Chemical –Induced Resistance against Brown Stem Rot in Soybean:

Journal of Applied Sciences Research, 4(12): 2046-2064, 2008
© 2008, INSInet Publication
Chemical –Induced Resistance against Brown Stem Rot in Soybean:
The Effect of Benzothiadiazole
1
E Nafie and M. 2M Mazen
1
Department of Botany, Faculty of Girls for Art,
Science and Education, Ain Shams University, Cairo, Egypt.
2
Plant Pathol. Res. Inst., Agric. Res. Center, Giza, Egypt.
Abstract: In the present work, the biochemical basis of tolerance in soybean to stem rot via its priming
with benzothiadiazole (BTH) was investigated. To evaluate the potentiation of BTH in this respect,
differences in the elements associated with the induction of defenses were traced before and after
subjecting soybean to biotic stress induced by its inoculation with Phialophora gregata. BTH priming of
non-inoculated soybeans was observed to increase percentage of seed germination, fresh and dry weights
of shoots and roots and photosynthetic pigments. Marked differences in the phenolics, lignin, flavonoids
and the enzymes involved in the regulation of their metabolism namely: phenylalanine ammonia- lyase
(PAL), Peroxidase POX and polyphenol oxidase (PPO) were recorded. Leaf tissues of soybeans which
were primed with BTH responded differently to pathogen inoculation with Phialophora gregata, compared
with both the control and BTH-primed and non-challenged ones. Appreciable increase in the activity level
of PAL, POX and PPO was observed in response to challenging of BTH-primed soybean, particularly on
applying it as both seed soaking followed by foliar spraying. On the other hand, catalase activity subjected
to marked increase in non-challenged, BTH-treated soybeans meanwhile it was obviously decreased upon
pathogen inoculation of BTH-treated plants. Appreciable increase in the different forms of phenolics (free,
conjugated, cell wall-bound phenolics and total soluble phenols) was recorded in response to BTH-priming
and challenging. Moreover, the same treatments induced obvious increase in the flavonoid content of
soybean leaves. Thus, a four- fold increase in leuteolin content was observed in treated tissues, compared
with the control. Also, the quercetin and genistein content subjected to marked increase in response to
BTH and challenging with Phialophora gregata. The bioassay for antifungal activity of phenolic
compounds obtained from BTH-primed and challenged soybeans revealed its high toxicity to fungal spore
germination. The marvelous changes induced in protein pattern in response to priming soybean with BTH
and its challenging with its pathogen, refer to that BTH act at the molecular level and that it induced
change at the transcriptional and translational levels.
Key words: Defense mechanisms- Flavonoids - Pathogensis related proteins, Phialophora gregata Oxidative enzymes - Systemic acquired resistance.
INTRODUCTION
Soybean (Glycine max L.Merr.) is one of the most
important leguminous crops. As it represents one of
important source of protein (about 40% of dry weight
of seeds), great efforts in Egypt are paid to increase
the cultivated area and to increase its productivity via
following programs concerned with pest control.
Soybean as other plants is challenged by a diverse
array of pathogenic microorganisms. Most protection
methods currently applied involve the use of chemicals
noxious to the environment, following crop rotation or
the use of resistant varieties. However, as plants can
acquire local and systemic resistance to pathogens
through various biological agents including pathogenic,
nonpathogenic and soil-borne rhizosphere bacteria and
fungi[1 ], treating soil or plant with these biocontrol
agents could provide an alternative, non-conventional
and ecologically- friendly approach for plant
protection [2 ]. Biocontrol of pathogens or herbivores
depends mainly on exploiting natural constitutive and
induced defense machinery of plants. Thus, treating
plants with pathogenic or non-pathogenic organisms
have been reported to sensitize plants to defend
themselves against pathogen attack by triggering
various defense mechanisms including production of
phytoalexins, synthesis of phenolics [3 ,4 ], accumulation
of pathogenesis related (PR)Proteins [5 ] and deposition
of structural barriers [6 ]. The defense gene products
peroxidases and polyphenol oxidases catalyze the
formation of lignin and phenylalanine ammonia -lyase
which is involved in the synthesis of phytoalexins and
phenolics [7 ]. Also, the pathogenesis related proteins, â1, 3 – glucanase and chitinases which degrade the
fungal cell wall and cause lysis of fungal cell walls are
markedly expressed. Furthermore, chitin and glucan
Corresponding Author: E Nafie, Department of Botany, Faculty of Girls for Art, Science and Education, Ain Shams
University, Cairo, Egypt.
E-Mail: [email protected]
2046
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
oligomers released during degradation of fungal cell
wall by the action of lytic enzymes act as elicitors that
trigger various defense mechanisms in plants [7 ].
The signaling pathway controlling systemic
acquired resistance (SAR) requires endogenous
accumulation of the stress hormone salicylic acid [8 ].
The expression of SAR, triggered by treatment with
salicylic acid or its functional analog benzothiadiazole
(BTH) is also tightly associated with the transcriptional
activation of genes encoding pathogenesis related
proteins [9 ,1 0 ]. So BTH has been used for induction of
SAR in wheat, soybean and barley against fungal and
bacterial pathogens [1 1 ]. However, the efficiency of BTH
in induction of SAR in soybean against the soil-borne
fungi Phialophora gregate has not been reported. This
fungus induces brown stem rot disease (BSR) of
soybean. BSR is now widespread where it could be
detected in most soybean growing areas. Moreover,
soybean yield losses caused by BSR occur regularly
and intensively [1 2 ,1 3 ]. So, strategies employed in BSR
control are divers and began earlier and depended on
following crop rotation or the use of resistant soybean
varieties or a combination of both [1 4 ].
BSR of soybean is among the diseases that are not
or weakly controlled by following the biocontrol
strategy. Moreover, the published studies in this regard
are too limited to allow critical assessment of this type
of control. So, the present work stand in the control of
BSR via using the chemical way through using B TH
following different types of application.
M ATERIALS AND M ETHODS
Soybean seeds [Glycine max L. (Merr.) cv. Clark]
were kindly provided from Legume Crop Research
Department, Field Crops Research Institute at the
Agriculture Research Centre, Cairo, Egypt. The
chemical Benzo-(1, 2, 3) thiadiazole-7-carbothioic acid
S-methyl ester( BTH) was obtained from Sigma.
Experimental Design: Pots filled with autoclaved soil
(clay: sand, 3:1) were prepared for sowing in 23 May
2007.The soybean seeds were surface sterilized in
sodium hypochlorite solution (5%, v/v) [1 5 ]. Before
sowing,the seeds were soaked for ten min either in
H 2 O (control,C) or in 40 ppm of BTH (low dose, l) or
in 60 ppm of BTH(high dose, h).After 16 days of
sowing, the stems of about half of the please add after
(high dose,h) the treated seed were sown in pots,5 seed
for each pots .3 replicates were used for each
treatments emerged plants were challenged with the
pathogen Phialophora gregata, where they were
designated with C+, l+ and h+, whereas the symbols,
C-, l- and h- were preserved to non-challenged groups.
In a second group of pots, sterilized soybean seeds
were sown. After 13and 46 days after sowing (DAS),
the emerged plants were subjected to foliar spray with
either 40 or 60 ppm of BTH. About half of these
plants were challenged with Phialophora gregata. They
accepted the same letters used above. In a third group
of pots, soybean seeds were soaked either in 40 or 60
ppm of BTH
plus
receiving
foliar
spraying
treatment at 13, 46 (DAS) with either 40, 60 ppm
BTH. About half of the developed plants were
inoculated with P.gregata. The differently treated plants
accepted the same symbols depicted above. Pointing to
different application methods seed soaking is
abbreviated to(s), foliar spraying to (f) and applying
both methods to (s+f).
Isolation and Preparation of the Fungal Culture:
The fungus Phialophora gregata used in this study was
isolated from naturally infected soybean plants collected
from Behaiera governorate. The isolate was purified
and identified according to [1 6 ].
Preparation of Inoculums and Challenge Plants:
Phialophora gregata was cultured in green bean extract
(GBE) medium (35 g/L ground frozen Phaseolus
vulgaris L. green pods, 20 g/L agar) supplemented with
50 mg/L ampicilline. Cultures were incubated at
25°C±1 in the dark until abundant spores were visually
evident. Conidia of Phialophora gregata were
suspended in 0.8% water agar (2.7 ×10 7 conidia/ml).
