Controlling of Xanthomonas axonopodis pv. phaseoli by induction of phenolic compounds in bean plants using salicylic and benzoic acids

Abstract

This study deals with the potentiality of salicylic (SA) and benzoic (BA) acids for controlling the common blight of beans (CBB) caused by Xanthomonas axonopodis pv. phaseoli (Xap). Impacts of the application of SA and BA (1.2 µg mL−1) on the plant biological parameters, bacterial count, disease severity, phenolic and salicylic acid contents as well as catalase activity in treated plants were investigated. In vitro, application of the both compounds at different concentrations (0.4, 0.8 and 1.2 µg mL−1) significantly suppressed growth of the pathogen. Under greenhouse conditions, application of BA and SA considerably reduced the disease development by 81 and 71%, respectively after 4 days of the application as compared to infected control. After 12 days of BA application, plants were protected 49.2% from disease as compared with SA (44.6%). SA-treated plants showed significant increases in the SA content and total phenolic content. Also, BA-treated plants showed an increment in the total phenolic content. Bean plants treated with SA showed higher catalase activity than those treated with BA. In conclusion, this study supports the use of SA and BA as abiotic elicitors to protect bean plants from the common blight disease. This protection may be attributed to the resistance induction, activation of defense enzymes as well as augmentation the phenolic content and salicylic acid in the host cells.

Full text

Vol.:(0123456789)
1 3
Journal of Plant Pathology
https://doi.org/10.1007/s42161-022-01102-5
ORIGINAL ARTICLE
Controlling ofXanthomonas axonopodis pv. phaseoli byinduction
ofphenolic compounds inbean plants using salicylic andbenzoic
acids
KamalA.M.Abo‑Elyousr1,2 · MuhammadImran1· NajeebM.Almasoudi1· EsmatF.Ali3· SabryHassan3·
NashwaMA.Sallam2· KhamisYoussef4,5 · IsmailR.Abdel‑Rahim6· HadeelM.M.KhalilBagy2
Received: 21 September 2021 / Accepted: 12 March 2022
© The Author(s) under exclusive licence to Società Italiana di Patologia Vegetale (S.I.Pa.V.) 2022
Abstract
This study deals with the potentiality of salicylic (SA) and benzoic (BA) acids for controlling the common blight of beans
(CBB) caused by Xanthomonas axonopodis pv. phaseoli (Xap). Impacts of the application of SA and BA (1.2 µg mL−1) on
the plant biological parameters, bacterial count, disease severity, phenolic and salicylic acid contents as well as catalase
activity in treated plants were investigated. Invitro, application of the both compounds at different concentrations (0.4, 0.8
and 1.2 µg mL−1) significantly suppressed growth of the pathogen. Under greenhouse conditions, application of BA and SA
considerably reduced the disease development by 81 and 71%, respectively after 4days of the application as compared to
infected control. After 12days of BA application, plants were protected 49.2% from disease as compared with SA (44.6%).
SA-treated plants showed significant increases in the SA content and total phenolic content. Also, BA-treated plants showed
an increment in the total phenolic content. Bean plants treated with SA showed higher catalase activity than those treated
with BA. In conclusion, this study supports the use of SA and BA as abiotic elicitors to protect bean plants from the com-
mon blight disease. This protection may be attributed to the resistance induction, activation of defense enzymes as well as
augmentation the phenolic content and salicylic acid in the host cells.
Keywords Bean plant· Benzoic acid· Common blight· Induce resistance· Salicylic acid
Introduction
Bean plants are attacked by several fungal and bacterial dis-
eases, but the most destructive seed-borne disease in com-
mon beans is the bacterial blight, caused by Xanthomonas
axonopodis pv. phaseoli (Xap), leading to sever economic
losses worldwide (Sallam 2011). In warmer zones, common
blight caused by Xap is the most serious, extensively dis-
tributed and one of the five major bacterial diseases of bean
causing significant yield losses (Silué etal. 2010). Basically,
common bacterial blight can be controlled by chemicals, soil
management, biological control, use of resistance cultivars, and
Kamal A. M. Abo-Elyousr, Muhammad Imran, Khamis Youssef,
and Ismail R. Abdel-Rahim contributed equally to this work.
