)
and illustrated in (Fig. 4) showed that the susceptible cultivars
cv. Giza 3 and Tribe White were recorded a low number of protein bands in all
treated groups (6, 4; 6, 6; 5, 6; 7, 10 and 6, 8 PBs), respectively, when inoculated
by BBTMV under SA treatments compared with control.
||Stimulation of susceptible faba bean leaf
cultivars cv. Giza 3 and Tribe White to BBTMV infection showing mosaic and
crinkling, size diminution and malformation compared with control (a) Susceptible
cultivars cv. Giza 3 and (b) Tribe white
Their Molecular Weights (MW) were arranged between 84 to 28 KDa in cv. Giza
3 and arranged between 162 to 29 KDa in cv. Tribe white. While, The results
tabulated in (Table 3 and 4) and illustrated
in (Fig. 5) showed that the tolerance cultivars cv. Giza 717
and Sakha l were recorded high number of protein bands (7, 11; 8, 12; 9, 12;
10, 14 and 8, 11 PBs) respectively, in all treated groups when inoculated by
BBTMV under SA treatments compared with control and (MW) were arranged between
265 to 28 KDa in cv. Giza 717 and arranged between 157 to 33 KDa in cv. Sakha1
and these results are in agreement with (Radwan et al.,
Stimulation of infected broad bean under SA application on percentage of protein
band amount (% of Amo.) in difference faba bean leave cultivars were presented
in (Table 1-4) and illustrated in (Fig.
4 and 5). In susceptible Giza 3 and Trible white cultivars
(% of Amo.) arranged between 1.758-18.257 and 3.245-16.349% in treated group1;
6.032 to 17.825 and 1.996-14.289% in treated group 2; 6.658-15.024 and 1.987-17.289%
in treated group 3; 7.008-21.929 and 1.720 to 21.389% in treated group 4 plus
6.203-18.937 and 1.542-19.242% in treated group 5, respectively while, in tolerance
Sakha1 and Giza 717 cultivars (% of Amo.) arranged between 2.417-21.133 and
5.853-16.239%; 3.634-23.695 and 5.397-21.341%; 3.663-26.038 and 5.702-23.750%;
3.484-35.996 and 5.990 to 35.451% also 3.581-30.193 and 3.512-32.747%, respectively.
Therefore, the percentage of protein amount bands in infected lanes were less
than that of the control in cv. Tribe white and Giza 3 but, more than in cv.
Sakha 1 and Giza 717.
||Stimulation of tolerance cultivars cv. Giza
717 and Sakha l to BYMV infection showing mild mosaic, size reduction and
light mottling compared with control (a) Tolerance cultivars cv. Giza 717
and (b) cv. Sakha 1
On the other hand, it can be distinguishable that sole bands include a high
percentage of the protein band amount were presented in infected cultivars under
all treatments by salicylic acid and absented in control healthy such as band
number 9 (MW. 52 and RF. 0.699) of cv. Giza 3 absent in treated group 1(control
healthy) and was recorded 12.321, 13.538, 15.822 and 13.367% in treated groups
(2-5), correspondingly. Alternatively, band number 7 (MW. 55 and RF. 0.659)
present in healthy control group 1 was recorded 12.862% and absent in treated
The bands number 16 (MW. 50 and RF. 0.737) and 19 (MW. 42 and RF. 0.803) of
cv. Tribe white were absent in treated group1 and were recorded (14.591, 13.137,
17.439 and 13.367%) and (13.755, 17.289, 21.389 and 19. 242%) in treated group
(2-5), in that order. Conversely, bands number 17 (MW. 47 and RF. 0.757) and
20 (MW. 41 and RF. 0.817) present in healthy control group 1 were recorded 19.296%
and 16.349, respectively and absent in treated group (2-5).
In addition to, the bands number 24 (MW. 115 and RF. 0.366), 39 (MW. 54 and
RF. 0.685) and 44 (MW. 33 and RF. 0.953 of cv. Sakha1 were absent in treating
group1 and were recorded (17.987, 26.038, 26.049 and 24.095%), (23.695, 24.461,
26.921 and 25.947%) and (16.779, 19.631, 29.321 and 19. 956%) in treated group
(2-5), correspondingly. On the other hand, bands number 27 (MW. 102 and RF.
