The Role of Compost and/or Bio Control Agents in Induction Resistance against Cucumber Root-Rot...

763 Middle East Journal of Agriculture Research, 3(4): 763-773, 2014 ISSN 2077-4605

Corresponding Author: El-Shahawy, I.E., Plant Pathology Department, National Research Centre, Cairo, Egypt. E-mail: [email protected]

The Role of Compost and/or Bio Control Agents in Induction Resistance against Cucumber Root-Rot Pathogens 1Kamel Kamal Sabet, 1Magdy Mohamed Saber, 2Mohamed Adel-Aziz El-Naggar, 2Nehal Samy El-Mougy, 2Hatem Mohamed El-Deeb and 2Ibrahim El-Saied El-Shahawy

1Plant Pathol. Dept., Fac. of Agric. Cairo Univ. Egypt. 2Plant Pathol. Dept., National Res. Center, Giza, Egypt.

ABSTRACT Two types of commercial compost i.e. and two biocontrol agents, i.e. P. fluorescens and T. harzianum were used to suppress the root-rot incidence caused by soil pathogens of (Fusarium solani (Mart.) App. and Wr, Pythium ultimum Trow, Rhizoctonia solani Khun and Sclerotium rolfsii Sacc.) of cucumber plants under greenhouse conditions.. The extent of enzymes induction in the cucumber plants was in the order of: plants treated with cucumber root-rot pathogens plus potted soil treated with compost and/or biocontrol agents > cucumber root-rot pathogens inoculation only > compost and/or biocontrol agents only Peroxidase, polyphenoloxidase, chitinase and β.1, 3-glucanase enzymes activities in all composts and/or biocontrol agents treatments were significantly higher than that in the control. Data also revealed that the protein bands (10 bands) i.e. 166.0, 80.0, 60.0, 59.0, 51.0, 33.0, 32.5, 32.0, 16.0 and 13.5 KDa were detected in protein profiles of some treated cucumber plants with composts and/or biocontrol agents, while not detected in the profile of control plants. These results suggested that composts and/or biocontrol agents induced resistance against cucumber root-rot pathogens. Key words: Biocontrol agents, compost, cucumber root-rot, induction resistance

Introduction

Fusarium solani, Pythium ultimum, Rhizoctonia solani, and Sclerotium rolfsii were considered the most important soilborne pathogens which cause cucumber root-rot disease (Roberts et al., 2005; Haikal-Nahed 2007; Jinghua et al., 2008; Abd-El-Kareem, 2009; Hasan-Nehal, 2011 and Yousef-Safaa et al., 2013). Suppression of these soilborne plant pathogens is considered an urgent need for present agriculture practices. Therefore, the uses of compost and/or biocontrol agents, viz. T. harzianum and P. fluorescens to suppress the root rot pathogens have been extensively reviewed by many workers (De Ceuster and Hoitink, 1999; Ryckeboer, 2001; Saravanakumar et al., 2007; Loganathan et al., 2010). Three main types of direct interactions may be characterized: parasitism, antibiosis and competition for nutrients. Recently however, another possible mechanism has been suggested by Inbar et al. (1994) namely, induced resistance in plants to fungal attack (Yedidia et al., 1999; Suprakash and Narayan, 2012 and Abd El-Razik-Sahar et al., 2012). The aims of this experiment were to investigate the role of different biocontrol agents and/or composts amended to potting soil to induction resistance in cucumber plants against root-rot pathogens. This aims were achieved by measuring oxidative enzymes i.e. peroxidase and polyphenoloxidase, and lytic enzymes i.e. chitinase and β-1, 3-glucanase. On the other hand, determination of pathogenesis related proteins (PR-Proteins) as SDS-PAGE polyacrylamide gel electrophoresis (SDS-PAGE) profile in cucumber plants association with suppressive effect of different biocontrol agents and/or compost to root-rot diseases. Materials and Methods Cucumber root-rot pathogens:

Fusarium solani, Pythium ultimum, Rhizoctonia solani and Sclerotium rolfsii, which considered the most virulent pathogens causing root-rot disease of cucumber, were selected based on their pathogenic ability in previous study (Sabet et al., 2013).

Composts and/or biocontrol agents:

The promising biocontrol agents as well as composts were selected based on their efficiency effects against cucumber root-rot pathogens (Sabet et al., 2013). Therefore, biocontrol agents i.e. T. harzianum and P. fluorescens as well as compost (A): a mixture of different aromatic plants, sugar beet and sugarcane and compost (B): a mixture of rice straw and animal wastes were used.

