Induction of Disease Defensive Enzymes in ... - Wiley Online Library

J Phytopathol 160:382–389 (2012) 2012 Blackwell Verlag GmbH

doi: 10.1111/j.1439-0434.2012.01915.x

Assiut University, Assiut, Egypt, and Agricultural Research Center, Giza, Egypt

Induction of Disease Defensive Enzymes in Response to Treatment with acibenzolar-S-methyl (ASM) and Pseudomonas fluorescens Pf2 and Inoculation with Ralstonia solanacearum race 3, biovar 2 (phylotype II) Kamal amal A. M. Abo bo-Elyousr lyousr1, Yasser asser E. Ibrahim brahim2 and Naglaa aglaa M. Balabel alabel3 AuthorsÕ addresses: 1Plant Pathology Department, Faculty of Agriculture, Assiut University, 71526 Assiut, Egypt; 2Plant Pathology Research Institute, Agricultural Research Center (ARC), Giza, 12619, Egypt; 3Potato Brown Rot Project (PBRP), Agricultural Research Center, Giza, Egypt (correspondence to Y. E. Ibrahim. E-mail: [email protected]) Received November 23, 2011; accepted March 31, 2012 Keywords: peroxidase, acibenzolar-S-methyl, benzothiadiazole, polyphenol oxidase, ß-Glucosidase, tomato bacterial wilt

Abstract Ralstonia solanacearum (Rs) race 3 biovar 2, the cause of bacterial wilt, is an economically important pathogen in tropical, subtropical and temperate regions of the world. We investigated the induced defence responses against tomato bacterial wilt by the application of acibenzolar-S-methyl (ASM) and Pseudomonas fluorescens (Pf2) alone or in combination. Seedling treatments of tomato plants with either Pf2 or ASM significantly reduced disease severity of bacterial wilt (58 and 56% disease reduction, respectively) of tomato plants. The highest disease reduction (72%) resulted from a combined application of both Pf2 and ASM. The application of ASM alone increased seedlings biomass relative to infected control with 64.3%. Changes in the activities of polyphenol oxidase (PPO), ß-glucosidase (ß-GL) and peroxidase (PO) in tomato after the application of ASM and Pf2 and inoculation with Rs were studied. Significant changes (P £ 0.05) in the activities of PPO, ß-GL and PO were found. These results indicate that the future integrated disease management programmes against tomato bacterial wilt may be enhanced by including foliar sprays and soil drench of ASM and P. fluorescens. This is the first report of the use of both ASM and Pf2 to control the tomato bacterial wilt disease under field conditions.

Introduction Ralstonia solanacearum strains are a heterogeneous group subdivided into five races based on host range. Apart from races, R. solanacearum strains have been classified into five biovars based on physiological and biochemical characteristics (Buddenhagen and Kelman1964; Hayward1991). Recently, Prior and Fegan (2005) proposed a new hierarchical classification scheme, based on DNA sequence analysis of the

internal transcribed spacer region. Four phylotypes were distinguished corresponding with their geographic origins (Prior and Fegan (2005). Each phylotype can be further subdivided into sequevars based on differences in the sequence of a portion of the endoglucanase (eg1) gene. Race 3 biovar 2 (phylotype II, sequevar 1) is pathogenic to potato, some weeds and to less extent on tomato, and some years ago, outbreaks of wilt caused by this race were described on Geranium (Pelargonium spp.) in Europe and North America (Hudelson et al. 2002; Janse et al. 2004; Elphinstone 2005). In the 1940–1950s, this bacterium was reported from the Mediterranean area, including Egypt. Moreover, the pathogen has been found in irrigation water (Farag et al. 1999). Bacterial wilt (BW) is a systemic disease that cannot be effectively managed with chemical treatments. Chemical control by soil fumigants such as methyl bromide (Enfinger et al.1979; Chellemi et al. 1997) antibiotics (Farag et al. 1982) and copper pesticides do not provide satisfactory disease control (Hartman and Elphinstone1994). Biological control has emerged as one of the most important methods in the management of soil-borne plant pathogens. Numerous actinomycetes and bacteria such as Stenotrophomonas maltophilia, Pseudomonas fluorescens, P. glumae, Burkholderia cepacia, Bacillus sp., Erwinia sp. and a Hrpmutant of R. solanacearum have been reported to be active control agents against R. solanacearum (Messiha 2001; Lopez and Biosca 2004; Ran et al. 2005; Messiha et al. 2007). Among the rhizosphere organisms, fluorescent pseudomonads are often selected for biological control agents because of their ability to utilize various substrates under different conditions, short generation time and motility that assist the colonization of plant roots (Bagnasco et al. 1998). Moreover, the modes of action in disease suppression by these bacteria include siderophore-mediated competition for iron (Jagadeesh et al. 2001), antibiosis, production of lytic enzymes

