Peroxidase and Polyphenoloxidase Activities as

Seleim et al., J Plant Physiol Pathol 2014, 2:1 http://dx.doi.org/10.4172/2329-955X.1000117

Journal of Plant Physiology & Pathology

Research Article

Peroxidase and Polyphenoloxidase Activities as Biochemical Markers for Biocontrol Efficacy in the Control of Tomato Bacterial Wilt Mohamed A Seleim1*, Kamal A Abo-Elyousr2, Abd-Alal A Mohamed1 and Hanan A Al-Marzoky3

a SciTechnol journal practice in many regions of the world. Although significant control of plant pathogens has been demonstrated by PGPR in laboratory and greenhouse studies, results in the field have been inconsistent. Recent progress in our understanding of their diversity, colonizing ability and mechanism of action, formulation and application should facilitate their development as reliable biocontrol agents against plant pathogens [5]. For many years, the role of oxidative enzymes and of their metabolic products in the defense mechanisms of infected plants have been studied [6]. Peroxidase activity in diseased plants and its effects on resistance or susceptibility in many host-pathogen interactions has been studied [7]. The objective of this research was to study peroxidase and polyphenoloxidase activities in tomato as biochemical markers after treatment with certain biocontrol agents in the control of tomato bacterial wilt.

Materials and Methods Abstract

Tomato plants

We have studied the effect of certain bioagents for the control of bacterial wilt of tomato under greenhouse and field conditions and the effect of these bioagents in induction of some enzyme activity in planta e.g. Peroxidase (PO) and polyphenoloxidase (PPO). Under greenhouse conditions the effect of Pseudomonas putida and P. fluorescens, and their combination were studied, and we found that both of them reduced the disease 60 and 66.67%, respectively and the combination treatment reduced the disease 53.33%. Also under field conditions P. putida was the best in reduction of the disease followed by the combination and then P. fluorescens. The P. fluorescens treatment recorded the highest percent yield increase in the two trails. The study also revealed that there was a significant increase in the activity of PO and PPO in tomato plants treated with PGPR strains P. fluorescens and P. putida. Our results show that peroxidase and polyphenoloxidase activities can be used as biochemical markers for biocontrol efficacy in the control of tomato bacterial wilt.

Tomato seeds (Lycopersicon esculentum Mill.) cv. G.S, were surface-sterilized with 2% sodium hypochlorite for 2 min. [8], thoroughly washed with sterilized water, and planted into pots of sterilized soil. After 4 weeks, seedlings were cultivated in the greenhouse in terracotta pots (diameter 30 cm) filled with experimental soil and grown at 25–35˚C.

Keywords

Source of rhizobacterial strains

Tomato bacterial wilt; Pseudomonas putida; P. fluorescens; Biochemical changes; Induced resistance

Pseudomonas fluorescens and Pseudomonas putida isolated from the tomto rhizosphere and identified by morphological and biochemical characterization [8,10-12] were used. Inhibitory activities of these rhizobacteria against R. solanacearum were confirmed by following the dual inoculation technique [13]. Rhizobacteria were cultured on nutrient agar (NA) supplemented with 5% sucrose and stored for further studies.

Introduction Bacterial wilt caused by Ralstonia solanacearum is a serious bacterial disease and a major constraint in the production of tomatoes [1]. Infected tomato plants may be stunted or completely wilted, resulting in poor fruit quality such as small fruit size and significant yield loss of about 10%. In Egypt, this disease was first identified in tomato in 2008 [2]. Bacterial wilt is among the most difficult disease to control [3] and the efficacy of current strategies for management of this disease is limited. No conventional pesticides are known to provide effective control of this soilborne pathogen [4]. The use of plant growth promoting rhizobacteria (PGPR) has become a common *Corresponding author: Mohamed A. Seleim, Department of Plant Pathology, Faculty of Agriculture, Al-Azhar University (Assiut Branch), 71524 Assiut, Egypt, E-mail: [email protected] Received: January 11, 2014 Accepted: March 04, 2014 Published: March 07, 2014

International Publisher of Science, Technology and Medicine

Source of R. solanacearum A virulent strain of R. solanacearum biovar 2 isolated from tomato plants, showing typical symptoms of bacterial wilt, identified by morphological and biochemical characteristics and whose pathogenicity on tomato plants had been confirmed in previous work was used in this study. R. solanacearum was isolated on tetrazolium chloride (TTC) agar [9] and cultured routinely on nutrient agar at 28°C for 48 h and temporarily stored in sterile distilled water at 4°C.