* Khamis Youssef
youssefeladawy@yahoo.com
Kamal A. M. Abo-Elyousr
kaaboelyousr@agr.au.edu.eg
Najeeb M. Almasoudi
nalmasoudi@kau.edu.sa
Ismail R. Abdel-Rahim
nashwasallam@aun.edu.eg
Hadeel M.M. Khalil Bagy
hadel_magdy@aun.edu.eg
1 Department ofArid Land Agriculture, King Abdulaziz
University, Jeddah80208, SaudiArabia
2 Faculty ofAgriculture, Department ofPlant Pathology,
University ofAssiut, Assiut71526, Egypt
3 Department ofBiology, College ofScience, Taif University,
P.O. Box11099, Taif21944, SaudiArabia
4 Agricultural Research Center, Plant Pathology Research
Institute, 9 Gamaa St., Giza12619, Egypt
5 Agricultural andFood Research Council, Academy
ofScientific Research andTechnology (ASRT), 101 Kasr Al
Aini St., Cairo, Egypt
6 Botany andMicrobiology Department, Faculty ofScience,
Assiut University, Assiut71516, Egypt

Journal of Plant Pathology
1 3
integrated disease management (IDM) but a complete eradica-
tion is not possible (Sallam 2011). Some cultural practices such
as elimination of weeds and removal of other possible hosts of
Xap reduced the disease incidence (Abo-Elyousr 2006). Foliage
infections have been controlled by application of the chemicals
potassium methyl dithiocarbamate, copper hydroxide and cop-
per sulphate. Copper-based bactericides may reduce the bacte-
rial population (Selamawit 2004). In addition, seed treatments
with strains of Rahnella aquatilis and Pseudomonas sp. sup-
pressed the Xap incidence (Sallam 2011). IDM is considered
one of the best favored approaches to control the disease in sev-
eral cropping systems; whereas varietal mixture and intercrop-
ping integration exhibited an effective control of the common
bacterial blight (Fikire 2004; Youssef etal. 2010).
Apparition of necrotic tissues on bean plants suggested
that Xap shifted from biotrophic to necrotrophic from 8 to
15days post inoculation (Foucher etal. 2020). The defense
response to biotrophic and hemibiotrophic pathogens is usu-
ally regulated by SA (Bürger etal. 2019). Herein, a global
induction of the SA pathway was linked to resistance, sug-
gesting that an adapted SA response is effective in BAT93
(resistant genotypes to CBB) after the infection by X. pha-
seoli pv. phaseoli. SA is involved in common bean resistance
to CBB (Foucher etal. 2020).
Several alternative control means have been proposed to
control plant diseases to reduce/replace the use of pesticides
(Hussien etal. 2018). Among these alternatives, salicylic acid,
also known as 2-hydroxybenzoic acid, is an inducer of sys-
temic acquired resistance (SAR) to plant diseases (Mur etal.
1996). The role of SA has been well approved in activation of
the defense responses against abiotic and biotic stresses (Ali
etal. 2018).Naturally, the benzoic acid (BA) occurs free as
well as bounded in several species of plants and animals is.
Indeed, benzoic acid restrains the fungal and bacterial growth
(Shahda 2000), it is used as a food preservative and a cure of
some fungal skin diseases (Shabana etal. 2008). Application
of BA, either as a seed treatment or in soil drenching, has been
found efficient with the range of 0.05–0.5mM for inducing
the tolerance to heat, drought, and chilling. BA is a general
segment in derivatives of SA to induce stress tolerance. More-
over, it is more effective even at lower concentrations than
salicylic acid and its derivatives as well (Tissa etal. 2003).
We hypothesized that BA and SA have the ability to con-
trol bean common blight directly by pathogen’s suppression
and indirectly by induction of the phenolic compounds and
pathogen defense-relatedenzymes that enhance the plant
resistance. Therefore, this research aimed to: (i) study the
effect of BA and SA on reduction of the pathogen growth
and disease severity of common blight of beans and (ii)
investigate the induction of phenolic compounds and their
possible role in the systemic acquired resistance of plants
against the pathogen.
Materials andmethods
Bacterial inoculum preparation andmethods
ofinoculation
For inoculum preparation, virulent bacterial isolate of Xan-
thomonas axonopodis pv. phaseoli (Xap3) was obtained
from the Department of Plant Pathology, Faculty of Agricul-
ture, Assiut University Egypt. Bacterial isolate was grown
in 25mL sterile tubes containing nutrient yeast extract broth
medium and at 27ºC, under shaking (200rpm) for 24h.
Subsequently, bacteria were pelleted by centrifugation for
5min at 15,000rpm and washed in saline solution. Con-
centration of the bacterial cells was adjusted to 108 colony
forming units (CFU) by dilution method. The bacterial sus-
pension (0.1mL) was injected in the middle vein of leaves
of 2-week-old plants (Klementet al. 1990). While, bacterial
free saline solution was used as a control treatment. Polyeth-
ylene bags were used to cover the inoculated plants for two
days at 25–27 ºC under greenhouse conditions. Then, the
plants were examined daily for development of the disease
symptoms (Abo-Elyousr 2006).
