Chitosan physico–chemical properties modulate defense responses and resistance in tobacco plants against the oomycete Phytophthora nicotianae

Pesticide Biochemistry and Physiology 100 (2011) 221–228

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Chitosan physico–chemical properties modulate defense responses and resistance in tobacco plants against the oomycete Phytophthora nicotianae Alejandro Bernardo Falcón-Rodríguez a, Daimy Costales a, Juan Carlos Cabrera b, Miguel Ángel Martínez-Téllez c,⇑ a

Grupo de Productos Bioactivos, Departamento de Fisiología y Bioquímica Vegetal, Instituto Nacional de Ciencias Agrícolas (INCA), Carretera a Tapaste Km. 3½, San José de las Lajas, La Habana 32700, Cuba Unité biotechnologie, Materia-Nova, Rue des Foudriers 1, 7822 Ghislenghien, Belgium c Centro de Investigación en Alimentación y Desarrollo, AC (CIAD), Coordinación de Tecnología de Alimentos de Origen Vegetal (CTAOV), Carretera a la Victoria Km. 0.6, AP 1735, Hermosillo 83000, Sonora, Mexico b

a r t i c l e

i n f o

Article history: Received 9 December 2010 Accepted 15 April 2011 Available online 24 April 2011 Keywords: Chitosan Induced resistance Tobacco Phytophthora nicotianae PAL Peroxidase b-1,3-Glucanase Molecular weight Degree of acetylation

a b s t r a c t Enzymatic defense responses and protection against Phytophthora nicotianae were studied in tobacco plants treated with chitosan of different molecular weights (MW) and degrees of acetylation (DA). The concentration and mode of chitosan application affected enzymatic induction in tobacco leaves. b-1,3Glucanase (EC 3.2.1.6) activity required 10 times the polymer concentration relative to the oligochitosan mixture to induce the highest activity above control when treated by foliar spray, indicating the influence of molecular weight in this response. PAL (EC 4.3.1.5) and POD (EC 1.11.1.6) activities increased above control as a result of the influence of polymer degree of acetylation when treatments were applied by foliar spray. A higher DA favored PAL activity, whereas a lower DA induced higher POD activity. Using an in vitro bioassay, it was found that the three chitosan compounds caused a reduction of the infection index of P. nicotianae in tobacco plants that was dependent on elicitor concentrations. There was a significant relationship between the reduction of the infection index and an increase in PAL activity when chitosan was applied by foliar spray and by substrate drench to the plant rhizosphere. These results demonstrate the influence of chitosan physico–chemical properties in plant-induced resistance and the relevance of particular responses in plant protection against pathogens. Ó 2011 Published by Elsevier Inc.

1. Introduction Plants are able to defend themselves against pathogen attacks by triggering defense reactions that included physical, chemical and enzymatic responses. This induced resistance can occur when plant cell membranes recognize general or specific signal molecules from pathogens during the pathogenesis interaction. In addition, the pathogen’s enzymatic arsenal can degrade the plant cell wall and liberate oligosaccharide fragments that, in turn, can amplify the resistance response in the plant. General pathogenderived signal molecules, which are known as pathogen-association molecular patterns (PAMP), include fungal cell wall fragments

Abbreviations: MW, molecular weight; DA, degree of acetylation; PAL, phenylalanine ammonio-lyase; POD, peroxidase; FW, fresh weight. ⇑ Corresponding author. E-mail addresses: [email protected] (A.B. Falcón-Rodríguez), daimy@inca. edu.cu (D. Costales), [email protected] (J.C. Cabrera), norawa@cascabel. ciad.mx (M.Á. Martínez-Téllez). 0048-3575/$ - see front matter Ó 2011 Published by Elsevier Inc. doi:10.1016/j.pestbp.2011.04.005

(oligosaccharins), lipopolysaccharides, peptidoglycans, and glycoproteins. When PAMPs are perceived by a host membrane receptor, they induce a non-host resistance in the plant that can reduce or eliminate plant infection by the pathogen [1]. Tobacco (Nicotiana tabacum L.) is a crop of great economic importance worldwide. Its cultivation involves a nursery phase lasting approximately 40 days, after which the most viable plantlets are moved to the field where they grow until they reach commercial size. Protection in the nursery is essential for obtaining healthy plants. During this phase, the plantlets are attacked by many soil-borne pathogens that need to be controlled by different means [2]. Phytophthora nicotianae is a pathogenic oomycete that resembles a fungus both morphologically and physiologically, but it is phylogenetically related to diatoms and brown algae. Oomycetes have a distinct physiology that causes most fungicides to be ineffective against them [3]. P. nicotianae is the causal agent of a common and destructive soil-borne disease of tobacco (Nicotiana tabacum L.) that primarily affects the roots and the stem basal region of the plant, although all parts of the plant can eventually be infected. This disease, commonly known as black shank, is one of

