Misaghi (1982) recorded that phenolic compounds, their
oxidative products (quinones) and derivatives (certain phytoalexins) exhibited
antibiotic properties and therefore were considered by some to play a role in
disease resistance. Halloin (1994) found that a histochemical
test employing nitrous acid, that produces red nitroso derivatives or some phenolic
compounds was modified for use in the field and was used to demonstrate localization
or phenolic compounds in healthy and Rhizoctonia solani infected sugar
beet (Beta vulgaris) plants. Nitroso-reactive materials were present
in petioles and around mature crown cavities or healthy plants. They were absent
from tap roots, except that they occasionally were observed in lateral root
tissue contained within tap roots.
Infection of tolerant/susceptible lentil (Lens esculentum) cultivars
by Fusarium oxysporum, F. solani, Sclerotium bataticola
and Rizoctonia solani led to a considerable increment in thio (Sulphur)
amino acids, methionine, cystine and cysteine were obtained in all tested cultivars
as a result of infection. The lowest decrease in thio-amino acids was recorded
after infection in susceptible cultivars compared with the tolerant and moderately
susceptible cultivars, El-Shaer (2002) found that.
Paranidharan et al. (2003) recorded that changes
in the activities of peroxidase, ascorbate peroxidase, catalase and superoxide
dismutase in rice in response to infection by R. solani were studied.
A significant increase in peroxidase activity was observed in R. solani inoculated
rice leaf sheaths 1 day after inoculation and the maximum enzyme activity was
recorded 3 days after inoculation at which period a 3 fold increase in peroxidase
activity was observed compared to the untreated control. Ascorbate peroxidase
and catalase activities significantly increased 1-2 day after inoculation and
the maximum enzyme activities were recorded 5 days after inoculation.
The aim of the present work was to study the cultivar reactions of nine common
commercial cultivars of potato to infection with Rhizoctonia solani under
controlled greenhouse conditions. Fractions of phenolic compounds by HPLC, oxidative
enzymes and thio amino acid contents in the described resistant and susceptible
potato cultivars were determined.
MATERIALS AND METHODS
Varietal reactions under greenhouse conditions: Nine potato cultivars
were screened for their reaction to black scurf and stem canker disease. The
tested cultivars were Draga, Cara, Diamont, Hermes, Nicola, Spunta, Mondial,
Monalisa and Lady Rosetta. Pure cultures of the selected R. solani isolates
(10 days old) were used for cultivars reaction tests. Soil infestation was conducted
with inocula of each isolate propagated on corn-meal sand medium. Loamy soil
was sterilized with formalin solution 5% and left for two weeks then soil infestation
was carried out at the level of 5% inoculum. Potato tubers of the nine cultivars
were surface sterilized by immersing in 0.2% Sodium hypochlorite for 2 min and
planting in pots was made in diameter 50 cm containing 1 tubers (Ranging in
size between 5-7.5 cc) and soil previously infested with R. solani (Anastomosis
group AG-3 and was obtained from Plant Pathology Research Institute, ARC, Egypt)with
4 replicates under greenhouse conditions were planted with each tested cultivar.
Infection percentage was recorded 90 days after planting.
Biochemical studies: Biochemical studies associated with healthy and
inoculated plants with R. solani on two cultivars Draga, the low susceptible
cv. and Lady Rosetta, the highly susceptible cv. were carried out. The investigated
parameter included changes in phenolic compounds, fraction of phenolic compounds,
oxidative enzymes and fraction of thio amino acid at 20, 40 and 60 days after
Determination of the phenolic compounds content: The phenolic compounds
were determined in leaves and roots of the tested cultivars of potato which
were inoculated separately with isolate of R. solani to determine total,
free and conjugated phenolic compounds. The phenolic compounds content was colorimetrically
determined using the folin reagent according to Snell and
Fractionation of phenolic compounds: HPLC system (HP1050) was used to
detect and to determine some phenolic compounds from the plant tissue (Coumarin,
Caffic acid, p. coumaric acid, Benzoic acid, Resoricinal and Apegenin). Ten
gm of fresh tissues of each sample were homogenized with methanol 40% and stirred
on a shaker. The extract was filtered through a whatman filter paper No. 1 and
the solvent was evaporated in vacuum. The dried residues containing phenol compounds
were dissolved in a solution consists of methanol: water: Acetic acid, 40: 59.3:
0.7, v: v: v) and stored in vials. The separation and determination were performed
on C18 column, according to Gertz (1990).