Plant materials
Plants of “Red Kidney” bean (Phaseolus vulgaris) variety
were used in this experiment. Under greenhouse, three plants
were grown in 20cm pots containing soil mixture fertilized
with 30mL NPK formulation (12:4:6) and incubated at
25 ± 5 ºC with lux light range 5000–14,000 and relative
humidity 68–80%. Plants were irrigated as per requirements.
In vitro assay ofthe antibacterial impact ofSA
andBA
SA and BA, used in this study, were of fine analytical grade
and purchased from Oxford Lab chem. Mumbai, India.
Amount of acids was dissolved in 1mL dimethyl sulfoxide
(DMSO) to prepare stock solution. The final concentrations
were adjusted to 0.4, 0.8, and 1.2µg mL−1.
Using the impregnated filter paper disc method, toxicity of
the both chemicals against Xap3 was tested. 0.1mL bacterial
suspension of Xap3 (1 × 108 CFUmL−1) of one day- old cultures
was poured in four Petri plates (9cm in diameter). Then, the
melted medium was added. After solidification of the medium,
sterilized Whatmann standard filter paper discs (9mm diameter,
1mm thick) saturated with 50 µL of each adjusted concentration
from SA or BA were placed in the middle of the seeded agar
surface. 200ppm of streptomycin and sterile water were used
as positive and negative control treatments, respectively. Four
replications were used for each treatment and they were repeated
twice. Inoculated plates were incubated at 27°C for 48h and the

Journal of Plant Pathology
1 3
inhibition zone diameter was measured around the compounds
(Abo-Elyousr and El-Hendawy 2008).
Effect ofSA andBA onbacterial count anddisease
severity
We tried to explore the suppressive effect of BA and SA on
multiplication of the pathogen inside the infected leaves.
Pathogen-inoculated plants were treated with BA and SA
(1.2 µgmL−1) at 2, 4, 6, 8, and 10days interval. Asepti-
cally, disc of 5mm diameter was removed from inoculated
region of leaves and homogenized in 1mL sterile solution of
0.06M NaCl. Solution was serially diluted and 0.1mL ali-
quots of the dilutions were placed on King’s B agar medium
plates. Plates were incubated for 48h at 26 ºC and emerging
colonies showing bacterial growth on all plates were counted
(Abo-Elyousr and El-Hendawy 2008). Two leaf discs were
used for each dilution. Four replicates were used and the
whole experiment was repeated twice.
To prepare sprinkles, SA (1.2 µgmL−1) was dissolved
in distilled water, while BA (1.2 µgmL−1) was dissolved in
1mL DMSO that was latterly diluted with water. Aqueous
solution of compounds (30mL per plant) was sprayed on
each plant. As a control treatment, plants injected with water
and then with pathogen were inoculated after two days of
treatment as mentioned previously.
Plants were categorized into four different groups based on
the treatments as represented in Table1 and each group con-
tained equal number of plants. In first group, plants were ini-
tially treated with SA, followed by the pathogen inoculation after
2days. In the second group, plants were earlier treated with BA
and after 2days the bacterial pathogen was inoculated. In the
third group, infected control plants were prepared. For infected
control, plants were firstly treated with water and after two days
the bacterial pathogen was inoculated. The fourth group rep-
resented the healthy control plants, whereas the plants were
treated only with water (Table1). Plants were irrigated properly
till 20days. To determine the disease severity, disease symp-
toms on the plants were reported after 20days of inoculation
based on the index described by Louws etal. (2001). Five grad-
ing disease index scale was used to score the plant as follow:
1-leaves with no symptom, 2-leaves with few necrotic spots (less
than10% affected leaf), 3-leaves having many necrotic spots
(10–20% affected leaf), 4- leaves having 20–50% affected leaf,
and 5-leaves with collapsed leaf. Experiment was conducted
twice using four replicates. Each pot containing three plants and
four pots for each replicate were used.
Methanolic extract preparation
The tip of treated plant leaves was immersed in liquid nitrogen
and then one gram of the plant materials was homogenized in
10mL 80% methanol for 24h. Samples were centrifuged at 4°C
and 15,000rpm for 30min. Thereafter, the pellet was discarded
and homogenate was vaporized three times in rotary evaporator.
The semi-solid residues were dissolved in 5mL distilled water.
Determination ofSA contents
For determination of SA contents, 500 µL of homogenate sam-
ples were mixed with 1000 µL methanol and 250 µL muriatic
acid (10N). Samples were incubated in water bath at 80°C for
2h. Then, 4–5 drops of 1M NaHCO3 were added to neutral-
ize along with 1000 µL of methanol. The SA contents were
assayed spectrophotometrically by measuring the OD at 254nm
(Baysal 2001). SA contents were represented as amount of total
salicylic acid = µg salicylic acid g−1 plant material.