The conidial suspension was thoroughly mixed by
tapping with sterile micropipette tips into a paste. After
16 days of sowing, control and BTH primed plants
were punctured approximately 2 cm above the soil line
with an 18-gauge needle with its bevel filled with the
inoculums paste [1 7 ].
Bioassays for Antifungal Activity: Fungitoxicity of
phenolics (glycoside-bound) extract obtained from BTH
-primed plants non challenged and primed challenged
and their control plants (stage 2 only) were tested
against spore germination of Phialophora gregata on
glass slides. 50 µL of each solution was added to wells
in Teflon microscope slides (BDH), evaporated, then
25 µL of spore suspensions were added. All slides
were incubated at 25 N Ñ for 24 h. Then percentage of
spore germination was recorded through microscopic
examination. Three replicates were used for each
treatment. Conditions and assessment of germination
were as described by [1 8 ].
Disease Assessment:
Foliar Symptom Assay: Foliage symptoms were
assessed, 48 DAS, Severity of foliar symptoms was
determined according to [1 7 ] Tabor et al. using the
formula: (stunted trifoliate leaflets+ necrotic trifoliate
leaflets + abscised trifoliate leaflets/total trifoliate
leaflets) × 100
Stem Discoloration Assay: Internal stem discoloration
was assessed 48 DAS, visually after splitting stems
longitudinally. A stem was considered discolored if
2047
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
there was any visible dark brown discoloration of the
vascular or pith tissue of the stem. Severity of
discoloration (percent stem length discolored) was
calculated by dividing highest point of discoloration by
total stem length × 100 [1 7 ].
H arvesting Time: At various times before and after
challenge, plants were harvested from each treatment at
(15, 26 and 48 DAS) referring to them as stages 1, 2
and 3respectively. The fresh weight of the roots and
shoots of the differently treated and untreated plants
were determined. They were then dried in oven at 70ºC
for dry weight determination.
Viability Assay on Germinating Soybean Seeds: To
evaluate the effect of BTH on soybean germination, ten
seeds were spread between two filter papers in two
different sets of Petri dishes and BTH was added at
either 40, or 60 ppm. A third group of Petri dishes
provided with water was prepared serving as control
the dishes were kept at room temperature. The
germination process and the survival rate were
observed daily for a period of 5 days.
Chemical Analysis:
Pigments Extraction and Determination: Chlorophyll
a, chlorophyll b and total carotenoids were extracted
from 0.5 g of longitudinal sections of fresh leaves in
85% acetone and measured spectrophotometrically
according to [1 9 ]
and their levels were calculated
according to the formula of[2 0 ].
Extraction and Assays of Certain Enzymes:
Extraction and Assaying the Activity Level of
Phenylalanine Ammonia- Lyase (PAL, EC 4.3.1.5):
Leaf samples (1g) were homogenized in 3 ml of icecold 0.1 M sodium borate buffer, pH 7.0, containing
1.4mM 2-mercaptoethanol and 0.1g insoluble polyvinylpyrrolidone. The extract was filtered through cheese
cloth and the filtrate was centrifuged at 16000 g at 4°C
for 15 min (Sigma, 3-18K). The supernatant was used
as the enzyme source [2 1 ]. Activity of PAL was
determined as the rate of conversion of L-phenylalanine
to trans-cinnamic acid as described by [2 1 ]. A sample
containing 0.5 ml of enzyme extract was incubated
with 0.5 ml of 0.1 M borate buffer, pH 8.8, and 0.5 ml
of 12 mM L-phenylalanine in the same buffer for 60
min at 30 °C. The reaction was terminated by the
addition of 35% (w/v) trifluoroacetic acid (2 2 ).The optical
density value was recorded at 290 nm.Enzyme activity
was expressed as change in readings min -1 g -1 f. wt.
Extraction and Assay of Peroxidase (POD, EC
1.11.1.7): Leaf samples (1 g) were homogenized in 2
ml 0.1 M phosphate buffer, pH 7.0, at 4°C. The
homogenate was centrifuged at 14000 g at 4 °C for 15
min and the supernatant was used as the enzyme
source. The reaction mixture consisted of 1.5 ml of
0.05 M pyrogallol, 0.5 ml of enzyme extract and 0.5
ml of 1% H 2 O 2 . The reaction mixture was incubated at
28 ± 2 °C for 5 min and reaction was stopped by
adding 1mL of 1M H Cl [2 3 ]. Changes in A 4 2 0 due to
pyrogallol oxidation were recorded. The enzyme
activity was expressed as changes in A min -1 g -1 f.
wt. [2 4 ].
Extraction of Polyphenol Oxidase (PPO) and
Catalase (CAT): Extraction was done following the (2 5 )
method. Leaf samples (1 g) were homogenized in 2 ml
0.1 M sodium phosphate buffer (pH 6.5) and
centrifuged at 15000 g for 15 min at 4 °C. The
supernatant was used as the enzyme source of
polyphenol oxidase and catalase.
Activity of PPO (EC 1.14.18.1): It was determined
following the method of [2 5 ]. The reaction mixture
consisted of 200 µl of the enzyme extract and 1.5 ml
of 0.1 M sodium phosphate buffer (pH 6.5). To start
the reaction, 200 µl of 0.01 M catechol were added
and the reaction was terminated after 10 min by adding
1 ml of 1M HCl [2 3 ].Change in A 4 9 5 was recorded. The
enzyme activity was expressed as change in oxidation
of catechol min -1 g -1 f. wt.
Catalase Assay (EC1.11.1.6): Catalase activity was
assayed following the method of [2 6 ]. In a quartz cuvette
(10-mm light path) we added 680 ìl of 50 mM
potassium phosphate buffer (pH 7.2) and 480 ìl of 40
mM hydrogen peroxide. The mixture was then
incubated for 2.5 min at 30 o C. After incubation, the
reaction was initiated by the addition of 200 ìl
enzyme extract, then the reaction was stopped by
vigorous boiling for 10 min [2 7 ].T he decrease in
absorbance at 240 nm was followed
spectrophotometrically. Activity of catalase was
represented as change in absorbance at 240 min -1
g -1 f.wt.
Extraction and Estimation of Phenolic Compounds:
Pooled leaf segments from different plants per time
point were homogenized and extracted with 80%, (v/v)
aqueous methanol [2 8 ,2 9 ]. Following re extraction of the
precipitates, the supernatants were combined
and
divided into three tubes to determine total soluble,
free,
and methanol soluble and glycoside-bound
(released after acid hydrolysis) phenolics concentration.
The remaining pellet was dried at 70 °C for 24 h.
The resulting alcohol insoluble residue (AIR) yielded
the cell wall material (CW M ) used to extract the esterbound cell wall phenolic acids after alkaline
hydrolysis. The concentration of the phenolic acids in
the extract was determined following [3 0 ] with FolinCiocalteau reagent and was expressed as mg tannic
acid per 100 g f.wt.
Estimation of Total Soluble Phenolic Acids: The
aliquoted supernatant for the total soluble phenolic
acids was concentrated under speed vac. The FolinCiocalteau reagent was used for estimation.
2048
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Non-conjugated Phenolic Acids: From the above
concentrated fraction certain volume was acidified by
1M HCl (1:3 v/v), before extraction using equal
volume of ethyl acetate for three successive times. The
extract was reduced to dryness under speed vac
and the resulting precipitate was re suspended in 80%
MeOH.This solution was used to determine the free
phenolic content using Folin-Ciocalteau reagent method.
G ly co side- B o un d P he no lics: T he aliq uoted
supernatant for M eOH soluble glycoside-bound
phenolic content was hydrolyzed in 1N HCl for 2 h at
96 ºC and then extracted, dried, re suspended and
estimated as in non-conjugate fraction. Fractions
extracted from plants (during the 2 n d stage only) were
tested for their antifungal effect (as previously stated)
and for HPLC estimation (certain compounds).
Determination of Wall-bound Phenolics: Residues
(AIR) previously extracted for soluble phenolics [2 8 ,2 9 ]
were washed three times with absolute ethanol, dried
and subjected to base hydrolysis for 18 hr in 10 ml of
1 N NaOH at room temperature. After centrifugation
(8000g at 4 °C for 15 min),the supernatant was
removed, acidified to pH 1.0 - 2.0 with 2 N HC1, and
extracted three times with an equal volume of ethyl
acetate. The organic phases were combined, taken to
dryness, and re suspended in 5 ml HPLC grade
methanol[3 1 ].
