Professional Documents
Culture Documents
PotatoBiotec 2009
PotatoBiotec 2009
Fruit, Vegetable and Cereal Science and Biotechnology ©2009 Global Science Books
ABSTRACT
Potato Late Blight is the main pathogen which affects potatoes. Due to unavailability of effective chemical control methods and the
intensive use of fungicides, the introduction of resistant cultivars represents a safe strategy. The sources of horizontal resistance to Late
Blight in the Argentinean Potato Breeding Programs have been varied. One of the interesting species to evaluate is Solanum tarijense
since its adaptation to long days is promissory, producing tubers of good size and aspect. In this study, the enzymatic activity of different
PR proteins in the specie S. tarijense was measured. These clones were more resistant to Late Blight than ‘Bintje’ and ‘Pampeana INTA’
cultivars. The activity of glucanases, chitinases, peroxidases and polyphenoloxidases were 15, 1.5, 2 and 3 times higher respectively, in S.
tarijense than in ‘Bintje’ and ‘Pampeana INTA’. These results suggest that some PR proteins can be used as biochemical markers to assist
breeders in the selection of horizontal resistant cultivars.
_____________________________________________________________________________________________________________
also involved in the formation of precursors of substances weekly readings were carried out beginning 7 days after the inocu-
that act as physical barriers (Avdiushko et al. 1993). PPO lation. For each clone it was determined the Area Under the Dis-
catalyzes the formation of highly reactive quinones (Mayer ease Progress Curve (AUDPC), as expression of the behaviour of
and Harel 1979). Quinones may act in several ways leading the plant in relation to the pathogen (Shaner and Finney 1977).
to protection of plants, for example, they can limit the n
development of diseases at the infected sites by accelerating AUDPC = ∑ ((X i+1 + Xi)/2) (ti+1 – ti)
the death of cells close to the infection site, by preventing i=1
the advance of infections and/or by generating a toxic where Xi, is the tissue percentage affected in the observation i,
environment, which will inhibit the growth of pathogens t, is the time in days of the observation i,
inside the cells (Bi and Felton 1995). Other enzymes such n, is the number of observations
as phenylalanine ammonia-lyase (PAL) (Beaudoin-Eagan The trials were carried out during the 2005/2006, 2006/2007
and Thorpe 1985; Dixon and Harrison 1991; Dalisay and and 2007/2008 cycles. During the 2006/2007 cycle resistance re-
Kuc 1995) are involved in phytoalexin or phenolic com- sults were not obtained in the field since the climatic conditions
pound biosynthesis. Such enzymes have been related with did not allow the development of the disease.
defense against pathogens in several plants, including to-
bacco (Beaudoin-Eagan and Thorpe 1985; Goy et al. 1992), Evaluation of in vitro resistance
tomato (Bashan et al. 1985), cucumber (Rasmussen et al.
1991) and rice (Rajappan 1995). Foliar infection with Late Blight was assessed in individual potato
The development of a system of biochemical markers, plants. The plants were cultivated in a growth chamber. During
involved in the horizontal resistance to P. infestans, may plant growth, the temperature ranged between 15-24°C and natural
help to implement a quick and economic technique to be daylight was supplemented by high-pressure sodium lamps (400
used in the selection of resistant clones. The quick identi- W) in 14-10 hours day-night cycle. Plants were irrigated with a
fication of resistant potato cultivars will contribute to the in- sprinkler system when needed. Three experiments with a ran-
tegrated management of the disease, with smaller economic, domized complete design were performed during 2006 and 2007.
environmental and human costs. Each experiment consisted of three replications each with 50
The objectives of this work were: i) to quantify proteins plants.
related with the horizontal resistance to P. infestans in po- The foliage of plants after 30 days of emergence (period of
tato clones and cultivars of well-known resistance and ii) to maximum expression of resistance genes) was cut and the leaves
select biochemical marker(s) that show a better correlation placed in 9 cm Petri dishes on 2% water agar. The leaves were
with the resistance response to Late Blight. The constitutive inoculated with P. infestans sporangial suspension (9.0 × 103 spo-
levels of the molecules related with the horizontal resis- rangia mL-1) under laboratory conditions (“detached-leaf” test)
tance may differ in resistant and susceptible potato clones to (Goth and Keane 1997). The assay was then placed in a growth
P. infestans. Therefore, the proteins profiles related with the chamber at 18-20°C and 80% relative humidity during the rest of
horizontal resistance in potato can provide evidence of the the experiment. Disease development was recorded daily from 4 to
metabolites that are responsible for the resistance and it 11 days after inoculation. Disease severity was estimated visually
may help to the selection of promising clones in breeding as the percentage foliage showing symptoms.
programs.