0.413) and 32 (MW. 76 and RF. 0.525) present in healthy control group 1 were
recorded 16.071% and 21.133, respectively and absent in treated group (2-5).
Finally, the bands number 10 (MW. 83 and RF. 0.548), 14 (MW. 65 and RF. 0.651)
and 15 (MW. 58 and RF. 0. 698 of cv. Giza 717 were absent in treated group1
and were recorded (21.341, 21.680, 28.538 and 27.998%), (13.234, 19.896, 33.047
and 21.063%) and (17.974, 17.721, 30.570 and 26.080%) in treated group (2-5),
respectively. Then again, bands number 9 (MW. 85 and RF. 0.536) and 20 (MW.
49 and RF. 0.772) present in healthy control group 1 were recorded 14.265% and
15.065, respectively and absent in treated group (2-5).
Orientation of lofty quantity of proteins was attended to cooperate a significant
position in resistance reply in opposition to viruses. Van
Loon (1985) confirmed that in more than a few plants groups infected by
many viruses the expansion of symptoms is go together with manifestation of
novel proteins, whose incidence from cooperation between specific pathogen and
|| Effect of salicylic acid treatments on protein bands in infected
faba bean Giza 3 cultivar after 21 days from inoculation by BBTMV
||Effect of salicylic acid treatments on protein
bands in infected faba bean Tribe white cultivar after 21 days from inoculation
||Effect of salicylic acid treatments on protein bands in infected
faba bean Sakha1 cultivar after 21 days from inoculation by BBTMV
||Effect of salicylic acid treatments on protein bands in infected
faba bean Giza717 cultivar after 21 days from inoculation by BBTMV
||Virus concentration, percentage of infection
and diseases severity between faba bean cultivars, BBTMV and SA treatments
calculated of (a) Virus concentration between faba bean cultivars, BBTMV
and SA treatments, (b) Diseases severity between faba bean cultivars, BBTMV
and SA treatments and (c) Percentage of infection between faba bean cultivars
BBTMV and SA treatments
||Electrophoretic profile of total soluble
protein extracted from treated and untreated four susceptible cultivars
cv. Giza 3 and Tribe White under five treatment groups. (M) Protein marker
||Electrophoretic profile of total soluble
protein extracted from treated and untreated four tolerance cultivars cv.
Giza 717 and Sakha l under five treatment groups. (M) Protein marker
Bean plants infected with BYMV were recorded higher percentage of protein contents
compared with healthy plants. As a general rule of SA application were observed
an increase in protein amounts (Radwan et al., 2010).
PVY infection was recorded higher whole comfortable of PR proteins compared
with control (Sapotsky et al., 2005).
Katoch (2007) at the PR proteins participate a significant
responsibility in plant tolerance aligned with pathogens.
The bands presented in 100 μM (SA+BBTMV) and 100 μM SA treatments
were greatly accumulated in treatments of tolerance cultivars. The protein subunits
were less or more build up depending on SA application.
Due to BBTMV infection, level of 50 and 100 μM (SA+BBTMV) and 100 μM
(SA) the protein outline was confirmed novel bands of weights compared to the
control in all broad bean leave cultivars.
In the main, the concentration of protein bands of infected lanes was less
than that of the control in susceptible cultivars but, more than resistance.
SA treatments have a very important role in the accumulation of many PR-proteins
responsible for induction of resistance and the presence of a line of defense
in plants against virus infection. Loake and Grant (2007)
and Radwan et al. (2010) mentioned that the high
increase from several novel protein subunit were detected. Infected \been leaves
by BYMV observed novel manufactured polypeptide called PRPs in rejoinder to
SA application under BYMV infection. In infected tobacco leaves by TMV PRPs
were accumulated under SA treatments (White, 1979). Newly,
pea leaves samples were collected after treatment with 5 mM salicylic acid (SA)
resulted in the appearance of a new protein band of 96.7 kDa 48 h after treatment
In conclusion, the outcome of the their exploration proposed that the protective
action of SA treatments may be associated with a reduction in concentration
of virus, infection percentage and severity of BBTMV disease with virus infection.
On the other hand, Overall, the BBTMV infection under SA applications caused
accumulation of protein contents and founded novel bands of polypeptides in
response to infection of BBTMV under SA application in Vicia faba L.