Determination of Total Phenolic Content (TP): Total
phenolic concentration was determined by the FolinCiocalteau reagent method [3 0 ]. Assay was conducted by
mixing 200 µl aliquot with 2 ml of 1 N FolinCiocalteau reagent (Sigma Chemical Co.) followed 3
min later by 2 ml of 1M Na 2 CO 3 [3 2 ]. After 60 min, A 7 5 0
was measured using Jenway 9600 spectrophotometer.
TP content was standardized against tannic acid and
expressed as mg tannic g -1 0 0 f.wt.
Extraction and Estimation of Lignin: Lignin was
extracted according to the method of [3 3 ]. Lignin content
was determined by digestion of AIR of leaves material
with 25% acetyl bromide in acetic acid [3 4 ]. Results were
expressed as the increase in A 3 1 0 per 5 mg of AIR.
Extraction and Estimation of Certain Flavonoids in
Leaf Tissue Using HPLC System: To hydrolyze any
possible isoflavone conjugates, 3 ml of 1 N HCl were
added to 1 ml of the methanolic extract and sample
was incubated at 96 °C for 2 h, followed by extraction
using equal volume of ethyl acetate three times.
Organic fractions were evaporated to dryness and the
residues were then solubilized in methanol and
analyzed by HPLC as reported by [3 5 ] . In brief, samples
were assayed on an H PLC system (model Agliant 11
00 ) on XDB- C18 column ( 150 × 4.6 mm), separated
by using a 10-min linear gradient from 20% methanol
/ 80% 100 mM ammonium acetate (pH 5.9) to 100%
methanol at a flow rate of 1 ml min -1 . Genistein and
quercetin were monitored by diode array detector at
A 2 8 0 while the luteolin at A 3 5 0 . The sample injection
volume was 20µl. For calibration, authentic genistein,
and luteolin (Sigma, St. Louis), were dissolved in
ethanol and used as standards whereas quercetin was
dissolved in dimethyl sulfoxide (DMSO). Peak areas
were converted to µg/g.
Protein Extraction and Electrophoresis: Soluble
proteins from soybean leaves (1g) were extracted
according to [3 6 ] from all treated and control plants
(stages 1, 2). Protein quantity was determined using the
method of[3 7 ]. To prepare protein samples for SDSPAGE, extract from different samples, were mixed
with equal amounts of 50 mM Tris buffer (pH 7.3)
containing 2% (w/v) SDS, 10% (v/v) glycerol, and
10% (v/v) 2-mercaptoethanol, and samples were
immersed in boiling water for 2 min. Proteins were
analyzed by SDS-PAGE on a slab gel (0.75 mm thick)
containing 11% (w/v) acrylamide, stabilized by a
10% sucrose [3 8 ]. The resolving polyacrylamide gel
was overlaid with a 6% (w/v) polyacrylamide stacking
gel. Electrophoresis was performed at room temperature
with a constant current 25 mA/gel for 2 to 2.5 h, and
then visualized using [3 9 ] silver stain method. Premixed
solutions from molecular weight marker proteins were
run simultaneously.
RESULTS AND DISCUSSION
Results:
Assessment of Disease Severity: As a result of BTH
treatment, seed soaking, foliar spray or
both,
significant decrease in the incidence of soybean stem
vascular discoloration was observed where it is lowered
from 73.33 (in control) to 11.72% in case of seed
soaking and foliar application using the higher dose
(Table 1and Figures 1A-C). Also, leaflet symptoms
were reduced significantly from 40.67 in control to
4.33% upon applying high concentration of BTH as
both seed soaking and foliar spray.
Bioassay for Antifungal Activity of Phenolic
Compounds: Fungitoxicity of phenolic compounds was
tested against spore germination on glass slides.
M ethanol soluble glucoside-bound phenols obtained
from B TH-primed challenged or non-challenged
soybeans showed its fungitoxicity to spore germination.
Table (2) shows that, all treatments significantly
reduced spore germination compared with untreated
control. The treatment which involves both seed
soaking and foliar spraying of emerged plants using
high concentration of BTH triggered the highest
reduction in fungal spore germination.
2049
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Effect of treating soybean {seed soaking ( s), foliar spraying (f) and both (s+f)} w ith BTH at the concentrations 40-60 ppm on
brown stem rot disease incidence (after actual infection (+) with Phialophora gregata) assessed by stem length vascular discoloration
(% ) and leaflet sym ptom s (% ) under green house condition.
Treatm ents
BTH Concentration
Stem vascular discoloration %
Leaf sym ptom s %
T+control
73.33
40.67
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ts+
40ppm
36.33
22.00
Tf+
32. 33
14.66
T(s+f)+
18.00
11.33
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ts+
60ppm
34.66
12.67
Tf+
14.33
8.00
T(s+f)+
11.72
4.33
L.S.D.
5.55
6.47
The difference between treatm ents is significant (P# 0.05% )
Table 1:
Table 2: The effect of using m ethonal soluble glucoside-bound extracted from (26 D AS plants) prim ed treated either (40 ppm ) or (60 ppm
BTH ) non challenged (-) leaves and from challenged(+), on spore germ ination reduction. {seed soaking (s), foliar spraying (f), and
both (s+f) applications}.
Spore germ ination
Spore germ ination
treatm ents
BTH Concentration
-------------------------------------------------treatm ents
--------------------------------------------------M ethanol glucosideReduction
M ethanol glucosideReduction
bound extract µg/g
bound extract µg/g
T-control
80.33
0.00
T+control
69.67
0.00
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ts40ppm
72.67
9.54
Ts+
51.33
26.32
Tf63.33
21.16
Tf+
38.00
45.46
T(s+f)66.00
17.84
T(s+f)+
33.67
51.67
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ts60ppm
70.67
12.03
Ts+
43.33
37.81
Tf61.67
23.23
Tf+
25.33
63.64
T(s+f)53.67
33.19
T(s+f)+
14.67
78.94
L.S.D. 5%
6.004
8.073
The difference between treatm ents is significant (P# 0.05% )
Effect of BTH on Soybean Seed Germination and
Seedling Growth: It is evident that soaking seeds in
BTH solution led to marked increase in percentage of
germination where it reached 76.6 and 70.0% in
response to the relatively low and high concentration
applied, respectively meanwhile, it recorded only 68.8%
in control untreated seeds. Seed soaking in BTH also
enhanced seedling growth where there was a marked
increase in fresh and dry weights of shoots and roots
(Figures 2A, B).
Effect of BTH on the Photosynthetic Pigments of
Soybean Plants: The changes induced by BTH
treatment in photosynthetic pigments of soybean at
different stages of growth are represented graphically
in Figures (3A-C). It is obvious from the figures that
there was an obvious increase in the content of the
photosynthetic pigments as a result of BTH treatment
via the different methods employed with a magnitude
of response being obtained on applying the relatively
high concentration as both seed soaking and foliar
application. Moreover, the promotive effect of B TH on
photosynthetic pigments was more pronounced in nonchallenged plants.
Changes in Activity Level of Some Enzymes:
Changes in the activity level of phenylalanine
ammonia-layse (PAL): The change in PAL activity in
response to BTH is shown in figures (4A, B). It is
clear that either seed soaking, foliar application or seed
soaking followed by foliar application of the two
different concentrations of BTH led to marked increase
in PAL throughout the duration of the experiment.
Change in Lignin Contents: Accumulation of lignin
in cell wall was assayed quantitatively. Detectable
increase in lignin was recorded (Figures 5A, B) for
primed tissues using low or high dose through
prolonged time from stage 1 to 3 using any of the
application methods if compared with the control. After
actual infection, challenged plants accumulate lignin
compared to their challenged non primed ones, during
the second and the third stage.
Changes in the Activity Level of Peroxidase (POX):
It was observed that POX activity was markedly
increased in soybean leaves at different stages of
growth and development in response to any of the
application patterns of BTH and in challenged or nonchallenged plants (figures 6A,B).
Changes in the Activity Level of Polyphenol O xidase
(PPO): In response to BTH treatment via different
application methods described, using low or high
concentration, thin challenging or not, soybeans, an
obvious increase in the activity level of PPO was
2050
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 1A: Soybean healthy control plants
Fig. 1B: Soybean diseased plants with stem rot symptoms, internal browning is evident on the stem on the
outside.
Fig. 1C: Brown stem rot on leaf, internal stem discoloration is characteristic symptoms of BSR.
2051
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 2A: The effect of using low dose (l) (40 ppm) and high (h) (60 ppm) via different application form (seed
soaking (s), foliar spraying (f) and both (s+f)) on shoot fresh &dry weight during stage (1) in non
challenged soybean plant (leaves) estimated as (g).