Extraction of β-1,3-glucanases and chitinases
MATERIALS AND METHODS
Leaves without infection, from each clone and cultivar were
Biological material homogenized with liquid nitrogen. The homogenized tissue was
rinsed with sodium acetate 50 mM pH 5.2 (anhydrous, CH2COONa,
A strain of P. infestans (race 2, 3, 6, 7, 8, 9; mating type A2), was J.T. Baker), 0.1% sodium metabisulfite (granular, Na2S2O5, Mal-
grown on rye-agar medium (Agar bacteriological, Agar Nº1, linkrodt Chemicals) and activated charcoal (Cat. C7606, Sigma-
LP0011, OXOID Ltd, Basingstoke, England) and on potato tuber Aldrich) and filtered through a 0-20 mm nylon filter. For each
slices. Slices were incubated in closed plastic boxes with water gram of tissue 4 volumes were used. The tissue extract was cen-
embedded paper (approximately 90% relative humidity), and were trifuged at 12.000 rpm for 10 min. The supernatant was dialyzed
maintained in darkness at 18°C. After 7 days the mycelium that with 50 mM sodium acetate pH 5.2 (Cat. S7899, Sigma-Aldrich).
developed on the slices was washed with distilled water, then fil- All operations were carried out at 0-4°C. The supernatant was
tered through 20 µm Nalgene filters and placed for 2 h at 4°C for transferred to a 1.5 ml vial and stored at -80°C.
the release of zoospores. The concentration of the inoculum was
determined by counting the sporangia in a Neubauer chamber and β-1,3-glucanase assay
finally the concentration was adjusted to assays.
Potato cultivars of Solanum tuberosum L., ‘Pampeana INTA’ β-1,3-glucanase activity was assessed by the rate of production of
and ‘Bintje’ and Solanum tarijense clones, Oka 7494.3 and Oka reducing sugar using laminarin (from Laminarina digitata,
5874.33, were provided by the Balcarce Experimental Station of EC232.712-4, Cat. L9634, Sigma-Aldrich) as substrate. The reac-
the Instituto Nacional de Tecnología Agropecuaria (INTA), Argen- tion mixture consisted of 0.1 ml of 500 mM sodium acetate pH 5.2
tina. Solanum tuberosum L. cv. ‘Pampeana INTA’ (MP1 59.789/12 containing 1% laminarin and 0.1 ml of enzyme extract. After 20
× ‘Huinkul MAG’) is a cultivar from the Argentine Breeding Prog- min incubation at 37°C the enzyme reaction was stopped by heat-
ram (INTA-Balcarce). ing in boiling water for 2 min. Aliquots (0.1 ml) were taken and
reducing sugar was determined as described by Ashwell (1957).
Evaluation of field resistance Glucose (puriss Cat. G8270, Sigma-Aldrich) was used as a stan-
dard. Activity is expressed in units per gram of fresh weight,
Clones Oka 7494.3, Oka 5874.33 and cvs. ‘Bintje’ and ‘Pampeana where 1 U = 1 µmol glucose released/h/ml enzyme.