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the most important problems for commercial tobacco crops, and farmers attempted to prevent infection by applying chemicals and using resistant tobacco varieties. However, current approaches are focused on decreasing the cost of crop production by reducing the cost of agrochemicals and improving ecological management in tobacco farming. Additionally, there are now several documented cases of P. nicotianae resistance against chemicals used to control it [4]. Chitosan can be considered a PAMP because it is a deacetylated derivative of chitin, an important structural polysaccharide in fungal cell walls that can be liberated in the pathogenesis interaction. Chitosans are a group of heteropolysaccharides comprising copolymers of b-1–4 linked D-glucosamine and N-acetyl Dglucosamine residues. Thus, the term describes a heterogeneous group of macromolecules differing in molecular weight (MW), viscosity, degree of acetylation (DA), pKa, and other variables [5,6]. These different physico–chemicals provide different chitosans with unique biological characteristics that allow a wide range of applications and, consequently, create high worldwide commercial interest. It is estimated that the world chitin production is approximately 1013 kg [6]. As a result of hundreds of research studies in the last 25 years, chitosan has become a commercial compound that is now used as a biomaterial for food [7], pharmaceutical [5], medical [8], textile [9], agricultural [10] and for environmental protection in some industries [11]. In agriculture, chitosan possess unique biological properties, making it an important component of a new generation of agro-products. Chitosan inhibits the growth and development of many pathogens, including fungi, oomycetes and bacteria [10,12– 14]. Chitosan molecules also induce defense responses in plants that improve plant basal resistance and, consequently, protect them against pathogenic diseases, including viral infections [10,15,16]. Moreover, these polymers have been reported to be enhancers and regulators of plant growth, development and yield in several crops [17–19]. According several reports from the literature, the physico– chemical features of chitosan derivatives, such as MW and DA, affect some of their biological activities, such as pathogen inhibition [10,12,20] and eliciting plant defense responses [21–24]. However, most of the research regarding the influence of MW and DA on plant response elicitation have been performed in cell suspensions [23] or in isolated plant organs, such as leaves [21,22], but never in whole plants. In a previous work, we demonstrated how MW and DA influence enzymatic defense responses in roots and leaves when chitosan treatments are directly applied to plant roots [24]. The aim of this work was thus to study the behavior of defensive enzymes activated in the leaves of whole tobacco plants when they are treated with different applications of chitosan derivatives with different MW and DA. In addition, we studied how application and different chitosan compounds affects tobacco plant protection against P. nicotianae and how defense responses are related to this protection.

Table 1 Chitosan derivatives and physico–chemical characterization. Derivative

Nomenclature

DPa

DAb

Polymer Polymer Oligochitosan

CH-36 CH-12 OLG

794 813 5–9

36.5 12 0–1c

a

Average degree of polymerization determined by viscosimetry. Degree of acetylation by potentiometry. c Degree of acetylation by MALDI-TOF [23]. Every oligosaccharide in the mixture coexists in two forms: A non-N acetylated one and the other with only one glucosamine N-acetylated. b

2.2. Plant material Experiments were performed using tobacco (Nicotiana tabacum L.) plants of the Cuban variety ‘‘Corojo’’ cultivated in a substrate mixture (Pro-Mix, Canada) containing peat 75–85% (Sphagnum canadiense), vermiculite, perlite, moisture agent and dolomitic and calcitic limestone for pH adjustment under semi-controlled conditions with a light/dark and a temperature regime of 16/8 h and 28/24 °C, respectively. 2.3. Plant applied treatments Three experiments were conducted according to three different methods of treatment application: foliar spray, soil addition as drench to grown plants and at seed planting. In the first experiment, chitosan and control treatments were applied as a foliar spray on the leaves of 25-day-old tobacco plants. In the second one, treatments were applied to the substrate of the same age tobacco plants. In both experiments, protein extractions and protective assays were performed at 72 h after treatment. In the third experiment, control and chitosan treatments were applied as drench to the substrate immediately after seed plantation. Leaf protein extractions and protective assays against P. nicotianae were conducted on 25-day-old tobacco plants. Chitosan treatments (CH36, CH-12 and OLG) were dissolved at 0.1, 1 and 2.5 g L 1 in potassium acetate pH 5.5, 0.01% Tween 80. Tween 80/potassium acetate solution was used as a control. 2.4. Plant protein extraction After the incubation period of each experiment, the true leaves were collected and ground in a porcelain mortar and pestle in liquid nitrogen. Powdered tissue was extracted in 50 mM sodium acetate buffer pH 5.2 containing 5 mM EDTA, 14 mM b-mercaptoethanol and 1.0 M NaCl at the rate of 1 g per 2 mL of buffer for leaves and 1 g per 1 mL for roots. The extract was then centrifuged at 12000 g for 15 min at 4 °C. The supernatant was collected in Eppendorf microtubes and stored at 60 °C for subsequent analysis. 2.5. Plant enzyme and protein determinations

2. Materials and methods 2.1. Chemicals To perform biological assays, three chitosan derivatives (CH-36, CH-12 and OLG) with different physico–chemical features were used. CH-36 and CH-12 are two chitosan polymers of similar molecular weight but different degrees of acetylation (Table 1) obtained by the basic de-acetylation of Cuban lobster chitin [25], whereas OLG is a mixture of chitosan oligosaccharides with a degree of polymerization (DP) ranging from 5 to 9 and obtained by enzymatic hydrolysis of the CH-12 polymer [23].