Fig. 2B: The effect of using low dose (l) (40 ppm) and high (h) (60 ppm) via different application form (seed
soaking (s), foliar spraying (f) and both (s+f) )on root fresh &dry weight during stage (1) in non
challenged soybean plant (leaves) estimated as (g ).
recorded at different stages of growth (figures7A,B).
Moreover, the results obtained revealed that the
enhanced effect of BTH on POX activity level was
more pronounced upon its application as both seed
soaking followed by foliar spraying.
Changes in the Activity Level of Catalase: The
results represented graphically in figures (8A, B)
revealed that the activity level of catalase subjected to
marked increase in BTH – primed non-challenged
soybean plant. However, upon their challenging,
appreciable decrease in catalase activity was detected
(Figures 8A, B).
Changes in the Content of Different Phenolic
Compounds: In general, there was an appreciable
increase in the different forms of phenolics (free,
conjugated and cell wall-bound phenolics and total
soluble phenols) as a result of BTH application and of
challenging soybean with the pathogen phialophora
gregata (Figures 9A-C).
Changes in the Content of Certain Flavonoids:
Marked increase in quercetin and genistein content of
soybean leaves in response to BTH priming and
challenging plants with the pathogen Phialophora
gregata was observed (Table 3). Luteolin content
subjected to four-fold increase, compared with the
control in BTH-primed and challenged soybeans.
Changes in Protein Pattern of Soybean Leaves:
Figures (10, 11) show the changes in protein banding
patterns
of soybean leaves in response to BTH
treatment and after challenging with phialophora
gregata. To evaluate the differences, total soluble
proteins were separated on SDS-polyacrylamide gel and
visualized by silver stain method. Scanning of the gel
revealed that before pathogen inoculation (stage 1)
BTH treatment effectively altered the protein banding
pattern of soybean leaves. Thus, proteins with M r 80,
58, 47, 40, 37, 27, 25and23 while were expressed in
untreated samples, disappeared completely in BTH
treated samples (Figure 10). On the other hand, de
novo bands (65, 60, 56, 44, 38, 36, 26,20KD) were
restricted to only BTH-treated plants. During the
second stage upon pathogen inoculation of BTH-treated
plants (Figure 11) there was pathogen inducible
proteins linked to the application method.In seed
soaking proteins 89,70,42,35 and26KD were recorded.
Discussion: Chemically induced resistance (IR) is a
suitable strategy to utilize natural defenses of the plant
to control pathogens. This phenomenon has been
studied at the molecular level and has proved to be
mediated by salicylic acid and associated with a
number of defense responses and genes [4 0 ].
Induced resistance was reported to be activated by
exogenous application of salicylic acid and its synthetic
2052
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 3A: The effect of using low dose (l) (40 ppm) and high (h) (60 ppm) via different application form (seed
soaking (s), foliar spraying (f) and both (s+f)) on photosynthetic pigments during stage (1) in non
challenged soybean plant (leaves) estimated as µg/g f.wt.
Fig. 3B: The effect of using low dose (l) (40 ppm) and high (h) (60 ppm) via different application form (seed
soaking (s) foliar spraying (f) and both (s+f)) on photosynthetic pigments during stage (2) in non
challenged (-) and challenged (+) soybean plant (leaves) estimated as µg/g f.wt.
Fig. 3C: The effect of using low dose(l) (40 ppm) and high (h) (60 ppm) via different application form (seed
soaking (s), foliar spraying (f) and both (s+f)) on photosynthetic pigments during stage (3) in non
challenged (-) and challenged (+) soybean plant (leaves) estimated as µg/g f.wt.
functional analog benzo [1 ,2 ,3 ] thiadiazol-7-carbothioic
acid-S methyl ester (BTH).
BTH is promoted as a safe, reliable and non
phytotoxic plant protection agent. It was recently
identified by scientists as a novel disease – control
compound. Despite the functional similarity between
SA and BTH, it was reported that induction of
systemic acquired resistance (SAR) gene expression by
BTH did not require the contribution of SA which
suggest that this compound could act as a secondary
messenger analog capable of activating SAR signal
transduction pathway independently of the accumulation
of other signal molecules[4 1 ]. Application of BTH to a
variety of plants before challenge with the pathogens
triggered a set of plant defense reactions that resulted in
the creation of a fungitoxic environment, which protect
them by different (physical and / or chemical means)
mechanisms. These observations raised the question of
2053
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 4A: The effect of using BTH 40 ppm dose (l) (seed soaking (s), foliar spray (f) and both (s+f)) on the
activity of phenylalanine ammonia- lyase (PAL) in both non challenged (-) and challenged (+) soybean
plants (leaves) throughout the different stages.
Fig. 4B: The effect of using BTH 60 ppm dose (h) (seed soaking (s), foliar spray (f) and both (s+f)) on the
activity of phenylalanine ammonia- lyase (PAL) in both non challenged (-) and challenged (+) soybean
plants (leaves) throughout the different stages.
Fig. 5A: The effect of using BTH 40 ppm (l) dose (soaked seed (s), foliar spray (f) and both (s+f)) on lignin
content in both non challenged (-) and challenged (+) soybean plants (leaves) throughout different stages.
Fig. 5B: The effect of using BTH 60 ppm (h) dose (soaked seed (s), foliar spray (f) and both (s+f)) on lignin
content in both non challenged (-) and challenged (+) soybean plants (leaves) throughout different stages.
2054
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 6A: The effect of using BTH 40 ppm dose (l) (seed soaking (s),foliar spray (f) and both (s+f)) on the activity
of peroxidase (POX) in both non challenged (-) and challenged (+) soybean plants (leaves) throughout
the three stages.
Fig. 6B: The effect of using BTH 60 ppm dose (h) (seed soaking (s),foliar spray (f) and both (s+f)) on the
activity of peroxidase (POX) in both non challenged (-) and challenged (+) soybean plants (leaves)
throughout the three stages.
Fig. 7A: The effect of using BTH 40 ppm (l) dose (soaked seed (s), foliar spray (f) and both (s+f)) on the
activity of polyphenol oxidase (ppo) in both non challenged (-) and challenged (+) soybean plants
(leaves) throughout the three stages.
Fig. 7B: The effect of using BTH 60 ppm (h) dose(soaked seed (s),foliar spray (f) and both (s+f)) on the activity
of polyphenol oxidase (ppo) in both non challenged (-) and challenged (+) soybean plants (leaves)
throughout the three stages.
2055
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Effects of applying either 40 ppm or 60 ppm through {soaked seed (s), foliar sprayed (f), and both (s+f)} on specific flavonoids
(quercetin, genistein, luteolin ) am ount during the second stage of soybean,non challenged (-) and challenged (+) plant (leaves)
growth estim ated as µg/g f. wt. using HPLC.
Treatm ents
BTH concentration
quercetin
genistein luteolin
Treatm ents
BTH concentration
quercetin
genistein
luteolin
T-control
38.467
6.17
543
T +control
25.26
16.77
285
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Ts 40 ppm
60.749
41.02
770
T s +
40 ppm
85.259
48.6
1550
Tf 57.201
9.2
1319
T f +
50.716
30.93
1325
T (s+f)44.951
11.36
966
T (s+f) +
34.414
6.6
1129
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------T s 60 ppm
70.521
16.25
1375
T s +
60 ppm
98.714
19.7
1395
T f 52.613
34.64
1296
T f +
30.679
8
1279
T (s+f) 94.968
6.93
664
T (s+f) +
41.119
5.32
566
Table 3:
Fig. 8A: The effect of using BTH 40 ppm (l) dose (soaked seed (s),foliar spray (f) and both (s+f)) on the activity
of catalase (CAT) in both non challenged (-) and challenged (+) soybean plants (leaves) throughout the
three stages.
Fig. 8B: The effect of using BTH 60 ppm (h) dose(soaked seed (s),foliar spray (f) and both (s+f)) on the activity
of catalase (CAT) in both non challenged (-) and challenged (+) soybean plants (leaves) throughout the
three stages.
to what extent BTH treatment of soybean plants before
and after their challenge with Phialophora gregata
could contribute in retarding or decreasing the rate of
incidence of infestation by the fungus Phialophora
gregata
which is responsible for marked loss in
soybean yield? The results obtained in the present work
referred to the potentiality of BTH as a priming agent
when applied before challenging soybean with
Phialophora gregata.
