Abstract

SHORT COMMUNICATION COMUNICAÇÃO

PR-protein activities in table beet against Cercospora beticola after spraying chitosan or acibenzolar-S-methyl

Ricardo B. Felipini; Robson M. Di Piero

Departamento de Fitotecnia, Universidade Federal de Santa Catarina, Cx. Postal 476, 88040-900, Florianópolis, SC, Brazil

^{Author for correspondence} Author for correspondence: Ricardo B. Felipini e-mail: ricardo_felipini@yahoo.com.br

ABSTRACT

Cercospora leaf spot (Cercospora beticola) is the most important disease of table beet in the world. In this study, the preventive application of chitosan or acibenzolar-S-methyl (ASM) to control the disease was evaluated, and the involvement of pathogenesis-related proteins in the plant-pathogen interaction was verified. Beet plants cultivar Early Wonder were sprayed with distilled water, HCl 0.05 N, chitosan at 2 mg/mL or 4 mg/mL and ASM at 25 mg/L, and 24 h or three days later, inoculated with the pathogen. Leaf samples were collected at different times after spraying and enzymatic activities were quantified spectrophotometrically. It was observed that chitosan and ASM reduced disease severity in table beet leaves by 76% and 68%, respectively, on the average of two experiments, and ASM promoted the accumulation of peroxidases and β-1,3 glucanases in beet leaves. Thus, both inducers reduce cercospora leaf spot in table beet, and the effect of ASM is associated with the activation of PR-proteins, whereas chitosan act by a still unknown mechanism.

Key words: Cercospora beticola, glucanases, peroxidases, phenylalanine ammonia lyase, resistance induction.

Cercospora leaf spot, caused by the fungus Cercospora beticola, is the main beet (Beta vulgaris) disease worldwide. In table beet, the disease besides resulting in decreased productivity, can depreciate the quality of the product due to spots on the leaves, when the plant is commercialized with the aerial part. The pathogen survives in plant debris, and environmental conditions such as high relative humidity and temperatures between 25ºC and 35ºC favor infection and disease development (Weiland & Koch, 2004). During colonization, the fungus occupies the intercellular spaces and uses reactive oxygen species as a weapon of attack in order to cause the collapse of the plant cells (Daub & Ehrenshaft, 2000). Among the plant defense mechanisms during the infectious process is the synthesis of pathogenesis-related proteins (PR-proteins), which participate in routes of the defense metabolism or act directly against the pathogen.

Peroxidases, for example, are enzymes involved in the oxidative elimination of reactive oxygen species produced both in the process of cellular respiration and in a pathogen attack. The increase of this enzymatic activity is associated with decreased severity of diseases such as Fusarium wilt in muskmelon caused by Fusarium oxyxporum f. sp. melonis (Madadkhah et al., 2012) and brown rot of peach by Monilinia fructicola (Ma et al., 2013). Phenylalanine ammonia lyase, in turn, is the key-enzyme in the phenylpropanoid metabolism (Hammerschimidt & Kagan, 2001). Through this metabolic pathway, plants synthesize polyphenols used in the process of lignification, and compounds of low molecular weight called phytoalexins that act against microbial infection, such as resveratrol on the grapevine downy mildew caused by Plasmopara viticola (Alonso-Villaverde et al., 2011). Another form of involvement of PR-proteins in plant defense is the direct effect, as it occurs with the β-1,3 glucanases, which hydrolyze β-glucans present in the cell wall of fungi.

The synthesis and accumulation of these PRproteins can be altered through the use of either abiotic or biotic elicitors. Chitosan, a polysaccharide obtained from deacetylation of chitin, extracted mainly from the shell of crustaceans, have the ability to activate biochemical defense responses by interacting with receptors on the surfaces of plant cells (Benhamou, 1992). Another mode of action of chitosan is the antimicrobial activity. Its positive charges may interact with negatively charged microbial surfaces, thereby restricting the movement of molecules for the microbial agent (Assis & Leoni, 2003). The ability of chitosan in reducing the severity of plant diseases has been demonstrated in peanut against Puccinia arachidis (Sathiyabama & Balasubramanian, 1998), common bean against Colletotrichum lindemuthianum (Di Piero & Garda, 2008) and apple against C. acutatum (Felipini & Di Piero, 2009).

On the other hand, acibenzolar-S-methyl (ASM) is a resistance inducer registered and commercially available for the prevention of fungal and bacterial diseases in crops such as bean (Phaseolus vulgaris) and tomato (Solanum lycopersicon) (MAPA, 2013). The molecule is absorbed and translocated by the plant and acts as a second messenger, resulting in the expression of genes related to systemic acquired resistance (Benhamou & Belanger, 1998). Its mode of action is mainly related to increased activity of phenylalanine ammonia lyase and the synthesis of phenolic compounds (Stadnik & Buchenauer, 1999).