INTA’ were planted in randomized complete blocks with two repli-
cates. Four plants per experimental unit (clone) were planted in Chitinase assay
two contiguous rows. Two flanking rows were planted with sus-
ceptible ‘Bintje’ and with moderate resistant ‘Pampeana INTA’, Chitinase activity was assessed by measuring the rate of N-acetyl-
both without major genes and used as reference cultivars. The glucosamine production using chitin purified powder from Crab
plants were inoculated 40 days after planting with a sporangia sus- Shells (Poly (N-acetyl-1,4-β-D-glucopyranosamine Cat. C9752,
pension of P. infestans. The concentration was adjusted at 9.0 × Sigma-Aldrich) as the substrate. The reaction mixture consisted of
103 sporangia/ml. Supplementary watering was applied in order to 60 µl of enzyme extract, 100 µl of partially hydrolyzed chitin in
give the appropriate conditions of humidity for the development of 50 mM sodium acetate buffer pH 5.2 and 80 µl of 50 mM sodium
the disease. The percentage of affected foliage was determined for acetate buffer pH 5.2. After 5 hrs of incubation at 37°C the en-
each plot using the scale of the International Potato Centre. Four zyme reaction was stopped by centrifugation for 10 min at 12,000
Biochemical markers involved in horizontal resistance in potato. Wolski et al.
× g. Aliquots (100 µl) of the supernatant were separated and 20 µl Table 1 Relative Area Under the Disease Progress Curves (AUDPC), in
of 0.8 M borate pH 9.1 (Buffer solution, puriss p.a. Cat. 82576, potato clones of S. tarijense and in potato cultivars. Seasons 2005/2006
Fluka AG, Switzerland) and 0.5 ml of the DMAB (4-dimethyl- and 2007/2008.
aminobenz-aldehyde, C9H11NO, puriss p.a. Cat. 02560, Fluka) Cultivar or Clone AUDPC 2005/6* AUDPC 2007/8**
were added. The mixture was incubated at 37°C for 20 min and Bintje 189.83 1853.5
absorbance at 585 nm was measured. N-actylglucosamine (N- Pampeana INTA 94.25 1343.4
Acetylglucosamine 6-sulfate sodium salt, H14NO9SNa, Cat. 44001, S. tarijense Oka 7494.3 38.38 1496.7
Sigma-Aldrich) was used as a standard (Reissig et al. 1955). Acti- S. tarijense Oka 5874.33 36.25 1380.6
vity is expressed in units per gram of fresh weight, where 1 U = 1 * Season with low disease incidence
µg of N-acetylglucosamine/h/ml of enzyme extract. ** Season with high disease incidence
50 Pampeana
Aldrich), (HCl, pro analysis, Merck, Argentina). The mixture was Oka 7494.3
incubated at 37°C for 70 min. The amount of trans-cinnamic acid 40
Oka 5874.33
formed from L-phenylalanine was measured spectrophotometric- 30
ally at 290 nm (UV). PAL (from Rhodoturala glutinis, Sigma-
Aldrich) was used as the standard. Activity is expressed in µg of 20
trans-cinnamic acid/g fresh weight/h. ns
10 ns
POX assay 0
48 72 96
Time post infection (hs)
POX activity (Chan and Tian 2006) was analyzed using guaiacol
as substrate (Yao and Tian 2005). The reaction mixture consisting
of 0.5 ml of crude extract, 2 ml of guaiacol (2-(CH3O)C6H4OH,
Cat. G5502, Sigma-Aldrich) substrate (100 mM sodium phosphate, B
pH 6.4 (monobasic, monohydrate, granular, NaH2PO4H2O, Millin-
ckrodt Chemicals) and 8 mM guaiacol) incubated for 5 min at
30°C. The increase in absorbance at 460 nm was measured spec-
trophotometrically after the addition of 1 ml H2O2 (24 mM) (hyd-
rogen peroxide solution, Cat. H3410, Sigma-Aldrich). Enzymatic
activities were defined as the increase of absorbance, and one unit
was defined as the increase in one absorbance unit per minute
under the conditions of the assay.
Statistical analysis
Data were evaluated by analysis of variance procedures and by
linear regression procedures using the routines included in SAS/
SAT (SAS Inst. 1990). For comparisons of averages Tukey’s test S. tarijense Oka 7494.3 S. tarijense Oka 5874.33
was used at P = 5%.
Fig. 1 “In vitro” evaluation of resistance. Detached leaves of plants after
RESULTS 30 days of emergence were inoculated with 5 µl of a suspension of 9.0 ×
103 sporangia/ml of P. infestans under laboratory conditions. (A) Progress
Evaluation of field resistance of the area of foliage injured after 24, 48 and 96 h post-infection. (*)
Statistically different at 5% according to Tukey’s test; (ns) not significant.