Determination of oxidative enzymes: The oxidative enzymes were determined
in leaves and roots of the tested cultivars of potato which were inoculated
separately with isolate of R. solani to determine peroxidase (PO) and
polyphenoloxidase (PPO) activity. The enzyme extraction from all samples was
prepared as recommended by Maxwell and Bateman (1967).
Peroxidase activity was estimated according to the method of Allam
and Hollis (1972) and the activity of polyphenoloxidase was measured using
the colorimetric method of Maxwell and Bateman (1967).
Fractionation of thio amino acids: HPLC system (HP1050) was used to
detect and determine thio amino acids from the plant tissue (Cysteine, cystine
and Methonine). Free amino acids were extracted according to the method proposed
by Shad et al. (2002). The separation and determination
were performed on C18 column according to Gertz (1990).
Statistical analysis: The statistical analysis was computed using analysis
of variance procedure described by Snedecor and Cochran (1980).
The significant differences between treatment means were separated by Duncan,s
Multiple Range Test (Duncan, 1955).
Reactions of different potato cultivars to R. solani: Nine Egyptian
potato cultivars, i.e., Diamont, Draga, Hermes, Cara, Lady Rosetta, Monalisa,
Mondial, Nicola and Spunta were evaluated, under greenhouse conditions, for
their reactions against the most pathogenic R. solani Sharkias
isolates code No. 18. Data presented in Table 1 show significant
differences between nine cultivars. The highest percentage of infection by stem
canker was obtained on Lady Rosetta (18.6%) and followed by Monalisa (18.1%).
||Reactions of potato cultivars against the highly pathogenic
Rhizoctonia solani isolate under green house conditions
|Each value represents the Mean±SE (Standard Error)
and mean of 4 replicates, Values in the same column with the same letter
are not significant at (p≤0)
The lowest percentage of infection was obtained with Draga (5.3%), Cara (6.4%)
and Diamont (6.6%). On the other hand, the same trend was observed in reaction
to black scurf symptom for the disease. The highest percentage of infection
by black scurf was obtained on Lady Rosetta (10.3%) followed by Monalisa (9.6%).
The lowest percentage of infection was obtained for Draga (3.3%) and Cara (3.5%).
Biochemical changes in susceptible and resistant potato cultivars:
Phenolic contents: Data presented in Table 2, reveal that
leaves and roots attained higher levels of free, conjugate and total phenol
contents in inoculated plants compared with uninoculated ones, in roots compared
to leaves and in resistant cultivar (Draga) compared with susceptible cultivar
(Lady Rosetta). Twenty days after inoculation the free, conjugated and total
phenols content were higher in inoculated treatments compared to the uninoculated
plants in each of Draga and Lady Rosetta cultivars. Forty days after inoculation,
similar trend was observed, high content of free (9.0 mg), (7.7 mg), conjugate
and total phenols (16.7) in Draga. The corresponding figures in healthy treatments
were 8.3, 7.0 and 15.3 mg, respectively. In Lady Rosetta the same trend was
observed in the inoculated leaves and root. Sixty days after inoculation, high
content of free, conjugate and total phenols in Draga was observed being 9.6,
7.6 and 17.2 mg, respectively. The corresponding figures were 9.1, 6.8 and 15.9
mg, respectively. In Lady Rosetta the same trend was observed in the inoculated
leaves and root.
Fractionation of phenolic compound: Data in Table 3,
reveal that leaves attained higher levels of six phenolic compounds in inoculated
plants compared to uninoculated ones. Twenty days after inoculation coumarin
and caffic acid content were higher in inoculated leaves compared to the uninoculated
ones in each of Draga and Lady Rosetta cultivars. Draga, the resistant cv, had
higher percentage of phenolic compound than that in Lady Rosetta i.e., the susceptible
Forty days after inoculation, a different trend was observed as the appearance
of four phenolic compound (p-coumaric acid, benzoic acid, resorcinol and apigenin)
in Draga and two phenolic compound (p-coumaric acid, benzoic acid) in Lady Rosetta
were recognized. High percentage of p-coumaric acid and Benzoic acid in inoculated
Draga leaves that showed 6.79 and 0.92 mg, compared to the uninoculated healthy
ones 4.83 and 0.74 mg, respectively. In Lady Rosetta the same trend was observed
in the inoculated leaves. Sixty days after inoculation, trend similar to that
of 40 days was observed as the appearance of four phenolic compounds (p-coumaric
acid, benzoic acid, resorcinol and apigenin) in Draga and two phenolic compounds
(p-coumaric acid, benzoic acid) in Lady Rosetta. High percentage of p-coumaric
acid and benzoic acid in Draga the inoculated leaves showed 2.87 and 0.59 mg,
respectively than the uninoculated 1.92 and 0.48 mg, respectively. In Lady Rosetta
the same trend was observed in the inoculated leaves but the amount and its
increase were limited, compared to Draga cultivar.