Determination ofthephenol content
The total phenol contents were measured as described by
McDonald (2001). Reaction mixture containing 2mL of
the plant extract, 0.5mL of Folin Ciocaulteau’s reagent and
1.0mL of distilled water was vortexed for three min and
then 2mL of 20% sodium carbonate solution was added. The
contents were mixed rigorously, placed in boiling water bath
for 1min and then cooled. The absorbance was measured
at 650nm in a spectrophotometer against the reagent blank.
Gallic acid was used to construct a calibration curve. Total
phenol contents of the extract were represented as mg Gallic
acid g−1 fresh weight. The experiment was performed twice
with four replicates.
Catalase activity
For preparation of the crude enzymes, 1g of fresh leaves treated
with liquid nitrogen, homogenized with 10mL of 0.1M sodium-
acetate buffer and the pH was adjusted 5.2. The mixture was
centrifuged at 1000rpm for 30min under 4°C. The supernatant
was collected to determine the enzyme activities. Protein content
Table 1 Group of plants and
treatment Group of Plants Treatments Time of inoculation
First Salicylic Acid Inoculation of bacterial suspension after 2days of treatment
Second Benzoic Acid Inoculation of bacterial suspension after 2days of treatment
Third Infected control First treated with water, after 2days bacterial suspension inoculation
Fourth Healthy Control Only treated with water

Journal of Plant Pathology
1 3
of the extract was measured as described by Bradford (1976)
using the Coomassie®- Protein assay regent.
The catalase activity was measured according to the
method described by Aebi (1984). 0. 1mL of the superna-
tant was mixed with 2.9ml of a reaction mixture comprising
20mM H2O2 and 50mM sodium phosphate buffer (pH 7.0).
On the other hand, 3mL of the Sorensen phosphate buffer
(pH 7.0) used as blank. Catalase activity was determined
at 240nm and the enzyme activity was represented as unit
mg−1 protein. One unit of enzyme activity was defined as the
decomposition of 1μmol of H2O2 per min.
Statistical analysis
A completely randomized block design was used for experi-
ments in the greenhouse. For all experiments, four replicates
were used for each treatment and the experiment was con-
ducted twice.. The significance of difference between the
mean values was calculated. Two-ways analysis of variance
(ANOVA) was used and the significance of difference among
the treatments was determined according to the least signifi-
cant difference (LSD) according to Gomez and Gomez (1984).
Results
Antibacterial activity ofBA andSA againstXap3
In vitro, the inhibition of Xap3 incident by using different
concentrations of SA and BA 0.4, 0.8 and 1.2 µgmL−1 as
well as streptomycin 200ppm were measured. As previously
described, filter paper disc method was applied and then the
inhibition zone (mm) of Xap3 was measured. The results
indicated that all used concentrations of BA have significant
inhibiting impacts against of Xap3. The maximum inhibition
rate was observed at 1.2 µgmL−1 comprising 10mm. On the
other hand, the minimum inhibition rate occurred by BA was
recorded at 0.4 µgmL−1 with 6mm. Furthermore, it was also
observed that all SA concentrations significantly suppressed the
growth of the pathogen (Fig.1). The inhibited rate of the patho-
gen at 1.2 µgmL−1 of SA was 9mm, which was less than those
investigated by BA (10mm) at same concentration. Moreover,
using of 0.4 µgmL−1 SA caused inhibition zone up to 5mm that
was lower than BA (6mm) at same concentration. Addition-
ally, using of the standard antibiotic agent streptomycin caused
inhibition zone up to 12mm. Indeed, BA at 1.2 µgmL−1 is
considered the most potent compound to suppress the pathogen
growth compring to SA (Fig.1).
Effect ofBA andSA onthebacterial multiplication
undergreenhouse conditions
The effect of BA and SA on the pathogen invasion of the
bean plant cells was observed by measuring the bacterial
count (CFU) at different day’s interval. Interestingly, there
was a significant suppressive effect on the bacterial multi-
plication inside the plant cells after treatment with BA and
SA. In details, we measured the bacterial counts inside the
plant cells after two days of BA and SA treatment and the
bacterial counts were reduced by 40 and 20%, respectively
comparing with the infected control. It is noteworthy; BA-
and SA-treated plant exhibited the maximum reduction on
the bacterial counts after 4days contributing 81 and 71.4%,
respectively. After 6days of plant treatments, there were
considerable decrement on the bacterial counts inside the
bean leaves reaching 75 and 60%, respectively.However,
the results revealed that the ability of BA and SA to induce
resistance of the bean plant cells against the pathogen
invasion was gradually declined after 6days of treatment.