Enzymatic activities were determined in the supernatant of leaf extracts. b-1,3-glucanase activity was determined using laminarin (Sigma, USA) as substrate and according to the methodology described by Boudart et al. [26]. In the assay, reducing sugars released from laminarin were quantified following the Somogyi method [27], and results were expressed as lg of glucose released per min per mg of protein (lg min 1 mg 1). Phenylalanine ammonia lyase (PAL) and peroxidase (POD) activity were determined using L-phenylalanine and guaiacol (Sigma, USA) as substrate, respectively, following the methodologies described by Vander and coworkers [21]. Enzymatic results of

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1.2

a

A

Control CH-36 CH-12 OLG

1.0

Description Healthy plant Roots affected Hypocotyls and cotyledons affected First and second leaves pair affected Dead plant

0.8 0.6

ab bcd

d

fd d

a a

abc

cd

b

c

b

c

e

A

a

a

a

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dd

d dd

d

0.4 0.2

2.6. Assessment of disease resistance in tobacco plants induced with chitosan derivatives In all experiments and after treatments and incubation, plants were gently removed from the substrate and placed in Eppendorf tubes containing Hoagland’s solution diluted 50 times and an agar culture plug (6 mm diameter) bearing mycelium of a pathogenic strain (227) of P. nicotianae placed at the bottom, allowing contact between plant roots and pathogen [29]. Plant-pathogen interaction was allowed to proceed for 5 days before determining plant infection. Fifteen plants were tested per treatment, and experiments were repeated twice with similar results. Plant infection was determined in each plant using a previously described [30] five-degree scale of pathogen invasion (Table 2). Data were analyzed with the Kruskal–Wallis non-parametric test, and all means were independently compared with the Mann– Whitney test for p 6 0.05, using the Bonferroni correction that allows comparison of more than two samples, through the statistical program SPSS 11.5 for Windows. In addition, the infection index was calculated for every treatment using the scale data. 2.7. Statistical relation analysis between enzymatic activities and infection index To determine if enzymatic activities were related to the infection index in inoculated tobacco plants, a standardized multiple linear regression was performed for each chitosan application using enzymatic activities as independent variables and infection index as the dependent variable. The regression was analyzed through the statistical program SPSS 11.5 for Windows. 3. Results 3.1. Accumulation of proteins and enzymatic activities induced in leaves of tobacco plants Foliar spray of the chitosan compounds tested caused increases in soluble proteins in tobacco leaves with all chitosan treatments relative to the control (Fig. 1A). The highest levels of proteins were detected with the two highest concentrations of the less acetylated polymer (CH-12), which doubled the amount of protein compared to control levels. When chitosan derivatives were applied as

0.0

mg protein. gr. FW-1

PAL were expressed as lg of transcinnamic acid formed per min per mg of protein (lg min 1 mg 1). Enzymatic results of POD were expressed as lmoles of product formed (oxidized guaiacol) per min per mg of protein (lmoles min 1 mg 1) and were calculated using the extinction coefficient of the product formed (26,000 Mol 1 cm 1). Protein determinations were performed following a micro Lowry assay [28] and expressed as mg of protein per fresh weight of plant tissue. Three determinations were performed per treatment, and experiments were repeated twice. Data were analyzed using a simple ANOVA with the statistical program Statgraphics plus 5.0 for WindowsÓ. Means with the same letters did not differ for p 6 0.05 in the Tukey test.

B

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abc bcd d66

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ab a a cde bcd abc ef def abc abc f c

0.1 bc

0.0 Control

0.1 g.L-1

1 g.L-1

2.5 g.L-1

Fig. 1. Changes of soluble protein contents in leaves of 25-day-old tobacco plants treated with different chitosan compounds and concentrations. Treatments were applied by foliar spray (A) (SEx = 0. 0131), soil drench to the plant rhizosphere (B) (SEx = 0. 0373) 72 h before foliar extraction or by soil drench to the substrate at seeding (C) (SEx = 0. 0092). Data are means ± SE of triplicate samples from one representative of two independent experiments. Different letters among treatments indicate significant differences in Tukey’s test (p 6 0.05).