Induction of Disease Defensive Enzymes

and induction of systemic resistance (Bakker et al. 2007). Recently, the plant activator Bion (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester), also known as benzothiadiazole (BTH) or acibenzolar-S-methyl (ASM), has shown activity against a number of bacterial diseases including bacterial spot (Xanthomonas axonopodis pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato) of tomato and pepper (Louws et al. 2001) and fire blight of apples (Erwina amylovora) (Momol et al. 1999). Interestingly, ASM significantly enhanced resistance of tolerant tomato cultivars against BW disease and resulted in increased tomato yield (Pradhanang et al. 2005). Another study showed that BTH effectively reduced BW incidence in susceptible tomato cultivars at low soil populations of R. solanacearum (Anith et al. 2004). Certain biochemical changes occurring after the application of resistance inducing agents can be used as markers for induced systemic resistance (Scho¨nbeck et al. 1980). These changes include the accumulation of certain enzymes and phenolic compounds (He et al. 2002). The purpose of this study was to assess the potential benefit of the activator ASM alone or in combination with P. fluorescens to suppress R. solanacearum under greenhouse and field conditions by using tomato plants as a model plant for plant–pathogen interactions (Arie et al. 2007). The possible modes of action of these agents via changes in growth and scavenger enzymes were also studied.

Material and Methods Bacterial culture and growth conditions

Potato tuber (Solanum tuberosum L. cv. Spunta) samples were collected from Minufiya governorate, Egypt, and deposited in the PBRP laboratory for inspection and isolation. A total of 10 Ralstonia solanacearum, biovar 2, race 3 strains designated as T1 to T10 were isolated from infected potato tubers showing internal brown rot symptoms on modified semi-selective media of South Africa (SMSA medium) according to Elphinstone et al. (1996). Typical virulent colonies were selected, and five of them (T1, T3, T5, T6 and T9) were purified on glucose nutrient agar (GNA). Pathogenicity of these five isolates was determined on 4-week-old tomato cv. Super Strain B by stem inoculation under greenhouse conditions as described by Janse (1988). Verification of R. solanacearum identity for the five isolates was performed serologically by using the immunofluorescent antibody staining (IFAS) method (Janse1988) and physiological and biochemical tests (Hayward 1964). One of five bacterial isolates (Rs1) was selected for further experiments because it was highly virulent. Inoculum preparation of R. solanacearum

Strain Rs1 was grown at 28C on casamino acid– peptone–glucose medium (CPG; DIFFCO, Michigan, USA) for 48 h. Bacterial cells were harvested and suspended in sterile distilled water, and the resulting

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suspension was adjusted turbidimetrically to approximately 107 CFU ⁄ ml. Isolation of antagonists