Whereas, the bacterial counts associated with BA- and SA-
treated plants re-increased at 10th day of treatment producing
Fig. 1 Effect of SA and BA on
growth of Xanthomonas axo-
nopodis pv. phaseoli in vitro.
Four replicates were used for
each treatment and the whole
experiment was repeated twice.
Bars indicate the standard error.
Columns with the same letters
are not significantly different
according to Fisher’s protected
least significant difference at
p ≤ 0.05
ed
c
d
cd
b
a
0
2
4
6
8
10
12
14
0.4mg/L0.8 mg/L 1.2mg/L0.4 mg/L 0.8mg/L1.2 mg/L 200ppm
Salicylic acid Benzoicaci
dS
treptomycin
Inhibitionzone (mm)
Treatments

Journal of Plant Pathology
1 3
an inconsiderable reduction rate of the pathogen up to 34 and
20%, respectively (Table2).
Disease severity
The capability of SA and BA to reduce the disease sever-
ity under greenhouse conditions was determined (Fig.2).
Indeed, Xap3 aggressively invaded the bean plants produc-
ing destructive symptoms on the leaves after 10th and 12th
day of pathogen inoculation with disease severity up to 42
and 65%, respectively. Interestingly, the results demonstrated
that SA and BA have a significant potentiality to reduce the
disease severity (Fig.2). SA-treated plants showed high
reduction level of the disease severity both at 10th and 12th
day of pathogen inoculation, whereas the disease severities
were 32 and 36%, respectively compared to the infected con-
trol (42 and 65%, respectively). The previous results empha-
sized the potential of SA to protect the plant and reduce the
disease severities at 10th and 12th day of pathogen inocula-
tion by 23.81 and 44.62%, respectively (Fig.2).
On the other hand, the plants treated with BA showed
disease severities up to 30 and 33% at the 10th and 12th day
of the pathogen inoculation, respectively compared to the
infected control (42 and 65%, respectively). Therefore, BA
had the potential to reduce the disease severities at 10th
and 12th day of pathogen inoculation by 28.57 and 49.23%,
respectively (Fig.2). It could be observed that BA and SA
showed significant performance to induce the plant resist-
ance for controlling the common blight disease of bean
undergreenhouse.
Effect ofSA andBA treatments onsalicylic acid
content
Firstly, in the healthy plant control that has not been attacked
by the pathogen, the salicylic acid content was slightly and
gradually decreased during the experimental time. Whereas,
the salicylic acid content in the healthy plant control was
1.30, 1.20, 1.0 and 0.95 µgg−1 plant material after 2, 4, 6 and
8days interval, respectively. On the other hand, in infected
Table 2 Effect of benzoic and
salicylic acids on CFUg−1 of
Xanthomonas axonopodis pv.
phaseoli on leaves
Values followed by the same letter are not significantly different as determined by the LSD test (P ≤ 0.05)
Daysafter
treatment
Infected Control Salicylic acid Benzoic acid
Count
CFUg−1 Count
CFUg−1 Reduction
%
Count
CFUg−1 Reduction
%
20.50 × 108 f 0.4 × 108 f 20 0.30 × 108 f 40
42.10 × 108 d 0.6 × 108 f 71.4 0.40 × 108 f 81
64.00 × 108ab 1.6 × 108 de 60 1.00 × 108 de 75
85.30 × 108 a 3.2 × 108 bc 39.6 2.3 × 108 d 56.6
10 5.00 × 108 a 4.00 × 108 ab 20 3.3 × 108 bc 34
Fig. 2 Effect of salicylic acid
and benzoic acid on CBB
percentage disease severity.
Four replicates were used for
each treatment and the whole
experiment was repeated twice.
Bars indicate the standard error.
Columns with the same letters
are not significantly different
according to Fisher’s protected
least significant difference at
p ≤ 0.05
e
d
c
b
a
e
d
ccc
e
d
ccc
0
10
20
30
40
50
60
70
4681
01
2
Disease severity (%)
Days after application
Infected control
Salicylic Acid
Benzoic acid

Journal of Plant Pathology
1 3
plant control that inoculated with the bacterial pathogen, the
salicylic acid content was increased in the beginning reach-
ing out 1.9 µgg−1 plant material at 2nd day of the pathogen
inoculation, followed by slightly decrease up to 1.5, 1.3,
and 1.3 at 4th, 6th and 8th day of the pathogen inoculation,
respectively (Fig.3).
