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Crop Protection 29 (2010) 163–167

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Crop Protection

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Superior bio-efficacy of a combined formulation of carbendazim and mancozebin inducing defense responses in chilli seedlings against Sclerotium rolfsii Sacc.in comparison with methyl jasmonate

Somnath Roy*, Amrita Banerjee, Jayanta Tarafdar, Samir Kumar SamantaFaculty of Agriculture, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur, Kalyani 741252, West Bengal, India

a r t i c l e i n f o

Article history:Received 27 March 2009Received in revised form15 September 2009Accepted 27 September 2009

Keywords:SclerotiumDefenseMethyl jasmonateCombination formulation of contact andsystemic fungicidesIsozyme

* Corresponding author. Tel.: þ91 9475164065; faxE-mail address: sroypbr@gmail.com (S. Roy).

0261-2194/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.cropro.2009.09.005

a b s t r a c t

The objective of this work was to investigate the efficacy of a prepackaged combined formulation,Companion (carbendazim 12%þmancozeb 63% WP), sole application of carbendazim 50% WP, manozeb75% WP and methyl jasmonate (MeJA), an inducer of systemic acquired resistance on disease severity andtheir role in post-infectional defense responses in chilli seedlings against Sclerotium rolfsii. Seeds weretreated for 8 h with MeJA (2.5 mM and 5.0 mM) and each of fungicides (500 ppm), and were sown in potscontaining soil and fungal inocula (95:5 w/w). At 15 days after sowing maximum defense against fungalinfection was exhibited by Companion comparably followed by the sole application of carbendazim andmancozeb. MeJA reduced percent mortality of S. rolfsii-infected chilli seedlings significantly as comparedto the inoculated control. Assessment of peroxidase (POX) and esterase (EST) at 15 days after sowingrevealed the increased activity under inoculated condition. Highest POX activity in MeJA treatments(5 mM> 2.5 mM) was followed by the Companion treatment. Highest EST activity was registered inCompanion treatment. The zymogram of POX isozymes showed over-expression of POX 2 and POX 4isoforms, and induction of POX 1 isoform in inoculated treatments. On the other hand, that of ESTisozymes showed induction of EST 1 isoform in Companion, carbendazim and MeJA treatments. All ESTisoforms were over-expressed in Companion-treated seedlings. Both fungicides and MeJA showedsignificant effects on disease severity, induction of defense enzymes and isozyme pattern in S. rolfsii-infected chilli seedlings. Contact and systemic fungicides under the experiment demonstrated differ-ential responses. The combination formulation was superior in disease control to application of thefungicide components individually. They compared favourably with MeJA in induction of defense-relatedenzyme activities. All these findings are new with respect to the chilli-S. rolfsii host–pathogen interactionsystem, S. rolfsii representing the sclerotial basidiomycetes in particular.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Plants possess a range of defense mechanisms activelyexpressed in response to pathogens, including fungi. Systemicacquired resistance and induced systemic resistance, two forms ofinduced resistance elicited by specific environmental stimuli, area physiological state of enhanced defense capacity (Vallad andGoodman, 2004). The classic form of systemic acquired resistancecan be triggered by exposing the plants to pathogens or artificiallywith chemical such as salicylic acid, jasmonic acid (JA), methyljasmonate (MeJA), inorganic salts etc. (Kessman et al., 1994; Sticheret al., 1997). It is well documented that signaling molecules such as,

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JA and MeJA are activators of plant defense responses (LiHong et al.,2004; Thaler et al., 2004; Shah, 2009). Plants react to pathogenattack through a variety of active and passive defense mechanismsprimarily related to the metabolism of phenolic compounds andoxidative metabolism. The whole process involves increasedexpression of several defense genes and activation of oxidativemetabolism (Vidhyasekaran, 2008). Production of reactive oxygenspecies (ROS), mainly hydrogen peroxide, superoxide anions (O2

�)and hydroxyl radicals (OH), is an important defense mechanism inplants against pathogens (Mittler, 2002). Excessive production ofROS may cause disruption of cellular functions, finally leading tocell death. Plants overcome these negative consequences of ROS byproducing antioxidative enzymes such as peroxidases, which reactwith ROS and keep them at low levels (Mittler, 2002).