Potential antagonists were selected from rhizosphere soils of tomato from Assuit governorate, Egypt. Tomato root samples were shaken vigorously to remove loosely adhering soil. Soil suspensions were serially diluted, and bacteria were isolated by adding approximately 1 g of soil to 9 ml of sterile phosphatebuffered saline (PBS, 3.0 g KH2PO4, 7.0 g Na2HPO4.7H2O, 4.0 g NaCl per litre of distilled water, pH 7.2 as described by Leben et al. (1968)). The solution was vortexed, allowed to settle for at least 20 min and then vortexed again. The supernatant was decanted in a clean tube and then centrifuged at 10 000 g at 15 min to collect the bacterial fraction. The pellets were dissolved in PBS, and aliquots from five serial 10-fold dilutions were made for each sample and spread on KingÕs medium B agar plates (KB: 20 g proteose peptone, 1.5 g K2HPO4, 1.5 g MgSO4, 20 g agar, 15 ml glycerol, in 1000 ml distilled water, King et al. (1956)) and incubated at 28C for 48 h. Colonies developed on inoculated plates were washed with sterile water (2 ml ⁄ isolate), purified and streaked over the middle of the surface of KB plates. The isolated P. fluorescens strains were further evaluated for their antagonistic activity by the dual culture technique. KB plates were prepared by mixing suspension of cells scraped from 48- to 72-h-old culture of the pathogen with warm and molten KB agar (42C). The agar bacterial suspension was then dispensed into Petri dishes and was spot inoculated with the P. fluorescens from a 24-h-old culture (Skathivel and Gnanamanickam 1987). The KB agar plates spot inoculated with sterile water were used as a negative control. Assay plates were maintained at 28C and observed for inhibition zones after 2–3 days (Kuarabachew et al. 2007). There were five replicates for each tested isolate, and the experiment was repeated two times during this study. The potential bacterial antagonists that showed strong (11–20 mm inhibition diameter) and very strong (over 20 mm inhibition diameter) degree of inhibition were labelled and maintained separately (Arsenijevic et al. 1998). The most effective in vitro inhibitors (>11 mm inhibition diameter) were further evaluated in the greenhouse. The identity of the isolated fluorescent bacteria based on morphological features was determined using KingÕs B agar medium. Oxidase, catalase; starch hydrolysis and levan formation tests were evaluated on media supplemented with 0.2% starch and 5% sucrose (Goszczynska et al. 2000). Fluorescein production, gelatin liquefaction, salt tolerance, siderophore detection and carbohydrate utilization tests were performed following the methods of Goszczynska et al. (2000) and Pickett et al. (1991). Inoculum preparation of antagonistic Pseudomonads

One strain identified as P. fluorescens (Pf 2) was selected for biocontrol tests. The 48-h-old culture on

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KB broth was centrifuged at 10 000 g for 10 min. Bacterial pellets were washed twice with sterilized distilled water by centrifugation. The optical density (OD) of the solution was adjusted to 0.45 (610 nm) to obtain l07 CFU ⁄ ml (Mortensen 1999). Greenhouse experiments