764 Middle East j. Agric. Res., 3(4): 763-774, 2014

Plant material:

Cucumber (Cucumis sativus L.) transplants c.v Beit-alpha was used in this study. Propagative trays were cleaned from any soil residues using plastic brush, and then sterilized by formalin solution. A vegetable propagative medium was prepared by mixing the nursery soil materials of peat and vermiculite, at the rate of (1:1, v/v), fertilizers (N, P and K) was added at the recommended doses. After that, the vegetable propagative mixtures were spread in the propagative trays. Cucumber seeds (c.v Beit-alpha) were surface sterilized using 5% sodium hypochlorite solution for 3 min then air-dried. Cucumber seeds were sown in seedling-type preformed trays consisting of 84 conical compartments each measuring 4.5 x 4.5 x 5.5 cm. Trays were distributed on the nursery bench. Seedlings were incubated at 24-30oC for 18 days under natural illumination and irrigated daily by spraying.

Pots experiments

Experments were carried out in the greenhouse at NRC, during 2011 season for controlling root-rot disease incidence of cucumber seedlings. Plastic pots, 30 cm in diameter were sterilized, by immersing it in formalin solution for 30 minutes and then air dried for 3 days. Sandy loam soil were sterilized, by drenching it with formalin solution and then covered with plastic sheet for 24 houres and then air dried until the formaldehyde odor was gone.

Preparation of cucumber root-rot pathogens inocula

Inocula of each F. solani, P. ultimum, R. solani and S. rolfsii, were prepared individually. Fungal mass production used for soil infestation was obtained by growing the tested isolates on sand-barley medium. This natural medium was prepared by mixing sand and barley (100 g ground barley grains mixed with 40 g washed sand and 60 ml distilled water) and then the mixture in 500-ml glass bottles with cotton plugs was sterilized at 121oC for 1 hour. The autoclaved medium was then inoculated individually with a 5-mm disk obtained from 5-day old culture of each tested fungal isolate and then incubated at 25 ± 1oC for two weeks (Singleton et al., 1992).

Preparation of biocontrol agent inocula

For fungal inoculation, T. harzianum was grown on PDA medium at 25 ± 1°C for 10 days, afterwards the mycelium with the spores were scraped from Petri plate and mixed with sterile distilled water (20 ml / plate) in a blender. The suspension was adjusted by heamocytometer slide to 106 propagules / ml as described by Morsy-Ebtsam et al., 2009.

For bacterial inoculation, one loopful of P. fluorescens was inoculated into Nutrient Broth medium (g/l): peptone 5 g, beef extract 3 g, sodium chloride 5 g, glucose 20g, pH 7; and incubated on a shaker incubator at 125 rpm for 48 h at 28 ± 1°C. Antagonistic bacterial cells were then harvested after 48 hours of growth in culture medium by centrifugation at 6,000 rpm for 15 min and resuspended in a phosphate buffer (0.01 M, pH 7.0). The concentration was adjusted by plate count technique to an approximately 108 colony forming unit CFU/ml as mentioned by Mosa et al. (1997).

Treatments

Sterilized pots were filled with 5 kg sterilized soil infested with the inoculum of each fungus,

separately, at the rate of 4%. Infested pots were irrigated one week intervals for two weeks. For compost treatment; each infested soil was amended with 25g / kg from compost at one week before transplanting. For biocontrol agent treatment, each infested potting soil was drenched by the antagonistic suspensions at the dose of 500 ml / pot. The individual treatments of T. harzianum and P. fluorescens, compost A and/or compost B amended-potting soil under soil infestation with cucumber root-rot pathogens, individually, were prepared. Infestation soil with cucumber root-rot pathogens, individually and/or un-inoculated sand-barley medium amended-potting soil were kept as control. Each pot was transplanting with five cucumber transplants (18 days old). Five pots were used as replicates for each treatment as well as the control.

Control of cucumber root-rot diseases:

Disease assessments were recorded as the percentages of dead plants (%) due to infection by root-rot at 30 days after transplanted.