A significant correlation (R2 = 0.67) was found between (B) 96 h post-infection.
both AUDPC seasons. Table 1 shows that for both seasons
S. tarijense clones Oka 7494.3 and Oka 5874.33 had a
lower AUDPC than ‘Bintje’ (susceptible) and ‘Pampeana
Fruit, Vegetable and Cereal Science and Biotechnology 3 (Special Issue 1), x-y ©2009 Global Science Books
A B
50000 7000 a
a
ß-1,3-Glucanases activity
a
6000
Chitinases activity
40000 b a
5000
a
(U/g FW)
(U/g FW)
30000 4000
20000 3000
c 2000
10000
c 1000
0 0
tj e
tj e
.3
33
.
an
.3
an
94
94
.
in
in
74
74
pe
pe
74
74
B
B
58
58
m
m
ka
ka
Pa
ka
Pa
ka
O
O
O
O
se
e
e
ns
e
en
ns
s
en
ije
rij
e
rij
ar
rij
ta
ta
ta
S.
S.
S.
S.
C D
20000 10000 a
a
POX activity (U/g FW)
b 6000
10000 b
4000 b
b
b
5000
2000
0 0
tj e
.3
33
tj e
an
.
an
.3
94
94
.
in
in
74
74
pe
pe
74
74
B
B
58
58
m
m
ka
ka
Pa
Pa
ka
ka
O
O
O
O
e
se
ns
e
e
s
en
ns
en
ije
r ij
ar
rij
rij
ta
ta
ta
S.
S.
S.
S.
Fig. 2 Constitutive levels of PR-proteins activities in potato cultivars and Solanum tarijense clones. (A) β-1-3 Glucanases activity, (B) Chitinases
activity, (C) Peroxidases activity, and (D) Polyphenoloxidases activity. Bars represent the mean values ± SD of three independent experiments, with five
replicates per experiment. Columns with the same letter are not statistically different at 5% according to Tukey’s test.
Constitutive levels of PR protein activities In addition, many authors have demonstrated that there
is a strong correlation between the levels of horizontal resis-
S. tarijense clones showed higher constitutive levels of PR- tance and the increase in the expression of genes that code
proteins activity than ‘Bintje’ and ‘Pampeana INTA’, which for PR proteins, suggesting that their constitutive expres-
were used as standards (Fig. 2). Significant differences sion or their induction could contribute to the horizontal re-
were observed in glucanase, chitinases, POX and PPO acti- sistance against P. infestans in Solanum species (Vleeshou-
vities among potato clones of S. tarijense and ‘Bintje’ and wers et al 2000; Tuzun 2001; Evers et al. 2003, 2004). Spe-
‘Pampeana INTA’. The enzymatic activities were 15, 1.5, 2, cifically, Vleeshouwers et al. (2000) reported a positive cor-
and 3 times higher, respectively for S. tarijense clones than relation between the constitutive levels of RNAm of: PR-1
for ‘Bintje’ and ‘Pampeana INTA’. (protein with antimicrobial activity), PR-2 (β-1,3-gluca-
A linear relationship was found between β-1,3-gluca- nases) and PR-5 (thaumatin like proteins) and the resistance
nase and PPO activity levels against AUDPC season 2007/ of potato cultivars to P. infestans, suggesting that the
2008 and in vitro resistance behaviour (Table 2). RNAm could serve as molecular markers in the breeding
Constitutive levels of PAL activity were not detected in programs. Recently, Ros et al. (2004) showed that the ex-
any of the cultivars and clones examined in this study. pression of PR-1, PR-2, PR-3 and PR-5 differed in suscep-
tible and moderately resistant potato cultivars to P. infestans.