Preparation of rhizobacterial inoculum Two day old cultures of two rhizobacteria (Pseudomonas fluorescens and Pseudomonas putida) were harvested from agar plates, suspended in sterile deionized water, and adjusted to a concentration of 106 cfu/ml as determined by a spectrophotometer at 600 nm.

Biocontrol of bacterial tomato wilt under greenhouse conditions In greenhouse trail, tomato seedling cv. GS roots were treated separately with each treatment for 30 minutes just before transplanting. Pathogen inoculum containing l08 cell ml-1 (100 mls) was added around tomato plants at the time of transplanting. Four

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Citation: Seleim MA, Abo-Elyousr KA, Mohamed AAA, Al-Marzoky HA (2014) Peroxidase and Polyphenoloxidase Activities as Biochemical Markers for Biocontrol Efficacy in the Control of Tomato Bacterial Wilt. J Plant Physiol Pathol 2:1.

doi:http://dx.doi.org/10.4172/2329-955X.1000117 weeks after inoculation, disease index percentage was recorded using the following formula: Disease index (%) = [∑ (ni × vi) ÷ (V × N)] ×100 Where ni = number of plants with respective disease rating; vi = disease rating; V = the highest disease rating (5); and N = the number of plants observed [4]. Disease rating was calculated as following scale: 1=no symptoms. 2=one leaf wilted. 3=two to three leaves wilted. 4=four or more leaves wilted. 5=whole plant wilted. Treatments were replicated seven times and the pots were arranged in completely randomized design.The treatments details are given below: T1 :Pseudomonas putida (Pp) T2 :Pseudomonas fluorescens (Pf) T3 :Pp + Pf T4 :Control (only Ralstonia solanacearum )

extract. To start the reaction, 200 μl of 0.1M catechol was added and the activity was expressed as change in absorbance at 495 nm at 30-s intervals for 3 min.The enzyme activity was expressed as changes in absorbance min-1 mg-1 of protein [16]. Biocontrol of bacterial tomato wilt under field conditions: This experiment was carried out in the experimental farm of the Plant Pathology Department, Faculty of Agriculture, Assiut University, Egypt. Treatments were distributed in a complete randomized block design with four replicates. The individual experimental plot area was 25 m2 containing fourteen rows, each row was 3.5 meter length and distance between rows was 50 cm and each plot contains 140 plants. Tomato seedling roots were treated by biocontrol agents at the time of transplanting as shown before; e.g 100 ml of pathogen inoculum containing l08 cell ml-1 was added around the tomato roots. Disease index was recorded as shown in the greenhouse trail. Tomato plants were grown using the recommended tomato production program of the Egyptian Ministry of Agriculture. At the end time of the experiment (90 days after transplanting), mature fruits harvested regularly and the total yield of each treatment was assessed per plant. Disease incidence was calculated as described before in greenhouse experiment.

Statistical analysis The data from the experiments were subjected to statistical analysis and interpreted by MSTAT-C using LSD test (P=0.05) [17].

Assay of defense related enzymes

Results

Sample collection: Samples were collected from individual treatments to study the induction of defense enzymes in response to biocontrol agent treatments under greenhouse conditions. Stems from different treatments were collected and used for analysis.