The aim of this study was to evaluate the effect of chitosan and ASM for the control of cercospora leaf spot and analyze the PR-protein accumulation in plants after treatment with inducers and inoculation with the pathogen.

Experiments were conducted under greenhouse conditions using table beet cultivar Early Wonder. Seeds were sown in 128-cell polystyrene trays filled with commercial substrates. When plants reached the four-leaf stage, each plant was individually transferred to 2 L pots containing mineral soil and organic matter at a 6:1 ratio. Plants were treated two weeks after transplanting, at the sixleaf stage.

The suspensions of chitosan with 85% deacetylation were prepared by dissolving the product in HCl 0.05 N, with constant stirring and pH adjustment to 5.6 using 2 N NaOH. The ASM solutions were prepared from dissolving the commercial product Bion 500 WG in sterile distilled water. The fungal inoculum was obtained by adding 10 mL sterile distilled water to Petri dishes containing 10day old cultures of C. beticola on V8 agar medium, which was subsequently scraped with a Drigalski spatula. In all experiments, inoculations were carried out by spraying a suspension containing 1x10⁴ conidia/mL on the beet seedlings to run off and the plants were kept in a moist chamber for 36 h.

In the first experiment, plants were treated with distilled water, 0.05N HCl pH 5.6, chitosan at 2 mg/mL and 4 mg/mL or ASM at 25 mg/L and after 24 h were inoculated with the pathogen. In the second bioassay, plants were sprayed with 0.05 N HCl pH 5.6, chitosan at 2 mg/mL or ASM at 25 mg/L and 72 h later were challenged with the pathogen as described above. In this experiment, leaf samples were collected at 2, 4, 6, 8 and 10 days after spraying in order to determine the activity of PRproteins. Disease severity was evaluated at 20 days after the inoculation through the computational program Quant v.1.0.2. (Vale et al., 2001).

For protein extraction, each sample composed of approximately 200 mg of leaf tissue was macerated in liquid nitrogen, and then homogenized in 1.5 mL of 100 mM phosphate buffer pH 7.0 containing 1 mM phenylmethanesulfonyl fluoride, 0.5% polyvinylpyrrolidone and 1 mM ethylenediaminetetraacetic acid. The homogenates were placed in 2 mL microtubes, and centrifuged at 20000 g for 30 min at 4ºC. The supernatant, called protein extract, was collected and used to determine enzyme activities. The protein content of the sample was evaluated by the method of Bradford (1976). β-1,3 glucanase (EC 3.2.1.39) activity was determined from 40 µL of protein extract added to 460 µL of 100 mM acetate buffer pH 5 containing laminarin at 1 mg/mL at 44ºC following 1 h. Each sample was compared to a blank control, which contained all reagents in the same amounts, with the exception of laminarin (Di Piero & Pascholati, 2004). After 1 h, the reducing sugars were quantified (Lever, 1972). The reaction product absorbance was measured at 595 nm. Reducing sugar content was determined through a standard glucose curve, and results were expressed as µkatal glucose formed from laminarin hydrolysis per mg protein (µkatal/mg protein). Peroxidase activity (EC 1.11.1.7) was measured from a reaction that included 45 µL of protein extract, and 2.55 mL of 50 mM phosphate buffer pH 6 containing 0.25% guaiacol and 0.1 M hydrogen peroxide. The reaction was carried out for 4 min at 40ºC, and the absorbance values recorded in a spectrophotometer Femto 700 Plus at 470 nm, in 30 s intervals, from the first reaction minute. The results were expressed as optical density units at 470 nm per min per mg protein (OD470nm/min/mg protein) (Hammerschmidt et al., 1982). The determination of phenylalanine ammonia lyase (EC 4.3.1.5) activity was carried out according Falcón et al. (2008). Therefore, 40 µL of protein extract were added to 460 µL of 100 mM sodium borate buffer pH 8.8 containing 50 mM phenylalanine. After 1 h incubation at 40ºC, the samples were immersed for 5 min in ice bath and the reaction stopped by adding 200 µL of 5 N HCl. Finally, 300 µL of distilled water were added to the mixture and the absorbance was measured at 290 nm. The experiments were conducted using a randomised complete design with five replicates per treatment; each plot was composed of a pot containing one plant. Analysis of variance (ANOVA), F-tests (P < 0.05), and Student-Newman-Keuls (SNK) (P < 0.05) were conducted in experiments to test for significant differences among means.