Assessment of disease severity refer to striking
differences in the rate and extent of fungal
colonization, where disease symptoms incidence (stem
vascular discoloration) were reduced from 73.33% in
control untreated plant to 18% (40 ppm BTH) and to
11.72% (60 ppm BTH). Moreover, enhanced soybean
resistance to stem rot disease was obviously expressed
in response to BTH upon following the method
employed both seed soaking and foliar spray.
The bioassay for antifungal activity (methanol
soluble glycoside – bound extracts) obtained from
BTH primed either challenged or non-challenged
soybean leaves showed marked toxicity where there
was appreciable reduction in fungal spore germination,
if compared with that extracted from control plants.
BTH application at low or high concentration
following seed soaking, foliar spray or a combination of
2056
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 9A: The effect of using 40 ppm (l) or 60 ppm (h) via different application form (soaked seed (s), foliar
spraying (f) and both (s+f)) on different phenolic groups{total soluble (TSP) , free, conjugate and
cell-wall }during stage (1) in non challenged soybean plant (leaves) estimated as mg phenolics/100
g f. wt.
Fig. 9B: The effect of using 40 ppm (l)or 60 ppm (h) via different application form (soaked seed (s), foliar
spraying(f) and both (s+f)) on different phenolic groups{total soluble (TSP), free, conjugate and cell-wall}
during stage (2) in non challenged (-) and challenged (+) soybean plant (leaves) estimated as mg
phenolics/100 g f. wt.
Fig. 9C: The effect of using 40 ppm (l)or 60 ppm (h) via different application form (soaked seed (s), foliar
spraying (f) and both (s+f)) on different phenolic groups{total soluble (TSP) , free, conjugate and
cell-wall }during stage (3) in non challenged (-) and challenged (+) soybean plant (leaves) estimated
as mg phenolics /100 g f. wt.
both of non-challenged soybean led to appreciable
increase in percentage of germination. Moreover, an
appreciable increase in fresh and dry weight of
seedlings in response to BTH was observed.
Application of BTH to soybean leaves before and
after challenge with the stem pathogen Phialophora
gregata led to pronounced increase, in general, in the
photosynthetic pigments chloropyll a,b and carotenoids.
Increased photosynthetic pigments in BTH- primed
soybean leaves before it’s challenging with the
pathogen P. gregata could contribute in increasing the
potentiation of BTH in flourishing induced resistance
(IR) in soybeans. Undoubtedly, IR exhausts the primary
metabolism, particularly the photosynthetic process.
BTH via increasing the photosynthetic pigments and
hence photosynthetic process could be a response to
compensate for the carbon skeletons engaged in the
biosynthesis of IR elements.
The role of carotenoids as an efficient antioxidant
agent is widely accepted. In the present work, in
2057
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 10: SDS PAGE of soluble proteins in soybean leaves (during stage 1). Content of the sample 20?l, M =Mol.
W t. marker (66, 42, 31).
band - 1 control non primed non challenged, band 2 - Treated plants as (seed soaked only in low dose
BTH (40ppm), band 3 - T reated plants as seed soaked only in high dose BTH(60ppm), band 4 -Treated
plants as foliar spray only with low BTH dose, band 5 - Treated plants as foliar spray only with the
high dose, band 6 -Treated plants as both type of application, seed soaking and foliar application using
low BTH dose, band 7, Treated plants as both type of application ,seed soaking and foliar application
using high BTH dose.
response to seed soaking in BTH solution followed by
foliar spray an obvious increase in carotenoids content
of leaves was recorded, compared with the control.
BTH effect in conditioning tissue may be via affecting
greatly carotenoid pigments to protect and prevent
singlet oxygen formation and its conversion to more
dangerous molecules such as hydroxyl and peroxyl
radicals that can lead to cellular injury such as protein
degradation, lipids peroxidation and DNA damage.
Carotenoids have the important antioxidant function of
quenching (deactivating) singlet oxygen, an oxidant
formed during photosynthesis [4 2 ]. Carotenoids can also
inhibit the oxidation of fats (lipid peroxidation)
increased under certain conditions [4 3 ].
As a result of seed soaking followed by foliar
application of BTH (48 DAS plants) a decreased rate
in incidence of soybean brown stem rot (BSR) disease
induced by the soil- borne fungus Phialophora gregata
was recorded. More than 70%of the untreated
soybean plants were subjected to infestation by this
fungus. The diseased plants were characterized by stem
pith tissue browning, necrosis of leaves and interveinal
chlorosis (Figures 1B, C). These observations strongly
suggest that BTH could play potential role in the
induction of resistance against BSR. To realize the
mode of action of BTH in this respect, the most
important elements involved in the induction of
resistance were analyzed.
It seems likely that increased activity of the
enzymes involved in defense reactions may be one of
the basic ways participate in the action of BTH in
inducing resistance in soybean against BSR. Thus,
phenylalanine ammonia- layse (PAL) was increased in
BTH – treated infected or non-infected, compared with
challenged and non – BTH control plants. Such
increase in PAL after P. gregata infection may be an
early defense response. In this connection, (4 4 ,7 ) stated
that an almost ubiquitous feature of plant responses to
incompatible pathogens or to elicitors is the activation
of phenylpropanoid metabolism pathway in which PAL
catalyses the first committed step of the core
pathway. In the present work, the increased activity
of PAL in primed and challenged soybean was
associated with obvious increase in phenolic
compounds especially the cell wall- bound and which
was reflected in lignin deposition. Lignin deposition in
cell walls is a well known defensive mechanism in
plants. This provides an effective barrier to mechanical
penetration by fungi physically shields the wall
polysaccharides from
degradation
by
fungal
enzymes and restricts
2058
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
Fig. 11: SDS PAGE of soluble proteins in soybean leaves (during stage 2 after challenge). Content of the sample
20ìl, M =Mol. W t. marker (66, 42 ).
band - 1 Control non primed non challenged, band 2 -Control non primed challenged,band-3 Treated
plants as (seed soaked only in low dose B TH (40ppm), band 4 -Treated plants as (seed soaked only in
low dose B TH (40ppm) after challenge, band 5 - Treated plants as seed soaked only in high dose
BTH(60ppm), band 6- Treated plants as seed soaked only in high dose BTH(60ppm) after challenge,
band 7 -Treated plants as foliar spray only with low BTH dose, band 8- Treated plants as foliar spray
only with low BTH dose after challenge, band 9 - Treated plants as foliar spray only with the high
dose, band 10- Treated plants as foliar spray only with the high dose after challenge, band 11 -Treated
plants as both type of application ,seed soaking and foliar application using low BTH dose , band 12Treated plants as both type of application ,seed soaking and foliar application using low BTH dose after
challenge, band 13 - Treated plants as both type of application, seed soaking and foliar application using
high B TH dose, band 14- Treated plants as both type of application, seed soaking and foliar application
using high BTH dose after challenge.
diffusion of enzymes and toxins from the fungus to the
host and of water and nutrients from the host to the
fungus [4 5 ]. In this regard, PAL was observed to increase
in BTH – treated wheat after infection with Blumeria
graminis and in cucumber plants infected with the
virulent fungi Colletotrichum orbiculare respectively
(4 6 ,4 7 )
.Moreover ,(4 8 ) found that soybean leaves inoculated
with Phytophthora sojae were characterized by high
rate of deposition of phenolic compounds after
infection. Recently BTH was reported to reduce the
vigor of Blumeria graminis in barley plants via up
regulating the phenolic compounds biosynthesis [4 9 ].
Also, [5 0 ] reported that the variation in the degree of
resistance observed in different sunflower genotypes
against rust incited by puccinia helianthi was mainly
due to the impairment of rust spore germination in
response to their differential excretion of coumarins and
probably of other phenolic compounds.
Among the enzymes which are involved in defense
reactions against plant pathogens, the oxidative
enzymes as peroxidase (POD) and polyphenol oxidase
(PPO), which catalyze the formation of lignin and
O.quinones that contribute to the formation of defense
barriers or reinforcing the cell structure [5 1 ]. Thus, these
enzymes have been correlated with defense against
pathogens in several plants, including rice [5 2 ] tomato [5 3 ],
wheat[5 4 ]; pepper [5 5 ].