Greenhouse experiments were conducted to determine the effect of ASM alone or in combination with fluorescent pseudomonad Pf2 in controlling R. solanacearum. Tomato seeds (cv. Super Strain B) were sown in trays with 2.5 · 2.5 cm cells containing sterilized peat moss. ASM was applied either as a drench (d) to the base of the plants or as a foliar spray (f). The initial foliar treatment was applied 2 weeks after seedling emergence, followed by a foliar and soil drench application 5 days prior to inoculation with the pathogen. For the foliar application, a concentration of 56 mg of ASM per litre of water was used. Leaves were atomized with a hand-held sprayer till run off. Tomato seedlings were drenched with 5 ml of ASM solution (28 mg ⁄ l) per cell of tomato transplant. P. fluorescens was applied as a spray (f) or a soil drench. For the (d) treatment, the inoculation suspension was adjusted to l07 CFU ⁄ g soil (Ciampi-Panno et al. 1989). Tomato seedlings with three to four expanded leaves were inoculated by applying 5 ml of 107 CFU ⁄ ml R. solanacearum strain 1 suspension into each transplant cell (Somodi et al. 1993). Seedlings were transplanted into 30-cm pots (2.5 kg soil), filled with a pasteurized mixture of soil and sand (4 : 1 w ⁄ w) 3 days after inoculation. Five millilitres of sterilized tap water was added into each tomato cell, which served as a negative control. Pots were placed in a saucer containing water to maintain high soil moisture to facilitate infection and wilt development. The plants were supplied with nitrogen–phosphorus–potassium (NPK) (12-4-6) at 10 days intervals at a rate of 7.5 g ⁄ l of water. Tomato plants were maintained in a controlled greenhouse (22–28C during the night and 30–35C during the day and 50– 70% relative humidity.) The treatments were designed as the following: 1-ASM (f); 2-Pf2 (f); 3-ASM (f) + Pf2 (f); 4-ASM (d); 5-Pf2 (d); 6-ASM (d) + Pf2 (d); 7-ASM (f) + Pf2 (d); 8-ASM (d) + Pf2 (f); 9- [ASM (f) + Pf2 (f)] + [ASM (d) + Pf2 (d)] and 10-Control (non-treated plants inoculated with R. solanacearum strain 1). The plants were kept under greenhouse conditions, and the disease severity was evaluated after 15 days. Plants with different treatments were removed, washed with water, blotted with tissue paper and dried at 60C for 72 h, and then dry weight was recorded. The experiment was conducted twice as a completely randomized design with four replications. Field experiments

The experiments were carried out during the summer of growing seasons 2009 and 2010 at the Experimental Farm of the Faculty of Agriculture, Assiut University, Egypt. Five rows of 15 plants each were used per

treatment. All treatments were carried out as described in the previous section of greenhouse experiments. The data of bacterial wilt severity and shoot and root weight experiments from field experiments were analysed using the anova procedures of the Statistical Analysis System (SAS Institute, Cary, NC, USA) for a split-plot randomized complete block design. Preparation of leaf samples for determining the activity of enzymes

Duplicate samples of tomato leaf tissues (1 g fresh weight) for enzyme extraction were harvested at 15 days after different treatments, weighed and immersed in liquid nitrogen. The frozen leaf segments for each sample were homogenized in an ice-cold mortar using m potassium phosphate buffer (pH 7. (1 : 5 w ⁄ v) 50 mm 0) containing 1 m NaCl, 1% polyvinylpyrrolidone, m EDTA and 10 mm m b-mercaptoethanol. Thereaf1 mm ter, the homogenates were centrifuged at 17 000 g for 20 min at 4C, and finally, the supernatant (crude enzyme extract) was collected and divided into 1.5-ml portions. Protein concentrations were determined using bovine serum albumin (BSA) as a standard according to Bradford (1976). The crude enzyme extract was then used to determine the activities of PO, Polyphenol Oxidase (PPO) and B-GL enzymes. Estimation of Peroxidase (PO) activity

To determine the peroxidase activity, 50 ll of the m crude enzyme extract was taken in 1 ml of 10 mm sodium phosphate buffer and mixed with 1 ml of m pyrogallol and 1 ml of 1% H2O2. The initial 100 mm rate of increase in absorbance was measured over 1 min at 470 nm. PO activity was expressed as units of PO ⁄ mg protein (Urbanek et al. 1991). Estimation of PPO activity

The activity of PPO was determined by adding 200 ll of the crude enzyme to 700 ll of sodium phosphate buffer (pH 6.0). Then, 100 ll of 0.2 m catechol was added, and the absorbance was read at 420 nm (rate of increase in absorbance for 1 min) and compared with the standard. The enzyme activity was expressed as lg PPO ⁄ mg protein (Gauillard et al. 1993). ß-Glucosidase (ß-GL) activity

ß-Glucosidase activity was determined using the method described by Zeller (1985). The reaction mixture was as follows: 0.5 ml of plant crude enzyme extract, 1.5 ml of phosphate buffer pH 6.5 (6.8 g KH2PO4, 8.99 g Na2HPO4.2H2O, 0.372 g ethylene diamine tetraacetic acid (EDTA) in 1000 ml of water and m p-nitrothe pH adjusted to 6.5) and 0.5 ml of 5 mm phenyl glucopyranosid. The mixture was incubated for 5 min at 30C and measured at 400 nm. ß-Glucosidase activity was determined according to the formula as enzyme unit ⁄ mg protein: ß-Glucosidase activity = mM p-Nitrophenol ⁄ mg protein.