Biocontrol of bacterial tomato wilt under greenhouse conditions

Enzyme extract: The stem sample, collected from treated and pathogen- inoculated tomato plants was immediately homogenized with liquid nitrogen. One g of powdered sample was extracted with 2 ml of sodium phosphate buffer, 0.1M (pH 7.0) at 4ºC. The homogenate was centrifuged for 20 min at 10,000 rpm. Protein extracts prepared from tomato tissues were used for estimation of defense enzymes (peroxidase (PO) and polyphenol oxidase (PPO)).The supernatants (crude enzyme extract) were immediately used for determination of enzyme activities and total protein. An aliquot of the extract was used to determine the protein content using bovine serum albumin as standard [14]. Assay of peroxidase (PO): Assay of PO activity was carried out as described; the reaction mixture consisted of 2.5 ml of a mixture containing 0.25 percent (v/v) guaiacol in 0.01M, sodium phosphate buffer, (pH 6.0) and 0.1M hydrogen peroxide. Enzyme extract (0.1 ml) was added to initiate the reaction which was followed colorimetrically at 470 nm. Crude enzyme preparations were diluted to give changes in absorbance at 470 nm of 0.1 to 0.2 absorbance units/min. Boiled enzyme was used as blank. Activity was expressed as the increase in absorbance at 470 nm min-1 mg-1 of protein [15]. Assay of polyphenoloxidase (PPO): A sample of one g was homogenized in 2 ml of 0.1 M sodium phosphate buffer (pH 6.5) at 4ºC. The homogenate was centrifuged at 20,000 rpm for 15 min. The supernatant served as enzyme source and polyphenoloxidase activity was determined as given; the reaction mixture consisted of 1.5 ml of 0.1 M sodium phosphate buffer (pH 6.5) and 200 μl of the enzyme

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Treatments were screened under greenhouse conditions for their control efficacy against R. solanacearum causing bacterial wilt in tomato and the results showed that all treatments reduced the disease significantly. Pseudomonas putida and Pseudomonas fluorescens, recorded 60 and 66.67% disease reduction, respectively. Under the combination treatment, the disease reduction percentage was 53.33% (Table 1).

Biocontrol of bacterial tomato wilt under field conditions In field trials, the disease control percentage varied from 51.76 % to 75.29% in 2011 trail and no variation among treatments in 2012 trail. Results in Table 2 showed that Pseudomonas putida controlled disease 69.41 and 70.59%, respectively in two trails and Pseudomonas fluorescens controlled disease 75.29 and 88.24%. Under the combination treatment, the disease control percentage was 51.76 Table 1: Disease incidence and disease reduction percentage of treated tomato plants under greenhouse conditions* Treatments

Disease incidence Disease reduction (%) (%)

Pseudomonas putida (Pp)

30b

60

Pseudomonas fluorescens (Pf)

25b

66.67

Pp + Pf

35b

53.33

Control

75a

-

LSD test (P=0.05)

28.34

* Values in the same column followed by a similar letter are not significantly different as determined by the LSD test (P=0.05).

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doi:http://dx.doi.org/10.4172/2329-955X.1000117 Table 2: Disease incidence and disease reduction percentage of treated tomato plants under field conditions* Season 2011

Treatments

Season 2012

Disease Disease Disease Disease incidence (%) reduction (%) incidence reduction (%) (%)


25c

69.41

25b

70.59


20c

75.29

10b

88.24

Pp + Pf

40b

51.76

25b

70.59

Control

85a

-

70a

-

LSD test (P=0.05)

11.92

19.22

* Values in the same column followed by a similar letter are not significantly different as determined by the LSD test (P=0.05). Table 3: Influence of different treatments on yield under field conditions* Season 2011

Season 2012

Treatments

Yield (kg per plant)

Yield increase (%)

Yield (kg per plant)

Yield increase (%)


0.852c

85.163

2.835b

128.629


1.102a

139.511

3.188a

157.056

Pp + Pf

0.996b

116.576

3.205a

158.468

Control

0.460

-

1.238c

-

LSD test (P=0.05)

0.047

d

0.23

*Values in the same column followed by a similar letter are not significantly different as determined by the LSD test (P=0.05). Table 4: Effect of different treatments on PO and PPO enzymes in tomato plants, inoculated with pathogen under greenhouse conditions* Treatment

PO

PPO

▲in absorbance**

IOC%*** ▲in absorbance**

IOC%***


0.1321b

49.97

0.0177a

62.61

Pseudomonas fluorescens(Pf)

0.1343a

52.44

0.0156b

42.66

Pp + Pf

0.1241c

40.86

0.0152b

39.08

Control

0.0881d

-

0.0109c

-

LSD test (P=0.05)

5.58

3.79

* Values in the same column followed by a similar letter are not significantly different as determined by the LSD test (P=0.05). ** Changes in absorbance / min/mg of protein, *** Per cent increase over control.