DISCUSSION On the other hand, the loci corresponding to genes related
to PAL and other genes of the defense mechanism in plants
It has been reported that the expression of PR proteins, PPO, were associated with the quantitative resistance to P. infes-
PAL, phytoalexins and phenols differs with the level of re- tans (Trognitz et al. 2002). In other Solanum species, for
sistance of the different potato cultivars. For example, example in tobacco, various resistant lines to Peronospora
cultivars with high resistance level to P. infestans showed tabacina, obtained from wild species through breeding,
an early expression and larger accumulation of these com- showed the presence of high glucanase and chitinase levels
pounds (Bi and Felton 1995; Dalisay and Kuc 1995; Tonón before the attack of the pathogen, as likewise the early in-
et al. 1998; Andreu et al. 2001; Conrath et al. 2001). duction of the same enzymes after the attack (Robertson
1995; Tuzun et al. 1997). Also, resistant tomato clones to
Alternaria solani, expressed high constitutive levels of β-
Table 2 Ordinate (a), slope (b) and coefficient of determination (R2) of 1,3-glucanases and chitinases compared to the susceptible
the linear regressions between β-1,3-glucanase, PPO, AUDPC and in plants (Lawrence et al. 1996, 2000). Therefore, the constitu-
vitro resistance. tive expression of PR proteins in plants with high levels of
a b R2 natural resistance to the disease is of great importance, sug-
PPO vs AUDPC 2007/2008 1802.4 -0.0577 0.52 gesting that these proteins would have a role as defense bar-
PPO vs in vitro resistance 50.4 -0.0074 0.56 riers (Edreva 2005). Also, it has been shown that chitinases,
β-1,3-glucanase vs AUDPC 2007/2008 1710.7 -0.0086 0.57 either alone or in combination with β-1,3-glucanases, can
β-1,3-glucanase vs in vitro resistance 38.3 -0.0011 0.58 inhibit the in vitro growth of different fungi (Mauch et al.
Biochemical markers involved in horizontal resistance in potato. Wolski et al.
1988). Tonon et al. (2002), suggest that β-1,3-glucanases Científicas y Tecnológicas (CONICET), authors gratefully ack-
may have a role in field resistance. Differential expression nowledge Dr. Mariana Laura Feldman from Universidad Nacional
of β-1,3-glucanases was observed between ‘Pampeana de Mar del Plata for their useful and critical comments to the
INTA’ and ‘Bintje’. In the moderately resistant cultivar original version and Agronomists Pablo Leandro Cicore from
(‘Pampeana INTA’), β-1,3-glucanases induction (four-fold Facultad de Ciencias Agrarias (UNMdP) and Patricia Alejandra
with respect to healthy tubers) occurred 14 hours after ino- Suarez from CONICET for their useful data analysis and revision
culation and remained over basal levels at 38 hours after of manuscript. E. A. Wolski is a postdoctoral fellow of CONICET.
inoculation with P. infestans. On the contrary, in susceptible S. Korgan is a fellow of ANPCYT. S. Capezio is a professor of
‘Bintje’, β-1,3-glucanases were induced at lower levels than UNMdP. M. A. Huarte is an established researcher from INTA. A.
those observed in ‘Pampeana INTA’. Also, β-1,3-glucanases B. Andreu is an established researcher from CONICET.
purified from ‘Pampeana INTA‘ showed antifungal activity
against P. infestans, by inhibiting the sporangia germination. REFERENCES
POX has been associated with resistance of potato to
Late Blight (Umareus 1959). An increase of POX activity Andreu AB, Oliva CR, Distel S, Daleo GR (2001) Production of phytoalexins,
upon infection by P. infestans has been described and rates glycoalkaloids and phenolics in leaves and tubers of potato cultivars with dif-
ferent degrees of field resistance after infection with Phytophthora infestans.
