- Email: [email protected]
Race-specific and ulvan-induced defense responses in bean (Phaseolus vulgaris) against Colletotrichum lindemuthianum
Physiological and Molecular Plant Pathology 78 (2012) 8e13
Contents lists available at SciVerse ScienceDirect
Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
Race-specific and ulvan-induced defense responses in bean (Phaseolus vulgaris) against Colletotrichum lindemuthianum Mateus B. de Freitas, Marciel J. Stadnik* Departamento de Fitotecnia, Rod. Admar Gonzaga, 1346, Universidade Federal de Santa Catarina, CP 476, 88040-900 Florianópolis, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 23 December 2011
The present work aimed to monitor and compare race-specific and ulvan-induced defense responses to race 73 of Colletotrichum lindemuthianum (Cl) in resistant and susceptible bean plants. Under greenhouse conditions, foliar spraying of ulvan, a water-soluble algal polysaccharide, reduced anthracnose severity locally and systemically in susceptible plants by 60 and 40% respectively. Neither race-specific resistance nor ulvan treatment affected both conidial germination and appressorial formation of Cl on leaves. Race 73-specific resistance was associated with a rapid recognition of the pathogen, expressed by a more frequent hypersensitive response in bean epidermal cells. Ulvan increased peroxidase activity in resistant, but not in susceptible plants, and glucanase activity in both resistant and susceptible plants inoculated or not with Cl. This is the first report of an increase in activity of plant defense-related enzymes by treatment of a polysaccharide before pathogen infection. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Colletotrichum lindemuthianum Phaseolus vulgaris Bean Anthracnose Ulvan Induced resistance Race-specific resistance Peroxidase Glucanase Hypersensitive response
1. Introduction Anthracnose caused by the hemibiotrophic fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Scrib (Cl) is one of the most important diseases of bean (Phaseolus vulgaris L.) causing serious crop losses worldwide. Up to now, resistant cultivars and fungicides application have been the main recommended measures for controlling this disease [1e4]. Anthracnose is a difficult disease to control because the pathogen shows high genetic variability, survives in cultural debris and can be efficiently transmitted through seeds [2e5]. Researches performed in Brazil have revealed the existence of great variability in pathogen population, where races 55, 65 and 73 are the most frequent in bean crops [4]. Currently, races of Cl are identified by inoculating isolates onto a universal set of 12 differential cultivars and named accordingly to a binary nomenclature system [2]. For example, race 73 is virulent to the following differential cultivars: Michelite (20), Cornell 49-242 (23) and Mexico 222 (26). Due to enormous genetic variability of pathogen and continuous emergence of new races,
Abbreviations: Cl, Colletotrichum lindemuthianum; DAB, diaminobenzidine; d.a.i, days after inoculation; GLU, glucanase; h.a.i, hours after inoculation; HR, hypersensitive reaction; POX, peroxidase. * Corresponding author. Tel.: þ55 48 3721 5423; fax: þ55 48 3721 5335. E-mail address: [email protected] (M.J. Stadnik). 0885-5765/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2011.12.004
monogenic resistance has been considered not durable for bean plants [3]. Bean race-specific resistance to anthracnose is conferred by different single, duplicate or complementary dominant genes [2,6]. Up to now, 13 resistance genes have been described (called Co genes) [7]. These genes and their alleles include Co-1, Co-11, Co-12, Co-13, Co-14, Co-15, Co-2, Co-3, Co-32, Co-4, Co-42, Co-43, Co-5, Co-6, Co-7, co-8, Co-9, Co-10, Co-11, Co-12 and Co-13 [2,8]. All of them are responsible for race-specific recognition, but none so far has been able to confer resistance to all known races [6]. Race-specific resistance is frequently associated with hypersensitive reaction (HR) characterized by a rapid death and browning of epidermal cells soon after penetration [9]. As a consequence, infection process can be efficiently blocked during the biotrophic phase of Cl [1]. The infection process of Cl begins with the germination of conidia on the plant surface producing germ-tubes and appressoria which penetrate the plant cuticle directly [1,3]. Following penetration, Cl produces infection vesicles and then primary hyphae inside host cells, between the plasma membrane and cell wall. By this way, the fungus can feed biotrophically on living host cells [1,3]. In the sequence, a necrotrophic mode of nutrition takes place where the pathogen produces narrow secondary hyphae which grow inter- and intracellularly ramifying rapidly through host tissues. At this stage, host protoplasts are killed and host cell walls degraded in advance of infection [1].
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
During an incompatible (host resistant) interaction between P. vulgaris and Cl, defense responses are induced with the beginning of appressoria maturation [3]. Induced defense responses include generation of reactive oxygen species like hydrogen peroxide (H2O2), cell wall reinforcement, synthesis of phytoalexins and accumulation of pathogenesis-related proteins like peroxidase and glucanase [9,10]. Peroxidase (POX) and glucanase (GLU) are two important enzymes involved in plant defense and their activity increases after pathogen attack or injuries. POX is likely to function in the cell wall lignification process using the novel H2O2 formed during oxidative burst. On the other hand, GLU hydrolyzes b-1,3-glucan polymers, one of the main component of fungal cell walls [9,10]. The current interest in the environment and human health together with an increase in the production costs and continuous breakdown of resistance in commercial cultivars have intensified the development of alternative control methods. In this scenario, algal polysaccharides arise as a convenient and environmentally friendly strategy [11]. Ulvan is a water-soluble heteropolysaccharide extracted from the cell walls of marine algae Ulva spp. representing from 8 to 29% of algal dry weight [11]. Recent studies have shown that it has potential to control rust (Uromyces appendiculatus) [12], anthracnose [11] and powdery mildew (Erysiphe polygoni) [13] on dry beans, and Glomerella leaf spot (C. gloeosporioides) on apple (Malus domestica) [14]. A growing number of scientific reports has also provided evidences that ulvan is able to induce resistance in both monocot and dicot plants [11e16]. Although some information on transcriptional and biochemical changes occurring in plants as a result of induced- or race-specificresistance is already available [3,9,10,15,16], knowledge on how the infection process of Cl is affected by race-specific as well as ulvaninduced resistance is still scarce. Therefore, the present work aimed to monitor the infection process of Cl, frequency of hypersensitive reaction and POX and GLU activities in resistant and susceptible bean plants treated with water or ulvan. 2. Material and methods 2.1. Biological material Near-isogenic resistant and susceptible lines derived from a segregating population of bean (P. vulgaris L.) cv. IPR Uirapuru [17] were used in the experiment. Ulvan was obtained as previously described by Paulert et al. [11]. Briefly, the ground dried algae (100 g) were autoclaved for 2 h at 110 C in distilled water (1 L). The resulting aqueous solution was filtered and the polysaccharide precipitated with ethanol for 48 h at 20 C. The precipitated ulvan was collected, dried and kept at 5 C until use in the biological assay. The strain MANE 003 of the physiological race 73 of C. lindemuthianum was used in this study. The isolate was kept on potato-dextrose agar (PDA) at 25 C and 12 h photoperiod. To obtain conidia, fungus was grown to sporulate on green bean pods at 25 C for 15 days. Thereafter, pods were flooded with 10 mL of distilled water, the conidial suspension was collected and filtered twice to remove mycelial fragments. The number of conidia was estimated using a haemocytometer and inoculum concentration was adjusted to 3 106 conidia mL1 with distilled water. 2.2. Plant growth conditions, treatment and inoculation Bean plants were cultivated in plastic pots containing a mixture of clay soil and organic compound (3:1; v/v). Nine days after sowing, seedlings were thinned out by leaving 3 plants in each pot. Plants were irrigated according to water needs of the culture.