It is worth mentioning that the SA-treated plants had a
significant increase on the salicylic acid content during the
experimental interval. Maximum level of the salicylic acid
(3.33 µgg−1 plant material) was detected at 2nd day of the
treatment. Furthermore, the salicylic acid contents on the
SA- treated plants were 2.7, 2.7 and 2.6 µgg−1 plant mate-
rial after 4, 6 and 8days of treatment, respectively (Fig.3)..
However, BA-treated plant did not exhibit significant
change on the salicylic acid content till the 6th day of the
experiment, followed by slight decrease on the 8th day of
the experiment up to 1.2 µgg−1 (Fig.3). Overall, the results
revealed that the cells of SA-treated plants showed a sig-
nificant increase of the salicylic acid which may play a vital
role in development of the resistance against the pathogen
against the pathogen.
Effect ofSA andBA onthetotal phenol content
(TPC)
The results primarily figured out that the treatments
enhanced the phenol content in the plant cells except in a
control. However, there were no significant changes in the
TPC in the health plant control during the experimental
time, whereas TPC ranged 2.1–2.3mg g−1 during the time
of the experiment (Fig.4). SA-treated plants showed a per-
sistent augmentation in the level of TPC and the highest
TPC (4.3mg g−1) was recorded on day 8 of the experiment.
Furthermore, TPC of the SA-treated plants were 3.2, 3.6 and
4.1mg g−1 after 2, 4 and 6days of treatment, respectively.
On the other side, BA-treated plants showed gradually rises
on the TPC reaching 2.1, 2.5, 3.2 and 3.7mg g−1 at 2nd,
4th, and 6th day of the experiment, respectively. Moreover,
TPC of the BA-treated plants was 3.3mg g−1 on day 8 of
the experiment. It is worth noting that although SA-treated
plants exhibited increases on TPC greater than BA-treated
plants, but without statistically differences. Plants attacked
by the pathogen (as an infected control) showed increases
on TPC till the 6th day of the treatment, followed by a sig-
nificant retreat on the 8th day of the experiment. It can be
observed that exogenous application of SA significantly
increased TPC in bean leaves (Fig.4). TPC were signifi-
cantly affected between 2–6days after the treatment with
SA and BA compounds (Fig.4).
Effect ofSA andBA treatments oncatalase activity
(CAT)
The results revealed that SA-treated plants showed signifi-
cant increases in CAT during the time of experiment. The
maximum catalase activity was measured on 6th day up to
2.9 unit mg−1 protein (Fig.5). Moreover, CAT of SA-treated
plants rose into constant level at 2, 4, and 8days of the treat-
ment comprising 2.2 unit mg−1 proteins. Indeed, the plant
control had significant increases in CAT after the second
day as 2.8, 2.2 and 2.3 unit mg−1 proteins at 4, 6 and 8days
of the treatment, respectively. BA-treated plants exhibited
insignificant changes in CAT during the experiment time,
displaying 2.1, 1.5, 1.7 and 1.9 unit mg−1 protein min−1 at
2, 4, 6 and 8days of the treatment, respectively. On the
other hand, the infected control (plants inoculated by the
Fig. 3 Effect of SA and BA
on salicylic acid contents in
inoculated bean plant with Xan-
thomonas axonopodis pv. pha-
seoli. Bars indicate the standard
error. Columns with the same
letters are not significantly
different according to Fisher’s
protected least significant differ-
ence at p ≤ 0.05 cd
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bb
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3
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02468
Salicylic acid contents (µgg-1 plant material)
Days after application
Salicylic Acid
Benzoic acid
Infected control
Healthy control

Journal of Plant Pathology
1 3
pathogen) showed significant responses and CAT increased
to 2.8, 2.2 and 2.3 unit mg−1 protein min−1 at the 4th, 6th and
8day of treatment, respectively. Overall, CAT in SA-treated
plants was higher as compared to BA-treated plants as well
as infected control (Fig.5).
Discussion
The main objectives of this study were to investigate the
effect of BA and SA against common blight of beans and to
explore their possible role in resistance of plants against the
pathogen. In this study, the influence of different concentra-
tions of BA and SA, on the induction of phenolic compounds
in bean plants was investigated. Results revealed that SA
and BA were effective against common blight disease of
bean plants. Several studies have been conducted to control
this disease following the applications of SA, BA and some
copper base bactericides (El-Tohamy etal. 2018).