Biochemical studies: Determination of enzymes activities

Cucumber leaves were collected after 3, 6, 9 and 12 days of transplanting. To extract the enzyme, plant leaves (g) was homogenized with 0.2 M Tris HCl buffer (pH 7.8) at 0°C containing 14 m M β- mercaptoethanol at the rate of 1/3 w/v. The extracts were obtained by filtering off the debris with a clean cloth and centrifuging at 3,000 rpm for 15 min. The supernatants were recovered and kept in a tube in an ice bath until assayed. The supernatant was used to determine the activity of peroxidase, polyphenol oxidase, chitinase and β-1,3-glucanase enzymes (Tuzun et al., 1989) by using using UV spectrophotometer.

Peroxidase enzyme assay

Peroxidase activity was assayed with guaiacol as the hydrogen donor as described by Hammerschmidt et al. (1984) and peroxidase activity was expressed as the increase in absorbance at 470 nm/g fresh weight according to the method described by Lee (1973). Polyphenoloxidase enzyme assay

Polyphenoloxidase enzyme activity was determined by measuring the rate of quinone formation as a results of oxidizing 3,4-dihydroxyphenylalanine (DOPA) and polyphenol oxidase activity was expressed as the increase in absorbance at 475 nm/g fresh weight according to the method described by Bashan et al. (1985). Chitinase enzyme assay

The determination of chitinase enzyme was carried out using colloidal chitin as substrate and dinitrosalicylic acid (DNS) as reagent to measure reducing sugar according the method described by Monreal and Reese (1969). Chitinase activity was expressed as mM N-acetyl glucose amine equivalent released/ gram fresh weight/ 60 minutes at 450nm. β.1,3-glucanase enzyme assay

β.1,3-glucanase was assayed based on the method described by Miller (1959). Laminarin (Sigma) was used as a substrate and dinitrosalicylic acid (DNS) as a reagent to measure reducing sugar. β.1,3-glucanase activity was expressed as mM glucose equivalent released/ gram fresh weight/ 60 minutes at 500nm. Electrophorotic studies

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (1970) and modified by Studier (1973). The procedures was done with the kind help of the Dept. of Genetic and Cytology, NRC. Vertical slab gel unit was used. The denatured gels prepared as separating gel 15% (1.5 M Tris-HCl, pH 8.8) and stacking gel 4% (0.5 M Tris-HCl , pH 6.8). The gels were prepared from monomer of 30% acrylamide and 0.8 % bis-acrylamide.

Cucumber leaves were collected after 12 day of transplanting. Cucumber leaves were grounded in liquid nitrogen using a mortar. The leaves powders of each treatment were mixed with 1 ml of sample extraction buffer, pH 7.5 (Tris-HCl, 0.61g; Glycerol (15%, v/v), 5m; β-mecaptoethanol (0.1%, v/v), 100µL and distilled water, 100mL) in eppendorf tube and then left in refrigerator overnight. The supernatant was obtained by centrifugation at 12,000 rpm at 4oC for 15 min. The supernatant was transferred to new eppendorf tube and kept at deep-freeze until use for electrophoretic analysis.

Fifty µL of cucumber leaves protein was added to the same volume of 2x Lan’s buffer in eppendorf tube. β-mercaptoethanol (5 µL) was added to the each tube and boiled in water bath for 10 min, quickly transferred to ice water, and then 10 µL bromophenol blue was added to each tube before samples loading.

Using loading tips, a 60 µL protein sample was loaded to each well and for control wells protein standard markers were loaded. For running, lower and upper chambers were filled with tank buffer. The electrodes was connected to power supply and run until the bormophenol blue had reached the bottom of the resolving gel at 2 mA for well. At the end of run, the gel was stained with (coomassie brilliant blue-R250, 0.5 g; methanol, 227.5 mL; acetic acid glacial, 45 mL and distilled water, 500 mL). After half hour the gel were removed from stained and immersed in the destining solution (methanol, 140 mL, acetic acid glacial, 40 mL and distilled water, 700 mL) on a shaker. The destining solution was changed until the protein bands can be visually seen. Gels were photographed using a 35 mm color film (200 ASA) and scanned with Bio-Rad video


densitometer Model 620 USA, at a wavelength of 577. Software data analysis for Bio-Rad Model 620 densitometer and computer were used as illustrated by the manufacture

Variation among protein profiles of cucumber leaves at different treatments as well as untreated control were evaluated from pair-wise comparisons for the proportion of shared bands, i.e. two times of number of common bands (shared fragments) divided by the total number of band for each pair of treatment against all markers

Statistical analysis:

Statistical analyses of all the previously designed experiments were carried out according to (ANOVA) procedures reported by Snedecor and Cochran (1982). Treatment means were compared by the least significant difference test “LSD” at 5% level of probability. Results Cucumber root-rot disease management:

Effects of composts and/or biocontrol agents i.e P. fluorescens and T. harzianum on cucumber root-rot disease incidence (%) are listed in Table 1. Data showed that all treatments reduced root-rot disease incidence (%) compared to the control. Under soil infestation with F. solani, R. solani, S. rolfsii and P. ultimum, individually, cucumber root-rot disease incidence (%) were (37.3, 30.7, 36.0 and 36.0%), (34.7, 26.7, 32.0 and 32.0%), (42.7, 36.0, 36.0 and 36.0%) and (40.0, 32.0, 38.7 and 38.7%) in potted soil amended with compost A, compost B, P. fluorescens and T. harzianum, compared to the incidence (%) of 58.7, 65.3, 69.3 and 76.0 % in the control, respectively.

Table 1. Effect of composts and/or biocontrol agents on cucumber root-rot incidence (%) under artificial infestation with F. solani, R.

solani , S. rolfsii and P. ultimum.

Treatment Cucumber dead plant (%) due to infection by root-rot

F. solani R. solani S. rolfsii P. ultimum

Compost A 37.3 34.7 42.7 40.0

Compost B 30.7 26.7 36.0 32.0

T. harzianum 36.0 32.0 36.0 38.7

P. fluorescens 36.0 32.0 36.0 38.7

Control 58.7 65.3 69.3 76.0

LSD 0.05 3.1 3.0 3.1 2.4

Enzymes activities Peroxidase enzyme activity

Data in Table 2 showed that the levels of peroxidase enzyme activity were significantly higher in all the treatments compared with the control.The dramatic induction of peroxidase activity in the cucumber plants was observed during the first 3 days after transplanting and the levels of peroxidase enzyme activity peaked at 3 days and then declined gradually. It was observed that after 3 days the magnitude of peroxidase induction in the plants treated with cucumber root-rot pathogens plus potted soil treated with biocontrol agents was significantly higher (range: 3.37 to 3.45 unit/g fresh weight) than in the plants treated with cucumber root-rot pathogens plus potted soil treated with compost (range: 3.21 to 3.34 unit/g fresh weight). However, after 12 days the magnitude of peroxidase induction in the plants treated with cucumber root-rot pathogens plus potting soil treated with compost was significantly higher (range: 1.11 to 1.29 unit/g fresh weight) than in the plants treated with cucumber root-rot pathogens plus potting soil treated with biocontrol agents (range: 0.91 to 0.98 unit/g fresh weight).

Polyphenoloxidase enzyme activity

Data in Table 3 showed that polyphenoloxidase enzyme activity in all the treatments were significantly higher than that in the control. The levels of polyphenoloxidase activity in the cucumber plants exhibited a wave-like induction pattern after transplanting and manifested two peaks at 3 and 6 days, respectively. It was observed that after 3 days the magnitude of polyphenoloxidase induction in the plants treated with cucumber root-rot pathogens plus potting soil treated with biocontrol agents was significantly higher (range: 2.81 to 2.85 unit/g fresh weight) than in the plants treated with cucumber root-rot pathogens plus potting soil treated with


compost (range: 1.92 to 2.16 unit/g fresh weight). However, after 12 days the magnitude of polyphenoloxidase induction was the same trend in the plants treated with cucumber root-rot pathogens plus potting soil treated with compost (range: 0.83 to 0.98 U) and in the plants treated with cucumber root-rot pathogens plus potting soil treated with biocontrol agents (range: 0.91 to 0.98 unit/g fresh weight). Table 2. Effect of compost and/or biocontrol agents amendments on peroxidase activity in cucumber plants grown under artificial

infestation with F. solani, R. solani , S. rolfsii and P. ultimum.