9
Powdery mildew fungus and insects were controlled by application of 80% sulfur (5 g L1) and the insecticides deltametrin (3 mL L1) and malathion (2.5 mL L1), every two weeks. Four-week-old bean plants showing the 3rd fully expanded trifoliate leaf were sprayed twice (i.e. 6 and 3 days before inoculation) with water (control) or ulvan (10 mg mL1), according to Paulert et al. [11]. A volume of 5 mL was delivered per plant. During the treatments the 3rd leaf was covered with a plastic bag to evaluate systemic effect. Plants were inoculated with a homogenous suspension of C. lindemuthianum and placed in a dark highly humid chamber (humidity >90%) for 48 h. After that, inoculated plants were placed back in greenhouse until evaluation of anthracnose symptoms. A mock-inoculation was performed by spraying plants with distilled water. 2.3. Disease evaluation The anthracnose severity was assessed on the 2nd (local effect) and 3rd (systemic effect) trifoliate leaves at intervals of 2 days beginning with the first symptoms until 11 days after inoculation (d.a.i). The scale of Rava et al. [5] with scores ranging from 1 (no symptoms) to 9 (almost all plants dead) was used to assess anthracnose severity. Values were transformed to percentage of necrotic leaf area as described by Marques Júnior et al. [18], and used to calculate the Area Under Disease Progress Curve (AUDPC) by the formula: AUDPC ¼ [((y1þy2)/2)*(t2-t1)], were y1 and y2 are two consecutive severity estimations realized at time points t1 and t2, respectively [19]. 2.4. Sampling The infection process and the detection of the hypersensitive reaction were assessed at 48 h after inoculation (h.a.i), whereas POX and GLU activities at 12, 24 and 48 h.a.i. For that, at each time point four pots (replications) were selected from each treatment and four discs (8 mm) from the central leaflet (1st trifoliate leaf) were collected for microscopic analysis. Left and right leaflets (1st trifoliate leaf) were collected, weighted and stored at 20 C to evaluate POX and GLU activities, respectively. Samples were collected from each plant of the replication. 2.5. Microscopic analysis To monitor infection process, collected discs (two per plant) were bleached and stained according to Stadnik and Buchenauer [20], as follows: discs were immediately put with upper side in a ethanol and acetic acid (3:1; v/v) solution to fix and bleach the tissues that was changed periodically during a three-day period. Finally, the solution was substituted for a conservation solution containing lactic acid, glycerol and water (1:1:1; v/v/v). Fungal infection structures were stained with Amann’s lactophenol and then, the percentage of conidial germination and appressorial formation were determined on 100 conidia. The frequency of hypersensitive reaction was determined as described by Hückelhoven et al. [21], with some modifications. Briefly, leaf discs were immediately placed on a diaminobenzidine solution (DAB; 1 mg mL1) for 12 h. Then, the DAB solution was switched to a trichloroacetic acid 0.15% in ethanol and chloroform (4:1; v/v) solution to bleach the tissues. After 24 h, the discs were placed in a conservation solution (lactic acid: glycerol: water; 1:1:1; v/v/v). Hypersensitive cells were considered those which exhibited reddish-brown color. The number of hypersensitive cells was evaluated over the surface of the leaf discs and expressed as number of hypersensitive cells/cm2.
10
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
Leaf discs were examined in an optical microscopy (400) and the fungal infective structures and hypersensitive cells were photographed using a digital camera (model DSC-W300, Sony, Japan). 2.6. Peroxidase activity POX activity was evaluated as described by Cipollini [22], with some modifications. Leaves were ground using a chilled mortar and pestle in liquid nitrogen to obtain a fine powder. Then, cold 100 mM sodium phosphate buffer (pH 7) was added in a ratio of 5 mL buffer/ g leaf fresh mass. The suspension was transferred to microtubes, centrifuged at 11,600 g for 15 min at 4 C and the supernatant was collected and placed in an ice bath until analysis. POX activity was determined spectrophotometrically in a reaction mixture (3 mL) containing 0.1 mL of enzyme extract and 2.9 mL of 10 mM sodium phosphate buffer (pH 6) containing 0.25% guaiacol (v/v) and 0.125% hydrogen peroxide. After the addition of the enzyme extract the variations in the absorbance (470 nm) were monitored for 3 min and recorded every 30 s. The POX activity was measured as the increase in the Optical Density/min./mg protein. Protein content of extracts was determined as described by Bradford [23]. 2.7. Glucanase activity GLU activity was evaluated as described by Wirth and Wolf [24], with some modifications. Leaves were ground using a chilled mortar and pestle in liquid nitrogen. Then, cold 80 mM sodium acetate buffer (pH 5) containing 1 mM EDTA was added in a ratio of 4 mL buffer/g leaf fresh mass. The suspension was transferred to microtubes, centrifuged at 10,000 g for 15 min and the supernatant was collected and placed at 20 C until analysis. GLU activity was determined in a reaction mixture (800 mL) containing 200 mL of leaf extract, 400 mL of 80 mM sodium acetate buffer (pH 5) and 200 mL of carboxymethyl curdlan remazol brilliant blue (CM-Curdlan-RBB; 4 mg mL1). A mixture containing only buffer and CM-Curdlan-RBB was used as blank. Reaction mixture was incubated in a water bath for 2 h at 40 C. After the reaction was stopped by pipetting 200 mL 2 N HCl, samples were cooled for 10 min at room temperature and then centrifuged at 10,000 g for 15 min. The absorbance was determined spectrophotometrically in the supernatant at 600 nm. GLU activity was expressed as absorbance at 600 nm/mg protein. 2.8. Experimental design and statistical analysis The experiment was carried out in a factorial completely randomized design with 3 factors: line (resistant or susceptible), treatment (water or ulvan) and inoculation (C. lindemuthianum- or mock-inoculated plants). The experiment was carried out with 12 replications (pots) each one containing three plants. After verification of homogeneity of the variances of the datasets, data were subjected to analysis of variance (ANOVA). Tukey’s or t tests were used for separation of means, both at 5% of significance level. The statistical analysis was performed using the software Statistica 6.0Ò (StatSoft). 3. Results and discussion In susceptible bean plants, first anthracnose symptoms appeared 5 days after inoculation (d.a.i) and the necrotic area of 2nd trifoliate leaf reached 56% at 11 d.a.i (Fig. 1A). The Area Under Disease Progress Curve (AUDPC) calculated for this leaf was 177 (Fig. 1B). In this situation, ulvan spraying reduced locally both leaf necrotic area and AUDPC by 60%. Accordingly, Paulert et al. [11]
Fig. 1. Percentage of leaf necrotic area at 11 days after inoculation (A) and Area Under Disease Progress Curve (AUDPC) (B) on the 2nd (local effect) and 3rd (systemic effect) trifoliate leaves of resistant and susceptible P. vulgaris plants sprayed with water (control) or ulvan. Lowercase and capital letters for 2nd and 3rd trifoliate leaves, respectively, indicate significant difference (t Test, p 0.05 or *p 0.08).
testing ulvan under similar conditions found a slightly lower disease reduction (40%) in bean anthracnose. Varying levels of protection by ulvan, ranging from 40% to nearly complete disease control, have, however, been reported in different foliar pathosystems, including bean diseases [11e13], Glomerella leaf spot (C. gloeosporioides) on apple [14], alfafa anthracnose (C. trifolii) [15] and powdery mildews on several crops plants [13,16]. Thus, ulvaninduced resistance seems to be consistent and able to provide an intermediate protection against anthracnose, at least in bean plants. Necrotic area of the 3rd trifoliate leaf in control plants at 11 d.a.i was 42% (Fig. 1A) and the AUDPC was 181 (Fig. 1B). Ulvan spraying reduced systemically both leaf necrotic area and AUDPC by 40%. The induction of resistance in non-treated tissues has been explained by the translocation of signals from treated leaves resulting in activation of defense genes and increase of resistance in distal leaves [25,26]. Indeed, ulvan can systemically elicit defense responses in beans and this effect has been described to be weaker than the local one [27]. It has also been known that, in general, systemic resistance provides a low, though significant, level of protection [26]. Induced resistance can protect plants against a broad spectrum of pathogens like fungi, bacteria and viruses [25,28]. Therefore, even for plants resistant to anthracnose, spraying of ulvan may have some advantages when controlling other diseases such as rust (U. appendiculatus) [12] and powdery mildew (E. polygoni) [13] or preventing race changes in the anthracnose population. This has been confirmed by Romero and Ritchie [29], who observed that treatment of bell pepper plants with acibenzolar-S-methyl induced resistance to bacterial spot and reduced the emergence of new
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
races of Xanthomonas axonopodis capable of overcoming existing resistance genes. This strategy is particularly important in beananthracnose pathosystem where the great genetic variability in the fungus inhibits the development of cultivars with durable resistance. Resistant plants sprayed with water or ulvan did not show any anthracnose symptoms, confirming the resistance to the race 73 of Cl. However, necrotic spots characterizing the hypersensitive response were observed 4 d.a.i (Fig. 2C). These spots had approximately 1 mm and were equally distributed (about 20 spots per leaflet) on the leaf internerval surface. Microscopic examination of spots revealed the presence of dead cell groups with no mycelial growth. The resistant line tested here was previously selected from a segregating population of the bean cv. IPR Uirapuru. IPR Uirapuru was obtained from the following cross: (IAPAR BAC 29 PR1711) [NEP 2 (Puebla 173 Icapijao)] and has been identified as susceptible to anthracnose [17]. Despite beans are self-pollinated plants and the commercial cultivars have a high degree of homozygosity, the emergence of segregants has been considered possible. For instance, Vallejo and Kelly [30] obtained segregating plants with a different resistant gene to anthracnose from cv. Black Magic. Another possibility would be the occurrence of a mutation producing the new resistance gene(s) present in the studied line [30]. Since the resistant line has shown similar agronomic characteristics to cv. Uirapuru, it could be used in cultivar mixtures for controlling bean anthracnose according to Ntahimpera et al. [31]. Nevertheless, further studies are necessary to evaluate the efficiency of such a strategy as well as to elucidate the genetic basis of this line. Microscopic analysis of the hostepathogen interaction revealed that neither genetic resistance nor ulvan treatment influenced both conidial germination and appressorial formation of fungus (Table 1; Fig. 2A and B). This result is consistent with previous reports [1,3,32], showing that conidial germination and appressorial formation of Cl are similar in both resistant and susceptible bean plants.