Peroxidases exist in plant tissues as multiple isozymes and aredistributed throughout the cell and catalyze the reduction of H2O2

Table 1Analysis of variance (mean sum of squares), SE and LSD values for mortality%,peroxidase (POX) and esterase (EST) activity assay in single factor experiment incompletely randomized design.

d.f Mean sum of square S.E LSD (P¼ 0.05)

Mortality % 5 293.093* 0.089 1.939POX activity 6 1.089* 0.037 0.080EST activity 6 0.00093* 0.003 0.005

*Significant at 1% level.

S. Roy et al. / Crop Protection 29 (2010) 163–167164

to H2O. An increase in total peroxidase activity is observed duringplant pathogenesis (Koike et al., 2001; Borden and Higgins, 2002).Esterases are a complex heterogeneous group of hydrolases whichcatalyze the cleavage and formation of carboxyl ester bonds. Inplants a large number of proteins with carboxylesterase activityhave been found to catalyze these apparently facile reactions(Gershater and Edwards, 2007).

For chemical disease management, various combinationformulations of contact and systemic fungicides have been manu-factured mainly to achieve synergy between them, to broaden theirspectrum of activity and to delay the build up of resistant fungalstrains especially since the introduction of single-site systemicfungicides (Levy et al., 1983; Deacon, 1997), and to promote greaterefficiency in chemical disease management. Certain fungicides, inaddition to their antifungal action, are involved in activating certaindefense responses of plants (Garcia et al., 2003). However, theraison d’etre of fungicide application as combination formulations,through phenol and oxidative processes are remain unexplored.

We artificially infected chilli seedlings with Sclerotium rolfsiiSacc., a devastating polytrophic soil-borne plant pathogenic fungusthat causes root rot, stem rot, cortical wilt, and foot rot on morethan 500 plant species including perhaps almost all the agriculturaland horticultural crops (Farr et al., 1989). This is a disease of warmsubtropical climate corresponding to the euhomotopic bio-geographical distribution of both host and pathogen. Thus thedisease caused by S. rolfsii is called ‘southern blight’ in the USA,because of its distribution in its southern warm subtropical region.The affected plants exhibit wilting owing to stem girdling, collarrotting and ultimately collapse. Seedlings being highly susceptibleto the disease die more quickly than the older plants.

In this study, we sought to compare the effects of seed treatmentwith MeJA, and mancozeb and carbendazim in a combinationformulation (Trade Name: Companion) and as sole applications interms of disease severity in S. rolfsii-infected chilli seedlings, as wellas the patterns of behavior of two groups of defense-relatedenzymes, peroxidase (POX) and esterase (EST) during pathogenesis.

2. Materials and methods

The chemical and fungicide formulations were obtained bycourtesy from the manufacturers: mentioned in parentheses: MeJA(95%) (Sigma–Aldrich Cat 39, 270-7), mancozeb (75% WP),carbendazim (50% WP) and Companion (carbendazim12%þmancozeb 63% WP) (Indofil Chemicals Company, Mumbai,India). Chilli seeds (cv. BCKV2) were surface sterilized with 1%mercuric chloride for 1 min. After drying, seeds were soaked for 8 hin solutions of MeJA (2.5 mM and 5.0 mM) and fungicides (500 ppmeach) and sterile water for both inoculated and uninoculatedcontrols. MeJA and fungicides treated seeds were sown in potsfilled with sandy loam soil (pre-sterilized by autoclaving) con-taining S. rolfsii inocula (95:5 w/w). Water treated seeds sown ininoculated and uninoculated pots served as controls. However,uninoculated pots were excluded from disease incidence scoring,because the efficacy of different treatments on disease severity onlycould be compared under inoculated condition. For each treatmentfifty seeds were sown per pot. No organic manure or fertilizer wasapplied to any of the pots. The experiment was laid out ina completely randomized design and for each treatment threereplicates were maintained. The pots were routinely watered withtap water and placed under controlled conditions in the glasshousewith a 16 h photoperiod, 350 mmol m�2 s�1 light intensity andtemperature of 25� 2 �C. At 15 days after sowing the diseaseintensity was scored, and leaves were taken for isozyme analyses.