In the present investigation, a marked increase in
POD activity was observed in BTH – treated,
challenged or non-challenged soybean leaves with a
magnitude being observed in challenged ones. The
concomitant enhanced resistance of soybean against
BSR with increased activity of POD refer to its
engagement in expressing the resistance as among the
physiological role of POD is the construction of crosslinks and deposition of lignin during secondary cell
2059
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
wall development which may limits the pathogen
ingress and spread in the host tissues [5 6 ]. Moreover, the
reactive compounds associated with the process of
lignifications may inhibit pathogen growth [5 7 ] . POD was
also observed to inhibit the spore germination and
mycelia growth of certain fungi[5 8 ].
Due to BTH treatment and challenging with the
pathogen Phialophora gregata a marked increase in the
activity level of polyphenol oxidase (PPO) of soybean
leaves was observed, this may enhance the oxidation of
phenolic compounds into the more toxic forms, Oquinones. A role for PPO in enhancing resistance
against pathogens is well documented. Activity of PPO
was observed to increase dramatically in rice inoculated
with Rhizoctonia solani and in peach plants inoculated
with penicillium expannsum and treated with BTH
respectively[5 2 ,5 9 ].
Still the strategies employed by BTH in enhancing
resistance are diverse. In this connection, BTH was
observed by [4 9 ] to create a hostile environment that
slowed down the fungal spreading in barley plants via
inducing oxidative burst which profoundly was
effective in inducing resistance in barley against
Blumeria graminis. Enhanced oxidative burst could be
triggered via regulating the activity level of many
oxidative enzymes, among them the catalase which has
a crucial role in this respect. The results obtained in
the present work revealed that BTH treatment of non
challenged soybean had increased the activity level of
catalase. Such increase in catalase would prevent the
increase of cytosolic H 2 O 2 which may create toxic
conditions leading to oxidative stress. In accordance
with our explanation, what[6 0 ] stated for catalase role.
After soybean inoculation, an obvious decrease in
catalase activity was recorded. As a result, hydrogen
peroxide is accumulated, creating toxic conditions
which prevent pathogen spreading and/or may play a
role as a secondary signal for defense gene expression
and activation of SAR.
The phenylpropanoid pathway fed plant tissues
with diverse groups of compounds having different
functions, the most important of them the isoflavonoid
phytoalexins which have antimicrobial activity.
Flavonoids are ubiquitously distributed and are widely
consumed secondary metabolites which have profound
pharmacological properties [6 1 ]. In the present work,
treating soybeans with BTH and their inoculation with
Phialophora gregata led to marvelous increase in the
flavone luteolin. Luteolin is one of the most potent
flavonoid inhibitors of soybean [6 2 ], but its role in plant
defense against pathogens remains to be established.
However,[6 3 ] reported that luteolin via controlling the
level of NO at the site of infection may retard the
spread of the pathogen. A more structurally related
compound is quercetin. In response to BTH and
challenging soybean, an appreciable increase in
quercetin was recorded associated with marked
inhibition of spore germination of P. gregata.Of the
isoflavones determined the genistein which showed
obvious accumulation in soybean leaves as a result of
treatment with BTH and inoculation with the pathogen
P. gregata. In this regard,[6 4 ] found that genistein could
behave as an antibiotic having antimicrobial activity
against Phytophthora sojae. Supporting the role of the
flavones and isoflavones in controlling pathogens, they
were identified in a variety of plant species including
soybean upon exposure to the fungal elicitor [6 3 ].
Thus, from a practical standpoint the genetic
manipulation of the phenylpropanoid pathway could be
a plausible and efficient approach to enhance plant
resistance to pathogens.
The present study demonstrates that treatment of
soybean with BTH activates high levels of resistance
against a severe isolate of Phialophora gregata and
activation of resistance is correlated with the
coordinated expression of pathogenesis related (PR)
proteins following treatment with BTH.
The biological or chemical activation of SAR is
correlated with systemic accumulation of PR
proteins [6 5 ,6 6 ]. In our present study; expression of PR
proteins was evaluated to understand the possible
mechanism of action of BTH against Phialophora
gregata. Soybean leaves soluble protein was extracted
and fractionated using SDS electrophoresis technique
on the 15 DAS samples (Figure 10) PR-2, PR-3, and
PR-5 were detected during this stage.
One of PR-3 members (26KD) concomitant with
36KD (PR-2) and 20KD (PR-5 member) protein were
expressed in almost BTH treated plants but not in the
control. W e can consider such protein expression may
enhance soybean responses to overcome future
pathogen infestation, as one of B TH mode of action
against pathogens attack. It is known that PR-3 proteins
belong to endochitinases that cleave cell wall chitin
polymers in situ, resulting in a weakened cell wall and
rendering fungal cells osmotically sensitive [6 7 ]. Chitinase
induction is often co-ordinate with the expression of
specific ß-1, 3-glucanases, PR-2 [6 7 ] which among its
function the degradation of fungal cell wall. PR-5
proteins are stabilized by eight disulfide bonds; this
highly stabilized structure allows PR-5 proteins to be
very resistant to protease degradation [6 8 ]. Three proteins
(89, 22,19KD) were expressed in response to BTHtreated and in control plants varying in intensity.
Difference in the protein amount in treated soybean
leaves may reflect BTH constitutive resistance effect.
On the other hand, repression in the synthesis of
certain polypeptides after BTH application is that the
gene (S) responsible for such certain proteins had been
either completely suppressed or temporally inhibited as
2060
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
result of tissue conditioning, or targeting the cell
metabolism toward specific cell defense compounds.
A marked overproduction or de novo protein bands
and pathogen inducible proteins were observed during
the 2 n d stage in response to BTH priming (Figure 11).
Comparing unchallenged control with high dose treated
plants (dual application), protein bands (26, 25,18KD)
were highly expressed.W e can assume that this
treatment efficiently prepare unfavorable area retarding
spore ingress and may directly affect the P. gregata
growth and limit the disease spread after pathogen
inoculation. In accordance with our results, [6 9 ] stated
that BTH effectively induced resistance to powdery
mildew in wheat, via altering its protein profile.
Proteins having (18,17KD) can be considered age
dependent where they were found after BTH priming
and before challenge and constitutively increased after
challenge. [7 0 ,7 1 ] respectively stated that 17KD and
18KD are PR-1, 10 members, both have antifungal
activity[7 2 ].
After infection with Phialophora gregata there was
certain pathogen inducible proteins linked with BTH
application. Expression of 74 and70 KD proteins in
primed after pathogen invasion in plants (received dual
BTH application, and seed soaking, respectively) may
be conceded as signaling molecules evoking interlink
of certain signaling pathways. In this connection,[7 3 ]
previously found that a membrane-bound, calciumdependent protein kinase 72-70 KD was implicated in
the signaling mechanisms involved in the induction of
plant defenses in tomato to the fungal pathogen
Cladosporium fulvum.
In light of the above recorded results, one may
suggest that BTH has the potential to sensitize soybean
plants to respond faster and to a greater extent to P.
gregata attack.
3.
ACKNOW LEDGM ENTS
10.
W e wish to express our thanks to Dr. Azza Elshafey, Professor of Plant Physiology, Ain Shams
University,Faculty of Science, Botany Department, for
her scientific support,encouragement and revising the
manuscript.
4.
5.
6.
7.
8.
9.
11.
REFERENCES
1.
2.
Van Loon, L, P Bakker and C Pieterse, 1998.
Systemic resistance induced by rhizosphere
bacteria. Annu Rev Phytopathol., 36: 453-483.
Veronese P, TR M aria, AC Maria, Agustin
Hernandez-Lopez, L Hyeseung, I José Ibeas, D
Barbara, MP José, MH Paul, AB Ray and LN
Meena, 2003.
Both plant sentinels and foot
soldiers need to know the enemy. Plant Physiol.
April, 131: 1580-1590.
12.
13.
2061
M'Piga, P, R Bélanger, T Paulitz and N
Benhamou, 1997. Increased resistance to Fusarium
oxysporum f. sp. radicis-lycopersici in tomato
plants treated with the endophytic bacterium
P se u d o m o n a s flu o re sc en s stra in 6 3 – 2 8 .
Physiological and Molecular Plant Pathology., 50:
301-320.
Chen, CQ, RR Belanger, N Benhamou and TC
Paulitz, 2000. Defense enzymes induced in
cucumber roots by treatment with plant growthpromoting rhizobacteria (PGPR) and Pythium
aphanidermatum. Physiological and M olecular
Plant Pathology., 56: 13-23.
Meena, B, V Ramamoorthy, J Banu, R Thangavelu
and M Muthusamy, 2000. Screening of rice
genotypes against sheath blight disease. J Ecol.,
12: 103-109.