11
In the present work, the number of hypersensitive cells/cm2 at 48 h.a.i was significantly one fold higher in resistant than in susceptible line (Table 1; Fig. 2D). This finding is in accordance to O’Connell et al. [1], Veneault-Fourrey et al. [3] and O’Connell and Bailey [32], who demonstrated that an incompatible interaction between Cl and P. vulgaris mostly leads to hypersensitive reaction. In such case, no infection vesicle is formed and fungal growth remains restricted to the penetrated cell [1,32]. On the other hand, resistance induced by ulvan did not affect the frequency of hypersensitive cells in both resistant and susceptible bean plants (Table 1). This is also in agreement with previous reports, showing that generalized induction of defense responses by ulvan is not correlated with induction of hypersensitive reaction in wheat against powdery mildew [16]. Although resistance conferred by major genes usually leads to hypersensitive reaction [2,3,8,21], in the present work we report for the first time the occurrence of hypersensitive response in a highly susceptible bean cultivar to a compatible race of Cl. Hypersensitive cells have been also detected by means of diaminobenzidine staining method in susceptible cereal plants to biotrophic pathogens. Accordingly, Hückelhoven et al. [21] observed hypersensitive cells in barley (Hordeum vulgare L.) susceptible to powdery mildew (Blumeria graminis), but less frequently than in the respective nearisogenic resistant line. Thus, these findings suggest an existence of a certain degree of incompatibility between susceptible plants and their aggressive races, leading to some hypersensitive cell responses, even in an interaction considered to be compatible like in this work. POX activity was increased by ulvan treatment in 1st trifoliate leaves (Fig. 3). Thus, in resistant mock-plants, ulvan treatment increased POX activity by 60% at 12 and 24 h.a.i. As far as we know, this is the first report of increase in enzymatic activity by application of an algal polysaccharide without pathogen inoculation. Usually, resistance inducers do not increase POX activity before inoculation [33]. In contrast, in the present work, ulvan applied twice 6 and 3
Fig. 2. C. lindemuthianum conidial germination (A), appressorial formation (B) and hypersensitive response in resistant P. vulgaris plants detected visually (C) and microscopically (D) using DAB. GT: germ tube; CO: conidium; AP: appressorium; HR: hypersensitive response; HC: hypersensitive cell. Bars ¼ 10 mm.
12
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
Table 1 Percentage of conidial germination, appressorial formation of C. lindemuthianum and number of hypersensitive epidermal cells determined at 48 h.a.i on 1st trifoliate leaves of resistant (R) and susceptible (S) P. vulgaris plants sprayed with water or ulvan. Hypersensitive cells/cm2a
Line
Conidial germination (%)
Appressorial formation (%)
H2O
Ulvan
x
H2O
Ulvan
x
H2O
Ulvan
xb
S R x
90.8 90.1 90.5
90.5ns 90.4ns 90.5ns
90.7 90.3NS
6.7 8.8 7.8
8.3ns 5.7ns 7.0ns
7.5 7.3NS
23 44 34
21ns 37ns 29ns
22 A 41 B
ns: not significant in the row; NS: not significant in the column. a Bean plants were treated twice at 6 and 3 days before inoculation with the race 73 of C. lindemuthianum. b Capital letters indicate significant difference (t Test, p 0.05) in the column.
days before inoculation increased POX activity in mock-inoculated resistant, but not in susceptible plants (Fig. 3). Therefore, it must be argued that ulvan can cause some metabolic alterations in dependence of major resistance genes prior to the inoculation. Accordingly, in near-isogenic lines of barley (H. vulgare), resistance induced by application of an avirulent isolate of powdery mildew was more effective in lines containing the gene Mla7 when compared to those that had gene Mla13 [34]. Basically, two hypotheses could be taken in account to explain these results: a) ulvan could have sensitized bean cells, increasing its ability to mobilize defense responses. Induced resistance is frequently associated with priming [35], where previously treated plants/cells become primed. Thus, faster and more intense defense responses are activated against abiotic stress or pathogens [16,25]. For instance, treatment of wheat (Triticum aestivum) cultured cells with exopolysaccharides from Erwinia herbicola increases POX activity by
100% [35]. Recently, Paulert et al. [16] demonstrated that ulvan is able to prime the oxidative burst in wheat (T. aestivum) and rice (Oriza sativa) cultured cells. Or, b) ulvan could provide nutrients for the microorganisms growing on the phylloplane, these, in turn, could attempt to invade bean epidermal cells and trigger defense responses such as an increase in POX activity. According to Madhaiyan et al. [36] foliar spraying of Methylobacterium spp., a non-pathogenic common bacterium, increases the activity of defense-related enzymes such as peroxidase and glucanase in rice. Interestingly, Methylobacterium spp. are widely distributed on the phyllosphere of plants, including P. vulgaris. The inoculation increased POX activity only at 48 h.a.i in resistant bean plants, but not in susceptible ones (Fig. 3). During a plantefungus interaction, race-specific resistance acts in a specific and fast recognition of the pathogen leading to the induction of defense responses [3,25]. Interaction of resistant bean plants with other kinds of pathogen has been reported to enhance
Fig. 3. Peroxidase activity in 1st trifoliate leaves at 12, 24 and 48 h after Cl- or mockinoculation of resistant and susceptible P. vulgaris (cv. IPR Uirapuru) plants previously sprayed with water or ulvan. *Differs from its respective control sprayed with water (t Test, p 0.05 or (a)p 0.07). Bars indicate the mean standard deviation.
Fig. 4. Glucanase activity in 1st trifoliate leaves at 12, 24 and 48 h after Cl- or mockinoculation of resistant and susceptible P. vulgaris (cv. IPR Uirapuru) plants previously sprayed with water or ulvan. *Differs from its respective control sprayed with water (t Test, p 0.05). ns: not significant. Bars indicate the mean standard deviation.
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
the activity of enzymes related to production and utilization of H2O2 such as POX [9]. Thus, the peak of highest activity at 48 h.a.i in resistant plants could contribute to some extent, in blocking the fungus via cell wall reinforcement. Coincidently, the number of hypersensitive cells was also higher in inoculated resistant plants. It has been also reported [37,38] that peroxidase can produce reactive oxygen species leading to a hypersensitive response. Indeed, in bean suspension-cultured cells treated with a cell wall elicitor from Cl, cell wall peroxidase rather than NADPH oxidase has been proposed to be the major source of reactive oxygen species [37]. Ulvan spraying (Fig. 4) and inoculation with Cl (t Test; p 0.05) enhanced GLU activity by 40% in both resistant and susceptible bean plants. In bean plants, enhanced GLU activity has been observed after ulvan treatment in moderately susceptible, but not in plants resistant to rust (U. appendiculatus) [12]. All of these data suggest that ulvan has the ability to intensify the activity of glucanase. In contrast, microarray studies using ulvan-treated alfalfa plants, has indicated that genes related to glucanase synthesis are not affected by the polysaccharide [15]. Thus, further studies are necessary to better understand ulvan effects on glucanase activity. In conclusion, the present results show that neither racespecific nor ulvan treatment affect both conidial germination and appressorial formation of Cl. Race-specific resistance of bean against Cl race 73 seems to be associated with a rapid recognition of the pathogen, expressed by a more frequent hypersensitive response. Although not affecting HR, ulvan is able to protect bean plants against C. lindemuthianum and to increase POX and GLU activities in mock-inoculated resistant bean plants. The protection levels reported here and the broad spectrum of action described in the literature for ulvan illustrate the potential application of this polysaccharide for disease management in agriculture.