Treatment Peroxidase activity*/g fresh weight

Days after transplanting 3 6 9 12

F. solani 2.04 1.32 1.11 0.66 F. solani + compost A 3.21 3.11 1.22 1.17 F. solani + compost B 3.30 3.16 1.62 1.25 F. solani + P.fluorescens 3.44 2.52 1.29 0.91 F. solani + T.harzianum 3.36 2.62 1.31 0.93 R. solani 2.04 1.34 1.15 0.71 R. solani + compost A 3.36 3.13 1.33 1.11 R. solani + compost B 3.34 3.18 1.64 1.24 R. solani + P.fluorescens 3.45 2.55 1.28 0.93 R. solani + T.harzianum 3.36 2.63 1.33 0.95 S. rolfsii 2.04 1.35 1.12 0.68 S. rolfsii + compost A 3.27 3.12 1.28 1.14 S. rolfsii + compost B 3.31 3.17 1.67 1.27 S. rolfsii + P.fluorescens 3.44 2.50 1.27 0.94 S. rolfsii + T.harzianum 3.40 2.64 1.36 0.98 P. ultimum 2.03 1.33 1.10 0.71 P. ultimum + compost A 3.30 3.11 1.35 1.11 P. ultimum + compost B 3.23 3.14 1.62 1.29 P. ultimum + P.fluorescens 3.47 2.50 1.29 0.92 P. ultimum + T.harzianum 3.37 2.65 1.35 0.97 Compost A 0.98 1.03 0.93 0.61 Compost B 0.98 1.14 0.98 0.62 P.fluorescens 0.95 0.86 0.43 0.33 T.harzianum 0.98 0.83 0.43 0.34 Control (un-treated soil) 0.33 0.34 0.33 0.30 LSD 0.05 0.07 0.02 0.03 0.03

(*) Peroxidase activity was expressed as the increase in absorbance at 470 nm/g fresh weigh.

Table 3.Effect of compost and/or biocontrol agents amendments on polyphenoloxidase activity in cucumber plants grown under artificial


Treatment Polyphenoloxidase activity*/g fresh weight


F. solani 1.82 1.99 0.91 0.61 F. solani + compost A 1.96 2.45 1.11 0.98 F. solani + compost B 2.10 2.51 1.41 0.93 F. solani + P.fluorescens 2.85 3.12 1.29 0.94 F. solani + T.harzianum 2.84 3.11 1.27 0.91 R. solani 1.83 1.99 0.90 0.61 R. solani + compost A 1.94 2.47 1.13 0.95 R. solani + compost B 2.13 2.53 1.42 0.93 R. solani + P.fluorescens 2.84 3.12 1.31 0.94 R. solani + T.harzianum 2.83 3.10 1.29 0.93 S. rolfsii 1.81 1.98 0.86 0.62 S. rolfsii + compost A 1.98 2.44 1.14 0.86 S. rolfsii + compost B 2.16 2.54 1.41 0.97 S. rolfsii + P.fluorescens 2.83 3.13 1.30 0.98 S. rolfsii + T.harzianum 2.81 3.12 1.29 0.94 P. ultimum 1.85i 1.99 0.88 0.65 P. ultimum + compost A 1.92 2.44 1.12 0.83 P. ultimum + compost B 2.15 2.57 1.40 0.98 P. ultimum + P.fluorescens 2.84 3.13 1.31 0.94 P. ultimum + T.harzianum 2.84 3.12 1.28 0.94 Compost A 0.65 0.82 0.75 0.52 Compost B 0.66 0.81 0.78 0.53 P.fluorescens 0.96 0.97 0.91 0.85 T.harzianum 0.95 0.99 0.93 0.81 Control (un-treated soil) 0.41 0.42 0.31 0.32 LSD 0.05 0.02 0.02 0.02 0.03

(*) Polyphenoloxidase activity was expressed as the increase in absorbance at 475 nm/g fresh weight.


Chitinase enzyme activity

The dramatic induction of chitinase enzyme activity in all the treatments compared with the control was observed during the first 3 days after transplanting and the levels of chitinase activity peaked at 3 days (Table 4). However, after 3 days of transplanting, some treatments i.e. Compost (A), Compost (B), P. fluorescens, T. harzianum, F. solani + Compost (A), F. solani + Compost (B), F. solani + P.fluorescens, F. solani+ T.harzianum, R. solani + Compost (A), R. solani + Compost (B), R. solani + P. fluorescens, R. solani + T.harzianum, S. rolfsii + Compost (A), S. rolfsii + Compost (B), S. rolfsii + P. fluorescens and S. rolfsii + T.harzianum resulted an increased gradually in chitinase activity after 6, 9 and 12 days after transplanting. The other treatments i.e. F. solani, R.solani, S. rolfsii, P.ultimum, resulted declined gradually in chitinase activity after 6, 9 and 12 days after transplanting.