||Free, conjugate and total phenol contents in inoculated and
uninoculated resistant potato cv. Draga (R) and susceptible cv. Lady Rosetta
(S) cultivars 20, 40 and 60 days after sowing under green house conditions
||Phenolic compounds in inoculated and uninoculated resistant
potato cv. Drage and susceptible cv. Lady Rosetta 20, 40 and 60 days after
sowing under green house conditions
|I*: Inoculated, H**: Healthy (uninoculated)
Peroxidase and polyphenoloxidase activities: Data presented in Table
4, show that roots exhibited higher levels of PO and PPO activities than
shoots in all sampling and in inoculated and uninoculated treatments. The activities
of PO and PPO were higher in either inoculated leaves or roots compared to the
healthy uninoculated plants, 20 days after inoculation in Draga and Lady Rosetta
cultivars. The resistant Draga, the resistant cultivar, had higher levels of
activities of PO and PPO than the susceptible Lady Rosetta one.
Forty days after inoculation, the PO and PPO activities in Draga were higher
in root samples than in the leaves after inoculation. In Lady Rosetta, the inoculated
leaves showed higher PO activity (2.52 mg) than the healthy uninoculated one
(2.23 mg). On the other hand, PPO activities were almost alike in inoculated
and uninoculated plants either in leaves or root samples. Sixty days after inoculation,
in Draga the activity of PO in inoculated leaves was higher (2.68 mg) than that
in uninoculated leaves (2.47 mg) and the same trend in inoculated and uninoculated
roots. In Lady Rosetta the activity of PPO in inoculated leaves was higher (2.48
mg) than that in healthy uninoculated leaves (2.27 mg) and the same trend in
inoculated and uninoculated roots. Generally, activity of PO was higher in the
3 intervals and in inoculated and uninoculated samples as well the activity
of PPO showed a tendency of an increase but to a limited extent.
Thio-amino acids fractionation: Three thio-amino acids cysteine, cystine
and methionine were chromatographically identified in the leaves and root of
Draga and Lady Rosetta cultivars using the High Performance Liquid Chromatography
These thio-amino acids cysteine, cystine and methionine were determined at
20, 40 and 60 days after inoculated and uninoculated plants with R. solani.
Data presented in Table 5 reveal that leaves and roots showed
higher levels of thio-amino acids cysteine, cystine and methionine content in
inoculated plants compared to uninoculated ones. Twenty days after inoculation,
the thio-amino acids content were higher in inoculated leaves or roots compared
to the uninoculated plants in each of Draga and Lady Rosetta cultivars. Forty
days after inoculation, a similar trend was observed as higher content of cysteine,
cystine and methionine in Draga, the inoculated leaves showed activity 0.78,
0.64 and 0.59 mg, respectively compared to the healthy uninoculated 0.67, 0.55
and 0.42 mg, respectively.
||Activity of peroxidase and polyphenoloxidase in inoculated
and uninoculated Draga, the resistant cv. and Lady Rosetta, the susceptible
one with R. solani 20, 40 and 60 days after inoculation
|PO*: Peroxidase, PPO**: Polyphenoloxidase
||Relative concentration of cysteine, methionine and cystine
in inoculated or uninoculated potato cultivars differed in their susceptibility
to R. solani 20, 40 and 60 days after sowing under green house conditions
The same trend was observed in the inoculated roots. In Lady Rosetta, similar
conclusion may be recognized in the inoculated leaves and roots.
Sixty days after inoculation, high content of cysteine, cystine and methionine
was observed in Draga, the inoculated leaves showed activity 0.51, 0.41 and
0.29, respectively than the healthy uninoculated 0.43, 0.36 and 0.22 mg, respectively
and the same trend was observed in the inoculated roots. In Lady Rosetta the
contents of cysteine, cystine and methionine were very low amount (tras) in
the inoculated and uninoculated leaves and roots. In general, leaves and roots
of Draga and Lady Rosetta cultivars recorded higher levels of thio-amino acids
(cysteine, cystine and methionine) in inoculated plants 20, 40 and 60 days after
planting, being more pronounced in the resistant Draga compared to the healthy
uninoculated plants and the susceptible cultivar, Lady Rosetta, respectively.