Benhamou, N, H Chamberland, G Ouellette and F
Pauzé, 1997. Ultrastructural localization of â-1,4-dglucans in two pathogenic fungi and in their host
tissues by means of an exoglucanase-gold complex.
Can J Microbiol., 33: 405-417.
Karthikeyan, M, K Radhika,, S Mathiyazhagan, R
Bhaskaran, R Samiyappan and R Velazhahan,
2006. Induction of phenolics and defencse- related
enzymes in coconut (Cocos nucifera L.) roots
treated with biocontrol agents.Braz.J.Plant Physiol.,
18(3): 367-377.
Nawrath, C and JP Métraux, 1999. Salicylic acid
induction-deficient mutants of Arabidopsis express
PR-2 and PR-5 and accumulate high levels of
camalexin after pathogen inoculation. Plant Cell.,
11: 1393-1404.
Van Loon, LC, 1997. Induced resistance in plants
and the role of pathogenesis-related proteins. Eur
J. Plant Pathol., 103: 753-765.
Maleck K, A Levine and T Eulgem, 2000. The
transcriptome of Arabidopsis thaliana during
systemic acquired resistance. Nature Genetics. 26:
403-410.
Kogel, KH, U Beckhove, J Dreschers, S Munch
and Y Romme, 1994. The resistance mechanism
induced by 2,6-dichloroisonicotinic acid is a
phenocopy of a genetically based mechanism
phenocopy of a genetically based mechanism Plant
Physiol., 106: 1264-1277.
Baraka, MA, AM Hassanein, AM El-Garhy and
MA Abdel-Al, 2004. Variation amang isolates of
Phialophora gregata. Egypt. J. Agric. Res., 82(2):
511-523.
W rather, JA and SR Koenning, 2006. Estimates of
disease effects on soybean yields in the United
States 2003-2005. Journal of Nematology. 38:
173-180.
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
14. Andreas, W , T Abney and G Shner, 2006.
Enhancing resistance to root rot pathogens of
soybean.Crop Production and Pest Control. New
Crops Center Purdue University Bulletin.
15. Doblinski, P, Ferrarese M de L, D Huber, C
Scapim, A Braccini de L and O Filho, 2003.
Peroxidase and Lipid Peroxidation of Soybean
Roots in Response to p-Coumaric and pHydroxybenzoic Acids. Brazilian Archives of
Biology and Technology. 46(2): 193-198.
16. Mengistu, A and DW Grau, 1986. Variation in
m o r p h o l o g ic al, c u ltu r a l a n d p a t h o lo g ic a l
characteristics of Phialophora gregata and
Acremonium sp. Recovered from soybean in
W isconsin. Plant Dis., 70: 1005-1009.
17. Tabor, G , S Cianzio, G Tylka, R Roorda and C
Bronson, 2006. A new greenhouse method to assay
soybean resistance to brown stem rot. Plant Dis.,
90: 1186-1194.
18. W illiams, J, S Hall, M Hawkesford, M Beale and
R Cooper, 2002. Elemental sulfur and thiol
accumulation in tomato and defense against a
fungal pathogen. Plant Physiology., 128: 150-159.
19. Metzner, H, H Raum and H Senger, 1965.
Unterschungen Zur Sychno-nisier-Barkeit einzeIner Pigmenmangel. M utantenovn. Chlorella.
Planta. 65: 186.
20. Lichtenthaler H
and A W ellburn 1983.
D e te r m in atio n s o f to ta l caro ten o id s a nd
chlorophylls a and b of leaf extracts in different
solvents. Biochemical Society Transactions., 11:
591-592.
21. Dickerson, D, S Pascholati, A Hagerman, L Butler
and R Nicholson, 1984. Phenylalanine ammonialyase and hydroxy cinnamate CoA ligase in maize
mesocotyls inoculated, with Helminthosporium
maydis or Helminthosporium carbonum. Physiol.
Plant Patho., 25: 111-123.
22. Rivero, R, J Ruiz, P Garcia, Lopez-Lefebre L, E
Sanchez and L Romero, 2001. Resistance to cold
and heat stress: accumulation in tomato and
watermelon plants. Plant Science., 160: 315-321.
23. Yingsanga P, V Srilaong, S Kanlayanarat, S
Noichinda and W McGlasson, 2008. Relationship
b e tw e e n b r o w n in g a nd re la te d e n z ym e s
(PAL,PPO,POD)in rambutan fruit (Nephelium
lappaceum Linn.) cvs. Rongrien and See-Chompoo.
Postharvest Biology and Technology, 50: 164-16.
24. Hammerschmidt R, E Nuckles and J Kuc, 1982.
Association of enhanced peroxidase activity with
induced systemic resistance of cucumber to
Colletotrichum lagenarium. Physiol. Plant Pathol.,
20: 73-82.
25. Mayer, AM , E Harel and RB Shaul, 1965. Assay
of catechol oxidase, a critical comparison of
methods. Phytochemistry., 5: 783-789.
26. Ueda, M , S Mozaffar and A Tanaka, 1990.
Catalase from Candida biodinii 2201. Methods
Enzymol., 188: 463-465.
27. Garge, S, H Dhawan, L Chawla and B
Nainawatee, 1999. Alternaria brassica induced
changes in the activity of cell wall degrading
enzymes in leaves of Brassica juncea, Bio. Plant.,
42: 475-8.
28. Cvikrová, M, J Nedelnik, J Eder and P Binarová
1993. Changes in pattern of phenolic acids induced
by culture filtrate of Fusarium oxysporum in
alfalfa plants differing in susceptibility to the
pathogen. J. Plant Physiol., 142: 1-5.
29. Torti, S, M
Dearing and T Kursar, 1995.
Extraction of phenolic compounds from fresh
leaves: A comparison of methods. J. Chem.
Ecol., 21: 117-125.
30. Singleton, V and J Rossi, 1965. Colorimetry of
to ta l p h eno lics w ith p h o sp h o m o lyb d ic phosphotungstic acid reagents. Am. J. Enol.Vitic.,
16: 144-158.
31. Howles, P, N Paiva, V Sewalt, N Elkind, Y Bate,
C Lamb and R Dixon, 1996. Overexpression of
L-phenylalanine ammonia-lyase in transgenic
tobacco plants reveals control points for flux into
phenylpropanoid biosynthesis. Plant Physiol., 112:
1617-1624.
32. Booker, F, S Anttonen and A Heagle, 1996.
Catechin, proanthocyanidin and lignin contents of
loblolly pine (Pinus taeda L) needles after chronic
exposure to ozone.New Phytologist., 132: 483-92.
33. Bruce, RJ and CA W est, 1982. Elicitation of
casbene synthetase activity in castor bean: The role
of pectic fragments of the plant cell wall in
elicitation by a fungal endopolygalacturonase.
Plant Physiol., 69: 1181-1188.
34. Sasaki, M, Y Yamamoto and H Matsumoto, 1996.
Lignin deposition induced by aluminum in wheat
(Triticum aestivum) roots. Physiol. Plantarum, 96:
193-198.
35. Jung, W , O Yu, S Lau, D keefe, J Odell, G Fader
and B McGonigle, 2000. Identification and
expression of isoflavone synthase, the key enzyme
for biosynthesis of isoflavones in legumes. Nat
Biotechnol., 18: 208-212, erratum Jung, W . Yu O.,
Lau, S.C., O.'Keefe, D.P., Odell, J., Fader, G.,
McGonigle, B., 2000. Nat Biotechnol. 18: 559.
36. W ittenbach, V, 1982. Effect of pod removal on
leaf senescence in soybeans. Plant Physiol. 70:
1544-1548.
2062
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
37. Bradford, M., 1976. A rapid sensitive method for
quantification of microgram quantities of protein
utilizing the principle of protein dye-binding.
Analytical Biochemistry. 72: 48-254.
38. Chua, N., 1980. Electrophoretic analysis of
chloroplastic proteins. Methods Enzymol., 69:
434-446.
39. Sammons, D, L Adams and E Nishizawa, 1981.
Ultrasensitive silver-based color staining of
polypeptides in po lyacrylamide gels.
Electrophoresis., 2: 135-139.
40. Ton, J, G Jakab, V Toquin, V Flors, A Iavicoli,
M Maeder, J Me´ traux and B Mauch-Mania,
2005. Dissecting the b-Aminobutyric Acid–Induced
Priming Phenomenon in Arabidopsis. The Plant
Cell., 17: 987-999.