β.1, 3-glucanase enzyme

The dramatic induction of β.1, 3-glucanase enzyme activity in all the treatments compared with the

control cucumber was observed during the first 3 days after transplanting and the levels of β.1, 3-glucanase activity peaked at 3 days (Table 5). However, after 3 days of transplanting, some treatments i.e. compost A, compost B, F. solani + compost A, F. solani + compost B, F. solani + P. fluorescence, F. solani + T. harzianum, R. solani + compost A, R. solani + compost B, R. solani + P. fluorescens, R. solani + T. harzianum, S. rolfsii + compost A, S. rolfsii + compost B, S. rolfsii + P. fluorescens, S. rolfsii + T. harzianum, P. ultimum + compost A, P. ultimum + compost B, P. ultimum + P. fluorescens and P. ultimum + T.harzianum resulted an increased gradually in β.1, 3-glucanase activity after 6, 9 and 12 days of transplanting. The other treatments i.e. F. solani, R.solani, S. rolfsii, P.ultimum, P. fluorescens and T. harzianum resulted declined gradually in β.1, 3-glucanase activity after 6, 9 and 12 days after transplanting. Table 4.Effect of compost and/or biocontrol agents amendments on chitinase activity in cucumber plants grown under artificial infestation

with F. solani, R. solani , S. rolfsii and P. ultimum.

Treatment Chitinase activity* /g fresh weight/60min.



(*) Chitinase activity was expressed as mM N-acetyl glucose amine equivalent released/ gram fresh weight/ 60 minutes.

Electrophorotic studies:

The electrophoretic data (Fig. 1. and Table 6) showing the protein profiles (protein fingerprints) of the total proteins of composts - and biocontrol agents - treated cucumber plants under artificially infection conditions with cucumber root-rot pathogens as well as the control plants using SD-PAGE. Data revealed that the protein fingerprints distinguished into 40 discreted protein bands and their molecular weight was in the range of 6.5 to 260.0 KDa. The protein bands (29 bands) i.e. 125.0, 120.0, 93.0, 91.0, 88.5,


82.0, 79.0, 75.0, 72.0, 65.0, 57.0, 52.0, 47.0, 42.0, 39.0, 35.0, 31.0, 30.5, 30.0, 28.0, 26.5, 24.0, 22.5, 20.0, 12.0, 11.5, 8.0, 7.5 and 6.5 KDa were the common protein bands in all protein profiles of treated cucumber plants as well as the control plants. On the other hand, the protein bands (10 bands) i.e.166.0, 80.0, 60.0, 59.0, 51.0, 33.0, 32.5, 32.0, 16.0 and 13.5 KDa were detected in protein profiles of some treated cucumber plants, while not detected in the profile of control plants. The protein band of 260.0 KDa only was detected in the protein profile of control plants as well as in some treated cucumber plants. Table 5.Effect of compost and/or biocontrol agents amendments on β.1, 3-glucanase activity in cucumber plants grown under artificial


Treatment β.1,3-glucanase activity*/g fresh weight/60min.



(*) β.1,3-glucanase activity was expressed as mM glucose equivalent released/ gram fresh weight/ 60 minutes.

Fig. 1. SDS-PAGE analysis for PR-proteins profile induced by biocontrol agents and/or compost in cucumber plants challenged with or

without root-rot pathogens. (a) Line M, molecular mass markers; line1, control plants; line 2, F.solani; line 3, R.solani; line 4, S.rolfsii and line 5, P.ultimum.(b) line 1, compost A; line 2, compost A + F.solani; line 3, compost A + R.solani; line 4, compost A + S.rolfsii and line 5, compost A + P.ultimum. (c) line 1, compost B; line 2, compost B + F.solani; line 3, compost B + R.solani; line 4, compost B + S.rolfsii and line 5, compost B + P.ultimum. (d) line 1, P. fluorescens; line 2, P. fluorescens + F.solani; line 3, P. fluorescens + R. solani; line 4, P. fluorescens + S.rolfsii and line 5, P. fluorescens + P.ultimum. and (e) line 1, T. harzianum; line 2, T. harzianum + F.solani; line 3, T. harzianum + R.solani; line 4, T. harzianum + S.rolfsii and line 5, T. harzianum + P.ultimum

1

C 2 3

C 4

C 5

C

KDs M C

a

1

C 1

C 1

C

b c d e

2 2 2 3

C

3

C

3

C

4

C

4

C

4

C

5

C 5

C

72.0

57.0

42.0

31.0

24.0

12.0

8.0

93.0 125.0 163.0 240.0

5 1

C

2

C

3

C

4

C

5

C


Table 6. Molecular weight (KDa) occurrence in protein profiles of cucumber plants treated with composts and/or biocontrol agents challenged with or without root-rot pathogens.