drench to the rhizosphere of tobacco plants, all treatments, except the CH-36 chitosan polymer at 2.5 g L 1, increased protein levels above the control (Fig. 1B). The largest increases were obtained with the two highest concentrations of CH-12 and the lowest concentration of CH-36, which accumulated more than twice the protein level of the control. The oligochitosans induced a similar change with all three concentrations tested. However, only three chitosan treatments caused protein levels to increase above the control levels in tobacco leaves when the treatments were applied to the soil at seed plantation; these protein levels were about 20% above the control level (Fig. 1C). Chitosan treatments applied by foliar spray to the plants caused increases in b-1,3-glucanase activity relative to the control. Oligochitosans induced higher enzymatic levels than controls with all the three concentrations tested, with the two lowest concentrations (0.1 and 1 g L 1) inducing the most change, whereas polymers only provoked increases in enzymatic activity above the control at 1 g L 1 (Fig. 2A). The addition of treatments as drench to plant rhizosphere induced enzymatic activity above the control only with the CH-36 (0.1 and 2.5 g L 1) and the CH-12 (0.1 g L 1) polymers. The highest concentrations of the less acetylated polymer (CH-12) caused decreases in enzymatic activity to below control levels (Fig. 2B). The addition of treatments at seed planting caused a decrease in enzymatic activity to below control levels with all the three oligochitosan treatments and with CH-36

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140

Control CH-36 CH-12 OLG

120 100 80 60

d

d d d

b cd A cd cd dd d d

10

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c c c cc d ddd

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ug cinnamic acid.min-1. mg protein. gr.FW-1

ug glucose. min-1. mg protein. gr. FW-1

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8 6

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a

Control CH-36 CH-12 OLG

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14 A

A

a

a

2.5 g.L-1

Fig. 2. Changes in b-1,3-glucanase enzymatic activity in leaves of 25-day-old tobacco plants treated with different chitosan compounds and concentrations. Treatments were applied by foliar spray (A) (SEx = 2,97), soil drench to the plant rhizosphere (B) (SEx = 1,66) 72 h before foliar extraction or by soil drench to the substrate at seeding (C) (SEx = 0712). Data are means ± SE of triplicate samples from one representative of two independent experiments. Different letters among treatments indicate significant differences in Tukey’s test (p 6 0.05).

polymer (1 g L 1), but these enzymatic reductions were less remarkable than in the former method of application (Fig. 2C). Only the polymer CH-36 (0.1 g L 1) induced b-1,3-glucanase activity relative to the control. When determining PAL enzymatic activity, the highest increases in activity after foliar spray treatments were found with the two highest concentrations (1 and 2.5 g L 1) of the more acetylated polymer (CH-36), more than three times above the control level (Fig. 3A). With this polymer, the enzymatic activity increased with the concentration of chitosan tested, whereas the less acetylated (CH-12) induced activity above the control level only with the highest concentration tested. No enzymatic activity was detected in tobacco leaves when treatments were applied as soil drench to the rhizosphere except for the polymers when applied at the lowest concentration (0.1 g L 1), which induced about three times the control level of enzymatic activity (Fig. 3B). The addition of treatments at seeding caused very large increases in PAL activity in almost all of the chitosan treatments tested (Fig. 3C). The highest enzymatic levels were detected with the chitosan polymers at 1 g L 1, which induced more than 20 times the enzymatic activity found in the control. The peroxidase activity in tobacco leaves treated with foliar spray increased with most of the chitosan treatments. The most dramatic changes were detected with the less acetylated polymer at 1 and 2.5 g L 1, the former inducing more than 10 times the

0 Control

d -1 -1 0.1 mg.L-1 1 mg.L 2.5 mg.L

Fig. 3. Changes in PAL enzymatic activity in leaves of 25-day-old tobacco plants treated with different chitosan compounds and concentrations. Treatments were applied by foliar spray (A) (SEx = 0.45), soil drench to the plant rhizosphere (B) (SEx = 1.18) 72 h before foliar extraction or by soil drench to the substrate at seeding (C) (SEx = 1.99). Data are means ± SE of triplicate samples from one representative of two independent experiments. Different letters among treatments indicate significant differences in Tukey’s test (p 6 0.05).

enzymatic activity found in the control (Fig. 4A). Oligochitosans induced similar, moderate levels of enzymatic activity with all concentrations tested, whereas the more acetylated polymer induced activity above control only at the highest concentration tested (2.5 g L 1). This treatment was also the only treatment that induced peroxidase activity when chitosan compounds were applied as soil drench to the plant rhizosphere (Fig. 4B). No differences in enzymatic activity between chitosan and control treatments were detected when they were applied at seeding (Fig. 4C). 3.2. Induced resistance in tobacco plants against Phytophthora nicotianae Foliar spray of chitosan compounds caused a reduction of the infection index in tobacco plants inoculated with P. nicotianae relative to control plants (Table 3). Plant protection was higher (36– 41%) with the more acetylated polymer (CH-36) at 1 and 2.5 g L 1, whereas the oligochitosan mixture (OLG) reduced the infection index by 27–33% at 0.1 and 1 g L 1. The highest percentage of healthy plants (Degree 1) was obtained with the higher concentrations of chitosan polymer (CH-36); at 2.5 g L 1 of CH-36, there were no dead plants. Conversely, control experiments showed no healthy plants (data not shown). Plants treated by soil drench, both as addition to the rhizosphere of 25 days old plantlets or as addition at seeding, caused