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Statistical analysis

Data were subjected to statistical analysis using analysis of variance, and means were compared using L.S.D. test according to Gomez and Gomez (1984).

Table 2 The effect of acibenzolar-S-methyl (ASM), Pseudomonas fluorescens (Pf2) and in combination on disease severity (%) for tomato plants in a bacterial wilt field experiment Method of application

Results The effect of ASM, Pf2 and in combination on disease severity (%) for tomato plants in bacterial wilt greenhouse and field experiments

Compared with the untreated control, the applications of ASM and Pf2 alone or in combination significantly reduced disease severity of tomato BW by 47–70% (Table 1). There were no statistically significant differences between ASM and Pf2 when they were applied separately (Table 1). However, significant statistical differences were observed in disease severity for the plants treated with ASM or Pf2 and in combination as a soil drench treatment. The highest reduction in disease severity (>70% reduction) was detected in tomato plants treated with ASM and Pf2 as a foliar spray or as a soil drench. The lowest disease reduction (47% reduction) was observed when the tomato seedlings were treated with ASM or pf2 as foliar sprays. The field experiments showed the same trend as the greenhouse experiments (Table 2). Tomato seedlings treated with a foliar and drench of ASM and Pf2 resulted in the highest reduction in disease severity (72% reduction) in comparison with the untreated control plants. The lowest reduction was achieved by foliar spray of both treatments (48, 50%).

Foliar (f) Soil drench (d) Foliar + soil drench (f + d)

Treatments ASM Pf2 ASM + Pf2 ASM Pf2 ASM + Pf2 ASM(f) + Pf2(d) ASM(d) + Pf2(f) (ASM + Pf2)(f) + (ASM + Pf2)(d) Control

Disease severity (%)

Reduction of disease %

52 b 52 b 50 c 44 d 45 d 45 d 33 e 32 e 28 f

48 48 50 56 55 55 67 68 72

56 a

44

Values in the column followed by different letters indicate significant differences among treatments.

treatment (15.5 and 1.2 g) and foliar application (10.3 and 1.1 g), respectively (Table 3). Generally, the combination of ASM with Pf2 caused significant increasing in weight of shoot and root dry weight compared with the other treatments. No significant differences were observed between ASM and Pf2 when applied separately as either a foliar spray or as a soil drench. Induction of defence enzymes against bacterial wilt pathogen

The dry weight of shoots and roots of infected and untreated control plants were significantly lower than those of treated ones with ASM or Pf2 or their combination. The highest dry weights of shoots and roots were obtained from plants treated with the combination of ASM and Pf2 as foliar and drench applications (19.9 and 1.9 g, respectively) followed by drench

The accumulation of defence enzymes was significantly higher in tomato plants treated with ASM and Pf2 or together compared with infected untreated control (Figs 1–3). Significant inductions of enzymes were observed in plants treated with ASM + Pf2 as a foliar and drench application. Based on peroxidase activity, tomato plants treated with ASM + Pf2 showed higher enzyme activity in all treatments followed by plants treated with ASM alone only (Fig. 1). The lowest peroxidase activity occurred in plants treated only with Pf2 (Fig. 1). When ASM or Pf2 was applied separately as foliar sprays, there was no statistically significant difference compared with the untreated control. The

Table 1 The effect of acibenzolar-S-methyl (ASM), Pseudomonas fluorescens (Pf2) and in combination on disease severity (%) for tomato plants in a bacterial wilt greenhouse experiment