No M.W

Control

Pathogens Compost Biological control agents

Compost A Compost B P. fluorescens T. harzianum

Fs Rs Sr Pu - Fs Rs Sr Pu - Fs Rs Sr Pu - Fs Rs Sr Pu - Fs Rs Sr Pu

1 260.0 + + + - + + + + + + + + + + + + + + + + + + - + +

2 166.0 - + + - - + - - - - - - + - - - - - - + - - - - -

3 125.0 + + + + + + + + + + + + + + + + + + + + + + + + +

4 120.0 + + + - + + + + + + + + + + + + + + + + + + + + +

5 93.0 + + + + + + + + + + + + + + + + + + + + + + + + +

6 91.0 + + + + + + + + + + + + + + + + + + + + + + + + +

7 88.5 + + + + + + + + + + + + + + + + + + + + + + + + +

8 82.0 + + + + + + + + + + + + + + + + + + + + + + + + +

9 80.0 - - - - - - + + + + + + + + + + - - - + - - - - +

10 79.0 + + + + + + + + + + + + + + + + + + + + + + + + +

11 75.0 + + + + + + + + + + + + + + + + + + + + + + + + +

12 72.0 + + + + + + + + + + + + + + + + + + + + + + + + +

13 65.0 + + + + + + + + + + + + + + + + + + + + + + + + +

14 60.0 - - - - + + + - - - + - + + + + + + + + - - - - +

15 59.0 - - + - - - - - - - - - - - - - - + + + - - - - +

16 57.0 + + + + + + + + + + + + + + + + + + + + + + + + +

17 52.0 + + + + + + + + + + + + + + + + + + + + + + + + +

18 51.0 - - - - - - - - - - - - - - - - + - - - - - - - -

19 47.0 + + + + + + + + + + + + + + + + + + + + + + + + +

20 42.0 + + + + + + + + + + + + + + + + + + + + + + + + +

Table 6. continued No. M.W

Control

Pathogens Compost Biological control agents

Compost A Compost B P. fluorescens T. harzianum

Fs Rs Sr Pu - Fs Rs Sr Pu - Fs Rs Sr Pu - Fs Rs Sr Pu - Fs Rs Sr Pu

21 39.0 + + + + + + + + + + + + + + + + + + + + + + + + +

22 35.0 + + + + + + + + + + + + + + + + + + + + + + + + +

23 33.0 - - - - - + + + + - + + + + + + + - - - - - - - -

24 32.5 - - - - - + + + + - + + + + + + + - - + - + - - +

25 32.0 - - - - - + + + + - + + + + + + + - - + - + - - +

26 31.0 + + + + + + + + + + + + + + + + + + + + + + + + +

27 30.5 + + + + + + + + + + + + + + + + + + + + + + + + +

28 30.0 + + + + + + + + + + + + + + + + + + + + + + + + +

29 28.0 + + + + + + + + + + + + + + + + + + + + + + + + +

30 26.5 + + + + + + + + + + + + + + + + + + + + + + + + +

31 24.0 + + + + + + + + + + + + + + + + + + + + + + + + + 32 22.5 + + + + + + + + + + + + + + + + + + + + + + + + + 33 20.0 + + + + + + + + + + + + + + + + + + + + + + + + + 34 16.0 - + - - - + - - - - + - + - + + + - - + - + + + + 35 13.5 - - - - - + - + - - + - + + + + + - - + - + + + + 36 12.0 + + + + + + + + + + + + + + + + + + + + + + + + + 37 11.5 + + + + + + + + + + + + + + + + + + + + + + + + + 38 8.0 + + + + + + + + + + + + + + + + + + + + + + + + + 39 7.5 + + + + + + + + + + + + + + + + + + + + + + + + + 40 6.5 + + + + + + + + + + + + + + + + + + + + + + + + + Fs = F. solani, Rs = R. solani, Sr = S. rolfsii and Pu = P.ultimum.