of accumulation may differ slightly between resistant and Potato Research 44, 1-9
susceptible clones (Dowley et al. 1991). Evers et al. (2004) Ashwell G (1957) Colorimetric analysis of sugars. Methods in Enzymology 3,
and Lozoya-Saldaña et al. (2007) detected an increase in 73-105
POX activity in clones with horizontal resistance after in- Avdiushko SA, Ye XS, Kuc J (1993) Detection of several enzymatic activities
fection by P. infestans. The increase was much more pro- in leaf prints cucumber plant. Physiological and Molecular Plant Pathology
nounced in the resistant clones as compared to the suscep- 42, 441-454
tible one. The relation of POX activity to resistance pheno- Bashan Y, Okon Y, Henis Y (1985) Peroxidase, polyphenol-oxidase, and phe-
mena could be explained by its function as a generator of nols in relation to resistance against Pseudomonas syringae pv. tomato in
toxic free radicals and its involvement in the production of tomato plants. Canadian Journal of Botany 65, 366-372
Beaudoin-Eagan LD, Thorpe TA (1985) Tyrosine and phenylalanine ammonia
lignin and phenolic compounds (Reuveni 1998). lyase activities during shoot initiation in tobacco callus cultures. Plant Physi-
Recently, Li and Steffens (2002) have obtained direct ology 78, 438-441
evidence of such role for PPO in plants. These authors ob- Bi JL, Felton GW (1995) Foliar oxidative stress and insect herbivory: primary
served that transgenic plants of tomato overexpressing PPO compounds, secondary metabolites, and reactive oxygen species as compo-
had a higher oxidizing capacity and displayed increased nents of induced resistance. Journal of Chemical Ecology 21, 1511-1530
resistance to Pseudomonas syringae pv. tomato. Black W (1970) The nature of inheritance of field resistance to Late Blight
Our results were in agreement with those obtained by (Phytophthora infestans) in potatoes. American Potato Journal 47, 279-288
the authors previously mentioned. The S. tarijense clones Chan Z, Tian S (2006) Induction of H2O2-metabolizing enzymes and total pro-
had higher resistance levels, than ‘Bintje’ (susceptible) and tein synthesis by antagonistic yeast and salicylic acid in harvested sweet
cherry fruit. Postharvest Biology and Technology 39, 314-320
‘Pampeana INTA’ (moderately resistant) cultivars (Table 1, Chen Ch, Belanger RR, Benhamou N, Paulitz TC (2000) Defense enzymes
Fig. 1), and showed higher constitutive levels of β-1,3-glu- induced in cucumber roots by treatment with plant growth-promoting rhizo-
canases, POX and PPO activities than the reference culti- bacteria (PGPR) and Pythium aphanidermatum. Physiological and Molecular
vars (Fig. 2). Plant Pathology 56, 13-23
PAL regularly exhibits greater activity or the novo syn- Conrath U, Thulke O, Katz V, Schwindling S, Kohler A (2001) Priming as a
thesis in the diseased tissues or in resistant genotypes and is mechanism in induced systemic resistance of plants. European Journal Plant
a precursor of most of the phenolic compounds, including Pathology 107, 113-119
phytoalexins and lignin (Dixon and Harrison 1991). The Dalisay RF, Kuc JA (1995) Persistence of induced resistance and enhanced
synthesis of these substances is related with the level of peroxidase and chitinases activities in cucumber plants. Physiological and
Molecular Plant Pathology 47, 315-327
genetic resistance of potato to the oomycete (Andreu et al. Distel S, Huarte MA (2000) Comparación en el tiempo de Poblaciones de Phy-
2001). Our results showed that constitutive levels of PAL tophthora infestans en Argentina. En: 19º Congreso de la Asociación Latino-
activity were not detected in none of the cultivars and americana de la Papa. La Habana, Cuba
clones tested. These results agree with those from Lozoya- Dixon RA, Harrison MJ (1991) Activation, structure, and organization of
Saldaña et al. (2007). However, these authors found that genes involved in microbial defense in plants. Advances in Genetics 28, 166-
after four weeks of infection by P. infestans, their action 234
increased suddenly in resistant cultivars and clones. The Dowley LJ, O’Sullivan E, Kehoe HW (1991) Development and evaluation of