The disease intensity in each replicate was assessed at 15 daysafter sowing as: Mortality%¼ (Number of dead plants/total number

of plants)� 100. Percentile reduction in mortality as compared tothe control in each treatment was also calculated to compare thebio-efficacy of the treatments.

2.1. Assay of enzymes

Enzymes were assayed from the leaf tissues of the seedlings at15 days after sowing. The activity of each enzyme was expressed asthe average of three replicates. Total POX activity was assayedfollowing the method of Thimmiah (2004). For enzyme extraction,100 mg of freshly harvested leaf samples were ground in liquidnitrogen and homogenized well in an ice-cold glass mortar-and-pestle with 1 ml of 0.1 M potassium phosphate buffer (pH 6.0).Extracted samples were centrifuged at 16,000 rpm for 20 min at0 �C and the supernatant was used for the enzyme assay. POXactivity was assayed by taking 1 ml of 0.01 M o-Dianisidine inmethanol (MeOH), to which were added 1 ml of potassium phos-phate buffer (pH 6.0) and 2.4 ml of double distilled water in testtubes. The reaction was started by adding 0.2 ml of enzyme extractand incubated at 30 �C for 5 min, was stopped by adding 1 ml of 2 NH2SO4 and the absorbance was read at 430 nm for 1 min. Thereaction mixture without enzyme extract served as a control. Theresults were expressed as units min�1 g�1 fresh wt. EST activity wasdetermined as described by Asperen (1962). For enzyme extraction,100 mg of fresh leaf tissue was ground with liquid nitrogen in anice-cold glass mortar-and-pestle. The powdered leaf tissues werethen homogenized with 10 ml phosphate buffer (0.2 M, pH 7.0). Thehomogenates were centrifuged at 10,000 rpm for 30 min at 0 �Cand supernatants were used for the assay. Activity of EST wasassayed by adding 1 ml of crude enzyme to 5 ml of reaction mixtureand kept at 37 �C for 1 h. The reaction mixture was prepared bydissolving 200 mg a-napthyl acetate in 10 ml of 50% acetone and200 mg Fast Blue RR salt in 90 ml of 0.2 M of phosphate buffer (pH7.0). These were mixed together and filtered through Whatman No.1 filter paper in the dark at 4 �C. The activity was measured at600 nm and expressed as units min�1 g�1 fresh wt.

Spectrophotometric assays were performed at room tempera-ture using a Bio Spectrophotometer (Elico-BL 198).

2.2. Polyacrylamide gel electrophoresis of isozymes

Polyacrylamide gel electrophoresis of POX and EST isoforms wasdone in 7.5% gel according to Kahler and Allard (1970). For viewing ofPOX isoforms, the gel was first soaked in staining solution con-taining 100 mg o-Dianisidine (dissolved in 1 ml acetic acid), 200 mldouble distilled water and 2 ml of 30% H2O2. The staining was donein the dark for 30 min with occasional shaking until reddish brownbands appeared. EST isozymes were stained by soaking the gel in100 ml Tris buffer (50 mM, pH 7.1) containing 100 mg Fast Blue RRsalt and 4 mg a-napthyl acetate (dissolved in 1 ml ethanol). The gelwas placed in the dark for 30 min with occasional shaking. When thebands appeared, the gel was washed with double distilled water.

Extraction for the enzymes for gel electrophoresis analyseswas essentially the same as that used for activity assays exceptsample and buffer quanta. A 100 mg of leaf tissue extracted with

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Control carbendazim mancozeb Companion MeJA (2.5 mM) MeJA (5.0 mM)

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Fig. 1. Effect of fungicides and methyl jasmonate on Sclerotium rolfsii disease severity in chilli seedlings. Values are means of three replicates.

S. Roy et al. / Crop Protection 29 (2010) 163–167 165

100 ml of buffer was taken for POX assay. For EST activity 100 mgleaf tissue was crushed with 150 ml of buffer. The extractions werecarried out in 1.5 ml microcentrifuge tubes with a micro pestle.The extraction buffer and the process used for POX and EST weresame as stated earlier.