Acknowledgments MBF is grateful for a master’s degree scholarship from CAPES/ REUNI. MJS is a research member of CNPq. This work was financially supported by CNPq Grant Number 481707/2007-7.
References [1] O’Connell RJ, Bailey JA, Richmond DV. Cytology and physiology of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum. Physiol Plant Pathol 1985;27:75e98. [2] Kelly JD, Vallejo VA. A comprehensive review of the major genes conditioning resistance to anthracnose in common bean. Hort Sci 2004;39:1196e207. [3] Veneault-Fourrey C, Laugé R, Langin T. Nonpathogenic strains of Colletotrichum lindemuthianum trigger progressive bean defense responses during appressorium-mediated penetration. Appl Environ Microbiol 2005;71: 4761e70. [4] Vidigal Filho PS, Gonçalves Vidigal MC, Kelly JD, Kirk WW. Sources of resistance to anthracnose in traditional common bean cultivars from Paraná, Brazil. J Phytopathol 2007;155:108e13. [5] Rava CA, Molina J, Kauffmann M, Briones I. Determinación de razas fisiológicas de Colletotrichum lindemuthianum en Nicaragua. Trop Plant Pathol 1993;18: 388e91. [6] Mahuku GS, Jara CE, Cajiao C, Beebe S. Sources of resistance to Colletotrichum lindemuthianum in the secondary gene pool of Phaseolus vulgaris and in crosses of primary and secondary gene pools. Plant Dis 2002;86:1383e7. [7] Gonçalves-Vidigal MC, Cruz AS, Garcia A, Kami J, Vidigal Filho PS, Sousa LL, et al. Linkage mapping of the Phg-1 and Co-14 genes for resistance to angular leaf spot and anthracnose in the common bean cultivar AND 277. Theor Appl Genet 2011;122:893e903. [8] Gonçalves-Vidigal MC, Vidigal Filho PS, Medeiros AF, Pastor-Corrales MA. Common bean landrace Jalo Listas Pretas is the source of a new Andean anthracnose resistance gene. Crop Sci 2009;49:133e8. [9] Milosevic N, Slusarenko AJ. Active oxygen metabolism and lignification in the hypersensitive response in bean. Physiol Mol Plant Pathol 1996;49:143e58. [10] Dann EK, Meuwly P, Métraux JP, Deverall BJ. The effect of pathogen inoculation or chemical treatment on activities of chitinase and b-1,3-glucanase and accumulation of salicylic acid in leaves of green bean, Phaseolus vulgaris L. Physiol Mol Plant Pathol 1996;49:307e19.
13
[11] Paulert R, Talamini V, Cassolato JEF, Duarte MER, Noseda MD, Smania Júnior A, et al. Effects of sulfated polysaccharide and alcoholic extracts from Green seaweed Ulva fasciata on anthracnose severity and growth of common bean (Phaseolus vulgaris L.). J Plant Dis Prot 2009;116:263e70. [12] Borsato LC, Di Piero RM, Stadnik MJ. Mecanismos de defesa eliciados por ulvana contra Uromyces appendiculatus em três cultivares de feijoeiro. Trop Plant Pathol 2010;35:318e22. [13] Jaulneau V, Lafitte C, Corio-Costet MF, Stadnik MJ, Salamagne S, Briand X, et al. An Ulva armoricana extract protects plants against three powdery mildew pathogens. Eur J Plant Pathol 2011;131:393e401. [14] Araujo L, Stadnik MJ, Borsato LC, Valdebenito-Sanhueza RM. Fosfito de potássio e ulvana no controle da mancha foliar da gala em macieira. Trop Plant Pathol 2008;33:148e52. [15] Cluzet S, Torregrosa C, Jacquet C, Lafitte C, Fournier J, Mercier L, et al. Gene expression profiling and protection of Medicago truncatula against a fungal infection in response to an elicitor from green algae Ulva spp. Plant Cell Environ 2004;27:917e28. [16] Paulert R, Ebbinghaus D, Urlass C, Moerschbacher BM. Priming of the oxidative burst in rice and wheat cell cultures by ulvan, a polysaccharide from green macroalgae, and enhanced resistance against powdery mildew in wheat and barley plants. Plant Pathol 2010;59:634e42. [17] Moda-Cirino V, Oliari L, Lollato MA. Fonseca Júnior NS. IPR88 Uirapuru e common bean. Crop Breed Appl Biotechnol 2001;1:205e6. [18] Marques Júnior OGM, Ramalho MAP, Ferreira DF, Santos JB. Viabilidade do emprego de notas na avaliação de alguns caracteres do feijoeiro (Phaseolus vulgaris L.). Revista Ceres 1997;44:411e20. [19] Araujo L, Valdebenito-Sanhueza RM, Stadnik MJ. Avaliação de formulações de fosfito de potássio sobre Colletotrichum gloeosporioides in vitro e no controle pós-infeccional da mancha foliar de Glomerella em macieira. Trop Plant Pathol 2010;35:54e9. [20] Stadnik MJ, Buchenauer H. Inhibition of phenylalanine ammonia-lyase suppresses the resistance induced by benzothiadiazole in wheat to Blumeria graminis f. sp. tritici. Physiol Mol Plant Pathol 2000;57:25e34. [21] Hückelhoven R, Fodor J, Preis C, Kogel KH. Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation. Plant Physiol 1999;119:1251e60. [22] Cipollini Jr DF. The induction of soluble peroxidase activity in bean leaves by wind-induced mechanical perturbation. Am J Bot 1998;85:1586e91. [23] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248e54. [24] Wirth SJ, Wolf GA. Micro-plate colourimetric assay for endo-acting cellulose, xylanase, chitinase, 1,3-b-glucanase and amylase extracted from forest soil horizons. Soil Biol Biochem 1992;24:511e9. [25] Walters D, Walsh D, Newton A, Lyon G. Induced resistance for plant disease control: maximizing the efficacy of resistance elicitors. Phytopathology 2005; 95:1368e73. [26] Sticher L, Mauchi-Mani B, Métraux JP. Systemic acquired resistance. Annu Rev Phytopathol 1997;35:235e70. [27] Stadnik MJ, Fernandes WS. Resistance induced by ulvan to Colletotrichum lindemuthianum in common bean (Phaseolus vulgaris). J Plant Pathol (supplement); 2008:261. [28] Hammerschimidt R. Induced disease resistance: how to induce plants stop pathogens? Physiol Mol Plant Pathol 1999;55:77e84. [29] Romero AM, Ritchie DF. Systemic acquired resistance delays race shifts to major resistance genes in bell pepper. Phytopathology 2004;94:1376e82. [30] Vallejo V, Kelly JD. Unexpected resistance genes for anthracnose uncovered. Annu Rep Bean Improv Coop 2005;48:74e5. [31] Ntahimpera N, Dillard HR, Cobb AC, Seem RC. Anthracnose development in mixtures of resistant and susceptible dry bean cultivars. Phytopathology 1996;86:668e73. [32] O’Connell RJ, Bailey JA. Differences in the extent of fungal development, host cell necrosis and symptom expression during raceecultivar interactions between Phaseolus vulgaris and Colletotrichum lindemuthianum. Plant Pathol 1988;37:351e62. [33] Stadnik MJ, Bettiol W. Association between lipoxygenase and peroxidase activity and systemic protection of cucumber plants against Podosphaera xanthii induced by Oudemansiella canarii extracts. J Plant Dis Prot 2007;114:9e13. [34] Martinelli JA, Brown JKM, Wolfe MS. Effects of barley genotype on induced resistance to powdery mildew. Plant Pathol 1993;42:195e202. [35] Ortmann I, Moerschbacher BM. Spent growth medium of Pantoea agglomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide and enhanced peroxidase activity upon elicitation. Planta 2006;224: 963e70. [36] Madhaiyan M, Poonguzhali S, Senthilkumar M, Seshadri S, Chung H, Yang J, et al. Growth promotion and induction of systemic resistance in rice cultivar Co-47 (Oryza sativa L.) by Methylobacterium spp. Bot Stud 2004;45:315e24. [37] Bolwell GP, Davies DR, Gerrish C, Auh CK, Murphy TM. Comparative biochemistry of the oxidative burst produced by rose and French bean cells reveals two distinct mechanisms. Plant Physiol 1998;116:1379e85. [38] Choi HW, Kim YJK, Lee SC, Hong JK, Hwang BK. Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense responses to bacterial pathogens. Plant Physiol 2007;145: 890e904.