Table 3 The effect of acibenzolar-S-methyl (ASM), Pseudomonas fluorescens (Pf2) and in combination on dry shoot and root weight for tomato plants in a bacterial wilt field experiment

Method of application treatments

Method of application

The effect of ASM, Pf2 and in combination on dry shoot and root weight of tomato plants inoculated with bacterial wilt pathogen under field conditions



Disease Reduction severity% % 50 b 50 b 53 b 44 c 42 c 40 c,d 36 d,e 35 e 30f

50 50 47 56 58 60 64 65 70

73 a

27




Shoots Roots weight (DW) weight (DW) 8.0 f 7.9 f 10.3 d,e 12.5 c 12. 0 c 15.5 b 11.2 c,d 13.5 c 19.9 a

1.1 e 0.6 f 1.1 e 1.3 c 1.1 e 1.2 d 1.7 b 1.7 b 1.9 a

8.1 f

0.8 g


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Fig. 1 The effect of ASM, Pf2 and in combination on Peroxidase (PO) activity for tomato plants in a bacterial wilt greenhouse experiment. Pf, Pseudomonas fluorescens; ASM, acibenzolar-S-methyl. Coulumns with the same letter are not significantly different. PO enzyme activity was calculated according to the change in absorbence and was expressed as enzyme unit ⁄ mg protein. 1, ASM(f); 2, Pf2(f); 3, (ASM + Pf2) (f); 4, ASM(d); 5, Pf2(d); 6, ASM + Pf2(d); 7, ASM(f) + Pf2(d); 8, ASM (d) + Pf2 (f); 9, (ASM + Pf2) (f) + (ASM + Pf2) (d); 10, Control

Fig. 2 The effect of ASM, Pf2 and in combination on Polyphenol oxidase (PPO) activity for tomato plants in a bacterial wilt greenhouse experiment. Pf, Pseudomonas fluorescens; ASM, acibenzolar-Smethyl. Coulumns with the same letter are not significantly different. PPO enzyme activity was calculated according to the change in absorbence and was expressed as enzyme unit ⁄ mg protein

expression of PPO was similar to the PO results (threefold increase in absorbance ⁄ min ⁄ g of tissue), while the control showed less induction. Drench treatments lead to an increase in the enzyme activity compared with untreated control, but there was no significant increase between the different treatments (Fig. 2). The applications of ASM or Pf2 separately or in combination as a foliar spray or soil drench lead to an increase in the ß-GL activity. The highest increase was observed after the infected plants treated with ASM + Pf2 as either a drench soil or a foliar spray (Fig. 3). Generally, increased enzyme responses were observed 15 days after treating the plants with ASM and Pf2 compared with the untreated control.

Discussion Controlling bacterial wilt under field conditions has been studied for decades (Kelman 1953), and the means available for controlling the disease are still limited (Pradhanang et al. 2005) due to the complex

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Fig. 3 The effect of ASM, Pf2 and in combination on ß-glucosidase (ß-GL) activity for tomato plants in a bacterial wilt greenhouse experiment. Pf, Pseudomonas fluorescens; ASM, acibenzolar-Smethyl. Coulumns with the same letter are not significantly different. B-GL enzyme activity was calculated according to the change in absorbence and was expressed as enzyme unit ⁄ mg protein