2.3. Statistical analysis

Data on percent mortality, enzyme assay were statisticallyanalyzed and analysis of variance (ANOVA) was determined foreach set (Gomez and Gomez, 1984). Fisher’s least significantdifference (LSD) at P¼ 0.05 was used to compare the treatmenteffects. Data reported in graphs are the means of three replicates formortality percent and for assay of enzymes the means of threereplicates with two repetitions are presented.

3. Results

ANOVA showed significant difference (P< 0.01) among thetreatment effects (Table 1).

3.1. Disease incidence

Seed treatment with fungicides and MeJA resulted in significantreduction in seedling mortality. Seedling mortality percent due to

0

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POX activity Percentile increase over contro

Fig. 2. Activity of peroxidase in fungicide- and methyl jasmonate-treated chilli se

S. rolfsii, in decreasing order, are shown in parentheses with respectto different treatments: control (74.3), 2.5 mM MeJA (50.3), 5.0 mMMeJA (46.8), mancozeb (37.5), carbendazim (34.8) and Companion(27.8). Percentile reduction in mortality was 62.9%, 53.5%, 49.9%,37.0% and 32.3% in Companion, carbendazim, mancozeb, MeJA(5.0 mM) and MeJA (2.5 mM) respectively (Fig. 1).

3.2. Enzyme activity

POX activity was lowest in the uninoculated control (2.65 uni-ts min�1 g�1 fresh wt.) and increased significantly in the inoculatedcontrol (3.46 units min�1 g�1 fresh wt.). Highest activity wasrecorded in MeJA-treated seedlings (4.36 units min�1 g�1 freshwt.). The activity of the enzyme did not vary significantly for thetwo concentrations of MeJA. For the fungicide treatments, thehighest activity was recorded for the Companion (4.11 uni-ts min�1 g�1 fresh wt.) and it was significantly higher than theseparate fungicides. Treatment with systemic fungicide resulted ina significant increase in enzyme activity over contact fungicide.Percentile increase in POX activity, lowest to highest, were: 30.9%,32.6%, 47.5%, 55.2%, 62.0% and 65.0% respectively in inoculatedcontrol, mancozeb, carbendazim, Companion, 2.5 mM MeJA and5.0 mM MeJA-treated chilli seedlings respectively (Fig. 2).

EST activity was significantly higher for all the treatmentsincluding inoculated control than the uninoculated control (Fig. 3).

eb Companion MeJA (2.5 mM) MeJA (5.0 mM)

ents

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edlings. Values are means of three replicates with duplicate determinations.

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Control Infected Control carbendazim mancozeb Companion MeJA (2.5 mM) MeJA (5.0 mM)

Treatments

ES

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-1 fre

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EST activity Percentile increase over control

Fig. 3. Activity of esterase in fungicide- and methyl jasmonate-treated chilli seedlings. Values are means of three replicates with duplicate determinations.

S. Roy et al. / Crop Protection 29 (2010) 163–167166

The enzyme activity increased in Companion- and carbendazim-treated seedlings, as in parentheses: Companion (0.173 uni-ts min�1 g�1 fresh wt.), carbendazim (0.146 units min�1 g�1 freshwt.). The carbendazim and MeJA treatments performed similarly.Further, mancozeb (0.138 units min�1 g�1 fresh wt.) did not registersignificant increase in the same over uninoculated control duringpathogen establishment (Fig. 3).

3.3. Isozyme analysis

Gel electrophoretic analysis of leaf POX showed six isoforms(POX# 1–6). The band intensities varied among the treatments(Fig. 4). In the uninoculated control, five bands were seen (POX# 2–6). Isoform POX 1 was expressed in all the inoculated treatments.The POX 3 isoform was intense only in the Companion and car-bendazim treatments and in the uninoculated control, thus indi-cating its specific association with carbendazim as well withnatural post-infectional defense response. Over-expression of all

Fig. 4. Peroxidase zymogram. Lane 1 – Companion, Lane 2 – carbendazim, Lane 3 –mancozeb, Lane 4 – uninoculated control, Lane 5 – inoculated control, Lane 6 – MeJA(2.5 mM) and Lane 7 – MeJA (5.0 mM).

enzyme isoforms indicates strong association of the given enzymewith defense system in a host–parasite relation. The POX 2 isoformwas over expressed in MeJA treatments along with Companion andcarbendazim but faint in mancozeb and controls. The POX 4 isoformwas intense in all inoculated treatments, which indicated its strongassociation with fungal infection. The relative position of POX 6varied among fungicides and MeJA treatments, being higher infungicide treated seedlings mainly in Companion and carbendazimtreatments.