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2000 1500

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Control CH-36 CH-12 OLG

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1000 500

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umol product.min-1. mg protein.FW-1

f 0

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b 200

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b b

b

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100

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a ab ab ab

400

abc abc abc

abc 300

bc

abc

abc c

200 100 0

Control

0.1 g.L-1

When analyzing the statistical relationship between the infection index and the enzymatic activities, it was found that b-1,3glucanase and PAL activities were inversely related to the infection index (Table 4) when treatments were applied by foliar spray, meaning that the highest levels of b-1,3-glucanase (OLG, 0.1 and 1 g L 1) and PAL (CH-36, 1 and 2.5 g L 1) detected in the experiment caused the lowest values of infection index in tobacco plants. Treatments applied as drench to the plant rhizosphere only showed an inverse relation between infection index and PAL enzymatic activity (CH-36 and CH-12, 0.1 g L 1), whereas in the experiment where treatments were applied as drench at seeding, there was no relationship between enzymatic activities and infection index (Table 4) because no significant regression coefficients were obtained.

1 g.L-1

2.5 g.L-1

Fig. 4. Changes in POD enzymatic activity in leaves of 25-day-old tobacco plants treated with different chitosan compounds and concentrations. Treatments were applied by foliar spray (A) (SEx = 66.01), soil drench to the plant rhizosphere (B) (SEx = 14.24) 72 h before foliar extraction or by soil drench to the substrate at seeding (C) (SEx = 41.16). Data are means ± SE of triplicate samples from one representative of two independent experiments. Different letters among treatments indicate significant differences in the Tukey’s test (p 6 0.05).

reduction of infection index with several chitosan treatments (Table 3). The highest reduction induced when treated plant rhizosphere (CH-36, 0.1 and 2.5 g L 1; CH-12, 0.1 g L 1; OLG, 1 g L 1) caused a protection between 24% and 32% related to control plants, while 25 days old plants, previously treated at seeding, induced a protection between 24% and 29% respect to control plants with some chitosan treatments (CH-36, 0.1 and 2.5 g L 1; CH-12, 1 g L 1; OLG, 2.5 g L 1).

4. Discussion Chitosan (b 1–4 linked N-glucosamine) has been shown to induce defense responses [21,22,31–33] and resistance against pathogens [15,31,34,35] in different plant species. In this study, chitosan compounds of different molecular weights and degrees of acetylation caused a differential response in the protein accumulation and enzymatic activity of particular defenses in tobacco leaves of 25-days-old plants; this response was dependent on the elicitor concentration and the method of application. Regarding protein accumulation, the time delay between treatment and protein determination was critical for both the levels of protein accumulation as well as increases in protein concentration in the chitosan treatments relative to the controls (Fig. 1). The highest protein levels were detected when protein levels were determined 72 h after treatment, regardless of whether the treatment was by foliar spray or as drench to the rhizosphere. This behavior could be related to the transient character of the defense responses mobilized after plant treatment with elicitors because plants, depending on elicitor concentrations, can usually only activate their defense signaling responses for a limited period of time due to the energy cost needed to keep up its basal resistance [36– 38]. Thus, protein levels induced by chitosan treatments at 72 h, representing proteins and enzymes related to the process of induced resistance, might be reduced to basal levels as time passed. This could be the reason why the protein levels in the leaves of 25day-old tobacco plants treated with chitosan derivatives at seeding are lower than in the same plants treated 72 h before protein determination. All three enzymes evaluated in this work were activated differentially in tobacco leaves when treated with the chitosan derivatives tested. b-1,3-glucanase is essential for tobacco protection against P. nicotianae [39] because this enzyme degrades b-1–3 glucan polymers, an important structure of the oomycete cell wall.

Table 3 Effect of different ways of application of chitosan compounds on the infection reached by Phytophthora nicotianae in tobacco plants of 25 days old. Results are shown as the mean of the ranks in the non-parametric Kruskal–Wallis test and as infection index for each treatment. Different letters means significant differences among treatments in the test of Mann–Whitney with the Bonferroni correction (p 6 0.05). Treatments

Control CH-36

CH-12

OLG

Concentration (g L

– 0.1 1 2.5 0.1 1 2.5 0.1 1 2.5

1

)