nature of soil-borne pathogens. Some biological methods were used successfully to control tomato bacterial diseases, but they were used mostly under greenhouse conditions (Jagadeesh et al. 2001; Anith et al. 2004; Guo et al. 2004; Ran et al. 2005; Kuarabachew et al. 2007; Messiha et al. 2007; Vanitha et al. 2009). In addition, several chemicals including methyl bromide, antibiotics and copper pesticides provide limited success only (Farag et al. 1982; Hartman and Elphinstone 1994). Recently, acibenzolar-S-methyl alone or in combination with Bacillus pumilus, Pseudomonas putida and two products containing plant growthpromoting rhizobacteria (PGPR) (Bio Yield and Equity) have been reported to reduce BW disease on tomato (Pradhanang et al. 2005; Anith et al. 2004). This is the first report of control of BW using a combination of ASM and a particular strain of P. fluorescens (Pf 2) in both greenhouse and field experiments, and our data show that the applications of these combinations significantly reduce BW severity as compared with the untreated tomato plants with more than 72%. The use of fluorescent Pseudomonas strains and compounds that induce systemic acquired resistance (SAR) in plants have been reported as effective alternative tools against a spectrum of pathogens and resulted in enhanced levels of protection against specific bacterial pathogens (Van Wees et al. 1999). Our data show that best protection is obtained when ASM and Pf2 are applied as soil drench compared with foliar application. This result agrees with those of Pradhanang et al. 2005 and Anith et al. 2004. Lopez and Lucas (2002) reported that a single soil drench application of ASM 10 days prior to inoculation with Colletotrichum gloeosporioides was sufficient to significantly reduce anthracnose disease on cashew (Anacardium occidentale) trees under field conditions in Brazil. The high efficacy of this application could also be due to the fact that ASM and Pf2 may prevent the colonization and movement of R. solanacearum in stems. ASM was reported to induce defence reactions in tomato plants through the accumulation of


pathogenesis-related proteins and by ultra-structural changes in root tissues (Benhamou and Beclanger 1998; Inbar et al. 1998). Our study also showed that P. fluorescens alone or in combination with ASM significantly reduced the BW severity under both greenhouse and field conditions. The combination of ASM and P. fluorescens provided an additional reduction in disease pressure. Pseudomonas fluorescens significantly reduced BW in tomato and potato under greenhouse and field conditions (Kloepper et al. 1980; Aspiras and De-la Cruz 1986; Guo et al. 2004; Kuarabachew et al. 2007; Vanitha et al. 2009). Theses combinations (ASM and P. fluorescens) also provided protection against bacterial spot of tomato (Wilson et al. 2002; Abo-Elyousr and El-Hendawy 2008).The ability of P. fluorescens to grow rapidly and to colonize plant root system would prevent the bacterial wilt pathogen from attaching to the point of entry and proceeding further into the vascular tissues. In addition, this bacterial agent might induce plant systemic resistance or it may have a direct inhibitory effect on the pathogen (Kloepper et al. 1980; Aspiras and De-la Cruz 1986; Van Loon et al. 1998). The fact that ASM and P. fluorescens can induce natural defence responses against other tomato pathogens (Wilson et al. 2002 and Abo-Elyousr and El-Hendawy 2008) increases the potential benefits of applying these treatments in integrated disease management. The positive impact of ASM alone or in combination with P. fluorescens strain Pf2 on plant growth has been shown in this study. Our result indicate that the combination of ASM with Pf2 causes significant increase in weight of shoot and root dry weight compared with the other treatments (64.3%). The role of ASM and P. fluorescens in promoting root and shoot growth of tomato and potato crops has been demonstrated before (e.g. Sivamani and Gwanamanickam 1988; Girish and Umesha 2005; Hacisalihoglu et al. 2007; Kavitha and Umesha 2007). Pathogenesis-related proteins such as peroxidase, ß-1-3-glucanases, proteinase inhibitors and chitinases are elicited in response to pathogen attack (Van Loon et al. 2006). In our case, the application of either a mixture of ASM and Pf2 or ASM and Pf2 alone induced a progressive increase (with 80, 75 and 64%, respectively, in case of peroxidase activity) of these enzymes in locally treated tissues. Close relationships were reported between ASM-induced resistance and the activity of peroxidase, a key enzyme responsible for the generation of active oxygen species in plants against fungal, bacterial and viral pathogens (Dalisay and Kuc 1995; Slusarenko 1996; Baysal et al. 2005) and also involved in several plants defence mechanisms, such as lignin biosynthesis, oxidative cross-linking of plant cell walls and also generation of oxygen species (Bestwick et al. 1998). Our data also showed that these treatments were characterized by increased PPO activity compared with the untreated plants. Induced resistance (IR) was evidenced by the enhancement of PPO at 1–72 h after