Potato cultivars differed in their susceptibility to stem canker and black
scurf. Cultivar reactions of the most commonly used commercial cultivars of
potato were tested. Data obtained from the nine tested cultivars were grouped
into three categories. Lady Rosetta was the most susceptible cultivars followed
by the cultivars Monalisa and Mondial. Spunta, Nicola and Hermes cultivars showed
moderate susceptibility while Draga, Cara and Diamant, were most resistance.
Cultivar variation in disease resistant may be attributed to the differences
in the plant morphology, anatomy and bio-chemical components of either tubers
or plants. The results reported herein are in accordance with those mentioned
by Hide et al. (1973), along with those of Carling
and Leiner (1990) and Demirci and Doken (1993) who
showed that most isolates of Rhizoctonia solani AG-3 were moderately
to highly virulent on King Edward and Resy potato cultivars.
This study was undertaken in the principal to studies different changes in
chemical components of the inoculated tubers and plants compared to the corresponding
Free, conjugated and total phenol contents were considered in leaves and root
samples of the highly resistant cv. Draga and the highly susceptible cv. Lady
Rosetta, 20, 40 and 60 days after planting and after inoculation with R.
solani. Results showed that free, conjugated and total phenol contents were
increased in the two potato cultivars but the increase in cv. Draga was higher
than cv. Lady Rosetta.
It is well established that among the measures by which plants express resistance
being through plant secondary metabolites. Among them, phenolic compounds are
known to impart resistance against fungal diseases. This could be explained
on base of the fact that phenolic compounds are toxic to several plant pathogens
Phenols and their oxidation products are involved in various stages of host
parasite relationship and are associated with disease syndrome of plant and
plant resistant phenomena. There was a clear correlation between the oxidative
enzymes, phenolic compound contents and resistance (Jindal,
Farkas and Kiraaly (1962) found that the participation
of an endogenous supply of phenolic compounds in plant disease resistance is
depended upon the activity of oxidative enzymes (polyphenol oxidase, peroxidase
and catalase). Phenols are oxidized to quinone or semi-quinones which play a
great role as antimicrobial agents. Some phenols have been found in all plants
after infection as response to the ingress of pathogen and their appearance
is considered as part of an active defense response. Since the first suggestion
that phenolic intermediates have a role in the active expression of resistance
the localization and timing of the host response (Stoessl,
Oxidative enzymes, i.e., peroxidase and polyphenoloxidase were considered in
leaves and root samples of the highly resistant cv. Draga and susceptible cv.
Lady Rosetta 20, 40 and 60 days after planting and inoculation with R. solani.
The obtained results showed that the inoculated potato plants showed a higher
peroxidase and polyphenoloxidase activity than the healthy uninoculated ones.
Also, the moderately resistant cultivar had higher peroxidase and polyphenoloxidase
activity than the susceptible one. These differences in enzymatic activities
might be attributed to that oxidative enzymes play a partial role in activating
inducible defence of plant (Vera-Estrella et al.,
The high increment in enzymatic activity happened in early stages in resistant
cultivar and successfully challenged the infection progress and by time, that
enzymatic activity is being elevated to the normal levels (like in the healthy).
While on the contrary, in the susceptible cultivar enzymatic activity increased
gradually with time after inoculation but with low levels and failed to challenge
the infection progress.
Fractionation of thio amino acids in leaves and root samples of resistant cv.
Draga and susceptible cv. Lady Rosetta 20, 40 and 60 days after planting and
inoculating with R. solani showed that thio amino acid contents increased
in the two potato cultivars but the increasing in cv. Draga was higher than
cv. Lady Rosetta.
The increasing in free amino acids contents in the inoculated tissues may be
ascribed to the decomposition of host protein or to the decrease in protein
synthesis (Sempio and Marte, 1968).
On other hand, the increase in amount of tyrosine and phenylalanine were observed
with inoculation, dont result of protein breakdown but the increase in
concentration of the aromatic amino acids due to specific synthesis for phenolic
compounds (Vir and Grewal, 1975).
Fractionation amino acids showed that the infection in general, caused several
changes in these fractions such as absence or presence of certain components
and increase or decrease in the amount of other ones. These data are in harmony
with those obtained by Farag et al. (1986).