Due to significant role in regulating physiological mecha-
nisms of different plants, SA and BA have been reported
to be the best plant growth regulators and non-enzymatic
antioxidants (Neelam etal. 2014; Salemet al. 2016). Many
studies reported that exogenous application of SA affects
Fig. 4 Effect of SA and BA
on total phenol contents in
inoculated bean plant with Xan-
thomonas axonopodis pv. pha-
seoli. Bars indicate the standard
error. Columns with the same
letters are not significantly
different according to Fisher’s
protected least significant differ-
ence at p ≤ 0.05 bc
a
a
aa
bc
b
a
a
a
bc
b
a
a
bc
bc bc bc bc bc
0
1
2
3
4
5
02468
Total phenols (mg g-1)
Days after application
Salicylic Acid
Benzoic acid
Infected control
Healthy control
Fig. 5 Effect of SA and BA on
catalase activity in inoculated
bean plant with Xanthomonas
axonopodis pv. phaseoli. Bars
indicate the standard error.
Columns with the same letters
are not significantly different
according to Fisher’s protected
least significant difference at
p ≤ 0.05
c
bb
a
b
c
c
c
c
c
cc
bcc
cc
a
b
b
0
0.5
1
1.5
2
2.5
3
3.5
02468
Catalase activity (unit mg-1 protein)
Days after application
Salicylic Acid
Benzoic acid
Infected control
Healthy control

Journal of Plant Pathology
1 3
various physiological aspects i.e., vegetative growth of
plant and their development, senescence, root initiation,
fruit yield, stomata closure, seed germination, flowering,
respiration, glycolysis, heat production, photosynthesis, the
alternative respiratory pathway and the Krebs cycle (Miura
and Tada 2014).The exogenous application of SA entices
local and systemic acquired resistance in different species
of plant against various pathogens, including Xanthomonas
spp, Alternaria alternata, Fusarium oxysporum, Magna-
porth egrisea, Colletotrichum gloeosporides, and some
viruses (Lachhabet al. 2015; Toanet al. 2017). It has been
reported that exogenous application of SA establishes SAR
that enhance nonspecific resistance to subsequent infection
and normally associated accumulation of PR protein in SA-
treated plants (Ward etal. 1991).
Indeed, the treatment with SA and BA induced resistance
of the plant cells that significantly reduced the multiplication
of the pathogen inside their cells. In the current study, BA-
treated plants exhibited higher resistance of the pathogen
multiplication and invasion than SA -treated plants. In SA-
treated plants, highest amount of CFU was recovered while
number of CFU in BA-treated plants was low as compared
to SA-treated plants. Recovery of CFU is mainly dependent
on rhizospheric conditions (Mohamed etal. 2020; Schreiter
etal. 2014) and type of inducer used against the pathogen.
The positive physiological action of benzoic acid could be
partially explained via the well admitted salicylic acid sign-
aling pathway whether we assume that benzoic acid is con-
verted to salicylic acid in bean plants as has been described in
tobacco (Yalpani etal. 1993). In this context, it is also worthy
to point out that in tomato leaves, exogenously applied ben-
zoic and salicylic acids were rapidly hydroxylated to gentisic
acid (2,5-dihydroxybenzoic acid) (Bellés etal. 1999).
In tomato plant, exogenous application of GA elicits
the accumulation of CEV d-induced antifungal pathogen-
esis-related (PR) proteins P23, P32, and P34 (Bellés etal.
1999) while, exogenous application of SA in tobacco plants
stimulates the expression of the pathogenesis-related (PR)
protein including PR1, PR2, and PR5 (Ali etal. 2018) that
ultimately reduce the survival of pathogen and also increase
the tolerance to abiotic stresses (Wu etal. 2016). After SA
and BA treatments, population of Xap remained stable up to
4days and after that a significant decline was observed. In
the current study, the reduction of Xap population after SA
application confers the development of resistance in plants.
Former studies reported that exogenous SA induces PR gene
expression, disease resistance or both in potato (Navarre
and Mayo 2004) and in different monocots (Takatsuji 2014).
Exogenously SA application inhibits the biosynthesis of
plant hormone stomatal closure, ethylene and uptake of
root ion (Raskin 1992). Previous studies also revealed that
exogenous SA application induced identical set of mRNAs
after pathogen infection (Sarowar etal. 2005; Shahda 2000).
SA is an endogenous defense signal and establishes resist-
ance to pathogens in several plants (Silva etal. 1989).It has
also been reported that SA has a key role in mediating SAR
(Ryals etal. 1997). In our study, application of SA and BA
demonstrated the satisfactory control against common blight
of beans since it increased the resistance at the concentra-
tions 1.2µgmL−1 of both compounds against the pathogen.
Our results consents with other studies viewing that treat-
ment of plants with SA may provide a considerable protec-
tion against pathogen of bacterial blight (Raskin 1992).