Soil drench to the rhizosphere

Soil drench at seeding

Mean of rank

Foliar spray Infection index

Mean of Rank

Infection index

Mean of Rank

Infection index

110 c 72.4 ab 58.43 a 52.33 a 102 bc 79.2 ab 74.0 ab 69.8 a 62.7 a 74.0 ab

81.33 60.0 52.0 48.0 77.3 64.0 61.3 58.7 54.7 61.3

101.8 b 62.33 a 76.8 ab 67.6 a 60.23 a 80.5 ab 77.5 ab 86.1 ab 69.7 a 72.6 ab

78.7 54.7 64.0 58.7 53.3 66.7 64.0 69.3 60.0 61.3

101.5 b 70.8 a 73.0 ab 67.2 a 73.0 ab 65.8 a 80.3 ab 82.8 ab 73.5 ab 67.2 a

78.7 60.0 61.3 57.3 61.3 56.0 65.3 66.7 61.3 57.3

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Table 4 Relation between enzymatic activities and the infection index in 25 days old tobacco plants. It is shown the standardized regression coefficients and the significant valor in every way of treatment application. Enzymatic variables

Foliar spray (R2 = 0.811) Standardized coefficient

b-1,3-glucanase PAL POD

0.43 0.786 0.111

p 0.026 0.002 0.472

Soil drench to the rhizosphere (R2 = 0.705)

Soil drench at seeding (R2 = 0.253)

Standardized coefficient

p

Standardized coefficient

p

0.384 0.012 0.080

0.551 2.256 0.000

0.602 0.065 1.000

0.290 1.076 0.428

PAL is crucial to opening several metabolic pathways in the phenylpropanoids for the synthesis of secondary metabolites such as phytoalexins, clorogenic and salicylic acids, and lignins, most of them being important defenses against pathogens [40]. The critical role of this enzyme against tobacco pathogens has been demonstrated by the expression of heterologous and antisense PAL genes [41,42]. In addition, it was demonstrated that the production of H2O2 and peroxidase activity are essential markers in the incompatible interaction between tobacco and P. nicotianae [43]. In this investigation, enzyme induction depended on chitosan molecular weight, its degree of acetylation and the method of chitosan application to the plants. When plant leaves were directly treated with the chitosan derivatives (foliar spray), activation of enzymatic activities in this organ depended on the elicitor concentrations (i.e., b-1,3-glucanase activities increased to its maximum at 0.1 g L 1 of oligochitosans (OLG) and at 1 g L 1 of the polymers (CH-36, CH-12)). In a previous work, a similar behavior was found in the roots of tobacco plants for b-1,3-glucanase and POD activities when the same chitosan derivatives were directly applied to tobacco roots [24]. From both results, it is possible to conclude that the difference in molecular weight between chitosan polymers and oligomers caused a 10-fold difference in the concentration needed to induce the highest enzymatic activity when these derivatives were directly applied to the plant organ. Polymers must have more difficulty than oligomers in passing plant physical barriers and accessing receptor centers at the plasma membrane in epidermal cells. In addition, it is possible that both derivatives are differently perceived at the plasma membrane, as proposed in previous studies [21,44,45]. The differences in the degree of acetylation between the polymers affected PAL and POD enzymatic activities when they were applied as foliar spray in tobacco leaves. The highest PAL activity was found with the more acetylated polymer (CH-36) in all three concentrations tested, whereas POD activity was above the control with all the three concentrations of the less acetylated polymer (CH-12). A similar behavior has been previously demonstrated for the same enzymatic markers in tobacco roots when the same chitosan polymers were directly applied to this organ in tobacco plants [24]. Thus, there is a clear influence of the degree of acetylation of chitosan on the induction of enzymatic defense responses, at least for these two enzymes, when chitosan derivatives are directly applied to the organ. In addition, in the present and prior studies, PAL and POD activities were affected differently with different applications [24]. Former authors reported that augmentation in the polymer’s degree of acetylation caused rise in enzymatic activity for PAL and POD in wheat leaves when directly applied [21]. The results of this study are in agreement with previous studies on PAL but are opposed for the influence on POD activation. This difference might be related to working with different plant species, especially when these species belong to different plant groups as, for instance, monocots and dicots. When chitosan was added to the substrate, changes in enzymatic activity in tobacco leaves were the result of the systemic induction of the defense signal because the elicitors were only in contact with the roots. When treatments were added to the plant