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spraying with ASM (Cavalcanti et al. 2006). In plants, PPO has been associated as well with lignification of cell walls, thus playing a protective role in injured plants against other organisms, due to reactive quinines produced from phenolic compound catalysis (Mayer and Staples 2002). In our study, P. fluorescens induced defence enzymes in tomato leaves compared with inoculated untreated control, which in turn reflected reduction of bacterial wilt. ß-Glucosidase activity increased nearly by the same factor in ASM- and Pf2-treated plants up to 15 days after application. An enhanced level of b-GL can decompose the binding of glucose to inhibitory substances so that the growth of the pathogen in an infected tissue is restricted. Pseudomonas fluorescens has been demonstrated to induce systemic resistance to a variety of diseases including wilt diseases, anthracnose, bacterial and viral diseases (Wei et al. 1991; Maurhofer et al. 1994; Liu et al. 1995). Tomato leaves showed increased synthesis of PO, PPO and ß-GL. Similar results of elevated levels of PO and PPO have been reported in cucumber plants treated with PGPR strains, which peaked 2–4 days after root treatment (Chen et al. 1995). Based on this study, integrated use of acibenzolarS-methyl and P. fluorescens may complement each other and can be used as an effective alternative management strategy against BW of tomato. Other means of control, such as cultural practices, crop rotation and use of available resistant cultivars should be part of a more comprehensive management strategy. References Abo-Elyousr KA, El-Hendawy HH. (2008) Integration of Pseudomonas fluorescens and acibenzolar-S-methyl to control bacterial spot disease of tomato. Crop Prot 27:1118–1124. Anith KN, Momol MT, Kloepper JW, Marios J, Olson SM, Jones JB. (2004) Efficacy of plant growth-promoting rhizobacteria, acibenzolar-S-methyl and soil amendment for integrated management of bacterial wilt on tomato. Plant Dis 88:669–673. Arie T, Takahshi H, Kodama M, Teraoka T. (2007) Tomato as a model plant for plant-pathogen interactions. Plant Biotechnol 24:135–147. Arsenijevic D, Girardier L, Seydoux J, Pechere JC, Garcia I, Lucas R, Chang HR, Dulloo AG. (1998) Metabolic cytokine responses to a secondary immunological challenge with LPS in mice with T. gondii infection. Am J Physiol 274:439–445. Aspiras RB, De-la Cruz AR (1986) Potential biological control of bacterial wilt in tomato and potato with Bacillus polymyxa FU6 and Pseudomonas fluorescens. In: Persley GJ. (eds) Bacterial Wilt Disease in Asia and the South Pacific. Canberra, ACIAR proceedings 13, ACIAR, pp 89–92. Bagnasco P, Fuente DL, Gualtieri G, Noya F, Arias A. (1998) Fluorescent pseudomonas spp. as biocontrol agent against forage legume root pathogenic fungi. Soil Biol Biochem 30:1317– 1372. Bakker PAHM, Pieterse CMJ, Van Loon LC. (2007) Induced systemic resistance by fluorescent Pseudomonas spp. Phytopathology 97:239–243. Baysal O¨, Gu¨rsoy YZ, Duru A, O¨rnek H. (2005) Induction of oxidants in tomato leaves treated with DL-b-Amino butyric acid (BABA) and infected with Clavibacter michiganensis subsp michiganensis. Eur J Plant Pathol 4:361–369. Benhamou N, Beclanger RR. (1998) Benzothiadiazole-mediated induced resistance to Fusarium oxysporum f.sp. radicis-lycopersici in tomato. Plant Physiol 118:1203–1212.

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