41. Lawton, KA, L Friedrich, M Hunt, K W eymann,
T Delaney, H Kessmann, T Staub and J Ryals
1996. Benzothiadiazole induces disease resistance
in Arabidopsis by activation of the systemic
acquired resistance signal transduction pathway.
Plant J., 10: 71-82.
42. Halliwell, B and JMC Gutteridge, 1999. Free
Radicals in Biology and M edicine. 3 th ed. New
York, NY: Oxford University Press.
43. Young, AJ and GM Lowe, 2001. Antioxidant and
prooxidant properties of carotenoids. Arch Biochem
Biophys., 385(1): 20-27.
44. Harllen, SAS, SR Reginaldo, M Dirceu, AH
Bernardo, CBP M aria and M Ann, 2004.
Rhizobacterial induction of systemic resistance in
tomato
plants:
Non-specific protection and
increase in enzyme activities. Biological Control,
29: 288-295.
45. Ride, JP, 1978. The role of cell wall alterations in
resistance to fungi. Ann. Appl. Biol., 89: 302-306.
46. Stadnik, MJ and H Buchenauer, 2000. Inhibition of
phenylalanine ammonia-lyase suppresses the
resistance induced by benzothiadiazole in wheat to
Blumeria graminis f. sp. tritici. Physiol. Mol. Plant
Pathol., 57: 25-34.
47. Cools HJ and H Ishii, 2003. Pre-treatment of
cucumber plants with acibenzo lar-S-methyl
systemically primes a phenylalanine ammonia lyase
gene (PAL1) for enhanced expression upon attack
with a pathogenic fungus.Physiological and
molecular Plant Pathology., 61: 273-280.
48. Mohr, PG and DM Cahill, 2001. Relative roles of
glyceollin, lignin and the hypersensitive response
and the influence of ABA in compatible and
incompatible interactions of soybeans with
Phytophthora sojae. Physiol. Mol. Plant Pathol.,
58: 31-41.
49. Faoro F, D Maffi, Dcantu and M Iriti, 2008.
Chemical-induced resistance against powdery
mildew in barley: the effects of chitosan and
benzothiadiazole. J. Bio Control, 53(2): 387-401.
50. Prats E, MJ Llamas, J Jorrin and D Rubiales
2007. Constitutive Coumarin Accumulation on
Sunflower Leaf Surface Prevents Rust Germ Tube
Growth and Appressorium Differentiation. Crop
Sci., 47: 1119-1124.
51. Avdiushko, SA, X S Ye and J Kuc, 1993.
Detection of several enzymatic activities in leaf
prints of cucumber plant. Physiological and
Molecular Plant Pathology, 42: 441-454.
52. Deborahs, D, A Palaniswami, P V idhyasekaran and
R Elazhahan, 2001. Time-course study of the
induction of defense enzymes, phenolics and lignin
in rice in response to infection by pathogen and
non-pathogen.Zeitschrift für Pflanzenkrankheiten
und Pflanzenschutz ISSN -0340-8159. 108(2): 204216 (1 p.3/4).
53. Borden, S and VJ Higgins, 2002. Hydrogen
peroxide plays a critical role in the defense
response of tomato to Cladosporicem fulvum.
Physiological and Molecular Plant Pathology. 61:
227-236.
54. Mohammadi, M and H Kazemi, 2002. Changes in
peroxidase and polyphenol activity in susceptible
and resistant wheat heads inoculated with
Fusarium graminearum and induced resistance.
Plant Science, 162: 491-498.
55. Jung, W J, YL Jin, YC Kim, KY Kim, RD Park
and TH Kim, 2004. Inoculation of Paenibacillus
illinoisensis alleviates root mortality, activates of
lignification-related enzymes, and induction of the
iso z ym e s i n p e p p e r p la n ts infe cte d b y
Phytophthora capsici. Biological Control, 30: 645652.
56. Reimers, PJ, A Guo and JE Leach, 1992. Increased
activity of a cationic peroxidase associated with an
incompatible interaction between Xanthomonas
oryzae pv. oryzae and rice (Oryza sativa). Plant
Physiol., 99: 1044-1050.
57. Chittoor, JM, JE Leach and FF W hite, 1997.
Differential induction of a peroxidase gene family
during infection of rice by X. oryzae pv. oryzae.
Mol. Plant-Microbe Interact., 10: 861-871.
58. Joseph, LM, TK T an and SM W ong, 1998.
Antifungal effect of hydrogen peroxide and
peroxidase on spore germination and mycelial
growth of Pseudocercospora species. Canadian
Journal of Botany., 76: 2119-2124.
59. Liu, H, W Jiang, Y Bi and Y Luo, 2005.
Postharvest BTH treatment induces resistance of
2063
J. Appl. Sci. Res., 4(12): 2046-2064, 2008
60.
61.
62.
63.
64.
65.
66.
67.
peach (Prunus persica L. cv. Jiubao) fruit to
infection by Penicillium expansum and
e n h a n c e s a c t i v i t y o f fr u it d e f e n s e
m echanism s. P o stharvest B iology and
Technology., 35: 263-269.
Srivalli B and CR Khanna, 2001. Induction of new
isoforms of superoxide dismutase and catalase
enzymes in the flag leaf of wheat during
monocarpic senescence. Biochem. Biophys. Res.
Commun., 288: 1037-1042.
W ollenber, E., 1988. Occurrence of flavonoid
aglycones in medicinal plants. Prog. Clin. Biol.
Res., 280: 45-55.
Ueda, H , C Yamazaki and M Yamazaki, 2002.
Luteolin as an anti-inflammatory and anti-allergic
constituent of Perilla frutescens. Biol Pharm Bull.,
25(9): 1197-1202.
M odolo, LV, FQ Cunha, MR Braga and I Salgado,
2002. Nitric Oxide Synthase-Mediated Phytoalexin
Accumulation in Soybean Cotyledons in Response
to the Diaporthe phaseolorum f. sp. meridionalis
Elicitor1. Plant Physiol., 130: 1288-1297.
Rivera-Vargas, LI, AF Schmitthenner and TL
Graham, 1993. Soybean flavonoid e€ects on and
metabolism by Phytophthora sojae.
Phytochemistry. 32: 851-857.
W hite, RF, 1979. Acetylsalicylic acid (aspirin)
induces resistance to tobacco mosaic virus in
tobacco. Virology., 99: 410-412.
Ziadi, S, S B arbedette, JF Godard, C Monot, DL
Corre and D Silue, 2001. Production of
pathogenesis related proteins in the cauliflower
(Brassica oleracea var. botrytis)-downy mildew
(Perenospora parasitica) pathosystem treated with
acibenzolar-S-methyl. Plant Pathol., 50: 579-586.
Claude, PS, 2001. Antifungal Proteins,minireview.
Applied and Environmental Microbiology. 67(7):
2883-2894.
68. Roberts, W and CP Selitrennikoff, 1990. Zeamatin,
an antifungal protein from maize with membranepermeabilizing activity. J. Gen. Microbiol., 136:
1771-1778.
69. Martinková, J, L Vìchet, M Šindeláøová and L
Burketová, 2004. Compounds Of Natural Origin
As Inducers Of Systemic Acquired Resistance To
Powdery Mildew In Wheat. Acta fytotechnica et
zootechnica, 7: Special Number, Proceedings of the
X V I. S lo va k a nd C z e c h.P la nt Protection
Conference organized at Slovak Agricultural
University in Nitra, Slovakia, 193-195.
70. Graham, MY, J W eidner, K W heeler, MJ Pelow
and TL Graham, 2003. Induced expression of
pathogenesis-related protein genes in soybean by
wounding and the Phytophthora sojae cell wall
glucan elicitor. Physiological and Molecular Plant
Pathology., 63: 141-149.
71. Pasternak, O, J Biesiadka, R Dolot, L Handschuh,
G Bujacz, MM Sikorski and M Jaskolski, 2005.
Structure of a yellow lupin pathogenesis-related
PR-10 protein belonging to a novel subclass.
Acta Crystallogr D Biol Crystallography., 61(Pt 1):
99-107.
72. Fritig, B, T Thierry Heitz and M Legrand, 1998.
Antimicrobial proteins in induced plant defense.
Current Opinion in Immunology., 10: 16-22.
73. Romeis, T, P Piedras and J Jones, 2000.
Resistance Gene-Dependent Activation of a
Calcium-Dependent Protein Kinase in the Plant
Defense Response. American Society of Plant
Physiologists. The Plant Cell., 12: 803-815.
2064