Three EST isoforms (EST# 1–3) were detected in MeJA andfungicide treatments (Fig. 5): EST 1 in Companion, carbendazimand MeJA treatments but not in mancozeb and controls. The rela-tive position of EST 1 in Companion was different and its intensitywas also very high. This indicates its strong association with MeJA,and the systemic fungicide, carbendazim in inducing systemicresistance in chilli seedlings. The EST 2 over-expressed itself in theCompanion and MeJA treatments. The EST# 1–3 over-expresseditself with the Companion (Fig. 5). The EST 3 had over-expressedwith MeJA 5.0 mM, indicating a higher biological dose of theinducer of systemic resistance for its promotion.

4. Discussion

The disease intensity in chilli seedlings due to S. rolfsii wasconsiderably reduced with all fungicides, and MeJA. The MeJA isa well-known signaling molecule triggering host defense responsesto diverse pathogens including fungi (El-Araby and Ahmed, 2004;

Fig. 5. Esterase zymogram. Lane 1 – uninoculated control, Lane 2 – inoculated control,Lane 3 – Companion, Lane 4 – carbendazim, Lane 5 – mancozeb, Lane 6 – MeJA(2.5 mM) and Lane 7 – MeJA (5.0 mM).

S. Roy et al. / Crop Protection 29 (2010) 163–167 167

LiHong et al., 2004; Thaler et al., 2004), but no such specific workcould be traced in the literature on S. rolfsii infection. While contactfungicides are toxic to cellular functions and also denature certainmetabolically significant proteins and enzymes, systemic fungi-cides inhibit fungal growth or induce systemic acquired resistance(Gisi et al., 1985).

Earlier the superior efficacy of combination formulation of car-bendazimþmancozeb in controlling collar and root rot of straw-berry caused by S. rolfsii has been reported (Raj and Sharma, 2005).But the effects of MeJA and contact and systemic fungicides ondisease severity as well as on plant’s natural defense mechanismwas not studied in case of infection by S. rolfsii in chilli.

In the present study, POX activity was increased in the infectedchilli seedlings. The POX activity, in decreasing order, was asfollows: MeJA>Companion> carbendazim>mancozeb. Generallythe POX activity is increased by MeJA as a common response toinfection by plant pathogens including fungi (Borden and Higgins,2002) but there is no specific evidence from any sclerotial basid-iomycete. However, MeJA induces POX (and cutinases) in melonseedlings due to Sclerotinia sclerotiorum (Lib.) de Burry 1884 (Fam.Sclerotiniaceae, Order Helotiales, Sub-class Discomycetes [inoper-culate], Class Ascomycetes) and other soil-borne plant pathogenicfungi (Buzi et al., 2004). In this experiment, POX 1 and POX 4 areassociated with pathogenesis. MeJA- and Companion-treated chilliseedlings over-expressed POX 2 resulting in greater resistanceagainst S. rolfsii.

The combination formulation (Companion) is either additive orsynergistic in action, combining the modes of action of mancozeband carbendazim, as indicated by distinct over-expression of ESTsin chilli defense systems against S. rolfsii. MeJA most stronglyinduced over-expression of the EST isoforms, resulting in reduceddisease intensity in chilli inoculated with S. rolfsii.

Overall, the results indicated strong effect of each fungicides,especially Companion, and MeJA on the reduction of diseaseseverity, induction of defense-related enzymes, and isozymepatterns, as well as strongly additive, if not synergistic action of thecontact and systemic fungicides in the combination formulationover each sole component in both disease management andinduction of defense-related enzymes, viz. POXs and ESTs in thehost. This is the first report of such evidences in any host–parasiterelation between a plant host and a sclerotial basidiomycete fungalpathogen. It is also important that further detailed study of theinduction of systemic resistance against plant pathogens bycombination formulation of contact and systemic fungicides isundertaken.