rhizosphere, only the polymers induced b-1,3-glucanase and PAL activities. The oligochitosan influence is thus reduced when mixed with the substrate, possibly due to the degradation of the oligomers to inactive fragments. This process could have less impact in polymers because of their high molecular weight, which can yield active fragments by degradation [30]. Interestingly, in this mode of application, the polycationic difference between both polymers caused different b-1,3-glucanase activity. It is likely that a higher polycationic character provokes a direct or indirect detriment of some defense response activity, possibly due to different interaction and perception at the cell membrane [44,45]. All three chitosan derivatives caused the reduction of the infection index in tobacco plants inoculated with P. nicotianae depending on the concentration and mode of application. The plant protection found was the unique result of induced resistance elicited in the plant, as demonstrated by bioassay, where the roots of the plant were in contact with the pathogen out of the substrate (in microtubes) after elicitor treatments. There was no contact at any time between the pathogen and chitosan derivatives. The highest reduction of infection in plants was achieved with the foliar spray of derivatives (Table 3). Based on the data, there was no difference in plant protection as a result of the different degrees of acetylation and molecular weight of the elicitors. Plant protection was apparently more related to chitosan concentration and the mode of application. The highest resistance induction found by foliar spray regarding to soil treatments, can be related to the direct interaction between the biological material and the elicitor compound, this is the case of the foliar spray, on the contrary, the substrate can prevent a suitable contact between chitosan elicitors and roots when the substrate is applied. As a matter of fact, a different behavior of enzymatic defenses was demonstrated by this lab when the same chitosan treatments where directly applied to the roots with no substrate interference [24]. However, it should not be discarded a difference in perception and in elicitor uptake inside the plant between both organs. In this support, a recent finding demonstrated that stomatal uptake of chitosan is determinant for antiviral defense induction in plant beans [46]. Papers from previous studies on the influence of chitosan molecular weight on plant resistance against pathogens generally reported that polymer degradation enhances plant protection against fungal and viral pathogens [34,47]. However, the protection of tomato fruits against rotting caused by Rhizopus was independent of the molecular weight of the chitosan derivative tested [48]. Recently, antiviral activity was demonstrated when tobacco plants were sprayed with an oligochitosan (DP 3–9) mixture [16]. To date, no research group had studied the effect of the physico– chemical features of chitosan on tobacco protection by induced resistance to fungal pathogens. From our results, tobacco plants could be protected against P. nicotianae by applying polymeric and oligomeric chitosan derivatives. Several enzymes play critical roles in tobacco resistance to pathogens. A former study, based on the constitutive gene cloning method, demonstrated that PAL is a key enzyme that protects tobacco against P. nicotianae [41]. The results of this study also dem-

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onstrate that the resistance against P. nicotianae found in tobacco plants induced with chitosan was significantly related to PAL activation; consequently, this result suggests that chemical defenses and signals synthesized through the phenylpropanoids pathway are critical for tobacco protection against P. nicotianae. POD is a group of essential enzymes in the formation and metabolism of reactive oxygen species (ROS) that create a highly toxic environment to the pathogens. In addition, they can have cell wall crosslinking activity (formation of lignin, intramolecular reticulation of phenols, extensin cross links and dityrosine bonds) and some ROS products (H2O2) are direct signals for the gene expression of plant defenses [49,50]. In tobacco, POD and ROS products have been shown to be involved in signaling and plant defensive response to oomycetes, especially in the incompatible plant-pathogen interaction [43,51]. From our results, POD seems to be unrelated to the reduction of the P. nicotianae infection index in tobacco plantlets (Table 4). However, because of the broad variety of POD enzymes that react with guaiacol and the multiple functions that they perform, their relation to tobacco resistance against P. nicotianae remains unclear. b-1,3-glucanase is considered a PR-protein that hydrolyses the b-1–3 glucan components of fungal cell walls [37,52]. This enzyme has been associated with tobacco resistance to oomycete pathogens, including P. nicotianae [39,53]. In this study, we found an inverse relationship between b-1,3-glucanase activity and the infection index by foliar spray application, and this mode of chitosan application also caused the highest degree of plant protection against P. nicotianae; in fact, the treatments that provoked the highest reduction of infection index by foliar spray also shared the highest levels of both PAL and b-1,3glucanase enzymatic activities. Thus, the best results of plant protection obtained by chitosan foliar spray may be due to synergistic activity between these two enzymes. The lasting activation of these three enzymes at levels two- to threefold the control suggests the activation of an induced resistance against pathogens and raises questions about the particular importance of each enzyme in tobacco protection against a specific pathogen. In the near future, we are interested in investigating if these enzymes could be induced to similar or higher levels in plants post-inoculation with P. nicotianae. In addition, the signaling pathways activated by chitosan in tobacco could be an interesting subject because, although defense expression induced by chitosan was demonstrated to be related to jasmonic acid signal transduction pathway, the large increases in PAL activity induced at different times by chitosan in our results suggests the possibility of a synergistic interaction with salicylic acid at certain periods of defense signal transduction. This idea is also supported by the reported synergistic interaction between salicylic and jasmonic acid, depending on the concentration, to induce defense responses [54]. From previous results of defense responses and resistance induction, it is well established that chitosan compounds are clearly recognized by the cell surfaces of leaves and roots [15,31,32,35]. Apparently, this perception is different for chitosan polymers and oligomers. According to former reports, the defense induction by chitosan polymers is the result of the interaction between positive charges of the polysaccharide (when dissolved in acidic solutions) and the negative charges exposed to the apoplast in the components of cell membrane [21,44,45]. In turn, it is possible that oligochitosans have protein receptors at plant cell membrane as previously reported [44,45]. It is likely that differences in perception at the cell membrane are one of the reasons for differences in the enzymatic and resistance response found in this work. However, a second reason could be the difficulty that polymers face when passing through the plant physical barriers. The difference in molecular weight between both compounds should affect their passage across leaf cuticles because the mobility of solutes