lowest activity was detected in the susceptible cultivar blight resistant potato cultivars. In: Lucas JA, Shattock RC, Shaw DS, Cooke
(Lozoya-Saldaña et al. 2007). LR (Eds) Phytophthora, Cambridge University Press, Cambridge, pp 373-
382
Edreva A (2005) Pathogenesis-Related Proteins: Research progress in the last
CONCLUSIONS 15 years. Genetic and Applied Plant Physiology 31, 105-124
Evers D, Ghislain M, Hausman JF, Dommes J (2003) Differential gene ex-
Solanum tarijense clones Oka 7494.3 and Oka 5874.33 pression in two potato lines differing in their resistance to Phytophthora
showed high levels of resistance to Late Blight as likewise infestans. Journal of Plant Physiology 160, 709-712
high levels of activity of constitutive proteins PR, such as, Evers D, Welschbillig N, Dommes J, Hausman JF (2004) Biochemical and
β-1,3-glucanases, POX and PPO. morphological characterization of potato clones differing in their resistance
These results and future studies on S. tarijense - P. to Late Blight. Potato Research 46, 105-115
infestans interaction, will allow us to introgress potato Goth RW, Keane J (1997) A detached-leaf method to evaluate Late Blight
resistance in potato and tomato. American Potato Journal 74, 347-352
genes from S. tarijense into S. tuberosum in breeding prog- Goy AP, Felix G, Métraux P, Meins F Jr. (1992) Resistance to disease in the
rams as well as use some PR proteins as biochemical mar- hybrid Nicotiana glutinosa x Nicotiana debneyi is associated with high cons-
kers for the selection of new resistant clones. titutive level of β-1,3-glucanases, chitinases, peroxidase and polyphenoloxi-
The development of a system using biochemical mar- dase. Physiological and Molecular Plant Pathology 41, 11-21
kers will be a fast and economic technique for the selection Huarte M (2002) Niveles disponibles de resistencia al tizón tardío en Latino-
of resistant clones. Finally, these results will contribute to américa. In: Centro Internacional de la Papa, 2002. Fernandez-Northcote EN
the selection of promissory cultivars and their use in the (Ed) Memorias del Taller Internacional Complementando la Resistencia al
breeding programs for integrated pest management. Tizón (Phytophthora infestans) en los Andes, Feb. 13-16, 2001, Cochabamba,
Bolivia, GILB, Taller Latinoamérica 1, Lima, Peru, pp 59-66
Johnson J (1984) A critical analysis of durable resistance. Annual Review of
ACKNOWLEDGEMENTS Phytopathology 22, 309-330
Lawrence CB, Joosten MH, Tuzun S (1996) Differential induction of patho-
Research supported by Agencia Nacional de Promoción Científica genesis-related proteins in tomato by Alternaria solani and the association of
y Tecnología (ANPCYT), Instituto Nacional de Tecnología Agro- a basic chitinase isozyme with resistance. Physiological and Molecular Plant
pecuaria (INTA), Banco Santander Rio, Universidad Nacional de Pathology 48, 361-377
Mar del Plata (UNMdP), Consejo Nacional de Investigaciones Lawrence CB, Singh NP, Qiu J, Gardner RG, Tuzun S (2000) Constitutive
Fruit, Vegetable and Cereal Science and Biotechnology 3 (Special Issue 1), x-y ©2009 Global Science Books
hydrolytic enzymes are associated with polygenic resistance of tomato to Inst., Cary, NC
Alternaria solani and may function as an elicitor release mechanism. Physio- Shaner G, Finney RE (1977) The effect of nitrogen fertilization on the expres-
logical and Molecular Plant Pathology 10, 211-220 sion of slow mildewing resistance in Knox wheat. Phytopathology 67, 1051-
Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic 1056
tomato plants results in enhanced bacterial disease resistance. Planta 215, Tonon C, Guevara G, Oliva C, Daleo G (2002) Isolation of a potato acidic 39
239-247 kDa β-1,3-glucanase with antifungal activity against Phytophthora infestans
Lozoya-Saldaña H, Rivera-Hinojosa R, Colinas-León MT (2007) Phenols, and analysis of its expression in potato cultivars differing in their degrees of
peroxidase and phenylalanine ammonia lyase: their relationship to the genetic field resistance. Journal of Phytopathology 150, 189-195
resistance against Late Blight (Phytophthora infestans Mont. De Bary) in po- Tonon CV, Andreu AB, Aued ME, Van Damme M, Huarte MA, Daleo GR