In the first experiment, there were reductions of 72%, 72% and 55% in cercospora leaf spot severity when chitosan was applied at 2 mg/mL or 4 mg/mL, and ASM at 25 mg/L, respectively, 24 h before inoculation. Spraying with HCl pH 5.6 did not differ from the control with distilled water (data not shown). The ability of the products to control the disease was confirmed in the second experiment, when applied 72 h before inoculation, with chitosan at 2 mg/mL reducing the disease severity in 80% and ASM at 25 mg/L in 91% (Figure 1A).

The reduction in disease by chitosan and ASM was reported in the literature for several foliar and fruit diseases. Sathiyabama & Balasubramanian (1998) obtained a reduction of peanut rust (Puccinia arachidis) by applying 1 mg/mL chitosan preventively. In addition, there was a decrease of the production of uredinospores in vivo. Ma et al. (2013) found that immersion of peach fruits in a solution with 0.5 mg/mL of chitosan reduced the incidence of Monilinia fructicola. In the case of ASM, the product is commercially recommended for different crops, such as cotton, potato, citrus, bean, melon and tomato (MAPA, 2013), However, there is no information on the use of chitosan and ASM for the control of cercospora leaf spot in table beet.

Beet leaves treated with ASM showed an increase in β-1,3 glucanases after inoculation, indicating that the plants were pre-sensitized by the product (Figure 1B).

These enzymes are able to hydrolyze β-1,3 glucan of the fungal cell wall. The basic forms are within the vacuoles and acid forms are found in cell walls (Pascholati & Leite, 1995). The latter defend the plant during the colonization of intercellular spaces by the pathogen, a place that C. beticola occupies during the initial process of colonization (Pal & Mukhopadhyay, 1984). This increase in β-1,3 glucanase activity may have contributed to lower disease levels in plants treated preventively with ASM. Increased levels of this enzyme after application of ASM has been shown to be associated with the reduction of diseases such as bacterial wilt in tomato (Xanthomonas campestris pv. campestris) (Cavalcanti et al., 2006), fusariosis in cowpea (Fusarium oxysporum f. sp. tracheiphilum) (Rodrigues et al., 2006), and leaf spot of cotton (Xanthomonas axonopodis pv. malvacearum) (Ishida et al., 2008).

Plants sprayed with ASM showed higher peroxidase activity before inoculation (Figure 1C). Peroxidases are related to greater stress tolerance and to the elimination of reactive oxygen species resulting from the metabolism (Resende et al., 2003). However, C. beticola uses reactive oxygen species as chemical weapons to destroy the cell membranes of plants (Daub & Ehrenshaft, 2000). Thus, the higher peroxidase activity may protect the plant by eliminating these highly cytotoxic molecules generated by the pathogen. The highest activity of this enzyme is related to the control of plant diseases in other crops. Viecelli et al. (2009) showed a reduction of the severity of angular leaf spot of bean caused by Pseudocercospora griseola when the plants were treated with 75 mg/L of ASM and observed that the control was associated with increased peroxidase activity.

The activity of phenylalanine ammonia lyase, in turn, increased after challenge with the pathogen, but did not differ between plants that received different treatments. One type of plant defense response against infection by pathogens is the production of defense molecules. This includes the phytoalexins, through the route of phenylpropanoids, catalyzed initially by phenylalanine ammonia lyase (Siegrist et al., 1998). The results reported here indicate that this was a normal reaction mechanism of the plant in contact with the pathogen, not influenced by the inducers tested and insufficient to prevent infection and disease development (Figure 1D).

Although several studies relate the ability of chitosan to induce the synthesis of defense enzymes, in the conditions of the present study it was observed that this polysaccharide did not promote an increase in the activity of the evaluated PR-proteins. Thus, the reduction of the cercospora leaf spot in beet by the application of chitosan may be due to the antimicrobial effect of the product on C. beticola or to the activation of other types of defense mechanisms unrelated to PR-proteins. The fungicidal effect of chitosan has been documented in the literature on Pythium aphanidermatum, C. acutatum, B. cinerea and P. expansum, among others (El Ghaouth et al., 1994; Liu et al., 2007; Felipini & Di Piero, 2009). Further studies need to be carried out to verify this effect on C. beticola.

Therefore, both chitosan and ASM reduce cercospora leaf spot in table beet. In the case of ASM, the effect occurs concomitantly to the increment of the peroxidase and β-1,3 glucanase activities, whereas chitosan act by a mechanism not revealed in our studies.

Received 4 July 2013

Accepted 17 September 2013

TPP 2013-0116

Section Editor: Jorge Teodoro de Souza

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