Contents lists available at SciVerse ScienceDirect
Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp
Race-specific and ulvan-induced defense responses in bean (Phaseolus vulgaris) against Colletotrichum lindemuthianum Mateus B. de Freitas, Marciel J. Stadnik* Departamento de Fitotecnia, Rod. Admar Gonzaga, 1346, Universidade Federal de Santa Catarina, CP 476, 88040-900 Florianópolis, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 23 December 2011
The present work aimed to monitor and compare race-specific and ulvan-induced defense responses to race 73 of Colletotrichum lindemuthianum (Cl) in resistant and susceptible bean plants. Under greenhouse conditions, foliar spraying of ulvan, a water-soluble algal polysaccharide, reduced anthracnose severity locally and systemically in susceptible plants by 60 and 40% respectively. Neither race-specific resistance nor ulvan treatment affected both conidial germination and appressorial formation of Cl on leaves. Race 73-specific resistance was associated with a rapid recognition of the pathogen, expressed by a more frequent hypersensitive response in bean epidermal cells. Ulvan increased peroxidase activity in resistant, but not in susceptible plants, and glucanase activity in both resistant and susceptible plants inoculated or not with Cl. This is the first report of an increase in activity of plant defense-related enzymes by treatment of a polysaccharide before pathogen infection. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Colletotrichum lindemuthianum Phaseolus vulgaris Bean Anthracnose Ulvan Induced resistance Race-specific resistance Peroxidase Glucanase Hypersensitive response
1. Introduction Anthracnose caused by the hemibiotrophic fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Scrib (Cl) is one of the most important diseases of bean (Phaseolus vulgaris L.) causing serious crop losses worldwide. Up to now, resistant cultivars and fungicides application have been the main recommended measures for controlling this disease [1e4]. Anthracnose is a difficult disease to control because the pathogen shows high genetic variability, survives in cultural debris and can be efficiently transmitted through seeds [2e5]. Researches performed in Brazil have revealed the existence of great variability in pathogen population, where races 55, 65 and 73 are the most frequent in bean crops [4]. Currently, races of Cl are identified by inoculating isolates onto a universal set of 12 differential cultivars and named accordingly to a binary nomenclature system [2]. For example, race 73 is virulent to the following differential cultivars: Michelite (20), Cornell 49-242 (23) and Mexico 222 (26). Due to enormous genetic variability of pathogen and continuous emergence of new races,
Abbreviations: Cl, Colletotrichum lindemuthianum; DAB, diaminobenzidine; d.a.i, days after inoculation; GLU, glucanase; h.a.i, hours after inoculation; HR, hypersensitive reaction; POX, peroxidase. * Corresponding author. Tel.: þ55 48 3721 5423; fax: þ55 48 3721 5335. E-mail address: [email protected] (M.J. Stadnik). 0885-5765/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2011.12.004
monogenic resistance has been considered not durable for bean plants [3]. Bean race-specific resistance to anthracnose is conferred by different single, duplicate or complementary dominant genes [2,6]. Up to now, 13 resistance genes have been described (called Co genes) [7]. These genes and their alleles include Co-1, Co-11, Co-12, Co-13, Co-14, Co-15, Co-2, Co-3, Co-32, Co-4, Co-42, Co-43, Co-5, Co-6, Co-7, co-8, Co-9, Co-10, Co-11, Co-12 and Co-13 [2,8]. All of them are responsible for race-specific recognition, but none so far has been able to confer resistance to all known races [6]. Race-specific resistance is frequently associated with hypersensitive reaction (HR) characterized by a rapid death and browning of epidermal cells soon after penetration [9]. As a consequence, infection process can be efficiently blocked during the biotrophic phase of Cl [1]. The infection process of Cl begins with the germination of conidia on the plant surface producing germ-tubes and appressoria which penetrate the plant cuticle directly [1,3]. Following penetration, Cl produces infection vesicles and then primary hyphae inside host cells, between the plasma membrane and cell wall. By this way, the fungus can feed biotrophically on living host cells [1,3]. In the sequence, a necrotrophic mode of nutrition takes place where the pathogen produces narrow secondary hyphae which grow inter- and intracellularly ramifying rapidly through host tissues. At this stage, host protoplasts are killed and host cell walls degraded in advance of infection [1].
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
During an incompatible (host resistant) interaction between P. vulgaris and Cl, defense responses are induced with the beginning of appressoria maturation [3]. Induced defense responses include generation of reactive oxygen species like hydrogen peroxide (H2O2), cell wall reinforcement, synthesis of phytoalexins and accumulation of pathogenesis-related proteins like peroxidase and glucanase [9,10]. Peroxidase (POX) and glucanase (GLU) are two important enzymes involved in plant defense and their activity increases after pathogen attack or injuries. POX is likely to function in the cell wall lignification process using the novel H2O2 formed during oxidative burst. On the other hand, GLU hydrolyzes b-1,3-glucan polymers, one of the main component of fungal cell walls [9,10]. The current interest in the environment and human health together with an increase in the production costs and continuous breakdown of resistance in commercial cultivars have intensified the development of alternative control methods. In this scenario, algal polysaccharides arise as a convenient and environmentally friendly strategy [11]. Ulvan is a water-soluble heteropolysaccharide extracted from the cell walls of marine algae Ulva spp. representing from 8 to 29% of algal dry weight [11]. Recent studies have shown that it has potential to control rust (Uromyces appendiculatus) [12], anthracnose [11] and powdery mildew (Erysiphe polygoni) [13] on dry beans, and Glomerella leaf spot (C. gloeosporioides) on apple (Malus domestica) [14]. A growing number of scientific reports has also provided evidences that ulvan is able to induce resistance in both monocot and dicot plants [11e16]. Although some information on transcriptional and biochemical changes occurring in plants as a result of induced- or race-specificresistance is already available [3,9,10,15,16], knowledge on how the infection process of Cl is affected by race-specific as well as ulvaninduced resistance is still scarce. Therefore, the present work aimed to monitor the infection process of Cl, frequency of hypersensitive reaction and POX and GLU activities in resistant and susceptible bean plants treated with water or ulvan. 2. Material and methods 2.1. Biological material Near-isogenic resistant and susceptible lines derived from a segregating population of bean (P. vulgaris L.) cv. IPR Uirapuru [17] were used in the experiment. Ulvan was obtained as previously described by Paulert et al. [11]. Briefly, the ground dried algae (100 g) were autoclaved for 2 h at 110 C in distilled water (1 L). The resulting aqueous solution was filtered and the polysaccharide precipitated with ethanol for 48 h at 20 C. The precipitated ulvan was collected, dried and kept at 5 C until use in the biological assay. The strain MANE 003 of the physiological race 73 of C. lindemuthianum was used in this study. The isolate was kept on potato-dextrose agar (PDA) at 25 C and 12 h photoperiod. To obtain conidia, fungus was grown to sporulate on green bean pods at 25 C for 15 days. Thereafter, pods were flooded with 10 mL of distilled water, the conidial suspension was collected and filtered twice to remove mycelial fragments. The number of conidia was estimated using a haemocytometer and inoculum concentration was adjusted to 3 106 conidia mL1 with distilled water. 2.2. Plant growth conditions, treatment and inoculation Bean plants were cultivated in plastic pots containing a mixture of clay soil and organic compound (3:1; v/v). Nine days after sowing, seedlings were thinned out by leaving 3 plants in each pot. Plants were irrigated according to water needs of the culture.