In beans leaves, a slight increase in SA contents was
observed earlier but after 2days, a significant increase from
1.6 to 3.33 µgg−1 was recorded as compared to BA and con-
trol which was decreases to 2.6 µgg−1 after 8days. Similar
increase has also been reported by (Baysal 2001) when the
seedlings of apple root stock were treated with plant extract
and benzothiadiazole against fire blight. Recent researches
demonstrated that during defense response, binding protein
of SA has a significant role in signal transmission which may
lead to change in physiological and biochemical states of
plant cells that are related to the defense response activation
of plant (Dhanya etal. 2020).
Phenolic contents were significantly higher in both SA- and
BA-treated plants as compared to control, which demonstrate
that pathogen was restricted, or resistance was developed.
Higher phenol contents were recorded in SA-treated plants after
6 and 8days of application that were 4.1 and 4.3mg g−1, respec-
tively. TPC were increases from 8.5 ± 0.3 to 68.5 ± 1.2mg g−1
in Thymus vulgaris L. plants treated with SA (Khalil etal.
2018). Phenolic contents were measure in fresh weight while
an increase was reported in dry weight plants. In our studies,
we recorded continues increase in TPC starting from 2nd day of
application to 8th day that were increase from 3.2 to 4.3mg g−1.
Previously, resistance development and raise in phenolic com-
pounds in strawberries and brown mustard seedlings has been
reported (Samadi etal. 2019; Sukhmeen etal. 2018).
SA-treated plant shows higher level of CAT as compared
to BA-treated plant and control as well. Increase in CAT in
SA-treated plants has also been reported (Ashis etal. 2017)
that support our results. Overall, SA-treated plants expressed
highest CAT. Recently, presence of salts along with SA also
increased the CAT by 34% in strawberries (Samadi etal.
2019). It has also been reported that SA inhibits CAT, lead-
ing to increase the level of H2O2 (Nuramalee etal. 2018).
Previous studies reported that SA application along with
their derivative compounds increases H2O2 concentration
in plants and change the antioxidant property of the particu-
lar plants (Dat etal. 1998; Hassan and Abo-Elyousr 2013).
This elevated level of H2O2 activates the oxidative/defense
enzymes such as polyphenol oxidase, ascorbate peroxidase,
glutathione reductase, peroxidase and catalase (Rahamah-
Bivi etal. 2014). Consequently, phenolic compounds have
capability to change the anti-oxidation properties of the host

Journal of Plant Pathology
1 3
(Hammerschmidt 2005). Therefore, CAT remains constant
in some treatments. SA protects beans from common blight
and suggests that SA induces the resistance by enhancing
chemical defenses in beans leaves. Hence, disease suppres-
sion increased due to the production of defense enzymes
that may increase phenolic contents and consequently, these
naturally occurring phenolic compounds have a significant
role for the activation of defense mechanism that protect the
beans plant.
Conclusion
We hypothesized that BA and SA may control the common
blight of bean by direct inhibition of Xanthomonas axonopodis
pv. phaseoli and indirectly by induction of the phenolic com-
pounds and pathogen defense-relatedenzymes that enhance
the plant resistance. This hypothesis was validated by our
results showing the significant potentiality of BA and SA to
reduce the disease incidence associated with pathogen suppres-
sion as well as enhancing of the catalase activity and elevation
of the phenolic content of the host cells. The obtained results
revealed that SA although being as a growth promoter, can
be used below an upper limit concentration beyond which it
effects become undesired. Further, SA could protect the beans
plant from common blight and reduce the incidence of dis-
ease. These findings suggested that SA and BA at 1.2µg mL−1
are promising compounds to control common blight of beans
since both compounds can induce natural host resistance by
enhancing the defense mechanism and also in term of defense
possibilities, plants may evolve signal molecules that are com-
plimentary to SA. Further experiments should be conducted
in the field scales on these compounds so that we can fully
recommend the use it and we are in the process of conducting
these experiments in the field.
Acknowledgements Authors are thankful to Taif University Research-
ers Supporting Project number (TURSP-2020/142), Taif University,
Taif, Saudi Arabia for providing the financial support and research
facilities.
Author contribution Kamal Abo-Elyousr, Mohamed S. Imaran, and
Esmat F. Ali suggested the idea of the work and contributed to data
curation and their validation as well as writing original draft. Youssef
Khamis, Najeeb Almasoudi, Nashwa Sallam, Ismail Abd-elrahim
and Hadeel M. M. Khalil Bagy contributed to the formal analysis
of the data all authors contributed to the reviewing and editing the
manuscript. All authors reviewed and approved the final version of
the manuscript.
Declarations
Ethics approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent All authors have reviewed the manuscript and
approved the final version of manuscript before submission.
Conflict of interest The authors declare that they do not have anyactual
or potential conflict of interest.
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