tato (Solanum tuberosum L.) clones. Agrociencia 41, 479-489 (1998) Defense reactions in two potato cultivars after infection with two
Malcolmson J, Black W (1966) New R genes in Solanum demissum and their races of Phytophthora infestans. Potato Research 41, 319-325
complementary races of Phytophthora infestans (Mont.) De Bary. Euphytica Trognitz BR, Trognitz F (2004) Increasing the efficiency of potato breeding
15, 199-203 through marker assisted selection-general thoughts. Molecular markers for
Mantecón J, Van Damme M, Zamudio N, Lobo R, Huarte M (1995) Estudio Late Blight resistance-when applied for breeders? Plant Breeding and Sci-
de la resistencia a metalaxyl en aislamientos argentinos de P. infestans ence 50, 95-105
durante 1993-1995. 8º Congreso Latinoamericano de Fitopatología (ALF). Trognitz F, Manosalva P, Gysin R, Niño-Liu D, Simon R, Herrera R, Trog-
Mérida, Venezuela nitz B, Ghislain M, Nelson R (2002) Plant defense genes associated with
Mauch F, Mauch-Mani B, Boller T (1988) Antifungal hydrolases in pea tissue quantitative resistance to potato Late Blight in Solanum phureja x Dihaploid
II: inhibition of fungal growth by combination of chitinase and β-1,3-gluca- S. tuberosum Hybrids. Molecular Plant-Microbe Interactions 15, 587-597
nases. Plant Physiology 88, 936-942 Tuzun S (2001) The relationship between pathogen-induced systemic resistance
Mayer AM, Harel E (1979) Polyphenol oxidases in plants. Phytochemistry 18, (ISR) and multigenic (horizontal) resistance in plants. European Journal
193-215 Plant Pathology 107, 85-93
Micheletto S, Boland R, Huarte M (2000) Argentine wild diploid Solanum Tuzun S, Gay PA, Lawrence CB, Robertson TB, Sayler RJ (1997) Biotech-
species as sources of quantitative late blight resistance. Theoretical and Ap- nological applications of inheritable and inducible resistance to diseases in
plied Genetics 101, 902-906 plants. In: Cresshoff PM (Ed) Technology Transfer of Plant Biotechnology,
Rajappan K, Vidhyasekaran P, Raguchander T (1995) Elicitation of enzyme CRC Press, New York, United State, pp 25-40
activity by nickel nitrate in suspension cultured rice. Plant Disease Research Umaerus V (1959) Relationship between peroxidase activity in potato leaves
10, 142-145 and resistance to Phytophthora infestans. American Potato Journal 36, 124-
Rasmussen JB, Hammerschmidt R, Zook MN (1991) Systemic induction of 131
salicylic acid accumulation in cucumber after inoculation with Pseudomonas Van Damme M, Capezio S, Huarte MA (1998) Phenotypes of Phytophthora
syringae pv. siringae. Plant Physiology 97, 1342-1347 infestans isolated in Argentina during 1994 1996: mating type, metalaxyl
Reissig J, Strominger J, Leloir LF (1955) A modified colorimetric methods reaction and isozyme analysis. Fitopatología 33, 53
for the estimation of N-acetylaminosugars. The Journal of Biological Che- Vleeshouwers V, van Dooijeweert W, Govers F, Kamoun S, Colon L (2000)
mistry 217, 959-966 Does basal PR gene expression in Solanum species contribute to non-specific
Reuveni M (1998) Relationships between leaf age, peroxidades and β-1,3-glu- resistance to Phytophthora infestans? Physiological Molecular Plant Patho-
canase activity, and resistance to downy mildew in grapevines. Journal of logy 57, 35-42
Phytopathology 146, 525-530 Wang B, Liu J, Tian Z, Song B, Xie C (2005) Monitoring the expression pat-
Robertson TL (1995) Hydrolytic enzyme accumulation in tobacco lines resis- terns of potato genes associated with quantitative resistance to Late Blight
tance to Peronospora tabacina, the causal agent of blue mold. MSc thesis, during Phytophthora infestans infection using cDNA microarrays. Plant
Auburn University, Auburn, AL, USA, 87 pp Science 169, 1155-1167
Ros B, Thümmler F, Wenzel G (2004) Analysis of differentially expressed Yao HJ, Tian SP (2005) Effect of pre and post-harvest application of salicylic
genes in a susceptible and moderately resistant potato cultivar upon Phytoph- acid or methyl jasmonate on inducing disease resistance of sweet cherry fruit
thora infestans infection. Molecular Plant Pathology 5, 1-11 in storage. Postharvest Biology and Technology 35, 253-262
SAS Institute (1990) User’s Guide. Statistics. Version 6, (Vol I, 4th Edn), SAS