9
Powdery mildew fungus and insects were controlled by application of 80% sulfur (5 g L1) and the insecticides deltametrin (3 mL L1) and malathion (2.5 mL L1), every two weeks. Four-week-old bean plants showing the 3rd fully expanded trifoliate leaf were sprayed twice (i.e. 6 and 3 days before inoculation) with water (control) or ulvan (10 mg mL1), according to Paulert et al. [11]. A volume of 5 mL was delivered per plant. During the treatments the 3rd leaf was covered with a plastic bag to evaluate systemic effect. Plants were inoculated with a homogenous suspension of C. lindemuthianum and placed in a dark highly humid chamber (humidity >90%) for 48 h. After that, inoculated plants were placed back in greenhouse until evaluation of anthracnose symptoms. A mock-inoculation was performed by spraying plants with distilled water. 2.3. Disease evaluation The anthracnose severity was assessed on the 2nd (local effect) and 3rd (systemic effect) trifoliate leaves at intervals of 2 days beginning with the first symptoms until 11 days after inoculation (d.a.i). The scale of Rava et al. [5] with scores ranging from 1 (no symptoms) to 9 (almost all plants dead) was used to assess anthracnose severity. Values were transformed to percentage of necrotic leaf area as described by Marques Júnior et al. [18], and used to calculate the Area Under Disease Progress Curve (AUDPC) by the formula: AUDPC ¼ [((y1þy2)/2)*(t2-t1)], were y1 and y2 are two consecutive severity estimations realized at time points t1 and t2, respectively [19]. 2.4. Sampling The infection process and the detection of the hypersensitive reaction were assessed at 48 h after inoculation (h.a.i), whereas POX and GLU activities at 12, 24 and 48 h.a.i. For that, at each time point four pots (replications) were selected from each treatment and four discs (8 mm) from the central leaflet (1st trifoliate leaf) were collected for microscopic analysis. Left and right leaflets (1st trifoliate leaf) were collected, weighted and stored at 20 C to evaluate POX and GLU activities, respectively. Samples were collected from each plant of the replication. 2.5. Microscopic analysis To monitor infection process, collected discs (two per plant) were bleached and stained according to Stadnik and Buchenauer [20], as follows: discs were immediately put with upper side in a ethanol and acetic acid (3:1; v/v) solution to fix and bleach the tissues that was changed periodically during a three-day period. Finally, the solution was substituted for a conservation solution containing lactic acid, glycerol and water (1:1:1; v/v/v). Fungal infection structures were stained with Amann’s lactophenol and then, the percentage of conidial germination and appressorial formation were determined on 100 conidia. The frequency of hypersensitive reaction was determined as described by Hückelhoven et al. [21], with some modifications. Briefly, leaf discs were immediately placed on a diaminobenzidine solution (DAB; 1 mg mL1) for 12 h. Then, the DAB solution was switched to a trichloroacetic acid 0.15% in ethanol and chloroform (4:1; v/v) solution to bleach the tissues. After 24 h, the discs were placed in a conservation solution (lactic acid: glycerol: water; 1:1:1; v/v/v). Hypersensitive cells were considered those which exhibited reddish-brown color. The number of hypersensitive cells was evaluated over the surface of the leaf discs and expressed as number of hypersensitive cells/cm2.
10
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
Leaf discs were examined in an optical microscopy (400) and the fungal infective structures and hypersensitive cells were photographed using a digital camera (model DSC-W300, Sony, Japan). 2.6. Peroxidase activity POX activity was evaluated as described by Cipollini [22], with some modifications. Leaves were ground using a chilled mortar and pestle in liquid nitrogen to obtain a fine powder. Then, cold 100 mM sodium phosphate buffer (pH 7) was added in a ratio of 5 mL buffer/ g leaf fresh mass. The suspension was transferred to microtubes, centrifuged at 11,600 g for 15 min at 4 C and the supernatant was collected and placed in an ice bath until analysis. POX activity was determined spectrophotometrically in a reaction mixture (3 mL) containing 0.1 mL of enzyme extract and 2.9 mL of 10 mM sodium phosphate buffer (pH 6) containing 0.25% guaiacol (v/v) and 0.125% hydrogen peroxide. After the addition of the enzyme extract the variations in the absorbance (470 nm) were monitored for 3 min and recorded every 30 s. The POX activity was measured as the increase in the Optical Density/min./mg protein. Protein content of extracts was determined as described by Bradford [23]. 2.7. Glucanase activity GLU activity was evaluated as described by Wirth and Wolf [24], with some modifications. Leaves were ground using a chilled mortar and pestle in liquid nitrogen. Then, cold 80 mM sodium acetate buffer (pH 5) containing 1 mM EDTA was added in a ratio of 4 mL buffer/g leaf fresh mass. The suspension was transferred to microtubes, centrifuged at 10,000 g for 15 min and the supernatant was collected and placed at 20 C until analysis. GLU activity was determined in a reaction mixture (800 mL) containing 200 mL of leaf extract, 400 mL of 80 mM sodium acetate buffer (pH 5) and 200 mL of carboxymethyl curdlan remazol brilliant blue (CM-Curdlan-RBB; 4 mg mL1). A mixture containing only buffer and CM-Curdlan-RBB was used as blank. Reaction mixture was incubated in a water bath for 2 h at 40 C. After the reaction was stopped by pipetting 200 mL 2 N HCl, samples were cooled for 10 min at room temperature and then centrifuged at 10,000 g for 15 min. The absorbance was determined spectrophotometrically in the supernatant at 600 nm. GLU activity was expressed as absorbance at 600 nm/mg protein. 2.8. Experimental design and statistical analysis The experiment was carried out in a factorial completely randomized design with 3 factors: line (resistant or susceptible), treatment (water or ulvan) and inoculation (C. lindemuthianum- or mock-inoculated plants). The experiment was carried out with 12 replications (pots) each one containing three plants. After verification of homogeneity of the variances of the datasets, data were subjected to analysis of variance (ANOVA). Tukey’s or t tests were used for separation of means, both at 5% of significance level. The statistical analysis was performed using the software Statistica 6.0Ò (StatSoft). 3. Results and discussion In susceptible bean plants, first anthracnose symptoms appeared 5 days after inoculation (d.a.i) and the necrotic area of 2nd trifoliate leaf reached 56% at 11 d.a.i (Fig. 1A). The Area Under Disease Progress Curve (AUDPC) calculated for this leaf was 177 (Fig. 1B). In this situation, ulvan spraying reduced locally both leaf necrotic area and AUDPC by 60%. Accordingly, Paulert et al. [11]
Fig. 1. Percentage of leaf necrotic area at 11 days after inoculation (A) and Area Under Disease Progress Curve (AUDPC) (B) on the 2nd (local effect) and 3rd (systemic effect) trifoliate leaves of resistant and susceptible P. vulgaris plants sprayed with water (control) or ulvan. Lowercase and capital letters for 2nd and 3rd trifoliate leaves, respectively, indicate significant difference (t Test, p 0.05 or *p 0.08).
testing ulvan under similar conditions found a slightly lower disease reduction (40%) in bean anthracnose. Varying levels of protection by ulvan, ranging from 40% to nearly complete disease control, have, however, been reported in different foliar pathosystems, including bean diseases [11e13], Glomerella leaf spot (C. gloeosporioides) on apple [14], alfafa anthracnose (C. trifolii) [15] and powdery mildews on several crops plants [13,16]. Thus, ulvaninduced resistance seems to be consistent and able to provide an intermediate protection against anthracnose, at least in bean plants. Necrotic area of the 3rd trifoliate leaf in control plants at 11 d.a.i was 42% (Fig. 1A) and the AUDPC was 181 (Fig. 1B). Ulvan spraying reduced systemically both leaf necrotic area and AUDPC by 40%. The induction of resistance in non-treated tissues has been explained by the translocation of signals from treated leaves resulting in activation of defense genes and increase of resistance in distal leaves [25,26]. Indeed, ulvan can systemically elicit defense responses in beans and this effect has been described to be weaker than the local one [27]. It has also been known that, in general, systemic resistance provides a low, though significant, level of protection [26]. Induced resistance can protect plants against a broad spectrum of pathogens like fungi, bacteria and viruses [25,28]. Therefore, even for plants resistant to anthracnose, spraying of ulvan may have some advantages when controlling other diseases such as rust (U. appendiculatus) [12] and powdery mildew (E. polygoni) [13] or preventing race changes in the anthracnose population. This has been confirmed by Romero and Ritchie [29], who observed that treatment of bell pepper plants with acibenzolar-S-methyl induced resistance to bacterial spot and reduced the emergence of new
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
races of Xanthomonas axonopodis capable of overcoming existing resistance genes. This strategy is particularly important in beananthracnose pathosystem where the great genetic variability in the fungus inhibits the development of cultivars with durable resistance. Resistant plants sprayed with water or ulvan did not show any anthracnose symptoms, confirming the resistance to the race 73 of Cl. However, necrotic spots characterizing the hypersensitive response were observed 4 d.a.i (Fig. 2C). These spots had approximately 1 mm and were equally distributed (about 20 spots per leaflet) on the leaf internerval surface. Microscopic examination of spots revealed the presence of dead cell groups with no mycelial growth. The resistant line tested here was previously selected from a segregating population of the bean cv. IPR Uirapuru. IPR Uirapuru was obtained from the following cross: (IAPAR BAC 29 PR1711) [NEP 2 (Puebla 173 Icapijao)] and has been identified as susceptible to anthracnose [17]. Despite beans are self-pollinated plants and the commercial cultivars have a high degree of homozygosity, the emergence of segregants has been considered possible. For instance, Vallejo and Kelly [30] obtained segregating plants with a different resistant gene to anthracnose from cv. Black Magic. Another possibility would be the occurrence of a mutation producing the new resistance gene(s) present in the studied line [30]. Since the resistant line has shown similar agronomic characteristics to cv. Uirapuru, it could be used in cultivar mixtures for controlling bean anthracnose according to Ntahimpera et al. [31]. Nevertheless, further studies are necessary to evaluate the efficiency of such a strategy as well as to elucidate the genetic basis of this line. Microscopic analysis of the hostepathogen interaction revealed that neither genetic resistance nor ulvan treatment influenced both conidial germination and appressorial formation of fungus (Table 1; Fig. 2A and B). This result is consistent with previous reports [1,3,32], showing that conidial germination and appressorial formation of Cl are similar in both resistant and susceptible bean plants.