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through this organ exponentially decreases with increases in molar volume [55] and even, through stomata uptake [46]. This theory is supported by the results of b-1,3-glucanase activity found when chitosan compounds were applied by foliar spray, where the highest increase in enzymatic activity was detected with oligochitosans at concentrations 10 times lower than the highest activity detected with polymers. In brief, this study proved that three different chitosan derivatives induced b-1,3-glucanase, PAL and POD activities in tobacco leaves, as local or systemic responses, on non-pathogen-inoculated plants. Tobacco protection against P. nicotianae infection by induced resistance was also shown. According to experimental results, plant resistance to this pathogen could be related to the degradation of the pathogen cell wall and to the damage caused by defenses produced in the phenylpropanoid pathway. In addition to the basic importance of understanding the differential activation of defense enzymes with different types of chitosans and modes of application, these results could also have importance in the development of future practical means to protect tobacco plantlets against P. nicotianae, the main soil-borne pathogen in tobacco seedbeds. Acknowledgements Thanks to CONACyT-Mexico and the International Foundation for Sciences (Project F-4446-2F) for its financial support. This research is part of a PhD thesis of the first author performed at the National Institute of Agricultural Science, La Habana, Cuba and the Center of Research in Food and Development, Hermosillo, Sonora, Mexico. Thanks to Olivia Briceño and Socorro Vallejo for their technical assistance. References [1] G.N. Agrios, How plants defend themselves against pathogens (Chapter six), in: Plant Pathology, fifth ed., Academic Press, New York, USA, 2005, pp 208. [2] D.C. Erwin, O.K. Erwin, Ribeiro Phytophthora nicotianae = Phytophthora parasítica, in: D.C. Erwin, O.K. Ribeiro (Eds.), Phytophthora diseases worldwide, The American Phytopathological Society, St Paul, Minnesota, USA, 1996, pp. 391–407. [3] R.P. Thakur, K. Mathur, Downy mildews of India, Crop Prot. 21 (2002) 333–345. [4] Jaarsveld, M. Wingfield, A. Drenth, Effect of metalaxyl resistance and cultivar resistance on control of Phytophthora nicotianae in tobacco, Plant Dis. 86 (2002) 362–366. [5] A.K. Singla, M. Chawla, Chitosan: some pharmaceutical and biological aspects— an update, J. Pharm. Pharmacol. 53 (2001) 1047–1067. [6] K.V.H. Prashanth, R.N. Tharanathan, Chitin/chitosan: modifications and their unlimited application potential-an overview, Trends in Food Sci. Techn. 18 (2007) 117–131. [7] J. Rhoades, S. Roller, Antimicrobial actions of degraded and native chitosan against spoilage organisms in laboratory media and foods, Appl. Environ. Microbiol. 66 (2000) 80–86. [8] Ueno, T. Mori, T. Fujinaga, Topical formulations and wound healing applications of chitosan, Adv. Drug Deliv. Rev. 52 (2001) 105–115. [9] K. Takai, T. Ohtsuka, Y. Senda, M. Nakao, K. Yamamoto, J. Matsuoka, Y. Hirai, Antibacterial properties of antimicrobial-finished textile products, Microbiol. Immunol. 46 (2002) 75–81. [10] S. Bautista-Baños, A.N. Hernández-Lauzardo, M.G. Velázquez-del Valle, M. Hernández-López, E. Ait Barka, E. Bosquez-Molina, C.L. Wilson, Chitosan as a potential natural compound to control pre and postharvest diseases of horticultural commodities, Crop Prot. 25 (2006) 108–118. [11] S. Babel, T.A. Kurniawan, Low-cost adsorbents for heavy metals uptake from contaminated water: a review, J. Hazard. Mater. 97 (2003) 219–243. [12] J. Xu, X. Zhao, X. Han, Y. Du, Antifungal activity of oligochitosan against Phytophthora capsici and other plant pathogenic fungi in vitro, Pest. Biochem. Physiol. 87 (2007) 220–228. [13] J. Palma-Guerrero, I.-C. Huang, H.-B. Jansson, J. Salinas, L.V. Lopez-Llorca, N.D. Read, Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy dependent manner, Fungal Gen. Biol. 46 (2009) 585–594. [14] D. Raafat, K. von Bargen, A. Haas, H.-G. Sahl, Insights into the mode of action of chitosan as an antibacterial compound, Appl. Environ. Microb. 74 (2008) 3764–3773. [15] R.G. Sharathchandra, S. Niranjan Raj, N.P. Shetty, K.N. Amruthesh, H.S. Shetty, A Chitosan ormulation ElexaTM induces downy mildew disease resistance, growth promotion in pearl Millet, Crop Prot. 23 (2004) 881–888.

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