References

Asperen, K., 1962. A study of housefly esterase by means of a sensitive colorimetricmethod. J. Ins. Physiol. 8, 401–416.

Borden, S., Higgins, V.J., 2002. Hydrogen peroxide plays a critical role in the defenseresponse to tomato to Cladosporium fulvum. Physiol. Mol. Plant Path. 61,227–236.

Buzi, A., Chilosi, G., Magro, P., 2004. Induction of resistance in melon seedlingsagainst soil-borne fungal pathogens by gaseous treatments with methyljasmonate and ethylene. J. Phytopathol. 152, 491–497.

Deacon, J.W., 1997. Modern Mycology, third ed. Blackwell Science, UK, pp. 281–284.El-Araby, M.M., Ahmed, H.F.S., 2004. Differential effect of jasmonic acid on the

defense of faba bean against Fusarium wilt: changes in protein and DNApatterns, peroxidase and esterase isozymes. Int. J. Agric. Biol. 6, 226–232.

Farr, D.F., Bills, G.F., Chamuris, G.P., Rossman, A.Y., 1989. Fungi on Plant and PlantProducts in the United States. American Phytopathological Society Press, St.Paul, MN.

Garcia, P.C., Rivero, R.M., Ruiz, J.M., Romero, L., 2003. The role of fungicides in thephysiology of higher plants: implications for defense responses. Bot. Rev. 69,162–172.

Gershater, M.C., Edwards, R., 2007. Regulating biological activity in plants withcarboxylesterases. Plant Sci. 173, 579–588.

Gisi, V., Binder, H., Rimbach, E., 1985. Synergistic interactions of fungicides withdifferent modes of action. Trans. Br. Mycol. Soc. 85, 299–306.

Gomez, K.A., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research.John Wiley and Sons, New York, USA.

Kahler, A.L., Allard, R.W., 1970. Genetics of isozyme variants in barley. I. Esterases.Crop Sci. 10, 444–448.

Kessman, H., Staub, T., Hofmann, C., Maetzke, T., Herzog, J., 1994. Induction ofsystemic acquired disease resistance in plants by chemicals. Annu. Rev. Phy-topathol. 32, 439–459.

Koike, N., Hyakumachi, M., Kageyama, K., Tsuyumu, S., Doke, N., 2001. Induction ofsystemic resistance in cucumber against several diseases by plant growthpromoting fungus: lignification and superoxide generation. Eur. J. Plant Pathol.107, 523–533.

Levy, Y., Levi, R., Cohen, Y., 1983. Buildup of a pathogen subpopulation to a systemicfungicide under various control strategies: a flexible simulation model. Phyto-pathology 73, 1475–1480.

LiHong, He, HongWei, He, JinHua, Bin, 2004. Effects of methyl jasmonate on theresistance of peanut seeds to Aspergillus flavus. J. South China Agric. Univ. 25,10–13.

Mittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends PlantSci. 7, 405–410.

Raj, H., Sharma, S.D., 2005. Integrated management of collar and root rot (Sclerotiumrolfsii) of strawberry. Acta Hort. (ISHS) 696, 375–379.

Shah, J., 2009. Plants under attack: systemic signals in defense. Curr. Opin. PlantBiol. 12, 459–464.

Sticher, L., Mauch-Mani, B., Metraux, J.P., 1997. Systemic acquired resistance. Annu.Rev. Phytopathol. 35, 235–270.

Thaler, J.S., Owen, B., Higgins, V.J., 2004. The role of the jasmonate response in plantsusceptibility to diverse pathogens with a range of lifestyles. Plant Physiol. 135,530–538.

Thimmiah, S.K., 2004. Standard Methods for Biochemical Analysis. KalyaniPublishers, Calcutta 700009, India, p. 58.

Vallad, G.E., Goodman, R.M., 2004. Systemic acquired resistance and inducedsystemic resistance in conventional agriculture. Crop Sci. 44, 1920–1934.

Vidhyasekaran, P., 2008. Fungal Pathogenesis in Plants and Crops: MolecularBiology and Host Defense Mechanisms, second ed. CRC Press, Taylor & FrancisGroup, New York.