11
In the present work, the number of hypersensitive cells/cm2 at 48 h.a.i was significantly one fold higher in resistant than in susceptible line (Table 1; Fig. 2D). This finding is in accordance to O’Connell et al. [1], Veneault-Fourrey et al. [3] and O’Connell and Bailey [32], who demonstrated that an incompatible interaction between Cl and P. vulgaris mostly leads to hypersensitive reaction. In such case, no infection vesicle is formed and fungal growth remains restricted to the penetrated cell [1,32]. On the other hand, resistance induced by ulvan did not affect the frequency of hypersensitive cells in both resistant and susceptible bean plants (Table 1). This is also in agreement with previous reports, showing that generalized induction of defense responses by ulvan is not correlated with induction of hypersensitive reaction in wheat against powdery mildew [16]. Although resistance conferred by major genes usually leads to hypersensitive reaction [2,3,8,21], in the present work we report for the first time the occurrence of hypersensitive response in a highly susceptible bean cultivar to a compatible race of Cl. Hypersensitive cells have been also detected by means of diaminobenzidine staining method in susceptible cereal plants to biotrophic pathogens. Accordingly, Hückelhoven et al. [21] observed hypersensitive cells in barley (Hordeum vulgare L.) susceptible to powdery mildew (Blumeria graminis), but less frequently than in the respective nearisogenic resistant line. Thus, these findings suggest an existence of a certain degree of incompatibility between susceptible plants and their aggressive races, leading to some hypersensitive cell responses, even in an interaction considered to be compatible like in this work. POX activity was increased by ulvan treatment in 1st trifoliate leaves (Fig. 3). Thus, in resistant mock-plants, ulvan treatment increased POX activity by 60% at 12 and 24 h.a.i. As far as we know, this is the first report of increase in enzymatic activity by application of an algal polysaccharide without pathogen inoculation. Usually, resistance inducers do not increase POX activity before inoculation [33]. In contrast, in the present work, ulvan applied twice 6 and 3
Fig. 2. C. lindemuthianum conidial germination (A), appressorial formation (B) and hypersensitive response in resistant P. vulgaris plants detected visually (C) and microscopically (D) using DAB. GT: germ tube; CO: conidium; AP: appressorium; HR: hypersensitive response; HC: hypersensitive cell. Bars ¼ 10 mm.
12
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
Table 1 Percentage of conidial germination, appressorial formation of C. lindemuthianum and number of hypersensitive epidermal cells determined at 48 h.a.i on 1st trifoliate leaves of resistant (R) and susceptible (S) P. vulgaris plants sprayed with water or ulvan. Hypersensitive cells/cm2a
Line
Conidial germination (%)
Appressorial formation (%)
H2O
Ulvan
x
H2O
Ulvan
x
H2O
Ulvan
xb
S R x
90.8 90.1 90.5
90.5ns 90.4ns 90.5ns
90.7 90.3NS
6.7 8.8 7.8
8.3ns 5.7ns 7.0ns
7.5 7.3NS
23 44 34
21ns 37ns 29ns
22 A 41 B
ns: not significant in the row; NS: not significant in the column. a Bean plants were treated twice at 6 and 3 days before inoculation with the race 73 of C. lindemuthianum. b Capital letters indicate significant difference (t Test, p 0.05) in the column.
days before inoculation increased POX activity in mock-inoculated resistant, but not in susceptible plants (Fig. 3). Therefore, it must be argued that ulvan can cause some metabolic alterations in dependence of major resistance genes prior to the inoculation. Accordingly, in near-isogenic lines of barley (H. vulgare), resistance induced by application of an avirulent isolate of powdery mildew was more effective in lines containing the gene Mla7 when compared to those that had gene Mla13 [34]. Basically, two hypotheses could be taken in account to explain these results: a) ulvan could have sensitized bean cells, increasing its ability to mobilize defense responses. Induced resistance is frequently associated with priming [35], where previously treated plants/cells become primed. Thus, faster and more intense defense responses are activated against abiotic stress or pathogens [16,25]. For instance, treatment of wheat (Triticum aestivum) cultured cells with exopolysaccharides from Erwinia herbicola increases POX activity by
100% [35]. Recently, Paulert et al. [16] demonstrated that ulvan is able to prime the oxidative burst in wheat (T. aestivum) and rice (Oriza sativa) cultured cells. Or, b) ulvan could provide nutrients for the microorganisms growing on the phylloplane, these, in turn, could attempt to invade bean epidermal cells and trigger defense responses such as an increase in POX activity. According to Madhaiyan et al. [36] foliar spraying of Methylobacterium spp., a non-pathogenic common bacterium, increases the activity of defense-related enzymes such as peroxidase and glucanase in rice. Interestingly, Methylobacterium spp. are widely distributed on the phyllosphere of plants, including P. vulgaris. The inoculation increased POX activity only at 48 h.a.i in resistant bean plants, but not in susceptible ones (Fig. 3). During a plantefungus interaction, race-specific resistance acts in a specific and fast recognition of the pathogen leading to the induction of defense responses [3,25]. Interaction of resistant bean plants with other kinds of pathogen has been reported to enhance
Fig. 3. Peroxidase activity in 1st trifoliate leaves at 12, 24 and 48 h after Cl- or mockinoculation of resistant and susceptible P. vulgaris (cv. IPR Uirapuru) plants previously sprayed with water or ulvan. *Differs from its respective control sprayed with water (t Test, p 0.05 or (a)p 0.07). Bars indicate the mean standard deviation.
Fig. 4. Glucanase activity in 1st trifoliate leaves at 12, 24 and 48 h after Cl- or mockinoculation of resistant and susceptible P. vulgaris (cv. IPR Uirapuru) plants previously sprayed with water or ulvan. *Differs from its respective control sprayed with water (t Test, p 0.05). ns: not significant. Bars indicate the mean standard deviation.
M.B. de Freitas, M.J. Stadnik / Physiological and Molecular Plant Pathology 78 (2012) 8e13
the activity of enzymes related to production and utilization of H2O2 such as POX [9]. Thus, the peak of highest activity at 48 h.a.i in resistant plants could contribute to some extent, in blocking the fungus via cell wall reinforcement. Coincidently, the number of hypersensitive cells was also higher in inoculated resistant plants. It has been also reported [37,38] that peroxidase can produce reactive oxygen species leading to a hypersensitive response. Indeed, in bean suspension-cultured cells treated with a cell wall elicitor from Cl, cell wall peroxidase rather than NADPH oxidase has been proposed to be the major source of reactive oxygen species [37]. Ulvan spraying (Fig. 4) and inoculation with Cl (t Test; p 0.05) enhanced GLU activity by 40% in both resistant and susceptible bean plants. In bean plants, enhanced GLU activity has been observed after ulvan treatment in moderately susceptible, but not in plants resistant to rust (U. appendiculatus) [12]. All of these data suggest that ulvan has the ability to intensify the activity of glucanase. In contrast, microarray studies using ulvan-treated alfalfa plants, has indicated that genes related to glucanase synthesis are not affected by the polysaccharide [15]. Thus, further studies are necessary to better understand ulvan effects on glucanase activity. In conclusion, the present results show that neither racespecific nor ulvan treatment affect both conidial germination and appressorial formation of Cl. Race-specific resistance of bean against Cl race 73 seems to be associated with a rapid recognition of the pathogen, expressed by a more frequent hypersensitive response. Although not affecting HR, ulvan is able to protect bean plants against C. lindemuthianum and to increase POX and GLU activities in mock-inoculated resistant bean plants. The protection levels reported here and the broad spectrum of action described in the literature for ulvan illustrate the potential application of this polysaccharide for disease management in agriculture.