% and 70.59 %, respectively in two trails. Yield-increasing percentages were calculated at the end of the experiments. As shown in Table 3, Pseudomonas fluorescens treatment increased yield the highest in the 2011 trail and the rest of the treatments showed a smaller yield increase. While, Pseudomonas fluorescens (Pf) and Pp+Pf recorded the highest increased yield percentage followed by Pseudomonas putida (Pp) in the 2012 trail.

Assay of defense related enzymes Activity of peroxidase (PO): Assay of peroxidase activity in tomato plants inoculated with R. solancearum showed differences among the various treatments. Significantly increased activity of PO was observed with all treatments upon challenge inoculation with the pathogen, when compared to the control (Table 4). PO activity varied from 40.86% to 52.44%. The plants treated with Pseudomonas fluorescens followed by those treated with Pseudomonas putida showed higher PO activity 52.44 and 49.97% respectively.

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Lowest PO activity was observed in the combination treatment with 40.86%.

Activity of polyphenoloxidase (PPO): Data in Table 4 showed that all treatments tested indicated significant increases in the activity of PPO when compared to control plants. PPO activity varied from 39.08% to 62.61%. Maximum PPO activity was noticed in the Pseudomonas putida treatment which showed a 62.61% increase over the control followed by Pseudomonas fluorescens and Pp+Pf, which showed a 42.66% and 39.08% increase over control, respectively.

Discussion Our biological efficacy studies conducted in the greenhouse indicated that plants treated with P. fluorescens and P. putida and their combination significantly reduced disease compared to the control. Similar results have been cited by other researchers [18-21]. Under field conditions, results illustrated that Pseudomonas fluorescens and P. putida and their combination significantly reduced the percentage of tomato bacterial wilt. Also, treatments were found to be effective in increasing yield per plant. Our results were in agreement with similar previous studies [22-26]. The mechanism involved in PGPRmediated plant growth promotion is through the production of plant growth regulators (auxins, cytokinins, gibberellins) and facilitation of the uptake of nutrients (nitrogen fixation, solubilization of phosphorus). The indirect promotion of plant growth occurs when PGPR lessen or prevent the deleterious effects of plant pathogens on plants by production of inhibitory substances (antibiotics, antifungal metabolites, iron-chelating siderophores, cell wall-degrading enzymes and competition for sites on roots) or by increasing the natural resistance of the host (induced systemic resistance) [27,28]. Induced resistance is a state of enhanced defensive capacity against a broad spectrum of pests and pathogens developed by a plant when appropriately stimulated [29]. The resulting elevated resistance due to biotic agents is referred to as induced systemic resistance (ISR) whereas that by other biological control agents is called systemic acquired resistance [30]. In our study, we concentrated on biotic inducers for generating the defense molecules against Ralstonia solanacearum in tomato. The ISR in this study was primarily focused for the defense related proteins, viz. PO and PPO. The results of the present study revealed that there was a significant increase in the activity of PO and PPO and in tomato plants treated with PGPR strains P. fluorescens and P. putida and their combination. Similar studies, which showed an increase in PO and PPO activity, were reported by others in increased activity of PO and PPO enzymes in tomato plants treated by PGPR against bacterial wilt caused by Ralstonia solanacearum [21]. The present study indicated enhanced activity of PO and PPO enzymes respond to test treatments, which might have prevented the establishment of R. solanacearum within the tomato roots. Addition, the results showed relatively correlation between PO, PPO and efficacy of test bioagents. Similar increase in plant growth and reduction in bacterial wilt in tomato plants treated with native isolates of P. fluorescens was observed and also increased activity of PO and PPO enzymes [31]. Our results support that peroxidase and polyphenoloxidase activities can be used as biochemical marker for biocontrol efficacy to control tomato bacterial wilt. References 1. Abo-Elyousr K, Asran M (2009) Antibacterial activity of certain plant extracts against bacterial wilt of tomato. Arch Phytopathology Plant Protect 42: 573578.

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Author Affiliations

Top

Department of Plant Pathology, Faculty of Agriculture, Al-Azhar University (Assiut Branch), 71524 Assiut,

1

Egypt Department of Plant Pathology, Faculty of Agriculture, Assiut University, 71526 Assiut, Egypt

2

Agricultural Botany Department, Faculty of Agriculture, Suez Canal University 41522 Ismailia, Egypt

3

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