Discussion

Cucumber plants, highly infected with damping-off and root-rot diseases caused by P. ultimum, S. rolfsii, R. solani and F. solani. In greenhouse experiment, potted soil amended with biocontrol agents and/or composts reduced cucumber root-rot disease incidence. Three main types of direct interactions between biocontrol agents and cucumber root-rot pathogens may be characterized: parasitism, antibiosis and competition for nutrients (Velazhahan et al., 1999; Saravanakumar et al., 2007; Wahyudi et al., 2011 and Olga et al., 2012). Disease control with compost is attributed to four factors: competition between beneficial organisms and the pathogen, antibiosis, parasitism and induced systemic resistance. Two classes of biological control mechanisms known as “general” and “specific” suppression have been described for compost-amended substrates (Bonanomi


et al., 2010). The “general suppression” phenomen is related to the total amount of microbiological activity in composts and is known to suppress pathogens such as Pythium spp. and Phytophthora spp. The second type of suppression, elicited by a specialist group of microorganisms capable of eradicating a certain pathogens such as R. solani, is referred to as “specific” suppression.

Recently however, another possible mechanism has been suggested by Inbar et al. (1994) namely, induced resistance in plants to fungal attack (Yedidia et al., 1999).

The current results, revealed that compost (A), compost (B), P. fluorescens and T. harzianum induction the levels of peroxidase, polyphenoloxidase, chitinase and β.1, 3-glucanase activities in cucumber plants. Our results also revealed that the treatments of compost (A), compost (B), P. fluorescens and T. harzianum effectively protected the cucumber from root-rot as judged by the markedly improved seedling survival rates at 3, 6, 9, 12 and 30 days after transplanting. This reduction in disease incidence may be due to an increase in defence-related enzymes such as peroxidase, polyphenoloxidase, chitinase and β-1, 3 glucanase (Kavroulakis et al., 2005). The oxidative enzymes play an important role in induced resistance by the oxidation of phenols to oxidized toxic products (quinine) which limite fungal activity. Peroxidases catalyze a number of reactions that fortify plant cell walls. These reactions include the incorporation of phenolics into cell walls and lignification and suberization of plant cell walls. On the other hand, the chitinase and β-1,3 glucanase enzymes play roles in plant defence against fungi by hydrolyze their cell wall. The amount of them significantly increase and play main role of defense reaction against fungal pathogen by degrading cell wall, because chitin and β-1,3-glucan is also a major structural component of the cell walls of many pathogenic fungi. These results also are in agreement with those obtained by Van Loon et al. (1998) and Abd El-Razik-Sahar et al. (2012).

The possible influence of the treatment with composts and/or biocontrol agents on the protein patterns of the healthy cucumber plant was also investigated. Our data revealed that the protein bands (10 bands) of 166.0, 80.0, 60.0, 59.0, 51.0, 33.0, 32.5, 32.0, 16.0 and 13.5 KDa were detected in protein profiles of some composts- and/or biocontrol agents- treated cucumber plants, while not detected in the control plants. It seems that the composts and/or biocontrol agents amended potted soil had slight influence on specific protein bands that resulted in some changes in the molecular weight and the protein amount of separated bands. There are recognized results in the activation of synthesis of pathogenesis-related (PR) proteins (Dowles, 1990). These results may be due to gene theory as reported by Harman et al. (2004) and Al-Sohaibani et al. (2011) which lead to changes in protein pattern. It was suggested that PR protein has specific function to the host pathogen interaction. The infected cucumber plants by P. ultimum, S. rolfsii, R. solani and F. solani and treated with composts and/or biocontrol agents were characterized with a different band. Some of these particular bands were not separated either in healthy plants or in the fungus alone. The present results could be considered as pathogenesis related protein, according to Dowles (1990) and Harman et al. (2004). They reported that species or strains of Trichoderma amended to the rhizosphere protect plants against infections including those of fungal pathogens, due to induction of resistance mechanisms similar to the hypersensitive response (HR), systemic acquired resistance (SAR), and induced systemic resistance (ISR) in plants. Also Abd El-Razik-Sahar et al. (2012) found that, electrophoratic analysis of extracts of healthy and diseased faba bean plants grown in composted soil at the rate of 10 gm/Kg soil and infested with the tested pathogens (R. solani and F. subglutinans; the causal of root-rot and F. oxysporum; the causal of wilt) indicated that a new proteins (MW 23.3 KDa), two chitinase (Chit-1 and Chit-6), one peroxidase (PRX-5) and one estrase (ESt-4) isoforms were induced by this treatment. References Abd-El-Kareem, F. 2009. Effect of acetic acid fumigation on soilborne fungi and cucumber root-rot disease

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