Acknowledgments MBF is grateful for a master’s degree scholarship from CAPES/ REUNI. MJS is a research member of CNPq. This work was financially supported by CNPq Grant Number 481707/2007-7.
References [1] O’Connell RJ, Bailey JA, Richmond DV. Cytology and physiology of infection of Phaseolus vulgaris by Colletotrichum lindemuthianum. Physiol Plant Pathol 1985;27:75e98. [2] Kelly JD, Vallejo VA. A comprehensive review of the major genes conditioning resistance to anthracnose in common bean. Hort Sci 2004;39:1196e207. [3] Veneault-Fourrey C, Laugé R, Langin T. Nonpathogenic strains of Colletotrichum lindemuthianum trigger progressive bean defense responses during appressorium-mediated penetration. Appl Environ Microbiol 2005;71: 4761e70. [4] Vidigal Filho PS, Gonçalves Vidigal MC, Kelly JD, Kirk WW. Sources of resistance to anthracnose in traditional common bean cultivars from Paraná, Brazil. J Phytopathol 2007;155:108e13. [5] Rava CA, Molina J, Kauffmann M, Briones I. Determinación de razas fisiológicas de Colletotrichum lindemuthianum en Nicaragua. Trop Plant Pathol 1993;18: 388e91. [6] Mahuku GS, Jara CE, Cajiao C, Beebe S. Sources of resistance to Colletotrichum lindemuthianum in the secondary gene pool of Phaseolus vulgaris and in crosses of primary and secondary gene pools. Plant Dis 2002;86:1383e7. [7] Gonçalves-Vidigal MC, Cruz AS, Garcia A, Kami J, Vidigal Filho PS, Sousa LL, et al. Linkage mapping of the Phg-1 and Co-14 genes for resistance to angular leaf spot and anthracnose in the common bean cultivar AND 277. Theor Appl Genet 2011;122:893e903. [8] Gonçalves-Vidigal MC, Vidigal Filho PS, Medeiros AF, Pastor-Corrales MA. Common bean landrace Jalo Listas Pretas is the source of a new Andean anthracnose resistance gene. Crop Sci 2009;49:133e8. [9] Milosevic N, Slusarenko AJ. Active oxygen metabolism and lignification in the hypersensitive response in bean. Physiol Mol Plant Pathol 1996;49:143e58. [10] Dann EK, Meuwly P, Métraux JP, Deverall BJ. The effect of pathogen inoculation or chemical treatment on activities of chitinase and b-1,3-glucanase and accumulation of salicylic acid in leaves of green bean, Phaseolus vulgaris L. Physiol Mol Plant Pathol 1996;49:307e19.
13
[11] Paulert R, Talamini V, Cassolato JEF, Duarte MER, Noseda MD, Smania Júnior A, et al. Effects of sulfated polysaccharide and alcoholic extracts from Green seaweed Ulva fasciata on anthracnose severity and growth of common bean (Phaseolus vulgaris L.). J Plant Dis Prot 2009;116:263e70. [12] Borsato LC, Di Piero RM, Stadnik MJ. Mecanismos de defesa eliciados por ulvana contra Uromyces appendiculatus em três cultivares de feijoeiro. Trop Plant Pathol 2010;35:318e22. [13] Jaulneau V, Lafitte C, Corio-Costet MF, Stadnik MJ, Salamagne S, Briand X, et al. An Ulva armoricana extract protects plants against three powdery mildew pathogens. Eur J Plant Pathol 2011;131:393e401. [14] Araujo L, Stadnik MJ, Borsato LC, Valdebenito-Sanhueza RM. Fosfito de potássio e ulvana no controle da mancha foliar da gala em macieira. Trop Plant Pathol 2008;33:148e52. [15] Cluzet S, Torregrosa C, Jacquet C, Lafitte C, Fournier J, Mercier L, et al. Gene expression profiling and protection of Medicago truncatula against a fungal infection in response to an elicitor from green algae Ulva spp. Plant Cell Environ 2004;27:917e28. [16] Paulert R, Ebbinghaus D, Urlass C, Moerschbacher BM. Priming of the oxidative burst in rice and wheat cell cultures by ulvan, a polysaccharide from green macroalgae, and enhanced resistance against powdery mildew in wheat and barley plants. Plant Pathol 2010;59:634e42. [17] Moda-Cirino V, Oliari L, Lollato MA. Fonseca Júnior NS. IPR88 Uirapuru e common bean. Crop Breed Appl Biotechnol 2001;1:205e6. [18] Marques Júnior OGM, Ramalho MAP, Ferreira DF, Santos JB. Viabilidade do emprego de notas na avaliação de alguns caracteres do feijoeiro (Phaseolus vulgaris L.). Revista Ceres 1997;44:411e20. [19] Araujo L, Valdebenito-Sanhueza RM, Stadnik MJ. Avaliação de formulações de fosfito de potássio sobre Colletotrichum gloeosporioides in vitro e no controle pós-infeccional da mancha foliar de Glomerella em macieira. Trop Plant Pathol 2010;35:54e9. [20] Stadnik MJ, Buchenauer H. Inhibition of phenylalanine ammonia-lyase suppresses the resistance induced by benzothiadiazole in wheat to Blumeria graminis f. sp. tritici. Physiol Mol Plant Pathol 2000;57:25e34. [21] Hückelhoven R, Fodor J, Preis C, Kogel KH. Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation. Plant Physiol 1999;119:1251e60. [22] Cipollini Jr DF. The induction of soluble peroxidase activity in bean leaves by wind-induced mechanical perturbation. Am J Bot 1998;85:1586e91. [23] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248e54. [24] Wirth SJ, Wolf GA. Micro-plate colourimetric assay for endo-acting cellulose, xylanase, chitinase, 1,3-b-glucanase and amylase extracted from forest soil horizons. Soil Biol Biochem 1992;24:511e9. [25] Walters D, Walsh D, Newton A, Lyon G. Induced resistance for plant disease control: maximizing the efficacy of resistance elicitors. Phytopathology 2005; 95:1368e73. [26] Sticher L, Mauchi-Mani B, Métraux JP. Systemic acquired resistance. Annu Rev Phytopathol 1997;35:235e70. [27] Stadnik MJ, Fernandes WS. Resistance induced by ulvan to Colletotrichum lindemuthianum in common bean (Phaseolus vulgaris). J Plant Pathol (supplement); 2008:261. [28] Hammerschimidt R. Induced disease resistance: how to induce plants stop pathogens? Physiol Mol Plant Pathol 1999;55:77e84. [29] Romero AM, Ritchie DF. Systemic acquired resistance delays race shifts to major resistance genes in bell pepper. Phytopathology 2004;94:1376e82. [30] Vallejo V, Kelly JD. Unexpected resistance genes for anthracnose uncovered. Annu Rep Bean Improv Coop 2005;48:74e5. [31] Ntahimpera N, Dillard HR, Cobb AC, Seem RC. Anthracnose development in mixtures of resistant and susceptible dry bean cultivars. Phytopathology 1996;86:668e73. [32] O’Connell RJ, Bailey JA. Differences in the extent of fungal development, host cell necrosis and symptom expression during raceecultivar interactions between Phaseolus vulgaris and Colletotrichum lindemuthianum. Plant Pathol 1988;37:351e62. [33] Stadnik MJ, Bettiol W. Association between lipoxygenase and peroxidase activity and systemic protection of cucumber plants against Podosphaera xanthii induced by Oudemansiella canarii extracts. J Plant Dis Prot 2007;114:9e13. [34] Martinelli JA, Brown JKM, Wolfe MS. Effects of barley genotype on induced resistance to powdery mildew. Plant Pathol 1993;42:195e202. [35] Ortmann I, Moerschbacher BM. Spent growth medium of Pantoea agglomerans primes wheat suspension cells for augmented accumulation of hydrogen peroxide and enhanced peroxidase activity upon elicitation. Planta 2006;224: 963e70. [36] Madhaiyan M, Poonguzhali S, Senthilkumar M, Seshadri S, Chung H, Yang J, et al. Growth promotion and induction of systemic resistance in rice cultivar Co-47 (Oryza sativa L.) by Methylobacterium spp. Bot Stud 2004;45:315e24. [37] Bolwell GP, Davies DR, Gerrish C, Auh CK, Murphy TM. Comparative biochemistry of the oxidative burst produced by rose and French bean cells reveals two distinct mechanisms. Plant Physiol 1998;116:1379e85. [38] Choi HW, Kim YJK, Lee SC, Hong JK, Hwang BK. Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense responses to bacterial pathogens. Plant Physiol 2007;145: 890e904.